The role of Mycobacterium avium ss paratuberculosis (MAP) in patients with Crohn's Disease

Research Doctorate - Doctor of Philosophy (PhD)The cause of chronic inflammation in the gut of subjects with Crohn’s Disease (CD) is unclear; however most would agree that 3 interacting factors are critical to mucosal inflammation: genetic susceptibility, enteric microflora and the host immune system. The most controversial theory is whether or not a particular microbe(s) infects and maintains in intestinal tissues, resulting in chronic inflammation. The most discussed microbe is Mycobacterium avium ss paratuberculosis (MAP). The aim of this thesis was to better document the natural history of MAP infection in subjects with Crohn’s disease, IBS, “non-Crohn’s colitis” and normal subjects, correlating clinical status with parameters relating to MAP, to the mucosal T cell response and to the genetic susceptibility gene CARD15/NOD2. MAP is an obligate intracellular pathogen, which causes chronic inflammation in the intestine of many species, including primates. MAP was first identified as the causative agent in Johnes Disease, a gastrointestinal disease in ruminants and primates. The infection of domestic livestock with MAP is now widespread, which has increased the risk of transmission of MAP to humans via milk products. The involvement of MAP in human patients with Crohn’s disease (CD) has been difficult to prove, due to the difficulties in isolating and detecting this organism. By using nested PCR (Polymerase Chain Reaction) on DNA extracted from fresh human intestinal mucosal biopsy samples the presence of MAP can be verified. Bacteria contain unique insertion elements, which play a role in virulence, pathogenicity and antibiotic resistance. Within MAP a unique 1.4 kb insertion element was identified, IS900. By using DNA primers for PCR within this unique insertion element we were able to identify MAP in tissue. There was found to be no statistical significance among the disease groups tested. The next objective was to determine whether the presence of MAP in gut biopsies was associated with a different cytokine secretion profile as observed in organ and whole blood culture, using an ELISA assay. Significantly higher levels of TNF-α were found in culture supernatants from organ culture for Crohn’s Disease when compared to ulcerative colitis (p<0.05), irritable bowel (p<0.01) and controls (p<0.0001). When TNF-α levels were correlated with the presence of MAP, significantly greater concentrations were only found in MAP positive Crohn’s Disease patients (p<0.05). In whole blood culture significantly higher levels of IL-4 (p<0.05) and IL-2 (p