The production of extracellular products by Aeromonas
salmonicida, in vitro, has been investigated. The results
indicated that the bacterium produces at least two haemolytic
activities in vitro. Unshaken cultural conditions favoured
the production of a haemolysin with a broad spectrum of
activity against various erythrocyte types (H-lysin), whilst
shaken cultural conditions favoured the production of a
haemolysin active against trout erythrocytes (T-lysin). The
effects of growth medium type and culture conditions on the
production of these haemolytic activities has been investigated.
The activity of the T-lysin appeared to be attributable to the
combined effects of an activity which caused incomplete lysis of
the erythrocytes (T1 activity) and caseinase. The T1 activity
appears to be found in culture supernate associated with fragments
of the bacterial cell wall or membrane resulting in apparent molecular
heterogeneity.
H-lysin activity appeared to be due to a single protein, which
did not require a divalent cation for the expression of activity.
The haemolysin was synthesised by the bacterium as an inactive
precursor molecule (pro-H-lysin) which was cleaved by the bacterial
protease to give the active haemolysin; other commercially available
proteases were also able to effect this activation. An unidentified
component of a variety of animal sera was also able to effect
conversion of the pro-H-lysin to the active form, however, this
conversion only occurred after the serum component had entered the
bacterial cell. The H-lysin was purified 1770 fold using freeze
fractionation, salt fractionation, ion exchange chromatography and gel
filtration chromatography. The partially purified protein possessed
erythrocyte lysing and glycerophospholipid:cholesterol acyltransferase
activities, however it was not clear whether these activities were
attributable to the same molecule. Investigation of the kinetics of
erythrocyte lysis by the partially purified H-lysin suggested that
the haemolysin possessed an enzymatic mode of action. In vitro the
haemolysin was active against both rainbow trout leucocytes and tissue
culture cells. However, in vivo the haemolysin had no obvious effect
on rainbow trout.Ministry of Agriculture,
Fisheries and Food, Weymout