Diagnosis of contagious caprine and contagious bovine pleuropneumonia by PCR and restriction enzyme analysis

Contagious caprine pleuropneumonia (CCPP) and contagious bovine pleuropneumonia (CBPP) are caused by Mycoplasma capricolum ssp. capripneumoniae and Mycoplasma mycoides ssp. mycoides, small colony type (SC), respectively. These species belong to the group of six mycoplasmas referred to as the Mycoplasma mycoides cluster. The members of the M. mycoides cluster are closely related, and some of them are very difficult to grow. Diagnosis based on identification of the causative agents by cultivation, biochemical reactions, or serology is therefore difficult, and improved diagnostic methods are sorely needed. Identification methods for M. capricolum ssp. capripneumoniae and M. mycoides ssp. mycoides SC, based on detection of the 16S rRNA genes from all members of the M. mycoides cluster by polymerase chain reaction (PCR) and differentiation of species by restriction enzyme analysis with PstI and AluI, respectively, have been developed in the Department of Bacteriology, National Veterinary Institute Laboratory, Sweden. The methods are based on the fact that members of the M. mycoides cluster have two rRNA operons and that there are sequence differences (polymorphisms) between the two 16S rRNA genes from the two operons. The majority of these polymorphisms are characteristic for each species, and some of them can be utilized for differentiation between the species. Techniques for extraction of DNA from lung tissue and from clinical material dried onto filter paper for preservation are described. Pleural fluid was found to be an ideal sample for direct analysis by PCR, but this material can also be dried onto filter paper for preservation and sent to another laboratory for the PCR analysis. These identification systems designed for M. capricolum ssp. capripneumoniae and M. mycoides ssp. mycoides SC have been used for diagnosis of CCPP and CBPP, respectively, by analysis of clinical samples brought to the laboratory as frozen lung tissue or preserved by drying onto a filter paper. (author)