DIFFERENTIATION OF ACTINOBACILLUS-PLEUROPNEUMONIAE STRAINS BY SEQUENCE-ANALYSIS OF 16S RDNA AND RIBOSOMAL INTERGENIC REGIONS, AND DEVELOPMENT OF A SPECIES-SPECIFIC OLIGONUCLEOTIDE FOR IN-SITU DETECTION

The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Ac tinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in sit u detection of A. pleuropneumoniae. Amplification and sequence analysi s of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the thr ee species showed the existence of two RIS's, differing by about 100 b p. Both sequences contained a region resembling the ribonuclease III c leavage site found in Escherichia coli. The smaller RIS contained a Gl u-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respecti ve tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoni ae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A . pleuropneumoniae. These sequences showed only minor base differences , indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the t arget species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA prob e labeled with fluorescein was used for in situ hybridization experime nts to detect A. pleuropneumoniae in biopsies of diseased porcine lung s.