Papel de las actividades superóxido dismutasa y catalasa en la ...

Journal of Fish Diseases 2006, 29, 355–364
PDíaz-Rosales et al. Superoxide dismutase and catalase in P. damselae ssp. piscicida
(a)
(b)
1 2 3 4 5 6 1 2 3 4 5 6
Figure 2 Detection of superoxide dismutase
(a) and catalase activity (b) in extracts of
Photobacterium damselae subsp. piscicida
(strain EPOY-8803-II) grown under different
conditions. Lane 1: growth until exponential
phase; 2: stationary growth phase;
3: exposure to hydrogen peroxide (20 lm
H 2 O 2 mid-exponential phase and 2 mm
H 2 O 2 early stationary phase); 4: addition of
methyl viologen (0.2 mm) to the culture
medium; 5: addition of 2,2¢-dipyridyl
(100 lm) to the culture medium; 6: addition
of FeCl 3 Æ6H 2 O (100 lm) to the culture
medium.
U SOD/mg prot.
20
15
10
5
0
EPOY-8803-II
Lgh41/01
Figure 3 Superoxide dismutase activity (U mg )1 protein) of
Photobacterium damselae subsp. piscicida strains grown under
different culture conditions. ( ) Growth until stationary phase;
( ) exposure to hydrogen peroxide (20 lm mid-exponential
phase and 2 mm early stationary phase); ( ) culture supplemented
with FeCl 3 Æ6H 2 O 100 lm and (h) culture supplemented
with the iron chelant 2,2¢-dipyridyl 100 lm. Data represent the
mean (SD) of three independent determinations.
U cat./mg prot.
140000
120 000
100000
80000
60000
40000
20 000
0
EPOY
Lgh41/01
Figure 4 Catalase activity (U mg )1 protein) of Photobacterium
damselae subsp. piscicida strains grown under different culture
conditions. ( ) Growth until stationary phase; ( ) exposure to
hydrogen peroxide (20 lm mid-exponential phase and 2 mm
early stationary phase); ( ) culture supplemented with FeCl 3 Æ
6H 2 O 100 lm and (h) culture supplemented with the iron
chelant 2,2¢-dipyridyl 100 lm. Data represent the mean (SD)
of three independent determinations.
In order to determine the influence of the levels
of SOD and catalase activity on the resistance to the
bactericidal activity of sole phagocytes, killing assays
were carried out with a virulent and non-virulent
strain of P. damselae ssp. piscicida. The percentages
of surviving bacteria after 5 h contact with sole
phagocytes are shown in Fig. 5. It can be observed
that survival of the virulent strain in contact with
phagocytes was significantly higher (P < 0.05) in
all cases compared with the non-virulent strain.
Despite this different survival rate, both strains
showed a similar behaviour depending on the
bacterial culture condition with highest rates
corresponding in both cases to growth in ironreplete
broths and lowest to growth under ironlimiting
conditions. In addition, a significant
increase in the survival percentages was observed
%Survival
100
90
80
70
60
50
40
30
20
10
0
EPOY-8803-II
Lgh41/01
Figure 5 Survival percentage of Photobacterium damselae subsp.
piscicida after 5 h in contact with sole phagocytes. ( ) Growth
until stationary phase; ( ) exposure to hydrogen peroxide (20 lm
mid-exponential phase and 2 mm early stationary phase); ( )
culture supplemented with FeCl 3 Æ6H 2 O 100 lm and (h) culture
supplemented with 2,2¢-dipyridyl 100 lm. Data represent the
mean (SD) of nine wells containing phagocytes from three fish
specimens.
Ó 2006
Blackwell Publishing Ltd
360