Proceedings of the 10th International Colloquium on Paratuberculosis

#130 Mycobacterium avium subsp. paratuberculosis detection in faeces and tissue samples
by quantitative real time PCR and by cultivation
Iva Slana, Marija Kaevska, Petr Kralik, Alena Kralova, Ivo Pavlik
Veterinary Research Institute, Czech Republic
Due to <strong>the</strong> fact that Mycobacterium avium subsp. paratuberculosis (MAP) is primarily an intestinal pathogen,
<strong>the</strong> most common matrix that is used for MAP detection by cultivation is faeces (and after slaughter <strong>of</strong> animal
is an intestinal mucosa). For culture, it is necessary to perform decontamination to eliminate <strong>the</strong> growth <strong>of</strong>
o<strong>the</strong>r bacteria. Although MAP is quite tolerant to sample decontamination procedures, <strong>the</strong> sensitivity <strong>of</strong> culture
is reduced. Methods based on PCR are suggested as an alternative tool for <strong>the</strong> diagnosis <strong>of</strong> paratuberculosis.
We focused on <strong>the</strong> introduction and optimization <strong>of</strong> an effective and reliable method for <strong>the</strong> DNA isolation
<strong>of</strong> MAP from faeces and from tissue samples. The developed techniques <strong>of</strong> <strong>the</strong> DNA isolation are based on a
slightly modified commercialy available kit. Detection <strong>of</strong> isolated DNA is made by two independent quantitative
real time PCR (qPCR) assays targeting two MAP specific elements: multicopy element IS900 and single copy
element F57. Both qPCR assays incorporate an internal amplification control amplified with <strong>the</strong> same primers
as <strong>the</strong> targets and <strong>the</strong> identical probe is used in both assays.
Faecal and tissue samples <strong>of</strong> different animal species suffering from paratuberculosis were collected over
a period <strong>of</strong> last 16 months and tested for <strong>the</strong> presence <strong>of</strong> MAP. More than 1691 faecal samples and 361 tissue
samples were examined by qPCR and simultaneously by culture. Based on <strong>the</strong>se results we determine <strong>the</strong> dependence
<strong>of</strong> concentration MAP DNA in faecal sample and <strong>the</strong> possibility to detect MAP DNA in tissue sample.
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