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The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The [DELTA]ascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. [sup.1]H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or [DELTA]ascC. The experimental infection by immersion with [DELTA]aopO was indistinguishable from that of the parental strain, that of [DELTA]aexT was delayed, whilst the virulence of [DELTA]aopH was reduced significantly but not abolished. By i.p. injection, [DELTA]aexT, [DELTA]aopH and [DELTA]aopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The [DELTA]ascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.

Aeromonas salmonicida subsp, salmonicida is a facultatively intracellular gram-negative bacterium that is the etiological agent of furunculosis, a bacterial septicemia of salmonids that causes significant economic loss to the salmon farming industry. The mechanisms by which A. salmonicida evades intracellular killing may be relevant in understanding virulence and the eventual design of appropriate treatment strategies for furunculosis. We have identified two open reading frames (ORFs) and related upstream sequences that code for two putative superoxide dismutases (SODs), soda and sodB. The soda gene encoded a protein of 204 amino acids with a molecular mass of approximately 23.0 kDa (SodA) that had high similarity to other prokaryotic Mn-SODs. The sodB gene encoded a protein of 194 amino acids with a molecular mass of approximately 22.3 kDa that had high similarity to other prokaryotic Fe-SODs. Two enzymes with activities consistent with both these ORFs were identified by inhibition of [O.sub.2.sup.-]-catalyzed tetrazolium salt reduction in both gels and microtiter plate assays. The two enzymes differed in their expression patterns in in vivo-and in vitro-cultured bacteria. The regulatory sequences upstream of putative soda were consistent with these differences. We could not identify other SOD isozymes such as sodC either functionally or through data mining. Levels of SOD were significantly higher in virulent than in avirulent strains old. salmonicida subsp, salmonicida strain A449 when cultured in vitro and in vivo.