2. Materials and methods
2.1. Prawns used P. Õannamei post-larvae were obtained from Centro de Investigacion in Alimentacion
Ž .
y desarrollo CIAD , Mazatlan, Mexico, and grown in the School of Ocean Sciences, University of Wales, Bangor, Menai Bridge, to a mean weight of 9.41 g in a
recirculatory raceway at 28 28C with 31–33‰ salinity. To avoid inadvertent stimula- tion of prawn immune systems by additives in formulated diets, they were fed only cut
squid and lugworms. Only apparently healthy, intermoult animals were used.
2.2. Bacterial species used and Õaccine preparation In both in vitro and in vivo bacterial killing assays, and for stimulation of the proPO
system of naıve P. Õannamei juveniles, non-pathogenic Vibrio harÕeyi strain DPEX,
¨
and pathogenic V. harÕeyi strains BP03, BP04, BP05, and IN7, isolated from both Ž
healthy and diseased P. monodon in Indonesia Prayitno, 1994; Prayitno and Latchford, .
1995 were obtained from bacterial collection of the School of Ocean Sciences. All Ž
. Vibrio sp. used were maintained on thiosulphate citrate bile sucrose TCBS agar plates
Ž . Ž
at 48C, and cultured in modified sea water complex SWC Reichelt and Baumann,
.
X
1973 at 288C for 24 h before use. In addition Escherichia coli strain XL1-Blue MRF Ž
. Ž .
y1
Bullock et al., 1987 Stratagene , resistant to 15 mg ml
tetracycline and with a recombinant plasmid providing resistance to 100 mg ml
y1
ampicillin, was maintained Ž
. on Luria-Bertani LB slants and was grown in SWC at 378C for 24 h before use.
Ž .
Aeromonas hydrophila strain PM from diseased P. monodon Prayitno, 1994 were stored on SWC agar plates at 48C and grown in SWC medium. Micrococcus lysodeikti-
Ž .
cus luteus obtained as a lyophilised isolate from Sigma , was stored at
y208C and similarly cultured in SWC medium at 288C for 24 h before use.
The concentrations of bacterial cells growing after 24 h was estimated for all the bacterial species used by spreading tenfold serial dilutions of 1 ml aliquots of bacterial
suspensions on SWC agar plates. Natural seawater was adjusted to 25‰ salinity, with Ž
. distilled water autoclaved in 9-ml volumes 1218C; 15 min; 15 psi and cooled to
ambient temperatures before using as dilution medium. The number of colony forming Ž
. units cfu of bacteria growing on three replicate agar plates per unit volume of added
bacterial suspension was determined for each experimental run. Incubation periods used were 24 and 48 h for all species and temperatures used were 378C for E. coli and 288C
for all other species used.
Vaccines were made by exposing log-phase cultures of the relevant bacterium to 0.5 formalin and incubating at 258C with shaking for 12 h. The formalin-killed
bacteria were centrifuged at 7000 = g for 2 min. The resulting pellet was rinsed three times and re-suspended in sterile 25‰ saline to the required concentrations before use.
2.3. Haemolymph extraction and separation Haemolymph was removed from the heart of juvenile P. Õannamei using 27-gauge
needles on 1 ml syringes containing 2 volumes of ice-cold sterile shrimp salt solution
Ž . Ž
. SSS
Vargas-Albores et al., 1993a . The procedures were carried out on ice and subsequent manipulations were performed at 48C. The withdrawn blood was centrifuged
in an Eppendorf microfuge at 13 000 = g for 2 min. The supernatant fluid was passed Ž
. through a 0.45-mm filter Gelman and stored at
y1778C for no more than 4 weeks before use.
2.4. Assays of proPO actiÕity To obtain the inactive proPO enzyme containing granules from the haemocytes, the
haemocyte pellets obtained after decanting the plasma supernatant fluids were resus- pended in sterile SSS and washed again. The haemocytes were resuspended in 3 ml of
Ž .
sodium cacodylate CAC buffer and homogenised on ice using a glass piston ho- mogeniser. The resulting suspension was centrifuged for 20 min at 30 000 = g and 48C
Ž .
to pellet the cell debris. The supernatant haemocyte lysate HLS was the enzyme source and was stored at
y1778C for no more than 4 weeks before use. Incubation procedures were performed in 96 well microtitre plates with 50 ml of HLS
mixed with 50 ml of each of the gram negative bacteria listed above which had been Ž
y1
. formalin-killed, lyophilised, and resuspended 2 mg ml
in CAC buffer. Blanks were Ž
. Ž
with CAC buffer alone and controls consisted of zymosan Sigma suspended 2 mg
y1
. ml
in CAC buffer q 50 ml HLS. Additional controls against spontaneous oxidation
of the substrate alone consisted of CAC buffer and zymosan. Plasma controls were included to ensure that no accidental activation of the HLS occurred during the assay
procedure. Batches with plasma controls showing proPO activity were discarded. Each experimental treatment had three replicates. The samples were incubated at 208C for 65
Ž .
Ž min, after which 100 ml of
L
-3,4-dihydroxyphenyl-alanine
L
-Dopa dissolved 3 mg
y1
. ml
in CAC buffer was added to each well and absorbance values at 490 nm were Ž
taken at 5 min intervals over 20 min in a microplate reader Biokinetics Reader EL-340, .
BIO-TEK Instruments . One unit of proPO activity was expressed as an increase in absorbance of 0.001 mg protein
y1
min
y1
. 2.5. Bacterial growth in plasma from naıÕe prawns
¨
To assess the bacterial growth in plasma from naıve juveniles, 25 ml of E. coli
¨
w Ž
.
5
x suspension mean total viable count TVC of bacteria
s 6.91 = 10 cfu were mixed with 150 ml of plasma and 25 ml of sterile SSS. In control samples, 150 ml of plasma
was replaced by 150 ml of SWC medium and all samples were incubated in sterile 1.5 ml Eppendorf tubes. Each of the treatments listed above had three replicates. To
determine initial bacterial concentrations, 50 ml aliquots from each replicate sample were spread on SWC agar plates at 0 h. All the samples were then incubated with gentle
shaking at 318C for 3 h, after which the tubes were rapidly chilled on ice, suitably diluted in sterile saline and 50 ml of each sample was again spread on SWC agar plates.
Plates were incubated at 378C for 24 h and the TVCs of bacteria were counted.
2.6. Assays of antibacterial and lysozyme actiÕity in cell free haemolymph of Õaccinated P. Õannamei
2.6.1. Effects of addition of lysozyme to E. coli suspensions Several methods of determining antibacterial activity in test samples may fail to
reveal a biological effect because the concentration of active ingredients present is too Ž
. low to operate with the minimum number of bacteria needed. Sung et al. 1996 detected
antibacterial activity in stimulated P. monodon only after concentrating the plasma by a factor of three. In our present study, lysozyme was added to increase the sensitivity of
the bacteria used in antibacterial assays to possible antibacterial factors present in the plasma. A lysozyme to sample ratio needed to be found which, while sensitizing the
bacterial cells to antibacterial action, will not, by itself, cause a decline in bacterial numbers. Hence, preliminary tests were performed to check the effects of the addition of
y1
Ž .
1.24 mg ml of egg white lysozyme Sigma to E. coli suspensions. Twenty five
Ž
5
. microlitres of E. coli suspension mean TVC
s 6.91 = 10 cfu was incubated with 25 Ž
y1
. ml of lysozyme 11.0 mg ml
in sterile SSS , 25 ml of SWC, and 125 ml of SSS. The controls consisted of 25 ml of E. coli suspension, 25 ml of SWC, and 150 ml of SSS.
Each treatment had three replicates. The samples were incubated in sterile 1.5 ml Eppendorf tubes and incubated with shaking at 318C for 3 h, after which, the bacterial
concentrations in each tube were estimated as described above.
2.7. Assays of antibacterial actiÕity Forty eight-hour cultures of non-pathogenic V. harÕeyi strain DPEX were centrifuged
Ž .
at 11 000 = g 5 min, 258C . The bacterial pellet was rinsed thrice and resuspended in Ž
.
5
sterile SSS pH s 7.3 . A total of 100 ml of the final suspension containing 2.22 = 10 –
3.00 = 10
5
cells at room temperature were injected into the ventral sinus of the first abdominal segment of each of three replicate prawns. Positive controls were injected
with 100 ml of sterile SSS and negative controls were uninjected. Test prawns were kept Ž
three each in net covered baskets with dimensions of 31.5 = 21.6 = 20.1 cm length = .
breadth = height suspended in a P. Õannamei rearing raceway as described above. Haemolymph was removed at 0, 6, 12, 24 and 48 h and 4, 7, 14 and 21 days and
cell-free plasma was obtained as described. Ž
. Log-phase E. coli and lyophilised M. luteus grown in SWC
pH s 6.4 were
Ž .
harvested by centrifugation 11 000 = g; 2 min; 258C and washed once in sterile SSS in Ž
. 0.1 M phosphate buffer pH
s 6.4 . The bacterial pellets were resuspended in sterile SSS, well shaken, and kept on ice. A stock solution of 11.2 mg ml
y1
lysozyme was prepared in 0.1 M phosphate buffered SSS, and incubation mixtures were prepared in
sterile 1.5 ml Eppendorf tubes on ice as follows: in antibacterial assays, 25 ml of E. coli suspension was mixed with 150 ml of plasma obtained from either bacteria injected or
sterile SSS injected prawns together with 25 ml of SWC and 25 ml of lysozyme. Controls consisted of plasma from uninjected prawns. To guard against accidental
bacterial contamination during assays, contamination controls were included with each batch, in which the bacterial suspension was replaced with SSS and the 150 ml of SSS
was replaced with 150 ml of plasma from naıve prawns. The addition of 25 ml lysozyme
¨
brought the final lysozyme concentration in the mixtures to 1.24 mg ml
y1
. The incubation treatments were repeated in triplicate at each time interval. Initial total cell
counts of the E. coli suspension was 6.91 = 10
5
1.90 = 10
4
cfu and antibacterial activity was regarded as a decrease in TVC compared to the initial TVC and ratio of
Ž . change in TVC R was calculated as
A y A
t
R s
, A
with A and A being the TVC at the beginning and the end of the reactions,
t
respectively. One unit of antimicrobial activity was computed as a difference of 0.01 Ž
. Ž
. between R control and R treatment .
2.8. Assays of lysozyme actiÕity Lysozyme assays were similarly carried out with M. luteus replacing the E. coli used
in the antibacterial assays and the egg white lysozyme was excluded. Initial total cell counts of the M. luteus suspension was 1.13 = 10
4
4.36 = 10
3
cfu and lysozyme activity was similarly regarded as a decrease in TVC compared to the initial TVC and
Ž . where this occurred, ratio of change in TVC R was calculated as
A y A
t
R s
, A
Ž .
Ž .
Ž .
and lysozyme activity units was computed as R control y R treatment .
2.9. In Õitro bacterial killing assays with plasma from Õaccinated, placebo-injected, and naıÕe prawns
¨
Ž .
P. Õannamei juveniles mean weight s 9.41 g were injected with 100 ml of thrice
washed vaccines of V. harÕeyi strains BPO4, DPEX and E. coli strain XL1-Blue MRF
X
. All vaccines were suspended in sterile SSS to give a wet weight concentration of about
10.88 mg ml
y1
which corresponded to total cfu of 7.46 = 10
6
, 7.38 = 10
6
, and 9.68 = 10
6
per 100 ml, respectively. Each vaccine preparation was injected into each of three replicate prawns. Positive controls were injected with sterile SSS and negative
controls were uninjected. Haemolymph was removed from the prawns’ hearts after 48 h and pooled for each group. The haemocytes were spun down in an Eppendorf mi-
crofuge, and the supernatant fluids passed through a 0.45 mm filter and stored at y1778C for no more than 4 weeks until used.
Triplicate challenge tests were carried out in sterile 1.5 ml Eppendorf tubes. The test mixtures contained 200 ml of plasma obtained from vaccinated, sterile SSS injected or
unvaccinated controls, and were reacted with 100 ml of live E. coli suspension Ž
5
. containing a mean total cfu
s 5.35 = 10 . A contamination control consisting of plasma from naıve prawns
q SSS was included. The bacterial cells in each group were
¨
sensitised by the addition of 25 ml of lysozyme to give a final concentration of 1.24 mg ml
y1
.
Fifty microliter samples were removed at 0, 3, 6, 12 and 24 h from each of the replicates and plated after suitable dilution. Antibacterial activity was recorded as
Ž . Ž
. survival index SI values Wardlaw and Unkles, 1978 calculated as
cfu at end SI
s =
100, cfu at start
with SI values greater than 100 indicating growth and lower than 100 indicating antibacterial activity.
2.10. In ÕiÕo bacterial killing assays in Õaccinated, sterile SSS injected and naiÕe prawns
2.10.1. Measurement of haemolymph Õolume To obtain the initial dilution of bacterial cells after injection into test prawns, the
haemolymph volume of P. Õannamei juveniles was estimated using the inulin dilution Ž
.
14
Ž
y1
. Ž .
method of Levenbook 1958 . Inulin carboxyl- C, 1.4 mCi g Sigma was diluted
4
Ž .
with sterile SSS to give a solution containing 6.0 = 10 disintegrations per minute dpm
y1
Ž .
ml . P. Õannamei juveniles n
s 12; weight range s 2.10–13.90 g , were injected with 50 ml of this inulin solution and held at 288C. At 1, 2 and 3 h, 40 ml of haemolymph
were removed from the pleopod base of the first abdominal segment of each prawn using a 20 ml auto-pipette equipped with an ultra thin microtip. A total of 100 ml of
Ž .
tissue solubilizer Soluene 350, Packard Canberra
were added to the removed haemolymph and the mixture was made up to 4015.00 ml in a high ionic strength liquid
Ž .
scintillation cocktail Hionic Flour, Packard Canberra . A set of standards were made up in SSS to reflect final dilutions in volumes of 1950–4950 ml. Blanks consisted of
haemolymph from uninjected prawns and SSS for the samples and the standards, respectively. Radioactivity was measured using a Beckman LS7500 liquid scintillation
counter.
2.11. Killing assays Ž
. Ž
. Lyophilised vaccines of V. harÕeyi strains BP04 virulent and DPEX avirulent , and
E. coli strain XL1-Blue MRF
X
were resuspended in SSS and the concentration of each was adjusted to 7.26 mg ml
y1
. A total of 100 ml of the final vaccine suspension corresponding to total cfu of
f 7.38 = 10
6
, 7.46 = 10
6
, and 9.68 = 10
6
for V. harÕeyi strains DPEX and BP04 and E. coli strain XL 1-Blue MRF
X
, respectively, were injected into the ventral sinus of the first abdominal segment of each of the nine replicate P.
Ž .
Õ annamei juveniles mean weight
s 9.41 2.23 g . Vaccinated prawns were stocked according to vaccines administered, with three prawns in 20 l of water in rectangular
stacking boxes. Bacterial challenge tests were carried out 48 h after vaccinations by injecting 100 ml
of each of the live bacteria from which the vaccines used were made into the ventral abdominal sinus of the target prawns. Each bacterial challenge treatment had three
replicate prawns per vaccine treatment group. Concentrations of injected bacteria were f 7.38 = 10
6
, 7.46 = 10
6
, and 9.68 = 10
6
total cfu for V. harÕeyi strains DPEX and BP04 and E. coli strain XL 1-Blue MRF
X
, respectively.
At 3, 8, 12, 24 and 48 h, 10 ml of haemolymph were removed from the pleopod base Ž
of the first abdominal segment of each prawn using a 10 ml auto-pipette Eppendorf .
Reference equipped with an ultra thin microtip. The withdrawn haemolymph was Ž
. immediately serially diluted 10-fold in sterile SSS, and spread on three replicate SWC
agar plates. The resulting bacterial colonies were counted following 24-h incubation at 288C.
2.12. Statistical analysis Statistical significance of differences obtained among measured parameters was
Ž .
computed using analysis of variance ANOVA . All data were tested for homogeneity of Ž
. variance using Bartlett’s test Sokal and Rohlf, 1995 prior to further analysis. When
Ž .
ANOVA indicated statistical significance a s 0.05 between factors, Tukey’s all-pair-
wise comparisons were applied to determine significant differences between individual treatment means.
All statistical calculations were performed with the minitab statistical software Ž
. package Minitab .
3. Results
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