Additionally they observed that the partially purified H lysin contained

Additionally they observed that the partially

This preview shows page 327 - 329 out of 594 pages.

stance, on the basis of results of the ultraviolet absorption spectrum. Additionally, they observed that the partially purified H-lysin contained detectable levels of GCAT. This enzyme possesses some similarities to the H-lysin, e.g. moleular weight of 23.2 and 25.9 kDa, respectively (Buckley et ai, 1982). Yet, the molecular weights were much smaller than the 200 kDa size of "salmolysin", the haemolytic toxin described by Nomura et al. (1988). The ionic strengths needed for the elution of GCAT and H-lysin from ion exchange gels were similar. These factors may complicate the isolation of pure H-lysin, assuming that GCAT and the haemolysins are separate entities, which appears to be the case. Thus, GCAT has not been reported to possess haemolytic activity, and is stable at room temperature (Buckley et al., 1982). Mem- brane filtration of the preparation failed to remove GCAT, whereas H-lysin activity was lost after the procedure (Titball and Munn, 1981). Other observations of H-lysin activity have indicated that haemolysis of horse erythrocytes occurs in two steps, namely a first stage in which there is no detectable cell lysis (this was termed the pre- lytic stage), followed by a second phase involving haemoglobin release and disruption of the cell membrane. Binding of the H-lysin to the erythrocytes during the pre-lytic stage does not occur. Together with the observation of an optimum temperature of 25-33°C for lysis, this suggests that the H-lysin has enzymic action on the erythrocyte membrane. Nevertheless, fish injected with H-lysin appeared to be unaffected, despite an apparent toxicity to rainbow trout gonad tissue cell Hnes. Titball and Munn (1981) concluded that the failure of H-lysin to elicit a response in the fish experiments was explained by the use of an unsuitable route of administration or the injection of too low a quantity of the material. In addition, these authors argued that possibly H-lysin is non-toxic to fish, with no important role in the pathological process. Several investigators have explained the relationship of haemolysins and pro- teases to virulence by using different strains of Aer. salmonicida. For example, Hackett et al. (1984) studied the possibiHty of a plasmid-encoded origin for these extracellular enzymes. Significantly, the team concluded that the loss of proteolytic and haemolytic activity in variants of wild-type Aer. salmonicida, obtained by treat- ment with ethidium bromide, did not correlate with loss of plasmid DNA. Moreover, there was no apparent change in the LD^QO between the virulent wild-type strain and its protease haemolysin-deficient variant. This implied that the extracellular activities were not essential for virulence or, indeed, pathogenicity, at least with regard to the acute form of furunculosis in rainbow trout. Two clones derived from another virulent strain, one of which was negative for protease and haemolysin production whereas the second derivative was positive for these attributes, were avirulent (LD50 increased by greater than four orders of magnitude). This was an
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308 Bacterial Fish Pathogens
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