Biopsy studies of Crohn's Disease Patients.

Obviously, if a micro-organism, such as a virus or bacterium, is the cause of a disease condition, then it should be possible to detect and isolate that micro-organism in the tissues of hosts infected with the disease. Attempts to isolate Mycobacterium paratuberculosis from the tissue of patients with Crohn's disease date back to the time when Crohn's disease was first recognized as a new disease by Thomas Kennedy Dalziel in 1913. Dalziel failed to isolate Mycobacterium paratuberculosis from his patients, and in 1932, because a bacterium could not be isolated from the patients, Crohn et al classified "Crohn's disease" as being an autoimmune disease.

First successful isolation

In 1984, Chiodini et al succeeded in isolating Mycobacterium paratuberculosis from the intestines of patients with Crohn's disease. They also threw light on the reasons why earlier researchers had failed to isolate the bacterium:- In humans, Mycobacterium paratuberculosis grows in a different form to that found in most animals. The form in which it grows in humans is the spheroplast form, which has no cell-wall (i.e. it is "Cell Wall Deficient"(CWD)), and is exceedingly difficult to culture. Only a handful of researchers have ever succeeded in culturing spheroplast Mycobacterium paratuberculosis, a feat which requires coaxing the bacterium to "revert" from the spheroplast form to the form normally isolated from animals, the bacillary form (also known as the "Cell Wall Intact" form). This "reversion" can only be achieved under extremely precise culture and decontamination conditions, and earlier researchers (e.g. Crohn et al) had failed to meet these stringent standards for culturing the bacteria. Also, the test that previous researchers had used to test for the presence of the bacterium, the Ziehl-Neelsen acid-fast stain test is incapable of detecting the spheroplast form Mycobacterium paratuberculosis.

Note that the spheroplast form of Mycobacterium paratuberculosis can also be found in sheep. Such sheep have OJD (Ovine Johnes Disease), but yet Mycobacterium paratuberculosis cannot be detected using the acid-fast stain test and in some of them, genetic testing with IS900 PCR also fails. See M. paratuberculosis infections in animals for more information.

The IS900 gene sequence

In 1991, researchers in England discovered a sequence of genes which is unique to Mycobacterium paratuberculosis. They termed this sequence the IS900 insertion sequence. If a bacterium tests positive for the IS900 sequence, then there is 100% certainty that the bacterium is Mycobacterium paratuberculosis. This discovery had three major consequences

1. Before the discovery of the sequence, it was never possible to identify whether or not a bacterium was Mycobacterium paratuberculosis or not, because Mycobacterium paratuberculosis is so similar to its mycobacterial cousins. The three strains of Mycobacterium paratuberculosis which Chiodini isolated from patients with Crohn's disease, strains Ben, Dominic and Linda, named for the Crohn's patients from whom they had been isolated, have since been tested with the IS900 sequence and they are all IS900 positive, i.e. they definitely are Mycobacterium paratuberculosis. As an aside, further testing has confirmed that strains Ben, Dominic and Linda are of the bovine subtype of Mycobacterium paratuberculosis, i.e. these strains originated in cattle.
2. It is no longer necessary to culture tissues to determine whether or not Mycobacterium paratuberculosis are present. If the IS900 sequence is found, then Mycobacterium paratuberculosis is there. Since culture of Mycobacterium paratuberculosis is an extemely difficult task, this represents a major advance in diagnostic techniques.
3. Mycobacterium paratuberculosis could be detected in extremely small numbers. The PCR techniques can detect as little as one bacterium. However, great care must still be taken when testing diseased tissues from animals or people, because the amount of host DNA can be greater than one million times the amount of MPTB DNA. Most of the host DNA must be discarded, but great care must be taken that the MPTB DNA is not also thrown away in the process, i.e. that "the baby is not thrown out with the bathwater".

First successful isolation of bacillary form from a human

In February 1998, Hermon-Taylor et al described the first successful isolation of bacillary form Mycobacterium paratuberculosis from a human. The patient in question was a young boy who developed a lump on his neck. The lump was surgically removed, and tested positive for Mycobacterium paratuberculosis, using both the IS900 sequence and Ziehl-Neelsen acid-fast staining. The boy went on to develop a clinical disease five years later which was indistinguishable from Crohn's disease. This provides further evidence that Mycobacterium paratuberculosis is in the process of crossing the species barrier into humans.

Kochs Postulates.

In dealing with infectious disease, the standard method of research is to apply Kochs Postulates to the disease. Basically, Kochs Postulates says that if

1. An organism can be extracted from the diseased tissue of a host suffering from a disease and
2. That organism can be cultured in the laboratory and
3. That organism causes disease when introduced into another previously uninfected host and
4. The organism can be re-isolated from that host

then that organism causes the disease, and the disease is an infectious disease.

Although there are many difficulties in relation to Crohn's disease, Mycobacterium paratuberculosis and Kochs postulates, as discussed below in Mycobacterial disease and Kochs Postulates, Kochs postulates have actually been fulfilled for Crohn's disease and Mycobacterium paratuberculosis on two separate occasions. For details, see the following abstracts

1. "Possible role of mycobacteria in inflammatory bowel disease. I. An unclassified Mycobacterium species isolated from patients with Crohn's disease."
2. "Experimental disease in young chickens induced by a Mycobacterium paratuberculosis isolate from a patient with Crohn's disease."

These fulfillments of Kochs postulates prove a cause/effect relationship between infection with Mycobacterium paratuberculosis and development of Crohn's disease.

Mycobacterial disease and Kochs Postulates.

It is difficult to apply Kochs postulates to mycobacterial diseases, for the following reasons.

1. There are two forms of mycobacteria. The first form is the "bacillary" form, which can be detected by Ziehl-Neelsen acid-fast staining, a simple chemical test. The second is the "spheroplast" form, which cannot be detected by Ziehl-Neelsen acid-fast staining. It can only be detected by searching for its genetic code with complex gene manipulation techniques. The "spheroplast" form is also sometimes referred to as the "cell-wall" deficient form, since it is missing the thick waxy cell wall that the more common bacillary form bacteria have. If disease is caused by spheroplast forms of mycobacteria, researchers may be searching for a mycobacterial genetic signal that makes up only one millionth, or less, of the volume of material they are searching.
2. Mycobacteria are notoriously difficult to culture. They are very slow growing, dividing as little as once a day. They also seem to have the ability of "suspending" their biological processes, effectively going into hibernation until conditions are again favourable for multiplication. It can take from two months to over a year to grow detectable quantities of some mycobacteria. Their nutritional requirements are complex and precise. Researchers have long sought the right nutritional mix. Mycobacteria seem also to be very sensitive to temperature, with many species preferring the temperature of the human body, 37 degrees C. The mycobacterium that causes leprosy, Mycobacterium leprae, has never been cultured in the laboratory. The only place it is known to grow outside the human body is in the footpads of the Armadillo, a south-american quadraped. Thus Kochs Postulates have never been fulfilled for Leprosy, a recognised infectious mycobacterial disease.
3. Inducing disease in another host using a mycobacterium can be a difficult task. The symptoms presented by infected hosts can be very different, since the symptoms are a direct result of the hosts immune reaction to the bacterium. Some hosts do not have any immune reaction, and go on to develop uncontrolled populations of the bacterium. This is the case for lepromatous leprosy and for miliary tuberculosis. Other hosts do have an immune reaction, one that contains the bacteria inside hard shells, called "granulomas" thus preventing the bacteria from further infecting the body. This latter group will have different symptoms, depending on the strength of the immune reaction, and the number of bacteria present. Yet other hosts will neither show immune reaction nor show symptoms of the disease. This is possible if there is a subset of the population that has a genetic "predisposition" to being affected by the bacteria. Hosts that do not have this genetic predisposition will not be affected by the bacterium in any way.

If you read the "Related information" links to the right of this page, please note that the paper "Crohn's disease and Mycobacterioses" was written in 1989, before the IS900 genetic sequence had been discovered. The discovery of this genetic sequence has revolutionized the identification of Mycobacterium paratuberculosis.

Genetic testing and Mycobacterium paratuberculosis.

Mycobacteria are difficult to find. If they exist in the "bacillary" form, where they have a cell wall, and they can be cultured successfully, then they can be identified using the Ziehl-Neelsen "acid-fast" staining technique. If they are in the "spheroplast" form, staining does not work and genetic techniques must be used. As mentioned earlier, the genetic code being searched for may constitute only a millionth of the total material being searched, or less. In 1991, a genetic sequence which is unique to M. paratuberculosis was discovered, the IS900 "insertion sequence". If DNA is found that matches this insertion sequence, then M. paratuberculosis is known to be present. However, the methods of testing for IS900 are widely open to interpretation. Each research group uses their experience and the equipment available to them to best implement the test. The success of the test varies widely. For example, one group from Japan found evidence for IS900 in 100% of patients with Crohn's Disease, and also found it in a large majority of control patients. On the other hand, two other research groups applied the technique to groups of 72 and 143 people, and found no evidence for IS900 in any of the people, but one. All of these studies concluded that M. paratuberculosis could not be the cause of CD, one because they found too much M. paratuberculosis, and the other two because they found too little.

Control groups.

When conducting studies to isolate an organism as a possible cause of disease, it is not enough proof to show that the organism can be isolated from sufferers of the disease. It must also be demonstrated that the organism cannot be isolated from "control" patients. "Control" patients are patients who are included in the study and who are known not to suffer from the disease. It is reasoned that if the organism is found in "control" patients, then it cannot be the cause of the disease, since it has not caused the disease in control patients. This reasoning does not apply if the sufferers have a genetic predisposition that makes them susceptible to the bacterium and the control patients do not have that genetic predisposition.

It is often the case that patients with Ulcerative Colitis are used as the control patients in studies of Crohn's Disease, as well as other patients who have required "resection" surgery, as a result of unrelated diseases. The fact that UC and CD are grouped together in this fashion reflects the fact that neither disease has ever been satisfactorily classified. Instead they are grouped together, because of the similarity of their symptoms, and generically known as "Inflammatory Bowel Disease".

Classification of mycobacteria.

The first mycobacterium that was discovered to cause disease was M. tuberculosis, the cause of the disease tuberculosis. At that time, all types of mycobacteria that were not M. tuberculosis were grouped together and simply called "atypical" mycobacteria. Knowledge of the various species has expanded over the years, and now many species are known. There has been confusion as to which species is which, and it is only with the advent of genetic testing that it has been possible to identify mycobacteria with certainty. In the paper "On the etiology of Crohn Disease", the researchers describe that a "control" bacterium that they were using in their search, which was named Mycobacterium avium, in fact was not Mycobacterium avium but was Mycobacterium paratuberculosis. The bacterium had been misclassified. Until the advent of genetic testing, it was never quite certain which mycobacteria were being dealt with.

Positive Post-IS900 studies.

Before the discovery of the IS900 genetic sequence, it was difficult to positively identify Mycobacterium paratuberculosis, because of its similarity to related mycobacteria. The PCR (Polymerase Chain Reaction) techniques used to detect the IS900 sequence are capable of detecting extremely small quantities of the gene sequence, and thus the only way of detecting Mycobacterium paratuberculosis when the bacterium is present in very small quantities. After the discovery of the sequence, the researchers who discovered it applied their techniques to the surgically removed intestines of Crohn's patients, Ulcerative colitis patients and control patients. They found that the bacterium was present in a substantial majority (65%) of Crohn's patients, in very few (4.3%) of Ulcerative Colitis patients and a small percentage (12.5%) of control patients. This led them to the conclusion that Mycobacterium paratuberculosis has some role in causing Crohn's disease. Since that time, their work has been replicated by six independent research groups in four countries.

Their conclusions were further strengthened by the discovery of Greenstein et al in 1996 that not only could Mycobacterium paratuberculosis DNA be found in Crohn's patients, but also Mycobacterium paratuberculosis RNA could be found. Since RNA is only produced by living organisms, and is chemically unstable (meaning it decays rapidly), this provides further evidence that infection with live Mycobacterium paratuberculosis is related to the development of Crohn's disease.

Negative Post-IS900 studies.

Three research groups have attempted to reproduce the finding of Mycobacterium paratuberculosis DNA in Crohn's patients and failed. One of the studies, conducted in Japan, found Mycobacterium paratuberculosis in 100% of Crohn's patients and also in a substantial proportion of control patients. The other two groups found hardly any Mycobacterium paratuberculosis DNA at all, with one group finding no MPTN DNA in 73 people, Crohn's and controls, and the other finding no MPTB DNA in Crohn's patients and in one control patient. The fact that these groups found no IS900 DNA at all in Crohn's patients is surprising, in light of the fact that six other independent research groups found that a significant majority of Crohn's patients did contain IS900 DNA as compared with controls, and leads one to question their experimental technique. Also, it must be remembered that absence of evidence is not evidence of absence.

Pre-IS900 studies.

• This paper was published in November 1984. It describes finding an "unclassified" mycobacterium which "may" be M. paratuberculosis. It reports a culture time for the organism of up eighteen months. "Characteristics of an unclassified Mycobacterium species isolated from patients with Crohn's disease."
• This paper was published in December 1984. It describes the isolation of an "unrecognized" mycobacterium, "closely related" to M. paratuberculosis, in bacillary form, from a Crohn's patient. This organism was cultured and caused a granulomatous disease when introduced into mice and a goat, but not in rats, guinea pigs, rabbits, or chickens. Note that inducing disease in a goat with the organism fulfills Kochs Postulates. "Possible role of mycobacteria in inflammatory bowel disease. I. An unclassified Mycobacterium species isolated from patients with Crohn's disease."
• This paper was published in August 1988. It describes the isolation of spheroplast mycobacteria, species unspecified, from intestinal tissue. The study population consisted of Crohn's, Ulcerative Colitis and non-IBD patients. By evaluating various culture media (i.e. food) for the mycobacteria, the researchers succeeded in growing the bacillus ("acid-fast" rods) form of the bacteria from the spheroplast form. "Progress in culture and subculture of spheroplasts and fastidious acid-fast bacilli isolated from intestinal tissues."
• This paper was published in September 1988. It does not find evidence for Mycobacterium paratuberculosis causing Crohn's Disease, but concludes that the test may not have been sensitive enough. "Investigation of mycobacteria in Crohn's disease tissue by Southern blotting and DNA hybridisation with cloned mycobacterial genomic DNA probes from a Crohn's disease isolated mycobacteria."
• This paper was published in April 1989. It uses immunological methods ( by using anti-bodies) to search for mycobacteria. The abstract mentions that they search for the spheroplast form of M. avium, but does not mention that they search for the spheroplast form of M. paratuberculosis. It finds no evidence for M. paratuberculosis in Crohn's patients. "Immunohistochemical examination for mycobacteria in intestinal tissues from patients with Crohn's disease."
• This paper was published in June 1989. It describes the isolation of five different species of mycobacteria from Crohn's patients. After culture times of 4 to 12 months, one was identified as a strain of M. chelonei and another as a strain of M. paratuberculosis. An attempt to induce disease in a goat with these organisms failed. "Preliminary report on isolation of mycobacteria from patients with Crohn's disease."

Source: http://archive.crohn.ie/biopsy.htm
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