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The use of immunostimulation in marine larviculture: possibilities and challenges
Aquaculture EISEVIER
Aquaculture 155 (1997) 401-417
The use of immunostimulation in marine larviculture: possibilities and challenges Olav Vadstein SINTEF Applied Chemistry
Center of Aquaculture.
* N-7034 Trondheim, Norway
Accepted 20 December I996
Abstract Inadequate microbial conditions are one of the main problems in rearing marine larvae. It is therefore an ultimate goal to develop methods for establishing microbial control at all stages of the cultivation process. In addition to measures improving environmental conditions, methods of improving the resistance of the larvae to bacterial infections need to be developed. One possibility is immunostimulation, which includes methods of enhancing the capacities of the specific and nonspecific immune systems. Although the information is limited, one must conclude that larval fish have a poorly developed immune system and that they primarily rely on nonspecific immune system. For specific defence, larvae have to rely on maternal immunity, which lasts only for a short period. Experiments have shown that maternal immunity may be manipulated by immunisation of the broodstock and that increased resistance to infections may be obtained. Direct immunostimulation of larvae must be aimed at the nonspecific part of the immune system, and several substances are known to have this ability. Experiments on nonspecific immunostimulation of fish suggest that the method has considerable potential for reducing losses in aquaculture, both during larva1 and on-growing stages. Reports on immunostimulation of larvae are, however, very limited. Further development of this method for larviculture will require the establishment of methods for the administration of the stimulant, and the adaptation of methods for detecting the response of the immune system. This last point is a particular challenge due to the small size and fragility of larvae. It is hypothesised that immunostimulation, together with other methods of achieving microbial control, will help to reduce the probability of microbial problems in
* Corresponding author. Norwegian University of Science and Technology, Trondhjem Biological station, Bynesveien 46, N-7018 Trondheim, Norway. Tel.: +(47)-7359 0204. Fax: +(47)-7359 1597. E-mail: [email protected]. 0044~8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO44-8486(97)001 14-2
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155 (19971401-417
larviculture. As a result, increased and more stable survival and growth are anticipated; resulting in the production of high-quality juveniles for the on-growth period. 0 1997 Elsevier Science B.V. Keywords:
Immunostimulation; Immunology; Larval rearing; Defence; Microbiology
1. Introduction Further development of the aquaculture industry requires diversification through the commercialisation of new species. Although considerable progress has been made during the past few years, the production of juveniles is still one of the main bottlenecks for several species. The problem seems to be the same for many species of both fish and crustaceans, with poor reproducibility in terms of survival, growth and quality as the main symptoms. This suggests that there is a lack of control of at least one factor. Nutritional factors and egg quality may be ruled out as the principal cause, because the lack of reproducibility is often manifested in replicate tanks with full sibling groups that are given the same treatment. This does not mean that nutritional factors and egg quality are optimal. Recent evidence and accumulated experience in commercial hatcheries suggest that bacteria normally present in hatcheries may be the principle cause of problems associated with production of juveniles (Vadstein et al., 1993). This is most likely due to opportunistic bacteria, because reported incidences of specific pathogens are low (Munro et al., 1995). In an attempt to overcome the microbial problems, many research laboratories and commercial hatcheries disinfect the water before use and/or apply antibiotics as a standard procedure. Both methods tend to destabilise the balance of bacterial populations (below), and, in the long run they only provide a temporary respite from the problem. The experience has been similar in the cultivation of shellfish and shrimp, with the prophylactic use of antibiotics as a normal procedure (D’ Angustino, 1972; Fitt et al., 1992). With the present increase in the frequency of antibiotic-resistant bacteria (Husev%g et al., 199 1; Andersen and Sandaa, 1994) and the recent discovery of the transfer of antibiotic resistance from the bacterial flora of domestic animals to the human microflora (Nikolich et al., 19941, there is an increasing public concern on the use of antibiotics by the aquaculture industry. The long-term use of antibiotics is also likely to affect the larvae negatively (Wishkovsky et al., 1987). Moreover, an industry that makes prophylactic use of antibiotics cannot be regarded as sustainable. Such use may ruin the health image that has such potential for fish as food. The establishment of a sustainable solution to the microbial problems in larviculture is therefore of utmost importance. In addition to countermeasures to improve environmental conditions, a strategy for microbial control must include measures to improve the resistance of the larvae to infection. Immunostimulation may be such a countermeasure, and include methods that increase the capacity of the specific and/or the nonspecific immune system. The aim of this review is to evaluate the status and the potential for immunostimulation as an element in the strategy for solving microbial problems in larviculture. The focus will be on fish, but the ideas are also applicable to other groups of organisms relevant to aquaculture.
0. Vndstrin/Ayuaculture
2. A strategy for microbial raising viable larvae
control
15.5 (19971
in larviculture:
401-417
increasing
403
the probability
of
The intensive larval rearing process is characterised by the exposure of larvae to considerable stresses of a chemical, physical and biological nature. In addition, some of these stressors occur in an intermittent manner or as pulses, which may amplify their negative effects. High loads of organic matter and bacteria are introduced with the live feed, and high densities of larvae and live feed organisms will due to defecation, result in a heavy internal load of organic matter and bacteria in the tanks. A substantial proportion of the organic matter will often occur in a patchy manner. It is well known from fundamental ecological principles that irregular supplies of organic matter tend to destabilise the bacterial community and select for opportunistic and pathogenic bacteria (e.g., Andrews and Harris, 1986). These conditions, in turn, produce conditions that may be detrimental to the larvae (Vadstein et al., 1993), and this situation is amplified by the fact that stress is a potent suppressor of the immune system of fish (Angelidis et al., 1987; Anderson, 1990). So far, the development of technology for production of juveniles from eggs and larvae has barely considered these factors. Microbial control cannot be regarded as absolute, but is more a question of probabilities. This is evident if one considers the three interacting factors that are required for the development of conditions that ensure good viability of the larvae (Fig. 1). These factors, which include the larvae, the biological and the physio-chemical environment, are in turn influenced by several conditions. Manipulating these conditions may increase the probability of producing viable larvae. It is clear that manipulation of one of the three factors may only have limited effect. Therefore, a strategy to achieve microbial control and improve viability of larvae makes it important to use a range of countermeasures directed towards these different aspects (Fig. 1). Vadstein et al. (1993) proposed a general strategy for microbial control that takes into account both the
A: Factors
determining
viabilitv
CP,,)
B: Manioulation
to increase
viability
lmmunostimulation
Genetics, Nutrition. Immune system
~~;~~~;~t~~lL;~l&.s _ ‘
Fig. I. (a) The three factors of significance for the probability of viable larvae (P,) and conditions that influence these fxtors; (b) the probability of viable larvae (P,) may be increased by using methods that push the circles towards each other. One such method is immunostimulation. Other methods are treated in Vadstein et al. (1993).
404
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/Aquaculture
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Table 1 The three elements in the strategy to achieve microbial control in the rearing of marine fish larvae, as proposed by Vadstein et al. (19931, with examples of possible methods for the different elements in the strategy Element 1. Nonselective reduction of bacteria l Surface disinfection of eggs 0 Reduction in input of organic matter 0 Removal of organic matter 0 Grazer control of bacterial biomass Element 2. Selective enhancement of bacteria 0 Selection for desirable bacteria 0 Addition of selected bacteria to tanks 0 Incorporation of selected bacteria in feed Element 3. Improvement of larval resistance against bacteria 0 Stimulation of general immune system 0 Modulation of maternal immunity
ecological conditions mentioned above and the various aspects described in Fig. 1. The strategy, which does not rely on the prophylactic use of antibiotics, is based on the concurrent use of three different elements (Table 1). Application of this strategy has so far produced good results (Vadstein et al., 1993; Salvesen and Vadstein, 1995; Skjermo et al., 1996, 1997). This review will focus on immunostimulation.
3. The immune
system of fish
The main function of the immune system is to protect the animal against diseasecausing microbes. The immune system of fish shows clear similarities to that of mammals, and several reviews have covered various aspects of the immune system of fish (Ellis, 1977, 1988; Ingram, 1980; Fletcher, 1982; Hart et al., 1988; Alexander and Ingram, 1992; Bly and Clem, 1992; Secombes and Fletcher, 1992; Siwicki et al., 1994). The immune system comprises both nonspecific and specific components, and involves both cellular and humoral factors. This division into four constituents is somewhat misleading because all components are interlinked and mutually dependent. Nonspecific defence mechanisms are part of all normal fish, and do not require prior contact with an antigen/pathogen to elucidate a response. On the other hand, the specific immune system requires activation; thus, there is a time delay between the first introduction to the antigen and the activation. This process is also temperature-dependent (Bly and Clem, 1992). It is believed that nonspecific immunity is phylogenetically older than specific immunity, and one might expect fish to be more reliant on nonspecific defences than higher vertebrates (Anderson, 1992). Such a reliance is expected to be even stronger for invertebrates. It is believed that larvae do not have the ability to develop specific immunity during the early stages of development (Ellis, 1988). In this respect, fish are reliant on passive immunisation from maternal antibodies. Although it has been reported that maternal transfer of specific immunity does not occur in salmonids (Ellis, 1988), this mechanism
0. Vadstein/Ayuaculture Table 2 Days post-hatch (negative numbers organs of four teleost species
are pre-hatch)
155 (19971401-417
when lymphocytes
405
appeared
in the developing
Organ
Salmon (4-7°C)
Rainbow trout (14°C)
Carp (22°C)
Sebasticus mrmoratus
Thymus Blood
-22 - 14
3-5 5-6
5 7-8
21 _
Kidney Spleen
- 14 42
5-6 21
7-8 8-9
30 44
Data from Ellis, 1988 (based on several sources) and Nakanishi,
lymphoid (2°C)
1991
has been shown in tilapias (Mar and Avtalion, 1990; Sin et al., 1994). In any given species, size rather than age seems to be the most critical determinant of the development of specific immunity. In salmonids, memory-dependent specific immunity has been achieved for sizes > 0.26 g (8 weeks of age), whereas in carp it was acquired at 9- 10 weeks of age (Ellis, 1988). The nonspecific immune system is probably the major defence against microorganisms in larvae. Although our understanding of the components of the innate defence system of fish is growing, relatively little is known about the functioning and the ontogeny of the general immune system in marine larvae (Ellis, 1988; Olafsen and Roberts, 1993). In the few fish species that have been studied, the major lymphoid organs are not fully developed at the time of hatching (Table 21, and the phagocytic activity is mainly associated with gills, skin and gut (Ellis, 1988). It is therefore possible that during the stages when the lymphoid organs are developing, the main cellular defence is by the phagocyte populations within the integument (Ellis, 1988). The nonspecific or innate immune system is regarded as the first line of defence in animals. Furthermore, it seems that the microbial problems in larviculture are most likely due to opportunistic bacteria rather than specific pathogens (Vadstein et al., 1993; Munro et al., 199.5). This emphasises the importance of nonspecific defence system for larvae under intensive hatchery conditions, and the need for more knowledge of the ontogeny and functionality of the nonspecific immune system of larvae.
4. Immunostimulation
of fish: definitions,
possibilities
and constraints
Immunotherapy comprises all methods that utilise immunological principles to prevent or treat diseases. In human and veterinary medicine, immunotherapy has already been applied, but it is still regarded as an area for growth (e.g., Hadden, 1993). Methods of transplanting immunologically active cells or transferring immunoglobulins are not relevant to larviculture, and therefore not treated further in this study. Immunomodulation, and in particular immunostimulation, seems to be the most suitable immunotherapeutic method for larviculture in the foreseeable future. Immunomodulation may be directed at both specific and nonspecific immunity. Vaccination is probably the best-known method of specific immunostimulation, and it entails increased resistance against a specific antigen/pathogen. Nonspecific immunos-
406
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timulation refers to a condition in which the immune response is changed to a condition with higher response towards a variety of antigens. An example of nonspecific immunostimulation is macrophage activation. An immunostimulant may be defined as an agent which stimulates the nonspecific immune mechanisms when given alone, or the specific mechanisms when given with an antigen. As mentioned above, eggs and larvae ‘inherit’ an immune defence, but they last for only a short period. A special case of immunostimulation may be to enhance the immune defence of larvae through immunostimulation of the mother (i.e. maternal immunity). Many different types or groups of substances are known to act as immunostimulants (below). The compounds may either be grouped by function or by origin (Anderson, 1992; Hadden, 1993; Secombes, 1994). Some immunostimulants, such as lipopolysaccharide (LPS) from Gram negative bacteria, may act by triggering a number of mechanisms; others (e.g., mannuronic acid polymers) may have a more restricted stimulatory effect (Espevik et al., 1993). It is important to consider the specificity of immunostimulants for two reasons. First, a stimulation of the immune system may be too intense, and may harm or even kill the host. This is well known in humans, where the activation caused by LPS in connection with infections may cause septic shock and death (Morrison et al., 1994). Second, the knowledge of the functions of different immunostimulators may be used to stimulate, more specifically, those parts of the immune system that may be more relevant in certain situations. Three factors are essential to consider in the design of an immunostimulation strategy. First, it is important to remember that in most cases we do not have a specific microbial problem (i.e. specific pathogens), but rather a general one involving large numbers of bacteria and a high proportion of opportunistic species. Secondly, the immune system of larvae is poorly developed, consisting mainly of nonspecific defence. Thirdly, the immune defences of maternal origin will be significant only during the earliest developmental stages. Although this period and hence the maternal immune defence, may be critical, aquaculturists cannot rely on this part of the immune defence of the larvae alone. These three factors mean that research aimed to develop methods for immunostimulation of larvae, should place the highest priority on stimulation of the nonspecific defence system. This work should principally involve stimulation of the nonspecific defence of the larvae itself, and should also include stimulation of nonspecific maternal defences if possible. In cases where specific pathogens are known to cause problems, stimulation of the specific defence may be considered through immunization of the broodstock.
5. Immunostimulation 5. I. Maternal
of fish: the state-of-the-art
immunity
Our understanding of the stimulation of maternal defence is very limited. In fact, this author is not aware of any attempts to stimulate the nonspecific defence of larvae by immunostimulation of the mother. In an attempt to increase specific antibodies in eggs and larvae of tilapias (Oreochromis aureus), Mor and Avtalion (1990) immunised
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407
female broodfish with three different proteins. A considerable increase in antibody activity was detected, with a maximum increase of IO-13 in log, units in embryo homogenates hatched 15-35 days post-immunisation. The optimum period between immunisation and hatching seemed to be dependent on the type of protein, and the antibody activity found was specifically against the protein used to immunise the mother. Sin et al. (19941, working with the same species, vaccinated broodfish with live tomites of the ciliated protozoa Ichthyophthirius multifiliis one month before spawning. Depending on whether or not the fish were allowed parental care of the larvae, a relative percent protection (RPP = 100% [ 1-(mortality in experimental group/mortality in control group)]) of 93% and 78% was obtained after challenge of fry with infective tomites. The protective immunity in the four groups (with and without vaccination/parental care) was correlated with titres of I. multijiliis antibodies in the fry and in the plasma of the fish. So far, no attempt has been made to evaluate the impact of using an immunostimulant (adjuvant) in connection with stimulation of specific maternal immunity. Use of adjuvant is a standard procedure to increase the response in normal vaccination. It is reasonable to believe that vaccination or secondary stimulation of mothers with appropriate vaccines before the spawning season could protect the larvae against diseases on the first days after hatching and until their own specific immune system is fully developed. 5.2. Direct stimulation Considerable data exist on stimulation of the nonspecific defence systems of fish, especially when we consider that the first studies appeared only 10 years ago. Direct stimulation of specific defence is not treated here because it is not considered to be relevant for larval stages. An attempt to summarise the available knowledge on stimulation of nonspecific immunity of fish is presented in Table 3. Although most studies were on adult fish, a high diversity in the studies is evident with respect to immunostimulants tested, species studied, mixture of in vivo and in vitro studies, and different response parameters used. The immunostimulants studied include crude and purified compounds of bacterial origin, various polysaccharides and synthetic chemicals with a range of freshwater, seawater and anadromous species. Response parameters include both humoral and cellular mechanisms, as well as challenges to pathogens. The positive effect of nonspecific immunostimulation, therefore, has been documented on several levels of the organisation. In addition to the wide diversity of the studies listed in Table 3, two other aspects are important. First, successful immunostimulation has been obtained by injection, bath and oral administration. If nonspecific immunostimulation could have practical use in larviculture, it is vital to be able to stimulate individuals without injection. Although it has been shown that injections with glucan and chitin gave a better resistance to Aerornonas salmonicida infection than administration by immersion (Anderson and Siwicki, 1994), too few conditions were tested to be able to draw any firm conclusions regarding the possible advantages of administration by injection. It is not known if immersion and oral administration entail complete stimulation of the nonspecific de-
of studies where immunostimulants
complete adjuuant
dipeptides
mykiss)
carpio)
Chanel catfish (Ictnlurus
Carp (Cyprinus
Glucans
mykiss)
punctatus)
Rainbow trout (Oncorhynchus Polysaccharides:
Peptidoglygan
Atlantic salmon (Salmo s&r)
Red sea bream (Pagrus major) Place (Plruronectes platessa) Channel catfish ( Ictalurus puncrams)
Lipopolysaccharide
Rainbow trout (Oncor/qnchus
Muramyl
Rainbow trout (Oncorhyrzchus
mykiss)
kisutch)
Brook trout (Salr’elinus~~ntinalis)
Coho salmon (Oncorhynchus
Freund’s
Bacteria and bacterial products:
Immunostimulant/species
Table 3 Summary
salmonicida,
A. hydrojila,
Vibrio ordalii
macrophage cultures
Increased phagocytic activity of kidney leucocytes, increased resistance to Edwnrdsiella torda. Stimulation of macrophages.
injected
Chen and Ainsworth,
1993
1992
Yano et al., 1989, 1991
Matsuo and Miyazono,
Solem et al., 1995
Increased respiratory burst of macrophages, stimulation of phagocytic activity and ability to kill bacteria Increased resistance to V. anguillarum.
Salati et al., 1987 MacArthur et al., 1985 Clem et al., 1985
Kodama et al.. 1993
Kajita et al., 1992
Oliver et al., 1986
Oliver et al., 1985
Reference
Enhanced phagocytic activity of blood leucocytes Enhanced migratory activity of macrophages Release of interleukine-1 -like molecule
Activation of phagocytic, respiratory burst and migratory activity of macrophages, increased disease resistance
Increased capacity for phagocytosis and killing of virulent A. salmonicidu Increased resistance to V. anguillurum, activation of leucocytes
Aeromonas
Increased resistance to
effects
oral
injected injected monocyte cultures macrophage cultures
injected
macrophage cultures injected
injected
Documented
were used to augment the general immune response of fish Administration
~alur)
Atlantic salmon (Salvo
Atlantic salmon (Sulmo salarl
Aminured polyglucose
Rainbow trout (Oncorhynchus
mykiss)
Brook trout (Sah~elinus fonrmalis)
Churn
maximus)
mykiss)
Turbot (Scophthalmus
hippoglossus)
Hugh-M or poly-M alginates Atlantic halibut (Hippoglossus
Brook trout (Sall,elinusfantinalis)
Rainbow trout (Oncorhynckus
injected
Atlantic salmon (Salma s&r)
macrophage cultures
injected and bath injected
macrophage cultures
orally
immersion
injected and harh
injected injected
macrophage cultures orally
injected
injected
Yellowtail (Seriola quinqueradiatn)
burst
Increased superoxide anion formatton, acid phosphatase activity and pmocytic activity
Increased resistance to A. salmonicida lasting l-2 weeks. Higher protection with injection than with bath Increased phagocytic and respiratory burst activity of leucocytes, increased resistance to V. anguillarum.
Increased survival of larvae during the one-month-long yolk sac period (cf. Fig. 3) Increased resistance to V. anguillurum. Administration via live feed (cf. Fig. 4) Increased phagocytic activity and respiratory
Increased activity of lysozyme and complement Increased ability of macrophages to kill A. salmonicida, increased respiratory burst of macrophages, increased serum lysozyme activity Increased resistance to A. salmonicida lasting 1-2 weeks. Higher protection with injection than with bath.
11992
and Robertsen,
1995
Sveinbjarnson
and Seljelid. 1994
Sakai et al., 1992
Anderson and Siwicki, 1994
Rokstad et al. unpubl.
Skjermo et al., 1996
Vadstein et al., 1993
Anderson and Siwicki, 1994
Engslad et al., 1992 Jetrgensen et al., 1993b
Raa et al
Increased resistance to V. angurllarum
and
Jergensen
V. ~olmonrctda.
et al., 1990
et al., 1992
Jprgensen et al., l993a
Robensen
Matsuyama
Accumulation of macrophages. neutrophils and trombocytes in peritoneal cavtty, increased ability of head kidney macrophages to kill Aeromonus salmonicido. Increased respiratory burst
and Yer.rrnia ruckrrr.
phagocytic activity of kidney leucocytes, levels of serum complement and lysozyme, resistance to Strep~~~cus sp. resistance to V anguillanrm,
V. .ralmonicida
Increased enhanced increased Increased
mykiss)
mykiss)
FK-565 Rainbow trout (Oncorhwchus
cnrpio)
Rainbow trout (Oncorhynchus
Carp (Cyprinus
Lrr~umisolr
Synthetic chemicals:
Immunostimulant/species
Table 3 (continued)
blood and spleen leucocyte cultures
injected
spleen cells in vitro
injected
oral
oral
immersion
injected
Administration
effects
Increased bactericidal and phagocytic activity of peritoneal and kidney phagocytes, resistance to A. sulmonicidu. Removal of suppressive effect of cyclophosphamide or hydrocortisone on phagocytic activity Increased respiratory burst and spreading
Increased blood neutrophil phagocytic, myeloperoxidase and migratory activity Increased growth of larvae at various concentrations (cf. Fig. 4) Enhancement of various nonspecific defence mechanisms after an immune suppression induced by trichlorfon Increased phagocytosis and production of oxygen radicals by blood neutrophila Increased phagocytic activity and chemolumineacence of kidney leucocytes, natural killer cell activity, serum complement, bactericidal activity, resistance to V. anguillnrum. Increased phagocytosis and production of oxygen radicals
Documented
1987
1989
1986
1991
Kitao and Yoshida,
1994
Kitao and Yoshida,
Siwicki et al., 1990
Kajita et al., 1990
Siwicki,
Siwicki and KorwinKossakowski, 1988 Siwicki and Studnicka,
Siwicki,
Reference
0.
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411
fence or stimulation of only the mucosal immune system. Intestinal uptake and organ redistribution of high-molecular-weight polysaccharides (glucan and poly-glucose) after oral administration have been documented (Dalmo et al., 1994; Sveinbjomsson et al., 1995). It is therefore possible that immersion and oral administration may result in complete stimulation, but with certain limitations depending on the immunostimulant used and species. Secondly, in addition to the positive effects as judged by immune parameters and challenge tests, immunostimulation has also been shown to abolish the negative effects of immune suppression. When rainbow trout were injected with the immunostimulator FK-565, the fish were able to overcome the suppressive effects of cyclophosphamide and hydrocortisone treatment (Kitao and Yoshida, 1986). Oral stimulation with levamisole has been shown to enhance the nonspecific defence of carp after immune suppression caused by trichlorfon (Siwicki and Studnicka, 1994). Most of the experiments presented in Table 3 are simple in design, and little is known about dose-response relationships and how long a stimulation may last. Both these aspects are important for the utilisation of immunostimulation in larval rearing. The study by Anderson and Siwicki (1994) showed that protection was reduced 2-3 weeks after stimulation, which may be regarded as long enough to be relevant in larviculture. Stimulation extended over at excessively long periods may have negative effects on the host, and this has been documented in a feeding trial (Onarheim, 1992). Only two of the studies in Table 3 specifically deal with larvae. In one (Vadstein et al., 1993) yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) were stimulated by immersion in an alginate rich in mannuronic acid. The stimulation lasted for the whole yolk sac period (28 days) and a considerable improvement in survival was obtained (Fig. 2), with a RPP of 47%. The second study on immunostimulation of larvae was with carp larvae using the synthetic immunostimulant levamisole (Siwicki and Korwin-Kossakowski, 1988). Four different concentrations were tested and the larvae were treated twice (days 1 and 3 after hatching) by immersion. After two weeks the effect of the stimulation was evaluated by growth measurements. For the three lowest concentrations tested, a significant increase in the size of the larvae was recorded (Fig.
70 60 3 2 P 2
40
50
g
30
g
20 10
Control
Stimulated
Fig. 2. The effect of immunostimulation of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) with an alginate rich in mannuronic acid on survival (mean i SEMI on day 28 after hatching. Larvae were stimulated by incubation in water with added immunostimulant at final concentrations of 25- 100 p,g I-’ just after hatching. Based on data from Vadstein et al. (1993).
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Concentration
155 (1997) 401-417
(mg dire-‘1
Fig. 3. Effect of stimulation with levamisole by immersion on day one and day three after hatching two-week-old carp larvae. Based on data from Siwicki and Korwin-Kossakowski (1988).
on size of
3). The survival was similar in all groups and above 93%. It is important to note that within the one order of magnitude variation in concentrations tested, no negative effects were recorded. A third study is relevant to larviculture because it included the use of Artemia as a carrier of the immunostimulant (Skjermo et al., 1996), an approach that is applicable to first-feeding of larvae. The immunostimulant (an alginate rich in mannuronic acid) was immobilised in beads made of a gel-forming alginate, and of a size appropriate for ingestion by Artemia. The beads were given to Artemia, which were fed in turn to 40-day-old turbot (Scophthalmus maximus) in several meals over 24 h. Two days later, the turbot were challenged by a pathogenic strain of Vibrio anguillarum. RPPs of 48% and 39% were obtained in two independent experiments (Fig. 4). The authors stated that there was no reason to believe that the conditions for stimulation were optimal during the experiments.
40 x g cd ‘;, 30 E F a, 20 $ a 10 0
1 Experiment
Fig. 4. Mortality (average+ SEM) in control groups and immunostimulated groups of juvenile turbot (Scophfhalmus maximus) after challenge with Vibrio unguillarum in two different experiments. The immunostimulant (an alginate rich in mannuronic acid) was immobilized in beads made of a gel-forming alginate and of a size appropriate for ingestion by Artemia. The beads were given to Arkmia, which again were given as food for 40.day-old turbot in several meals over 24 h. Two days later, the turbot was challenged by a pathogenic strain of Vibrio anguillarum. Based on data from Skjermo et al. (1996).
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(1997)
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It is concluded that although still in its infancy, immunostimulation of fish shows considerable potential as a method for reducing losses in aquaculture. This includes both the larval and on-growing stages. Both stimulation of nonspecific defence mechanisms and stimulation of specific immunity of maternal origin have been documented. Experience with larvae is, however, still very limited.
6. Immunostimulation
in larviculture:
future challenges
As the review above demonstrates, more knowledge is needed before a strategy for specific and nonspecific immunostimulation can be developed for larviculture. The few studies on stimulation of specific maternal immunity and the more numerous studies of stimulation of nonspecific defence of fish suggest that there is a good possibility of developing efficient methods for immunostimulation in larviculture in the near future. A strategic decision that has strong implications is determining what types of immunostimulants the research should focus on. Attention should be focused on a limited number of immunostimulants in order to obtain maximum information from the research. Two criteria might serve as guidelines in the selection of suitable immunostimulants. Firstly, several substances with documented immunostimulatory effects are unlikely to be approved for use, either because they are poorly defined, or because of their source and adverse effects. Secondly, considerable research on immunostimulation is being carried out in veterinary and human medicine. Research in fish biology and aquaculture is unlikely to have access to funds comparable to those available to human medicine; therefore research should focus on the immunostimulators that have already been intensively studied in other areas. The knowledge and the spinoff from these other areas represents a considerable resource for aquaculture research. For stimulation of maternal immunity, it is important to clarify whether it is possible to stimulate nonspecific immunity in a way that is relevant to larviculture. Due to the shorter duration of such a stimulation, nonspecific stimulation through the maternal pathway may not be feasible, but a clarification of this point is necessary before any final conclusion can be drawn. Stimulation of specific immunity is the most promising method for applying maternal stimulation. As a general method, it is not suitable, but in the cases where specific problem organisms have been identified, it should be possible to evaluate the potential of the method. Such an evaluation should include optimisation of immunisation procedures, and evaluations in both challenge experiments and under production conditions. It is important that evaluations are done under both sets of conditions, because even if stimulation of maternal immunity does not have significant positive effects in a challenge test, in which the problem organism occurs at high densities, it may do so under production conditions. The method is not restricted to bacteria (Sin et al., 1994). The challenges related to nonspecific immunostimulation of the larva itself are more diverse than those related to maternal immunity. Reliable larval experiments are expensive, due to the complexity and the poor reproducibility of such experiments. More information is required on the developmental stage at which immunostimulation is possible, and for this, additional evaluation criteria other than challenge tests will be
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needed. The research on the ontogeny of the nonspecific immune system should focus not only on the development and the functioning of relevant organs, but should also include studies of the immune system in the integument/mucosa. The development of methods of assessing stimulation is a special challenge, due to the small size of larvae. Priority should be given to the establishment of methods for determining parameters that occur early in the cascade of reactions triggered by immunostimulants. The research directly related to the establishment of an immunostimulation strategy should include evaluation of administration procedures, dose-response relationships, and evaluation of the duration of a stimulation.
7. Conclusion Immunostimulation stimulation origin seem to be the most promising knowledge, it is concluded timulation both larval needed
if immunostimulation
Direct Based on available still in its infancy, immunoslosses in aquaculture, during
would
Acknowledgements The ideas presented in this paper evolved while working on several projects financed by the Research Council of Norway and two industrial partners (Fina Exploration Norway and Norsk Hydro). The scientific atmosphere provided by my colleagues working with larviculture is highly appreciated, and the scientists at the Norwegian Biopolymer Laboratory at the University of Science and Technology - Trondheim for introducing me to the fascination of immunostimulation. Kjell Inge Reitan and Yngvar Olsen provided helpful comments on an earlier version of the manuscript. Linguistic help was provided by Allen Market. This study forms part of a project financed by the Research Council of Norway (contract 1 lOSSO/ 120).
References Alexander, J.B., Ingram, G.A., 1992. Noncellular nonspecific defence mechanisms of fish. Ann. Rev. Fish Dis. 2, 249-279. Andersen, S.R., Sandaa, R.-A., 1994. Distribution of tetracycline resistance determinants among Gram-negative bacteria isolated from polluted and unpolluted marine sediments. Appl. Environ. Microbial. 60, 908-912. Anderson, D.P., 1990. Immunological indicators: effects of environmental stress on immune protection and disease outbreaks. Am. Fish. Sot. Symp. 8, 38-50.
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Anderson, D.P., 1992. Immunostimulants, adjuvants, and vaccine carriers in fish: applications to aquaculture. Ann. Rev. Fish Dis. 2, 281-307. Anderson, D.P., Siwicki, A.K., 1994. Duration of protection against Aeromonas salmonicida in brook trout immunostimulated with glucan or chitosan by injection or immersion. The Progressive Fish Culturist 56, 258-26 1. Andrew% J.H., Harris, R.F., 1986. r- and K-selection in microbial ecology. Adv. Microb. Ecol. 9, 99-147. Angelidis, P., Baudin-Laurencin, F., Youinou, P., 1987. Stress in rainbow trout, Salmo gairdneri: effects upon phagocyte chemoluminescence, circulating leucocytes and susceptibility to Aeromonas salmonicidu. J. Fish Biol. 31 (Suppl. A), 113-122. Bly, J.E., Clem, L.W., 1992. Temperature and teleost immune functions. Fish and Shellfish Immunol. 2, 159-171. Chen, D., Ainsworth, A.J., 1992. Glucan administration potentiates immune defence mechanisms of channel catfish, Ictulurus punctutus Rafinesque. J. of Fish Diseases 15, 295-304. Clem, L.W., Siaemore, R.C., Ellsaesser, C.F., Miller, N.W., 1985. Monocytes as accessory cells in fish immune responses. Dev. Comp. Immunol. 9, 803-809. D’Angustino, A., 1972. Antibiotics in cultures of evertebrates. In: Smith, W.L., Chanley, M.H. (Eds.), Culture of marine evertebrate animals. Plenum, pp. 109-133. Dalmo, R.A., Ingebrigtsen, K., Horsberg, T.E., Seljelid, R., 1994. Intestinal absorption of immunomodulatory laminaran and derivatives in Atlantic salmon, Salmo s&r L. J. Fish Dis. 17, 579-589. Ellis, A.E., 1977. The leucocytes of fish: a review. J. Fish Biol. 11, 453-491. Ellis, A.E., 1988. Ontogeny of the immune system in teleost fish. In: Ellis, A.E. (Ed.), General principles of fish vaccination. Academic Press, pp. 20-3 1. Engstad, R.E., Robertsen, B., Frivold, E., 1992. Yeast glucan induces increase in lysozyme and complementmediated haemolytic activity in Atlantic salmon blood. Fish and Shellfish Immunol. 2, 287-297. Espevik, T., Otterlei, M., Skjak-Brmk, G., Ryan, L., Wright, S.D., Sundan, A., 1993. The involvement of CD14 in stimulation of cytokine production by uranic acids polymers. Eur. J. Immunol. 23, 255-261. Fitt, W.K., Heslinga, GA., Watson, T.C., 1992. Use of antibiotics in the mariculture of giant clams (F. triducnidae). Aquaculture 104, l-10. Fletcher, T.C., 1982. Nonspecific defence mechanisms of fish. Dev. Comp. Immunol. (Suppl. 21, 123-132. Hadden, J.W., 1993. Immunostimulants. Immunol. Today 14, 275-280. Hart. S., Wrathmell, A.B., Harris, J.E., Grayson, T.H., 1988. Gut immunology in fish: a review. Dev. Comp. Immunol. 12, 453-480. Husevag, B.. Lunestad, B.T., Johannessen, P., Enger, 0.. Samuelsen, O., 1991. Simultaneous occurrence of Vibrio salmonicidu and antibiotica resistant bacteria in sediments at abandoned aquaculture sites. J. Fish Dis. 14, 63 t-640. Ingram, G.A.. 1980. Substances involved in the natural resistance of fish to infection - a review. J. Fish Biol. 16, 23-60. Jorgensen, J.B., Lunde, H., Robertsen, B., 1993a. Peritoneal and head kidney cell response to intraperitoneally injected yeast glucan in Atlantic salmon, Salmo s&r L. J. Fish Dis. 16, 3 13-325. Jorgensen, J.B., Robertsen, B., 1995. Yeast P-glucan stimulates respiratory burst activity of Atlantic salmon (Salmu s&r L.) macrophages. Developmental Comp. Immunol. 19, 43-57. Jorgensen, J.B., Sharp, G.J.E., Secombes, C.J., Robertsen, B., 1993b. Effect of a yeast-cell-wall glucan on the bactericidal activity of rainbow trout macrophages. Fish and Shellfish Immunol. 3, 267-277. Kajita, Y., Sakai, M., Atsuta, S., Kobayashi, M., 1990. The immunomodulatory effects of levamisole on rainbow trout Oncorhyzchus mykiss. Fish Pathol. 25, 93-98. Kajita, Y., Sakai, M.. Atsuta, S., Kobayashi, M., 1992. Immunopotentiation activity of Freund’s complete adjuvant in rainbow trout Oncorhychus mykiss. Nippon Suisan Gakkaishi 58, 433-437. Kitao, T., Yoshida, Y., 1986. Effect of an immunopotentiator on Arromonas salmonicida infection in rainbow trout (Salmom gairdneri). Vet. Immunol. Immunopathol. 12, 287-296. Kitao, T., Yoshida, T., 1991. Immunostimulation of immunoactive peptide to fish leucocytes. Dev. Comp. Immunol. 15 (Suppl. 11, 53. Kodama, H., Hirota, Y., Mukamoto, M., Baba, T., Azuma, I., 1993. Activation of rainbow trout (Onrorhwchus mykiss) phagocytes by muramyl dipeptide. Dev. Comp. Immunol. 17, 129-140.
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J.I., Thomson, A.W., Fletcher, T.C., 1985. Aspects of leucocyte migration in the plaice, platessa L. J. of Fish Biol. 21, 661-616. Matsuo, K., Miyazono, I., 1993. The influence of long-term administration of peptidoglycan on disease resistance and growth of juvenile rainbow trout. Nippon Suisan Gakkaishi 59, 1377- 1379. Matsuyama, H., Mangindaan, R.E.P., Yano, T., 1992. Protective effect of schizophyllan and scleroglucan against Streptococcus sp. infection in yellowtail (Seriola quinqueradiata). Aquaculture 101, 197-203. Mor, A., Avtalion, R.R., 1990. Transfer of antibody from immunized mother to embryo in tilapias. J. Fish Biol. 37, 249-255. Morrison, D.C., Dinarello, C.A., Munford, R.S., Natanson, C., Danner, R., Pollack, M., Spitzer, J.J., Ulevitch, R.J., Vogel, S.N., McSweegan, E., 1994. Current status of bacterial endotoxins. ASM News 60, 479-484. Munro, P., Barbour, A., Birkbeck, T.H., 1995. Comparison of the growth and survival of larval turbot in the absence of culturable bacteria with those in the presence of Vibrio anguillarum, Vibrio alginolyticus or a marine Aeromonas sp. Appl. Environ. Microbial. 61, 4425-4428. Nakanishi, T.. 1991. Ontogeny of the immune system in Sebasticus marmoratus: histogenesis of the lymphoid organs and effects of thymectomy. Environ. Biol. Fishes 30, 135-145. Nikolich, M.P., Hong, G., Shoemaker, N.B., Salyers, A.A., 1994. Evidence for natural horizontal transfer of tetQ between bacteria that normally colonize humans and bacteria that normally colonize livestock. Appl. Environ. Microbial. 60, 3255-3260. Olafsen, J.A., Roberts, R.J., 1993. Salmon disease. In: Heen, K., Monahan, R.L., Utter, F. (Eds.), Salmon Aquaculture. Fishing News Books, Oxford, pp. 166-186. Oliver, Cl., Evelyn, T.P.T., Laillier, R., 1985. Immunity to Aeromonas salmonicida in coho salmon (Oncorhynchus kisutch) induced by modified Freund’s complete adjuvant: its nonspecific nature and the probable role of macrophages in the phenomenon. Dev. Comp. Immunol. 9, 419-432. Oliver, G., Eaton, C.A., Campbell, N., 1986. Interaction between Aeromonas salmonicida and peritoneal macrophages of brook trout (Saluelinus fontinalis). Vet. Immunol. Immunopathol. 12, 223-234. Onarheim, A.M., 1992. Now a yeast extract to fortify fish. Fish Farmer, May 1992, p. 45. Raa, J., Rorstad, G., Engstad, R., Robertsen, B., 1992. The use of immunostimulants to increase resistance of aquatic organisms to microbial infections. Diseases of Asian Aquaculture 1, 39-50. Robertsen, B., Rorstad, G., Engstad, R., Raa, J., 1990. Enhancement of nonspecific disease resistance in Atlantic salmon, Salmo salar L., by a glucan from Saccharomyces cerecisiae cell walls. J. of Fish Diseases 13, 391-400. Sakai, M., Kamiya, H., Ishii, S., Atsuta, S., Kobayashi, M., 1992. The immunostimulating effects of chitin in rainbow trout, Oncorhynchus mykiss. Diseases in Asian Aquaculture 1, 413-417. Salati, F., Hamaguchi, M., Kusuda, R., 1987. Immune response of red sea bream to Edwardsiella tarda antigens. Fish Pathol. 22, 93-98. Salvesen, I., Vadstein, 0.. 1995. Surface disinfection of eggs from marine fish. Evaluation of four chemicals. Aquaculture International 3, 155-171. Secombes, C.J., 1994. Enhancement of fish phagocyte activity. Fish and Shellfish Immunol. 4, 421-436. Secombes, C.J., Fletcher, T.C., 1992. The role of phagocytes in the protective mechanisms of fish. Ann. Rev. of Fish Dis. 2, 53-71. Sin, Y.M., Ling, K.H., Lam, T.J., 1994. Passive transfer of protective immunity against ichthyophthiriasis from vaccinated mother to fry in tilapias, Oreochromis aureus. Aquaculture 120, 229-237. Siwicki, A., 1987. Immunomodulatory activity of levamisole in carp spawners, Cyprinus carpio L. J. of Fish Biol. 3 IA, 245-246. Siwicki, A.K., 1989. Immunostimulating influence of levamisole on nonspecific immunity in carp (Cyprinus carpio). Dev. Comp. Immunol. 13, 87-91. Siwicki, A.K., Anderson, D.P., Dixon, O.W., 1990. In vitro immunostimulation of rainbow trout (Oncorhynchus mykiss) spleen cells with levamisole. Dev. Comp. Immunol. 13, 87-91. Siwicki, A.K., Anderson, D.P., Studnicka, M., 1994. The immune system of fish. Archives of Polish Fisheries 2, 61-79. Siwicki, A.K., Korwin-Kossakowski, M., 1988. The influence of levamisole on the growth of carp (Cyprinus carpio L.1 larvae. J. Appl. Ichthyol. 4, 178-181. Pleuronectes
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Siwicki, A.K., Studnicka, M., 1994. Stimulation of nonspecific immunity after immunosuppression induced by chemical stress in carp (Cyprinus carpio). In: Mlller, R., Lloyd, R. (Eds.), Sublethal and chronic effects of pollutants on freshwater fish. Fishing News Books, Oxford, pp. 148-152. Skjermo, J., Defoort, T., Dehasque, M., Espevik, T., Olsen, Y., Skjik-Brrek, Cl., Sorgeloos, P., Vadstein, O., 1996. Immunostimulation of juvenile turbot (Scophthalmus maximus L.) with high mannuronic acid (M) alginate administered through the live food organism Artemia. Fish and Shellfish Immunol. 5, 53 l-534. Skjermo, J., Salvesen, I., 0ie, G., Olsen, Y., Vadstein, O., 1997. Microbially maturated water: a technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae. Aquaculture International 5, 13-28. Solem, S.T., Jorgensen, J.B., Robertsen, B., 1995. Stimulation of respiratory burst and phagocytic-activity in Atlantic salmon (S&no s&r L.1 macrophages by lipopolysaccharide. Fish and Shellfish Immunol. 5, 475-49 1. Sveinbjornson, B., Seljelid, R., 1994. Aminated p-1, 3-ppolyglucose activates salmon pronephros macrophages in vitro. Vet. Immunol. Immunopathol. 41, 113-123. Sveinbjornsson, B., Smedsrod, B., Berg, T., Seljelid, R., 1995. Intestinal uptake and organ distribution of immunomodulatory aminated p-1, 3-o-polyglucose in Atlantic salmon (S&no s&r L.). Fish and Shellfish Immunol. 5, 39-50. Vadstein, O., @ie, G., Olsen, Y., Salvesen, I., Skjermo J., Skjlk-Brsek, G., 1993. A strategy to obtain microbial control during larval development of marine fish. In: Reinertsen, H., Dahle, L.A., Jorgensen, L., Tvinnereim, K. (Eds.), Fish Farming Technology. A.A. Balkema Publishers, pp. 69-75. Wishkovsky, A., Robertson, B.S., Hetrick, F.M., 1987. In vitro suppression of the phagocytic response of fish macrophages by tetracyclines. J. Fish Biol. 3 I (Suppl. A), 61-65. Yano, T., Mangindaan, E.P., Matsuyama, H., 1989. Enhancement of the resistance of carp Cyprinus carpio to experimental Edwardsiella tat-da infection, by some P-l$glucans. Nippon Suisan Gakkaishi 55, 18151819. Yano, T., Matsuyama, H., Mangindaan, R.E.P., 1991. Polysaccharide-induced protection of carp, Cyprinus carpio L., against bacterial infection. J. Fish Dis. 14, 577-582.
Aquaculture 155 (1997) 401-417
The use of immunostimulation in marine larviculture: possibilities and challenges Olav Vadstein SINTEF Applied Chemistry
Center of Aquaculture.
* N-7034 Trondheim, Norway
Accepted 20 December I996
Abstract Inadequate microbial conditions are one of the main problems in rearing marine larvae. It is therefore an ultimate goal to develop methods for establishing microbial control at all stages of the cultivation process. In addition to measures improving environmental conditions, methods of improving the resistance of the larvae to bacterial infections need to be developed. One possibility is immunostimulation, which includes methods of enhancing the capacities of the specific and nonspecific immune systems. Although the information is limited, one must conclude that larval fish have a poorly developed immune system and that they primarily rely on nonspecific immune system. For specific defence, larvae have to rely on maternal immunity, which lasts only for a short period. Experiments have shown that maternal immunity may be manipulated by immunisation of the broodstock and that increased resistance to infections may be obtained. Direct immunostimulation of larvae must be aimed at the nonspecific part of the immune system, and several substances are known to have this ability. Experiments on nonspecific immunostimulation of fish suggest that the method has considerable potential for reducing losses in aquaculture, both during larva1 and on-growing stages. Reports on immunostimulation of larvae are, however, very limited. Further development of this method for larviculture will require the establishment of methods for the administration of the stimulant, and the adaptation of methods for detecting the response of the immune system. This last point is a particular challenge due to the small size and fragility of larvae. It is hypothesised that immunostimulation, together with other methods of achieving microbial control, will help to reduce the probability of microbial problems in
* Corresponding author. Norwegian University of Science and Technology, Trondhjem Biological station, Bynesveien 46, N-7018 Trondheim, Norway. Tel.: +(47)-7359 0204. Fax: +(47)-7359 1597. E-mail: [email protected]. 0044~8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SOO44-8486(97)001 14-2
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larviculture. As a result, increased and more stable survival and growth are anticipated; resulting in the production of high-quality juveniles for the on-growth period. 0 1997 Elsevier Science B.V. Keywords:
Immunostimulation; Immunology; Larval rearing; Defence; Microbiology
1. Introduction Further development of the aquaculture industry requires diversification through the commercialisation of new species. Although considerable progress has been made during the past few years, the production of juveniles is still one of the main bottlenecks for several species. The problem seems to be the same for many species of both fish and crustaceans, with poor reproducibility in terms of survival, growth and quality as the main symptoms. This suggests that there is a lack of control of at least one factor. Nutritional factors and egg quality may be ruled out as the principal cause, because the lack of reproducibility is often manifested in replicate tanks with full sibling groups that are given the same treatment. This does not mean that nutritional factors and egg quality are optimal. Recent evidence and accumulated experience in commercial hatcheries suggest that bacteria normally present in hatcheries may be the principle cause of problems associated with production of juveniles (Vadstein et al., 1993). This is most likely due to opportunistic bacteria, because reported incidences of specific pathogens are low (Munro et al., 1995). In an attempt to overcome the microbial problems, many research laboratories and commercial hatcheries disinfect the water before use and/or apply antibiotics as a standard procedure. Both methods tend to destabilise the balance of bacterial populations (below), and, in the long run they only provide a temporary respite from the problem. The experience has been similar in the cultivation of shellfish and shrimp, with the prophylactic use of antibiotics as a normal procedure (D’ Angustino, 1972; Fitt et al., 1992). With the present increase in the frequency of antibiotic-resistant bacteria (Husev%g et al., 199 1; Andersen and Sandaa, 1994) and the recent discovery of the transfer of antibiotic resistance from the bacterial flora of domestic animals to the human microflora (Nikolich et al., 19941, there is an increasing public concern on the use of antibiotics by the aquaculture industry. The long-term use of antibiotics is also likely to affect the larvae negatively (Wishkovsky et al., 1987). Moreover, an industry that makes prophylactic use of antibiotics cannot be regarded as sustainable. Such use may ruin the health image that has such potential for fish as food. The establishment of a sustainable solution to the microbial problems in larviculture is therefore of utmost importance. In addition to countermeasures to improve environmental conditions, a strategy for microbial control must include measures to improve the resistance of the larvae to infection. Immunostimulation may be such a countermeasure, and include methods that increase the capacity of the specific and/or the nonspecific immune system. The aim of this review is to evaluate the status and the potential for immunostimulation as an element in the strategy for solving microbial problems in larviculture. The focus will be on fish, but the ideas are also applicable to other groups of organisms relevant to aquaculture.
0. Vndstrin/Ayuaculture
2. A strategy for microbial raising viable larvae
control
15.5 (19971
in larviculture:
401-417
increasing
403
the probability
of
The intensive larval rearing process is characterised by the exposure of larvae to considerable stresses of a chemical, physical and biological nature. In addition, some of these stressors occur in an intermittent manner or as pulses, which may amplify their negative effects. High loads of organic matter and bacteria are introduced with the live feed, and high densities of larvae and live feed organisms will due to defecation, result in a heavy internal load of organic matter and bacteria in the tanks. A substantial proportion of the organic matter will often occur in a patchy manner. It is well known from fundamental ecological principles that irregular supplies of organic matter tend to destabilise the bacterial community and select for opportunistic and pathogenic bacteria (e.g., Andrews and Harris, 1986). These conditions, in turn, produce conditions that may be detrimental to the larvae (Vadstein et al., 1993), and this situation is amplified by the fact that stress is a potent suppressor of the immune system of fish (Angelidis et al., 1987; Anderson, 1990). So far, the development of technology for production of juveniles from eggs and larvae has barely considered these factors. Microbial control cannot be regarded as absolute, but is more a question of probabilities. This is evident if one considers the three interacting factors that are required for the development of conditions that ensure good viability of the larvae (Fig. 1). These factors, which include the larvae, the biological and the physio-chemical environment, are in turn influenced by several conditions. Manipulating these conditions may increase the probability of producing viable larvae. It is clear that manipulation of one of the three factors may only have limited effect. Therefore, a strategy to achieve microbial control and improve viability of larvae makes it important to use a range of countermeasures directed towards these different aspects (Fig. 1). Vadstein et al. (1993) proposed a general strategy for microbial control that takes into account both the
A: Factors
determining
viabilitv
CP,,)
B: Manioulation
to increase
viability
lmmunostimulation
Genetics, Nutrition. Immune system
~~;~~~;~t~~lL;~l&.s _ ‘
Fig. I. (a) The three factors of significance for the probability of viable larvae (P,) and conditions that influence these fxtors; (b) the probability of viable larvae (P,) may be increased by using methods that push the circles towards each other. One such method is immunostimulation. Other methods are treated in Vadstein et al. (1993).
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/Aquaculture
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Table 1 The three elements in the strategy to achieve microbial control in the rearing of marine fish larvae, as proposed by Vadstein et al. (19931, with examples of possible methods for the different elements in the strategy Element 1. Nonselective reduction of bacteria l Surface disinfection of eggs 0 Reduction in input of organic matter 0 Removal of organic matter 0 Grazer control of bacterial biomass Element 2. Selective enhancement of bacteria 0 Selection for desirable bacteria 0 Addition of selected bacteria to tanks 0 Incorporation of selected bacteria in feed Element 3. Improvement of larval resistance against bacteria 0 Stimulation of general immune system 0 Modulation of maternal immunity
ecological conditions mentioned above and the various aspects described in Fig. 1. The strategy, which does not rely on the prophylactic use of antibiotics, is based on the concurrent use of three different elements (Table 1). Application of this strategy has so far produced good results (Vadstein et al., 1993; Salvesen and Vadstein, 1995; Skjermo et al., 1996, 1997). This review will focus on immunostimulation.
3. The immune
system of fish
The main function of the immune system is to protect the animal against diseasecausing microbes. The immune system of fish shows clear similarities to that of mammals, and several reviews have covered various aspects of the immune system of fish (Ellis, 1977, 1988; Ingram, 1980; Fletcher, 1982; Hart et al., 1988; Alexander and Ingram, 1992; Bly and Clem, 1992; Secombes and Fletcher, 1992; Siwicki et al., 1994). The immune system comprises both nonspecific and specific components, and involves both cellular and humoral factors. This division into four constituents is somewhat misleading because all components are interlinked and mutually dependent. Nonspecific defence mechanisms are part of all normal fish, and do not require prior contact with an antigen/pathogen to elucidate a response. On the other hand, the specific immune system requires activation; thus, there is a time delay between the first introduction to the antigen and the activation. This process is also temperature-dependent (Bly and Clem, 1992). It is believed that nonspecific immunity is phylogenetically older than specific immunity, and one might expect fish to be more reliant on nonspecific defences than higher vertebrates (Anderson, 1992). Such a reliance is expected to be even stronger for invertebrates. It is believed that larvae do not have the ability to develop specific immunity during the early stages of development (Ellis, 1988). In this respect, fish are reliant on passive immunisation from maternal antibodies. Although it has been reported that maternal transfer of specific immunity does not occur in salmonids (Ellis, 1988), this mechanism
0. Vadstein/Ayuaculture Table 2 Days post-hatch (negative numbers organs of four teleost species
are pre-hatch)
155 (19971401-417
when lymphocytes
405
appeared
in the developing
Organ
Salmon (4-7°C)
Rainbow trout (14°C)
Carp (22°C)
Sebasticus mrmoratus
Thymus Blood
-22 - 14
3-5 5-6
5 7-8
21 _
Kidney Spleen
- 14 42
5-6 21
7-8 8-9
30 44
Data from Ellis, 1988 (based on several sources) and Nakanishi,
lymphoid (2°C)
1991
has been shown in tilapias (Mar and Avtalion, 1990; Sin et al., 1994). In any given species, size rather than age seems to be the most critical determinant of the development of specific immunity. In salmonids, memory-dependent specific immunity has been achieved for sizes > 0.26 g (8 weeks of age), whereas in carp it was acquired at 9- 10 weeks of age (Ellis, 1988). The nonspecific immune system is probably the major defence against microorganisms in larvae. Although our understanding of the components of the innate defence system of fish is growing, relatively little is known about the functioning and the ontogeny of the general immune system in marine larvae (Ellis, 1988; Olafsen and Roberts, 1993). In the few fish species that have been studied, the major lymphoid organs are not fully developed at the time of hatching (Table 21, and the phagocytic activity is mainly associated with gills, skin and gut (Ellis, 1988). It is therefore possible that during the stages when the lymphoid organs are developing, the main cellular defence is by the phagocyte populations within the integument (Ellis, 1988). The nonspecific or innate immune system is regarded as the first line of defence in animals. Furthermore, it seems that the microbial problems in larviculture are most likely due to opportunistic bacteria rather than specific pathogens (Vadstein et al., 1993; Munro et al., 199.5). This emphasises the importance of nonspecific defence system for larvae under intensive hatchery conditions, and the need for more knowledge of the ontogeny and functionality of the nonspecific immune system of larvae.
4. Immunostimulation
of fish: definitions,
possibilities
and constraints
Immunotherapy comprises all methods that utilise immunological principles to prevent or treat diseases. In human and veterinary medicine, immunotherapy has already been applied, but it is still regarded as an area for growth (e.g., Hadden, 1993). Methods of transplanting immunologically active cells or transferring immunoglobulins are not relevant to larviculture, and therefore not treated further in this study. Immunomodulation, and in particular immunostimulation, seems to be the most suitable immunotherapeutic method for larviculture in the foreseeable future. Immunomodulation may be directed at both specific and nonspecific immunity. Vaccination is probably the best-known method of specific immunostimulation, and it entails increased resistance against a specific antigen/pathogen. Nonspecific immunos-
406
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timulation refers to a condition in which the immune response is changed to a condition with higher response towards a variety of antigens. An example of nonspecific immunostimulation is macrophage activation. An immunostimulant may be defined as an agent which stimulates the nonspecific immune mechanisms when given alone, or the specific mechanisms when given with an antigen. As mentioned above, eggs and larvae ‘inherit’ an immune defence, but they last for only a short period. A special case of immunostimulation may be to enhance the immune defence of larvae through immunostimulation of the mother (i.e. maternal immunity). Many different types or groups of substances are known to act as immunostimulants (below). The compounds may either be grouped by function or by origin (Anderson, 1992; Hadden, 1993; Secombes, 1994). Some immunostimulants, such as lipopolysaccharide (LPS) from Gram negative bacteria, may act by triggering a number of mechanisms; others (e.g., mannuronic acid polymers) may have a more restricted stimulatory effect (Espevik et al., 1993). It is important to consider the specificity of immunostimulants for two reasons. First, a stimulation of the immune system may be too intense, and may harm or even kill the host. This is well known in humans, where the activation caused by LPS in connection with infections may cause septic shock and death (Morrison et al., 1994). Second, the knowledge of the functions of different immunostimulators may be used to stimulate, more specifically, those parts of the immune system that may be more relevant in certain situations. Three factors are essential to consider in the design of an immunostimulation strategy. First, it is important to remember that in most cases we do not have a specific microbial problem (i.e. specific pathogens), but rather a general one involving large numbers of bacteria and a high proportion of opportunistic species. Secondly, the immune system of larvae is poorly developed, consisting mainly of nonspecific defence. Thirdly, the immune defences of maternal origin will be significant only during the earliest developmental stages. Although this period and hence the maternal immune defence, may be critical, aquaculturists cannot rely on this part of the immune defence of the larvae alone. These three factors mean that research aimed to develop methods for immunostimulation of larvae, should place the highest priority on stimulation of the nonspecific defence system. This work should principally involve stimulation of the nonspecific defence of the larvae itself, and should also include stimulation of nonspecific maternal defences if possible. In cases where specific pathogens are known to cause problems, stimulation of the specific defence may be considered through immunization of the broodstock.
5. Immunostimulation 5. I. Maternal
of fish: the state-of-the-art
immunity
Our understanding of the stimulation of maternal defence is very limited. In fact, this author is not aware of any attempts to stimulate the nonspecific defence of larvae by immunostimulation of the mother. In an attempt to increase specific antibodies in eggs and larvae of tilapias (Oreochromis aureus), Mor and Avtalion (1990) immunised
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female broodfish with three different proteins. A considerable increase in antibody activity was detected, with a maximum increase of IO-13 in log, units in embryo homogenates hatched 15-35 days post-immunisation. The optimum period between immunisation and hatching seemed to be dependent on the type of protein, and the antibody activity found was specifically against the protein used to immunise the mother. Sin et al. (19941, working with the same species, vaccinated broodfish with live tomites of the ciliated protozoa Ichthyophthirius multifiliis one month before spawning. Depending on whether or not the fish were allowed parental care of the larvae, a relative percent protection (RPP = 100% [ 1-(mortality in experimental group/mortality in control group)]) of 93% and 78% was obtained after challenge of fry with infective tomites. The protective immunity in the four groups (with and without vaccination/parental care) was correlated with titres of I. multijiliis antibodies in the fry and in the plasma of the fish. So far, no attempt has been made to evaluate the impact of using an immunostimulant (adjuvant) in connection with stimulation of specific maternal immunity. Use of adjuvant is a standard procedure to increase the response in normal vaccination. It is reasonable to believe that vaccination or secondary stimulation of mothers with appropriate vaccines before the spawning season could protect the larvae against diseases on the first days after hatching and until their own specific immune system is fully developed. 5.2. Direct stimulation Considerable data exist on stimulation of the nonspecific defence systems of fish, especially when we consider that the first studies appeared only 10 years ago. Direct stimulation of specific defence is not treated here because it is not considered to be relevant for larval stages. An attempt to summarise the available knowledge on stimulation of nonspecific immunity of fish is presented in Table 3. Although most studies were on adult fish, a high diversity in the studies is evident with respect to immunostimulants tested, species studied, mixture of in vivo and in vitro studies, and different response parameters used. The immunostimulants studied include crude and purified compounds of bacterial origin, various polysaccharides and synthetic chemicals with a range of freshwater, seawater and anadromous species. Response parameters include both humoral and cellular mechanisms, as well as challenges to pathogens. The positive effect of nonspecific immunostimulation, therefore, has been documented on several levels of the organisation. In addition to the wide diversity of the studies listed in Table 3, two other aspects are important. First, successful immunostimulation has been obtained by injection, bath and oral administration. If nonspecific immunostimulation could have practical use in larviculture, it is vital to be able to stimulate individuals without injection. Although it has been shown that injections with glucan and chitin gave a better resistance to Aerornonas salmonicida infection than administration by immersion (Anderson and Siwicki, 1994), too few conditions were tested to be able to draw any firm conclusions regarding the possible advantages of administration by injection. It is not known if immersion and oral administration entail complete stimulation of the nonspecific de-
of studies where immunostimulants
complete adjuuant
dipeptides
mykiss)
carpio)
Chanel catfish (Ictnlurus
Carp (Cyprinus
Glucans
mykiss)
punctatus)
Rainbow trout (Oncorhynchus Polysaccharides:
Peptidoglygan
Atlantic salmon (Salmo s&r)
Red sea bream (Pagrus major) Place (Plruronectes platessa) Channel catfish ( Ictalurus puncrams)
Lipopolysaccharide
Rainbow trout (Oncor/qnchus
Muramyl
Rainbow trout (Oncorhyrzchus
mykiss)
kisutch)
Brook trout (Salr’elinus~~ntinalis)
Coho salmon (Oncorhynchus
Freund’s
Bacteria and bacterial products:
Immunostimulant/species
Table 3 Summary
salmonicida,
A. hydrojila,
Vibrio ordalii
macrophage cultures
Increased phagocytic activity of kidney leucocytes, increased resistance to Edwnrdsiella torda. Stimulation of macrophages.
injected
Chen and Ainsworth,
1993
1992
Yano et al., 1989, 1991
Matsuo and Miyazono,
Solem et al., 1995
Increased respiratory burst of macrophages, stimulation of phagocytic activity and ability to kill bacteria Increased resistance to V. anguillarum.
Salati et al., 1987 MacArthur et al., 1985 Clem et al., 1985
Kodama et al.. 1993
Kajita et al., 1992
Oliver et al., 1986
Oliver et al., 1985
Reference
Enhanced phagocytic activity of blood leucocytes Enhanced migratory activity of macrophages Release of interleukine-1 -like molecule
Activation of phagocytic, respiratory burst and migratory activity of macrophages, increased disease resistance
Increased capacity for phagocytosis and killing of virulent A. salmonicidu Increased resistance to V. anguillurum, activation of leucocytes
Aeromonas
Increased resistance to
effects
oral
injected injected monocyte cultures macrophage cultures
injected
macrophage cultures injected
injected
Documented
were used to augment the general immune response of fish Administration
~alur)
Atlantic salmon (Salvo
Atlantic salmon (Sulmo salarl
Aminured polyglucose
Rainbow trout (Oncorhynchus
mykiss)
Brook trout (Sah~elinus fonrmalis)
Churn
maximus)
mykiss)
Turbot (Scophthalmus
hippoglossus)
Hugh-M or poly-M alginates Atlantic halibut (Hippoglossus
Brook trout (Sall,elinusfantinalis)
Rainbow trout (Oncorhynckus
injected
Atlantic salmon (Salma s&r)
macrophage cultures
injected and bath injected
macrophage cultures
orally
immersion
injected and harh
injected injected
macrophage cultures orally
injected
injected
Yellowtail (Seriola quinqueradiatn)
burst
Increased superoxide anion formatton, acid phosphatase activity and pmocytic activity
Increased resistance to A. salmonicida lasting l-2 weeks. Higher protection with injection than with bath Increased phagocytic and respiratory burst activity of leucocytes, increased resistance to V. anguillarum.
Increased survival of larvae during the one-month-long yolk sac period (cf. Fig. 3) Increased resistance to V. anguillurum. Administration via live feed (cf. Fig. 4) Increased phagocytic activity and respiratory
Increased activity of lysozyme and complement Increased ability of macrophages to kill A. salmonicida, increased respiratory burst of macrophages, increased serum lysozyme activity Increased resistance to A. salmonicida lasting 1-2 weeks. Higher protection with injection than with bath.
11992
and Robertsen,
1995
Sveinbjarnson
and Seljelid. 1994
Sakai et al., 1992
Anderson and Siwicki, 1994
Rokstad et al. unpubl.
Skjermo et al., 1996
Vadstein et al., 1993
Anderson and Siwicki, 1994
Engslad et al., 1992 Jetrgensen et al., 1993b
Raa et al
Increased resistance to V. angurllarum
and
Jergensen
V. ~olmonrctda.
et al., 1990
et al., 1992
Jprgensen et al., l993a
Robensen
Matsuyama
Accumulation of macrophages. neutrophils and trombocytes in peritoneal cavtty, increased ability of head kidney macrophages to kill Aeromonus salmonicido. Increased respiratory burst
and Yer.rrnia ruckrrr.
phagocytic activity of kidney leucocytes, levels of serum complement and lysozyme, resistance to Strep~~~cus sp. resistance to V anguillanrm,
V. .ralmonicida
Increased enhanced increased Increased
mykiss)
mykiss)
FK-565 Rainbow trout (Oncorhwchus
cnrpio)
Rainbow trout (Oncorhynchus
Carp (Cyprinus
Lrr~umisolr
Synthetic chemicals:
Immunostimulant/species
Table 3 (continued)
blood and spleen leucocyte cultures
injected
spleen cells in vitro
injected
oral
oral
immersion
injected
Administration
effects
Increased bactericidal and phagocytic activity of peritoneal and kidney phagocytes, resistance to A. sulmonicidu. Removal of suppressive effect of cyclophosphamide or hydrocortisone on phagocytic activity Increased respiratory burst and spreading
Increased blood neutrophil phagocytic, myeloperoxidase and migratory activity Increased growth of larvae at various concentrations (cf. Fig. 4) Enhancement of various nonspecific defence mechanisms after an immune suppression induced by trichlorfon Increased phagocytosis and production of oxygen radicals by blood neutrophila Increased phagocytic activity and chemolumineacence of kidney leucocytes, natural killer cell activity, serum complement, bactericidal activity, resistance to V. anguillnrum. Increased phagocytosis and production of oxygen radicals
Documented
1987
1989
1986
1991
Kitao and Yoshida,
1994
Kitao and Yoshida,
Siwicki et al., 1990
Kajita et al., 1990
Siwicki,
Siwicki and KorwinKossakowski, 1988 Siwicki and Studnicka,
Siwicki,
Reference
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fence or stimulation of only the mucosal immune system. Intestinal uptake and organ redistribution of high-molecular-weight polysaccharides (glucan and poly-glucose) after oral administration have been documented (Dalmo et al., 1994; Sveinbjomsson et al., 1995). It is therefore possible that immersion and oral administration may result in complete stimulation, but with certain limitations depending on the immunostimulant used and species. Secondly, in addition to the positive effects as judged by immune parameters and challenge tests, immunostimulation has also been shown to abolish the negative effects of immune suppression. When rainbow trout were injected with the immunostimulator FK-565, the fish were able to overcome the suppressive effects of cyclophosphamide and hydrocortisone treatment (Kitao and Yoshida, 1986). Oral stimulation with levamisole has been shown to enhance the nonspecific defence of carp after immune suppression caused by trichlorfon (Siwicki and Studnicka, 1994). Most of the experiments presented in Table 3 are simple in design, and little is known about dose-response relationships and how long a stimulation may last. Both these aspects are important for the utilisation of immunostimulation in larval rearing. The study by Anderson and Siwicki (1994) showed that protection was reduced 2-3 weeks after stimulation, which may be regarded as long enough to be relevant in larviculture. Stimulation extended over at excessively long periods may have negative effects on the host, and this has been documented in a feeding trial (Onarheim, 1992). Only two of the studies in Table 3 specifically deal with larvae. In one (Vadstein et al., 1993) yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) were stimulated by immersion in an alginate rich in mannuronic acid. The stimulation lasted for the whole yolk sac period (28 days) and a considerable improvement in survival was obtained (Fig. 2), with a RPP of 47%. The second study on immunostimulation of larvae was with carp larvae using the synthetic immunostimulant levamisole (Siwicki and Korwin-Kossakowski, 1988). Four different concentrations were tested and the larvae were treated twice (days 1 and 3 after hatching) by immersion. After two weeks the effect of the stimulation was evaluated by growth measurements. For the three lowest concentrations tested, a significant increase in the size of the larvae was recorded (Fig.
70 60 3 2 P 2
40
50
g
30
g
20 10
Control
Stimulated
Fig. 2. The effect of immunostimulation of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) with an alginate rich in mannuronic acid on survival (mean i SEMI on day 28 after hatching. Larvae were stimulated by incubation in water with added immunostimulant at final concentrations of 25- 100 p,g I-’ just after hatching. Based on data from Vadstein et al. (1993).
412
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Fig. 3. Effect of stimulation with levamisole by immersion on day one and day three after hatching two-week-old carp larvae. Based on data from Siwicki and Korwin-Kossakowski (1988).
on size of
3). The survival was similar in all groups and above 93%. It is important to note that within the one order of magnitude variation in concentrations tested, no negative effects were recorded. A third study is relevant to larviculture because it included the use of Artemia as a carrier of the immunostimulant (Skjermo et al., 1996), an approach that is applicable to first-feeding of larvae. The immunostimulant (an alginate rich in mannuronic acid) was immobilised in beads made of a gel-forming alginate, and of a size appropriate for ingestion by Artemia. The beads were given to Artemia, which were fed in turn to 40-day-old turbot (Scophthalmus maximus) in several meals over 24 h. Two days later, the turbot were challenged by a pathogenic strain of Vibrio anguillarum. RPPs of 48% and 39% were obtained in two independent experiments (Fig. 4). The authors stated that there was no reason to believe that the conditions for stimulation were optimal during the experiments.
40 x g cd ‘;, 30 E F a, 20 $ a 10 0
1 Experiment
Fig. 4. Mortality (average+ SEM) in control groups and immunostimulated groups of juvenile turbot (Scophfhalmus maximus) after challenge with Vibrio unguillarum in two different experiments. The immunostimulant (an alginate rich in mannuronic acid) was immobilized in beads made of a gel-forming alginate and of a size appropriate for ingestion by Artemia. The beads were given to Arkmia, which again were given as food for 40.day-old turbot in several meals over 24 h. Two days later, the turbot was challenged by a pathogenic strain of Vibrio anguillarum. Based on data from Skjermo et al. (1996).
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It is concluded that although still in its infancy, immunostimulation of fish shows considerable potential as a method for reducing losses in aquaculture. This includes both the larval and on-growing stages. Both stimulation of nonspecific defence mechanisms and stimulation of specific immunity of maternal origin have been documented. Experience with larvae is, however, still very limited.
6. Immunostimulation
in larviculture:
future challenges
As the review above demonstrates, more knowledge is needed before a strategy for specific and nonspecific immunostimulation can be developed for larviculture. The few studies on stimulation of specific maternal immunity and the more numerous studies of stimulation of nonspecific defence of fish suggest that there is a good possibility of developing efficient methods for immunostimulation in larviculture in the near future. A strategic decision that has strong implications is determining what types of immunostimulants the research should focus on. Attention should be focused on a limited number of immunostimulants in order to obtain maximum information from the research. Two criteria might serve as guidelines in the selection of suitable immunostimulants. Firstly, several substances with documented immunostimulatory effects are unlikely to be approved for use, either because they are poorly defined, or because of their source and adverse effects. Secondly, considerable research on immunostimulation is being carried out in veterinary and human medicine. Research in fish biology and aquaculture is unlikely to have access to funds comparable to those available to human medicine; therefore research should focus on the immunostimulators that have already been intensively studied in other areas. The knowledge and the spinoff from these other areas represents a considerable resource for aquaculture research. For stimulation of maternal immunity, it is important to clarify whether it is possible to stimulate nonspecific immunity in a way that is relevant to larviculture. Due to the shorter duration of such a stimulation, nonspecific stimulation through the maternal pathway may not be feasible, but a clarification of this point is necessary before any final conclusion can be drawn. Stimulation of specific immunity is the most promising method for applying maternal stimulation. As a general method, it is not suitable, but in the cases where specific problem organisms have been identified, it should be possible to evaluate the potential of the method. Such an evaluation should include optimisation of immunisation procedures, and evaluations in both challenge experiments and under production conditions. It is important that evaluations are done under both sets of conditions, because even if stimulation of maternal immunity does not have significant positive effects in a challenge test, in which the problem organism occurs at high densities, it may do so under production conditions. The method is not restricted to bacteria (Sin et al., 1994). The challenges related to nonspecific immunostimulation of the larva itself are more diverse than those related to maternal immunity. Reliable larval experiments are expensive, due to the complexity and the poor reproducibility of such experiments. More information is required on the developmental stage at which immunostimulation is possible, and for this, additional evaluation criteria other than challenge tests will be
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needed. The research on the ontogeny of the nonspecific immune system should focus not only on the development and the functioning of relevant organs, but should also include studies of the immune system in the integument/mucosa. The development of methods of assessing stimulation is a special challenge, due to the small size of larvae. Priority should be given to the establishment of methods for determining parameters that occur early in the cascade of reactions triggered by immunostimulants. The research directly related to the establishment of an immunostimulation strategy should include evaluation of administration procedures, dose-response relationships, and evaluation of the duration of a stimulation.
7. Conclusion Immunostimulation stimulation origin seem to be the most promising knowledge, it is concluded timulation both larval needed
if immunostimulation
Direct Based on available still in its infancy, immunoslosses in aquaculture, during
would
Acknowledgements The ideas presented in this paper evolved while working on several projects financed by the Research Council of Norway and two industrial partners (Fina Exploration Norway and Norsk Hydro). The scientific atmosphere provided by my colleagues working with larviculture is highly appreciated, and the scientists at the Norwegian Biopolymer Laboratory at the University of Science and Technology - Trondheim for introducing me to the fascination of immunostimulation. Kjell Inge Reitan and Yngvar Olsen provided helpful comments on an earlier version of the manuscript. Linguistic help was provided by Allen Market. This study forms part of a project financed by the Research Council of Norway (contract 1 lOSSO/ 120).
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