To the Editor:

Crohn's disease (CD) is a relapsing transmural chronic inflammatory bowel disease (IBD). Its etiology remains uncertain, and infectious causes have been proposed as an integral part of this process together with genetic and environmental factors. Mycobacterium avium subspecies paratuberculosis (MAP), an acid-fast bacillus causing a chronic granulomatous enteritis (Johne's disease) in ruminants, has been implicated in the pathogenesis of CD, although critics of the mycobacterial theory argue that MAP is a secondary invader rather than a causal factor.1

The gold standard for MAP detection is the isolation of the organism through culture methods; however, due to the difficulties in growing MAP in vitro, molecular and serological methods have been developed. The detection of the insertion sequence (IS)900 by polymerase chain reaction (PCR) is the most common and widely used molecular technique,1 and the IS1311 PCR/restriction endonuclease analysis of PCR products allows differentiation between different MAP strains (cattle, or Type II, and sheep, including Type I and Type III).2 A recent meta-analysis of 28 case–control studies has shown that tests positive for MAP are more common in patients with CD, independently of whether PCR in tissue samples or enzyme-linked immunosorbent assay (ELISA) in serum is used, in comparison to patients with ulcerative colitis (UC) and non-IBD subjects.3

In the present study we enrolled 70 patients with CD (Table 1). The diagnosis of CD was based on conventional clinical, endoscopic, and histologic criteria and disease activity was assessed with the Crohn's Disease Activity Index (CDAI). Fifty-nine patients affected by ulcerative colitis (UC) (36 males and 23 females; mean age 48.4 years, range 23–68) and 71 healthy volunteers (37 males and 34 females; mean age 42.7 years, range 19–68) were also examined as controls.

Table 1. Clinical Features of Patients with Crohn's Disease (n=70)
Characteristics and ParametersNo.Median (Range)
  1. CDAI, Crohn's Disease Activity Index; 6-MP, 6-mercaptopurine.

Age (years) 34.2 (15-63)
Intestinal location  
 Small bowel and colon62 
 Colon only8 
Clinical phenotype  
Duration of disease (months) 113.5 (1-223)
CDAI 125 (28-433)
 Mesalazine only20 
 Mesalazine + topical steroids8 
 Mesalazine + antibiotics4 
 Mesalazine + azathioprine/6-MP/methotrexate15 
 Mesalazine + adalimumab3 
 Mesalazine + infliximab6 
 Mesalazine + systemic steroids7 
 Adalimumab only1 
 Infliximab only1 

All the plasma samples were screened by nested PCR for MAP-specific DNA IS900, while the IS1311 PCR/restriction endonuclease analysis of PCR products was used to differentiate between cattle and sheep MAP strains in the IS900-positive CD patients. The MAP-specific antibody production to MAP MptD peptide (a cell envelope protein), MAP heparin-binding hemagglutinin adesin (HBHA) (a protein required for extrapulmonary dissemination), and MAP lysate were assessed by ELISA.4 Data were analyzed by Fisher's Exact Test and a level of P < 0.05 was considered statistically significant. Each patient and control subject who took part in the study was enrolled after appropriate Ethics Committee approval and informed consent was obtained in all cases.

The number of IS900-positive CD patients (68.6%) was significantly (P < 0.0001) higher in comparison to UC patients (11.9%) and healthy volunteers (26.8%), without any significant difference between the last two groups. No difference was found in the percentage of IS900-positive CD patients when considering disease duration, disease activity assessed by CDAI, intestinal location, or the clinical phenotype. Among the 48 IS900-positive CD patients, 38 were positive for the cattle strain and three for the sheep. Finally, the percentages of CD patients showing a humoral response to MAP peptides (MptD: 5.7%; HBHA: 6.4%) and lysate (2.9%) did not significantly differ from those of UC patients (MptD: 6.8%; HBHA: 8.5%; lysate 0%) and controls (MptD: 7.0%; HBHA: 4.2%; lysate 2.8%).

Controversial data have been reported when humoral reactivity to MAP is searched in different populations, even when analyzed in the same study.5 We previously reported that in sheeps with paratuberculosis, no single MAP antigen tested showed a consistently strong antibody response over the 1-year study period in any single animal.6 In keeping with the findings by Collins et al,7 we found a poor correlation between molecular results and serological response to MAP. We suggest that ELISA tests have poor sensitivity, and an interferon-γ assay measuring the specific lymphocyte response to MAP antigens would be more sensitive and specific.8


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  • 1
    Mendoza JL, Lana R, Díaz-Rubio M. Mycobacterium avium subspecies paratuberculosis and its relationship with Crohn's disease. World J Gastroenterol. 2009; 15: 417422.
  • 2
    Möbius P, Fritsch I, Luyven G, et al. Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III. Vet Microbiol. 2009; 139: 398404.
  • 3
    Feller M, Huwiler K, Stephan R, et al. Mycobacterium avium subspecies paratuberculosis and Crohn's disease: a systematic review and meta-analysis. Lancet Infect Dis. 2007; 7: 607613.
  • 4
    Rosu V, Ahmed N, Paccagnini D, et al. Specific immunoassays confirm association of Mycobacterium avium Subsp. paratuberculosis with type-1 but not type-2 diabetes mellitus. PLoS One. 2009; 4: e4386.
  • 5
    Bernstein CN, Blanchard JF, Rawsthorne P, et al. Population-based case control study of seroprevalence of Mycobacterium paratuberculosis in patients with Crohn's disease and ulcerative colitis. J Clin Microbiol. 2004; 42: 11291135.
  • 6
    Bannantine JP, Rosu V, Zanetti S, et al. Antigenic profiles of recombinant proteins from Mycobacterium avium subsp. Paratuberculosis in sheep with Johne's disease. Vet Immunol Immunopathol. 2008; 122: 116125.
  • 7
    Collins MT, Lisby G, Moser C, et al. Results of multiple diagnostic tests for Mycobacterium avium subsp. paratuberculosis in patients with inflammatory bowel disease and in controls. J Clin Microbiol. 2000; 38: 43734381.
  • 8
    Olsen I, Tollefsen S, Aagaard C, et al. Isolation of Mycobacterium avium subspecies paratuberculosis reactive CD4 T cells from intestinal biopsies of Crohn's disease patients. PLoS One. 2009; 4: e5641.

Antonio Di Sabatino MD*, Daniela Paccagnini PhD†, Francesca Vidali MD*, Valentina Rosu PhD†, Paolo Biancheri MD*, Andrea Cossu PhD†, Stefania Zanetti PhD†, Gino R. Corazza MD*, Leonardo A. Sechi PhD†, * First Department of Medicine S. Matteo Hospital, Centro per lo Studio e la Cura delle Malattie Infiammmatorie Croniche Intestinali University of Pavia Pavia, Italy, † Dipartimento di Scienze Biomediche Sezione di Microbiologia Clinica e Sperimentale Università di Sassari Sassari, Italy.