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Mycobacteria and the aetiology of Crohn's disease.

J. Hermon-Taylor, M. Tizard, J. Sanderson, K. Kempsell, N. Sumar, D. Milar, M. Loughlin, J. Ford and S. Withey.

"Inflammatory Bowel Disease", 1994.

Contents.

Introduction.

Crohn's disease (CD) emerged perceptibly in Western Europe and North America in the late 1940s and early 1950s. The incidence then increased progressively in both continents to a level which in some areas such as northeast Scotland (11.6/100,000 per year), now approaches that of an epidemic. With a substantial proportion of research in this field currently engaged in the experimental pursuit of disease mechanisms, our real knowledge of the principal underlying causes of CD, means of prevention and specific chemotherapy are all hopelessly inadequate. Here we consider experimental and other evidence which, although as yet incomplete, suggests that CD is mostly "paratuberculosis" in humans, a chronic inflammation of the intestine due to infection by the specific chronic enteric pathogen Mycobacterium paratuberculosis. The disease has features in common with tuberculoid leprosy.

Since its first description in 1895 as the cause of chronic enteritis in a German cow, Mycobacterium paratuberculosis has been shown to be capable of causing chronic inflammation of the intestine in many species including primates. Its involvement in chronic inflammation of the intestine in humans, proposed in 1913, should not come as much of a surprise.

What is "paratuberculosis"?

"Paratuberculosis" is chronic inflammation of the intestine caused by the specific chronic enteric pathogen Mycobacterium paratuberculosis. This mycobacterium has a very broad host range and high degree of tropism, causing chronic enteritis experimentally even when administered intravenously. Its name suggests a close similarity with "tuberculosis", but an examination of its biology and host relationships emphasizes that this is far from being so.

The natural habitat for mycobacteria is in the environment, where they have probably been for some 2 billion years or more. Mycobacterium tuberculosis, which remains a contemporary threat to animal and human populations, is well known to us as it has evolved into an obligate pathogen. It has become unable to survive and replicate successfully in the environment and is instead dependent upon a temperature-regulated existence within animal and human bodies. In general our cells have a malignant, kill-or-be-killed relationship with this organism. The distancing of Mycobacterium tuberculosis from its harsh environmental origins over millions of years has resulted in it becoming susceptible to physical stress and the offensive chemical products of its former near-neighbour environmental competitors. This in turn results in the virtually ensured destruction of Mycobacterium tuberculosis during pasteurization or by antimycobacterial agents such as those from its environmental relative streptomyces, unless resistance in Mycobacterium tuberculosis is re-established as a result of inappropriate or prolonged exposure to these compounds.

Mycobacterium paratuberculosis on the other hand, has remained within the principal original body of environmental mycobacteria. It can survive and probably replicate in the soil, on the surface of plants, and in ground and river waters. A local amplification cycle consisting of multiplication in susceptible intensely farmed domestic herds, contamination of pastures and drinking troughs, reingestion by adults and the infection of successive generations of young animals, occurs. The relatively benign relationship between Mycobacterium paratuberculosis and the cells of the host, the ability of Mycobacterium paratuberculosis to exist in tissues without causing disease and the prolonged interval of abundant faecal shedding before clinical disease becomes obvious, are characteristic of "paratuberculosis" in temperate climates and serve to diminish awareness of the scale of the problem.

Environmental mycobacteria in temperate regions are manifestly able to survive the climatic range of heat and cold, light and dark, wet and dry, acid and alkali. The destruction of Mycobacterium paratuberculosis in milk by pasteurization is by no means assured. If Mycobacterium paratuberculosis was readily susceptible to streptomyces products such as rifamycins, cycloserine, streptomycin, kanamycin or erythromycin, it is unlikely that it would be with us now. The chemotherapeutic eradication of "paratuberculosis" from sick animals, prohibited in any case by cost, is vitually impossible using the present range of antimycobacterial drugs.

"Paratuberculosis" in animals.

The classical picture of "paratuberculosis" of Johne's disease in animals, particularly cattle, deer and some sheep, is characterized by the visible presence in the diseased intestine of millions of acid-fast bacillary form Mycobacterium paratuberculosis together with macrophages, but little additional inflammatory cell infiltrate. Mycobacterium paratuberculosis can be isolated from the gut or faeces of about two-thirds of sick animals after 2-4 months in conventional in-vitro culture. This classical picture of "paratuberculosis" in animals is, however, far from constant, and paucibacillary (pauci is latin for "few") forms occur characterized by the absence of visible mycobacteria in the affected tissues, an exaggerated granulomatous inflammatory response, and the virtual inability to isolate Mycobacterium paratuberculosis by culture from the tissues or faeces. This is particularly seen in sheep in dry climates, as well as in other species. This pluribacillary/paucibacillary range in the clinicopathological spectrum of "paratuberculosis" in animals is compellingly reminiscent of the extremes represented by the lepromatous and tuberculoid forms of leprosy in humans.

The conventional recognition of disease caused by Mycobacterium tuberculosis in animals and humans is in general ultimately dependent upon the isolation of the reponsible pathogen by laboratory culture with a variety of liquid or solid media routinely available for this purpose. Identification of the specific organism depends upon the achievement of its bacillary-form with intact lipid-rich cell walls forming visible colonies with a recognizable morphology. With Mycobacterium tuberculosis this can usually be achieved within a few weeks. Mycobacterium paratuberculosis by contrast is far more fastidious, requiring shallow liquid media, or solid media enriched with additional iron-scavenging proteins such as mycobactin and other special conditions. Where growth does occur it may take many months or years to achieve a bacillary form that can be characterized by conventional means. In many cases of "paratuberculosis" in animals the organism cannot be isolated at all.

"Paratuberculosis" in humans.

The usual ways of establishing that a particular microorganism causes an illness are the ability to see it microscopically within diseased tissue, to culture it in the laboratory from affected individuals, to identify characteristic immunological responses to it and to cause a similar illness by administering the organism experimentally. As with tuberculoid leprosy, few of these methods work for Mycobacterium paratuberculosis in humans. Acid-fast Mycobacterium paratuberculosis are not visible in CD tissue or granulomata. They are also very difficult to see in natural paucibacillary "paratuberculosis" in animals. Months or years are needed for the most experienced laboratories in the field to culture recognizable bacillary-form Mycobacterium paratuberculosis from CD tissues, and then only at most in up to about 3% of cases. Immunoassays using purified extracts of in-vitro cultured Mycobacterium paratuberculosis containing hundreds of different epitopes are unable to distinguish responses to Mycobacterium paratuberculosis from the inevitable immune recognition of very closely related environmental Mycobacterium avium-intracellulare. These organisms do not cause chronic enteritis in immunocompetent hosts and are common inhabitants of the healthy animal and human intestine. When eventually cultured from a few cases of chronic enteritis in humans, bacillary-form Mycobacterium paratuberculosis do indeed cause chronic enteritis when given to animals. This is, however, only to be expected, as veterinary research has repeatedly demonstrated over many years.

Difficulties with the laboratory culture of Mycobacterium paratuberculosis and its differentiation from closely related Mycobacterium avium species are the reasons why research into diseases caused by this potential pathogen has made such limited progress over many years. The advent of DNA-based methods for detecting microorganisms using highly specific target DNA sequences and amplification procedures of exquisite sensitivity has fragmented this experimental stalemate.

Polymerase chain reaction (PCR) and other techniques such as ligase chain reaction (LCR) are methods for the amplification of target DNA millions of fold. IS900 is a DNA insertion element, the first to be identified and characterized in mycobacteria. It has so far proved highly specific for Mycobacterium paratuberculosis which has about 18 copies conveniently integrated into its genome. PCR assays based on IS900 using carefully selected oligonucleotide primers and reaction conditions can detect a single Mycobacterium paratuberculosis organism. This extreme sensitivity is reduced two or three orders of magnitude when IS900 PCR is required to work in the Sargasso Sea of competing human DNA which, together with other inhibitory factors, characterizes the microenvironment of unselected DNA extracts of human intestine in the test. With a detection limit in the range 300-1000 Mycobacterium paratuberculosis per gram of tissue, IS900 PCR demonstrates this chronic enteric pathogen in the diseased intestine of two-thirds of a sample population of people with CD in southern England, in 12% of non-IBD controls and 4.3% of people with ulcerative colitis. The predominance in CD is highly significant (chi-squared = 0.0001). Other PCR assays we have developed based in the 32 kDa antigen for mycobacteria in general, show no difference between CD and histologically normal intestine.

Direct PCR assays based on IS902, specific for the other known mycobacterial chronic enteric pathogen Mycobacterium avium subsp. silvaticum, do not identify this organism at all. IS902 PCR performed on DNA enriched for target sequences by hybridization capture from the initial intestinal DNA extracts, reveals Mycobacterium avium subsp. silvaticum about equally (12-18%) in CD, ulcerative colitis (UC) and non-inflammatory bowel disease (nIBD) samples. Taken together these data suggest that the presence of Mycobacterium paratuberculosis in low abundance in the substantial majority of CD patients is specific to the disease, with a causal relationship strongly implied by its known broad host range and well established enteric pathogenicity. Further experimental evidence is likely to derive from the parallel application of immunological tests using chemically defined epitopes on components which are also highly specific for Mycobacterium paratuberculosis and expressed in it spheroplast state.

An unexpected finding in our carefully performed studies was the presence of Mycobacterium paratuberculosis in the colon of about 12% of people without IBD. How are we being exposed to this organism? Milk supplies and waters draining from heavily grazed pastures are obvious places to look. Reliable experimentally evidence in relation to pasteurized milk supplies througout central and southern England, using meticulously applied DNA-based methodologies, is accumulating.

Recognition and reversal of the "paratuberculosis problem".

The emerging picture is consistent with a simultaneous and progressive amplification of Mycobacterium paratuberculosis in Western Europe and North America during this century, the reasons for which we do not understand. This has led to there being a larger pool of these chronic enteric pathogens in our domestic livestock than hitherto suspected or recognized. Mycobacterium paratuberculosis is being carried to the human population in certain foods and in some areas in water supplies. Most of us will have at some time been exposed to these agents as benign and possibly transient intestinal or respiratory cohabitants. But in those of us with an inherited susceptibility (well known in CD), an intercurrent microbial collaborating coinfection (well known in the initiation of CD) or in whom permissive conditions are created by psychological factors mediated through the neuroendocrine and immune systems in the gut (well known with the emergence of Johne's disease in stressed animals), the benign enteric cohabitation of Mycobacterium paratuberculosis converts to parasitism and the development of clinical disease.

Because of the special ambiguity of the Mycobacterium paratuberculosis-host relationship in terms of pathogenicity, the clinical balance of the resulting chronic inflammatory disease swings with episodes of exacerbation and remission, particularly before the disease progression becomes further established due to the consequential effects of irreversible tissue damage. During this early period substantial but usually temporary clinical improvement may be achieved by immune modulation in a manner which would be disastrous in the contrasting kill-or-be-killed host relationship with Mycobacterium tuberculosis.

If the reasoning and interpretation of the currently available reliable experimental and other evidence considered here is mostly correct, the progressive reversal of the "paratuberculosis problem" will require the involvement of several agencies. These would include those responsible for agriculture, environmental monitoring, food safety, public health, human health services, and the veterinary and medical professions. At the centre of the response would be the academic and commercially funded science from which new precise diagnostics, effective vaccines and pharmaceuticals could be expected eventually to flow. The combined effect of remedial measures would, over some years, reverse the "paratuberculosis problem". Until this happens up to 4000 new people in Britain, and many more tens of thousands especially elsewhere in Europe, North America and the equivalent temperature zones in the southern hemisphere, will continue to each year to acquire the organism which some years later will ruin their lives, hurt their families and cost their overburdened health services millions.


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