Detection and verification of Mycobacterium avium subsp. paratuberculosis in fresh ileocolonic mucosal biopsy specimens from individuals with and without Crohn's disease

Abstract

Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900. Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis. In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.

FIG. 1.

(a) Alignment of DNA sequences from IS900 and related elements amplified with the second-round primers AV1 and AV2 in the nested IS900[L/AV] PCR, demonstrating sequence differences between these elements; (b) alignments of first- and second-round primers used in the IS900[L/AV] PCR and IS900[TJ1-4] PCR, demonstrating sequence differences in hybridization to target regions of related elements. Dots indicate the same base, and hyphens indicate deleted bases. For L1, L2, AV1, and AV2, no sequence data were available for strain 22850 and parts of strains WA-1, WA-2, V-1, and 2333. For TJ1, TJ2, TJ3, and TJ4, no sequence data were available for strains 22850 and V-1.

FIG. 2.

PCR products from the IS900[L/AV] nested PCR (upper panel) and the IS900[TJ1-4] nested PCR (lower panel) obtained with DNA from the following sources: mucosal biopsy specimen from a patient with CD (lane 2), culture of Mycobacterium sp. strain 2333 (lane 3), culture of Mycobacterium sp. strain 28850 (lane 4), culture of Mycobacterium sp. strain WA-1 (lane 5), culture of M. avium subsp. avium containing IS901 (lane 6), culture of M. avium subsp. avium containing IS1613 (lane 7), a plasmid containing a single full copy of IS1626 (lane 8), and a mucosal biopsy specimen from a patient without CD (lane 9). Lane 1, 100-bp ladder.