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Kochs Postulates

Mycobacterial disease and Kochs Postulates

Genetic Testing and Mycobacteria

Control groups

Classification of mycobacteria

Positive Post-IS900 studies

Negative Post-IS900 studies

Pre-IS900 studies

 

Biopsy studies of Crohn's Disease Patients.

Obviously, if an 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.


  Related Information

Mycobacteria and Crohn's Disease: a historical perspective

History of Johne's Disease

Ruminant paratuberculosis:- Current Status and Future Prospects

Working Knowledge:- Polymerase Chain Reaction

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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.


  Related Information

Crohn's disease and the mycobacterioses:- Cultural data

Crohn's disease and the mycobacterioses:- Animal Model Data

Crohn's disease and the mycobacterioses:- Histochemical Data

Ruminant paratuberculosis:- Transmission and Mode of Infection

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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.


  Related Information

Crohn's disease and the mycobacterioses:- Discussion of Cultural data

Crohn's disease and the mycobacterioses:- Discussion of Animal Model Data

Crohn's disease and the mycobacterioses:- Discussion of Histochemical Data

Ruminant paratuberculosis:- Susceptibility and Resistance

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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.


  Related Information

Laboratory Diagnosis of Johne's Disease

Ruminant paratuberculosis:- Diagnostic testing

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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".


  Related Information

Ruminant paratuberculosis:- Susceptibility and Resistance

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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.


  Related Information

Laboratory Diagnosis of Johne's Disease

How are bacteria identified?

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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.

DateCountryDetails
1998 England Hermon-Taylor et al described the first ever isolation of bacillary form M. paratuberculosis from a human being. The patient in question was a seven year old boy, who developed a M. paratuberculosis infection in the lymph nodes of his neck, resulting in a lump on his neck. This lump was surgically removed, and on examination was found to contain large numbers of bacillary form M. paratuberculosis. This was confirmed by Ziehl-Neelsen acid-fast staining. The patient went on to develop an intestinal condition that was indistinguishable from Crohn's disease, after an incubation period of five years. Mycobacterium paratuberculosis Cervical Lymphadenitis followed five years later by terminal ileitis similar to Crohn's Disease.
1996 USA Since this paper is an important one, I have put it on a separate page, with comments. "On the etiology of Crohn disease".
1995 New Zealand This paper was published in 1995. "Mycobacterium paratuberculosis and inflammatory bowel disease: frequency distribution in serial colonoscopic biopsies using the polymerase chain reaction."
1994 Denmark This paper describes finding M. paratuberculosis in a higher proportion of CD patients than UC and non-IBD patients, and concludes that the subject is worthy of further study. "Mycobacterium paratuberculosis in intestinal tissue from patients with Crohn's disease demonstrated by a nested primer polymerase chain reaction."
1994 England This paper describes finding M. paratuberculosis in 4 out of 31 CD patients and none out of 30 UC and non-IBD patients. All of the positive CD samples contained granulomas, and none contained "acid-fast" (bacillary form) bacteria. "Specific detection of Mycobacterium paratuberculosis DNA associated with granulomatous tissue in Crohn's disease".
1994 France This paper describes the search for M. paratuberculosis in tissues surgically removed from 53 children with various disorders :-( The results are stated in the abstract. "Detection of Mycobacterium paratuberculosis by polymerase chain reaction in children with Crohn's disease."
1993 England This paper describes searching for various species of mycobacteria in 30 unidentified intestinal samples. M. paratuberculosis was identified in six of these samples, all from patients with Crohn's disease. Various "unidentified" mycobacteria were also found in all samples. "Identification of spheroplast-like agents isolated from tissues of patients with Crohn's disease and control tissues by polymerase chain reaction."
1992 England This paper was published in December 1992. It conjectures that M. paratuberculosis may be the cause of some cases of Crohn's Disease and that a related species, M. avium, might be the cause of some cases of Inflammatory Bowel Disease. "Mycobacteria in Crohn's disease: DNA probes identify the wood pigeon strain of Mycobacterium avium and Mycobacterium paratuberculosis from human tissue."
1992 England This paper was published by the team who discovered the IS900 sequence. It describes finding M. avium with equal prevalence in CD, UC and non-IBD patients. However, it finds M. paratuberculosis in one-third of CD patients, in no UC patients and in one-sixth of non-IBD control patients. Since the signals found were not strong, and since they were unsuccessful at visually identifying the organisms, they concludes that M. paratuberculosis exists in humans in a form that is difficult to detect and to culture. "Polymerase chain reaction detection of Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum in long term cultures from Crohn's disease and control tissues."
1992 England This paper was published by the team who discovered the IS900 sequence. It tests a large study population, comprising 40 CD patients, 23 UC patients and 40 non-IBD control patients. M. paratuberculosis DNA is found in 65% of Crohn's disease samples, in 4.3% of ulcerative colitis samples, and in 12.5% of non-IBD controls. It concludes that the reason for finding M. paratuberculosis in the control patients may be because M. paratuberculosis is endemic to the population, a fact which may have gone unnoticed. It concludes that M. paratuberculosis has a role in the aetiology of Crohn's Disease. "Mycobacterium paratuberculosis DNA in Crohn's disease tissue."
1991 England This paper describes the IS900 insertion sequence, and the genetic techniques which are capable of detecting it. It states that the techniques are not only highly effective at identifying M. paratuberculosis, but are also highly effective at discerning M. paratuberculosis from other closely related species of mycobacteria, such as M. avium. "Specific detection of Mycobacterium paratuberculosis by DNA hybridisation with a fragment of the insertion element IS900."


  Related Information

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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.

1996 Belgium This paper describes finding no M Paratuberculosis at all, out of a total group size of seventy two people. "No Mycobacterium paratuberculosis found in Crohn's disease using polymerase chain reaction."
1995 England This paper was published in November 1995. It describes finding M Paratuberculosis in one person out of a total group size of one hundred and forty three people, with that one being a non-IBD "control" patient. "Mycobacterium paratuberculosis DNA not detected in Crohn's disease tissue by fluorescent polymerase chain reaction."
1995 Japan This paper was published in January 1995. It describes how M Paratuberculosis was found in all tissues from Crohn's patients, but also in a large proportion of tissues from non-Crohn's patients. Since there is no statistically significant difference between the two groups, it concludes that M Paratuberculosis has no role in the aetiology of Crohn's disease. "Mycobacteria in the intestine of Japanese patients with inflammatory bowel disease."


  Related Information

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Pre-IS900 studies.


Source: http://archive.crohn.ie/biopsy.htm
Follow this link to the top level page
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  Related Information

Crohn's disease and the mycobacterioses:- Cultural data

Crohn's disease and the mycobacterioses:- Animal Model Data

Crohn's disease and the mycobacterioses:- Histochemical Data