Fecal Detection of Mycobacterium avium Paratuberculosis Using the IS900 DNA Sequence in Crohn’s Disease and Ulcerative Colitis Patients and Healthy Subjects

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused by Mycobacterium avium subsp. paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences of M. avium subsp. paratuberculosis in patients with Crohn's disease has been described; it is of interest to rapidly detect M. avium subsp. paratuberculosis in milk for human consumption. IS900 insertion is used as a target for PCR amplification to identify the presence of M. avium subsp. paratuberculosis in biological samples. Two target sequences were selected: IS1 (155 bp) and IS2 (94 bp). These fragments have a 100% identity among all M. avium subsp. paratuberculosis strains sequenced. M. avium subsp. paratuberculosis was specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU of M. avium subsp. paratuberculosis can be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU of M. avium subsp. paratuberculosis per ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.

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“…IS900 has been detected by PCR-based methods in 60% of patients with this disease (39). Although the role of M. avium subsp.…”

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confidence: 99%

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

Mundo

Gilardoni

Hoffman

et al. 2013

Appl Environ Microbiol

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“…In a previous systematic review and meta-analysis of the association of CD and MAP, 28 casecontrol studies were analyzed and although the pooled odds ratio [OR] from studies using PCR in tissue samples showed a strong association with CD compared to healthy controls, similar fi ndings were found in UC [ 72 ]. However, in a more recent study of the fecal detection of MAP using IS900 DNA sequences, the results were similar in CD, UC, and healthy subjects, 68 %, 65 %, and 48 %, respectively [ 73 ]. The precise mechanisms by which normal commensal bacteria, even in the presence of altered equilibrium, could cause IBD in genetically susceptible subjects still [ 74 ].…”

Section: Mycobacterium Avium Subspecies Paratuberculosis [Map] Causesmentioning

confidence: 85%

Microbes in Colon Cancer and Inflammatory Bowel Disease

Fong

2014

The Role of Microbes in Common Non-Infectious Diseases

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Colorectal cancer [CRC] is the third most common tumor and fourth most common cause of cancer death in the world [ 1 ]. It is prevalent in many countries but more common in developed nations. In the United States [US] CRC estimated new cases in 2008 were 77,250 in men and 71,560 in women [ 1 ]. The higher incidence of CRC in developed countries has been attributed to dietary and lifestyle habits, and the infl uence of genetic predisposition.

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“…*Significant differences with HC; & significant differences with the corresponding active IBD group (CDA or UCA); # significant differences with CIR patients. * ,& or # p < 0.050; ** ,&& or ## p < 0.010; *** , &&& or ### p < 0.001 lymph node tissues [57] and stool cultures [56,58]. AIEC strains possessing enhanced invasive ability [33] have also been detected and isolated from ileal mucosa of CD patients [59] expressing specific receptors [40].…”

Section: Discussionmentioning

confidence: 99%

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease

Nazareth

Magro

Machado

et al. 2015

Med Microbiol Immunol

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Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn's disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

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Fecal Detection of Mycobacterium avium Paratuberculosis Using the IS900 DNA Sequence in Crohn’s Disease and Ulcerative Colitis Patients and Healthy Subjects

Cited by 34 publications

References 30 publications

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping

Microbes in Colon Cancer and Inflammatory Bowel Disease

Prevalence of Mycobacterium avium subsp. paratuberculosis and Escherichia coli in blood samples from patients with inflammatory bowel disease

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