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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Aquaculture has potential to support the food supply of increasing world population. Flavobacterial diseases pose a serious problem in wild and aquacultured fish stocks throughout the world. Flavobacterium columnare, F. branchiophilum, and F. psychrophilum are well-known Flavobacterium species that cause important fish losses. Recently, new Flavobacterium species, isolated from diseased fish, have been reported, but their virulence mechanisms are not clear. Thus, the goal of this study was to understand pathogenicity of Flavobacterium species. To this goal, 86 Flavobacterium genomes were analyzed by comparative genomics. Predicted virulence genes were identified for all genomes. For each species, unique and shared virulence genes were determined. For all genomes, unique and common predicted antibiotic resistance genes were identified as well. Secreted proteins are important virulence factors. Thus, all encoded secretion and related systems were determined. By using different genomics approaches, F. columnare genomovar I (highly virulent to cold-water fish species like trout) and genomovar II (extremely virulent to warm-water fish species such as catfish and tilapia) genomes were analyzed, and transposon mutants using Tn4351 in six F. columnare genomovar II strain 94-081 were generated. The hemolysin and glycine cleavage protein mutants had 15% and 10% mortalities, respectively while wild-type strain caused 100% mortality. Potential virulence genes, unique proteins, and other genomic features of F. columnare genomovars were determined. Mutants targeting unique genes in valine-leucine-isoleucine biosynthesis pathway were constructed. The virulence of Fcol(DELTA)leuD and Fcol(DELTA)ilvD mutants exhibited reduced virulence.

The role of reactive oxygen and nitrogen intermediates in the immune response of rainbow trout to R.s. was investigated. The early events occurring when the pathogen interacted with trout macrophages were assessed in terms of the respiratory burst elicited. Live R.s. elicited a respiratory burst, which was enhanced by heat-killed microorganism. This phenomenon, though, was not observed using UV-killed bacteria. Both responses were enhanced when a combination of LPS and TNF was used to activate the macrophages prior to contact R.s. Further studies demonstrated that both compounds synergised to enhance superoxide (O2) production, and that this was correlated with the ability to kill the pathogen. Opsonisation of R.s. with serum factors also increased the respiratory burst, but no difference was found between normal serum and heat-inactivated serum. The role of NO in the immune response of rainbow trout is also studied. Though no evidence of NO production was found in vitro, i.p. injection of live R.s. produced higher NO levels in serum as compared to controls. Fish injected with a virulent strain showed higher levels of NO than controls and than fish injected with an avirulent strain and other strains of unknown virulence. Fish vaccinated with killed R.s. and FIA also showed a significant increase in NO levels, but only four days after vaccination, decreasing thereafter, at both doses of vaccine tested. Injected of Brivax II, an attenuated strain of Aeromonas salmonicida, did not produce a significant increase of NO. RT-PCR was used to detect the expression of the iNOS in different tissues of rainbow trout. iNOS expression was seen only in gill and kidney after i.p. injection. iNOS was detected in the gills 6 h after injecting live R.s. and the expression was still present at day 5. iNOS was detected in the kidney 24 h after injection but was switched off at day 3. After bath challenge with the bacterium, iNOS was expressed in gill, gut and kidney, but the expression varied in each fish. No iNOS expression was found in macrophages isolated from challenged fish.