Molecular characterisation of Mycoplasma mycoides subsp. mycoides SC

Mycoplasma mycoides subsp. mycoides SC (M. mycoides SC), is the causative agent of contagious bovine pleuropneumonia (CBPP), a severe respiratory disease in cattle. The disease has spread alarmingly across the African continent during the last decade and the problems with eradication are many. Existent diagnostic systems lack specificity and sensitivity, and the available vaccines are not efficient. Hitherto, very little is known about the pathogenicity ofthis organism and new information is sorely needed to control the disease.The first study in the thesis comprises the development of two diagnostic PCR systems that detect M. mycoides SC in various specimens. One system is designed for automated analysis of the PCR products in which the amplicons are detected by laser induced fluorescence to give a high sensitivity and accuracy. The second method can be performed with standard equipment for PCR and agarose gel electrophoresis and the CBPP agent can be distinguished after restriction ofthe amplicon.CBPP agent can be distinguished after restriction ofthe amplicon. Insertion sequences are transposable DNA elements that have been shown to be efficient markers for epidemiology. In the second study, a new IS element ofM. mycoides SC was discovered and characterised, ISMmy/. IS-typing with an ISMmy/-specific probe indicates that this insertion sequence can be a useful marker to differentiate naturally infected CBPP cases and vaccinated animals. ISMmy/ was also found in another bovine pathogen, Mycoplasma bovis and our data suggests that horizontal transfer has occurred at some stage.In the third investigation, a putative virulence factor, Vmm, was identified and characterised. Vmm is a small lipoprotein located at the surface of the bacterial membrane, which undergoes reversible phase variation. By comparing the vmm gene regions from subcloned phenotypic variants, it was shown that expression of Vmm is regulated at the transcriptional level by insertions and deletions in the promoter spacer. The whole genome of M. mycoides SC was sequenced in the fourth project. It is a bacterial genome of 1.14 Mbp with very low G+C content (24%). Altogether, 1,067 open reading frames were identified and the putative genes were annotated and correlated to a function or metabolic process, wherever possible.