177
importance of haemolysins were based upon work with protease-defi cient mutants,
the ECP from which was more toxic to recipient fi sh than from wild-type cultures.
Conversely, Thune et al. ( 1982a , b ) obtained a fi sh-toxic fraction, which possessed
proteolytic but not haemolytic activity. Moreover, in a comparison of ECP from
virulent and weakly virulent isolates, Lallier et al. ( 1984 ) noted that both were haemolytic, enterotoxigenic and dermonecrotic, but the weakly virulent isolate produced twenty-fold more haemolysin than the virulent organism. Yet, only cell-free
supernatants from virulent isolates produced toxic (oedematous) effects in fi sh.
Following detailed chemical analyses, this heat-labile toxic factor was separated on
Sephacryl S-200 from the haemolysin. These data suggest that factors other than
haemolysins and proteases may be relevant in fi sh pathology. Indeed, after studying
numerous isolates, Hsu et al. ( 1981 , 1983 ), Shotts et al. ( 1984 ) and Paniagua et al.
( 1990 ) correlated virulence with extracellular proteolytic enzymes, notably caseinase and elastase. Santos et al. ( 1987 ) reported a relationship between virulence in
fi sh and elastase and haemolysin (of human erythrocytes) production and fermentation of arabinose and sucrose. On this theme, Hsu et al. ( 1983 ) associated virulence
with gas production from fructose, glucose, mannitol, mannose, salicin and trehalose, and the possession of resistance to colistin.
Extracellular metallo- and serine-proteases of Aer. hydrophila (strain B 5 ) have
been characterised, and deemed to be heat (to 56 °C) (Leung and Stevenson 1988 )
and cold-stable (to −20 °C) (Nieto and Ellis 1986 ). Most activity was inhibited by
EDTA. Overall, there were many differences in the proteases (4 or 5 were present)
described by Nieto and Ellis ( 1986 ) to reports from other workers. This may be
explained by the work of Leung and Stevenson ( 1988 ), who examined the proteases
from 47 Aer. hydrophila isolates. Of these isolates, 27 produced both metallo- and
serine-proteases, 19 produced only metallo-proteases, and ATCC 7966 produced
only a serine-protease. The differences in these 47 isolates may well explain the
apparent confl icting reports which result from the examination of only single isolates. Certainly, it seems that there are pronounced differences in the characteristics
of the ECP and thus protease composition between strains.
It has been suggested that the proteases may be involved in protecting the pathogen against serum bacteriocidal effects, by providing nutrients for growth following
the destruction of host tissues, and by enhancing invasiveness (Leung and Stevenson
1988 ). Also, proteases may be involved with the activation of haemolysin (Howard
and Buckley 1985 ). In a signifi cant development, it was determined that isolates
with aerolysin ( AerA ), cytotoxic enterotoxin ( alt ) and serine protease genes ( ahp )
were most frequently virulent with lower LD 50 doses in zebra fi sh than isolates with
only one or two of these virulence genes (Li et al. 2011 ).
A further study identifi ed acetylcholinesterase (a 15.5 kDa polypeptide) in the
ECP, and regarded the enzyme as a major lethal factor, possibly with neurotoxic
activity (Nieto et al. 1991 ; Rodriguez et al. 1993a , b ; Peréz et al. 1998 ). The minimal lethal dose of the compound was given as 0.05 μg/g of fi sh.
Aeromonas hydrophila
importance of haemolysins were based upon work with protease-defi cient mutants,
the ECP from which was more toxic to recipient fi sh than from wild-type cultures.
Conversely, Thune et al. ( 1982a , b ) obtained a fi sh-toxic fraction, which possessed
proteolytic but not haemolytic activity. Moreover, in a comparison of ECP from
virulent and weakly virulent isolates, Lallier et al. ( 1984 ) noted that both were haemolytic, enterotoxigenic and dermonecrotic, but the weakly virulent isolate produced twenty-fold more haemolysin than the virulent organism. Yet, only cell-free
supernatants from virulent isolates produced toxic (oedematous) effects in fi sh.
Following detailed chemical analyses, this heat-labile toxic factor was separated on
Sephacryl S-200 from the haemolysin. These data suggest that factors other than
haemolysins and proteases may be relevant in fi sh pathology. Indeed, after studying
numerous isolates, Hsu et al. ( 1981 , 1983 ), Shotts et al. ( 1984 ) and Paniagua et al.
( 1990 ) correlated virulence with extracellular proteolytic enzymes, notably caseinase and elastase. Santos et al. ( 1987 ) reported a relationship between virulence in
fi sh and elastase and haemolysin (of human erythrocytes) production and fermentation of arabinose and sucrose. On this theme, Hsu et al. ( 1983 ) associated virulence
with gas production from fructose, glucose, mannitol, mannose, salicin and trehalose, and the possession of resistance to colistin.
Extracellular metallo- and serine-proteases of Aer. hydrophila (strain B 5 ) have
been characterised, and deemed to be heat (to 56 °C) (Leung and Stevenson 1988 )
and cold-stable (to −20 °C) (Nieto and Ellis 1986 ). Most activity was inhibited by
EDTA. Overall, there were many differences in the proteases (4 or 5 were present)
described by Nieto and Ellis ( 1986 ) to reports from other workers. This may be
explained by the work of Leung and Stevenson ( 1988 ), who examined the proteases
from 47 Aer. hydrophila isolates. Of these isolates, 27 produced both metallo- and
serine-proteases, 19 produced only metallo-proteases, and ATCC 7966 produced
only a serine-protease. The differences in these 47 isolates may well explain the
apparent confl icting reports which result from the examination of only single isolates. Certainly, it seems that there are pronounced differences in the characteristics
of the ECP and thus protease composition between strains.
It has been suggested that the proteases may be involved in protecting the pathogen against serum bacteriocidal effects, by providing nutrients for growth following
the destruction of host tissues, and by enhancing invasiveness (Leung and Stevenson
1988 ). Also, proteases may be involved with the activation of haemolysin (Howard
and Buckley 1985 ). In a signifi cant development, it was determined that isolates
with aerolysin ( AerA ), cytotoxic enterotoxin ( alt ) and serine protease genes ( ahp )
were most frequently virulent with lower LD 50 doses in zebra fi sh than isolates with
only one or two of these virulence genes (Li et al. 2011 ).
A further study identifi ed acetylcholinesterase (a 15.5 kDa polypeptide) in the
ECP, and regarded the enzyme as a major lethal factor, possibly with neurotoxic
activity (Nieto et al. 1991 ; Rodriguez et al. 1993a , b ; Peréz et al. 1998 ). The minimal lethal dose of the compound was given as 0.05 μg/g of fi sh.
Aeromonas hydrophila