Abstract :
The objective of the present study was to optimise and evaluate a procedure for detection of
Mycobacterium avium subsp. paratuberculosis in archived formalin-fixed, paraffin embedded
tissue sections from cases of naturally occurring paratuberculosis in goats. A pilot study
assessed 3 procedures for extraction of DNA for detection by PCR. The procedure that gave
the most consistent results involved removal of paraffin by treatment with xylene and
ethanol, disruption of tissue pellets by beating with zirconium/silica beads, extraction of
DNA using a DNeasy kit (Qiagen) with overnight proteinase K digestion and final ethanol
precipitation. This procedure was used to analyse 82 paraffin embedded tissues (44 small
intestine, 38 mesenteric lymph node) with various grades of histological lesions of paratuberculosis
and acid-fast bacilli (AFB) loads. The overall sensitivity of the PCR was about 72%
of all samples including both paucibacillary and multibacillary lesions. The sensitivity of
the assay was 87.5% (42/48) in all paraffin sections having clearly and easily demonstrable
AFB. Fifty percent of the tissue sections with rarely detectable AFB were positive by PCR.
There was no significant difference (<0.05) between the sensitivity of the PCR analyses carried
out on intestinal and mesenteric lymph node tissues. The results of this study suggest
that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and
important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues
for bacterial culture or PCR are not available due to problem of maintaining a cold chain
system.
