ISRAEL JOURNAL OF
VETERINARY MEDICINE
Vol.
59 (4)
2003
IS MYCOBACTERIUM AVIUM
SUBSP.PARATUBERCULOSIS AN
ETIOLOGICAL FACTOR IN CROHN'S
DISEASE?
Rudoler N.
Visit of Infectious Diseases, Soroka Medical
Center, Beer-Sheva, Israel
Abstract
Paratuberculosis is a chronic inflammation of the intestine in
animals caused by the slow growing and fastidious bacterium,
Mycobacterium avium subsp.paratuberculosis (MAP). The main
clinical features of the disease include chronic diarrhea, a drop in
milk yield, and progressive, afebrile, weight loss that leads to
emaciation. Crohn's disease is a chronic inflammation of the
intestine in humans whose etiology is unknown. Immunological
factors, infectious agents, genetic and environmental factors are
proposed as possible causes. Crohn's disease is characterized by
chronic diarrhea, abdominal pain, fever, anorexia and weight loss.
The pathological and clinical similarities of the two diseases have
raised the hypothesis that MAP may be involved in the
pathogenesis or is a direct cause of Crohn's disease.
In this literature review we summarize the published data on
the possible connection between MAP and Crohn's disease. The
review is sub-divided according to clinical and laboratory
findings. The clinical evidence is based on trials in which
antimycobacterial therapy was prescribed for Crohn's disease
patients. The laboratory evidence consists of several methods
(primarily PCR) used to identify MAP from Crohn's disease
patients. The accumulated data shows that there is no clear-cut
relationship between MAP and Crohn's disease. Prospective
studies are needed to test this hypothesis over a wider range of
possible etiologies that include combination of infectious,
immunological and genetic factors.
Introduction
Paratuberculosis (Johne’s disease) is a chronic digestive tract
disorder of both wild and domestic ruminants. The disease is
characterized by granulomatous enteritis that induces a progressive
weight loss and terminates in the animals' death. Paratuberculosis is
both a contagious and enzootic disease of ruminants caused by the
multiplication of a specific bacterium, Mycobacterium avium subsp
paratuberculosis (MAP) in the intestinal mucous membrane. The
infection can be transmitted by either direct or indirect contact of
infected animals with susceptible animals, and occurs mainly by the
fecal - oral route. Bacilli are ingested in large numbers most commonly
when young animals nurse on teats contaminated by feces of infected
animals. Infection is also caused by ingestion of contaminated feed and
water, the organism can also be shed in the colostrum and milk of
infected cows, and intrauterine infections have also been described.
Infection is acquired early in life, but clinical signs rarely develop in cattle
less than 2 years old. There appears to be an age-related resistance to
the development of new paratuberculosis infections. Animals infected in
the first months of life are the most susceptible, while adult animals are
quite resistant. Whether or not the exposed calf becomes infected
depends on the number of bacilli ingested and the host defense
mechanisms (1,2,3).
MAP is an obligate intracellular pathogen; it lacks the iron-chelating
compound mycobactin, and thus can only survive in a host that can
provide the iron required for growth (4). It is closely related to other
M.avium bacteria, sharing some antigenic determinants(1). MAP is
differentiated from the other mycobacterial species which are common
in the environment by its ability to cause bowel disease in cattle and
other animal species (3). The main distinguishing feature of MAP is its
slow growth and the dependency on exogenous mycobactin for in vitro
growth (1).
Crohn’s disease is a chronic inflammatory bowel disease of which
the cause or causes are not yet established. Chronic diarrhea with
abdominal pain, fever, anorexia, weight loss and a right lower quadrant
mass are the most common presenting features of Crohn’s disease.
Although primarily considered a disease of the small intestine it can
affect any part of the gastrointestinal tract and sometimes extraintestinal sites (the most commonly affected site is the distal ileum and
the colon). Current concepts regarding the cause of Crohn’s disease
emphasize a dysfunction of the immune system resulting in a prolonged
and intense process of inflammation. The damage to the bowel appears
to be due to this inflammatory process (3). The pathogenesis of Crohn’s
disease probably involves an interaction of genetic and environmental
factors, but the precise mechanism responsible for initiating chronic
intestinal inflammation remains unclear. Three theories of disease
mechanisms in Crohn’s disease are currently under consideration: (i)
reaction to a persistent intestinal infection, (ii) existence of a defective
mucosal barrier to luminal antigens, (iii) a dysfunctional host immune
response to ubiquitous antigens, cytokine imbalance and the breakdown
of tolerance to gut microbial flora. Cigarette smoking has also been
linked to the long term course of Crohn’s disease. It is possible that an
infectious agent is also involved in the etiology of Crohn’s disease, and
of those incriminated as possible causes, Campylobacter jejuni,
Campylobacter faecalis , Listeria monocytogenes,Escherichia coli,
Mycobacteria spp are prominent.
In 1913, Dalziel (4) reported several cases of chronic intestinal
enteritis in humans that were similar to intestinal tuberculosis but did not
feature acid-fast bacilli. He speculated that these human cases and
Johne’s disease (=paratuberculosis in ruminants) might have the same
etiology. In 1932, what is now known as Crohn’s disease was
distinguished from intestinal tuberculosis, but suspicion remained that it
might have a mycobacterial origin. Crohn’s disease in humans and
Johne’s disease have similar clinical and pathological features. Both are
characterized by chronic diarrhea and weight loss and by
granulomatous appearance of the diseased tissue. Interest has
remained high since 1984 when Chiodini repeated Ward Van Patter's
findings from the 1950’s that employed techniques to isolate MAP from
animals (4). Chiodini reported the isolation of uncharacterized
mycobacteria from tissues of three patients with Crohn’s disease (4).
The main way that humans become infected is by ingestion of infected
water or milk. Several reports have emphasized the crucial role of MAP
surviving high-temperature, short time pasteurization if they are present
in raw milk in sufficient numbers (more than 10 cells/ml) (5,6,7,8). In
addition, the epidemiology of the disease, whose incidence is rising in
western societies, concurrent with the low rates of Crohn’s disease in
developing nations over the second half of the 20th century and high
rates among immigrants to western societies, is consistent with the
possibility that a critical infection may be acquired from cattle or sheep
via milk or meat ingestion, and the emergence of Crohn’s disease in
patients with the appropriate genetic predisposition (9).
The aim of the article
The objective of the review is to examine the evidence that Crohn’s
disease is associated with MAP infection. The importance of this issue
to public health is clear: it is a pathogen with a prevalence rate of
infection ranging between 21 and 70% in herds of Western Europe and
North America (10). It can survive commercial pasteurisation conditions
in routine use (subclinically infected cows may secrete MAP in milk). It is
found in the potable water supply of large cities in industrialized
countries and also survives higher concentrations of chlorine (two parts
per million) than the 1.1 parts per million routinely present in first-use
municipal water in the USA (10,11). These issues are of extreme
epidemiological and clinical importance since specific anti-mycobacterial
therapy may improve the quality of life of Crohn’s disease patients.
Methods
The review was based on a PubMed search of publications between
1966 and 2004. Only articles that provided sufficient clinical or
laboratory information on the association between MAP and Crohn’s
disease were included.
The following key words were used: Mycobacterium paratuberculosis;
Crohn’s disease; etiology; therapy.
Results
The review will discuss the laboratory and clinical evidence. The
laboratory evidence includes 3 articles dealing with diagnosis by culture,
11 studies employing PCR as the method of diagnosis, 2 articles on
serological methods and 15 articles that present clinical evidence of a
link between MAP and Crohn’s disease.
Laboratory evidence
Cultural detection of MAP in Crohn’s disease
Three articles dealt with a possible connection between MAP and
Crohn’s disease and are based on culturing of the organism. In these
articles the culture times were very long (the shortest took 12 weeks
(9),the median was between 14 and 88 weeks (13), and the longest
yielded acid fast bacilli or spheroplasts after between 2 and 6 years
(12)). In the first study, seven breast milk samples comprising 2 Crohn’s
disease patients and 5 control mothers were cultured. All five control
samples were negative for MAP in all cultures, while MAP was isolated
from the cultures of the Crohn’s disease patients which were were also
confirmed by hybridization (9). In the second study (12), seven of 31
cultures were MAP-positive, and six of these were from Crohn’s disease
patients. Another positive culture of MAP was detected in a sample
taken from a non-inflammatory bowel disease patient. This positive
culture also grew acid-fast bacilli which were not MAP. The third article
that MAP was detected in 14 of 33 (42%) Crohn’s disease patients, and
3 of 33 (9%) of non-inflammatory bowel disease controls (13).
Detection of MAP in Crohn’s disease by PCR
The extremely long incubation periods and the very close similarity
with M.avium, (a species ubiquitous in the environment, and also in the
human gut), has stimulated the use of DNA probe technologies. The
discovery of IS900 , a species-specific, repeating DNA insertion element
in the MAP genome was an important step forward. The use of the
polymerase chain reaction (PCR) to amplify fragments of IS900 permits
rapid and accurate detection of MAP DNA from tissue without the need
for laborious and time consuming culture (14,15).The review dealing
with detection of MAP by PCR and culture includes 11 publications . We
have summarized them according to whether they support or disclaim
the involvement of MAP in Crohn’s disease.
Four of the 11 studies do not support the hypothesis that MAP is
involved in Crohn’s disease. In the study of Rowbotham and others,
samples of intestinal tissue were obtained from patients undergoing
either colonoscopy or surgical resection. These included 68 patients
with Crohn’s disease,49 patients with ulcerative colitis, and 26 non-
inflammatory bowel disease controls. In no case was MAP detected in
any of the inflammatory bowel disease tissue samples while only one
non-inflammatory bowel disease case was positive(14). In Suenaga and
others' study, three biopsy specimens were obtained from the
rectosigmoidal colonic mucosa using colonoscopy in 10 patients with
Crohn’s disease and 18 patients with ulcerative colitis. Sixteen noninflammatorybowel disease controls were also examined. IS900
sequences were detected in all of 10 patients with Crohn’s disease, in
11 of 18 patients with ulcerative colitis, and in 14 of 16 control patients
with non-inflammatory bowel disease (16).
In another article, mycobacteria were found in 17 out of 36 (47%)
patients with Crohn’s disease, 6 out of 13 (46%) in patients with
ulcerative colitis, and 13 out of 23(57%) control tissue samples. No MAP
was detected in any of the samples (17). In addition, other results which
failed to support the involvement of MAP stress in 25 specimens tested
from 21 Crohn’s patients: only 1 positive specimen was noted, whereas
the 8 specimens from the 5 ulcerative colitis patients and 11 samples
from control patients were negative by polymerase chain reaction. None
of the cultures grew MAP (18).
Five reports support the hypothesis that MAP is involved in Crohn’s
disease. In the first study, PCR was applied to DNA extracts of full
thickness samples of intestine removed at surgery from 40 patients with
Crohn’s disease, 23 patients with ulcerative colitis, and 40 control
patients without inflammatory bowel disease. MAP was identified in 26
of 40 (65%) Crohn’s disease, in 1 of 23 (4.3%) ulcerative colitis, and in 5
of 40 (12.5%) control tissues (19). PCR was also done on the positivecultured milk samples, and provided 99% homology with the IS 900
sequence (9). Dellísola and others used the IS 900 sequences of MAP
and found them in 13 of 18 samples (72%) from patients with Crohn’s
disease, in 1 of 5 with ulcerative colitis, in 2 of 6 with severe unclassified
colitis (20). Nine of 36 Crohn’s disease patients were found positive by
PCR in comparison with 3 of 43 patients with ulcerative colitis and noninflammatory bowel disease according to the study of Hulten and others
(21). The most convincing evidence concerning the link between MAP
and Crohn’s disease that was challenged by the PCR, is demonstrated
by Bull and others (13). In this study, MAP was detected in inflamed ileal
and colonic mucosae of Crohn’s disease patients in 92% of cases,
regardless of whether granulomata were found microscopically.
The last three studies employing PCR and culture as the method of
choice for detecting MAP did not reach a clear- cut conclusion
concerning the involvement of MAP in Crohn’s disease. Four of 31
Crohn’s disease tissues and none of 30 control and the ulcerative colitisderived tissues amplified MAP (15). The second study indicated that 6 of
18 of Crohn’s disease cultures and one of six of non-inflammatory bowel
disease control were MAP- positive. The intensity of the IS 900 PCR
signals indicated very low numbers of MAP organisms and bore no
relation to visible spheroplastic or bacillary mycobacterial growth (12).
The third study failed to identifiy M.avium in tissue samples taken from
Crohn’s disease patients but were identified in the control group,
however (22).
Several possible explanations for the discrepancies in PCR
experiments have been raised. The first is geographical variability in
MAP infections. It is conceivable that Japan, an area with a large
number of PCR-positive Crohn’s disease patients, has a high
prevalence of MAP enteritis. Another explanation is differences in the
PCR technique. A third reason for the discrepancy in PCR experiments
is sampling error. In addition, in situ sampling techniques, specifically
the presence of granulomata, have been implicated as a variable in the
detection of MAP by PCR (18). Granulomatous Crohn’s disease was
significantly more likely to contain MAP than non-Crohn’s disease tissue
controls. There are a number of reasons why Crohn’s disease tissues
with granulomata can contain more MAP DNA. The most obvious is that
that this organism causes Crohn’s disease in a subgroup ( 20%) of
patients with this histopathological appearance. Another possibility is
that granulomatous tissue is more likely to develop infection with this
organism but is not its cause (15).
Two articles (13,21)report that IS900 sequences were detected in
65% Crohn’s tissues and in 25% of children without inflammatory bowel
disease. A low prevalence rate of IS 900 DNA was detected in tissues
from controls suggesting an unexpected prevalence of this pathogen in
the alimentary tract of normal healthy humans. These studies also
suggested that MAP could be spread from infected cattle to humans
through ingestion of milk or water (16).
Serological detection of MAP in Crohn’s disease
MAP is very closely related to other MAC (Mycobacterium avium
complex) and organisms of M. intracellulare complex. MAC's are widely
distributed in the environment and can be isolated by fecal culture from
healthy persons. A serological study in 1980 attempted to identify
agglutination of three strains of MAP by means of Crohn’s disease sera.
No response was seen with two of the strains, while the agglutination
observed with the third MAP strain showed no difference between
Crohn’s disease and normal sera(1). Stainsby and others examined
whether a specific serum antibody response to mycobacteria occurs in
Crohn’s disease or ulcerative colitis. Sera from 38 patients with Crohn’s
disease, 15 with ulcerative colitis, and 30 healthy control population
were assayed in an enzyme immune linked immunosorbent assay
(ELISA). In addition, IgG, IgM, and IgA levels to MAP were determined
in sera from patients with active and inactive Crohn’s disease and the
control population. There was strong evidence of contact with
enviromental mycobacteria in all patients and control population , with
the greatest responses to M.avium, M.tuberculosis, and M.kansasii.
There were no differences in antibody levels to MAP in patient and
control groups . Although a subset of patients with active Crohn’s
disease (25%) had IgG concentrations that exceeded the control mean
by more than 2 SD, this finding may not be specific to Crohn’s disease
(24). In another study of the seropositivity rate among Crohn’s disease
patients, ulcerative colitis, healthy controls and unaffected siblings, the
researchers found no differences between the groups( 37.8%,
34.7%,33.6%,34.1% ) of the Crohn’s disease patients, ulcerative colitis,
healthy controls and the unaffected siblings,respectively (23).Both
sources agree that serological investigations cannot provide consistent
and convincing evidence of raised serum antibody levels to
mycobacteria in Crohn’s disease. The raised levels reported in some
studies may represent sensitivity as a result of exposure to ubiquitous
environmental strains of mycobacteria. It is well documented that
patients with Crohn’s disease often have an increased antibody
response to other microorganisms , including enterobacterial species
and Saccharomyces cerevisiae (23). As such contact with these
organisms and environmental mycobacteria is via the oral route,
presumably through a defective barrier function of the gut mucosa, it is
proposed that the organisms may be just “passing through”. Given the
taxonomic similarities between mycobacterial species, it is not surprising
that there is extensive cross-reactivity between MAP and both M.
kansasii and M.tuberculosis. In addition, it has been suggested that the
lack of a distinct immune response to certain mycobacterial antigens
may be the result of a lack of these antigens in the cell wall-deficient
forms thought to cause Crohn’s disease (24).
Clinical evidence
Treatment of Crohn’s disease by anti-mycobacterial drug
We found 15 articles that dealt with the effects of anti-MAP therapy
in the clinical course of patients with Crohn’s disease. Of these, 7 were
controlled trials ,3 were prospective open label trials, 2 were case
reports and 3 studies were unclassified (Table 1).
Clinical improvement as a result of successful treatment, may be
the best evidence for the existence of some link between MAP and
Crohn’s disease. The main parameter used to asses the clinical
condition in the above studies was a Crohn’s disease activity index. This
index was improved in 7 of 10 trials that used it.Only 3 trials (29,34,38)
reported lack of improvement or the absence of statistically significant
results between the control and active treatment groups. The main
criterion is variable length remission, and complete resolution was
achieved only in one case (36). Complete anatomical healing is difficult
to achieve because this needs specific drug combinations that are not
always well tolerated by the patients, the length of the treatment, and
the patient's characteristics. A longer period of treatment is generally
necessary for cure of some mycobacterial diseases, while multiple drug
resistance commonly develops during treatment. Also Johne’s disease
of cattle is incurable to date (30). Another possible explanation for anti-
MAP therapy to induce remission could be due to the possibility that
Crohn’s disease has several causes involving an interaction between
infectious or environmental triggers, the immune response and genetic
susceptibility. MAP may cause Crohn’s disease in only a proportion of
patients while some unknown etiology is responsible for initiating
disease in other patients (25). In contrary to the above successful
results some claim that drug-induced clinical improvement of Crohn’s
disease should be interpreted with caution for several reasons. First,
antibiotics have been shown to be effective in controlling the activity of
Crohn’s disease. Second, as the course of the disease is variable and
“spontaneous” decrease of disease activity may be seen in 26-42% of
patients (31).
Discussion
In this review, we have tried to evaluate whether MAP is the etiologic
factor involved somehow in the pathogenesis of Crohn’s disease or acts only as
a microbial bystander . Those who support the view that MAP may be involved
or cause the disease claim that when treating Crohn’s disease patients with
specific anti-mycobacterial drugs, either clinical remission (25,28,,30,36,37,), a
decrease in Crohn’s disease activity index (26, 27, 28, 30, 33, 39, 41) or
complete resolution (37) can be achieved. In addition, in Crohn’s disease
patients, MAP DNA has been detected by PCR in up to 90% of cases, by in-situ
hybridization in 70% of cases, and by RT-PCR for MAP RNA in 100% of cases
(21,42). MAP has also been cultured from human milk (12), feces, intestinal
tissues, and peripheral blood of patients with Crohn’s disease (43). A recent
development is attributed to the finding of NOD2, a family of cytosolic proteins
implicated in intracellular recognition of bacterial components. This finding
indicates that the presence of certain bacteria in the face of permissive NOD2
variants (as they occur in some Crohn’s disease patients) permits the
establishment of a persistent mycobacterial infection and the development of
Crohn’s disease (43).
In contrary to these arguments, there are some other propositions dealing
with the role of MAP in Crohn’s disease. First, there are competing etiologies
for Crohn’s disease, such as genetic predisposition, enviromental factors within
the gut related to dietary factors or the microbiological enviroment and an
abnormal immune reaction. For the infectious disease hypothesis to be accepted
for any organism, Koch’s postulates need to be fulfilled. To date, there is no
evidence that high risk groups susceptible to the disease (e.g. families of cattle
and sheep farmers, abattoir workers, etc) exist with a higher incidence of
Crohn’s disease. Another claim is that MAP has been isolated from individual
cases of Crohn’s disease whereas it has been grown from individuals without
the disease (10,16). Other arguments include failure to detect MAP markers in
Crohn’s patients when compared to ulcerative colitis patients (10,14 ,16,17) and
failure of clinical improvement in patients treated with anti-mycobaterial drugs
(14, 31, 34, 35, 38). There are pathological similarities between Johne’s disease
and Crohn’s disease but there are also many features that are absent (1) . Van
Kruiningen (10) suggests that the closest human disease to Johne’s disease is
the Mycobacterium avium intracellular infections of AIDS patients, rather than
Crohn’s disease. According to the above sources (5,6,7,8,9,11,23) the potential
exists of MAP as an emerging zoonosis which is transmitted through milk,
undercooked beef and water. Three articles (3,11, 42) that stress the steps taken
in the UK to minimize the entry of MAP into the food chain and the policy that
the USA should consider this threat have been published. At present, most
agree that Crohn’s disease is best considered as a syndrome with different
etiologic factors with similarities in presentation (1,43). The organism is
relatively common and it is likely that many people have come into contact
with it. The organism may have a role either as a causal agent , a secondary
invader which exacerbates the disease, or as a non-pathogenic colonizer
because of changed bowel conditions (1).
Up to date, the possible link between MAP and Crohn’s disease as an
etiological factor of the disease has not been confirmed. There is need of better
prospective studies to elucidate this connection. According to these findings, it
is impossible to recommend any specific anti-MAP therapy to Crohn’s disease
patients.
LINKS TO OTHER ARTICLES IN THIS ISSUE
References
1. Possible links between Crohn’s disease and paratuberculosis. Report of the
scientific committee on animal health and animal welfare, Europe. eu.int/
comm/ food/ fs sc/ scah/ out_38 pdf.
2. Johne’s disease (paratuberculosis); The Merck Veterinary Manual, eighth
edition, Merial, p.537-539, 1998.
3. Mycobacterium paratuberculosis: Does it contribute to Crohn’s disease?
Food Safety Authority of Ireland, www.fsai.ie/publications/other/myco.pdf.
4. El-Zaatari F.A.K, Osato S.M and Graham D.Y. Etiology of Crohn’s disease:
the role of Mycobacterium avium paratuberculosis. Trends in molecular
medicine, 2001.
5. Grant I.R, Hitchings E.J, McCarthney A. Effect of commercial -scale hightemperature, short-time pasteurisation on the viability of M.Paratuberculosis in
naturally affected cow’s milk. Applied and environmental microbiology, 2002;
68:602-607.
6. Miller D., Ford J., Senderson J. IS900 PCR to detect M.Paratuberculosis in
retail supplies of whole pasteurized cows’ milk in England and Wales. Applied
and environmental microbiology, 1996; 62:3446-3452.
7. Sung N. and.Collins M.T., Thermal tolerance of M. Paratuberculosis.
Applied and environmental microbiology, 1998; 64:999-1005.
8. Grant I.R, Ball H.J, Neil S.D and Rowe M.T. Inactivation of
M.paratuberculosis in cows' milk at pasteurization temperatures. Applied and
enviromental microbiology, 1996; 62:631-636.
9. Naser S.A, Schwartz D., Shafran I. Isolation of Mycobacterium avium subsp
paratuberculosis from breast milk of Crohn’s disease patients.
AJG,2000;95:1094-1095.
10. Hermon-Taylor J., Quirke P. Mycobacterium avium subspecies
paratuberculosis is a cause of Crohn’s disease. Gut 2001; 49:755-760.
11. Greenstein R.J. Is Crohn’s disease caused by Mycobacterium? Comparisons
with leprosy, tuberculosis, and Johne’s disease. The Lancet infectious diseases
2003; 3:507-514.
12. Gross M.T, Sanderson J.D, Tizard M.L.V, Hermon-Taylor J.,.El-Zaatari
F.A.K.,Maresich D.C,.Graham D.Y. Polymerase chain reaction detection of
Mycobacterium paratuberculosis and Mycobacterium avium subsp silvaticum
in long term cultures from Crohn’s disease and control tissues. Gut, 1992;
33:1209-1213.
13. Bull T.J,.McMinn E.J, Sidi-Boumedine K.,.Skull A., Durkin D., Neild P. et
al. Detection and verification of Mycobacterium avium subsp. Paratuberculosis
in fresh ileocolonic mucosal biopsy specimens from individuals with and
without Crohn’s disease. JCM, 2003; 41 :2915-2923.
14. Rowbotham D.S, Mapstone N.P, Trejdosiewicz L.K ,.Howdle P.D, Quirke
P., Mycobacterium paratuberculosis DNA not detected in Crohn’s disease
tissue by fluorescent polymerase chain reaction. Gut ,1995; 37:660-667.
15. Fidler H.M, Thurell W., McI Johnson N.,.Rook G.A,.McFadden J.J.
Specific detection of Mycobacterium paratuberculosis DNA associated with
granulomatous tissue in Crohn’s disease. Gut, 1994;35:506-510.
16. Suenaga K., Yokoyama Y., Okazaki K.,.Yamamoto Y. Mycobacteria in the
intestine of Japanese patients with inflammatory bowel disease. AJG ,1995;90:
76-80.
17. Dumonceau J.M,. Gossum A.V, Adler M., .Fonteyene P.A,.Vooren J.P.V,
Deviere J. No Mycobacterium paratuberculosis found in Crohn’s disease using
the Polymerase Chain Reaction. Digestive Diseases and Sciences, 1996; 41:
421-426.
18. Clarkston W.K,.Presti M.E,.Petersen P.F,.Zachary P.E, Fan W.X,.Leonardi
C.L et al., Role of Mycobacterium paratuberculosis in Crohn’s disease. Dis
Colon Rectum, 1998; 41: 195-199.
19. Sanderson J.D, Moss M.T, Tizard M.L.V,.Hermon-Taylor J.
Mycobacterium paratuberculosis DNA in Crohn’s disease tissue. Gut, 1992;
33:890-896.
20. DellÍsola B., Poyart C., Goulet O. ,.Mougenot J.F, Sadoun-Journo E.,
Brousse N. et al. Detection of Mycobacterium paratuberculosis by Polymerase
Chain Reaction in children with Crohn’s disease. JID ,1994;169:449-451.
21. Hulten K.,.El-Zimaity H.M.T,.Karttunen T.J,.Almashrahrawi A., Schwartz
M.R, Graham D.Y et al. Detection of Mycobacterium avium subspecies
paratuberculosis in Crohn’s disease tissues by In Situ Hybridization. AJG,
2001; 96:1529-1535.
22. Bernstein C.N, Nayar G., Hamel A., Blanchard J.F, Study of animal-borne
infections in the mucosae of patients with inflammatory bowel disease and
population —based controls. JCM , 2003 ;41, :4986-4990.
23. Bernstein C.N,.Blanchard J.F, Rawsthorne P., Collins M. Population-based
case control study of seroprevalence of Mycobacterium paratuberculosis in
patients with Crohn’s disease and ulcerative colitis. J. clin. Microbiol., 2004;
42:1129-1135.
24. Stainsby K.J, Lowes J.R, Allan R.N,.Ibbotson J.P, Antibodies to
Mycobacterium paratuberculosis and nine species of environmental
mycobacteria in Crohn’s disease and control subjects. Gut, 1993; 34:371-374.
25. Borody T.J, Leis S.,.Warren E.F, Surace R. Treatments of severe Crohn’s
disease using antimycobacterial triple therapy-approaching a cure? Digest.
Liver Dis. 2002; 34:29-38.
35. Jarnerot G., Rolny P., Wickbom G., Alemayehu G. Antimycobacterial
therapy ineffective in Crohn’s disease after a year. The Lancet, 1989; 164-165.
36. Schultz M.G, Reader H.L, Hersh T., Riepe S. Remission of Crohn’s disease
with antimycobacterial chemotherapy. The Lancet, 1987; 1391-1392.
37. Graham D.Y, Al-Assi M.T, Robinson M. Prolonged remission in Crohn’s
disease following therapy for Mycobaterium paratuberculosis.
Gastroenterology,1995;108:A826.
38. Woodgame R.W, Kimball k., Akram S, Graham D.Y, Ou C.N. Randomized
controlled trial of clarithromycin and ethambutol in the treatment of Crohn’s
disease. Gastroenterology,1999;116(suppl):A725.
39. Borody T J, Pearce L,Bampton P A ,Leis S. Treatment of severe Crohn’s
disease using rifabutin-macrolide-clofazimine combination:interim report.
Gastroenterology,1998;114:A938.
40. Behr M.A, Semret M., Poon A., Schurr E. Crohn’s disease, mycobacteria,
and NOD2. The Lancet infectious diseases, 2004; 4:136-137.
41. Greenstein R.J, Collins M.T. Emerging pathogens: is Mycobacterium avium
subspecies paratuberculosis zoonotic? The Lancet ,2004;364:396-397.
42. Borgaonkar M.R, MacIntosh D.G, Fardy M.J.A meta-analysis of
antimycobacterial therapy for Crohn’s disease. AJG,2000;95:725-728.
43. Hulten K, Almashhrawi A., El-Zaatari F.A.K, Graham D.Y. Antibacterial
therapy for Crohn’s disease: a review emphasizing therapy directed against
mycobacteria. Digestive diseases and Sciences, 2000; 45: 445-456.