Motile Aeromonas septicaemia of farmed Rana spp

Abstract

The bacteria associated with septicaemic disease of farmed frogs in Thailand were investigated. A group of motile aeromonads, designated Au {Aeromonas unspeciated), was unusually homogeneous in their biochemical reactions for a geographically diverse collection, notably none of the Au isolates utilised sucrose. Bacteria conforming to the phenotypic reactions of Aeromonas hydrophila and Aeromonas sobria were isolated from the skin and intestine of disea.sed and clinically normal frogs but only Au isolates were found internally in septicaemic frogs. The DNA relatedness of the collection of aeromonads was examined using randomly amplified polymorphic DNA (RAPD) and 16s rDNA sequencing. RAPD analysis produced very consistent banding patterns for the Au isolates whilst producing scattered profiles for type strains and other aeromonads from Thailand. The RAPD profiles showed that the Au isolates were all closely related tOi4. hydrophila (HGl) but were more closely related to each other. 16s rDNA sequence analysis revealed that in the hypervariable region V3 of the 16s rDNA gene all the Au isolates were identical and differed from all previously published Aeromonas sequences. The phenotypic and genotypic findings strongly suggest that Au is a previously unspeciated motile aeromonad. Histological examination of tissues from frogs affected by acute Au septicaemia revealed widespread vascular congestion, severe cardiac myopathy and pulmonary, renal, hepatic and splenic necrosis. In pathogenicity studies, clinically normal animals challenged with Au, by both injection and bath challenge, developed acute septicaemic disease and yielded pure cultures of Au on bacteriological examination. Haemolysin activities against frog RBC were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila) and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any other Thai aeromonads or type strains tested. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophilia. The selection of suitable assay substrates is therefore very important; cells from homeotherms may be insensitive to cytotoxins associated with pathogenic processes in poikilotherms