8

th
International Symposium on Tilapia in Aquaculture 2008
EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS
AND DISEASE RESISTANCE OF NILE TILAPIA
(OREOCHROMIS NILOTICUS)
NEVIEN K. M. ABDELKHALEK
1
, VIOLA H. ZAKI
2
AND M. A. A. YOUSEF
2
1. Present address: Laboratory of Marine Biochemistry !aculty of A"riculture
#yushu $ni%ersity &apan.
2. 'epartment of Internal Me(icine Infectious an( !ish 'iseases !aculty of
)eterinary Me(icine *l Mansoura $ni%ersity *"ypt.
Abstract
Different immunostimulants were investigated to detect
changes in the activity of innate non-specific immune response,
disease resistance and growth performance of Nile Tilapia
+reochromis niloticus. In this experiment, fish fed on diets
supplemented with levamisole HCl (! mg"#g diet$, Immunoton
%
(a
mixture of vitamins & ('!( mg"#g diet$ and C ('(') mg"#g diet$
and control diet for wee#s) Total leu#ocyte count (T*C$,
Differential leu#ocyte count (D*C$, phagocytosis have +een
measured as indicators for cellular innate immune response)
*yso,yme and protein electrophoresis have +een measured as
indicators for humoral innate immune response, in addition to
immunocompetence assay against Aeromonas hy(rophila and
growth performance test) The o+tained results indicated that,
addition of levamisole HCl to the diet of +. niloticus significantly
enhanced +oth cellular and humoral innate immune responses and
increased resistance to A. hy(rophila infection although they had a
little growth promoting activity) -hile addition of vitamins C and & to
+. niloticus higher than the re.uirements needed for growth lead to
maximum growth compared to levamisole +ut little enhancement of
the immune response as well as resistance to A. hy(rophila
infection)
Key w!"#$ Immunostimulants/ Innate immune response/
01
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32 NI*& TI*79I7 (+,*+-.,+MIS /IL+TI-$S$
+reochromis niloticus)
%
C!!e#&'"(') *+,-!
Nevien :amel 5ohamed 7+del#hale#, 9hD
*a+oratory of 5arine ;iochemistry,
Department of ;ioscience and ;iotechnology,
<raduate 4chool of ;ioresource and ;ioenvironmental 4cience,
:yushu 6niversity, 2u#uo#a ='-=!=', >apan)
Tel? @='-1-A0-=10 2ax? @='-1-A0-=1B mo+ile? @='- 1(- =0(0-=1(A
&-mail) nevienC#Dagr)#yushu-u)ac)Ep
INTRODUCTION
-ith the worldwide fish production and intensive cultivation system, fish are
su+Eected to many diseases that lead to great losses and decrease in fish production)
The lac# of effective disease control has the potential of +eing the chief limiting factor
of the reali,ation of highly sta+le fish production (9hillip et al. ((($) 7.uatic animal
diseases control in &gypt includes a limited num+er of <overnment-approved
anti+iotics and chemotherapeutics, +eside limited vaccines that can +e used to assist
the environmental management (7ly et al. ((=$) However, the approach which
concentrates on treatment once disease develops using anti+iotic are sometimes of
little value or less successful due to emergence of anti+iotic resistant micro-
organisms which ma#es this method of control less successful (7nderson, '11 and
4tos#opf, '11F$)
Immunotherapy is an approach that has +een actively investigated in recent
years as a method for disease prevention) It does not involve recognition of a specific
antigen or targeting the immune response towards a specific pathogen, +ut causes
an overall immune response that hastens recognition of foreign proteins (Campos et
al), '11F/ 4ecom+es, '110 and 4ordello et al., '11B$) 4o the use of
immunostimulants for prevention of diseases in fish is considered an alternative and
promising area (4a#ai, '111$)
*evamisole, originally synthesi,ed as an anti-helminthic, has +een widely used as
an immunomodulator in fish either +y inEection (4iwic#i, '1=B$, immersion (4iwic#i
and :orwin-:oss#ows#i, '1==$, oral administration (4iwic#i, '1=1/ 4iwic#i and
4tudnic#a, '110/ 5ulero et al. '11=+ 7lvare, et al., ((A$ or in vitro
immunostimulation (4iwic#i et al. '11$)
!(
N&GI&N :) 5) 7;D&*:H7*& et al.
6se of vitamins as immunostimulant has +een used in many fish including
7tlantic salmon (-aag+o et al. '11$, rain+ow trout (GerIhac et al. '11=$ and
gilthead sea +ream (5ulero et al. '11=a, 3rtuno et al. '111, ((( and Cuesta et al.
(('$ although not so much data for tilapia) The aim of the present wor# is to study
the effect of dietary inta#e of levamisole and Immunoton
%
(vitamins C and &$ on
innate immune system, resistance to diseases and growth performance of Nile
Tilapia 0+reochromis niloticus1)
MATERIALS AND METHODS
FISH
7 total num+er of F(( apparently healthy +reochromis niloticus o+tained
from a private fish farm in &* Da#ahlia <overnorate with average +ody weight of
=)'(H!$ g transported alive to the la+oratory of Department of 2ish Diseases and
5anagement, 2aculty of Geterinary 5edicine, &l 5ansoura 6niversity) They were
#ept for wee#s under o+servation for acclimati,ation in glass a.uaria (0( I A( I
'(( cm$) The water of the a.uaria was removed daily, and its temperature was
maintained at ! H ' JC)
IMMUNOSTIMULANTS
Le.*/(#0e
1 HCL

ItKs a commercial product availa+le in the mar#et manufactured +y 5emphis
9harmaceutical, Cairo, &gypt) ItKs used as anti helminthic and immunostimulant for
large animals in &gyptian farms) &ach ml contains ()'g levamisole HC*) The dose
was calculated to +e !mg "#g diet then mixed with the +asal diet and pellets were
made) The pellets were prepared +iwee#ly, air dried at room temperature and stored
in a refrigerator (0 JC$ for daily use)
I//+','
1
ItKs a commercial product availa+le in the mar#et manufactured +y &l 4afa 9harm
for manufacturing, 7l Nu+aria, &gypt) ItKs used as immunostimulant for poultry in
&gyptian farms) &ach one #g contains (vitamin & '!( g, vitamin C '(') g, sodium
selenite mg, folic acid !' mg and *actose up to one '((( g$) It is mixed with
the +asal diet at a dose of 'g of the product"#g diet and pellets were made) The
!'
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32 NI*& TI*79I7 (+,*+-.,+MIS /IL+TI-$S$
pellets were prepared +iwee#ly, air dried at room temperature and stored in a
refrigerator (0 JC$ for daily use)
E2PERIMENTAL DESI3N
2ish were divided into F groups ('(( fish"group$) 2irst group served as a control
group fed on +asal diet,
nd
group fed on *evamisole
1
supplemented diet for wee#s
then fed on +asal diet till the end of the 0
th
wee#) F
rd
group fed on Immunoton
1

supplemented diet for wee#s then fed on +asal diet till the end of the 0
th
wee#) &ach
group su+divided into 0 su+groups (! fish"su+group$)
2ish were fed on +asal diet of F((( #cal"#g digesti+le energy and F(L protein either
control (+asal diet only$ or immunostimulant supplanted diets at a feeding rate of F( g
diet"#g +iomass"day, divided into two feeding times for wee#s then fed on
immunostimulant free diet (+asal diet only$ till the end of experimental period (the end
of the 0
th
wee#$ with the same feeding rate and feeding time) The re.uired diets were
prepared +iwee#ly and stored in a refrigerator (0 JC$ for daily use)
ASSESSMENT OF 3RO4TH PERFORMANCE.
2ish samples were collected from each treatment and control groups at '
st
and
=
th
days of the experiment, then weighted for determining (7verage +ody weight,
;ody weight gain, 7verage daily gain (7D<$, condition factor (C2$ and specific
growth rate according to R(56e!, (1787) using the following e.uations?
a$Total gain (g " fish$ M -t N -o
+$ 7verage daily gain (g "fish "day$ M -t N -o"n
b c$ Condition factor ( C2$ M

d$ 4pecific growth rate)
4<8M (*in -t N *in -o$ '(( " n
-o? Is the initial fish weight (gm$ at the start of the experiment)
-t? Is the final fish weight (gm$ at the end of the experiment)
n? Is the duration period of the experiment in day)
*in? Is the natural logarithm)
DETERMINATION OF NON SPECIFIC INNATE IMMUNE RESPONSE
7 total num+er of ( +lood samples were collected from the caudal vein of (
fish in each group (! samples from each replicate$ on days '
st
, '0
th
, '
st
and =
th
of
the experimental period according to 8owley, ('11($) &ach sample divided into
haves (one after adding anticoagulant for examination of total leu#ocyte count (T*C$
!
-eight (g$ x '((
*ength (cm$ 9
N&GI&N :) 5) 7;D&*:H7*& et al.
according to 4chaperclaus, ('11$, differential leu#ocyte count (D*C$ according to
4tos#opf, ('11F$ using <iemsa stain) 9hagocytosis assay (phagocytic activity and
phagocytic index$ was determined according to :awahara et al. ('11'$ using
-an(i(a albicans culture) -hile the other half allowed to clot at 0 JC in a refrigerator
for 0hrs then centrifuged at '!(( xg for '(minits) 2ollowing centrifugation, the serum
was collected and fro,en at - =( JC until used for *yso,yme (lysoplate$ assay
according to &llis, ('11($ using !( Og"ml Micrococcus lyso(ictecus an( 4D4-
polyacrylamide serum protein electrophoresis according to *aemmli, ('1B($)
IMMUNOCOMPETANCE TEST (DISEASE RESISTANCE)
Two wee#s after last administration of the medicated diets, ( fish from each of
the tested groups (! fish"replicate$ were inEected I"9 with ()' ml"0 hrs +roth culture
of A. hy(rophila strain containing F I '(
B
via+le cells"ml according to (Pa#i , '11'$,
Clinical signs, moralities were recorded daily for B days) The relative level of
protection (8*9$ among the challenged fish was determined according to
8uangroupan et al. ('1=A$)
STATISTICAL ANALYSIS
Data were statistically analy,ed using one- way or two- way analysis of
variance (7N3G7$ (SAS, '11A$) Duncan multiple range test was used to test the
significance among the means (4nedecor and Cochrang, '1=1$. Differences were
considered significant when 2 Q()(!)
RESULTS
ASSESSMENT OF 3RO4TH PERFORMANCE
<rowth performance (;ody weight again, 7D<, 4<8, C2$ of +. niloticus fed on
Immunoton showed a significant increase (at p Q ()(!$ compared to levamisole or
control groups +y the end of the experimental period (=days$ (ta+le '$)
Ta+le ') <rowth performance +y the end of experiment of +. niloticus fed on different
immunostimulants for wee#s
5eans in a column with different superscripts different significantly at 9Q()(!)
P*!*/e,e!# C',!0 Le.*/(#0e I//+','
B

"
y

w
e
(
)
-
,

(
)
/
:
;
(
#
-
)
Initial weight =)(= H ()'F B)10 H ()A =)F(H()B
2inal weight =)! H ')10
<
=)A0 H )(=
<
F)FAH')'A
*
-eight gain ')F( H ()'
5
)0 H()00
*<
F)(AH()!A
*
AD3
(/) : ;(#- : "*y)
()(0B H()('
5
()(=AH()(
*<
()'(1H()(
*
S3R (= : "*y) ()'A H ()(!
<

()FA H ()(1
*
()FB=H()(1
*
CF (=) ()(=1 H ()(F
<
()'F H ()(=
*
()F(AH()(B
*
!F
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32 NI*& TI*79I7 (+,*+-.,+MIS /IL+TI-$S$
ASSESSMENT OF CELLULAR INNATE IMMUNE RESPONSE
TOTAL LEUKOCYTES COUNT (TLC)
There was a significant increase (at p Q ()(!$ in T*C through out the experimental
period reached the highest level at end of 0
th
wee# of experiment in group
supplemented with levamisole followed +y Immunoton supplemented group (fig) '$)
DIFFERENTIAL LEUKOCYTES COUNT (DLC)
*evamisole and Immunoton significantly increased lymphocytes L (at p Q ()(!$
when compared to control that pea#ed after the 0
th
wee#, while induced a non-
significant effect on the other cell types (fig)$)
PHA3OCYTOSIS
There was a non-significant difference in phagocyte activity (9)7$ in all
immunostimulants supplemented groups and control throughout the experimental
period) -hile phagocyte index (9)I$ showed a significant increase during the
experimental period pea#ed after 0
th
wee# for levamisole supplemented group +ut did
not show a significant increase with Immunoton supplemented group or control
(fig)F$)
5
<
*
"
2ig) ') Total leu#ocytes count (cells x'(
F
cell"Rl$ of +. niloticus fed on different
immunostimulants for wee#s)
Columns with the different letter are significantly different at 9Q()(!)
!0
N&GI&N :) 5) 7;D&*:H7*& et al.
*
<<
5
<<
*
5
C',!0 Le.*/(#0e I //+','
2ig) ) Differential leu#ocyte count of +. niloticus fed on different immunostimulants
for wee#s)
Columns with the different letter are significantly different at 9Q()(!)
*
<
5
"
2ig) F) 9hagocytosis of +.niloticus fed on different immunostimulants for wee#s)
!!
P-*)5y,e# *5,(.(,y
P-*)5y,e# ('"e>
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32 NI*& TI*79I7 (+,*+-.,+MIS /IL+TI-$S$
Columns with the different letter are significantly different at 9Q()(!)
ASSESSMENT OF HUMORAL INNATE IMMUNE RESPONSE
LYSOZYME CONCENTRATION
There was a significant increase in *yso,yme concentration started at the end of
F
rd
wee# with levamisole supplemented group while Immunoton supplemented group
showed a non significant difference trough out the experimental period (fig) 0$)
PROTEIN ELECTROPHORESIS
S- <lo+ulin concentration significantly increased at the end of the 0
th
wee# with
levamisole (at p Q ()(!$ in fig)A) -hile Immunoton was non significantly different
(fig)!$) Total al+umin, T and U glo+ulin didnKt show a significant difference either in
each treatment through out the experimental period, or +etween different treatments
including control)
*
< <
5
2ig) 0) *yso,yme concentration (Og"ml$ of +.niloticus fed on different
immunostimulants for wee#s)
Columns with the different letter are significantly different at 9Q()(!)
!A
N&GI&N :) 5) 7;D&*:H7*& et al.
"
C',!0 Le.*/(#0e
I //+','
*
<
5
2ig) !) 9rotein electrophoresis of +. niloticus fed on different immunostimulants for
wee#s)
Columns with the different letter are significantly different at 9Q()(!)
IMMUNOCOMPETANCE TEST (DISEASE RESISTANCE)
5ortality rates have +een decreased with levamisole supplemented to '!L and
high relative protection level (=F)F$ while was 0(L with Immunoton supplemented
group with moderate 89* (!!)A$ compared to 1(L mortalities and no protection in
control group ta+le ($)
Clinical pictures of the experimentally infected fish were nearly similar in all
treatments including control +ut varied in the severity of the developed lesions) They
included poor appetite, loss of e.uili+rium with erratic movement of some fish,
swimming with head down due to a+dominal distension and finally loss of all reflexes
Eust prior to death) Hemorrhage of all fins, caudal peduncle and ventral a+dominal
wall with hemorrhagic and protruded anal opening were seen (fig) A$)
Ta+le ) Disease resistance of +. niloticus fed on different immunostimulants for wee#s
2ish groups No)
of
fish
8oute of
inEection
Type of
Inoculate
Dose
(ml$
Died fish during B days after inEection) 5ortality
(L$
8*9
( ' F 0 ! A B
Control ( I"9 A. hy(rophila ()' C 0 F F F ' 1( (
*evamisole ( I"9 A. hy(rophila ()' C C ' C C C C '! =F)F
Immunoton ( I"9 A. hy(rophila ()' C ' F C C C 0( !!)A
!B
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32 NI*& TI*79I7 (+,*+-.,+MIS /IL+TI-$S$
.
2ig) A) +. niloticus experimentally infected with A. hy(rophila with hemorrhage at all fins
(arrow$ and caudal peduncle (a$ compared to apparently normal healthy fish (+$)
DISCUSSION
During the last decade there was an increasing interest in the modulation of the
non specific immune response of fish to elevate the general defense +arriers and
hence increase resistance against diseases through use of immunostimulants (8aa,
((( and 4ahoo and 5u#herEee, (($)
2rom the presented results, use of levamisole as feed additive at a dose of
(! mg " #g of diet for wee#s has shown to +e a powerful activator of non specific
innate immune response) This increase in total leu#ocyte count, lymphocyte count
and phagocytosis after wee#s from the last administration of levamisole could +e
attri+uted to stimulation of leu#iopoiesis and lymphocyte proliferation +y levamisole
(Courntney and 8o+ertsen, '11!$ although in our study levamisole did not
significantly increased monocytes and neutrophils num+ers +ut increasing their
activity to phagocytose can(i(a albicans) 4imilarly 4iwic#i, ('1=1$ reported an
increase in T*C and lymphocyte count with dietary inta#e of levamisole to common
carp at a dose of ! mg"#g +ody weight for '! days) 2urthermore, increased
phagocytic activity (9)7$ and phagocytic index (9)I$ were prominent after wee#s
from last administration of levamisole in fish #ept at
o
C and lasted for F months)
7lso 5ulero et al., ('11= +$ o+served increased (9)7$ and (9)I$ in gilt head sea +ream
fed on levamisole supplemented diet for '( days especially at low doses ('!, !(
mg"#g diet$ while a dose of !(( mg"#g diet had a suppressive effect)
2rom our presented data, dietary inta#e of levamisole also increased serum
lyso,yme concentrations of +. niloticus) 4imilar results in carp o+tained +y 4iwic#i,
!=
* <
<
*
N&GI&N :) 5) 7;D&*:H7*& et al.
('1=1$ who o+served an increase in serum lyso,yme concentrations in +oth fish
groups #ept at '
o
C and
o
C +ut the highest serum lyso,yme concentrations was
o+served in fish #ept at
o
C) 7nother study in rohu fed on levamisole in a dose of !
mg"#g +ut in ! consecutive occasions for A( days recorded a significant increase in
lyso,yme concentrations (4ahoo and 5u#herEee, (('$) 7s it is clear that lyso,yme is
mainly produced +y activated macrophages (<ordon et al), '1B0$, so the increase in
lyso,yme concentration may +e attri+uted to the increase in the activity phagocytic
cells)
9rotein electrophoresis is availa+le tool in assessing humoral immunity
(#ane#o et al), '11B$) 2rom our result, S-glo+ulin (the immunity related protein
fraction$ was the only to +e increased after levamisole stimulation while total protein
and total al+umin levels remain unchanged, which shows the important role of S-
glo+ulin in immune response)
4upporting this findings, results o+tained +y 4ahoo and 5u#herEee, ((('$ in
which total protein levels remain unchanged irrespective to aflatoxin" levamisole
exposure, although the increased al+umin " glo+ulin ratio noted due to aflatoxin
treatment was restored +y levamisole feeding) 7nother study +y &*-;oushy and &*-
7shram, ((($ in 7frican catfish reported that levamisole found to restore the total
protein level in immunocompromised fish and increasing S-glo+ulin levels in healthy
fish)
It is well #nown that the ideal conditions are always prevailing in commercial
farms that ma#e fish continuously stressed) 6nder these conditions, the re.uirements
for certain nutrients will almost certainly increase due to an increased activity of the
immune system) 2rom the present study the com+ination of vitamins C and & could
cause little enhancement of the cellular innate immune response while had a non
significant effect on humoral one) This may +e attri+uted to the low doses of vit) C
('(') mgV#g diet$ and & ('!(mgV#g diet$ present in the supplemented diet and so it
could not induce maximum immunity +ut induce enhanced immune response through
leu#opoiesis and enhanced lymphocytes proliferation) 4imilar results on rain+ow trout
o+tained +y -ahli et al., ('11B$ who reported that, com+ination of vit)C and &
significantly increase lymphocyte proliferation) In contrast, 5ulero et al), ('11=a$
found that in vitro addition of either vitamin C or & individually had no effect on the
phagocytic activity of gilt head sea +ream leu#ocytes and even com+ination of +oth
vitamins failed to further increase such activity at any of the tested concentrations ('-
!1
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32 NI*& TI*79I7 (+,*+-.,+MIS /IL+TI-$S$
'(( Rg " ml$ vitamin C or (()('- '( Rg " ml$ vitamin & for 0= hrs)
8egarding to resistance to infection, +oth of the tested immunostimulants had
+een found to increase resistance of +. niloticus against A. hy(rophila infection
although it was little with Immunoton) This resistance may +e due to the increased
activity of phagocytic cells with su+se.uent increase in lyso,yme activity and
+actericidal activity of fish phagocytes"macrophages (8o+ertsen et al), '110$) 5any
studies similarly, recorded increased resistance against A.hy(rophila challenge after
stimulation with levamisole (3liver et al., '1=!/ ;a+a et al), '11F/ >eney and
7nderson, '11F and 4ahoo and 5u#herEee, (('$, while the lower resistance in case
of Immunoton could +e attri+uted to the low doses of Git) C ('(') mg V #g diet$ and
vit) & ('!( mg V #g diet$ presents in the supplemented diet and so it could not induce
maximum immunity) 7lthough these doses were a+ove the minimal levels needed for
growth in addition to presence of lactose in the product that could help in growth
promoting activity with maximum feed utili,ation) This explains why it showed the
highest growth stimulator compared to levamisole) 8esults o+tained +y 3rtuno et al),
('111 and ((($ showed that, a moderately high dietary dose of vit) & together with
ade.uate vitamin & " C ratio are needed for maximum growth and enhanced immune
response)
4o in conclusion, dietary inta#e of levamisole to +. niloticus enhances non
specific innate immunity rather than growth performance) -hile addition of a
com+ination of vitamins C and & could significantly enhance growth performance +ut
higher doses are needed for maximum immunity)
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&d), 7cademic 9ress, 4an Diego, *ondon, To#yo and
Toronto)
'F) :awahara, &) T), :) N) 6eda and 4) 5) Nomura) '11') In vitro phagocytic
activity of white-spotted char +lood cells after inEection with Aeromonas
salmonici(a extra cellular products) <yo+yu :en:yu, A? 'F-'0)
'0) *aemmli, 6) :) '1B() Cleavage of structural proteins during the assem+ly of
the head of +acteriophage T0) Nature B? AB(-A=!)
'!) 5ulero, G), 5) 7) &ste+an and >) 5eseguer) '11=a) &ffects of in vitro addition
A'
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32 NI*& TI*79I7 (+,*+-.,+MIS /IL+TI-$S$
of exogenous vitamins C and & on gilthead sea +ream 0Sparus aurata L.1
phagocytes) Geterinary Immunology and Immunopathology AA? '=!-'11)
'A) 5ulero, G), 5) 7) &ste+an, >) 5uno, and >) 5eseguer) '11=+) Dietary inta#e
of levamisole enhances the immune response and disease resistance of the
marine teleost gilthead sea+ream 0Sparus aurata L.1 2ish Y 4hellfish
Immunology =? 01-A)
'B) 3liver, <), T) 9) &velyn, 8) *allier) '1=!) Immunity to Aeromonas Salmonici(a
in coho salmon (+ncorhynchus 4isutch1 induced +y modified 2reundZs complete
adEuvant? Its non-specific nature and the pro+a+le role of macrophages in the
phenomenon) Dev) Comp) Immunol) 1 (F$? 0'1-0F)
'=) 3rtuno, >), 5) 7) &ste+an and >) 5eseguer) '111) &ffect of high dietary
inta#e of vitamin C on non-specific immune response of gilthead sea
+reamSparus aurata L., 2ish and 4hellfish Immunology, 1, 01-00F)
'1) 3rtuno, >), 5) 7) &ste+an and >) 5eseguer) ((() High dietary inta#e of T-
tocopherol acetate enhances the non-specific immune response of gilthead
sea+ream 0Sparus aurata L.1.2ish and 4hellfish Immunology) '(? 1F-F(B)
() 9hillip, H) #), 7) C) 4hoema#er and >) >) &vans) ((() Gaccination? 7 health
management practice for preventing diseases caused +y 4treptococcus in Tilapia
and other cultured fish) In? Tilapia a.uaculture in the '
st
Century) 9roceeding
from the fifth International 4ymposium on Tilapia 7.uaculture, 8io de >aneiro,
;ra,il) 4eptem+er F-B, Gol ($) !!=-!A0)
') 8aa, >) ((() The use of immunestimulants in fish and shellfish feeds) In?
Cru,-4uare,, *)&)/ 8ic.ue-5arie, D)/ Tapia-4ala,ar, 5)/ 3lvera-Novoa, 5)7)W)
and Civera-Cerecedo, 8) (&ds)$) 7vances en Nutrici[n 7cu\c[la G) 5emorias del
G 4imposium International de Nutrici[n 7cu\c[la) '1- Noviem+re, ((()
5erida, Wucatan, 5exico)
) 8ic#er, -) &) '1B1) <rowth rates and models) In? fish 9hysiology (Hoar,
-)4)/ 8ondal, 9)>) and ;rett, >)8) &ds)$ pp) ABB-B0F) New Wor#? 7cademic 9ress)
F) 8o+ertsen, ;), 8) &) &ngstad and >) ;) >orgensen) '110) U) glucan as
immunostimulants in fish) In? 5odulators of 2ish Immune 8esponses) 4tolen,
>)4) and 2letcher, T)C) (&ds)$, Gol) ', 434 9u+lications, 2air Haven, N>, 99)=F-
11)
0) 8owley, 7) 2) '11() Collection, 4eparation and Identification of fish
*eucocytes) In? Techni.ues in 2ish Immunology, >)4) 4tolen, T)C) 2letcher, D)9)
A
N&GI&N :) 5) 7;D&*:H7*& et al.
7nderson, ;)4) 8o+ertson, -);) van 5uiswin#el (eds)$, 434) 9u+lications, 2air
Haven, 647) Chapter '0, pp ''F-''!)
!) 4ahoo, 9) :) and 4) C) 5u#herEee) ((') Dietary inta#e of levamisole
improves non-specific immunity and disease resistance of healthy and aflatoxin
induced immunocompromised roho 0Labeo rohita1. >ournal of 7pplied
7.uaculture, ''(0$, '! N !)
A) 4ahoo, 9) :) and 4) C) 5u#herEee) (() The effect of dietary
immunomodulation upon *(5ar(siella tar(a vaccination in healthy and
immunocompromised Indian maEor carp (Labeo rohita$) 2ish and 4hellfish
Immunology) ' ('$? '-'A)
B) 4a#ai, 5) '111) Current research status of fish Immunostimulants)
7.uaculture 'B? AF-1)
=) 474) '11A) Copyright] '11A 474 institute Inc) Car), NC B!'-=(( 6)4)7)
4oftware release (version$ A)' 474 " 4T7T6 user is guide) A
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ed)
1) 4chaperclaus, -) '11) Hematological and serological techni.ues) In? 2ish
Diseases (4chaperclaus, -)/ :ulow, H) and 4chrec#en+ach, :) eds)$, vol '? B'-
'(B)
F() 4ecom+es, C) >) '110) &nhancement of fish phagocytic activity) 2ish and
4hellfish Immunology) 0? 0'-0FA)
F') 4iwic#i, 7) '1=B) Immunomodulatory activity of levamisole in carp spawners,
-yprinus cariop L. >ournal of 2ish ;iology (4upp) 7$ F'? 0-0A)
F) 4iwic#i, 7) :) '1=1) Immunostimulating influence of levamisole on non-
specific immunity in carp 0-yprinas carpio1. Dev) Comp) Immunol) 'F, =B-1')
FF) 4iwic#i, 7):) and 5) :orwin-:ossa#ows#i) '1==) The influence of levamis-
-ole on the growth of carp 0-yprinus -arpio1 larvae) >ournal of 7pplied
Ichthyology 0? 'B=-'=')
F0) 4iwic#i, 7):) and 5) 4tudnic#a) '110) 4timulation of non specific immunity
after immunosuppression induced +y chemical stress in carp 0-yprinus carpio1.
In? 5uller, 8 and *ioyd, 8 (&ds$, 4u+lethal and chronic effects of pollutants on
freshwater fish) 2ishing news ;oo#s, 3xford, 99) '0=-'!)
F!) 4iwic#i, 7), D) 9) 7nderson and 3) -) Dixon) '11) In vitro effect of
levamisole on the neutrophil activity in rain+ow trout 0+ncorhynchus my4iss1)
7rch) Immunol) Ther) &xp) 0((!-A$) !F-!A)
FA) 4nedecor, <) w) and -) <) Cochran) '1=1) 4tatistical 5ethods, =
th
(&d)$, low
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state 6niv), 9ress 7mes, I3-7, 6)4)7)
FB) 4ordello, *) 5), -) :) 4hafer and D) De 8osa) '11B) Immuno+iology of the
mammary gland) >ournal of dairy science, =(, '=!'-'=A!)
F=) 4tos#opf, 5) :) '11F) 2ish 5edicine) -);) 4aunders, 9hiladelphia,
9ennsylvania)
F1) Gerlhac, G), 7) 3+ach, >) <a+audan, -) 4chuep and 8) Hole) '11=)
Immunomodulation +y dietary vitamin C and glucan in rain+ow trout
0+ncorhynchus my4iss1. 2ish Y 4hellfish Immunology =, 0(1-00)
0() -aag+o, 8), >) <lette), &) 8aa-Nilsen and :) 4andnes) '11) Dietary vitamin
;A and vitamin C) Influence on immune response and disease resistance in
7tlantic salmon 0Salmo Salar1. 7nn N W 7cad 4ci) AA1?FB1-=)
0') -ahli, T), G) Gerlhac), >) <a+audan), -) 4ch^ep and -) 5eier) '11B)
Influence of com+ined vitamins C and & on non-specifics immunity and disease
resistance of rain+ow trout, +ncorhynchus my4iss (-al+aum$) >ournal of 2ish
Diseases '? 'B-'FB)
0) Pa#i, G) H) '11) 4ome studies on motile aeromonas in fresh water fish with
special emphasis to their toxogenic prolife) 5)G)4c) Thesis) 2ac) of Get) 5ed)
7lex) 6niv)
0F) 8uangroupan, *), T) :itao and T) Woshida) '1=A) 9rotective efficacy of
Aeromonas hy(rophila vaccine in Nile tilapia) Get) Immun) and Immunopath)
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