THE MOLECULAR DIFFERENTIATION OF RENIBACTERIUM SALMONINARUM ISOLATES
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Studies were undertaken to examine the extent of molecular variation among isolates of the fish pathogen Renibacterium salmoninarum. As many isolates as possible were gathered from diagnostic laboratories within the UK, and checked for viability and contamination. The isolates were derived mainly from infected rainbow trout and Atlantic salmon that were farmed in England, Scotland and Wales, subject to the requirements of statutory legislation. Incomplete histories were available for the sources of the isolates, which spanned a time scale of 36 years, from 1962-1998. Molecular variation between the isolates was examined using two strategies. Firstly, defined regions of the genome were examined for polymorphisms. PCR analysis of previously characterised genes, msa, hly, and rsh, revealed no length polymorphisms among 43 UK isolates of R. salmoninarum. The 23S and 5S rRNA genes were sequenced and sequence analysis of the 16S- 23S (ITS I) and 23S-SS (ITS2) rRNA regions was performed. Sequence analysis confirmed the correct taxonomic placement of R. salmoninarum within the Micrococcus/Arthrobacter subdivision of the Actinomycetes. Some isolates possessed small sequence variations within ITS1 that can provide an indication of their geographical origins. Sequence variation also exists in the ITS2 region but was found only within a single isolate from an outlying region of Canada. Ribotyping was found to be of limited use for discriminating among isolates of R. salmoninarum probably because R. salmoninarum possesses only two copies of the rRNA operon with no length polymorphisms in the ITS regions. Additionally, the discovery and analysis of a genetic locus containing a 51 bp exact tandem repeat unit (designated ETR-A), revealed that some isolates of R. salmoninarum could be distinguished by the possession of one rather than two copies of this repeat unit. Finally, PCR amplification of length polymorph isms in the tRNA gene spacer regions (tDNA-PCR) was developed using consensus tRNA gene primers to enable tRNA genes and spacer regions to be cloned and sequenced. Subsequently, specific tRNA gene primers were designed and enabled the discrimination of 43 UK isolates into I 5 groupings. tDNA-PCR proved to be one of the most powerful typing methods applied to this organism. Secondly, typing methods that analysed the whole genome for the presence of molecular variation were employed. A putative insertion sequence IS994, was used as a probe in a RFLP-based study to discriminate between 52 isolates of R. salmoninarum from different countries. This method showed great potential for distinguishing between isolates and separated the 52 isolates that were examined into 12 groupings. Randomly amplified polymorphic DNA (RAPD) was also used to examine 28 isolates of UK origin. This method was found to be highly discriminatory, with 28 isolates generating 12 different banding patterns, which appeared to reflect geographical source. Pulsed field gel electrophoresis was also used to investigate the genomic diversity of isolates. Due to technical difficulties in obtaining pure, undamaged DNA the preparation of suitable macro restriction profiles was never achieved. However, preliminary findings suggested that the R. salmoninarum chromosome was linear and approximately 4.5-6Mb in size. Finally, repetitive element PCR was evaluated using 3 different approaches (ERIC, REP and BOXA-2) but proved to have a limited capacity for defining molecular variation between different isolates. Ultimately, RAPD, tDNA-PCR and 1S994 RFLP profiling proved to be most useful for the molecular differentiation of R. salmoninarum. A comparison of the groupings that resulted from each of these methods revealed substantial areas of agreement. The use of a multifactorial approach not only resulted in a greater discrimination of isolates but also provided increased confidence in the outcome. It is recommended that for typing purposes such an approach should be adopted. Epizootiological interpretations of groupings were hampered by the general lack of background information attached to each isolate. However, the application of multiple typing methods reveal that, despite the highly conserved nature of this bacterium, UK isolates of R. salmoninarum possess genetic diversity, with geographically related isolates often being grouped together. Overall there was little evidence to suggest the regular introduction of genetically distinct R. salmoninarum into the UK and the results suggest that some isolates may be relatively localised despite the international trade in fish stocks.
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