Mycobacterium avium subspecies paratuberculosis and its relationship with Crohn's disease

The hypothesis postulating that Mycobacterium avium paratuberculosis (MAP) is the cause of Crohn's disease (CD) has been circulating for many years. Advances in molecular techniques, such as polymerase chain reaction and culture methods, have enabled researchers to demonstrate that there is an association between MAP and CD. Recently, genome-wide association studies have identified novel susceptibility genes for CD, which are critical for generation of an adaptive immune response that is protective against intracellular pathogens,including M. tuberculosis infection. However,the role of MAP as a cause of CD suffered a setbackwith the report that administration of antimycobacterial therapy failed to lead to a sustained response in CD patients. Accordingly, this review sought neither to confirm nor refute this, but instead to survey recent literature on the role of MAP in CD.     

Mycobacterium avium subspecies paratuberculosis causes Crohn's disease in some inflammatory bowel disease patients

Crohn’s disease (CD) is a chronic inflammatory condition that plagues millions all over the world. This debilitating bowel disease can start in early childhood and continue into late adulthood. Signs and symptoms are usually many and multiple tests are often required for the diagnosis and confirmation of this disease. However, little is still understood about the cause(s) of CD. As a result, several theories have been proposed over the years. One theory in particular is that Mycobacterium avium subspecies paratuberculosis (MAP) is intimately linked to the etiology of CD. This fastidious bacterium also known to cause Johne’s disease in cattle has infected the intestines of animals for years. It is believed that due to the thick, waxy cell wall of MAP it is able to survive the process of pasteurization as well as chemical processes seen in irrigation purification systems. Subsequently meat, dairy products and water serve as key vehicles in the transmission of MAP infection to humans (from farm to fork) who have a genetic predisposition, thus leading to the development of CD. The challenges faced in culturing this bacterium from CD are many. Examples include its extreme slow growth, lack of cell wall, low abundance, and its mycobactin dependency. In this review article, data from 60 studies showing the detection and isolation of MAP by PCR and culture techniques have been reviewed. Although this review may not be 100% comprehensive of all studies, clearly the majority of the studies overwhelmingly and definitively support the role of MAP in at least 30%-50% of CD patients. It is very possible that lack of detection of MAP from some CD patients may be due to the absence of MAP role in these patients. The latter statement is conditional on utilization of methodology appropriate for detection of human MAP strains. Ultimately, stratification of CD and inflammatory bowel disease patients for the presence or absence of MAP is necessary for appropriate and effective treatment which may lead to a cure.     

Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is a member of the M avium complex (MAC). It differs genetically from other MAC in having 14 to 18 copies of IS900 and a single cassette of DNA involved in the biosynthesis of surface carbohydrate. Unlike other MAC, MAP is a specific cause of chronic inflammation of the intestine in many animal species, including primates. The disease ranges from pluribacillary to paucimicrobial, with chronic granulomatous inflammation like leprosy in humans. MAP infection can persist for years without causing clinical disease. The herd prevalence of MAP infection in Western Europe and North America is reported in the range 21% to 54%. These subclinically infected animals shed MAP in their milk and onto pastures. MAP is more robust than tuberculosis, and the risk that is conveyed to human populations in retail milk and in domestic water supplies is high. MAP is harboured in the ileocolonic mucosa of a proportion of normal people and can be detected in a high proportion of full thickness samples of inflamed Crohn's disease gut by improved culture systems and IS900 polymerase chain reaction if the correct methods are used. MAP in Crohn's disease is present in a protease-resistant nonbacillary form, can evade immune recognition and probably causes an immune dysregulation. As with other MAC, MAP is resistant to most standard antituberculous drugs. Treatment of Crohn's disease with combinations of drugs more active against MAC such as rifabutin and clarithromycin can bring about a profound improvement and, in a few cases, apparent disease eradication. New drugs as well as effective MAP vaccines for animals and humans are needed. The problems caused by MAP constitute a public health issue of tragic proportions for which a range of remedial measures are urgently needed.     

Mycobacterium avium subspecies paratuberculosis, Crohn's disease and the Doomsday scenario

Johne's disease is chronic inflammation of the intestine caused by Mycobacterium avium subspecies paratuberculosis. Infection and disease are mainly in domestic livestock but can affect many species including primates. Johne's is a new disease which emerged at the turn of the 19^th and 20^th centuries and principally involved Europe and North America. It has since spread to former low incidence regions to become a global problem. Crohn's disease is a chronic inflammation of the intestine in humans which emerged in Europe and North America mid 20^th century and increased to become a major healthcare problem. It has now spread to former low incidence regions. Infected animals shed Mycobacterium avium subspecies paratuberculosis in milk and into the environment. Human populations are widely exposed. Outcomes maybe influenced by microbial phenotype. Exposure to extracellular forms of these pathogens may confer some natural protection; exposure to intracellular forms which have passaged through milk macrophages or environmental protists may pose a greater threat to humans particularly individuals with an inherited or acquired susceptibility. Hot spots of human disease such as in Winnipeg which sits on rock at the junction of two rivers may result from local exposure to high levels of waterborne pathogens brought down from farmland. When appropriate methods are used most people with Crohn's disease are found to be infected. There are no data which demonstrate that these pathogens are harmless to humans. An overwhelming balance of probability and Public health risk favours the conclusion that Mycobacterium avium subspecies paratuberculosis is also pathogenic for people. A two tier co-operative pathogenic mechanism is proposed in Crohn's disease. Intracellular infection with the primary pathogen widely distributed throughout the gut causes an immune dysregulation and a specific chronic enteric neuropathy with loss of mucosal integrity. Segments of gross inflammatory disease result from the perturbed neuroimmune response to penetration into the gut wall of secondary pathogens from the lumen. These include both normal gut organisms and educated members of the enteric microbiome such as more aggressive E. coli. More new diseases may arise from failure to apply a range of remedial measures to this longstanding zoonotic problem.     

Detection of Mycobacterium avium subspecies paratuberculosis in surgicalpathology blocks from patients with Crohn's disease in China

AIM To determine whether MAP can be detected in archival paraffin embedded full thickness samples ofintestinal tissue from patients in China with Crohn's disease (CD), ulcerative colitis (UC), and in controlsubjects (NIBD) having surgery for bowel cancer.METHODS Optimized procedures for the removal of paraffin, recovery of tissue and access to MAP DNA,followed by MAP-specific nested IS900 PCR. Confirmation of specific amplification by Southern blotting andDNA sequencing.RESULTS IS900 PCR positive tests identified MAP in 9 (69%) of 13 CD, 1 of 3 UC and 2 (14%) of 14NIBD in the presence of correctly reporting positive and negative sample and reagent control reactions. DNAsequence analysis of the 298bp IS900 PCR amplification product from MAP in 2 Chinese CD patientsdemonstrated 99% homology with the GenBank IS900 sequence accession number X16293.CONCLUSION Although larger numbers of Chinese samples need to be studied, these initial results areconsistent with an exposure of human populations in China to MAP, and an involvement of this pathogen inchronic inflammation of the intestine of the Crohn's disease type. The results are in agreement with similarpositive studies reported from China, from Western Europe and elsewhere.     

Presence and characterization of Mycobacterium avium subspecies paratuberculosis from clinical and suspected cases of Crohn's disease and in the healthy human population in India.

OBJECTIVES: To investigate and characterize Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease, attendants of animals with suspected infection, and healthy humans, using multiple diagnostic tests. METHODS: A total of 119 samples (35 stool, 76 serum, three blood clots, and five biopsies) were collected from five patients with Crohn's disease, eight attendants of animals with Johne's disease, and 93 apparently normal control subjects (Agra region) from North India. Samples were screened for the presence of MAP by smear examination, culture of stool, blood clot and biopsies, and ELISA. Colonies obtained by culture were further characterized using polymerase chain reaction (PCR) with IS900 MAP-specific primers. RESULTS: Using all diagnostic modalities, MAP and/or MAP antibodies were identified in 100% (5/5) of subjects with Crohn's disease; 75.0% (6/8) of attendants of MAP infected animals were positive and 38.0% (27/71) of apparently normal controls were also positive. Most sensitive test was ELISA (100%, 5/5), followed by culture (80.0%, 4/5), and acid-fast staining. Ziehl-Neelsen staining was positive in 37.5% (3/8) of subjects with active animal husbandry practices. In 71 serum samples from control subjects, seroprevalence of MAP was 38.0% using indigenous protoplasmic antigens (PPA) and 36.6% using commercial PPA. Of the serum samples from the Crohn's disease patients, 100% (5/5) were positive by ELISA using indigenous PPA and 40.0% (2/5) were positive by ELISA using commercial PPA. IS900 PCR was used to characterize tiny colonies of MAP that grew extremely slowly on Herrold's egg yolk medium, and of 15 (42.8%) cultures, 14 (93.3%) were typed as MAP. CONCLUSIONS: Paper documented the presence of MAP in all patients with Crohn's disease, in some animal attendants who had the history of working with goat herds infected with Johne's disease and in few normal healthy individuals. Presence of Ziehl Neelsen positive MAP. In the stool of attendants working with MAP-infected animals was unique to humans. ELISA based on antigens derived from indigenous MAP 'bison type' genotype of goat origin was most sensitive modality for screening Crohn's disease patients.     

Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis

This systematic review assesses the evidence for an association between Mycobacterium avium subspecies paratuberculosis (MAP) and Crohn's disease. We analysed 28 case-control studies comparing MAP in patients with Crohn's disease with individuals free of inflammatory bowel disease (IBD) or patients with ulcerative colitis. Compared with individuals free of IBD, the pooled odds ratio (OR) from studies using PCR in tissue samples was 7.01 (95% CI 3.95-12.4) and was 1.72 (1.02-2.90) in studies using ELISA in serum. ORs were similar for comparisons with ulcerative colitis patients (PCR, 4.13 [1.57-10.9]; ELISA, 1.88 [1.26-2.81]). The association of MAP with Crohn's disease seems to be specific, but its role in the aetiology of Crohn's disease remains to be defined.     

Incidence of Mycobacterium avium subspecies paratuberculosis in a population-based cohort of patients with Crohn's disease and control subjects

OBJECTIVE:  To define the incidence of Mycobacterium avium subspecies paratuberculosis (MAP) in patients with Crohn's disease (CD) and in control subjects.
METHODS:  Blood samples from 361 CD patients from a previously described population-based inflammatory bowel disease (IBD) cohort and 200 blood donor controls, of known NOD2 genotype, were screened by PCR for MAP-specific IS900 DNA. These results were correlated with NOD2 genotype.
RESULTS:  The PCR assay was capable of detecting 20 fg of purified MAP DNA, equivalent to roughly 100 MAP cells/mL of blood. MAP-specific IS900 DNA was detected in 33.8% of CD cases and 21.5% of controls (OR 1.86, 95% CI 1.247–2.785, P = 0.002). All study participants were genotyped for the NOD2 mutations 2104C>T (R702W), 2722G>C (G908R), and 3020insC (1007fs). Carriage of one or two NOD2 mutations was not associated with a significantly higher risk of CD (OR 0.75, 95% CI 0.465–1.207, P = 0.234). No significant association was seen in the CD cohort for carriage of one or two NOD2 mutations and MAP status (OR 0.883, 95% CI 0.494–1.579, P = 0.675).
CONCLUSIONS:  Screening peripheral blood using IS900 PCR indicated that MAP DNA could be detected in a significant proportion of CD cases from a large population-based cohort, and also, in control subjects. The over-representation of MAP DNA in CD suggests either a role or a probable role for MAP in the etiology of CD.