Final Progress Report
Proposal No. IBD-0207R
Principal Investigator: Saleh A. Naser, Ph.D.
Applicant Organization: University of Central Florida (Orlando, U.S.A.)
Project Title: Role of Mycobacterium paratuberculosis (MAP) in family members with Crohn’s disease: genetic link and environmental influence
Period of Award: January 1, 2008 – February 28, 2011
Lay Summary:
Mycobacterium avium subspecies paratuberculosis (MAP) association with IBD especially Crohn’s disease has been investigated since 1913. Despite the significant amount of literature which supports MAP role in Crohn’s disease pathogenesis, the association continued to be controversial; it is accepted by many experts in the IBD field and totally rejected by others. Our group had repeatedly emphasized that correct methodology is the key for investigating MAP in IBD because of the sole fact that MAP in human is different than MAP in animals. In human, MAP lacks the bacterial cell wall which is critical for the survival of the microorganism in a laboratory specimen and for electing host’s immune response.
In this study, we validated our acclaimed culture-nested PCR MAP test through investigating coded blood samples from IBD patients and controls by our lab and two additional well-respected independent laboratories. Each patient provided three blood tubes and each laboratory followed the same protocol for detection of viable (live) MAP. At the conclusion of the study, MAP was detected in human blood by the three laboratories which confirmed that correct methodology is key for detection of MAP in human blood. Because of the potential sampling error, it is clinically accepted that multiple blood samples from a single patient are collected to test for any bacteremia. In this study, MAPbacteremia (MAP in the blood) was detected at least in 1 out of 3 blood tubes in 55% of IBD patients compared to 22% controls (at least one of the three laboratories identified the sample to be MAP positive). Moreover, MAPbacteremia was detected in 2 out of 3 blood tubes in 41% IBD patients compared to zero controls. As expected, we did not detect MAP in all three tubes from a single patient (no sample was positive by all three laboratories). The data clearly confirms that MAP in IBD is real and it can be detected by any laboratory if the correct methodology is used. It also supports multiple samplings for accurate detection.
Other aims in this study focused on investigating the association between recently discovered genes in IBD patients and the risk of acquiring microbial infection such as MAP. We confirmed that mutations in four key genes (NOD2, ATG16L1, IRMG and IL23R1) occurred significantly more in IBD patients than in controls. However, there was no statistical significance between the mutations of these key genes and the presence of MAP in the blood of IBD patients. Moreover, we identified 21 families who consented and provided blood samples for investigating gene mutations and the influence of environmental factors (such as diet, travel, exposure to animals, etc) on acquiring MAP in patients with IBD. Of the 21 family groups, we identified 16 biological families (a parent and offspring or blood related siblings) and 5 non-biological families (husband and wife). As expected, MAPbacteremia was detected more in relatives with IBD than in healthy family members. However, it was surprising to see that the genetic susceptibility in key IBD genes did not correlate with MAP incidence in patients compared to healthy relatives. Clearly, there was no association between the use of immunosuppressant and MAP in the blood of IBD patients. Unlike detection of MAP in IBD intestinal lesions, the detection of MAP in the blood of IBD patients is alarming, serious and should be treated accordingly (with appropriate antibiotics).