4th Annual BMRP Investigator Meeting - Abstract
Detection of Mycobacterium avium paratuberculosis in Crohn’s tissue
Mathy Jeyanathan, Makeda Semret, David Alexander and Marcel Behra
Department of Medicine, McGill University Health Centre (Montreal, Quebec, Canada)
Introduction: A recent series of reports and reviews has re-opened the debate about Mycobacterium avium paratuberculosis (MAP) in Crohn’s disease (CD). Unfortunately, many techniques applied to search for MAP have not been adequately validated prior to use in these epidemiologic studies. In our laboratory, we have performed a number of studies to first define and then detect MAP. The properties of an assay that we aim for are sensitivity (able to detect small numbers of organisms present in infected tissue) and specificity (the method fails to generate positive signals in uninfected tissue). We have therefore compared various methods used to look for MAP organisms in tissue samples for which the infection state is well defined as a step towards CD studies.
Materials and Methods: To generate infected tissue, we have artificially infected C57BL/6 mice i.v. and i.p. with MAP strain K10, M. avium strain 104 and M. tuberculosis complex strains (M. tuberculosis H37Rv and M. bovis BCG). Infection state of these tissue samples was confirmed and quanitified by culturing livers and spleens after four to eight weeks. Sections of these organs then were examined by tissue PCR and a variety of staining methods targeting the cell wall (Ziehl-Neelsen and Auramine Rhodamine) or nucleic acids (in situ hybridization and indirect in situ PCR).
Result: Colony counts revealed that infected tissues providing a model for a paucibacillary Mycobacterial infection. Liquid-phase PCR of tissue for the IS900 element is specific for MAP, but extraction methods are critical and amplicons must be sequence-confirmed to identity with the K10 genome sequence. By microscopy, all methods revealed that MAP organisms are smaller and less rod-shaped than tubercle bacilli; individual organisms only can be detected with 100x oil-immersion microscopy. ZN and Auramine-based stains afforded comparable sensitivity, but the fluorescent method was prone to non-specific signal. ISH targeting 16s rRNA provided comparable sensitivity to ZN as determined by enumerating bacillary forms in serial sections. In situ PCR targeting the IS900 element and ISH using the IS900 probe resulted in non-specific signals, observed in TB-infected and uninfected tissues.
Conclusion: Results of this comparative study of staining techniques show: 1) detection of paucibacillary MAP infection requires 100x oil immersion microscopy, 2) 16s rRNA-ISH provides comparable sensitivity and specificity to ZN staining and 3) IS900-based probes, whether used directly or after in situ PCR, are prone to non-specific hybridization signals. This analysis suggests that the microscopic detection limit for Mycobacterium avium paratuberculosis in tissue is governed more by bacterial burden than by staining method. The use of these methods in Crohn’s tissue will be described at the investigator’s meeting.
aPrincipal Investigator