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Doctoral thesis, 2001

Phylogeny, diversity, detection : multiple uses of 16S rRNA genes in veterinary bacteriology

Heldtander Königsson, Malin;


Ribosomal RNA in general and the 16S unit in particular has proven very useful for phylogenetic as well as identification purposes in microbiology. Among the advantages in using the 16S rRNA molecule and its genes can be mentioned the fact that it is present and has the same function in all self-replicating cells, and that regions with different variability, from universal to hypervariable, occur and are homologous between organisms. This thesis, based on five scientific publications, describes the use of 16S rRNA genes as a tool for the establishment of phylogenies and the investigation of intraspecific variation in several mycoplasma species and as target region for a species-specific PCR for detection ofR. salmoninarum, the causative agent of bacterial kidney disease.

The phylogeny of four goat mycoplasmas and three seal mycoplasmas was established by sequence analysis ofthe 16S rRNA genes of the type strains ofthe respective species. All three seal mycoplasmas and two goat mycoplasmas belonged to different clusters of the hominis group, and the other two goat mycoplasmas belonged to the M. mycoides cluster ofthe spiroplasma group.

M. capripneumoniae causes contagious pleuropneumonia in goats, a severe disease that is responsible for great economic losses in Africa and Asia. The 16S rRNA genes in M. capripneumoniae have an unusually high number of nucleotide differences between the two operons within a strain, as well as between the homologous operons of different strains. The molecular evolution and epidemiology of 12 M. capripneumoniae strains from three neighbouring African countries were investigated by 16S rDNA sequence analysis.

M. agalactiae and M. bovis, the causative agents ofmastitis and respiratory problems in small ruminants and cattle, respectively, also have high degrees of variability in their 16S rRNA genes. The intraspecific variation in 17 and 8 strains of the respective species was investigated. No distinct evolutionary patterns could be distinguished, despite a high degree of variation. Some conclusions could still be drawn from the data, particularly for M. agalactiae, where the non-European strains shared three characteristic nucleotides, and European strains from the same or neighbouring countries were very similar.

The 16S rRNA gene sequences from 8 strains of/?, salmoninarum were analysed, and sensitive and specific PCR primers and a set of oligonucleotides for DNA preparation by sequence capture were constructed. An internal standard, a mimic molecule, was developed to identify false negative results due to inhibition ofthe amplification reaction.

This work demonstrates that the 16S rRNA still is a powerful tool in many areas of microbiology. It is often a suitable target in detection by PCR for diagnostic purposes and it can be used for studies of genetic diversity and sometimes even for molecular epidemiology. As for molecular phylogeny, the 16S rRNA might well be the most powerful tool, at least so far, simply because ofits versatility.


l6S rRNA; Evolution; Genetic diversity; Mollicutes',Mycoplasma; Phylogeny; PCR; Renibacterium salmoninarum

Published in

Acta Universitatis Agriculturae Sueciae. Veterinaria

2001, number: 102
ISBN: 91-576-5925-7
Publisher: Swedish University of Agricultural Sciences

Authors' information

Heldtander Königsson, Malin
Swedish University of Agricultural Sciences, Department of Veterinary Microbiology
Heldtander Königsson, Malin
National Veterinary Institute (SVA)

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