Sakai (1999)

.
Aquaculture 172 1999 6392

Current research status of fish immunostimulants
Masahiro Sakai
)
Faculty of Agriculture, Miyazaki Uniersity, Miyazaki 889-21, Japan
Accepted 30 October 1998
Abstract
Immunostimulants are valuable for the control of fish diseases and may be useful in fish
culture. The immunostimulatory effects of glucan, chitin, lactoferrin and levamisole for fish and
shrimp have been reported. Nutritional factors such as Vitamins B and C, growth hormone and
prolactin have also been reported to be immunostimulators. These immunostimulants mainly
facilitate the function of phagocytic cells and increase their bactericidal activities. Several
immunostimulants also stimulate the natural killer cells, complement, lysozyme and antibody
responses of fish. The activation of these immunological functions is associated with increased
protection against infectious disease. Resistance to bacterial pathogens such as Vibrio anguil-
larum, V. salmonicida, Aeromonas salmonicida, Yersinia rukeri and Streptococcus spp. and to
parasitic infections such as white spot disease can be increased by administration of immunostimu-
lants, but not to intracellular pathogens such as Renibacterium salmoninarum and Pasteurella
piscicida. The most effective method of administration of immunostimulants to fish is by
injection. Oral and immersion methods have also been reported, but the efficacy of these methods
decreases with long-term administration. Overdoses of several immunostimulants induce immuno-
suppression in fish. The side effects of immunostimulants have not been well-studied. Growth-
promoting activity has been noted in fish or shrimp treated with glucan or lactoferrin. Immunos-
timulants can overcome immune suppression by sex hormones. Thus, the influence of immunos-
timulants in mature fish should be studied. In conclusion, immunostimulants can reduce the losses
caused by disease in aquaculture; however, they may not be effective against all diseases. For the
effective use of immunostimulants, the timing, dosages, method of administration and the
physiological condition of fish need to be taken into consideration. q1999 Elsevier Science B.V.
All rights reserved.
Keywords: Immunostimulant; Non-specific immunity; Macrophage; Fish; Aquaculture
)
Fax: q81-985-58-2884
0044-8486r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
.
PII: S0044- 8486 98 00436- 0
( )
M. Sakai rAquaculture 172 1999 6392 64
1. Introduction
Fish culture is an important industry. Various kinds of marine and freshwater fish
have been cultured and the worldwide production of cultured fish increases every year.
Fishes are usually cultured in enclosed spaces such as ponds or net cages and efforts
have been made to increase productivity per unit space. Overcrowding tends to
adversely affect the health of cultured fish. These conditions tend to produce poor
physiological environment for fish and increase susceptibility to infections.
Various chemotherapeutics have been used to treat bacterial infections in cultured
fish for about the last 20 years. However, the incidence of drug-resistant bacteria has
.
become a major problem in fish culture Aoki, 1992 . Vaccination is a useful prophy-
laxis for infectious diseases of fish and is already commercially available for bacterial
infections such as vibriosis, redmouth disease and furunculosis and for viral infection
such as IPN. Vaccination may be the most effective method of controlling fish disease.
Furthermore, the development of vaccines against intracellular pathogens such as
Renibacterium salmoninarum has not so far been successful. Therefore, the immediate
control of all fish diseases using only vaccines is impossible.
Immunostimulants increase resistance to infectious disease, not by enhancing specific
immune responses, but by enhancing non-specific defense mechanisms. Therefore, there
is no memory component and the response is likely to be of short duration. Use of these
immunostimulants is an effective means of increasing the immunocompetency and
disease resistance of fish. Research into fish immunostimulants is developing and many
agents are currently in use in the aquaculture industry.
Use of immunostimulants, in addition to chemotheraputic agents and vaccines, has
been widely accepted by fish farmers. Many questions about the efficacy of immunos-
timulants from users still remain for example, whether immunostimulants can protect
.
against all infectious diseases . In this review, the current status of immunostimulant
research will be discussed.
2. Fish defense system enhancement by immunostimulants
.
The fish defense system Iwama and Nakanishi, 1996 is basically similar to that
described in mammals. For cellular defense systems, teleosts have phagocytic cells
.
similar to macrophages, neutrophils, and natural killer NK cells, as well as T and B
lymphocytes. Teleosts also have various humoral defense components such as comple-
.
ment classical and alternative pathways , lysozyme, natural hemolysin, transferrin and
C-reactive protein. The existence of cytokines interferon, interleukin 2, macrophage

. .
activating factors has also been reported Secombes et al., 1996 .
Fishes treated with immunostimulants usually show enhanced phagocytic cell activi-
w .
ties glucan Yano et al., 1989; Chen and Ainsworth, 1992; Jrgensen et al., 1993a ,
. .
lactoferrin Sakai et al., 1993 , levamisole Kajita et al., 1990 , FK-565 Kitao and
. . .x
Yoshida, 1986 , chitin Sakai et al., 1992 , EF203 Yoshida et al., 1993 . The activities
of phagocytic cells can be detected by phagocytosis, killing and chemotaxis. Enhance-
ment of pathogen killing is most important in the macrophages of fish treated with
( )
immunostimulants. Killing mechanisms of macrophages can be broadly categorized as
oxygen-dependent or oxygen-independent. Oxygen-dependent killing mechanisms as
.
mediated by reactive oxygen species ROS can be detected by the chemiluminescence
. .
Kajita et al., 1990 and the NBT test Sakai et al., 1995e . Recently, the importance of
.
reactive nitrogen species RNS in macrophage killing, especially in the killing of
.
intracellular pathogens, has been indicated Nathan and Hibbs, 1991 . However, there is
no report of the activation of RNS in the macrophages of fish treated with immunostim-
ulants.
Lymphocytes are also activated by immunostimulants. Immunostimulants enhance
the mitogen activities induced by concanavalin A or lipopolysaccharides and produce
.
macrophage activating factors Hardie et al., 1991; Siwicki et al., 1996 . Complement
.
activity can also be activated by several immunostimulants. Li and Lovell 1985 and
.
Hardie et al. 1991 reported that fish given the large amounts of vitamin C increased
levels of complement activity. Atlantic salmon injected with yeast glucan also showed
.
increased complement activities Engstad et al., 1992 . Lysozyme activity is also
influenced by the administration of immunostimulants Engstad et al., 1992; Jrgensen

. . .
et al., 1993a . NK cells can be activated by growth hormone GH Kajita et al., 1992b
.
and levamisole Kajita et al., 1990 .
Antibody production can also be enhanced by many immunostimulants. Channel
catfish injected with yeast glucan responded to subsequent Edwardsiella ictaluri immu-
nization with higher serum antibody titers relative to controls Chen and Ainsworth,
. .
1992 . Aakre et al. 1994 reported that Atlantic salmon injected with a mixture of
Aeromonas salmonicida bacterin and yeast glucan showed enhanced antibody responses.
The enhancement of antibody responses to A. salmonicida bacterin was also reported
.
with administration of vitamin C Thompson et al., 1993 and FK-565 Kitao et al.,
.
1987 .
3. Immunostimulants studied in fish and shellfish
Table 1 lists those immunostimulants reported to be effective in fish and shellfish. In
this review, immunostimulants include chemical agents, bacterial components, poly-
saccharides, animal or plant extracts, nutritional factors and cytokines.
3.1. Synthetic chemicals
3.1.1. Leamisole
Levamisole is an anthelminthic used for the treatment of nematode infections in man
and animals. Incidental observations suggested a state of enhanced resistance to various
kinds of infection upon treatment. Levamisole in mammals enhances the metabolic and
phagocytic activities of neutrophils, and increases the number of phagocytes and
.
leucocytes and the level of lysozyme in serum Symoens and Rosenthall, 1977 . Animal
.
studies also suggested a stimulatory effect on tumor immunity Fidler and Spitler, 1975 .
Coho salmon, Oncorhynchus kisutch, injected with levamisole mixed with modified
.
Freunds complete adjuvant MFCA showed increased resistance to A. salmonicida
( )
Table 1
Immunostimulants used in fish and shrimp
Synthetic chemicals
Levamisole
FK-565
.
MDP Muramyl dipeptide
Biological substances
.
1 Bacterial derivatives
b-glucan
.
Peptidoglucan Breibacterium lactofermentum; Vibrio sp.
FCA
EF203
.
LPS lipopolysaccharide
Clostridium butyricum cells
Achromobacter stenohalis cells
.
Vibrio anguillarum cells Vibrio vaccine
.
2 Polysaccharides
Chitin
Chitosan
Lentinan
Schizophyllan
Oligosaccharide
.
3 Animal and plant extracts
.
Ete Tunicate
.
Hde Abalone
Firefly squid
.
Quillaja saponin soap tree
.
Glycyrrhizin licorice
.
4 Nutritional factors
Vitamin C
Vitamin E
.
5 Hormones, cytokines and others
Lactoferrin
Interferon
Growth hormone
Prolactin
. .
Olivier et al., 1985 and Siwicki 1987, 1989 reported that carp injected with
levamisole showed enhanced phagocytic activity and myeloperoxidase activity in neu-
.
trophils, increased leucocyte numbers and serum lysozyme levels. Kajita et al. 1990
reported that rainbow trout injected with levamisole showed increased protection against
Vibrio anguillarum, caused by the enhancement of non-specific immune responses such
as phagocytic activity, chemiluminescance responses of leucocytes and NK cell activi-
ties. Concentration of complement also increased in levamisole-injected rainbow trout.
.
Levamisole can stimulate immune responses in vitro. Siwicki et al. 1990 reported
that in vitro immunization of spleen cells cultured with levamisole enhanced phagocytic
activity, the NBT reaction and increased the number of plaque forming cells antibody-
.
producing cells against Yersinia ruckeri O-antigen. The immunostimulatory effects of
( )
levamisole by oral and immersion administration have also been reported. Siwicki
.
1989 reported that oral administration of levamisole increased the number of leuco-
cytes, lysozyme activities in serum and the stimulated NBT reduction and phagocytic
.
index of phagocytic cells. Baba et al. 1993 reported that carp immersed in a levamisole
.
bath 10 mgrml, 24 h showed enhanced resistance against Aeromonas hydrophila, and
its phagocytes increased chemotactic ability, phagocytic activity and chemilumines-
.
cence. Jeney and Anderson 1993a also showed that bathing rainbow trout for 30 min in
.
levamisole solution 5 mgrml before a 2 min bath in A. salmonicida O-antigen
bacterin elevated both the non-specific defense mechanisms phagocytic activity and
. .
index and specific antibody titers . Although the immunostimulating efficacy of
levamisole has been reported in fish, the optimum doses should be considered. Kajita et
. .
al. 1990 indicated that rainbow trout injected with high doses 5 mgrkg of levamisole
did not show stimulated chemiluminescence responses of head kidney leucocytes,
.
compared with fish injected with the optimum dose 0.10.5 mgrkg . The same effect
.
was also observed in vitro. Siwicki et al. 1990 demonstrated that in vitro immunization
with Y. ruckeri O-antigen cultured with 50 mgrml of levamisole did not stimulate the
antibody forming cells in the spleen of rainbow trout, and the antibody production was
completely suppressed by the high doses of levamisole. The immunostimulating effects
of levamisole have been clearly shown in fish, and this agent can be administered by
various methods. However, the safety of levamisole still remains to be determined,
especially when administered high doses.
3.1.2. FK-565
. . .
FK-565 heptanoyl-y-D-glutamyl- L -meso-diaminopimelyl- D -alanine is a peptide
.
related to lactoyl tetrapeptide FK-156 isolated from cultures of Streptomyces oli-
aceogriseus and has been shown to be active against microbial infection in mice Mine
. .
et al., 1983 . Kitao and Yoshida 1986 applied FK-565 to fish and demonstrated that the
injection of FK-565 in to rainbow trout increased their resistance to A. salmonicida,
following the activation of phagocytic cells. The activation of phagocytic cells was also
.
observed in yellowtail Yoshida, personal communication . Numbers of splenic anti-
body-producing cells and humoral antibody titers can be elevated during immunization
in rainbow trout when Y. ruckeri or A. salmonicida O-antigen preparations are mixed
.
with FK-565 Kitao et al., 1987 .
3.2. Bacterial deriaties
3.2.1. MDP
. .
Muramyl dipeptide MDP N-acetyl-muramyl-L-alanyl-D-isoglutamine , derived from
Mycobacterium, elicits an immunostimulatory effect; e.g., the activation of macrophages,
.
B lymphocytes and alternative pathway of complement in mice. Olivier et al. 1985
reported that coho salmon injected with a mixture of MDP and modified Freunds
incomplete adjuvant should a 47-fold increase in resistance to A. salmonicida. Kodama
.
et al. 1993 reported that intraperitoneal injection of rainbow trout with MDPLys
increased the phagocytic activities, respiratory burst and migration activities of kidney
leucocytes as well as resistance of the fish to A. salmonicida challenge. Furthermore,
( )
they demonstrated that sera of fish injected with MDPLys could stimulate colony
.
formation of kidney cells Kodama et al., 1994 .
3.2.2. LPS
.
LPS lipopolysaccharide is a cell wall component of gram-negative bacteria. LPS
can stimulate B cell proliferation, and, LPS injected into red sea bream Pagrus major
.
has been demonstrated to enhance macrophage phagocytic activity Salati et al., 1987 .
.
MacArthur et al. 1985 reported that plaice, Pleuronectes platessa, injected with LPS
showed increased macrophage migratory activity. In vitro, LPS stimulates phagocytosis
and the production of superoxide anions in Atlantic salmon macrophages Solem et al.,
.
1995 . Similarly, LPS stimulates the production of macrophage activating factor in
.
goldfish lymphocytes Neumann et al., 1995 and the production of interleukin 1 like
.
molecules in catfish monocytes Clem et al., 1985 .
3.2.3. FCA
.
Freunds complete adjuvant FCA , a mineral oil adjuvant containing killed My-
cobacterium butyricum, enhances immune responses and increases the efficacy of
. .
vaccination in fish e.g., Paterson and Fryer, 1974 . Olivier et al. 1985 reported that
.
coho salmon, O. kisutch, injected with FCA increased protection 450 times in LD
50
against A. salmonicida challenge. This increased protection was also seen against A.
hydrophila and V. ordalii. Immunologically, they demonstrated that the activation of
.
macrophages phagocytosis and killing in fish injected with FCA caused increased
. .
resistance to A. salmonicida Olivier et al., 1986 . Adams et al. 1988 also reported that
FCA-injected rainbow trout appeared to show increased protection against furunculosis,
.
vibriosis and redmouth disease. Kajita et al. 1992a confirmed that rainbow trout
injected with FCA showed increases in respiratory burst, phagocytic and NK cell
activity of leucocytes and protection against V. anguillarum infection. However,
.
Kawakami et al. 1998 showed that yellowtail injected with FCA did not show
increased resistance to P. piscicida infection, although the adjuvant effect of FCA on a
P. piscicida vaccine was observed in fish.
3.2.4. Vibrio bacterin
V. anguillarum bacterin is the most successful vaccine for salmonid fish, and this
efficacy is seen following administration by injection, oral dosing and immersion
.
methods. Sakai et al. 1995c reported that rainbow trout immersed in V. anguillarum
.
bacterin solution showed increased protection 12 times in LD to Streptococcus sp.
50
.
infection. Norqvist et al. 1989 also showed that vaccination of rainbow trout with
attenuated V. anguillarum induced protection against A. salmonicida challenge. How-
ever, no immunostimulation of V. anguillarum bacterin was seen in yellowtail infected
.
with P. piscicida Kawakami et al., 1998 . The immunostimulant effects of vibrio
.
bacterins were also reported in Kuruma prawns Penaeus japonicus. Itami et al. 1989
reported that prawns injected or immersed with the formalin-killed vibrio bacterin
experienced reduced mortalities when they were challenged by Vibrio injection 30 days
.
later. Horne et al. 1995 also reported the efficacy of vibrio vaccines in black tiger
shrimp. The migration of hemocytes treated with vibrio bacterin increased, compared to
( )
.
non-treated controls Itami et al., 1989 . This fact suggested the immunostimulation of
vibrio bacterin in prawn and shrimp, as invertebrates do not have specific immune
responses. It is still unknown what components of V. anguillarum cells stimulate
non-specific immune responses. However, as bacterial LPS stimulated non-specific
immunity, especially macrophage activation, V. anguillarum LPS may act as an
immunostimulant.
3.2.5. A. stenohalis and C. butyricum
A. stenohalis is a gram-negative aerobic organism which has been isolated from sea
water. The LPS of this bacteria activates mouse macrophages and B-lymphocytes Isogai
. . .
et al., 1989 and shows anti-tumor effects Chang, 1966 . Kawahara et al. 1994
reported that white-spotted char, Salelinus leucomaenis, injected with inactivated A.
stenohalis showed enhanced chemiluminescent responses of kidney cells, complement
activation and increased protection against A. salmonicida challenge.
A butyric acid bacterium, C. butyricum, has been used clinically to prevent distur-
bances of microbial flora in the human intestine, and oral administration of the spores of
this bacterium ameliorate diarrhoea, constipation and abdominal distension in man. C.
butyricum shows immunostimulatory effects such as stimulation of macrophages and
.
NK cells and enhances protection against Candida infection Young et al., 1987 . Sakai
.
et al. 1995a,b showed enhancement of resistance to vibriosis in rainbow trout by oral
administration of C. butyrium bacterin mediated by leucocyte activation, including
phagocytosis and increased superoxide anion production.
3.2.6. Chitin and chitosan
Chitin is a polysaccharide forming the principal component of crustacean and insect
.
exoskeletons and the cell walls of certain fungi. Sakai et al. 1992 reported that rainbow
trout injected with chitin showed stimulated macrophage activities and an increased
resistance to V. anguillarum infection. Yellowtail injected with chitin alone also showed
increased protection against P. piscicida challenge which continued until 45 days after
.
treatment Kawakami et al., 1998 . Compared with the increase in non-specific protec-
tive immunity in yellowtail injected with chitin, chitin did not show adjuvant effects
.
Kawakami et al., 1998 , as had been was demonstrated in mice and guinea pigs
.
Nishimura et al., 1985 .
Chitosan, de-N-acetylated chitin, also showed immunostimulatory effects. Brook
trout, Salelinus fontinalis, injected or immersed in chitosan solution showed increased
.
protection against A. salmonicida infection Anderson and Siwicki, 1994 as did
.
rainbow trout administered chitosan orally Siwicki et al., 1994 . Rainbow trout treated
with chitosan by injection or immersion showed increases in immunological parameters
in the blood such as NBT, potential killing activity, myeloperoxidase and total Ig
.
concentration Anderson et al., 1995 .
3.2.7. EF203
EF203 is the fermented product of chicken eggs. The oral administration of EF203 to
rainbow trout stimulates the activity of leucocytes such as phagocytosis and chemilumi-
nescence, and increases protection against Streptococcus infection Yoshida et al.,

(
)
M
.
S
a
k
a
i
r
A
q
u
a
c
u
l
t
u
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1
7
2
1
9
9
9
6
3
9
2
7
0
Table 2
Immunostimulant effects in fish and shrimp
Agent Authors Fish Administration Results Resistance to pathogens
.
Levamisole Siwicki 1987 carp ip phagocytosis
.
Siwicki 1989 carp oral NBT
.
Siwicki et al. 1990 trout in vitro phagocytosis
NBT
.
Kajita et al. 1990 trout ip phagocytosis V. anguillarum
CL
complement
.
Jeney and Anderson 1993a trout im A. salmonicida
.
Baba et al. 1993 carp im phagocytosis A. hydrophila
CL
.
FK-565 Kitao and Yoshida 1986 trout ip phagocytosis A. salmonicida
.
Kitao et al. 1987 trout in vitro antibody
.
MDP Kodama et al. 1993 trout ip phagocytosis V. anguillarum
CL
.
LPS Clem et al. 1985 catfish in vitro IL1
.
MacArthur et al. 1985 plaice ip macrophage migration
.
Salati et al. 1987 red sea bream ip phagocytosis
.
Neumann et al. 1995 goldfish in vitro MAF
.
Solem et al. 1995 salmon in vitro phagocytosis
NBT
.
Dalmo and Seljelid 1995 salmon in vitro phagocytosis
NBT
.
FCA Olivier et al. 1985 coho ip A. salmonicida
A. hydrophila
V. ordarii
.
Olivier et al. 1986 brook ip phagocytosis
.
Adams et al. 1988 trout ip A. salmonicida
V. anguillarum
Y. ruckeri
.
Kajita et al. 1990 trout ip V. anguillarum
.
Kawakami et al. 1998 yellowtail ip P. piscicida
(
)
M
.
S
a
k
a
i
r
A
q
u
a
c
u
l
t
u
r
e
1
7
2
1
9
9
9
6
3
9
2
7
1
.
Vibrio Norqvist et al. 1989 trout im A. salmonicida
.
bacteria Sakai et al. 1995c trout im Enterococcus seriolicida
.
Itami et al. 1989 prawn ip Vibrio sp.
im
.
Horne et al. 1995 shrimp ip Vibrio sp.
im
oral
.
C. butyricum Sakai et al. 1995a,b trout oral phagocytosis V. anguillarum
NBT
.
A. stenohalis Kawahara et al. 1994 char ip CL A. salmonicida
complement
.
Chitin Sakai et al. 1992 trout ip phagocytosis V. anguillarum
lysozyme
.
Chitosan Siwicki et al. 1994 trout oral NBT A. salmonicida
phagocytosis
.
Anderson et al. 1995 trout ip NBT
im killing
.
EF203 Yoshida et al. 1993 trout oral phagocytosis Streptococcus sp.
CL
.
Sakai et al. 1995f trout oral phagocytosis R. salmoninarum
.
BKD vaccine adjuvant NBT
antibody
.
Soybean Rumsey et al. 1994 trout oral NBT
protein phagocytosis
killing
.
PS-K Park and Jeong 1996 tilapia oral phagocytosis E. tarda
protein-bound
polysaccharide
.
Oligosaccharide Yoshida et al. 1995 catfish oral NBT
.
Spiralina Duncan and Klesius 1996b catfish oral CL E. ictaluri
antibody
(
)
M
.
S
a
k
a
i
r
A
q
u
a
c
u
l
t
u
r
e
1
7
2
1
9
9
9
6
3
9
2
7
2
.
Table 2 continued
.
Yeast glucan Robertsen et al. 1990 salmon ip V. anguillarum
V. salmonicida
Y. ruckeri
.
Raa et al. 1992 salmon oral V. anguillarum
V. salmonicida
.
Engstad et al. 1992 salmon ip complement
lysozyme
.
Chen and Ainsworth 1992 catfish ip phagocytosis E. ictaluri
killing
antibody
.
Jrgensen et al. 1993a trout ip lysozyme
killing
y
O
2
.
Jrgensen et al. 1993b trout ip NBT
killing
.
Rrstad et al. 1993 salmon ip A. salmonicida
.
adjuvant
A. salmonicida
.
Sung et al. 1994 shrimp im phenoloxidase V. ulnificus
lysozyme
.
Song and Hsieh 1994 shrimp in vitro NBT
CL
.
Aakre et al. 1994 salmon ip antibody A. salmonicida
.
adjuvant
.
Thompson et al. 1995 trout ip lysozyme V. anguillarum
.
Yoshida et al. 1995 catfish oral NBT
.
Jrgensen and Robertsen 1995 salmon in vitro NBT
killing
(
)
M
.
S
a
k
a
i
r
A
q
u
a
c
u
l
t
u
r
e
1
7
2
1
9
9
9
6
3
9
2
7
3
.
Baulny et al. 1996 turbot oral lysozyme V. anguillarum
.
adjuvant
complement
CL V. anguillarum
.
Duncan and Klesius 1996a catfish oral CL E. ictaluri
migration
.
yeast glucan Verlhac et al. 1996 trout oral CL
qVitamin C complement
lysozyme
.
VitaSim Nikl et al. 1991 coho ip A. salmonicida
oral A. salmonicida
.
Nikl et al. 1993 chinook oral A. salmonicida
im A. salmonicida
.
Ainsworth et al. 1994 catfish oral antibody E. ictaluri
.
Peptidoglucan Matsuo and Miyazano 1993 trout oral V. anguillarum
.
Boonyaratpalin et al. 1995 shrimp oral Yellow-head baculovirus
.
Itami et al. 1996 yellowtail oral phagocytosis E. seriolocida
.
Lentinan Yano et al. 1989 carp ip phagocytosis E. tarda
Schizophyllan
.
Scleroglucan Yano et al. 1991 carp ip A. hydrophila
.
Schizophyllan Matsuyama et al. 1992 yellowtail ip complement E. seriolocida
Scleroglucan phagocytic index P. piscicida
lysozyme
.
Schizophyllan Itami et al. 1994 prawn oral phagocytosis Vibrio sp.
.
polyglucose Sveinbjornsson and Seljelid 1994 salmon in vitro NBT
pinocytosis
acid phosphatase
. .
glucan Sigma Jeney and Anderson 1993b trout im phagocytosis
ip NBT
( )
. .
1993 . Sakai et al. 1995f reported that R. salmoninarum-vaccinated rainbow trout
receiving EF203 showed higher phagocytic activities and NBT responses in kidney
leucocytes when compared to vaccinated fish without EF203 treatment or to unvacci-
nated fish. However, the serum agglutinating antibody titers of vaccinated fish did not
show a significant increase between the EF203 and control groups, and the vaccinated
fish treated with EF203 showed slightly increased survival in comparison with the other
groups following R. salmoninarum challenge.
3.2.8. Glucan
.
The immune stimulatory effects of glucan have been well-studied Table 2 . The
.
effects of several types of glucan; e.g. yeast glucan, peptide-glucan b-1,3, glucan VST ,
have been investigated in fish. Yeast glucan is the most extensively studied of these
.
glucans. Intraperitoneal injection of yeast glucan b-1,3- and b-1,6-linked glucan
prepared from cell walls of Saccharomyces cereisiae into Atlantic salmon resulted in
increased resistance to V. anguillarum, V. salmonicida and Y. ruckeri Robertsen et al.,

. .
1990 . Chen and Ainsworth 1992 reported that catfish injected with yeast glucan
.
showed increased resistance to E. ictaluri. However, Thompson et al. 1995 reported
that rainbow trout injected with yeast glucan did not show enhanced protection against
V. anguillarum infection. Yeast glucan has been applied by immersion and oral
.
administration methods. Raa et al. 1992 reported that oral administration of yeast
glucan to Atlantic salmon increased protection against V. anguillarum and V. salmoni-
.
cida. Tiger shrimp immersed in yeast glucan solution 0.5 and 1 mgrml showed
.
enhanced protection against V. ulnificus infection Sung et al., 1994 . Although channel
catfish injected with yeast glucan showed increased protection to E. ictaluri Chen and
.
Ainsworth, 1992 , oral administration did not show such an effect Ainsworth et al.,
.
1994; Duncan and Klesius, 1996a .
The adjuvant effects of yeast glucan have also been demonstrated. Injection of A.
salmonicida vaccine and yeast glucan into Atlantic salmon enhanced antibody responses
Notes to Table 2:
: increase.
: no change.
trout: rainbow trout.
salmon: Atlantic salmon.
coho: coho salmon.
brook: brook trout.
prawn: kuruma prawn.
shrimp: black tiger shrimp.
chinook: chinook salmon.
ip: intraperitoneal injection.
im: immersion.
NBT: nitroblue tetrazolium reaction.
CL: chemiluminescent response.
IL1: interleukin 1 production.
MAF: macrophage activating factor.
killing: bactericidal activity of macrophage.
O
y
: production of superoxide anion.
2
( )
.
Aakre et al., 1994 and induced significantly increased protection against furunculosis
.
over vaccines without yeast glucan Rrstad et al., 1993 . The injection of yeast glucan
.
alone did not show protection. Baulny et al. 1996 reported that oral administration of
yeast glucan to turbot immersed in V. anguillarum bacterin also increased protection
compared with bacterin alone. As in the experiment above, yeast glucan alone did not
enhance protection against V. anguillarum infection.
Yeast glucan enhances the lysozyme activity in Atlantic salmon, rainbow trout and
turbot Engstad et al., 1992; Jrgensen et al., 1993a; Thompson et al., 1995; Baulny et
. .
al., 1996 , complement activity Engstad et al., 1992 , bacterial killing activity of
macrophages in rainbow trout, Atlantic salmon and catfish Chen and Ainsworth, 1992;
.
Jrgensen et al., 1993a,b; Jrgensen and Robertsen, 1995 and the production of
superoxide by macrophages or hemocytes in rainbow trout, tiger shrimp, catfish and
Atlantic salmon Jrgensen et al., 1993a; Song and Hsieh, 1994; Yoshida et al., 1995;
.
Baulny et al., 1996 .
.
b-1, 3 glucan VST derived from Schizophyllum commune has also been evaluated
as a protection against disease. Coho salmon treated with VST by injection or oral routes
.
showed enhanced protection against A. salmonicida Nikl et al., 1991, 1993 . However,
chinook salmon immersed in a solution of this glucan did not show protection Nikl et
. .
al., 1993 . Ainsworth et al. 1994 reported that catfish fed VST did not show increased
serum lysozyme activity, although they showed increased production of superoxide
anion by macrophages and reduced mortality when challenged with E. ictaluri. Further-
more, this glucan showed no adjuvant effect on E. ictaluri bacterins despite increased
.
antibody titers Ainsworth et al., 1994 .
Peptidoglucan prepared from Breibacterium lactofermentum increased phagocytosis
.
in yellowtail and resistance to Enterococcus seriola infection Itami et al., 1996 . The
efficacy of peptidoglucan was also demonstrated against vibriosis in rainbow trout
.
Matsuo and Miyazano, 1993 and yellow-head baculovirus infection in black tiger
.
shrimp Boonyaratpalin et al., 1995 . The efficacies of glucan supplied by Sigma Jeney
.
and Anderson, 1993b , Lentinan, Schizophyllan and Scleoglucan Yano et al., 1989,
.
1991; Matsuyama et al., 1992; Itami et al., 1994 have also been demonstrated in
rainbow trout, carp, yellowtail and prawn.
3.3. Animals and plant components
3.3.1. Animal extracts
The extracts from some invertebrates have immunostimulatory effects. An extract
.
from the marine tunicate, Ecteinascida turbinata Ete and a glucoprotein fraction of
.
water extract Hde from abalone, Haliotis discus hannai, enhanced the killing of tumor
.
cells in vitro and inhibited tumor growth in vivo Sigel et al., 1970; Uchida et al., 1987 .
Eel injected with Ete showed enhanced phagocytosis and increased survival following
. .
A. hydrophila challenge Davis and Hayasaka, 1984 . However, Stanley et al. 1995
demonstrated that the survival after infection with E. ictaluri decreased in channel
catfish injected with Ete, although immunoenhancement was observed. Rainbow trout
injected with Hde also showed enhanced phagocytic and NK cell activities, and showed
.
increased survival against V. anguillarum infection Sakai et al., 1991 . The heat extract
( )
from firefly squid, Watasenia scintillans, also stimulated the immune system of rainbow
trout such as the production of superoxide anion, potential killing activities by
macrophages and the lymphoblastic transformation of lymphocytes in vitro Table

. .
3 Siwicki et al., 1994 .
3.3.2. Glycyrrhizin
Glycyrrhizin is a glycosylated saponin, containing one molecule of glycyrretinic acid,
which has anti-inflammatory and anti-tumor activities, mediated by its immunomodula-
. .
tory activities Wada et al., 1987; Zhang et al., 1990 . Edahiro et al. 1990, 1991
reported that yellowtail treated orally with glycyrrhizin showed increased protection
against E. seriola infection, although lysozyme activity of blood and phagocytic
.
activities of macrophages were not enhanced. However, Jang et al. 1995 reported that
in vitro treatment with glycyrrhizine enhanced the respiratory burst activity of
macrophages and the proliferative responses of lymphocytes from rainbow trout.
3.3.3. Other immunostimulants
Other immunostimulants are also shown in Table 2. Rainbow trout treated orally with
soybean protein showed increased leucocyte activities such as phagocytosis, bacterial
.
killing and the production of superoxide Rumsey et al., 1994 . The bath administration
of Quil A saponin with Y. ruckeri vaccine enhanced the in vitro bactericidal activities in
. .
rainbow trout Grayson et al., 1987 . Ninomiya et al. 1995 reported that oral
administration of Quil A saponin increased leucocyte migration in yellowtail. The
.
immunostimulant effects of PS-K protein-bound polysaccharide , Spiralina, acid pep-
tide fractions from fish protein hydrolysate were also reported in tilapia, Oreochrmis
. .
niloticus, by Park and Jeong 1996 , channel catfish by Duncan and Klesius 1996b and
.
Atlantic salmon by Gildberg et al. 1996 , respectively.
3.4. Diet components
Dietary vitamin C is essential for normal growth and for several physiological
.
functions in most fishes Halver, 1989 . High levels of dietary vitamin C are reported to
increase resistance to Edwardsiella tarda and E. ictaluri infection in channel catfish
.
Durve and Lovell, 1982; Li and Lovell, 1985; Liu et al., 1989 , to V. anguillarum, IHN
and Ichthyophthirius multifiliis in rainbow trout Navarre and Halver, 1989; Suzuki and
.
Ai, 1989; Wahli et al., 1995 and to A. salmonicida and V. salmonicida in Atlantic
. .
salmon Erdal et al., 1991; Hardie et al., 1991 . Li and Lovell 1985 and Hardie et al.
.
1991 showed that treatment with high doses of vitamin C increased complement
.
activities in catfish or Atlantic salmon. However, Yano et al. 1988 and Liu et al.
.
1989 did not observe such effects in catfish or red sea bream, respectively. On the
.
other hand, the activation of macrophages was reported by Thompson et al. 1993 in
. .
Atlantic salmon and by Roberts et al. 1995 in turbot. Hardie et al. 1991 reported that
treatment of fish with high doses of vitamin C stimulated macrophage activating factors,
.
followed by lymphocyte proliferation Hardie et al., 1993 . These results show that
.
fishes fed with high doses more than 1000 mgrkg of vitamin C have protective
.
immune responses Table 4 .
(
)
M
.
S
a
k
a
i
r
A
q
u
a
c
u
l
t
u
r
e
1
7
2
1
9
9
9
6
3
9
2
7
7
Table 3
Animal and plant extracts used as fish immunostimulants
. .
Tunicate Ete Davis and Hayasaka 1984 eel ip phagocytosis A. hydrophila
.
Stanley et al. 1995 catfish ip antibody E. ictaluri x
phagocytosis
. .
Abalone Hde Sakai et al. 1992 trout ip CL V. anguillarum
phagocytosis
NK
.
Firefly squid Siwicki et al. 1996 trout ip NBT
.
mitogen Con A, LPS
killing
.
Acid peptide fractions from Gildberg et al. 1996 salmon in vitro NBT
fish protein hydrolysate
.
Quil A saponin Ninomiya et al. 1995 yellowtail oral leucocyte migration
.
Grayson et al. 1987 trout im serum bactericidal activity
.
Glycyrrhizin Edahiro et al. 1990 yellowtail oral complement E. seriolicida
.
Edahiro et al. 1991
. .
Jang et al. 1995 trout in vitro mitogen Con A, LPS
MAF
y
O
2
: increase.
: no change.
x: decrease.
NK: natural killer cell.
Con A: concanavalin A.
im: immersion.
( )
Table 4
Immunostimulant effects of diet components in fish
Authors Doses Fish Results Resistance to
.
mgrkg pathogens
Vitamin C
.
Durve and Lovell 1982 150 catfish E. tarda
14 w
.
Li and Lovell 1985 3000 catfish complement E. tarda
antibody
phagocytic index
macrophage killing
.
Liu et al. 1989 4000 catfish complement E. tarda
9 w antibody
.
Yano et al. 1988 1000 Red sea complement
bream
.
Suzuki and Ai 1989 550 trout IHNV
10 d
.
Navarre and Halver 1989 2000 trout V. anguillarum
12 w
.
Erdal et al. 1991 4000 salmon V. salmonicida
52 d
.
Hardie et al. 1991 2750 salmon complement A. salmonicida
26w NBT
phagocytosis
MAF
.
Johnson and Ainsworth 1991 1000 catfish neutrophil
.
7 w phagocytosis
. .
Hardie et al. 1993 in vitro 244 trout proliferation
16 w NBT
MAF
.
Thompson et al. 1993 3170 salmon NBT
16 w killing
.
A. salmonicida
migration
antibody
.
A. salmonicida
.
Roberts et al. 1995 2000 turbot phagocytic index
127 d lysozyme
.
Wahli et al. 1995 2000 trout I. multifiliis
30 d
Vitamin E
.
Blazer and Wolke 1984 low levels trout antibody x
.
Hardie et al. 1990 800 salmon NBT A. salmonicida
20 w complement
lysozyme x
.
Wise et al. 1993a 240 catfish NBT
120 d
Se 0.8
.
Wise et al. 1993b 2500 catfish phagocytic index
180 d antibody
( )
.
Table 4 continued
Authors Doses Fish Results Resistance to
.
mgrkg pathogens
Vitamin E
.
Pulsford et al. 1995 500 turbot phagocytosis
12 w
.
Taksdal et al. 1995 low levels salmon IHNV x
Vitamin A
.
Thompson et al. 1994 salmon anti-protease activity A. salmonicida
migration
w: week.
d: day.
: increase.
: no change.
x: decrease.
Se: selenium.
Vitamin E also enhances both humoral and cellular defenses in mammals Panush
. .
and Delafuente, 1985 . Blazer and Wolke 1984 reported that specific and cell-mediated
immunity and macrophage phagocytosis were all compromised in rainbow trout fed
.
vitamin E-depleted diets. Furthermore, Hardie et al. 1990 reported that Atlantic salmon
fed vitamin E-depleted diets had significantly increased mortality rate following A.
salmonicida infection compared to fish on the commercial diet. However, only comple-
.
ment activity was compromised in these vitamin E-depleted fish. Wise et al. 1993a,b
showed that catfish fed high doses of vitamin E had an increased phagocytic indices and
.
superoxide anion production by leucocytes Table 4 .
Other dietary components such as lipids, other vitamins, trace elements, etc. can
affect immune responses in fish. The importance of diet in fish immune responses was
. .
reviewed by Landolt 1989 and Blazer 1992 .
3.5. Hormones, cytokines and others
3.5.1. Hormones
The relationship between neuroendocrine regulation and the immune system has
recently become the subject of intense investigation Berczi and Nagy, 1987; Kelley,
.
1989; Gala, 1991; Auernhammer and Strasburger, 1995 . It is also known that GH and
.
PRL directly affect immunocompetent cells macrophages, lymphocytes and NK cells
. .
Table 5 . In fish, exogenous growth hormone GH has mitogenic activity on lympho-
.
cytes and activates NK cells Kajita et al., 1992b; Sakai et al., 1996b . In addition, Sakai
.
et al. 1995d, 1996a reported that exogenous GH given to rainbow trout increased the
.
production of superoxide anion in leucocytes. Calduch-Giner et al. 1995 detected GH
receptors in monocytes and lymphocytes of gilthead sea bream, Sparus aurata. Re-
( )
Table 5
Hormones and cytokines used as fish immunostimulants
.
LF Sakai et al. 1993 trout oral phagocytosis V. anguillarum
CL Streptococcus sp.
.
Sakai et al. 1995b trout in vitro CL
NBT
.
Kakuta et al. 1996 red sea oral lectin Cryptocaryon
bream lysozyme
.
GH Kajita et al. 1992b ip NK
.
Sakai et al. 1995d trout ip CL
.
Sakai et al. 1996a trout in vitro NBT
mitogen
.
Sakai et al. 1997 trout ip phagocytosis V. anguillarum
.
PRL Sakai et al. 1996a trout in vitro NBT
.
INF Tamai et al. 1993 hirame in vitro HRV
LF: lactoferrin.
GH: growth hormone.
PRL: prolactin.
INF: interferon.
: increase.
: no change.
NK: natural killer cell.
.
cently, Sakai et al. 1996c reported that injection of rainbow trout with GH increased
serum hemolytic activities. Furthermore, the injection of GH increased protection
.
against V. anguillarum in rainbow trout Sakai et al., 1997 .
. .
Prolactin PRL also has immunostimulatory activities. Sakai et al. 1996b reported
that the addition of homogeneous PRL to chum salmon, Oncorhynchus keta, induced
lymphocyte mitogenic responses and PRL increased the production of superoxide anion
. .
by leucocytes in rainbow trout Sakai et al., 1996a . Sakai et al. unpublished also
reported that b-endorphin activated the phagocytic activity of rainbow trout leucocytes.
3.5.2. Cytokines
Cytokines are polypeptides or glycoproteins which act as modulators in the immune
system. The existence of several cytokines has been reported in fish Secombes et al.,
.
1996 . The structures of cytokines such as interleukin 2 have already been reported
.
Tamai et al., 1992 . Several cytokines can be used as immunostimulants. Tamai et al.
.
1993 reported that a recombinant interferon-like protein produced from Japanese
flatfish, Paralichthys liaceus, tha amino acid homology was less than 20%, with
.
murine interferon a or b molecule , inhibited infection of EPC cells by hirame
rhabdovirus. Although the use of cytokines as immunostimulants has only been de-
.
scribed in one report by Tamai et al. 1993 , cytokines may be useful as powerful
( )
immunostimulants if their structures can be identified and recombinant molecules
prepared.
3.5.3. Lactoferrin
Lactoferrin, which consists of a single peptide chain with a molecular weight of about
87,000 and possessing two Fe-binding sites per molecule, is widely distributed in the
physiological fluids of mammals. Many biological functions have been attributed to
lactoferrin including antibacterial activity Arnold and Brewer, 1980; Bortner et al.,
. .
1986 , regulation of Fe absorption Kawakami et al., 1988 and growth promotion of
.
macrophages Amberuso and Johnston, 1981 . It has also been found to regulate
hydroxyl radical production by macrophages, granulocytes and neutrophil leucocytes
. .
Amberuso and Johnston, 1981; Gahr et al., 1991 . Sakai et al. 1993 reported that
rainbow trout orally treated with bovine lactoferrin showed enhanced phagocytic activi-
ties and the production of superoxide anion by macrophages, and demonstrated high
.
resistance 40-fold increase in LD to V. anguillarum infection but marginal increase
50
. .
in resistance 4-fold increase in LD to Streptococcus sp. Sakai et al., 1993 .
50
.
Lactoferrin can activate macrophages in vitro Sakai et al., 1995b . Recently, Kakuta
. .
and Kurokura 1995 and Kakuta et al. 1996 reported that red sea bream, P. major,
orally administered bovine lactoferrin showed increased numbers of granulocytes and
lymphocytes in the blood, elevated secretion of body mucus and resistance to Crypto-
caryon irritans infection.
4. Therapeutic regiments for immunostimulant application
The use of immunostimulants can protect fish from several infectious diseases and
.
decrease mortality rates Table 6 ; however, fish cannot be protected against all
infectious diseases by immunostimulants. Fishes receiving immunostimulants show
increased resistance against infection by bacteria such as V. anguillarum, V. salmoni-
cida, A. salmonicida, and Streptococcus sp., viral infection such as IHN and yellow-head
baculovirus, and parasite infection such as white spot disease. Positive effects of
immunostimulants against many virus infections such as VHS, IHN, etc. and other
bacterial infections such as Cytophaga infections have not been reported.
Immunostimulants do not increase resistance against R. salmoninarum, P. piscicida
.
or E. ictaluri infection Table 7 . These bacteria are resistant to phagocytosis and can
survive within macrophages Nelson et al., 1989; Baldwin and Newton, 1996; Guten-
.
berger et al., 1997 . As already indicated, the main immunological function increased by
immunostimulants is the activity of phagocytic cells. However, macrophage-resistant
bacteria may escape from activated macrophages and thus in these situations, immunos-
timulants do not appear effective against such infections.
5. Timing of immunostimulants administration
The effect of timing the administration on immunostimulant function is a very
important issue. Usually, the most effective timing of antibiotics is upon the occurrence
( )
Table 6
Effectiveness of the use of immunostimulants on infectious diseases
Effectie
Bacterial infections
.
V. ulnificus yeast glucan
V. anguillarum yeast glucan, lactoferin, HDe, levamisole, chitin, Vitamin C,

.
C. butyricum cells GH, MDP, PG
.
V. salmonicida yeast glucan, Vitamin C, Vitamin E
.
A. salmonicida Vitamin C, FK-565, yeast glucan, A. stenohalis cells, chitosan, levamisole, VST
.
A. hydrophila levamisole, Ete
.
Y. ruckeri yeast glucan
.
Streptococcus sp. EF203
.
E. seriolocida Schizophyllan, Scleroglucan, Glycyrrhizin, PG
.
E. ictaluri yeast glucan
.
E. tarda Vitamin C, PS-K, Lentinan, Schizophyllan, Scleroglucan
Viral infections
.
IHN Vitamin C
.
Yellow-head baculovirus yeast glucan
Parasite infections
.
I. multifiliis Vitamin C
.
Cryptocaryon sp. lactoferrin
Non-effectie
Bacterial infections
.
A. salmonicida yeast glucan
.
V. anguillarum yeast glucan
.
E. ictaluri Ete, yeast glucan, VST, Spiralina
.
P. piscicida Schizophyllan, Scleroglucan, FCA, yeast glucan, chitin
.
R. salmoninarum EF203
of disease, and they cannot often be used prophylactically due to risk of fostering the
.
development of drug-resistant bacteria. Anderson 1992 proposed that immunostimu-
lants should be applied before the outbreak of disease to reduce disease-related losses.
Immunostimulants can promote recovery from immunosuppression states caused by
.
stress. Kitao and Yoshida 1986 reported that rainbow trout injected with cyclophospha-
mide or hydrocortisone showed suppressed phagocytic activity of peritoneal and kidney
leucocytes, and this suppression of the phagocytosis was reversed by injection of
.
FK-565. Boonyaratpalin et al. 1995 also showed that peptidoglycan-fed black tiger
shrimp exhibited a higher tolerance to dissolved oxygen, salinity, and stress than those
fed the control diet.
Table 7
A comparison of characteristics of chemotherapeutics, vaccines and Immunostimulants
Chemotherapeutics Vaccine Immunostimulants
When Therapeutically Prophylactically Prophylactically
Efficacy Excellent Excellent Good
Spectrum of activity Middle Limited Wide
Duration Short Long Short
( )
The effective timing of administration of immunostimulants such as Ete is very
difficult. Ete exerted a protective effect in eels injected intraperitoneally 2 days after
challenge with A. hydrophila. However, the protection was not seen when Ete was
administered intraperitoneally 2 days before or concurrently with the bacteria Davis and
.
Hayasaka, 1984 .
6. Route of administration
Many authors reported that the injection of immunostimulants enhances the function
of leucocytes and protection against pathogens. However, this method is labor intensive,
relatively time-consuming and becomes impractical when fishes weigh less than 15 g.
Thus, another method such as oral administration or immersion should be used. Oral
administration of immunostimulants has already been reported for glucans, EF203,
lactoferrin, levamisole and chitosan. This method is non-stressful and allows mass
administration regardless of fish size. Oral administration of these immunostimulants
results in enhancement of leucocyte function and protection against infectious diseases
such as furunculosis, vibriosis, streptococcosis. The immersion method can also used.
.
Baba et al. 1993 reported that carp immersed in levamisole solution 10 mgrml for 24
.
h showed activated phagocytic activities, chemotactic abilities and the production of
active oxygen in head kidney phagocytes and enhanced protection against A. hy-
.
drophila. This effect lasted for at least 2 weeks. Anderson et al. 1995 demonstrated
that rainbow trout immersed with glucan or chitosan showed increased protection
against A. salmonicida after treatment for 3 days. However, this effect was transient and
was not present after 14 days. Adjuvant effects of immunostimulants administered by
.
immersion have also been reported by several authors. Jeney and Anderson 1993a
reported that rainbow trout bathed in A. salmonicida O-antigen in combination with
immunostimulants levamisole, quarternary ammonium compound, polypeptide of fish

. .
extract 30 min immersion enhanced phagocytosis by leucocytes and antibody titers
.
against A. salmonicida, and showed adjuvant effects with vaccination. Nikl et al. 1993
compared the adjuvant effects of glucan against A. salmonicida vaccine oral delivery 7
. .
days administration and immersion 15 min . No adjuvant effects were seen with the
immersion treatment, although the fish administered glucan orally showed enhanced
vaccine effects. The efficacies of immersion administration of immunostimulants has
been demonstrated by several authors. However, the dilution and the levels of efficacy
require more complete investigation.
7. Doses
Immunostimulants increase the immune responses and enhance protection against
.
pathogens which raises the question of dose-dependency. Kajita et al. 1990 showed
that the chemiluminescent effects of phagocytic cells in rainbow trout were increased by
injection of levamisole at 0.1 and 0.5 mgrkg. However, they also reported that the
injection of 5 mgrkg of levamisole did not produce this immunostimulant effect.
( )
.
Robertsen et al. 1994 reported that the increase in respiratory burst activity of
glucan-treated macrophages was maximal at glucan concentrations of 0.11 mgrml,
whereas at 10 mgrml no effect was seen and at 50 mgrml glucan was inhibitory.
.
Comparable effects on function were also observed in lymphocytes. Kitao et al. 1987
.
reported that high doses 10 mgrkg of FK 565 did not increase the numbers of
. .
plaque-forming cells PFC against Y. rukeri, although the optimum dose 5 mgrkg
increased PFC. The effects of immunostimulant are therefore not directly dose-depen-
dent, and high doses may not enhance and may inhibit the immune responses.
8. The effects of long-term administration
Oral administration is the most practical method for delivery of immunostimulants;
however, the effects of long-term oral administration of immunostimulants are still
.
unclear. Matsuo and Miyazano 1993 reported that rainbow trout treated with pepti-
doglucan orally for 56 days did not show protection after challenge with V. anguillarum,
.
although fish treated for 28 days showed increased protection. Yoshida et al. 1995 also
demonstrated that the number of NBT-positive cells in African catfish increased
following oral administration of glucan or oligosaccharide over 30 days, but not over 45
days. Although the reasons for these decreases in immune responses in fish by long-term
oral administration of immunostimulants is still unknown, negative feedback systems
against immunostimulation may function in fish, and the immune response may revert to
a previous state. Thus, the effective administration period should be investigated for
each immunostimulant.
9. The additional effects of immunostimulants
Several authors have reported relationships between immunostimulation and growth-
.
promoting activity. Boonyaratpalin et al. 1995 reported that black tiger shrimp fed with
peptidoglycan-supplemented feed showed better growth and feed conversion rates than
those fed a normal diet. This effect was observed with 0.01% peptidoglycan supplemen-
. .
tation, but not with the highest level administered 0.1% . Sung et al. 1994 also
showed that black tiger shrimp grew faster with glucan immersion at the 0.5, 1, and 2
mgrml than at 0.25 mgrml or in control solutions. However, Matsuo and Miyazano
.
1993 reported that peptidoglucan did not influence rainbow trout growth after 60 days
of oral administration. It is well-known that GH promotes the growth of fish Rand-

. .
Weaver and Kawauchi, 1993 . Sakai et al. 1995d, 1996a,b showed that GH also
functions as an immunostimulant and enhances macrophage activities of fish. Rainbow
trout injected with GH exhibited increased resistance against V. anguillarum challenge.
Thus, there may be a close correlation between growth and immunostimulation.
On the other hand, the mere deleterious side effects of immunostimulants have not
been investigated. As already described in this review, overdoses and long-term adminis-
tration of immunostimulants reduce their efficacy. In aquaculture, the maturation and
spawning of fish are very important processes. No research has yet been performed
( )
concerning the influence of immunostimulants at these stages. When fish spawn, the
.
immune systems become suppressed by sex hormones Wang and Belosevic, 1995 .
Although the use of immunostimulants could cause recovery of the immune systems
suppressed by sex hormones, they may disturb sexual maturation and other essential
functions associated with spawning.
10. Conclusions
In this review, the benefits of immunostimulants for aquaculture were discussed. The
characteristics of immunostimulants are shown in Table 7. There are mainly three
methods for control of fish disease: vaccination, chemotheraputics and immunostimu-
lants. Both immunostimulants and vaccines are used to prevent infectious diseases.
However, we cannot expect the marked or long-term effects observed with vaccines to
occur with immunostimulants. Although immunostimulants may be used for treatment of
some infectious diseases, they are not as effective as many chemotheraputics. Immunos-
timulants are, however, not inferior to vaccines and chemotherapeutics. Antibiotic-re-
sistant bacteria can prevail in the aquaculture environment and the treatment of fish
disease using chemotherapeutics has thus become more difficult. Although vaccination
is the most reliable method to control fish diseases, there are, as yet, no effective
vaccines against BKD or most viral infections. Immunostimulants may be able to
compensate for these limitations of chemotherapeutics and vaccines. Immunostimulants
are thought to be safer than chemotheraputics and their range of efficacy is wider than
vaccination. The combination of vaccination and immunostimulant administration may
also increase the potency of vaccines. Thus, with a detailed understanding of the
efficacy and limitations of immunostimulants, they may become powerful tools to
control fish diseases.
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Aquaculture 172 1999 6392

C-reactive protein. The existence of cytokines interferon, interleukin 2, macrophage

influenced by the administration of immunostimulants Engstad et al., 1992; Jrgensen

nescence, and increases protection against Streptococcus infection Yoshida et al.,

increased resistance to V. anguillarum, V. salmonicida and Y. ruckeri Robertsen et al.,

macrophages and the lymphoblastic transformation of lymphocytes in vitro Table

V. anguillarum yeast glucan, lactoferin, HDe, levamisole, chitin, Vitamin C,

immunostimulants levamisole, quarternary ammonium compound, polypeptide of fish

of oral administration. It is well-known that GH promotes the growth of fish Rand-

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