Review

Vaccines for fish in aquaculture

Ingunn Sommerset, BjrnK rossy,EirikBieringand Petter Frost
Vaccination plays an importan t role in large-scal e commerc ial fish farming an d has bee n a key reason for the succes s of salmon cultivation. In addi tion to salmo n and trout, commerc ial vacc ines are avail able for channel catfish, Europe an seaba ss and seabream , Japan ese ambe rjack and yellowtail, tilapia and Atlant ic cod . In gener al, empi rically develo ped vaccine s base d on inac tivated bacterial path ogen s have proven to be very effica cious in fish. Fewer commercia lly available viral vaccines and no parasit e vaccine s exist. Substa ntial effica cy data are availabl e for new fish vaccines and ad vanc ed technolo gy has be en implem ented. However, before such vacc ines can be succe ssfully commerc ialized, several hurdles have to be overcom e regardi ng the produ ction of cheap but effective antige ns and adju vant s, while bea ring in mind environmental and associated regulatory concerns (e.g., those that limit the use of live vaccines). Pharmace utica l compa nies have performe d a considerable amo unt of research on fish vaccines, however, limited information is available in scientific pub lications. In add ition, salmonid s domin ate both the literature an d commercial focu s, de spite their relatively sma ll co ntribution to the total volume of farmed fish in the world. This review provides an overview of the fish vaccines that are currently commercially availab le and som e viewpoint s on how the field is likely to evolve in the nea r future.
Expert Rev. Vaccines 4(1), 89101 (2005) Aquac ulture: agriculture of the oceans

CONTENTS
Aquaculture : ag riculture of the ocea ns Vaccines & diseas e cont rol Administratio n metho ds & factors affectin g effica cy Limitation s in vaccin e developme nt Bacte rial fish vaccines Viral fish vaccines Fish vaccines against parasites Expert opinion & five-year view Key issues References Affiliations

Author for co rrespondenceIntervet Norbio AS, Thormhlensgate 55, N-5008 Bergen,Norway Tel.: +47 55 543 958 Fax: +47 55 960 135 ingunn.so merset@ in rvet.com m te KEYWORDS: aquaculture, bacteria, disease, fish, immunology, parasites, salmon, vaccination, vaccine, virus

Aquaculture is growing more rapidly than all other food animal-producing sectors; its contri- bution to global supplies of fish, crustaceans and molluscs increased from 3.9% of total production by weight in 1970 to 29% in 2001 [101]. As the capture fishing industry has declined and wild stocks diminished, the aqua- culture industry has become an important source of seafood. China is the largest fish pro- ducer, with 70% of the total volume and 53% of total value in 2002 [102,103]. Today, carps, barbels and other members of the cyprinid family dominate fish aquaculture (FIGURE 1). These fish species have a relativel low value compared y with other types of farmed fish and are typically raised in simple pond systems where they are a food source for families in developing countries. The tendencies are, how- ever, that all fish farming will become more industrialized with the increasing investment

from multinational companies. Today, high- value species, such as salmon and trout, account for 7% of total volume and 16% of total value of cultivated fish worldwide. Atlan- tic salmon is one of the most intensively farmed fish in the world, with Norway, Chile, the UK and Canada being the major produc- ers. Farming of highvalue marine species such as European seabass and seabream, halibut, flounder, cod, tuna, eel and amberjack/ ellow- tail will y probably increase as appropriate inten- sive aquaculture systems are developed. Although crustaceans (e.g., shrimp), molluscs (e.g., clams and oysters) and aquatic plants are important in global aquaculture, they do not possess an adapted or specific immune system. Therefore, vaccines (at least by their traditional definition) are not appropriate for inverte- brates and plants, and prophylactics in the farming of these aquatic organisms will not be reviewed in this article.

ww w.futu re-d rug s.com

10.1586/14760584.4.1.89

2005 Future Drugs Ltd.

ISSN 1476-0584

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Somme rset, Kross y, Biering & Frost

antigens in one oil-adjuvanted vaccine was the product of choice. The excellent efficacyof these vaccines soon resulted in their extensive use and an immediate and permanent reduction in the use of antibiotics, concurrent with a three fold increase in fish production (FIGURE 2). With the exception of the introduc- tion of a recombinant virus vaccine in 1995 [4], vaccination strategies in the salmon farming industry have been more or less unchanged over the last 10 years. Proper fish management with good hygiene and limited stress are key factors in the prophylaxis of infectiou diseases s and are also a necessity for the optimal effect of vaccine [57]. s Today, vac- cination is an integratal part of most salmon farms and the use of antibiotics is very limited, at least in Northern Europe and North America. The development of fish vaccines is, as with the devel- opment of human and veterinary vaccines, an ongoing interac- tion between academia, the pharmaceutical industry and regula- tory authorities. Until the early 1990s, most fish vaccines were develope and d commercialized by small local companies. During the 1990s and to date, five multinational animal health compa- nies have acquired, or formed, joint venture companies with the smaller companies specializin in the field of aquaculture g vac- cines. The major producers of fish vaccines are now: Intervet International (The Netherlands), Novartis Animal Health (Swit- zerland), Schering-Plough Animal Health (USA), Pharmaq (Norway; was part of Alpharma Animal Health until 2004) and Bayer Animal Health (Bayotek)/Microtek, Inc. (Germany/Can- ada). The major commercial markets for these companies are currently the salmon and trout industries in Northern Europe, Chile, Canada and the USA, where the value of a healthy population justifies the price paid per vaccine dose. Vaccines used Administr ation in Japan are mostly developed and distribthe feed would such vac- cines. However, poor and & be the ideal inconsistent responses have been reported uted by Japanese companies. Commercial method s vaccines are also available for the catfish facto rs affecting method of vac- by conven- tional oral vaccine due to s effica cy industry in the USA and, on a smaller scale, Oral cine delivery to antigen destruc- tion in the gut [8,9]. for European seabream, seabass and tilapia. vaccination fish and much Different approaches to protect the Some limited-use, locally developed vac- with antigen effort has been antigen from degradation, such as cines are also available in countries such as included in put into the entrapping in liposome or alginate bead. s China, Russia, Spain and Germany. development of neutralization of gastric secretions or application of biofilm vaccin [12], es have demonstrated some promising results. In all forms of intensive culture, where single or multiple spe- cies are reared at high densities, infectious disease agents are easily transmitted between individuals. Fish such as carp, often farmed in muddy ponds, appear to be more robust than, for instance, Atlantic salmon that are adapted to spend their early life in clean, running fresh water. Independent of high- or low- technology farming, good environmental conditions are impor- tant to maintain a healthy fish population. For species reared in nets in an open aquatic environment, exposure to pathogens is impossible to avoid. Due to the effectiveness of pathogen trans- portation in water and the high density of animals used in com- mercial large-scale farming, pathogens quickly spread within a population of cultured fish. During the 1980s, salmon farming in Norway experienced huge losses due to bacterial diseases(mostly Vibrio spp.) and a total crash in the industry was only prevented by the use of vast amounts of antibiotics (FIGURE 2). Fish immersion vaccines based on formalin-inactivated broth cultures had proven to be effective against vibriosis in the USA in the 1970s [3] and similar vaccines were quickly developed against the salmonid Vibrio diseases. The good efficacy of these vaccines immediately resulted in a decline in the use of antibi- otics. However, a new bacterial disease, furunculosis (Aerom- onas salmonicida) appeared and, as immersion vaccines proved ineffecti e against this pathogen, injectable vaccines containing v adjuvants were developed in the early 1990s. Following a few years of testing with different vaccine adjuvants and a range of different antigen combinations, it soon became evident that all

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Expert Rev. Vaccines 4(1), (2005)

However, a large quantity of antigen is usually necessary and the protection achieved is generally weak and of short duration. Therefore, the two main methods of vaccine delivery to fish are still, immersion in a diluted suspension of the vaccine or injecting it into the body cavity (typically by intraperitoneal injection). Immersion vaccines are effectiv for e a number of bacterial patho- gens and are cheap and easy to administer to small fish. In con- trast, vaccination of fish by injection is labor intensive and

requires the fish to be over a certain size, making vaccination of fry difficult. Nevertheless, many fish vaccin today are es multicompo- nent injection vaccines, because at least one of the components requires injection and an oil adjuvant to be effective. The advan- tages of injection vaccines is that the volume of vaccine needed is relatively low and that every fish is vaccinated and with the correct dose. In industrial salmonid farming, professional vaccination teams or automated vaccination machines are used (FIGURES 3 & 4).

Fish are cold-blooded animals with a body temperature that equals their surroundings. Dependent upon fish species and tem- perature, vaccination must be performed within a certain mini- mum period of time before the risk of their exposure to pathogens [6]. The onset of an immune response is faster in warm- water species than in cold-water species. At an optimal tempera- ture for Atlantic salmon (10 12C), antibodies are typically not detected until at least 46 weeks after vaccination, whereas a warm-water species such as seabass, with an optimal temperature of 22C, can have detectable antibodies 1 week after immuniza- tion [13]. In addition to temperature, stress caused by environmen- tal or man-made factors such as photoperiod, seasona changes, l salinity, heavy metals, crowding, handling and transport, can induce immune suppression and be a limiting factor for vaccine efficacy [14]. Unlike wild fish exposed to similar stressors, farmed fish cannot escape from these factors, and both acute and chronic stress can render fish susceptible to different opportunistic as well as pathogenic microorganisms.

exists as a tetramer in its secreted form [17]. No isotypes corre- sponding to mammalian IgG, IgA or IgE have been identified in fish and the secondary humoral immune response in fish is, if present at all, less prominent than in mammals. Due to ease of measurement, the presence and titer of specific antibodies is a fre- quently used immunologic assay. Assays to measure specific T-cell responses are still in their infancy, however, cell lines from cloned fish lines (matching major histocompatibility complex [MHC] molecules are now available for carp and rainbow trout [18]. ) Even among the relatively few fish species that are commercially farmed today, it is important to keep in mind that fish immunology can- not be considered a homogenous system. The number of fish spe- cies is much greater than the number of mammalian species and the evolutionary distance between different teleost (bony fish) families are considerably larger than between mammals. Further- more, there are more than 25,000 species of fish living in habitats ranging from polar regions to the tropics, from muddy fresh

Limitations in vacci ne development

The major goal of vaccination is to induce a specific long-term protection against a cer- tain disease. It has been debated whether the effectiv e long-term protection of oil-adju- vant injection vaccine is due to immuno- logic memory s in the fish or constant stimu- lation from the antigen depot. As the existing empirically developed vaccines can induce protection after a single administra- tion and until the fish are harvested, less effort has been put into the investigation of the actual mechanisms behind the protec- tion. In evolutionary terms, fish are the first group of animals with the basic aspects of the immune system of higher vertebrates. Although fish immune systems are primi- tive compared with mammals, there seem to be more similarities than differences [15,16]. In the limited number of fish species stud- ied, the major antibody type is an immu- noglobulin (Ig)M-like isotype that typically

water to the ocean, from dark high-pressure depths to bright low- pressure surface areas. It is therefore likely that different species have major differences in how they combat pathogens and also in their response to vaccines. Lack of detailed knowledge of the immune systems in different fish species limits the possibilities to study both pathogen and vaccine-induced immunity. As with all veterinary vaccines, cost effectiv eness in the field is an essentia limitation to commercial vaccine l development. For example, the calculations would be very different for a population of sturgeon used to produce Russian caviar versus a population of carp in a Chinese village pond. Fish generally need a large antigen dose compared with terrestrial animals and cost-effectiv e inactivated viral vaccines have proven diffi- cult to develop. In some species all types of injection vaccines (or even , immersion vaccines) are simply too expensive. Other species are too vulnerabl to handle the stress induced during the e vaccination or may develop severe side effects post vaccination. Yet, in other species the major disease problems , may appear in the larval or fry stages, before the animal is large enough to be vaccinated or have even developed a functional immune system. The apparent lack of maternal immunity in fish also limits the possibilities to protect offspring by parental vaccination [19,20].
Bacterial fish vaccines

pathogens, with losses of up to US$6080 million annually in the USA [22].

At present, vaccines are available against many of the serious bacterial diseases causing problems for the fish farming indus- tries (TABLE 1) [21]. The first commercialy l available bacterial vac- cines were against enteric redmouth disease (ERM, yersiniosis) and vibriosis, introduced in the USA in the late 1970s [3]. These vaccines were based on inactivated whole-cell formula- tions and were administered by immersion. Such vaccines have proven efficacious in preventing many of the major bacterial diseasesand the fact that these vaccines can be produced at low cost makes them ideal for use in the aquaculture industry. Vibriosis has a global distribution in a wide range of fish spe- cies. The bacterial species causing classical vibriosis is Listonella anguillarum (previous nomenclature, Vibrio anguil- larum), with more than ten serotypes characterized so far. For the salmonid and cold-water marine fish species such as cod and halibut, different serotypes might be involved in disease outbreaks; thus, vaccines designed for salmonids cannot automatically be used for the marine species. In addition to using the right serotypes, the main challenges for vaccination of marine species are that vaccination often has to be performed on very small juveniles/arvae and that outbreaks occur l during the entire lifecycle. This probably means that in some species revaccination will be necessa if the fish are to be ry protected during the entire production period. Vibriosis is an example of a disease for which the simple inac- tivated bacterin vaccines works well but other bacteria have proven more difficult to control by means of vaccination. In the channel catfish industry, Edwardsiella ictaluri and Flavobacte- rium columnare are two of the most important

Vaccines containing E. ictaluri and F. columnare bacterins have low efficac and the use of live attenuated isolates is a y new and promising approach [23,24]. A live attenuated E. ictaluri vaccine, licensed in the USA a few years ago, has proven efficaciou by immersion of fish as early as 710 days s post hatching [25]. A live vaccine has also been licensed for use in salmonids in North America and Chile. This vaccine is based on the cross- protective property of Arthrobacter spp. against bacterial kidney disease (BKD) caused by the intracellular bacterium Renibacte- rium salmonina rum [26]. Another intracellular bacterium for which development of vaccines has proven difficult is Piscirick- ettsia salmonis. This is the causative agent of salmonid rickett- sial septicemia (SRS, pisciricketsiosis), which is the most seri- ous disease t problem in the Chilean aquaculture industry. Inactivated P. salmonis bacterins are available, however, due to low efficacy, development of new vaccines based on recombinant proteins is ongoing [27]. These new approaches might offer a solution for diseases where inactivated bacterins are inef- ficient, although the long-term performance of the vaccines remains to be documented.
Viral fish vacc ines

Today, most available virus vaccines for aquaculture are based on inactivated virus or recombinant subunit proteins. Inacti- vated/killed viral vaccine are generally not efficacious s unless deliv- ered by injection and, as relatively high doses are needed to achieve protection, cost-effectiv inactivated viral e vaccines are difficult to develop. Live viral vaccines have been tested with good results in fish [2830] and should be the optimal

regarding protection, admin- istration and price. However, as discusse below, ecosafet aspects of live virus vaccines are d y considered major drawbacks and currently hinder their use as commercial vaccines. The first viral vaccine for fish was produced by a Czechoslova- kian company (Bioveta) in 1982. The vaccine was against a carp rhabdovirus, causing spring viremia of carp (SVC) and was based on two inactivated strains of SVC virus emulsified in oil and administered by injection. TABLE 2 summarize the viral fish vac- cines commercially available s today. Carp production is huge in Asia, both for food (common carp) and ornamental (koi carp) purposes. Although some severe viral diseases exist (e.g., caused by koi herpes virus, grass carp hemorrhage disease virus and SVC virus), few viral vaccine trials have been published. To the authors knowledge, the only commercial carp vaccine in Asia is an inactivated grass carp hemorrhage disease virus (a reovirus) vaccine, which has been widely used in China. A koi herpesvirus vaccine based on an attenuated strain of carp interstitial nephritis and gill necrosis virus is available in Israel [30,104]. In salmonid farming, commercial vaccines exist for some of the most common viral diseases However, few, if any, of . the viral vaccines are as efficaciou as the bacteria vaccines. s l Disease caused by the birnavirus, infectious pancreatic necrosis virus (IPNV), is a major problem in the salmonid industry [31]. Commercially available IPNV vaccines are based on either inactivated cell cul- ture-propagated virus or recombinant structural proteins. Most IPNV vaccine exist as s polyvalent oil-adjuvante vaccines where d

the IPNV antigen(s) is mixed with one or several bacterins; this appears to improve the efficacy compared with monovalent IPNV vaccines [31]. Despite good efficacy data in laboratory trials and extensive vaccination, IPN is still a problem, partly due to the complex nature of this disease. Aquatic birnaviruses (also called marine IPNV strains) are pathogenic for a number of farmed marine fish species, such as halibut, turbot, European sea- bass, yellowtail and Japanese eel, however, vaccines are currently not available.

An inactivated viral vaccine against pancreas disease (PD, caused by an aquatic alphavirus) is available in Ireland under a provisional license [32,33], and a vaccine against infectious salmon anemia (ISA, caused by an orthomyxovirus) is available in Can- ada and the USA [105], but not in Europe, due to preventative leg- islation (stamping-out policy). The rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic sep- ticemia virus (VHSV), cause devastating and highly infectious diseases, predominantly affecting cultured salmonids in western

North America (IHNV), France (VHSV and IHNV) and Den- mark (VHSV). For more than 30 years, several attempts have been made to develop efficacious vaccines using the conventional approaches of killed or live virus vaccines. Despite their ability to induce good protection in laboratory studies, the live vaccines were demonstrated to be unsafe for field use and inactivated vac- cines required high doses [34,31]. Different recombinant subunit vaccines based on the IHNV and VHSV membrane glycoprotein have been less successful [3538]. However, DNA vaccines encod- ing the same viral glycoproteins are remarkably efficacious [3941]. Indeed, these DNA vaccines are protective when used at small doses (as little as 10 ng in trout fry) and efficaciou as early as s 48 days and for up to 2 years post vaccination [42 46]. Viral nervous necrosis, caused by betanodaviruses, is a major problem in the farming of several marine fish species (e.g., Euro- pean seabass Atlantic halibut, barramundi and , several groupe [47], yet commercial vaccines are unavailable. rs) A few publications have demonstrate the effect of d recombinant subunit formulations [4850]. However, in most marine species, disease caused by betano- daviruses strike early in the lifecycle of the fish (larval or fry stages), before injection of these formulations are protective or applicable.
Fish vaccines against parasites

Instead, large amounts of antiparasitic pharmaceutical are s used with potentially negative environmental effects In . general, fish possess both humoral and cell-mediated defense mechanisms against many parasites [5153] and there are many reports on immunity/i creased resistance among n fish surviving natural par- asite infections [5458]. Therefore, there are no principal biologic limitations hindering vaccine development against at least some parasiti diseases in fish. c Experimental parasite vaccines based on whole pathogens have been reported and live vaccines appear to be superior to killed vaccine [5964]. s Cultivation of parasite for potential killed or live vaccines s is even more expensive than virus cultivation, as a host population rather than cell cultures are usually required. In addition to the high costs, the use of natural hosts for cultivation of the parasite would create major problems with respect to safety documenta- tion. Therefore, identification and production of protective antigens is probably the most feasible strategy towards commer- cial parasite vaccines, at least for low-cost vaccines The success- fully developed . vaccine against cattle tick (Boophilus microplus) [6567], a terrestrial ectoparasite of cattle, demonstrates that this strategy is indeed feasible albeit very difficult. ,
Expert opinion & five-yea r view

There is a wide range of parasites in both wild and cultured fish stocks. Although parasitic diseases such as amoebic gill disease, white spot disease, whirling disease, proliferative kidney disease (PKD) and salmon lice infestation create severe problems in fish farming, no parasite vaccine are commercially s available (TABLE 3).

To date, trial and error has been the main strategy for the devel- opment of fish vaccines and this empirical approach will probably continue in the short term. Development of vaccines for newly cultivated species (or new diseases in already domesticate fish) is usually restricted by a limited d knowledge of the fish immune

systems, the pathogenesi of the disease and the pathogen. Hows ever, the trial and error approach is less expensive for fish vaccine development than for other veterinary vaccines. For most dis- eases, the efficacy of candidate vaccine formulations can be tested on large groups of animals in relatively cheap challeng experi- ments where protection can be evaluated based e on counts of dead and surviving fish. With a few exceptions, vaccination against the most serious bacterial diseases of large-sca commercial farmed fish has le been quite succes sful. Besides small improvements to some of the existing products, no major changes are expected to take place within the next 5 years, at least for the salmonid vaccines. New vaccines against the intracellular pathogens R. salmoninarum and P. salmonis are now in use and field performance over the next few years will demonstrate if these vaccines can bring the diseases under control. A trend that has been observed and that is expected to continue, is the use of more sophisticated tech- niques in the development of bacterial fish vaccines. Develop- ment of vaccines using attenuated strains (e.g., E. ictaluri) or recombinant protein technology (e.g., P. salmonis) has been introduced as inactivated bacterins have demonstrated limited

effect. The use of DNA technology has also been tested against P. salmonis where fish for example, have been vaccinated with a crude genomic library [68]. Sequencing of several bacterial genomes are currently ongoing and information obtained from such projects can be used in the development of new vaccines. For obvious practical reasons, there is a reluctance to include more than one vaccination during a production cycle. One of the most extensivel used vaccines for y salmon contains antigens for six different pathogens and this number is likely to increase as new diseases emerge. However, a con- stant demand for more complex products will sooner or later result in antigens being immunologically incompatible, or there may be a limit to the amount of antigens that can be included in the water phase of an oilwater emulsion. Fur- thermore, the increasing numbers of antigens makes the development, production and registration of such vaccines more difficult. Manufacturers may become reluctant to make small improvements, as any change usually requires a huge effort in providing new documentation for licensing. It is also difficult to constantly develop multivalent products that keep pace with the changing disease situation in the field. It

is likely that this development will sooner or later make it necessary to apply more than one vaccination, thus enabling the development of vaccines with fewer antigens. Adverse effects following vaccination with adjuvant vac- cines are common problems and fish vaccines are no excep- tion to this rule [69]. As injection of nonadjuvant vaccines or adjuvant without antigens results in little or no side effects, it is clear that it is the combination of adjuvant and antigens, particularly crude bacterial broth antigens, which creates problems. Adverse effects have been taken very seroiusl by vaccine manufactu y rers in recent years. Products with a greater benefit to risk ratio are now on the market and the quest for new adjuvants with satisfactory efficacy and minimal side effects will continue. As bacterial antigens contribute signifi- cantly to the side effects, another solution to this problem would be to identify and separate the protective antigens from those that induce side effects. For already effective bac- terin vaccines, this is not likely to happen in the near future, as the process of identifying and separating these components is expensive and laborious. Injection vaccines developed for some of the new marine spe- cies in aquaculture may require adjuvants other than oil. In many species both the liver and the eggs are valuable food , sources (the liver is also an important source of fish oil) and remnants of oil adjuvants in these products will be undesirable. Delivery systems for effective oral or immersion administration would be a major improvement to fish vaccination in the future. Recently, a Cana- dian company has made interesting progress regarding oral administration of antigens [106], but it is unlikely that this approach will result in effective commercially available vaccines within a 5year view. Even in humans, only the live polio vaccine has been widely used with oral administration [70]. The lack of effective viral vaccines is one of the main problems facing fish vaccinolog Within the next 5 years, new y. and (or) improved virus vaccines will probably be developed for ISA, PD, VHS and IHN. Experimental IHN and VHS DNA vaccines are highly effective and the DNA vaccine against IHN is currently being tested in controlled field trials in Canada. It is likely that an IHN vaccine will be the first licensed DNA vac- cine for fish. For aquaculture applications, DNA vaccines are safer than attenuated live vaccines, but varying governmental attitudes towards both DNA and genetically modified organ- ism vaccines (North America being more liberal than Europe) may limit the use of both these vaccine types. Live attenuated virus vaccines would be the optimal fish vaccines if cost, efficacy and ease of administration were the only concerns. However, commercial fish vaccine develop- ment is limited by safety concerns to the consumers, and to the environment. Most cultured fish live in ponds or nets with no physical barriers to wild stocks. This limits the use of live vaccines, whether they are attenuated by classica means or by use of molecular l biology. Although nonpathogenic to the target animal, a live vaccine may have to be proven non- pathogenic to all

other relevant species in the aquatic environ- ment. Such safety data, if possible to generate at all, will

demand an enormous cost and effort to collect. However, for high-value species reared in closed indoor tanks with sterilized effluent water, attenuated virus vaccines may become accepta- ble. In addition, if live virus vaccines are to become a realistic alternative also for species reared in nets in the ocean, they need to have a high level of safety, for example, by develop- ment of a replication-deficient virus (infective but unable to produce new infective virus). These viruses or other modified microorganisms might be used as vehicles for other protective antigens. Based on reverse genetics methodology, a new live recombinant IHNV vaccine strain has been developed by a French group [71,72]. As the small nonvirion protein Nv proved to be nonessential for the recombinant IHNV, the Nv gene may be used as a site of insertion for foreign gene expres- sion and potentially serve as a vector for expressing additional antigens in the host. An interesting aspect of the fish rhabdovirus glycoprotein DNA vaccines is their stimulation of strong innate antiviral responsesand the ability to induce early protection against heterologous viruses [41,44,45,73]. In fish, the innate immune systems probably play a very important role in the protection against infectious diseases [74]. More attention should be paid to aspects of the innate immune responses in the search for specific immune stimulants and adjuvants. Recently, Jrgensen and coworkers demonstrated that short DNA fragments (CpG DNA) protect salmon, to a certain extent, from IPNV chal- lenge, suggesting that CpG DNA may be an effectiveadjuvant in some viral vaccines [75]. With the use of modern genomics, strategies for fish para- site vaccine development should be feasible. The commercial Boophil s vaccine in cattle is an example of how u a single pro- tein expressed in the tick gut, a protein that is not exposed to the host during a natural infection (i.e., a concealed antigen), can be used as a vaccine antigen, at least in parasites that feed on host tissue that contains components of the immune sys- tem. To study a parasite in such detail that new essential bio- logic processes are identified, is a major task, both financialy and scientific l ally. This will probably limit this kind of vac- cine development to high-value fish species where a single parasite causes huge financial or ecologic problems. In our opinion, the ectoparasitic copepods L. salmonis (salmon louse) and Caligus spp., are currently the only fish parasites that fit this description. On salmon ongrowing farms these copepods are the major parasite problem and in the northern hemisphere salmon lice alone cause 50100 million annual losses through mortality, growth reduction, quality reduction and pharmaceutical costs [76]. Caligus spp. currently causes severe problems for the huge salmon industry in Chile. Unlike the salmon lice, Caligus spp., are not salmonid spe- cific and are found globally, and most cultured marine fish species, present and future, are likely to experience Caligus problems, as production is intensified. In addition, the phar- maceuticals currently used against ectoparasites may sooner or later induce drug resistance. Therefore, efforts in basic research and commercial vaccine development against these

ectoparasite is likely to be intensified. As long as the s market potential is large enough and vaccine development feasible, an effort towards product developmen is likely to t take place.

assistance from colleagues in Intervet. In particular William Enright, Oscar Parra, Luc Grisez, Marian McLoughlin, Roy Olav Hovlid and Kari Thors are thanked for their effort. Dag H Knappskog, Frank Nilsen and Stephane Villoing are acknowledged for valuable Acknowledge ments comments during the prepara- tion of this manuscript and Information regarding commercial fish vaccines worldwide Irene Nygrd for making the FIGURE 3 collage. would have been difficult to obtain without the invaluable Key issues
Most of todays fish vaccineshave been developed and commer ialized for the salmon and trout farming industry, but vaccines c are also available for other high-value fresh water and marine species. Currentl , most vaccinesare based on simple empirically developed inactivated pathogens. A few recombinant subunit vaccines y are also available. Vaccines against bacterial diseases are, with a few exceptions, highly effective while more variable efficacyis apparent for viral vaccines. No vaccinesagainst fish parasites exist. Injectable multivalentoil emulsion vaccinesfor salmonids dominate the commercial fish vaccine market. A relativelyhigh antigenic mass is needed in most fish vaccinescompared with similar vaccinesused in higher vertebrates. Environmental safety concerns currently hinder the development and use of live virus vaccinesin fish. Speciesdiversity and limited knowledge of immune systems in fish limit the developmentof vaccinesor new vaccine deliverysystems based on nonempiricalstrategies.