Effect of Dietary Immunostimulants on Some Innate Immune ...
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Aquaculture Science 54(2), 153-162 (2006)
Effect of Dietary Immunostimulants on Some Innate Immune
Responses and Disease Resistance against Edwardsiella tarda
Infection in Japanese Flounder (Paralichthys olivaceus)
Jorge GALINDO-VILLEGAS1'*, Haruhisa FUKADA2, Toshiro MASUMOTO2 and Hidetsuyo HOSOKAWA2
Abstract: Six commercial substances, known for its immunostimulatory effects in several fish
species, were evaluated for their ability to enhance innate immune response and disease resistance
against Edwardsiella tarda in Japanese flounder. Each substance (ƒ¿-tocopherol, astaxanthin,
ƒÀ-glucan , DL-arginine, L-ascorbic acid or peptidoglycan) was mixed with a practical diet and fed
daily for three weeks to duplicate fish groups. Growth, hematology , humoral (lysozyme) and
cellular (chemotaxis, phagocytic and respiratory burst) activities were examined after one , two and
three weeks. Oral administration of these substances did not affect growth or fish health status but
caused enhancement in bacterial lytic and phagocytic responses. After one and two weeks , arginine
and astaxanthin groups, respectively, showed some significant enhanced immune responses .
However, by the end of the third week, only peptidoglycan-treated fish showed significantly
higher level of lysozyme and leucocytes activities. After three weeks of dietary treatment with
immunostimulants, the fish were challenged by intraperitoneal injection with E . tarda, where
peptidoglycan and ƒ¿-tocopherol-treated fish demonstrated significantly increased survival rate.
Peptidoglycan appears to enhance resistance against infection in Japanese flounder with increased
lysozyme and phagocytic activities. Alfa-tocopherol seems to have induced protection by enhancing
some other resistant mechanisms than those measured in this study .
Key words: Paralichthys olivaceus; Edwardsiellosis; Immunostimulant; Innate immune response
In recent years, marine aquaculture has been
expanded, intensified and diversified. However,
on the contrary disease has become a primary
constrain for sustainable aquaculture produc-
tion and product trade.
In economical terms, Japanese flounder
Paralichthys olivaceus, is one of the most impor-
tant marine farmed fish species in Japan. In
culture facilities, edwardsiellosis, caused by
the bacterium Edwardsiella tarda, is one of
the main causes of huge economical losses
for farmers of this species. Traditionally, anti-
microbial drugs have been employed for pro-
phylactic and therapeutic purposes. But, the
growing-promoting levels of antibiotics are no longer expected in Europe and elsewhere.
Therefore, the way to replace them has been investigated. Vaccination may be the most ideal method to control infectious diseases. Nevertheless available vaccines are limited,
there is no effective vaccine against all sero-logical strains of E. tarda until today (Kawai et al. 2004) . Further, E. tarda a member of the Enterobacteriaceae family, is an intra cellular
pathogen. The activation of leucocytes and macrophage in innate immune system must be needed to digest phagocyted E. tarda. Since
the use of the killed vaccine sometimes cause
Received October 24, 2005: Accepted January 25, 2006.1 The United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, Ehime 790-8566, Japan.2 Laboratory of Fish Nutrition, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan.* Corresponding author: Tel & Fax: +81-088-755-3628; E-mail: [email protected]
154 J. Galindo-Villegas, H. Fukada, T Masumoto and H. Hosokawa
the proliferation of phagocyted E. tarda in non-
activated leucocytes and/or macrophage, which
has a possibility to deprave the disease condi-
tion. From this back ground, the effective use
of immunostimulatory substances (immunos-
timulant) should contribute to prevent Japanese
flounder from edwardsiellosis.
Many studies have been looked into the
modulation of immune response in order to pre-
vent from diseases when outbreaks are present
(Sakai 1999; Sealey and Gatlin 2001). Several
substances, chemical agents, bacterial compo-
nents, polysaccharides, animal-derived, plant
extracts, nutritional factors and cytokines, have
been reported to be effective as Immunostimu-
lants in fish (Yano et al. 1989; Anderson 1992;
Raa et al. 1992; J, rgensen et al. 1993; Ainsworth
et al. 1994; Neumann et al. 1995; Roberts et al.
1995; Buentello and Gatlin III 1999; Castro et
al. 1999; Sakai 1999; Clerton et al. 2001; Kono
and Sakai 2001; Halver 2002). However, very
few Immunostimulants have been tested in
Japanese flounder (Ashida et al. 1999; Kono and
Sakai 2001; Yokoyama et al. 2005) . Therefore,
the aim of the present study was to determine
whether the dietary intake of six arbitrarily
selected substances (ƒ¿-tocopherol acetate,
astaxanthin, ƒÀ-glucan, DL-arginine, L-ascorbic
acid and peptidoglycan) was effective for the
innate immune response and disease resistance
against E. tarda in Japanese flounder.
Materials and Methods
Fish
Fingerling Japanese flounder grown bacteria-
free were obtained from a local hatchery in
Iyo City, Ehime Prefecture. Animals were
transported to the facility of Kochi University
in Akaoka town. The fish were maintained on
a commercial diet until desired weight was
gained. Seven duplicate groups of 30 fish, each
weighing about 60 g, were reared in fourteen
800 l tanks. Tanks were randomly distributed
and supplied with running sea water (aver-
age temperature of 21.0•Ž) at a flow rate of
20 l/mm.
ImmunostimulantsThe peptidoglycan, PG-AQUA, derived from
Brevibacterium lactofermentum, was obtained from Ajinomoto (Tokyo, Japan). Beta glucan MacroGard(R), an insoluble yeast glucan from Saccharomyces cerevisiae, was purchased from ZINPRO Animal Nutrition (Tokyo, Japan). Astaxanthin as carophyll pink was obtained from DMS Nutrition (Tokyo, Japan). Alfa tocopherol acetate, L-ascorbic acid and DL-arginine were
purchased from Wako Pure Chemical Industries (Osaka, Japan).
Preparation of diets and feeding
To acclimatize the fish to experimental con-
ditions, all groups were fed to satiation for ten
days with a same basal diet indicated in Table
1. Following acclimation, six experimental diets
based on the common basal were prepared by
mixing practical dry ingredients and immunos-
timulants. The supplemented doses of Immuno-
stimulants were: PG-AQUA, 3.0; MacroGard(R),
3.0; Carophyll Pink, 0.1; L-ascorbic acid, 6.1;
a-tocopherol, 0.6 and DL-arginine, 1.5 g/kg.
Doses were determined from previous experi-
ments carried-out in this laboratory (unpub-
lished data). To dry mixtures, 40% water was
added to allow extrusion with a food grinder.
Subsequently, pellets were cut into suitable size
in regard to fish mouth. Diets were prepared
weekly and stored frozen in vinyl bags at -20•Ž
until use. Fish were fed for three weeks with its
Table 1. Composition of the basal diet.
1Supplied (g/kg diet): thiamine hydrochloride (0.022), riboflavin
(0.022), pyridoxine hydrochloride (0.023), nicotinic acid (0.096),
phantothenic acid (0.072), myo-inositol (0.6), biotin (0.0014),
folic acid (0.024), choline chloride (3.0), cyanocobalamine
(0.0004), ascorbic acid (0.112) , palmitate (0.011) and tocopherol
acetate (0.119).2Supplied (g/kg diet): KH
2PO4 (4.12), Ca(H2PO4)2• H2O (6.18),
Ca-lactate (2.82), Fe-fumaric acid (1.6), ZnSO4•E7H2O (0.1765),
MnSO4•E4H2O (0.081), CuSO4.5H2O (0.0155), CoC12•E6H2O
(0.0005) and KIO3 (0.0015).
155Immunostimulants against E, tarda in Japanese Flounder
respective diet twice a day at a total rate of 2.5%
biomass/day. Each group of fish was weighed
weekly, and amount of fed diet adjusted accord-
ingly.
Sample collection
After one, two and three weeks of feeding,
five fish from each of the replicate tanks were
randomly collected and killed immediately with
a lethal dose of MS-222 (ethyl 3-aminobenzoate
methanesulfonate salt, Sigma, USA). Samples
were then collected for hematological and
innate immune activities determination. Blood
was drawn from the caudal vein of each fish
using non-heparinized syringes, transferred to
microcentrifuge tubes and allowed to clot for
8 h. After centrifugation, supernatants were col-
lected and stored at - 80•Ž for later analysis.
Hematological parameters of individual fish
were immediately determined from aliquots
of fresh blood kept in heparinized venoject II
(Terumo, Japan). Analyses were carried out
using an automated blood analyzer Celltac
MEK 5105 (Nihon Koden, Japan) calibrated
with Japanese flounder mean blood values.
After bleeding, the head kidney of each fish was
aseptically removed and immersed in tubes con-
taining 5 ml of cold RPMI-1640 medium (Sigma,
USA) supplemented with 10.3 ml/l penicillin-
streptomycin-L-glutamine (Sigma, USA), 0.1%
heparin (Sigma, USA) and 2% fetal bovine
serum (ICN, USA). Cell suspensions were
obtained by forcing tissue fragments through a
100ƒÊm nylon cell strainer (BD Falcon, USA).
Resulting suspensions were centrifuged on 51%
discontinuous Percoll (Sigma, USA) density
gradients. Leucocytes were collected from a
leucocyte-rich layer and washed in Hanks' bal-
anced salts solution (Sigma, USA). The cells
were re-suspended, counted and adjusted in
the supplemented medium to 1•~106 cells/ml.
Leucocyte viability was 98% or more as deter-
mined by the trypan blue exclusion test.
Immunological analyses
The chemotaxis activity of head-kidney leu-
cocytes was examined using blind well cham-
bers (Nucleopore Co., USA) as described by
Ninomiya et al. (1995). Two hundred micro-
liters of Zymosan-activated normal Japanese
flounder serum was placed in the lower well
and used as the chemotactic agent. Dried
Zymosan powder was activated following the
method of Scott and Klesius (1981). Nucleopore
polycarbonate membrane filters (5ƒÊm pore
size) were placed onto the wells and fixed
with screwed upper chambers, where 200ƒÊl
of the head kidney cell suspension was added.
Chambers were incubated at 25•Ž for 3 h.
Thereafter, filters were washed, removed from
chambers and stained with Giemsa's solu-
tion (Merck, Germany). Migrated leucocytes
were counted in 30 random optical fields at
100 X magnification with a microscope BX-40
(Olympus, Japan).
Phagocytic activity of head-kidney leuco-
cytes was determined in 24 multi-well plates
(Sumilon, Japan) as described by Blazer and
Wolke (1984). One hundred microliters of head-
kidney leucocyte suspensions adjusted to 1•~
106 cells/ml were delivered on a 13 mm pore
size Thermanox plastic cover slip (Nunc, USA)
in each well. Leucocytes were allowed to adhere
to the plastic slip for 2 h at 25•Ž, and then non-
adherent cells were removed from the slips
by washing with medium. Immediately, five
hundred microliters of supplemented medium
including 1•~108 fluorescent yellow-green
Latex beads (2ƒÊm diameter) (Sigma, Japan),
opsonized by incubation for 1 h at 25•Ž with
homologous fish serum, were delivered in each
well. Plates were incubated at 25•Ž for 1 h to
allow phagocytosis. After washing, the cover
slips were fixed in methanol and stained with
Diff Quik differential staining solution (Sysmex,
Japan). From each stained sample, 200 cells
were examined under microscope. Phagocytic
ability was defined as the percentage of cells
with one or more ingested Latex beads within
the total cell population, and the phagocytic
capacity as the average number of ingested
Latex per phagocyte.
Respiratory burst activity of head-kidney
leucocytes was quantified by the reduction of
nitro blue tetrazolium as a measure of super-
oxide anion release following the technique
156 J. Galindo-Villegas, H. Fukada, T Masumoto and H. Hosokawa
described by Chung and Secombes (1988).
One hundred microliters of 1•~107 cells/ml in
supplemented medium were plated in a 96 well
microtiter plate (Nunc, USA). The cells were
incubated for 2h at 25•Ž and washed twice,
and a one hundred microliters of RPMI-1640
containing 1 mg/ml NBT and 1ƒÊg/ml phor-
bol 12-myristate 13-acetate (Sigma, USA) was
dispensed in each well containing leucocytes
monolayer. After lh incubation, reaction was
stopped and fixed with methanol. Resulting
formazan was dissolved with KOH and DMSO.
Immediately, absorbance was measured at
630 nm by a micro plate reader Model 550 (Bio-
Rad, Japan).
Serum lysozyme activity was assayed by
the turbidimetric method based on the lysis of
Micrococcus lysodeikticus (Sigma, USA) origi-
nally described by Ellis (1990) and modified by
Villamil et al. (2003). Fifty microliters of undi-
luted serum were plated in triplicate into each
well of a 96-well microtiter plate, and 150ƒÊl of
M. lysodeikticus suspension in 0.1 M phosphate
buffer (pH 6.2) was added to each well. After
rapid mixing at 37•Ž, the change in turbidity
was measured every 30s for 5 min at 450 nm
using the micro plate reader. A lysozyme unit
was defined as the activity of enzyme produc-
ing a decrease in absorbance of 0.001 min -1.
Lysozyme units present in sera were obtained
from a standard curve made with hen egg white
lysozyme (Sigma, USA).
Experimental infection
At the end of the feeding trial, challenge was
performed by intraperitoneal injection with
0.1 ml of 2.4•~105 CFU/ml E. tardy suspen-
sion in physiological saline. E. tardy serological
strain was EF-1. Bacterium was kindly sup-
plied by Mr. Tsuyoshi Yamamoto from the
Fish Disease Department, Kochi University.
Duplicate groups of 10 fish in each 200 l tanks
were supplied with running sea water (aver-
age temperature of 22.5•Ž) at a flow rate of
approximately 5 l/min. Dead fish were removed
twice a day from the tanks and the number was
recorded for 15 days. Kidney samples from the
dead fish were examined by the API 20E iden-
tification kit (Biomerieux, France) to verify the
cause of death.
Statistical methods
To compare differences in immunological responses between groups fed immunostimu-lants and control groups, the data of immune
parameters were subjected to Student's t-test (JMP 5.0.1, SAS Institute Inc.). Mean survival rates, by challenge test were subjected to chi
square test. Significance level was determined at P<0.05.
Results
Growth and blood characteristics Along the feeding trial, all groups showed similar growth rates, and no abnormal symp-tom was observed. Furthermore, any marked differences in red blood cells, hematocrit and hemoglobin contents were not detected among different groups (data not presented).
Serum lysozyme activityThe serum lysozyme activity of peptidoglycan-
a-tocopherol-and DL-arginine-treated fish was significantly higher (P<0.05) than that of the control fish after one week (Fig. 1). After two weeks, peptidoglycan-and DL-arginine-treated fish still showed significantly higher lysozyme activity. At the end of the trial, after three weeks, only the peptidoglycan-treated fish showed significantly higher activity. For the astaxanthin and ascorbic acid groups, a surpris-ing decreased activity in this parameter was recorded along the feeding period.
Phagocytic activity of head kidney leucocytes
Supplementation with peptidoglycan, ƒÀ-
glucan or DL-arginine, significantly (P<0.05)
enhanced the phagocytic ability of head kidney
leucocytes after one week of administration. After
two weeks, significant higher phagocytic ability
was detected in the astaxanthin-treated group.
At the end of the trial, increased activity were
observed in most groups, however statistical sig-
nificance was recorded in the peptidoglycan-and
astaxanthin-treated groups (Fig. 2A).
157Immunostimulants against E, tarda in Japanese Flounder
Phagocytic capacity showed little change
along the feeding trial. Significantly higher
values were observed only after one week in
the peptidoglycan and ,ƒÀ-glucan-treated groups
and, after two weeks, in the astaxanthin-treated
group (Fig. 2B).
Chemotaxis activity of head kidney leucocytes
Leucocytes migration, expressed as chemo-
taxis, was significantly (P<0.05) enhanced
throughout the trial by the peptidoglycan-treated
group. In addition, significant enhancements
(P<0.05) were detected in the ƒÀ-glucan-and
astaxanthin-treated groups after two weeks, and
after three weeks, in the astaxanthin-treated
group, (Fig. 3A).
Respiratory burst activity of head kidney leuco-
cytes
The peptidoglycan-and DL-arginine-treated
groups showed significantly higher (P<0.05)
respiratory burst activity than the control group
after one week. The activity increased accord-
ing to time in most groups, and then after
three weeks, significantly higher activity was
observed in all the dietary groups (Fig. 3B).
Survival after challenge
The cumulative survival rate of the experi-
mental fish following E. tarda intraperitoneal
challenge is shown in Fig. 4. Peptidoglycan-and
ƒ¿-tocopherol-treated fish groups showed sig-
nificantly (P<0.05) higher survival rate after
Fig. 1. Lysozyme activity of Japanese flounder serum.
Data represent the mean •} S.D, of two replicas. Statistical
significant differences (P<0.05) from control group are
indicated by asterisks over the bar graphs.
Fig. 2. Phagocytic ability (a) and phagocytic capacity (b)
of Japanese flounder head kidney leukocytes. Data repre-
sent the mean •} S.D. of two replicas. Statistical significant
differences (P<0.05) from control group are indicated by
asterisks over the bar graphs.
Fig. 3. Chemotaxis (a) and respiratory burst as NBT (b)
of Japanese flounder head kidney leukocytes. Data repre-
sent the mean •} S.D. of two replicas. Statistical significant
differences (P<0.05) from control group are indicated by
asterisks over the bar graphs.
158 J. Galindo-Villegas, H. Fukada, T Masumoto and H. Hosokawa
Fig. 4. Effect of immunostimulants administration on
survival of Japanese flounder juveniles experimentally
infected with 0.1 ml/fish of 2.4•~105 E. tardy CFU/ml on
day 22 after first feeding. Statistical significant differences
(P<0.05) between supplemented treatments and control
group are indicated by asterisks.
15 days. Fish in all groups started to die from the third day following injection and most of them kept dying until the end of the observa-tion period, nevertheless, for the peptidoglycan
group, mortality stopped on day nine after injec-tion.
Discussion
Vaccination may be the ideal method to con-trol infectious diseases. However the use of killed vaccine sometimes occur the deprivation of the edwardsiellosis, since the phagocyted E. tarda proliferates in non-activated leucocytes and/or macrophage. The fully activation of leu-cocytes and macrophage is needed to prevent
Japanese flounder from the edwardsiellosis. Thus, the use of immunostimulant should be an effective method for increasing disease resis-tance in Japanese flounder.
In this experiment no significant growth variations were observed in any group com-
pared with control. However, this lack of enhanced growth promotion activity has also been observed after dietary immunostimulant trials with a wide range of similar substances in different species (Hardie et al. 1990; Maeland et al. 1999; Cook et al. 2002; Amar et al. 2004). Hematological parameters analyzed were not affected by the dietary administration of all
tested substances during the three weeks.
This suggests that supplementation of the sub-
stances in Japanese flounder diet for a short
period should not have any abnormal physiolog-
ical effect.
There are experimental evidences, report-
ing that several substances supplemented as
immunostimulants in fish diet can be the means
of stimulating innate immune system, and such
treatment may lead to increase disease resis-
tance against bacterial infections (Ortuno et al.
2000; Cook et al. 2001; Gannam and Schrock
2001; Sealey and Gatlin III 2001; Bagni et al.
2005). Until now, however for Japanese floun-
der, little is known about the effect of immuno-
stimulants. The studies on the effect of the oral
administration of polysaccharides in combina-
tion with formalin-killed bacteria and the analy-
sis of the expressed genes after peptidoglycan
injection have been reported (Ashida et al. 1999;
Kono and Sakai 2001). However, more informa-
tion is required to determine the potential effect
of substances from different sources by dietary
administration on the innate immune response
and disease resistance against E. tarda in
Japanese flounder. In the present study, six sub-
stances (the fat-soluble vitamins ƒ¿-tocopherol
acetate and astaxanthin; the water-soluble
L-ascorbic acid; the bacterial-derived polysac-
charides ƒÀ-glucan and peptidoglycan; and the
amino acid DL-arginine) previously known as
immunostimulants in several fish species were
examined by dietary administration.
Present study revealed that dietary supple-
mentation of tested substances induced some
enhancement in immune parameters during the
three weeks feeding period. However, increased
disease resistance was shown only in the pepti-
doglycan-and ƒ¿-tocopherol-treated groups by
challenge test.
The use of peptidoglycan or ƒ¿-tocopherol
acetate as disease resistance enhancers in
different fish species has shown a variety of
results. After the short term administration of
peptidoglycan, increased resistance against
Vibrio anguillarum and Lactococcus garvieae
in rainbow trout Onchorhynchus mykiss or yel-
lowtail Seriola quinqueradiata, respectively,
159Immunostimulants against E. tarda in Japanese Flounder
were observed (Matsuo and Miyazono 1993;
Itami et al. 1996). For the administration of
ƒ¿-tocopherol, only few challenge tests have
been carried out, where increased resistance
to bacterial jaundice has been reported for yel-
lowtail (Ito et al. 1999) . The in vitro addition
of ƒ¿-tocopherol resulted in ultraestructural
changes and increased migration and phagocy-
tosis of gilthead seabream leucocytes (Mulero
et al. 1998). However, in the present study, the
administration of ƒ¿-tocopherol did not induce
consistent enhancement of immune responses.
And obviously high immune parameter was not
detected at the time of challenge test (three
weeks). This suggests that ƒ¿-tocopherol may
increase disease resistance by boosting some
other immune responses more likely relating
with acquired immunity through lymphocytes.
Such idea is based on the knowledge that
ƒ¿-tocopherol is absorbed from the gut and
being taken into systemic circulation via lym-
phatic system in mammals (Gallo-Torres 1980).
In mice, it is reported that ƒ¿ -tocopherol stimu-
lates helper T-cell activity (Tanaka et al. 1979).
And for fish, it was suggested that the enhance-
ment of immune response is due to co-operative
effect between T and B cells (Blazer and Wolke
1984; Anderson 1996). The direct extrapolation
between different fish species is not possible.
However, the administration of vitamin E did
not increase resistance against A. salmonicida
(Lall 1988). This may indicate that vitamine E
(ƒ¿-tocopherol) behaves as a nutritional factor
which has an effect to disease resistance.
Lysis capacity of serum lysozyme was signifi-
cantly enhanced after one week of treating fish
with peptidoglycan, ƒ¿-tocopherol or arginine
when compared with control group. Afterwards,
only the peptidoglycan-treated fish kept an
enhanced response until the end of the trial. In
addition, ascorbic acid-or astaxanthin-treated
fish presented remarkably low activities along
the three weeks treatment period. Suppressed
activities of ascorbic acid-or astaxanthin-
treated fish suggest a negative dose interac-
tion between the water soluble vitamins and
the lysozyme production mechanisms. On the
other hand, increased lysozyme activity from
the peptidoglycan-treated fish may be the direct
result of macrophage activation (Mulero and
Meseguer 1988).
Leucocytes phagocytic ability and migra-
tion were significantly higher in fish fed with
peptidoglycan and astaxanthin along the feed-
ing trial. This suggests that peptidoglycan and
astaxanthin can trigger an enhanced response
between the system of signaling process and
engulfment target. In addition, after three
weeks the respiratory burst through activated
leukocytes was significantly enhanced in these
fish groups, suggesting that the effect on the
membrane of leukocyte triggers the production
of bacterial killing oxygen free radicals or reac-
tive oxygen specie. Previous report showed the
significantly increased activity of super oxide
anion in kidney cells after the intraperitoneal
injection of peptidoglycan in Japanese flounder
(Kono and Sakai 2001).
The yeast cell wall component ,ƒÀ-glucan has
been recognized as a potent enhancer of innate
immunity in several fish species (Jorgensen
et al. 1993; Robertsen et al. 1994; Castro et al.
1999; Bagni et al. 2005). In this study, some
significant enhancements in phagocytosis, che-
motaxis and respiratory burst were recorded.
However, ƒÀ-glucan-treated fish did not show
high survival rate in challenge test. The lack of
ƒÀ-glucan's ability to increase disease resistance
suggests that yeast polysaccharide can activate
the innate immune system only partially in this
species. Different doses, source of ƒÀ-glucan or
the administration period should be tested to
improve effect.
Amino acid arginine, previously reported
positive influence on the health of mice and
fish (Buentello and Gatlin III 1999; Alam et al.
2002; Moreira et al. 2004) was also observed in
the present experiment. However, by the end
of third week's trial, only respiratory burst was
enhanced. This should be the result of arginine
involvement in the synthesis of nitric oxide
which is inducible in fish (Gatlin III 2002).
Nitric oxide was not measured in this study.
However, the effect of nitric oxide seems to
be innocuous against infection with E. tarda
because disease resistance was not observed.
160 J. Galindo-Villegas, H. Fukada, T. Masumoto and H. Hosokawa
Generally it is possible to observe increase in the values of many immunological parameters in the early time of administration with immuno-stimulants and decrease after long term admin-istration. Such kind of fast enhancement and later drooping down was observed also in other fish species (Cook et al. 2003).
In conclusion, only administration with pep-tidoglycan induced resistance against E. tarda infection with considerably induced immuno-logical parameters. So far, for Japanese floun-der peptidoglycan seems to be a prospect to increase disease resistance against E. tarda. More research should be carried-out to deter-mine optimal dose, timing and suitable adminis-tration therapy.
Acknowledgments
This study was supported in part by grant-
in-aid from the Ministry of Education, Culture,
Sports, Science and Technology, Japan;
and, The National Council of Science and
Technology, (CONACYT), Mexico. We thank
Dr. Daud Kassam of Kochi University for his
suggestions and critical review of the manu-
script.
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ヒ ラ メParalichthys olivaceusの 自然 免 疫 応 答 とEdwardsiella tarda感 染
に対 す る抗 病 性 に お け る免 疫 賦 活 物 質 の 効 果
Jorge GALINDO-VILLEGAS・ 深 田 陽 久 ・益 本 俊 郎 ・細 川 秀 毅
これまで数魚種 で免疫賦活効果が認 め られている6つ の物質の ヒラメにお ける自然免疫応答 と
Edwardsiella tardaに 対する抗病性向上能を評価 した。実験魚 には,そ れぞれの物質(α-ト コフェロー
ル,ア スタキサ ンチ ン,β-グ ルカン,DL-ア ルギニ ン,L-ア スコル ビン酸,ペ プチ ドグリカン)を 混
合 した試験飼料 を3週 間給与 した。成長,血 液性状,液 性免疫活性(リ ゾチーム活性)お よび細胞性
免疫活性(走 化性,貧 食能,活 性酸素産生能)を 実験開始時から1,2,お よび3週 間後に測定 した。
上記の6物 質の経口投与はいずれ も成長 と健康度には影響を及ぼさず,溶 菌能(リ ゾチーム活性)と
貧食能を亢進 した。第1週 と第2週 では,ア ルギニ ンお よびアス タキサ ンチ ン給与群はい くつかの免
疫指標で有意な応答を示 した。ペプチ ドグリカン給与群のみで全ての免疫反応が3週 間後 まで に亢進
されていた。E.taydaの 腹腔内投与においてペ プチ ドグリカンとα-トコフェロール給与群は高い生存
率 を示 した。以上のことか ら3週 間のペプチ ドグリカ ン給与 は炎症性の免疫応答 を活性化 させエ ドワ
ジェラ症 に対す るヒラメの抵抗力 を向上 させ ることが明らか となった。 また,α-ト コフェロールは
本研究で測定 した指標 とは異 なる免疫機構 によって防御能 を亢進させ ていると考え られた。