Effect of Dietary Immunostimulants on Some Innate Immune

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


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.


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。

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