1. Why are bacteria relevant to Crohn’s? Animal model data suggests the inflammation of CD results from an abnormal response to the intestinal flora Animal model data suggests the inflammation of CD results from an abnormal response to the intestinal flora Transgenic animal models do not develop CD if raised in sterile environment Transgenic animal models do not develop CD if raised in sterile environment CD relapses have been associated with gut pathogens like Campylobacter, Salmonella, Shigella, Yersinia, pathogenic E. coli CD relapses have been associated with gut pathogens like Campylobacter, Salmonella, Shigella, Yersinia, pathogenic E. coli Controversy is whether a specific etiologic agent is required vs. any general flora Controversy is whether a specific etiologic agent is required vs. any general flora Pathogenesis of other mycobacterial infections (TB, leprosy, MAC in AIDS) shares features of CD Pathogenesis of other mycobacterial infections (TB, leprosy, MAC in AIDS) shares features of CD

2. Infectious agents implicated in IBD Diplostreptococci (UC) Diplostreptococci (UC) Bacteroides necrophorum Bacteroides necrophorum Bacteroides fragilis Bacteroides fragilis Pseudomonas maltophilia Pseudomonas maltophilia Helicobacter hepaticus or pylori species and, Helicobacter hepaticus or pylori species and, Shigella Shigella Chlamydia Chlamydia Listeria Listeria measles measles Wolinella Coxsakie A, B Reovirus Polio virus Norwalk virus Influenza herpes virus Paramyxovirus pathogenic E. coli

3. Is Crohn’s disease due to an infectious etiology? Ballard et al. IDCP; Nov 2002 n=393 March 2000 Ballard et al. IDCP; Nov 2002

4. Is Crohn’s Disease caused by MAP? n=393

5. What is MAP? M. avium M. avium subsp paratuberculosis subsp paratuberculosis subsp lepraemurium subsp lepraemurium subsp silvaticum and subsp silvaticum and strains not belonging to any of these species that possibly form new species strains not belonging to any of these species that possibly form new species Very slow grower, fastidious, and intracellular pathogen Very slow grower, fastidious, and intracellular pathogen Resistant to chlorine Resistant to chlorine Survives pasteurization Survives pasteurization UK survey 7% of cartons positive UK survey 7% of cartons positive Has been detected in human breast milk of CD patients Has been detected in human breast milk of CD patients Ubiquitous in water supply, etc Ubiquitous in water supply, etc UK has established guidelines to eradicate in food supply UK has established guidelines to eradicate in food supply First suggested as a cause of Crohn’s by Dalziel (described Crohn’s) in 1913 First suggested as a cause of Crohn’s by Dalziel (described Crohn’s) in 1913 1986 isolated from Crohn’s patients (Chiodini et al. J Clin Microbiol. 1984;20:966-971)- spheroplasts 1986 isolated from Crohn’s patients (Chiodini et al. J Clin Microbiol. 1984;20:966-971)- spheroplasts Case report of child w/paraTB scrofula, then Crohns, which resolved on rifabutin and clarithromycin Case report of child w/paraTB scrofula, then Crohns, which resolved on rifabutin and clarithromycin Mixed results (0-100%) regarding presence of MAP in tissue samples from CD patients Sanderson JDet al. Mycobacterium paratuberculosis DNA in Crohn's disease tissue. Gut. 1992;33:890-896. Mixed results (0-100%) regarding presence of MAP in tissue samples from CD patients Sanderson JDet al. Mycobacterium paratuberculosis DNA in Crohn's disease tissue. Gut. 1992;33:890-896. High seroprevalence in CD but variable results (p35 and p36 antibodies), and recent sero study inconclusive High seroprevalence in CD but variable results (p35 and p36 antibodies), and recent sero study inconclusive

6. What is Johne’s disease ? chronic inflammatory disease of ruminants (6% of US cattle), and also primates chronic inflammatory disease of ruminants (6% of US cattle), and also primates Ileitis with chronic diarrhea and wasting Ileitis with chronic diarrhea and wasting Easy to detect MAP Easy to detect MAP Not segmental, no fistula/strictures/abscesses/ulcers Not segmental, no fistula/strictures/abscesses/ulcers caused by MAP caused by MAP UK 2002 guidelines for eradication (published 8/04) UK 2002 guidelines for eradication (published 8/04)

7. Koch’s postulates 1890 German physician/bacteriologist Robert Koch 1890 German physician/bacteriologist Robert Koch Koch's postulates are as follows: Koch's postulates are as follows: The organism must be present in every case of a specific disease, but not in healthy subjects. The organism must be present in every case of a specific disease, but not in healthy subjects. The organism must be isolated from the host with the disease and grown in pure culture. The organism must be isolated from the host with the disease and grown in pure culture. The specific disease must be reproduced when a pure culture of the organism is inoculated into a healthy susceptible host The specific disease must be reproduced when a pure culture of the organism is inoculated into a healthy susceptible host The organism must be recoverable from the experimentally infected host The organism must be recoverable from the experimentally infected host may not hold if: may not hold if: bacteria (M. leprae, R. rickettsiae) cannot be "grown in pure culture" in the laboratory-but PCR and other molecular techniques allow identification bacteria (M. leprae, R. rickettsiae) cannot be "grown in pure culture" in the laboratory-but PCR and other molecular techniques allow identification no animal model of infection exists no animal model of infection exists organism may also cause disease if: organism may also cause disease if: acquired extra virulence factors making it pathogenic acquired extra virulence factors making it pathogenic gains access to deep tissues via trauma, surgery, an IV line, etc gains access to deep tissues via trauma, surgery, an IV line, etc infects an immunocompromised patient. infects an immunocompromised patient. Ignores subclinical infection Ignores subclinical infection Many examples of accepted infectious etiologies that do not fit the model Many examples of accepted infectious etiologies that do not fit the model Cholera Cholera leprosy leprosy

8. Are they met? The bacteria must be present in every case of the disease. The bacteria must be present in every case of the disease. Essentially yes for MAP in Johne’s disease Essentially yes for MAP in Johne’s disease Not established for CD, although most recent papers have shown higher correlation Not established for CD, although most recent papers have shown higher correlation The bacteria must be isolated from the host with the disease and grown in pure culture The bacteria must be isolated from the host with the disease and grown in pure culture First isolated in 1984 (Chiodini) First isolated in 1984 (Chiodini) Has been isolated from Crohn’s patients, and recently from blood (?less chance of environmental contamination) Has been isolated from Crohn’s patients, and recently from blood (?less chance of environmental contamination) The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host The specific disease must be reproduced when a pure culture of the bacteria is inoculated into a healthy susceptible host Yes but not repeatedly Yes but not repeatedly The bacteria must be recoverable from the experimentally infected host The bacteria must be recoverable from the experimentally infected host Yes for Johne’s (strains of human origin) Yes for Johne’s (strains of human origin)

10. Romero et al, IBD: Volume 11(2), February 2005, pp 116-125 Detection of MAP DNA in Tissue by FISH Using CSLM and Nested PCR

11. Serologic evidence for MAP in CD Highly variable Highly variable Bernstein et al, JCM: Mar 2004; 42 (3) 1129-35 Bernstein et al, JCM: Mar 2004; 42 (3) 1129-35 Population based trial in Manitoba (high Crohn’s rate) Population based trial in Manitoba (high Crohn’s rate) ELISA to MAP ELISA to MAP >900 subjects, 283 w/CD >900 subjects, 283 w/CD No correlation between MAP seropositivity and CD, among siblings, UC pts, CD pts, normal controls No correlation between MAP seropositivity and CD, among siblings, UC pts, CD pts, normal controls No epidemiologic/demographic correlation with high likelihood of MAP exposure No epidemiologic/demographic correlation with high likelihood of MAP exposure

12. Most recent data Bull et al. Detection and verification of Mycobacterium avium subsp paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease. J Clin Microbiol. 2003;41:2915-2923. Bull et al. Detection and verification of Mycobacterium avium subsp paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease. J Clin Microbiol. 2003;41:2915-2923. 92% of intestinal biopsies + for MAP (vs. 26%) 92% of intestinal biopsies + for MAP (vs. 26%) Naser et al (Lancet 2004;364:1039-44)-blasted by editorials Naser et al (Lancet 2004;364:1039-44)-blasted by editorials Small (n=28 w/CD, 15 controls, some w/other bowel disease) Small (n=28 w/CD, 15 controls, some w/other bowel disease) Isolated MAP from blood of 50% of CD, 22% of UC and none of non-IBD pts Isolated MAP from blood of 50% of CD, 22% of UC and none of non-IBD pts No other organisms isolated (so not just leaky mucosa) No other organisms isolated (so not just leaky mucosa) But 20% of non-IBD pts (n=15) had MAP DNA in blood But 20% of non-IBD pts (n=15) had MAP DNA in blood sequenced strains sequenced strains Sechi LA - Am J Gastroenterol - 01-JUL-2005; 100(7): 1529-36 Sechi LA - Am J Gastroenterol - 01-JUL-2005; 100(7): 1529-36 Survey of patients having ileocolonoscopy in Sardinia Survey of patients having ileocolonoscopy in Sardinia 83% of CD had MAP by PCR vs 10% of controls (OR= 43) 83% of CD had MAP by PCR vs 10% of controls (OR= 43) MAP was isolated in 63% vs 10% MAP was isolated in 63% vs 10% Initial form isolated was Z-N negative, then reverted to the Z-N positive form. Initial form isolated was Z-N negative, then reverted to the Z-N positive form.

13. Antibiotics in Crohn’s Variable data for metronidazole, although overall appears to have activity in Crohn’s, but poorly tolerated Variable data for metronidazole, although overall appears to have activity in Crohn’s, but poorly tolerated Case reports in 1980’s reported dramatic improvement in CD when pts rx for concomitant TB Case reports in 1980’s reported dramatic improvement in CD when pts rx for concomitant TB MAP is resistant to many anti-TB agents, and macrolides are the most active agent MAP is resistant to many anti-TB agents, and macrolides are the most active agent Lowest MICs Lowest MICs High intracellular concentrations High intracellular concentrations At least 17 negative trials, but none included macrolides At least 17 negative trials, but none included macrolides Small open label study w.clari alone reported dramatic benefit Small open label study w.clari alone reported dramatic benefit N=25 N=25 4 trials with clarithromycin show benefit Sample sizes <60 Follow-up range 12-54 mo Triple drug therapy Enpoints variable (CR vs improved scores) All open label single controlled trial (Goodgame et al) N=30, randomized Only 12mo f/u and 3mo rx No benefit

14. For NOD2 defect predisposes to infection with intracellular bacterial infections and is associated with CD [Girardin SE et al. Trends Immunol. 2003;24:652]. NOD2 defect predisposes to infection with intracellular bacterial infections and is associated with CD [Girardin SE et al. Trends Immunol. 2003;24:652]. MAP RNA has been detected in a large proportion of CD patients, indicating viability MAP RNA has been detected in a large proportion of CD patients, indicating viability organism has been cultivated from stool, intestinal tissue, and peripheral blood in patients with Crohn disease [Bull TJ et al. J Clin Microbiol. 2003;41:2915]. organism has been cultivated from stool, intestinal tissue, and peripheral blood in patients with Crohn disease [Bull TJ et al. J Clin Microbiol. 2003;41:2915]. Clinical trials suggest benefit of effective combination therapy, albeit mostly uncontrolled small trials. Duration of rx and follow-up may have been inadequate. Clinical trials suggest benefit of effective combination therapy, albeit mostly uncontrolled small trials. Duration of rx and follow-up may have been inadequate. Negative Ziehl-Neelson staining may reflect pathogenic non-cell wall forms. Confirmed by culture data. Negative Ziehl-Neelson staining may reflect pathogenic non-cell wall forms. Confirmed by culture data.

15. Against Finding of MAP DNA may reflect non-viable MAP coincidentally passing through the GI tract Finding of MAP DNA may reflect non-viable MAP coincidentally passing through the GI tract Isolation from blood may reflect only increased intestinal permeability as a result of CD Isolation from blood may reflect only increased intestinal permeability as a result of CD Johne’s is more similar to MAC wasting syndrome than UC Johne’s is more similar to MAC wasting syndrome than UC Data from seroprevalence studies has been highly variable Data from seroprevalence studies has been highly variable If MAC is cause, then why don’t immunosuppressants exacerbate the infection? If MAC is cause, then why don’t immunosuppressants exacerbate the infection? In trials where organism was isolated, finding did not correlate with immunosuppression In trials where organism was isolated, finding did not correlate with immunosuppression Increased MAP detection not reports with infliximab and other immunosuppressants Increased MAP detection not reports with infliximab and other immunosuppressants MAP has been isolated from normals and UC patients and may represent epiphenomenon MAP has been isolated from normals and UC patients and may represent epiphenomenon No epidemiological evidence that CD is associated with exposure to ruminents No epidemiological evidence that CD is associated with exposure to ruminents

16. Future Results of Australian RPCT of 2 years’ rx of 214 pts with clari/rifabutin/clofazimine pending Results of Australian RPCT of 2 years’ rx of 214 pts with clari/rifabutin/clofazimine pending Complete genome of MAP just published (Proc Natl Acad Sci U S A - 30-AUG-2005; 102(35): 12344-9) Complete genome of MAP just published (Proc Natl Acad Sci U S A - 30-AUG-2005; 102(35): 12344-9) RPCT’s with long term follow-up using drugs known to be active against MAP RPCT’s with long term follow-up using drugs known to be active against MAP ?Australian study ?Australian study Better case definitions and more uniform therapeutic endpoints Better case definitions and more uniform therapeutic endpoints More basic science More basic science Pre-exposure histology, cultures and PCR then post-exposure Pre-exposure histology, cultures and PCR then post-exposure Immunogenetics Immunogenetics Better epidemiology (?trace a strain from cow to human) Better epidemiology (?trace a strain from cow to human)

17. Antibiotics in Crohn’s 32. Thomas GA, Swift GL, Green JT, et al. Controlled trial of antituberculous chemotherapy in Crohn's disease: a five year follow up study. Gut. 1998;42:497-500. Bibliographic Links [Context Link] 32. Thomas GA, Swift GL, Green JT, et al. Controlled trial of antituberculous chemotherapy in Crohn's disease: a five year follow up study. Gut. 1998;42:497-500. Bibliographic Links [Context Link]Bibliographic Links[Context Link]Bibliographic Links[Context Link] 33. Prantera C, Kohn A, Mangiarotti R, et al. Antimycobacterial therapy in Crohn's disease: results of a controlled, double-blind trial with a multiple antibiotic regimen. Am J Gastroenterol. 1994;89:513-518. Bibliographic Links [Context Link] 33. Prantera C, Kohn A, Mangiarotti R, et al. Antimycobacterial therapy in Crohn's disease: results of a controlled, double-blind trial with a multiple antibiotic regimen. Am J Gastroenterol. 1994;89:513-518. Bibliographic Links [Context Link]Bibliographic Links[Context Link]Bibliographic Links[Context Link] 34. Rutgeerts P, Geboes K, Vantrappen G, et al. Rifabutin and ethambutol do not help recurrent Crohn's disease in the neoterminal ileum. J Clin Gastroenterol. 1992;15:24-28. Bibliographic Links [Context Link] 34. Rutgeerts P, Geboes K, Vantrappen G, et al. Rifabutin and ethambutol do not help recurrent Crohn's disease in the neoterminal ileum. J Clin Gastroenterol. 1992;15:24-28. Bibliographic Links [Context Link]Bibliographic Links[Context Link]Bibliographic Links[Context Link] 35. Hampson SJ, Parker MC, Saverymuttu SH, et al. Quadruple antimycobacterial chemotherapy in Crohn's disease: results at 9 months of a pilot study in 20 patients. Aliment Pharmacol Ther. 1989;3:343-352. Bibliographic Links [Context Link] 35. Hampson SJ, Parker MC, Saverymuttu SH, et al. Quadruple antimycobacterial chemotherapy in Crohn's disease: results at 9 months of a pilot study in 20 patients. Aliment Pharmacol Ther. 1989;3:343-352. Bibliographic Links [Context Link]Bibliographic Links[Context Link]Bibliographic Links[Context Link] 36. Shafran I, Kugler L, El-Zaatari FA, et al. Open clinical trial of rifabutin and clarithromycin therapy in Crohn's disease. Dig Liver Dis. 2002;34:22-28. Bibliographic Links [Context Link] 36. Shafran I, Kugler L, El-Zaatari FA, et al. Open clinical trial of rifabutin and clarithromycin therapy in Crohn's disease. Dig Liver Dis. 2002;34:22-28. Bibliographic Links [Context Link]Bibliographic Links[Context Link]Bibliographic Links[Context Link] 37. Hermon-Taylor J. Treatment with drugs active against Mycobacterium avium subspecies paratuberculosis can heal Crohn's disease: more evidence for a neglected public health tragedy. Dig Liver Dis. 2002;34:9-12. [Context Link] 37. Hermon-Taylor J. Treatment with drugs active against Mycobacterium avium subspecies paratuberculosis can heal Crohn's disease: more evidence for a neglected public health tragedy. Dig Liver Dis. 2002;34:9-12. [Context Link][Context Link][Context Link] 38. Borody TJ, Leis S, Warren EF, et al. Treatment of severe Crohn's disease using antimycobacterial triple therapy: approaching a cure? Dig Liver Dis. 2002;34:29-38. [Context Link] 38. Borody TJ, Leis S, Warren EF, et al. Treatment of severe Crohn's disease using antimycobacterial triple therapy: approaching a cure? Dig Liver Dis. 2002;34:29-38. [Context Link][Context Link][Context Link] 39. Hulten K, Almashhrawi A, El-Zaatari FA, et al. Antibacterial therapy for Crohn's disease: a review emphasizing therapy directed against mycobacteria. Dig Dis Sci. 2000;45:445-456. Bibliographic Links [Context Link] 39. Hulten K, Almashhrawi A, El-Zaatari FA, et al. Antibacterial therapy for Crohn's disease: a review emphasizing therapy directed against mycobacteria. Dig Dis Sci. 2000;45:445-456. Bibliographic Links [Context Link]Bibliographic Links[Context Link]Bibliographic Links[Context Link] 40. Borgaonkar MR, MacIntosh DG, Fardy JM. A meta-analysis of antimycobacterial therapy for Crohn's disease. Am J Gastroenterol. 2000;95:725-729. 40. Borgaonkar MR, MacIntosh DG, Fardy JM. A meta-analysis of antimycobacterial therapy for Crohn's disease. Am J Gastroenterol. 2000;95:725-729. ?since then ?since then ?4 studies ?4 studies

18. Hermon-Taylor J, Bull TJ, Sheridan JM, et al. Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis. Can J Gastroenterol. 2000;14:521-539. Bibliographic Links [Context Link] Hermon-Taylor J, Bull TJ, Sheridan JM, et al. Causation of Crohn's disease by Mycobacterium avium subspecies paratuberculosis. Can J Gastroenterol. 2000;14:521-539. Bibliographic Links [Context Link]Bibliographic Links[Context Link]Bibliographic Links[Context Link] Hermon-Taylor J. Protagonist: Mycobacterium avium subspecies paratuberculosis is a cause of Crohn's disease. Gut. 2001;49:755-756. Hermon-Taylor J. Protagonist: Mycobacterium avium subspecies paratuberculosis is a cause of Crohn's disease. Gut. 2001;49:755-756.

19. Summary of MAP and Crohn’s The NOD2 genetic defect predisposes to infection with intracellular bacterial infections and is associated with Crohn disease [Girardin SE et al. Trends Immunol. 2003;24:652]. The NOD2 genetic defect predisposes to infection with intracellular bacterial infections and is associated with Crohn disease [Girardin SE et al. Trends Immunol. 2003;24:652]. The DNA of M. avium ss paratuberculosis has been detected by PCR in 90% of cases, by in situ hybridization in 70% of cases, and by RT-PCR in up to 100% of cases [Sechi LA et al. J Clin Microbiol. 2001;39:4514; Mishina D et al. Proc Natl Acad Sci USA. 1996;93:9816]. The DNA of M. avium ss paratuberculosis has been detected by PCR in 90% of cases, by in situ hybridization in 70% of cases, and by RT-PCR in up to 100% of cases [Sechi LA et al. J Clin Microbiol. 2001;39:4514; Mishina D et al. Proc Natl Acad Sci USA. 1996;93:9816]. This organism has been cultivated from stool, intestinal tissue, and peripheral blood in patients with Crohn disease [Bull TJ et al. J Clin Microbiol. 2003;41:2915]. This organism has been cultivated from stool, intestinal tissue, and peripheral blood in patients with Crohn disease [Bull TJ et al. J Clin Microbiol. 2003;41:2915]. Serologic tests support this association, but results have been variable [Collins MT et al. J Clin Microbiol. 2000;38:4373]. Serologic tests support this association, but results have been variable [Collins MT et al. J Clin Microbiol. 2000;38:4373]. Multiple antibiotic trials with Crohn disease have been negative including those using antituberculous agents. However, treatment of this pathogen requires macrolides, and since 1997, 4 trials with macrolides have shown complete clinical remission in up to 66% [Greenstein RJ. Lancet Infect Dis. 2003;3:507]. Multiple antibiotic trials with Crohn disease have been negative including those using antituberculous agents. However, treatment of this pathogen requires macrolides, and since 1997, 4 trials with macrolides have shown complete clinical remission in up to 66% [Greenstein RJ. Lancet Infect Dis. 2003;3:507]. This organism causes a similar, but not at all identical, disease in cattle characterized by granulomatous inflammation of the intestine [Mortensen HJ. Dairy Sci. 2004;87: 2108]. This organism causes a similar, but not at all identical, disease in cattle characterized by granulomatous inflammation of the intestine [Mortensen HJ. Dairy Sci. 2004;87: 2108].