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 The withdrawn haemolymph was Ž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
3.1. Bacterial growth in plasma from naıÕe prawns
¨
TVCs for E. coli increased approximately threefold from 6.91 = 10
5
1.90 = 10
4
to 2.38 = 10
6
8.38 = 10
4
cfu and about 2.3 times to 1.62 = 10
6
3.21 = 10
4
cfu after 3 h incubation in plasma from naıve prawns and SWC, respectively. Upper tailed
¨
two-sample t-tests revealed that the differences in the final concentrations were signifi- Ž
. cantly different
p - 0.0001, t s y34.09 .
3.2. Effects of addition of lysozyme to E. coli suspensions Final total TVCs obtained when E. coli suspensions were incubated with and without
the addition of 1.24 mg ml
y1
of lysozyme were 1.28 = 10
6
2.41 = 10
5
and 1.50 = 10
6
1.90 = 10
5
cfu, respectively. Lower tailed t-tests revealed that these differences Ž
. were not statistically significant
p s 0.13, t s y1.30 indicating that the addition of
egg white lysozyme to give a final concentration of 1.24 mg ml
y1
had no significant effect on bacterial growth.
3.3. Measurement of antibacterial actiÕity in plasma of Õaccinated P. Õannamei In assays for antibacterial activity, plasma obtained from naıve prawns increased
¨
Ž .
bacterial counts over 13-fold compared with initial bacterial counts. E. coli concentra- tions increased from 6.91 = 10
5
1.90 = 10
4
cfu at 0 h to 9.26 = 10
5
1.22 = 10
5
cfu for naıve control plasma samples used in the assay. Upper tailed two-sample t-tests
¨
Ž .
revealed this increase to be significant p s 0.0071, t s 3.69, 95 CI s 1.09–1.63 cfu ,
indicating that the plasma of naıve P. Õannamei contains no background antibacterial
¨
activity when analysed under present conditions. In contrast, a marked antibacterial Ž
activity expressed as a decline in TVC values compared with initial bacterial concentra- .
tions appears within 6 h in both vaccinated and sterile SSS injected juveniles and rises Ž
. to maximum values at 12 h Fig. 1 . Antibacterial activity was detectable for 7 days after
vaccination at which time ANOVA revealed no significant differences in antibacterial activities obtained in the vaccinated treatment and those of the sterile SSS injected and
Ž .
control groups f
s 0.407, p s 0.683 . At all times, when antibacterial activity was detected, the levels were higher in the vaccinated treatments compared with the sterile
Ž .
SSS injected treatments and the untreated controls Fig. 1 , suggesting that antibacterial activity observed in prawn plasma was stimulated in the prawns not only by vaccination,
but also by sterile SSS injection.
3.4. Measurement of lysozyme actiÕity in plasma of Õaccinated P. Õannamei Ž
In lysozyme assays, TVCs of M. luteus obtained in all the treatments vaccinated, .
sterile SSS injected and controls after 3 h incubation, were consistently higher than the
Fig. 1. Effect of vaccination on antibacterial activity in plasma of P. Õannamei. Haemolymph was removed from vaccinated, placebo injected, and naıve prawns at various times after
¨
treatment and the haemocytes were spun down in an Eppendorf microfuge. A total of 150 ml of plasma from each treatment group were then mixed with 25 ml of sea water complex nutrient medium, 25 ml of 11.24 mg
y1
Ž
4
. ml
lysozyme and 25 ml of E. coli suspension mean TVC s 2.76=10 cfu , and incubated for 3 h at 318C.
Ž . Antibacterial activity is regarded as a decrease in TVC compared to the initial TVC and ratio of change R in
Ž .
TVC was calculated as R s A y A r A . With A and A being the TVC at the beginning and the end of
t t
the reactions, respectively. One unit of antimicrobial activity was computed as a difference of 0.01 between R Ž
. Ž
. control and R treatment . I: Antibacterial activity in plasma from prawns vaccinated by injection of 100 ml
of non pathogenic Õibrio harÕeyi strain DPEX containing , 2.61=10
5
cells; `: antibacterial activity in
Ž .
plasma from SSS injected prawns. Prawns were injected with 100 ml of shrimp salt solution SSS ; :
antibacterial activity in plasma from naive prawns.
Ž .
initial TVCs at 0 h Fig. 2 , indicating a lack of lysozyme activity in all the treatments as defined for this study. Despite of this, ANOVA indicated that the increase in TVC
obtained in plasma from vaccinated prawns 6 h after vaccination was not significant, suggesting a definite suppression of M. luteus growth when compared with plasma
Ž .
samples from sterile SSS injected and naıve prawns f
s 5.76, p s 0.018 . After 6 h,
¨
TVCs obtained in all treatments were significantly higher than initial log TVCs up to 48 Ž
. h Fig. 2 .
3.5. Measurement of in Õitro killing actiÕity in plasma of Õaccinated P. Õannamei Results of the in vitro E. coli killing assays in plasma from prawns injected with
Ž .
vaccines made from various bacterial strains Fig. 3
reveals that SI values fell consistently in all vaccinated treatments up to 24 h where SI values of 11.92 5.23,
11.94 3.64, and 27.25 7.78 were obtained for the E. coli and V. harÕeyi strains DPEX and BP04 treatments, respectively. SI values obtained in the control and sterile
SSS injected treatments decreased only up to 6 and 12 h, respectively, after which they
Ž .
both exhibited rises in SI values until the termination of the experiment at 24 h Fig. 3 . As some of the SI values obtained here contained percentage values higher than 100,
ANOVA, followed by Tukey’s pairwise comparisons, were performed on TVC values
Fig. 2. TVC of M. luteus cells after 3 h incubation in plasma withdrawn at different times from vaccinated and naive prawns. Control prawns were injected with sterile SSS.
Haemolymph was removed from vaccinated, placebo injected, and naıve prawns at various times after
¨
treatment and the haemocytes were spun down in an Eppendorf microfuge. A total of 150 ml of plasma from each treatment group were then mixed with 25 ml of SWC nutrient medium, 25 ml of M. luteus suspension
and incubated for 3 h at 318C. Lysozyme activity is regarded as a decrease in TVC compared to the initial Ž .
Ž .
TVC and ratio of change R in TVC was calculated as R s A y A r A . With A and A being the TVC
t t
at the beginning and the end of the reactions, respectively. One unit of lysozyme activity was computed as a Ž
. Ž
. difference of 0.01 between R control and R treatment . I: plasma from vaccinated prawns; `: plasma
from prawns injected with sterile shrimp salt solution; : plasma from naive prawns.
Fig. 3. Survival index values of E. coli strain XL1-Blue MRF
X
suspensions’ various times after incubation in plasma from P. Õannamei juveniles which had been previously vaccinated by injection with 100 ml of a 10.88
y1
Ž .
Ž .
Ž .
mg ml wet weight suspension of V. harÕeyi strains BPO4 virulent and DPEX avirulent and E. coli
strain XL1-Blue MRF
X
in SSS. Injury controls were injected with 100 ml of SSS and plasma controls were uninjected. Haemolymph was removed from prawns and plasma separated from the haemocytes 48 h after
vaccination, and 200 ml of plasma from each treatment group were reacted with 100 ml of live E. coli suspension containing a mean total TVC of about 5.35=10
5
cfu and sensitized by addition of lysozyme to give a final concentration of 1.24 mg ml
y1
. A total of 50 ml aliquots were removed from each treatment at 0, 3, 6, 12 and 24 h, serially diluted in sterile saline, and spread on seawater nutrient agar plates. Bacterial
colonies growing after 24 h incubation at 318C were counted and antibacterial activity was recorded as SI Ž
. values calculated as SI
s cfu at endrcfu at start =100. SI value greater than 100 indicates growth and one lower than 100 indicating antibacterial activity. I; plasma from prawns vaccinated with V. harÕeyi strain
BP04: `; plasma from prawns vaccinated with V. harÕeyi strain DPEX: ; plasma from prawns vaccinated
with E. coli strain XL-1 Blue MRF
X
: q; plasma from SSS injected prawns: N; plasma from unvaccinated
Ž .
control prawns.
obtained after 24 h. This revealed significant reductions in the survival of E. coli when incubated in plasma from all vaccinated prawns. In addition, bacteria incubated in all
plasma samples from vaccinated prawns displayed significantly lower concentrations when compared with bacteria incubated in sterile SSS and naıve plasma controls,
¨
respectively. The concentrations of surviving bacteria in these two treatments were not Ž
. significantly different from each other ANOVA: f
s 22.65, p - 0.001 . 3.6. In Õitro actiÕation of the proPO actiÕating systems of naiÕe P. Õannamei juÕeniles
by formalin-killed Õaccines from different bacterial species Ž
. Scatterplot analyses of results of the kinetic assays of phenoloxidase PO activation
in haemocytes of naıve P. Õannamei juveniles over 20 min by the different bacterial
¨
2
Ž .
strains used consistently displayed low R goodness of fit values , indicating non-lin-
earity of the observed increases in absorbencies observed over time. PO activation
values were therefore calculated per milligram of protein per minute for each time Ž
. interval Table 1 . Results revealed that for all the bacterial species tested, PO activation
Ž .
detected was higher in the initial 5 min after the addition of L-Dopa compared with Ž
. subsequent times after which, the activities generally leveled out or declined Table 1 .
The highest PO activation was recorded upon stimulation by V. harÕeyi strain IN7, followed by A. hydrophila, V. harÕeyi strain BP04, zymosan, and V. harÕeyi strains
DPEX, BP03, and BP05 with mean values of 26.57 3.20, 22.75 5.30, 20.76 3.32, 15.28 9.84, 12.63 2.01, 11.79 0.29, and 11.29 4.20 units, respectively. E. coli
Ž .
gave the lowest mean PO activity value of 5.65 1.60 units over 5 min Table 1 . 3.7. Estimation of the initial concentration of injected bacteria
Total dpm obtained in the standards gave a linear correlation with a prepared set of Ž
2
. Ž .
standard volumes r s 92.6 . Variance ratios f and p-values obtained were f s 25.05
and p s 0.038 and normal plots of the residuals and the fits gave a straight line.
Haemolymph volumes in the sample prawns were estimated from the total. Some error may have been introduced into the estimation of haemolymph volumes by the use of the
Ž inulin dilution method, which may measure both intra and intercellular fluid Levenbook,
. 1958 and give correspondingly higher values. Despite this, regression of estimated
haemolymph volume against body weight of sample prawns indicated a linear equation with r
2
s 71.9, and f-statistic and p-values of 15.33 and 0.008, respectively. In addition, normal plots of the residuals and the fits gave a straight line. The estimated
Ž .
total fluid volumes inulin haemolymph were therefore utilised in the calculations of initial bacterial dilutions.
Ž .
The volume SD of haemolymph corresponding to the mean weight of the prawns used in in vivo killing assays was estimated from this regression line and this was used
Table 1 Ž
. PO activity
meanSD exhibited by haemocyte lysates of P. Õannamei elicited on exposure to A.
hydrophila, E. coli, various strains of V. harÕeyi and zymosan A total of 50 ml of haemocyte lysate from naıve P. Õannamei was reacted for 1 h with 50 ml of 2 mg ml
y1
of
¨
each of the listed bacteria suspended in sodium cacodylate buffer and 100 ml of
L
-Dopa was added. proPO activation was measured as dopa chrome formation by absorbance at 490 nm at 5 min intervals over 20 min.
One unit of proPO activity is expressed as an increase in absorbance of 0.001 mg protein
y1
min
y1
. Elicitor
N 5 min
10 min 15 min
20 min Zymosan
3 15.289.84
7.725.46 5.653.87
4.533.06 A. hydrophila
3 22.755.28
13.124.06 9.472.90
7.062.46 V. harÕeyi strain BP03
3 11.790.29
6.310.80 4.321.09
3.160.83 V. harÕeyi strain BP04
3 20.763.32
12.620.76 8.411.84
6.101.08 V. harÕeyi strain BP05
3 11.294.24
5.982.64 4.151.45
3.531.23 V. harÕeyi strain DPEX
3 12.622.01
6.151.01 4.650.72
3.410.44 V. harÕeyi strain IN7
3 26.573.20
15.031.77 10.801.69
8.261.65
X
E. coli strain XL1-Blue MRF 3
5.651.60 2.332.26
1.272.16 y0.081.82
Cell free plasma 3
3.8212.82 y2.826.43
y4.154.51 y4.943.58
Control 3
y16.111.04 y15.281.25
y13.730.85 y13.120.71
to estimate the initial mean concentrations of bacteria in the haemolymph following injection.
Ž .
The mean weights of prawns used 9.41 2.23 g , corresponded to an estimated volume of 5.63 0.63 ml. Initial concentration of injected bacteria after dilution in
haemolymph was therefore 1.31 = 10
3
, 1.32 = 10
3
and 1.72 = 10
3
cfu ml
y1
for prawns injected with V. harÕeyi strains DPEX and BP04 and E. coli strain XL1-Blue MRF
X
, respectively.
3.8. In-ÕiÕo killing assays In all the treatments, injection of bacteria into the haemolymph was followed by a
reduction in the mean TVC of bacteria within 3 h, although these reductions were not Ž
. always significant Fig. 4 . Prior immunostimulation with vaccines was not implicated in
these initial decreases, as for each challenge treatment group, ANOVA revealed no significant differences in the concentrations of surviving bacteria obtained in both
vaccinated and sterile SSS injected prawns at 3 h.
Ž .
The initial decline phase lasted from 3 to 8 h Table 2 . Following this, a second Ž
. phase of bacterial multiplication occurs, the duration 8–12 h of which is both strain-
and vaccine-dependent. These multiplication phases generally lasted only up to 8 h in
Ž .
X
Fig. 4. Survival meanSD of cells of V. harÕeyi strains BP04 and DPEX and E. coli strain XL1-Blue MRF at various times after injection into P. Õannamei juveniles which had been vaccinated 48 h prior to challenge,
with formalin-killed cells of V. harÕeyi strains BP04 or DPEX or E. coli strain XL-1 Blue MRF
X
. Controls Ž
. were injected with sterile SSS. I: challenged with V. harÕeyi strain BP04 virulent ; `: challenged with V.
Ž .
X
harÕeyi strain DPEX avirulent ; : challenged with E. coli strain XL-1 Blue MRF ; A: injected with sterile
SSS; B: vaccinated with V. harÕeyi: DPEX; C: vaccinated with V. harÕeyi BP04; D: vaccinated with E. coli strain XL-1 Blue MRF
X
.
Table 2 Lower tailed one-sample t-tests on the mean survival in bacterial numbers of cells of V. harÕeyi strains BP04
and DPEX and E. coli strain XL1-Blue MRF
X
per microlitre, 3 h after injection into P. Õannamei juveniles which had been previously vaccinated with formalin-killed cells of V. harÕeyi strains BP04 or DPEX or E.
coli strain XL1-Blue MRF
X
. Controls were injected with SSS. Bacterial challenge was carried out 48 h after vaccination
Bacterial counts ml
y1
were compared to initial mean bacterial counts of 1310, 1320, and 1720 cells ml
y1
for prawns injected with V. harÕeyi strains DPEX and BP04 and E. coli strain XL1-Blue MRF
X
, respectively. Challenged
Vaccinated N
Mean SD
SE mean t
p-Value Significant
by with
reduction? DPEX
SSS 3
185.3 163.4
94.4 y4.66
0.022 yes
DPEX DPEX
3 745.0
337.0 194.0
y2.07 0.087
no DPEX
BP04 3
411 204.0
118.0 y3.66
0.034 yes
DPEX E. coli
3 327
188.0 108.0
y3.42 0.038
yes E. coli
SSS 3
761 262
151.0 y3.82
0.031 yes
E. coli DPEX
3 490
277 160.0
y3.20 0.043
yes E. coli
BP04 3
818 178
103.0 y5.64
0.015 yes
E. coli E. coli
3 397
296 171.0
y2.50 0.065
no BP04
SSS 3
330 161.5
93.3 y5.53
0.016 yes
BP04 DPEX
3 507.0
77.4 44.7
y11.33 0.0038
yes BP04
BP04 3
449.3 153.2
88.5 y4.97
0.019 yes
BP04 E. coli
3 456.0
154.2 89.0
y5.14 0.018
yes
prawns vaccinated with bacterin from V. harÕeyi strains BP04 and DPEX, in contrast to prawns vaccinated with the sterile SSS and E. coli bacterin where these multiplication
phases generally lasted up to 12 h. Bacterial concentrations then declined in all vaccinated treatments up to 24 h, after which this decline either continued or bacterial
multiplication occurred.
When prawns were vaccinated with V. harÕeyi strain BP04 bacterin, and challenged with non-virulent V. harÕeyi strain DPEX or E. coli, the second phase of bacterial
multiplication was absent as the initial decline continued up to 24 h, after which it either Ž
. Ž
. Ž continued DPEX bacterin or bacterial multiplication occurred E. coli bacterin
Fig. .
4 .
4. Discussion
