Supra dietary levels of vitamins C and E enhance antibody
production and immune memory in juvenile milkfish,Chanos
chanos (Forsskal) to formalin-killed Vibrio vulnificus
I.S,Azad
*
,J,Syama Dayal,M,Poornima,S.A,Ali
Central Institute of Brackishwater Aquaculture,75 Santhome High Road,R.A,Puram,Chennai 600028,India
Received 26 May 2006; revised 16 September 2006; accepted 29 September 2006
Available online 10 October 2006
Abstract
Juveniles of milkfish,Chanos chanos (Forsskal),were fed two independent supra dietary levels of vitamins C (500 and
1500 mgkg
C01
feed,T1 and T2) and E (50 and 150 mgkg
C01
,T3 and T4),Milkfish fed diets with supra (in addition to the vitamins
present in the control diet) and normal levels (T5 containing 90 and 1.2 mg of vitamins C and E,respectively,kg
C01
of feed) of
vitamins were immunized (ip) with formalin-killed Vibrio vulnificus (FKVV),Priming and booster antibody responses to the
injected bacterin were significantly (P<0.05) better in the milkfish juveniles fed supra dietary levels,Survival response of the
experimental fish fed supra dietary levels of vitamins (T1,T2 and T3) was significantly (P<0.01) better than that of the control
set,Protective response against virulent bacterial challenge of the vaccinated fish fed vitamin-supplemented diets (T2 and T3) was
better than the control (T5) and T1 and T4,Memory factor reflecting immunological memory was superior in the fish fed vitamin-
supplemented diets,Diets supplemented with either 1500 mg of Vitamin C or 50 mg of Vitamin E kg
C01
produced the best antibody
responses,final survival and protective response upon challenge,No conclusive inferences could be drawn on the growth responses
from the experiment.
C211 2006 Elsevier Ltd,All rights reserved.
Keywords,Milkfish; Immune response; V,vulnificus; Vitamins C and E; Memory factor
1,Introduction
The biological role played by vitamins C and E is very vital for the sustained growth and health of many living
organisms,These vitamins exhibit antioxidant properties that scavenge reactive oxygen species in membranes [1]
andbiologicalfluids[2].Vitamindeficienciesinfishunderaquacultureareknowntoproducebiochemicaldysfunction
leading to tissue and cellular level clinical manifestations,Several morphological and functional abnormalities have
been reportedinvarious fish species deprivedof vitamins.Properties of diseaseresistance in fish fed ascorbicacidand
Vitamin E have been reported by several researchers [3e7],Dietary vitamins were reported to have antibody
Fish & Shellfish Immunology 23 (2007) 154e163
www.elsevier.com/locate/fsi
* Corresponding author,Present address,Mariculture and Fisheries Department,Kuwait Institute for Scientific Research,P.O,Box No,1638,
Salmiya 22017,Tel.,t965 5711295; fax,t965 5711090.
E-mail address,azadis@rediffmail.com (I.S,Azad).
1050-4648/$ - see front matter C211 2006 Elsevier Ltd,All rights reserved.
doi:10.1016/j.fsi.2006.09.014
enhancement effects in salmon [4,7],Disease resistance and humoral antibody production in rainbow trout was
directly and positively related to the levels of Vitamin C in the trout diet [8],Interaction between these vitamins is
also known to influence the beneficial effects they induce in cultured fish,Vitamin C/E sparing action in channel
catfish was studied to explain the variability observed in its sensitivity to Vitamin E deficiency [9],A dose dependant
protection of dietary Vitamin C against dietary deficiency of Vitamin E was demonstrated in Atlantic salmon [10].
With the persistent losses due to diseases in shrimpaquaculture,coastal aquaculture farmers in India are constantly
on the look-out for sustainable aquaculture and mixed farming of fish with shrimp,Milkfish is one such fish species
that is traditionally harvested from extensivepaddyefish culture systems [11],Information generated on nutrition and
disease management will not only help enhance productivity from milkfish aquaculture but make it possible to tackle
the disease problems that are increasingly becoming a part of aquaculture,Private shrimp farms in India use commer-
cial feeds with vitamins C and E supplementations,The present investigation was carried out with an aim of obtaining
information on the immune response of milkfish to supra dietary vitamins C and E,This study also is aimed at obtain-
ing the information on the protective response and immunological memory.
2,Materials and methods
2.1,Fish
Fingerlings of milkfish (0.87C60.01 to 1.08C60.04 g) collected from the coastal waters off north Chennai,India,
were stocked in 10-tonne cement tanks supplied with filtered aerated seawater (Salinity e 32e34 ppt; DO e 6.2e
7.4 ppm) for acclimatisation.
The experiments were conducted in two sets of rearing systems,Set-I was used for evaluating the effect of supra
dietary levels of vitamins C and E on the growth and survival after 6 weeks of feeding,Set-II was used to immunize
(priming and booster) and evaluate the efficacy of supra dietary vitamins on the antibody production and protective
response.
Set-I,Fish were stocked (30 per tank) in fibre glass reinforced plastic (FRP) tanks of 0.5 tonne capacity and con-
ditioned to experimental environment and control diet for a week,Five treatments were randomly laid out each with
three replicates.
Set-II,Fish were stocked in 200-l FRP tanks,Duplicates of primed and booster sets (5C22C22) containing 12 fish
in each tank were immunized and fed as stated above,All the tanks were supplied with filtered aerated seawater with
more than 80% daily replenishment.
2.2,Feed preparation
Vitamin incorporated feed was prepared using locally available feed ingredients (Table 1),The ingredients such as
thedry fish (Anchovy sp.),squid(Loligosp.),mantis shrimp(Oratosquilla nepa),Acetes and soyacakewereground in
amicropulveriser,passedthrougha300-mmmeshscreenandmixedwithbinder(Aquastab)inanelectricblender.Fish
oil was added into the blender and thoroughly homogenized,Feed ingredients were mixed with additional levels of
stable Vitamin C (SD Fine Chemicals,India,T1 and T2 with 500 and 1500 mgkg
C01
feed,respectively)and Vitamin E
(Merck,India,T3 and T4 with 50 and 150 mgkg
C01
feed,respectively) for the supra dietary supplementation,Control
feed,as per the ingredients (Table 1),contained 90 mg of Vitamin C and 1.2 mg of Vitamin E kg
C01
in the prepared
diet,The ingredients were kneaded into a dough (to an approximate moisture level of 30%),steamed at atmospheric
pressure for 5 min and pelletized (2 mm diameter) in a bench top pelletizer,The pellet was dried in a hot air oven at
40
C14
C for 2e3 days to a uniform moisture level of 9e10%.
Proximate composition of the feed was analysed as per the AOAC [12] methods.
2.3,Immunization
V,vulnificus,isolated from diseased wild collections of gray mullet collected from the backwaters of Muttukadu,
south of Chennai,India was grown in braineheart infusion broth (Hi Media,India) with a final salt concentration of
1.5% at 32
C14
C for 34 h,The bacterium was harvestedby spinning the suspension at 13,000C2g for 10 min; the process
was repeated three times with sterile phosphate buffered saline (PBS,pH 7.2) as the resuspension medium,The final
155I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
suspension of the bacterium corresponded to a cell density of 10
8
CFUml
C01
and the count was confirmed through
spread plate enumeration,Formalin inactivation was carried out adjusting the final formalin concentration in the bac-
terial suspension to 0.5% (v/v of formalin,for 24 h),Inactivated cells were harvested as explained above and the final
suspension (in sterile PBS) offormalin-killed V,vulnificus (FKVV) was used for immunization and for estimating the
antibody levels.
Fish of set-II were conditioned to experimental diet for a week,anaesthetized using 20 ppm crude clove oil (from
alocalpharmacy)emulsioninfilteredseawater,injectedintraperitoneally(0.1 mlcorrespondingto10
7
CFU)withthe
stock suspension of FKVV,Set-II had two subsets of experimental fish one each for priming and booster vaccination.
The booster sets were injected with FKVV,as stated above,at 21 days post-priming (dpp),All the treatment groups
(T1,T2,T3,T4 and T5) of set-II were vaccinated as detailed above and fed respective designated diets for 6 weeks.
2.4,ELISA for anti-FKVV antibodies
Random samples of five fish from different treatments (set-II) were drawn at 0,7,14,21,28 (7 days post-booster,
dpb),35 (14 dpb) and 42 (21 dpb) dpp,The fish were anaesthetized as explained above and the blood was drawn using
1-ml sterile disposable tuberculin syringe via caudal vein,Blood was allowed to clot at room temperature for 1 h and
held overnight at 4
C14
C,centrifuged at 3500C2g for 5 min and the serum was used for the ELISA,Rabbit polyclonal
antiseraagainstFKVVbacterinpreviouslyproducedinthelaboratorywereusedforquantitativetitrationofantibodies
in the fish serum using standard sandwich ELISA protocols,Briefly,100 ml of test serum from milkfish in 1:100
dilutions with sterile carbonate bicarbonate coating buffer (CBC,pH 9.6) was loaded in 96 well ELISA plates for
overnight incubation at 4
C14
C,The plates were flipped off to remove unbound serum from the wells and washed three
times with 1% tween-20 in sterile phosphate buffered saline (TePBS,pH 7.4),The plates were inverted on paper
towels to remove excess moisture and unbound sites of thewells in the plates were blocked using 0.5% BSA in sterile
Table 1
Ingredient and composition of feed used in milkfish feeding trial
Ingredients % composition Vitamins C and E (mgkg
C01
diet)
Treatments C E
Fish meal 35 T1 0590 01.20
Squid 05 T2 1590 01.20
Squilla 10 T3 0090 50.00
Acetes 05 T4 0090 150.00
Soya cake 15 T5 0090 01.20
Wheat flour 22
Binder
a
01
Fish oil 02
Lecithin 02
Vitamin mixture
b
01
Mineral mixture
c
02
Proximate components % composition
Moisture 09.65
Crude protein 39.81
Ether extract 06.94
Crude fibre 04.01
Nitrogen free extract
d
24.91
Ash 14.68
Controlfeed (T5);T1and T2e500 and1500 mgkg
C01
,respectively,ofadditionalstableVitaminC;T3andT4e50and150 mgkg
C01
,respectively,
of additional Vitamin E.
a
Poly methyl carbomide.
b
Vitamin mixture (mg/100 g),Vitamin A 2.0,Vitamin D 0.4,Vitamin E 12.0,Vitamin K 6.0,Choline chloride 600.0,Thiamine 18.0,Riboflavin
24.0,Pyridoxine 18.0,Niacin 108.0,Pantothenic acid 72.0,Biotin 0.2,Folic acid 3.0,Vitamin B
12
0.015,Inositol 150.0,Vitamin C 900.0.
c
Mineral mixture (g/kg),CaCO
3
e28.0,NaHPO
4
e22.0,K
2
SO
4
e10.0,MgSO
4
e12.5,CuSO
4
e0.2,FeCl
3
e0.5,MnSO
4
e0.5,KIe0.01,
ZnSO
4
e 1.0,CoSO
4
e 0.01,Cr
2
SO
4
e 0.05,Bread flour e 7.14.
d
NFE?100 C0 (moisture %tCrude protein %tCrude fibre %tEther extract %tAsh %).
156 I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
PBS for 1 h,The blocking solution was flipped off,washed with TePBS three times and the wells were loaded with
100 ml of FKVV bacterial suspension,incubated for 1 h and washed as above,Thewells were then loaded with 100 ml
of rabbit anti-FKVV polyclonal serum (1:500 dilutions in sterile PBS,pH 7.4) and incubated for 1 h,After the next
washing,100 ml of goat anti-rabbit HRP conjugated antibody (Bangalore Genei,India) was added,incubated for 1 h,
washed and 50 ml of the substrate (TMBeH
2
O
2
,Bangalore Genei,India) was added,The colour development was
stopped after 20 min using 1 N HCl and optical density (OD) of colour developed was measured at 450 nm using
an ELISA reader,Negative control consisted of similarly treated wells where un-immunized rabbit serum was
used instead of anti-FKVV polyclonal serum,The difference in ODs of treatment sets and that of the negativecontrols
was taken as the anti-FKVV titre of the test serum.
2.5,Challenge and relative percent survival (RPS)
A random sample of eight fish from each replicates of primed and boosted groups of set-II and 8 fish from unvac-
cinated control (T5) of set-I were challenged,at the end of the experiment using live bacterial culture of V,vulnificus.
The bacterium was grown in braineheart infusion broth (1.5% NaCl) to get a final cell suspension of
10
7
CFUml
C01
,Challengewas carried out in 100-l FRP tanks with eight fish from each tank,The fish were maintained
for recording mortalities till 10 days post-challenge,Relativepercent survival (RPS) was calculated following Amend
[13] as:
RPS?
C18
1C0
% mortality in vaccinated group
% mortality in C-I or C-II
C19
C2100
where C-I and C-II are control fish (T5) from set-I and set-II.
2.6,Memory factor (MF)
Evaluation of the efficacy of booster response to quantify the immunological memory [14] was carried out using
the ELISA titres measured at different time intervals and the memory factor (MF) was calculated as follows:
MF?
TbexTC0TerT
TpexT
whereTb(x):titreofboostedfishatxdpb;T(r):titreofprimedfishonthedayofbooster;andTp(x):titreofprimedfish
at x dpb.
2.7,Statistical analysis
The data on antibody titres was subjected to ANOVA for testing the significance of difference between treatment
parameters,Pair-wise multiple comparisons for final growth and survival were made following Dennet’s test using
PEPI-404 statistical software,Tukey’s HSD test was used for comparing the ELISA titres due to treatments,due to
days post-immunization and protective response of different treatment groups.
3,Results
3.1,Growth and survival
Information on the stocking,growth and survival of milkfish juveniles is presented in Table 2a,Growth of milkfish
juveniles fed higher levels of Vitamin E (T4) was lower than those of T2 and T3; however,the ANOVA of weight gain
datarevealedthattheparametersdidnotdiffersignificantly(P>0.05)fromoneanother.Itwasinterestingtonotethat
all the treatments differed significantly (P<0.01) in their survival responses from that of the control (Table 2b),Fish
from the control group (T5) showed the lowest survival (80%) followed by those fed higher dietary Vitamin E levels
(150 mgkg
C01
).
157I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
3.2,Antibody response
Antibody titres elicited by the fish fed supra dietary levels of vitamins were significantly (P<0.05) higher than
thoseofthecontrolfish(Tables 3aand3b),Thetrends(Fig.1)inantibody production,withtime,under different treat-
ments indicated a clear superior response of fish fed both levels of vitamins C and E,However,the best booster titres
were noticed in T2 and T3 followed by T1 and T4.
Memory factor (MF),depicting the efficacy of booster immunization,at 21 dpb was the highest in T2 followed by
T3 and T1,while the MF recorded for T4 was significantly (P<0.05) lower than that of T1,T2 or T3 (Fig,2).
3.3,Protective response
Complete mortality of unvaccinated control fish (T5) from set-I was recorded (100% in all replicates) upon
challenge,All the vaccinated groups showed significant (P<0.05) protective response compared to the unvaccinated
control fish from set-I,Challenge tests conducted on fish of set-II revealed a significantly (P<0.05) higher protective
responses of T2 and T3 compared to that of T1,T4 and T5 (Fig,3,Table 3c),Though the challenged fish were kept
under observation for 10 days,mortality was noticed only up to 5 days post-challenge,The relative protective
responses in comparison with unvaccinated and vaccinated control fish (sets I and II,respectively,designated as
RPS-I and RPS-II) depicted efficacy of vaccination with different levels of vitamins and efficacy of different levels
of vitamins within vaccinated groups,There was no significant (P>0.05) difference between the higher level of
Vitamin C (T2) and lower level of Vitamin E (T3) in their protective responses.
4,Discussion
The dietary protein level (39%) used in the present investigation is higher than that suggested by Borlongan and
Satoh [15].Theyrecommended 24%dietaryprotein foreconomicgrow-outproduction ofmilkfish.However,a higher
level of protein was used in the present study; keeping in view the commercial shrimp diet used in shrimp production
ponds and aquaculture potentials of fish-shrimp mixed farming as done in many of the traditional farms of West
Bengal and Kerala in India,Growth responses due to additional supplementation of vitamins C and E,in the present
study,did not result in a statistically significant improvement compared to that of the control,Supra dietary levels of
Vitamin C in yellow perch (Perca flavescens) resulted in better growth and feed efficiency [16],Similar growth
promoting properties of sufficient dietary levels of Vitamin C are well documented [17e20],Short duration of the
experiment was probably responsible for the statistically insignificant difference in growth enhancement between
thegroups,though,therewas an apparentimprovement in treatments T1,T2and T3compared to that of T5.However,
Table 2a
Stocking and growth details of juveniles of Chanos chanos fed vitamin-supplemented diets
Treatments Nt (N0) Wi (gC6SE) Wf (gC6SE) Growth (g) Survival (% C6SE)
T1 79 (90) 0.916C60.138 1.606C60.256 0.69 87.78C61.93
T2 80 (90) 1.082C60.036 1.777C60.118 0.69 88.89C61.93
T3 86 (90) 0.995C60.117 1.682C60.173 0.69 95.56C61.93
T4 84 (90) 0.868C60.011 1.411C60.117 0.54 93.33C63.33
T5 72 (90) 0.952C60.107 1.562C60.217 0.61 80.00C63.33
Nt e number at termination; N0 e Number at start; Wi e initial average weight; Wf e final average weight.
Table 2b
Multiple comparisons for survival response (Dennett test)
Comparison treatments Difference S.E,of difference Two-tailed P
1 vs,5 7.778 0.667 <0.01
2 vs,5 8.889 <0.01
3 vs,5 15.556 <0.01
4 vs,5 13.333 <0.01
158 I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
higher levels of Vitamin E (T4) produced growth retardation,Higher levels of vitamins are required by fish in tropical
aquaculture due to increased physiological stress [21,22],Enhanced growth and survival responses of hybrid striped
bass juveniles fed vitamins C and E was noticed by Sealy and Galtin [23],It is evident from the present study that the
supra dietary levels of both vitamins C and E resulted in a better survival response of milkfish juveniles compared to
the fish in control group.
Role of dietary vitamins in the contextof disease resistance offarmed fish has beenverywell established,Ascorbic
acid deficiency in rainbow trout [24],channel catfish [25] and Atlantic salmon [26] was found to increase disease
Table 3a
ANOVA of ELISA titres in milkfish fed Vitamins (C and E)-supplemented diets
Source of variation SS df MS FP-value F crit
Duration 0.009471 4 0.002368 151.002 1.83E-41 2.462613
Treatments 0.006016 4 0.001504 95.91786 2.41E-33 2.462613
Interaction 0.003905 16 0.000244 15.56677 1.88E-20 1.745647
Within 0.001568 100 1.57E-05
Total 0.02096 124
Table 3b
Multiple comparisons (Tukey’s HSD) of mean ELISA titres in different treatments at different time intervals after priming or booster vaccination
Days post-priming/booster Tukey’s HSD ‘q’ Treatments Difference between mean antibody titres
T1 T2 T3 T4 T5
0 dpp 0.00725 T1 0 0.0004 0.002 0.0008 0.0012
T2 0 0.0016 0.0004 0.0008
T3 0 C00.0012 C00.0008
T4 0.0004
T5 0
7 dpp 0.00738 T1 0 C00.0052 0.0022 0.0104* 0.0128*
T2 0 0.0074* 0.0156* 0.018*
T3 0 0.0082* 0.0106*
T4 0 0.0024
T5 0
14 dpp 0.00511 T1 0 C00.013* C00.003 0.0076* 0.0104*
T2 0 0.01* 0.0206* 0.0234*
T3 0 0.0106* 0.0134*
T4 0.0028
T5 0
21 dpp 0.00579 T1 0 C00.006* C00.0052 0.003 0.0072*
T2 0 0.0008 0.009* 0.0132*
T3 0 0.0082* 0.0124*
T4 0 0.0042
T5 0
42 dpp 0.01065 T1 0 C00.006 C00.0066 0.0058 0.007
T2 0 C00.0006 0.0118* 0.013*
T3 0 0.0124* 0.0136*
T4 0 0.0012
T5 0
7 dpb 0.01045 T1 0 C00.0048 C00.004 0.0104 0.00672
T2 0 0.0008 0.0152* 0.01152*
T3 0 0.0144 0.01072
T4 0 C00.00368
T5 0
21 dpb 0.003462 T1 0 C00.0112* C00.006* 0.0203* 0.0238*
T2 0 0.0052* 0.0315* 0.035*
T3 0 0.0263* 0.0298*
T4 0 0.0035*
T5 0
*Significant at P?0.05.
159I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
susceptibility,Supra dietary levelsof vitamins C and E were found to have direct influence onthe immune response of
juvenile milkfish as evident from the enhanced anti-FKVVantibody titres (T1,T2 and T3) in the present study,Supra
dietary levels of Vitamin C probably helped in neutralizing the stress responses [4,22] of confinement in the present
study and thus,resulted in enhanced antibody production,Similar enhancements in antibody production of channel
catfish against Edwardsiella ictaluri [25] and in rainbow trout against V,anguillarum [4] have been reported,Working
on grouper (Epinehelus awoara),Wei and co-workers [27] reported higher antibody production in the fish fed supra
dietary levels of Vitamin C.
Protective response of milkfish in the present study following vaccination was significant compared to the unvac-
cinated fish,High protective response and immunological memory in the present study can be attributed to a combi-
nation of enhanced specific antibody production and probable elevation of non-specific immune responses as reported
in many fish species receiving varying levels of dietary Vitamin C [23,28e31],High specific antibody response and
enhanced protective responses to bacterial challenge exhibited by milkfish juveniles is supported by the enhanced
immunological memory,Similar findings were reported by Wei et al,[27] who tested higher levels of dietary Vitamin
C in grouper resulting in enhanced specific antibody response against injected formalin-killed V,vulnificus and
protective response against the bacterium,delivered live via injection/bath.
Fig,1,Immune response (ELISA) of juveniles of Chanos chanos fed different vitamin (C and E) enriched diets to Vibrio vulnificus.
Fig,2,Memory factor (MF) as an index of specific immunity in vaccinated juveniles of Chanos chanos fed vitamin (C and E) enriched diets.
Treatment plot points with common letter-lable are not significantly (P?0.05) different from one another.
160 I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
Booster vaccination was rendered highly efficient in milkfish juveniles fed high Vitamin C (T2) and low Vitamin E
(T3) indicating a probable interaction between the two vitamins,It is widely accepted that Vitamin C,in the water
phase,spares Vitamin E and helps in its regeneration from the radical form [32],Lower antibody titres and protective
response of milkfish juveniles fed higher Vitamin E (T4) are probably due to the reduced lymphoproliferation
responses as reported in rainbow trout from a feeding trial with varying combinations of vitamins C and E [28].
Protective response of milkfish juveniles followed closely the results of antibody production with high levels of
Vitamin C not differing significantly from that of low levels of Vitamin E fed fish,Rainbow trout fed double deficient
or double low vitamin (vitamins C/E) diets recorded high mortalities upon challenge with Yersinia ruckeri [28].Low
protective response of T5 is probably due to the negligible levels of vitamins C and E (90 mgkg
C01
and 1.2 mgkg
C01
of
diet,respectively) in the control diet and these levels were not enough to make the fish overcome the vaccination
stress,It has been very well shown by previous researchers that sampling and confinement stress can be managed
with Vitamin C supplementation [20,21],Stress is known to reduce the immune response and disease resistance in
fish [33]; hence,low levels of vitamins C and E (T5) resulted not only in the reduced antibody production but also
in protective response upon challenge with live V,vulnificus,High protective response of T2 and T3 in the present
study is probably due to both enhanced specific immune response and non-specific immune response.
Results of the present study open up new avenues of making milkfish an alternative for mixed crop species in
shrimp aquaculture ponds utilizing the high nutrient feed and supplementing additionally to keep the immune system
fit to fight diseases.
Fig,3,Protective response of vaccinated juveniles of Chanos chanos fed vitamin (C and E) enriched diets to Vibrio vulnificus (plot points of
treatment means sharing common labels are not significantly (P?0.05) different from one another,RPS (C-I),protective response relative to
unvaccinated control,RPS (C-II),protective response relative to the vaccinated control.
Table 3c
Multiple comparisons (Tukey’s HSD) of average percentage of mortality in different treatments following challenge with live Vibrio vulnificus
Treatments Calculated difference between means
T1 T2 T3 T4 T5
T1 0.00 25.00* 27.78* 5.56 13.89
T2 0.00 2.78 30.56* 38.89*
T3 0.00 33.33* 41.67*
T4 0.00 8.33
T5 0.00
Tukey’s HSD at a?0.05 16.48
*Difference between means significant at P<0.05.
161I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
Acknowledgements
TheauthorsarethankfultotheDirector,CIBAforhisconstantsupportandcriticalsuggestions.Thanksarealsodue
to Joseph Sahay Rajan,Technician of CIBA for the help in laboratory analysis.
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e-ronpaku/2003/17BORLONGAN.pdf>; 2003,p,1e42 [accessed 28.10.06].
[16] Lee K-J,Dabrowski K,Interaction between vitamins C and E affects their tissue concentrations,growth,lipid oxidation,and deficiency
symptoms in yellow perch (Perca flavescens),Br J Nutr 2003;89:89e596.
[17] Dabrowski K,El-fiky N,Kock G,Wieser W,Requirement and utilization of ascorbic acid and ascorbic sulphate in juvenile rainbow trout.
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[18] Dabrowski K,Moreau R,El-saidy D,Ontogenic sensitivity of channel catfish to ascorbic acid deficiency,JAquat Anim Health 1996;8:22e7.
[19] Lee K-J,Kim K-W,Bai SC,Effects of different dietary levels of L-ascorbic acid on growth and tissue vitamin C concentrations in juvenile
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[20] Shiau S-Y,Hsu T-S,Quantification of vitamin C requirement for juvenile hybrid tilapia,Oreochromis niloticusC2Oreochromis aureus,with
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[21] Woodward W,Dietary vitamin requirements of cultured young fish,with special emphasis on quantitative estimates for salmonids.
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[22] Montero D,Marrero M,Izquierdo MS,Robaina L,Vergara JM,Tort L,Effect of vitamin E and C dietary supplementation on some immune
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[23] Sealey WM,Galtin DM,Dietary vitamin C and vitamin E interact to influence growth and tissue composition of juvenile hybrid striped bass
(Morone chrysops \C2M,saxatilis _) but have limited effects on immune response,J Nutr 2002;132:748e55.
[24] Blazer VS,Nutrition and disease resistance in fish,Ann Rev Fish Dis 1992;1:309e23.
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[26] Hardie LJ,Fletcher TC,Secombes CJ,The effect of dietary vitamin C on the immune response of Atlantic salmon,Aquaculture
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[28] Verlhac V,Gabaudan J,Obach A,Schuep W,Hole R,Influence of dietary glucan and vitamin C on non-specific and specific immune
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resistance of rainbow trout,Oncorhynchus mykiss (Walbaum),J Fish Dis 1998;21:127e37.
[30] Anbarasu K,Chandran MR,Effect of ascorbic acid on the immune response of the catfish,Mystus gulio (Hamilton),to different bacterins of
Aeromonas hydrophila,Fish Shellfish Immunol 2001;11:355.
[31] Chen R,Lochmann R,Goodwin A,Praveen K,Dabrowski K,Lee K-J,Effects of dietary vitamins C and E on alternative complement
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163I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
production and immune memory in juvenile milkfish,Chanos
chanos (Forsskal) to formalin-killed Vibrio vulnificus
I.S,Azad
*
,J,Syama Dayal,M,Poornima,S.A,Ali
Central Institute of Brackishwater Aquaculture,75 Santhome High Road,R.A,Puram,Chennai 600028,India
Received 26 May 2006; revised 16 September 2006; accepted 29 September 2006
Available online 10 October 2006
Abstract
Juveniles of milkfish,Chanos chanos (Forsskal),were fed two independent supra dietary levels of vitamins C (500 and
1500 mgkg
C01
feed,T1 and T2) and E (50 and 150 mgkg
C01
,T3 and T4),Milkfish fed diets with supra (in addition to the vitamins
present in the control diet) and normal levels (T5 containing 90 and 1.2 mg of vitamins C and E,respectively,kg
C01
of feed) of
vitamins were immunized (ip) with formalin-killed Vibrio vulnificus (FKVV),Priming and booster antibody responses to the
injected bacterin were significantly (P<0.05) better in the milkfish juveniles fed supra dietary levels,Survival response of the
experimental fish fed supra dietary levels of vitamins (T1,T2 and T3) was significantly (P<0.01) better than that of the control
set,Protective response against virulent bacterial challenge of the vaccinated fish fed vitamin-supplemented diets (T2 and T3) was
better than the control (T5) and T1 and T4,Memory factor reflecting immunological memory was superior in the fish fed vitamin-
supplemented diets,Diets supplemented with either 1500 mg of Vitamin C or 50 mg of Vitamin E kg
C01
produced the best antibody
responses,final survival and protective response upon challenge,No conclusive inferences could be drawn on the growth responses
from the experiment.
C211 2006 Elsevier Ltd,All rights reserved.
Keywords,Milkfish; Immune response; V,vulnificus; Vitamins C and E; Memory factor
1,Introduction
The biological role played by vitamins C and E is very vital for the sustained growth and health of many living
organisms,These vitamins exhibit antioxidant properties that scavenge reactive oxygen species in membranes [1]
andbiologicalfluids[2].Vitamindeficienciesinfishunderaquacultureareknowntoproducebiochemicaldysfunction
leading to tissue and cellular level clinical manifestations,Several morphological and functional abnormalities have
been reportedinvarious fish species deprivedof vitamins.Properties of diseaseresistance in fish fed ascorbicacidand
Vitamin E have been reported by several researchers [3e7],Dietary vitamins were reported to have antibody
Fish & Shellfish Immunology 23 (2007) 154e163
www.elsevier.com/locate/fsi
* Corresponding author,Present address,Mariculture and Fisheries Department,Kuwait Institute for Scientific Research,P.O,Box No,1638,
Salmiya 22017,Tel.,t965 5711295; fax,t965 5711090.
E-mail address,azadis@rediffmail.com (I.S,Azad).
1050-4648/$ - see front matter C211 2006 Elsevier Ltd,All rights reserved.
doi:10.1016/j.fsi.2006.09.014
enhancement effects in salmon [4,7],Disease resistance and humoral antibody production in rainbow trout was
directly and positively related to the levels of Vitamin C in the trout diet [8],Interaction between these vitamins is
also known to influence the beneficial effects they induce in cultured fish,Vitamin C/E sparing action in channel
catfish was studied to explain the variability observed in its sensitivity to Vitamin E deficiency [9],A dose dependant
protection of dietary Vitamin C against dietary deficiency of Vitamin E was demonstrated in Atlantic salmon [10].
With the persistent losses due to diseases in shrimpaquaculture,coastal aquaculture farmers in India are constantly
on the look-out for sustainable aquaculture and mixed farming of fish with shrimp,Milkfish is one such fish species
that is traditionally harvested from extensivepaddyefish culture systems [11],Information generated on nutrition and
disease management will not only help enhance productivity from milkfish aquaculture but make it possible to tackle
the disease problems that are increasingly becoming a part of aquaculture,Private shrimp farms in India use commer-
cial feeds with vitamins C and E supplementations,The present investigation was carried out with an aim of obtaining
information on the immune response of milkfish to supra dietary vitamins C and E,This study also is aimed at obtain-
ing the information on the protective response and immunological memory.
2,Materials and methods
2.1,Fish
Fingerlings of milkfish (0.87C60.01 to 1.08C60.04 g) collected from the coastal waters off north Chennai,India,
were stocked in 10-tonne cement tanks supplied with filtered aerated seawater (Salinity e 32e34 ppt; DO e 6.2e
7.4 ppm) for acclimatisation.
The experiments were conducted in two sets of rearing systems,Set-I was used for evaluating the effect of supra
dietary levels of vitamins C and E on the growth and survival after 6 weeks of feeding,Set-II was used to immunize
(priming and booster) and evaluate the efficacy of supra dietary vitamins on the antibody production and protective
response.
Set-I,Fish were stocked (30 per tank) in fibre glass reinforced plastic (FRP) tanks of 0.5 tonne capacity and con-
ditioned to experimental environment and control diet for a week,Five treatments were randomly laid out each with
three replicates.
Set-II,Fish were stocked in 200-l FRP tanks,Duplicates of primed and booster sets (5C22C22) containing 12 fish
in each tank were immunized and fed as stated above,All the tanks were supplied with filtered aerated seawater with
more than 80% daily replenishment.
2.2,Feed preparation
Vitamin incorporated feed was prepared using locally available feed ingredients (Table 1),The ingredients such as
thedry fish (Anchovy sp.),squid(Loligosp.),mantis shrimp(Oratosquilla nepa),Acetes and soyacakewereground in
amicropulveriser,passedthrougha300-mmmeshscreenandmixedwithbinder(Aquastab)inanelectricblender.Fish
oil was added into the blender and thoroughly homogenized,Feed ingredients were mixed with additional levels of
stable Vitamin C (SD Fine Chemicals,India,T1 and T2 with 500 and 1500 mgkg
C01
feed,respectively)and Vitamin E
(Merck,India,T3 and T4 with 50 and 150 mgkg
C01
feed,respectively) for the supra dietary supplementation,Control
feed,as per the ingredients (Table 1),contained 90 mg of Vitamin C and 1.2 mg of Vitamin E kg
C01
in the prepared
diet,The ingredients were kneaded into a dough (to an approximate moisture level of 30%),steamed at atmospheric
pressure for 5 min and pelletized (2 mm diameter) in a bench top pelletizer,The pellet was dried in a hot air oven at
40
C14
C for 2e3 days to a uniform moisture level of 9e10%.
Proximate composition of the feed was analysed as per the AOAC [12] methods.
2.3,Immunization
V,vulnificus,isolated from diseased wild collections of gray mullet collected from the backwaters of Muttukadu,
south of Chennai,India was grown in braineheart infusion broth (Hi Media,India) with a final salt concentration of
1.5% at 32
C14
C for 34 h,The bacterium was harvestedby spinning the suspension at 13,000C2g for 10 min; the process
was repeated three times with sterile phosphate buffered saline (PBS,pH 7.2) as the resuspension medium,The final
155I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
suspension of the bacterium corresponded to a cell density of 10
8
CFUml
C01
and the count was confirmed through
spread plate enumeration,Formalin inactivation was carried out adjusting the final formalin concentration in the bac-
terial suspension to 0.5% (v/v of formalin,for 24 h),Inactivated cells were harvested as explained above and the final
suspension (in sterile PBS) offormalin-killed V,vulnificus (FKVV) was used for immunization and for estimating the
antibody levels.
Fish of set-II were conditioned to experimental diet for a week,anaesthetized using 20 ppm crude clove oil (from
alocalpharmacy)emulsioninfilteredseawater,injectedintraperitoneally(0.1 mlcorrespondingto10
7
CFU)withthe
stock suspension of FKVV,Set-II had two subsets of experimental fish one each for priming and booster vaccination.
The booster sets were injected with FKVV,as stated above,at 21 days post-priming (dpp),All the treatment groups
(T1,T2,T3,T4 and T5) of set-II were vaccinated as detailed above and fed respective designated diets for 6 weeks.
2.4,ELISA for anti-FKVV antibodies
Random samples of five fish from different treatments (set-II) were drawn at 0,7,14,21,28 (7 days post-booster,
dpb),35 (14 dpb) and 42 (21 dpb) dpp,The fish were anaesthetized as explained above and the blood was drawn using
1-ml sterile disposable tuberculin syringe via caudal vein,Blood was allowed to clot at room temperature for 1 h and
held overnight at 4
C14
C,centrifuged at 3500C2g for 5 min and the serum was used for the ELISA,Rabbit polyclonal
antiseraagainstFKVVbacterinpreviouslyproducedinthelaboratorywereusedforquantitativetitrationofantibodies
in the fish serum using standard sandwich ELISA protocols,Briefly,100 ml of test serum from milkfish in 1:100
dilutions with sterile carbonate bicarbonate coating buffer (CBC,pH 9.6) was loaded in 96 well ELISA plates for
overnight incubation at 4
C14
C,The plates were flipped off to remove unbound serum from the wells and washed three
times with 1% tween-20 in sterile phosphate buffered saline (TePBS,pH 7.4),The plates were inverted on paper
towels to remove excess moisture and unbound sites of thewells in the plates were blocked using 0.5% BSA in sterile
Table 1
Ingredient and composition of feed used in milkfish feeding trial
Ingredients % composition Vitamins C and E (mgkg
C01
diet)
Treatments C E
Fish meal 35 T1 0590 01.20
Squid 05 T2 1590 01.20
Squilla 10 T3 0090 50.00
Acetes 05 T4 0090 150.00
Soya cake 15 T5 0090 01.20
Wheat flour 22
Binder
a
01
Fish oil 02
Lecithin 02
Vitamin mixture
b
01
Mineral mixture
c
02
Proximate components % composition
Moisture 09.65
Crude protein 39.81
Ether extract 06.94
Crude fibre 04.01
Nitrogen free extract
d
24.91
Ash 14.68
Controlfeed (T5);T1and T2e500 and1500 mgkg
C01
,respectively,ofadditionalstableVitaminC;T3andT4e50and150 mgkg
C01
,respectively,
of additional Vitamin E.
a
Poly methyl carbomide.
b
Vitamin mixture (mg/100 g),Vitamin A 2.0,Vitamin D 0.4,Vitamin E 12.0,Vitamin K 6.0,Choline chloride 600.0,Thiamine 18.0,Riboflavin
24.0,Pyridoxine 18.0,Niacin 108.0,Pantothenic acid 72.0,Biotin 0.2,Folic acid 3.0,Vitamin B
12
0.015,Inositol 150.0,Vitamin C 900.0.
c
Mineral mixture (g/kg),CaCO
3
e28.0,NaHPO
4
e22.0,K
2
SO
4
e10.0,MgSO
4
e12.5,CuSO
4
e0.2,FeCl
3
e0.5,MnSO
4
e0.5,KIe0.01,
ZnSO
4
e 1.0,CoSO
4
e 0.01,Cr
2
SO
4
e 0.05,Bread flour e 7.14.
d
NFE?100 C0 (moisture %tCrude protein %tCrude fibre %tEther extract %tAsh %).
156 I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
PBS for 1 h,The blocking solution was flipped off,washed with TePBS three times and the wells were loaded with
100 ml of FKVV bacterial suspension,incubated for 1 h and washed as above,Thewells were then loaded with 100 ml
of rabbit anti-FKVV polyclonal serum (1:500 dilutions in sterile PBS,pH 7.4) and incubated for 1 h,After the next
washing,100 ml of goat anti-rabbit HRP conjugated antibody (Bangalore Genei,India) was added,incubated for 1 h,
washed and 50 ml of the substrate (TMBeH
2
O
2
,Bangalore Genei,India) was added,The colour development was
stopped after 20 min using 1 N HCl and optical density (OD) of colour developed was measured at 450 nm using
an ELISA reader,Negative control consisted of similarly treated wells where un-immunized rabbit serum was
used instead of anti-FKVV polyclonal serum,The difference in ODs of treatment sets and that of the negativecontrols
was taken as the anti-FKVV titre of the test serum.
2.5,Challenge and relative percent survival (RPS)
A random sample of eight fish from each replicates of primed and boosted groups of set-II and 8 fish from unvac-
cinated control (T5) of set-I were challenged,at the end of the experiment using live bacterial culture of V,vulnificus.
The bacterium was grown in braineheart infusion broth (1.5% NaCl) to get a final cell suspension of
10
7
CFUml
C01
,Challengewas carried out in 100-l FRP tanks with eight fish from each tank,The fish were maintained
for recording mortalities till 10 days post-challenge,Relativepercent survival (RPS) was calculated following Amend
[13] as:
RPS?
C18
1C0
% mortality in vaccinated group
% mortality in C-I or C-II
C19
C2100
where C-I and C-II are control fish (T5) from set-I and set-II.
2.6,Memory factor (MF)
Evaluation of the efficacy of booster response to quantify the immunological memory [14] was carried out using
the ELISA titres measured at different time intervals and the memory factor (MF) was calculated as follows:
MF?
TbexTC0TerT
TpexT
whereTb(x):titreofboostedfishatxdpb;T(r):titreofprimedfishonthedayofbooster;andTp(x):titreofprimedfish
at x dpb.
2.7,Statistical analysis
The data on antibody titres was subjected to ANOVA for testing the significance of difference between treatment
parameters,Pair-wise multiple comparisons for final growth and survival were made following Dennet’s test using
PEPI-404 statistical software,Tukey’s HSD test was used for comparing the ELISA titres due to treatments,due to
days post-immunization and protective response of different treatment groups.
3,Results
3.1,Growth and survival
Information on the stocking,growth and survival of milkfish juveniles is presented in Table 2a,Growth of milkfish
juveniles fed higher levels of Vitamin E (T4) was lower than those of T2 and T3; however,the ANOVA of weight gain
datarevealedthattheparametersdidnotdiffersignificantly(P>0.05)fromoneanother.Itwasinterestingtonotethat
all the treatments differed significantly (P<0.01) in their survival responses from that of the control (Table 2b),Fish
from the control group (T5) showed the lowest survival (80%) followed by those fed higher dietary Vitamin E levels
(150 mgkg
C01
).
157I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
3.2,Antibody response
Antibody titres elicited by the fish fed supra dietary levels of vitamins were significantly (P<0.05) higher than
thoseofthecontrolfish(Tables 3aand3b),Thetrends(Fig.1)inantibody production,withtime,under different treat-
ments indicated a clear superior response of fish fed both levels of vitamins C and E,However,the best booster titres
were noticed in T2 and T3 followed by T1 and T4.
Memory factor (MF),depicting the efficacy of booster immunization,at 21 dpb was the highest in T2 followed by
T3 and T1,while the MF recorded for T4 was significantly (P<0.05) lower than that of T1,T2 or T3 (Fig,2).
3.3,Protective response
Complete mortality of unvaccinated control fish (T5) from set-I was recorded (100% in all replicates) upon
challenge,All the vaccinated groups showed significant (P<0.05) protective response compared to the unvaccinated
control fish from set-I,Challenge tests conducted on fish of set-II revealed a significantly (P<0.05) higher protective
responses of T2 and T3 compared to that of T1,T4 and T5 (Fig,3,Table 3c),Though the challenged fish were kept
under observation for 10 days,mortality was noticed only up to 5 days post-challenge,The relative protective
responses in comparison with unvaccinated and vaccinated control fish (sets I and II,respectively,designated as
RPS-I and RPS-II) depicted efficacy of vaccination with different levels of vitamins and efficacy of different levels
of vitamins within vaccinated groups,There was no significant (P>0.05) difference between the higher level of
Vitamin C (T2) and lower level of Vitamin E (T3) in their protective responses.
4,Discussion
The dietary protein level (39%) used in the present investigation is higher than that suggested by Borlongan and
Satoh [15].Theyrecommended 24%dietaryprotein foreconomicgrow-outproduction ofmilkfish.However,a higher
level of protein was used in the present study; keeping in view the commercial shrimp diet used in shrimp production
ponds and aquaculture potentials of fish-shrimp mixed farming as done in many of the traditional farms of West
Bengal and Kerala in India,Growth responses due to additional supplementation of vitamins C and E,in the present
study,did not result in a statistically significant improvement compared to that of the control,Supra dietary levels of
Vitamin C in yellow perch (Perca flavescens) resulted in better growth and feed efficiency [16],Similar growth
promoting properties of sufficient dietary levels of Vitamin C are well documented [17e20],Short duration of the
experiment was probably responsible for the statistically insignificant difference in growth enhancement between
thegroups,though,therewas an apparentimprovement in treatments T1,T2and T3compared to that of T5.However,
Table 2a
Stocking and growth details of juveniles of Chanos chanos fed vitamin-supplemented diets
Treatments Nt (N0) Wi (gC6SE) Wf (gC6SE) Growth (g) Survival (% C6SE)
T1 79 (90) 0.916C60.138 1.606C60.256 0.69 87.78C61.93
T2 80 (90) 1.082C60.036 1.777C60.118 0.69 88.89C61.93
T3 86 (90) 0.995C60.117 1.682C60.173 0.69 95.56C61.93
T4 84 (90) 0.868C60.011 1.411C60.117 0.54 93.33C63.33
T5 72 (90) 0.952C60.107 1.562C60.217 0.61 80.00C63.33
Nt e number at termination; N0 e Number at start; Wi e initial average weight; Wf e final average weight.
Table 2b
Multiple comparisons for survival response (Dennett test)
Comparison treatments Difference S.E,of difference Two-tailed P
1 vs,5 7.778 0.667 <0.01
2 vs,5 8.889 <0.01
3 vs,5 15.556 <0.01
4 vs,5 13.333 <0.01
158 I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
higher levels of Vitamin E (T4) produced growth retardation,Higher levels of vitamins are required by fish in tropical
aquaculture due to increased physiological stress [21,22],Enhanced growth and survival responses of hybrid striped
bass juveniles fed vitamins C and E was noticed by Sealy and Galtin [23],It is evident from the present study that the
supra dietary levels of both vitamins C and E resulted in a better survival response of milkfish juveniles compared to
the fish in control group.
Role of dietary vitamins in the contextof disease resistance offarmed fish has beenverywell established,Ascorbic
acid deficiency in rainbow trout [24],channel catfish [25] and Atlantic salmon [26] was found to increase disease
Table 3a
ANOVA of ELISA titres in milkfish fed Vitamins (C and E)-supplemented diets
Source of variation SS df MS FP-value F crit
Duration 0.009471 4 0.002368 151.002 1.83E-41 2.462613
Treatments 0.006016 4 0.001504 95.91786 2.41E-33 2.462613
Interaction 0.003905 16 0.000244 15.56677 1.88E-20 1.745647
Within 0.001568 100 1.57E-05
Total 0.02096 124
Table 3b
Multiple comparisons (Tukey’s HSD) of mean ELISA titres in different treatments at different time intervals after priming or booster vaccination
Days post-priming/booster Tukey’s HSD ‘q’ Treatments Difference between mean antibody titres
T1 T2 T3 T4 T5
0 dpp 0.00725 T1 0 0.0004 0.002 0.0008 0.0012
T2 0 0.0016 0.0004 0.0008
T3 0 C00.0012 C00.0008
T4 0.0004
T5 0
7 dpp 0.00738 T1 0 C00.0052 0.0022 0.0104* 0.0128*
T2 0 0.0074* 0.0156* 0.018*
T3 0 0.0082* 0.0106*
T4 0 0.0024
T5 0
14 dpp 0.00511 T1 0 C00.013* C00.003 0.0076* 0.0104*
T2 0 0.01* 0.0206* 0.0234*
T3 0 0.0106* 0.0134*
T4 0.0028
T5 0
21 dpp 0.00579 T1 0 C00.006* C00.0052 0.003 0.0072*
T2 0 0.0008 0.009* 0.0132*
T3 0 0.0082* 0.0124*
T4 0 0.0042
T5 0
42 dpp 0.01065 T1 0 C00.006 C00.0066 0.0058 0.007
T2 0 C00.0006 0.0118* 0.013*
T3 0 0.0124* 0.0136*
T4 0 0.0012
T5 0
7 dpb 0.01045 T1 0 C00.0048 C00.004 0.0104 0.00672
T2 0 0.0008 0.0152* 0.01152*
T3 0 0.0144 0.01072
T4 0 C00.00368
T5 0
21 dpb 0.003462 T1 0 C00.0112* C00.006* 0.0203* 0.0238*
T2 0 0.0052* 0.0315* 0.035*
T3 0 0.0263* 0.0298*
T4 0 0.0035*
T5 0
*Significant at P?0.05.
159I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
susceptibility,Supra dietary levelsof vitamins C and E were found to have direct influence onthe immune response of
juvenile milkfish as evident from the enhanced anti-FKVVantibody titres (T1,T2 and T3) in the present study,Supra
dietary levels of Vitamin C probably helped in neutralizing the stress responses [4,22] of confinement in the present
study and thus,resulted in enhanced antibody production,Similar enhancements in antibody production of channel
catfish against Edwardsiella ictaluri [25] and in rainbow trout against V,anguillarum [4] have been reported,Working
on grouper (Epinehelus awoara),Wei and co-workers [27] reported higher antibody production in the fish fed supra
dietary levels of Vitamin C.
Protective response of milkfish in the present study following vaccination was significant compared to the unvac-
cinated fish,High protective response and immunological memory in the present study can be attributed to a combi-
nation of enhanced specific antibody production and probable elevation of non-specific immune responses as reported
in many fish species receiving varying levels of dietary Vitamin C [23,28e31],High specific antibody response and
enhanced protective responses to bacterial challenge exhibited by milkfish juveniles is supported by the enhanced
immunological memory,Similar findings were reported by Wei et al,[27] who tested higher levels of dietary Vitamin
C in grouper resulting in enhanced specific antibody response against injected formalin-killed V,vulnificus and
protective response against the bacterium,delivered live via injection/bath.
Fig,1,Immune response (ELISA) of juveniles of Chanos chanos fed different vitamin (C and E) enriched diets to Vibrio vulnificus.
Fig,2,Memory factor (MF) as an index of specific immunity in vaccinated juveniles of Chanos chanos fed vitamin (C and E) enriched diets.
Treatment plot points with common letter-lable are not significantly (P?0.05) different from one another.
160 I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
Booster vaccination was rendered highly efficient in milkfish juveniles fed high Vitamin C (T2) and low Vitamin E
(T3) indicating a probable interaction between the two vitamins,It is widely accepted that Vitamin C,in the water
phase,spares Vitamin E and helps in its regeneration from the radical form [32],Lower antibody titres and protective
response of milkfish juveniles fed higher Vitamin E (T4) are probably due to the reduced lymphoproliferation
responses as reported in rainbow trout from a feeding trial with varying combinations of vitamins C and E [28].
Protective response of milkfish juveniles followed closely the results of antibody production with high levels of
Vitamin C not differing significantly from that of low levels of Vitamin E fed fish,Rainbow trout fed double deficient
or double low vitamin (vitamins C/E) diets recorded high mortalities upon challenge with Yersinia ruckeri [28].Low
protective response of T5 is probably due to the negligible levels of vitamins C and E (90 mgkg
C01
and 1.2 mgkg
C01
of
diet,respectively) in the control diet and these levels were not enough to make the fish overcome the vaccination
stress,It has been very well shown by previous researchers that sampling and confinement stress can be managed
with Vitamin C supplementation [20,21],Stress is known to reduce the immune response and disease resistance in
fish [33]; hence,low levels of vitamins C and E (T5) resulted not only in the reduced antibody production but also
in protective response upon challenge with live V,vulnificus,High protective response of T2 and T3 in the present
study is probably due to both enhanced specific immune response and non-specific immune response.
Results of the present study open up new avenues of making milkfish an alternative for mixed crop species in
shrimp aquaculture ponds utilizing the high nutrient feed and supplementing additionally to keep the immune system
fit to fight diseases.
Fig,3,Protective response of vaccinated juveniles of Chanos chanos fed vitamin (C and E) enriched diets to Vibrio vulnificus (plot points of
treatment means sharing common labels are not significantly (P?0.05) different from one another,RPS (C-I),protective response relative to
unvaccinated control,RPS (C-II),protective response relative to the vaccinated control.
Table 3c
Multiple comparisons (Tukey’s HSD) of average percentage of mortality in different treatments following challenge with live Vibrio vulnificus
Treatments Calculated difference between means
T1 T2 T3 T4 T5
T1 0.00 25.00* 27.78* 5.56 13.89
T2 0.00 2.78 30.56* 38.89*
T3 0.00 33.33* 41.67*
T4 0.00 8.33
T5 0.00
Tukey’s HSD at a?0.05 16.48
*Difference between means significant at P<0.05.
161I.S,Azad et al,/ Fish & Shellfish Immunology 23 (2007) 154e163
Acknowledgements
TheauthorsarethankfultotheDirector,CIBAforhisconstantsupportandcriticalsuggestions.Thanksarealsodue
to Joseph Sahay Rajan,Technician of CIBA for the help in laboratory analysis.
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