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
intervals by a spectrophotometer (Hitachi U-2000:
Hitachi, Tokyo, Japan). The amount of enzyme
resulting in 50% of maximum inhibition of NBT
reduction was determined.
Detection, characterization and quantification
of catalase activity
Catalase activity was visualized on non-denaturing
acrylamide gels following the methodology of
Woodbury, Spencer & Stahmann (1971). After
electrophoresis, gels were washed three times in
distilled water for 20 min and soaked in a solution
of 0.015% H 2 O 2 (30%) (Merck, Darmstadt,
Germany). Then, the activity was visualized by
transferring the gels to a solution of 1% (w/v)
ferric chloride (Panreac Quimica, Barcelona,
Spain) and 1% (w/v) potassium ferricyanide
(Sigma). Regions corresponding to catalase activity
were identified as clear yellow bands on a dark
green background.
The metal cofactor of the catalase produced by
P. damselae ssp. piscicida was determined by
enzymatic inhibition studies according to Barnes,
Bowden, Horne & Ellis (1999b). Lysates of
P. damselae ssp. piscicida strains were incubated
for 1 h with either 100 and 50 mm potassium
cyanide (KCN; Sigma), 1 and 0.5 mm mercuric
chloride (HgCl 2 ; Sigma), 25 and 12.5 mm sodium
azide (NaN 3 ; Sigma) or 50 mm phosphate buffer as
control. Equal volumes of treated extracts were
electrophoresed and gels stained for catalase activity
(Woodbury et al. 1971). Catalases with manganese
as metal cofactor are resistant against sodium azide
and potassium cyanide and sensitive to mercuric
chloride (Kono & Fridovich 1983; Allgood &
Perry 1986; Barnes et al. 1999b). Control wells
inoculated with extracts of Escherichia coli (ATCC
13706) containing a ferric catalase retained the
activity after treatment with mercuric chloride but
not with sodium azide.
Catalase activity was measured spectrophotometrically
by monitoring the decrease in absorbance at
240 nm due to decomposition of hydrogen peroxide.
One unit of catalase was defined as the activity
causing the hydrolysis of 1 lmol of hydrogen
peroxide per minute (Aebi 1984). Briefly, bacterial
extracts were diluted (1:100) in 50 mm potassium
phosphate buffer, pH 7.0 and the absorbance of
the sample containing 660 lL of lysate and
340 lL of H 2 O 2 was measured against a blank
with buffer. The decrease in absorbance at 240 nm
(Hitachi U-2000) was monitored during a 10-min
period.
Bactericidal activity of sole phagocytes
Monolayers of sole phagocytes were prepared
following the methodology of Secombes (1990).
Briefly, the kidneys of 100–300 g sole were dissected
and pressed through a 100 lm nylon mesh with
L-15 medium (Gibco, Gaithersburg, MD, USA)
containing 2% fetal calf serum (FCS; Sigma), 1%
penicillin/streptomycin (Sigma), 0.1% gentamicin
sulphate (50 mg mL )1 distilled water; Sigma) and
10 U mL )1 sodium heparin. The resultant suspension
was layered onto a 30–51% (v/v) Percoll
(Amersham Pharmacia, Piscataway, NJ, USA) density
gradient and the band of cells lying at the 30–
51% interface was collected. The cell suspension
was washed and adjusted to 10 7 cells mL )1 in L-15
medium with antibiotics. The viability was determined
by the exclusion test with trypan blue
(Sigma) (0.5% in PBS). A volume of 100 lL per
well was added to 96-well microtitre plates.
Monolayers were maintained at 22 °C overnight
until bactericidal assays were performed.
Bacterial culture conditions to determine the
ability to resist the bactericidal activity of phagocytes
included growth until stationary phase, addition
of two hydrogen peroxide pulses and growth in
replete or reduced iron medium as previously
described. The bacterial concentration was adjusted
to 1 OD 600 , corresponding to 10 8 bacteria per mL.
The methodology employed to test bacterial survival
after contact with phagocytes was according to
Secombes (1990).
Phagocyte monolayers were washed twice with
L-15 and the cells were then supplemented with
100 lL L-15, 5% FCS per well. Bacterial suspensions
(20 lL) were added to triplicate wells containing
macrophages. The microtitre plate was shaken
and centrifuged at 150 g for 5 min to bring the
bacteria into contact with cells and subsequently
incubated at 22 °C for 0 and 5 h. At the end of the
incubation period, the supernatants were removed
and the killing stopped by lysing the phagocytes with
50 lL of cold sterile distilled water. Subsequently,
100 lL of TSBs was added to support the growth of
the surviving bacteria for 18–20 h at 22 °C.
The number of surviving bacteria was quantified
colorimetrically following the methodology of Peck
(1985) as modified by Graham, Jeffries &
Secombes (1988). Briefly, 10 lL of 3 [4,5-di-
Ó 2006
Blackwell Publishing Ltd
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