275
A LPS-free phospholipase has also been recovered from the ECP, and demonstrated to cause disease signs, upon injection, into Atlantic salmon (Wong et al.
1989 ; Huntly et al. 1992 ). Death resulted overnight following injection of 10 μg/g
body weight of fi sh. Disease signs included lethargy, melanosis, and other defects
characteristic of furunculosis. In addition, erythema was noted on the undersurfaces, particularly around the vent, at the bases of the pectoral and pelvic fi ns, and
head (Huntly et al. 1992 ). Erythrocyte membranes were degraded (= haemolytic
activity). It was concluded that this phospholipase exhibited GCAT activity.
Production of haemolysins by Aer. salmonicida may also contribute to the pathogenesis of furunculosis, insofar as it has been established that ECP contain components with pronounced haemolytic activity for trout erythrocytes (Munro et al.
1980 ). Titball and Munn ( 1981 ) carried out the fi rst extensive study of haemolysin
production by Aer. salmonicida. These authors reported the existence of two distinct
haemolytic activities. Essentially, they determined that the supernatant from
unshaken broth cultures contained haemolytic activity against erythrocytes from a
diverse range of vertebrate species, with maximal activity against horse red blood
cells. Titball and Munn termed this ‘H’ activity. If cultures were shaken, however,
the resulting supernatant yielded an activity against trout erythrocytes only (this was
designated the ‘T’ activity). Furthermore, the H lysin was reported as unstable in
culture supernatants, sensitive to heat after exposure to 56 °C for 5 min, and became
membrane bound when solutions were fi ltered. In contrast, the T lysin was stable in
supernatants, and was inactivated by normal rainbow trout serum. Nomura and
Saito ( 1982 ) also studied the extracellular haemolytic toxin, which was recorded as
cytotoxic for sheep and salmonid erythrocytes. These investigators observed that
the production of haemolysin was stimulated by the addition of enzymic hydrolysates of protein, but suppressed by carbohydrates, such as glucose or sucrose.
Moreover, bivalent metal ions, e.g. Ca
2+ , Co
2+ and Mn
2+ , and phosphate ion
((HPO 4 )
2−
) were necessary for production of the haemolysin. The optimum pH
range and optimum temperature for toxin production was 7.5–8.0 and 20 °C,
respectively. Nomura and Saito ( 1982 ) concluded that the haemolysin was produced
during the stationary phase of the growth cycle, and was relatively heat labile, being
inactivated at 60 °C. These observations coincided with those of Titball and Munn
( 1981 ).
In continued studies of the T and H lysins, Titball and Munn ( 1983 , 1985a ) purifi ed the components, and examined properties of the haemolytic activity. Thus the
T lysin activity was separated into two factors, namely a caseinase and another,
apparently membrane-associated (T 1 ) activity, which by itself caused only incomplete lysis. In fact, complete lysis of trout erythrocytes occurred only in the presence
of both T 1 activity and the caseinase (also see Rockey et al. 1988 ). Titball and Munn
( 1983 ) believed that this phenomenon was due to the co-operative effect of both
activities on the red blood cell membrane, rather than the conversion of T 1 to T lysin
by caseinase. This opinion was reached because the inhibition of caseinase resulted
in the loss of complete lytic potential from supernatant fl uids containing T lysin.
Titball and Munn ( 1985a ) regarded the H lysin to be a proteinaceous substance, on
the basis of results of the ultraviolet absorption spectrum. Additionally, they
Aeromonas salmonicida
A LPS-free phospholipase has also been recovered from the ECP, and demonstrated to cause disease signs, upon injection, into Atlantic salmon (Wong et al.
1989 ; Huntly et al. 1992 ). Death resulted overnight following injection of 10 μg/g
body weight of fi sh. Disease signs included lethargy, melanosis, and other defects
characteristic of furunculosis. In addition, erythema was noted on the undersurfaces, particularly around the vent, at the bases of the pectoral and pelvic fi ns, and
head (Huntly et al. 1992 ). Erythrocyte membranes were degraded (= haemolytic
activity). It was concluded that this phospholipase exhibited GCAT activity.
Production of haemolysins by Aer. salmonicida may also contribute to the pathogenesis of furunculosis, insofar as it has been established that ECP contain components with pronounced haemolytic activity for trout erythrocytes (Munro et al.
1980 ). Titball and Munn ( 1981 ) carried out the fi rst extensive study of haemolysin
production by Aer. salmonicida. These authors reported the existence of two distinct
haemolytic activities. Essentially, they determined that the supernatant from
unshaken broth cultures contained haemolytic activity against erythrocytes from a
diverse range of vertebrate species, with maximal activity against horse red blood
cells. Titball and Munn termed this ‘H’ activity. If cultures were shaken, however,
the resulting supernatant yielded an activity against trout erythrocytes only (this was
designated the ‘T’ activity). Furthermore, the H lysin was reported as unstable in
culture supernatants, sensitive to heat after exposure to 56 °C for 5 min, and became
membrane bound when solutions were fi ltered. In contrast, the T lysin was stable in
supernatants, and was inactivated by normal rainbow trout serum. Nomura and
Saito ( 1982 ) also studied the extracellular haemolytic toxin, which was recorded as
cytotoxic for sheep and salmonid erythrocytes. These investigators observed that
the production of haemolysin was stimulated by the addition of enzymic hydrolysates of protein, but suppressed by carbohydrates, such as glucose or sucrose.
Moreover, bivalent metal ions, e.g. Ca
2+ , Co
2+ and Mn
2+ , and phosphate ion
((HPO 4 )
2−
) were necessary for production of the haemolysin. The optimum pH
range and optimum temperature for toxin production was 7.5–8.0 and 20 °C,
respectively. Nomura and Saito ( 1982 ) concluded that the haemolysin was produced
during the stationary phase of the growth cycle, and was relatively heat labile, being
inactivated at 60 °C. These observations coincided with those of Titball and Munn
( 1981 ).
In continued studies of the T and H lysins, Titball and Munn ( 1983 , 1985a ) purifi ed the components, and examined properties of the haemolytic activity. Thus the
T lysin activity was separated into two factors, namely a caseinase and another,
apparently membrane-associated (T 1 ) activity, which by itself caused only incomplete lysis. In fact, complete lysis of trout erythrocytes occurred only in the presence
of both T 1 activity and the caseinase (also see Rockey et al. 1988 ). Titball and Munn
( 1983 ) believed that this phenomenon was due to the co-operative effect of both
activities on the red blood cell membrane, rather than the conversion of T 1 to T lysin
by caseinase. This opinion was reached because the inhibition of caseinase resulted
in the loss of complete lytic potential from supernatant fl uids containing T lysin.
Titball and Munn ( 1985a ) regarded the H lysin to be a proteinaceous substance, on
the basis of results of the ultraviolet absorption spectrum. Additionally, they
Aeromonas salmonicida