Aeromonas salmonicida and aeromonas hydrophila (liquefaciens) as pathogens of salmonid fish. A. Selective aeromonas medium. B. Comparative characteristics of virulent and avirulent strains of aeromonas salmonicida. C. Effect of water temperature on aeromonas infections Public Deposited

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  • Studies were directed toward the formulation of a selective Aeromonas medium that would permit the isolation of Aeromonas species in the presence of other common bacterial flora. The final composition of this medium, PBG Agar, in grams per liter, is: Bacto-peptone, 10; Bacto-beef extract, 10; glycogen, 4; NaCl, 5; sodium lauryl sulfate, 0. 1; brom thymol blue, 0. 1; agar, 15. The pH of the medium after autoclaving is 6. 9-7. 1. Any organism isolated from natural sources that gives characteristic yellow colonies in this medium under the specified conditions may presumptively be identified as either Aeromonas, Vibrio, or Pleisomonas.. Aeromonas colonies are frequently surrounded by a yellow acidic halo in the green medium, and occasionally produce a small bubble of gas under the non-nutrient agar overlay. Biochemical and antigenic analyses were performed in an attempt to differentiate virulent (LD₅₀ from one to 10³ colony forming units per fish) from avirulent (LD₅₀ greater than 10⁶ cfu) strains of A. salmonicida. The virulent and avirulent representatives gave similar responses in the following tests: onset and duration of bacteremia in juvenile coho salmon after intramuscular injection; time of recovery of the organism from fish kidney during an acute infection; production of a leucocytolytic agent active on juvenile steelhead trout white blood cells at a dilution of 1;3000; a factor cytotoxic to cultured chinook embryo cells at a dilution of 1:320; formation of eight extracellular enzymes including elastase, several proteases and hemolysins; and the failure to produce hyaluronidase. There were two real differences observed between a virulent strain (As-SS-70) and an avirulent strain (As-Sil) of Aeromonas salmonicida. When grown on 1% peptone agar containing 1. 5% NaCl, colonies of As-SS-70 were opaque, convex, and granular while those of As-Sil were translucent, less convex, and non-granular. An attenuated strain of As-SS-70 gradually assumed avirulent colony characteristics with continued passage on artificial media. Sonicates of As-SS-70 and As-Sil were concentrated about 20 fold by dialysis against Polyethylene Glycol 4000. Use of these concentrated antigens and As-SS-70 rabbit antiserum demonstrated a unique antigen in the As-SS-70 sonicate by immunodiffusion techniques. This unique precipitin line was not observed in the As-Sil sonicate, or when anti-As-Sil rabbit antiserum was used. This was the second difference found between the virulent and avirulent strains. Groups of juvenile coho salmon were injected intramuscularly with about two LD₅₀ doses of virulent A. salmonicida and held at temperatures of 74°F, 69°F, 64°F, 59°F, 54°F, 49°F, 44°F, and 39°F. Water temperatures of 59°F and above produced high mortality rates, with losses exceeding 40 percent at 54°F and 49°F. Mortality rates were very low at temperatures of 44°F and 39°F. The mean time to death was estimated to be 3.5 days at 69°F, with steady increases as water temperature decreased, to a maximum of 31 days at 39°F. Thus, the disease process was accelerated progressively as water temperature increased. When juvenile spring chinook salmon were intraperitoneally injected with about 1.5 LD₅₀ doses of A. salmonicida, the mean time to death was estimated to be 2.9 days at 74°F, and was found to progressively increase as water temperature decreased, to a maximum of 18.4 days at 39°F. The effect of temperature on the growth rate of A. salmonicida in vitro appeared to be similar to its effect on the rate of progress of the infection in juvenile salmonids. The percentage of fatal infections among juvenile steelhead trout and coho salmon injected with Aeromonas hydrophila was high at temperatures of 64°F and above, moderate at 59°F, and zero at 49°F and below.
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