Proceedings of the 10th International Colloquium on Paratuberculosis

#111 Detection and molecular confirmation <strong>of</strong> Mycobacterium avium subsp. paratuberculosis
in drainage water and forage after application <strong>of</strong> dairy cattle manure on agricultural soils
Miguel Angel Salgado, Marta Andrea Alfaro, Francisco Javier Salazar, Robert Söderlund, Göran Bölske, Monica
Viviana Pradenas, Armin Fabian Mella, Juan Kruze
Universidad Austral de Chile, Chile; Institute for Agricultural Research, Chile; National Veterinary Institute (SVA),
Sweden
Mycobacterium avium subsp. paratuberculosis (Map) is considered an obligate pathogen. In <strong>the</strong>ory it can be
eradicated by <strong>the</strong> removal <strong>of</strong> all infected animals. However, Map can survive for long periods outside <strong>the</strong> host.
Land spreading <strong>of</strong> manure can cause water pollution, ei<strong>the</strong>r through direct aerosol contamination or through
run<strong>of</strong>f from manure applied on <strong>the</strong> fields. There is no published data on Map survival in soils after manure application.
The aim <strong>of</strong> this study was to detect <strong>the</strong> presence <strong>of</strong> Map after application <strong>of</strong> contaminated manure
onto agricultural soils.
A longitudinal study was carried out by extracting intact soil columns that were kept under controlled
environmental conditions. Spiked manure with a known bacterial concentration (108 Map/ml) and manure from
Map positive animals was applied to <strong>the</strong> soil and both drainage water and forage samples were analyzed
bacteriologically. An automated liquid culture system (MGIT) was used for Map detection. All positive tubes
were confirmed by a robust real-time PCR for IS900 and F57 targets, including effective mechanical cell
disruption, DNA extraction and removal <strong>of</strong> PCR inhibitors.
Preliminary results show that Map could be detected by MGIT and confirmed by IS900 and F57 PCR in
drainage water 2 to 4 months after manure application. Map presence in forage and top soil could be confirmed
8 months after manure application. Regarding <strong>the</strong> cycle threshold values (Ct) from <strong>the</strong> real-time PCR
(where <strong>the</strong> Ct cut-<strong>of</strong>f value is above 40 for a negative result), positive drainage water samples showed on average
33 Ct, compared with 25 Ct <strong>of</strong> positive samples from forage and top soil.
These results are suggestive <strong>of</strong> forage and top soil being a more favourable environment for Map survival
than can be provided by <strong>the</strong> subsoil, implying a higher risk for grazing animals than for pollution <strong>of</strong> well water.
176