Detection of typical Aeromonas salmonicida during clinical infections

Under ordinary circumstances, typical A. salmonicida can be recovered from clinically diseased fish, especially from the kidney and surface lesions, where present (Austin and Austin, 1993). However, culturing samples from several organs, rather than kidney alone, has been demonstrated to increase the detection rate in fish populations where the incidence of infection is low (Bernoth, 1997b). The recommended diagnosis of the presence of A. salmonicida in clinically diseased fish is based on isolation of the organism from the kidney on either tryptone soya agar (TSA) or brain-heart infusion agar (BHIA) (Department of Fisheries and Oceans, 1984; Shotts, 1984). The brown water-soluble pigment produced by typical A. salmonicida on TSA after 2-4 days' incubation at 20-25°C is used as a presumptive identification. However, caution must be exercised when pigmentation on TSA is used as a presumptive identification of A. salmonicida. Other bacteria have also been found to produce a brown diffusible pigment on TSA, such as mesophilic aeromonads and Pseudomonas fluorescens (McCarthy, 1975; Frerichs and Holliman, 1991). Neither TSA nor BHIA is selective for A. salmonicida, allowing the growth of competing organisms, which may inhibit pigmentation or the growth of A. salmonicida (Austin and Austin, 1993). Inhibition of growth may result from the ability of faster-growing organisms to sequester the available nutrients in the medium (particularly iron) or from the production of inhibitory substances by these competing organisms (Cornick et al., 1969; Michel and Dubois-Darnaudpeys, 1980; Smith and Davey, 1993). Supplementing TSA with 0.01% (w/v) Coomassie brilliant blue (CBB) (CBB agar) (CBBA) has been found by these and other authors to aid in the preliminary differentiation of A. salmonicida from competing bacteria (Cipriano and Bertolini, 1988; Markwardt et al., 1989; Cipriano et al, 1992). On this medium, A-layer-positive A. salmonicida colonies stain deep blue to navy and can be easily distinguished, the intensity of staining being dependent on the source of the dye and the batch of TSA. However, CBBA cannot be totally relied upon, because bacteria other than A. salmonicida can produce dark blue colonies on CBBA (Teska and Cipriano, 1993). None the less, the use of CBBA as a primary plating medium reduces the numbers of bacteria that need to be screened to ensure definitive identification.

Occasional failure to isolate typical A. salmonicida from diseased fish with macroscopic and histological signs of furunculosis has been noted. There are a number of reasons why A. salmonicida may fail to yield colonies on solid media, even in the absence of competing bacteria. Firstly, the number of detectable cells present in the original sample may be below the lower detection limit of cultural isolation (Bernoth, 1997b). Attempts to overcome this limitation, by incorporating a pre-enrichment step, carried out in liquid media, prior to plating on solid media, were reported by Daly and Stevenson (1985). Pre-enrichment of kidney samples in tryptone soya broth (TSB) for 48 h more than doubled the A. salmonicida detection rate in a fish population undergoing a furunculosis epizootic. A second reason for lack of growth might be the unsuitability of laboratory media, such as TSA and BHIA, to support the growth of A. salmonicida. Little is known about the specific nutrient requirements of typical A. salmonicida, other than that it requires methionine and arginine (Nerland et al., 1993). Most artificial media have been formulated for the isolation of medically important bacteria and do not, therefore, present an ideal environment for terrestrial and aquatic organisms. Another potential problem with the use of TSA as the primary isolation medium is that some batches may occasionally fail to support the growth of typical A. salmonicida which will grow on BHIA (Power et al., 1987) or blood agar (Bernoth and Artz, 1989). To exclude this possibility, the Galway laboratory now routinely checks the ability of each TSA batch to support the growth of a positive control A. salmonicida.

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