Introduction.

Detection of Mycobacterium avium subsp. paratuberculosis with routine histopathology examination and Ziehl-Neelsen staining is not specific enough for conclusive diagnosis of paratuberculosis if lesions are not well developed e.g. in the subclinical cases of the disease. In samples that already were formalin-fixed the culture examination is impossible. A rapid and specific method for the isolation and detection of M. avium subsp. paratuberculosis DNA from archival samples with IS900-based polymerase chain reaction (PCR) is described.

Material and methods.

In this study 70 archival samples of formalin-fixed paraffin-embedded tissue blocks of cattle, sheep and goat intestines and lymph nodes were used. For PCR procedure several paraffin tissue sections were transferred in microcentrifuge tube, deparaffinized with xylene, transferred to 100% ethanol and air dried. Isolation of genomic DNA was performed using High Pure PCR Template Preparation Kit (Boehringer, Mannheim). PCR was performed with IS900 specific primers in 40 cycles at 94ºC, 62ºC and 72ºC. PCR-products were separated on electrophoresis and analysed by scanning and visualisation system (BIO-Rad).

Results and discussion.

All 26 histologically positive samples were positive with IS900-based PCR, also 10 of 20 inconclusive samples, and one of 24 histologically negative samples. In this study an agreement between the results of histological examination and IS900-based PCR in formalin-fixed paraffin-embedded tissues was obtained.

Conclusions.

These results show that the procedure used in our laboratory for the isolation of M. avium subsp. paratuberculosis DNA from formalin-fixed paraffin-embedded tissues is useful for the final diagnosis of paratuberculosis, especially in the inconclusive cases.