There is an uncertainty surrounding the role of Mycobacterium avium subsp. paratuberculosis (Map) in Crohn's disease. This has been compounded by possible contamination from Map present in the lumen microflora.
Researchers from Ireland used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas.
These were isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn's disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel).
The team examined the effect of amplicon size on reliability of PCR from formalin fixed samples by amplifying 435 bp and 133 bp sequences of the human APC gene.
They then designed nested primers to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn's granulomas.
LCM isolated granulomas from Map culture positive bovine intestine was used as positive control.
Furthermore, PCR product specificity was confirmed by direct DNA sequencing.
The researchers found that the smaller fragment of the APC gene amplified reliably in all samples.
|Mycobacterium avium subsp paratuberculosis DNA found in 6 out of 15 Crohn's granulomas.|
Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in 6 out of 15 microdissected Crohn's granulomas. However, there were no positive results for any of the 12 disease control granulomas.
Dr Ryan's team concluded that, "LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn's disease.
"However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection.
"Detection of Map DNA within granulomas might suggest an infectious etiology in a subset of patients.
"Alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses".