Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of the serious disease paratuberculosis in ruminants. It is a chronic enteric disease that causes considerable economic losses worldwide. Paratuberculosis leads to reduced milk production and eventually, diarrhoea, weight loss and death. Its slow development, the inappropriate immune response of the host and the fastidiousness of the bacteria all contribute to the difficulty of early diagnosis, necessary to restrict spread of the disease. Thanks to rigorous control measures, paratuberculosis is rare or absent in Sweden. However, occasional import-related outbreaks have occurred, during which all animals in the infected herds were culled. Freedom from paratuberculosis is mainly monitored by slow culture methods. In some situations, fast and reliable alternative methods are needed, for instance, when semen is imported for breeding purposes. Donor bulls may be asymptomatic carriers of MAP and the risk for venereal transmission of the disease is insufficiently investigated. In this thesis, the development and sensitivity assessment of a protocol for detection of MAP in bovine semen by real-time PCR is described. Beadbeating with zirconia/silica beads and phenol/chloroform extraction was used to purify the bacterial DNA and it was shown to successfully remove PCR inhibiting substances. A method for accurately evaluating the analytical sensitivity was also developed. By analysis of artificially infected samples, a sensitivity of 10 organisms per 100 µl sample was assessed. The target gene, for the method described here and for many other PCR methods for detection of MAP, is the insertion element IS900. Although specific for MAP, it has been shown to share similarities with genes in other mycobacteria. Positive PCR results must, therefore, be confirmed by an alternative method. Three novel real-time PCR systems were designed and validated on 267 strains and 58 positive clinical faecal and tissue samples. Two systems were based on IS900 and one system on the MAP-specific gene F57. The latter was the most specific for MAP and is therefore the recommended system for confirmation, but as it was slightly less sensitive when tested on pure DNA, the other systems may be applied when necessary
