Introduction.

Cultivation of M. paratuberculosis is the golden standard of paratuberculosis diagnosis although the method is time consuming, laborious and shows low sensitivity in subclinically infected animals. Detection of M. paratuberculosis by PCR is often hampered by the lack of efficient methods for sample treatment. Faecal material contains an especially high content of PCR inhibitors, and different methods have been performed to avoid such factors. The aim of the present study was to achieve a sensitive method to detect M. paratuberculosis in fecal specimens.

Material and methods.

To assess the optimal sensitivity of the PCR and detection system chosen, serial dilutions of M. paratuberculosis genomic DNA were set up and analysed by PCR and dot blot hybridisation. In addition, serial dilutions of bacterial genomic DNA were analysed by sequence capture-PCR followed by dot blot hybridisation. Furthermore, known amounts of whole M. paratuberculosis bacteria, defined as colony-forming units (CFU), were analysed by sequence capture-PCR and dot blot hybridisation. Finally, faecal samples spiked with known amounts of bacteria, were subjected to buoyant density centrifugation in Percoll(r) prior to subsequent analysis by sequence capture-PCR and dot blot hybridisation. To measure the loss of bacteria by buoyant density centrifugation, known amounts of whole M. paratuberculosis bacteria were submitted to the density centrifugation procedure prior to sequence capture-PCR.

Results and discussion.

By using buoyant density centrifugation, sequence capture-PCR, and dot blot hybridisation, we achieved a sensitivity of 10³ colony forming units (CFU) per g faeces. The detection limit by culture was assessed to 10² CFU per g of faeces. We conclude the described protocol to be a fast and sensitive alternative to bacterial culture of faecal samples.