A short history of research on immunity to infectious diseases in fish

Accepted Manuscript A short history of research on immunity to infectious diseases in fish Willem B. Van Muiswinkel, Miki Nakao PII: DOI: Reference:

S0145-305X(13)00243-7 http://dx.doi.org/10.1016/j.dci.2013.08.016 DCI 2023

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Developmental & Comparative Immunology

Please cite this article as: Van Muiswinkel, W.B., Nakao, M., A short history of research on immunity to infectious diseases in fish, Developmental & Comparative Immunology (2013), doi: http://dx.doi.org/10.1016/j.dci. 2013.08.016

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Title page for DCI 2023 A short history of research on immunity to infectious diseases in fish Willem B. Van Muiswinkel a,*, Miki Nakao b a Cell Biology & Immunology Group, Department of Animal Sciences, Wageningen UniversityWUR, Wageningen, The Netherlands b Laboratory of Marine Biochemistry, Department of Bioscience & Biotechnology, Kyushu University, Fukuoka, Japan *Corresponding author. Address: Cell Biology & Immunology Group (CBI), Department of Animal Sciences, Wageningen University-WUR, P.O. Box 338, 6700AH Wageningen, The Netherlands. Tel.: +31 222 365 869. E-mail address: [email protected] Abstract This review describes the history of research on immunity to infectious diseases of fish in the period between 1965 and today. Special attention is paid to those studies, which are dealing with the interaction between immune system and invading pathogens in bony fish. Moreover, additional biographic information will be provided of people involved. In the 1960s and 1970s the focus of most studies was on humoral (Ig, B-cell) responses. Thorough studies on specific cellular (T-cell) responses and innate immunity (lectins, lysozyme, interferon, phagocytic cells) became available later. In the period between 1980 and today an overwhelming amount of data on regulation (e.g. cell cooperation, cytokines) and cell surface receptors (e.g. T-cell receptor; MHC) was published. It became also clear, that innate responses were often interacting with the acquired immune responses. Fish turned out to be vertebrates like all others with a sophisticated immune system showing specificity and memory. These basic data on the immune system could be applied in vaccination or in selection of disease resistant fish. Successful vaccines against bacterial diseases became available in the 1970s and 1980s. Effective anti-viral vaccines appeared from the 1980s onwards. There is no doubt, that Fish Immunology has become a flourishing science by the end of the 20th century and has contributed to our understanding of fish diseases as well as the success of aquaculture. Keywords: Immunology, Vaccination, Diseases, Fish, History, Biography

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1. Introduction This review describes the history of research on fish immunity to infectious diseases during the last 50 years. Special attention is paid to those studies, dealing with the interaction between the immune system and invading pathogens in bony fish. It is not the intent of this review to evaluate the scientific merit of the work discussed, but to provide the reader with an idea how our present knowledge did develop over the years and to give biographic information on the people behind these studies. A historical review on fish health research in the USA was published by Mitchell in 2001. This extensive report covers a period of almost 3 centuries between 1609 and 1969! Another review focusing on the history of fish vaccinology was published by Evelyn (Fig. 1) in 1997. He describes, that initially the diseases in aquaculture were treated with “chemotherapy”, because of the relative low price of antibiotics and other drugs. It was only in the mid to late 1970s that the attention turned to the possibility of vaccination in fish farming. A 3rd review on the early days of fish immunology and vaccination describing the period between 1850 and 1965 appeared a few years ago (Van Muiswinkel, 2008). Only after 1940 the 1st researchers can be found, who were devoting their career mainly to fish immunology and/or comparative immunology, e.g. Gongarov, Cushing, Fänge, Ambrosius, Hildemann, Sigel, Clem, Lukýanenko, Anderson and Marchalonis. For more information on these early pioneers see previous review (Van Muiswinkel, 2008). For practical reasons we have limited ourselves in this publication to the period from 1965 till present. Taking into account, that recent technical and scientific progress (gene cloning, genome sequencing) is amazingly fast, it implies that this historical review offers only a limited picture of the past. The other contributions to this special issue of Developmental & Comparative Immunology provide us with an overview of recent developments in this field. We will refer to these publications in our introductory paper, when appropriate. 2. Innate and acquired immunity 2.1. Epithelial barriers The skin, gills and gut are examples of these epithelial barriers. It is of prime importance for fish to maintain the integrity of covering epithelia because they are important in defense and for osmoregulation. Normal epithelia are covered by a mucus layer, which is secreted by goblet cells. It has been shown, that an increase of bacterial load in the surrounding water is stimulating the production and release of high molecular weight glycoproteins of carp skin mucus (Van der Marel et al., 2010). The most important function of mucus is probably to prevent the attachment of bacteria, fungi, parasites and viruses to epithelial surfaces. During amoebic infections in salmon hyperplasia of epithelial and mucous cells is observed as described in the contribution from the group of Nowak in this special issue. Their paper provides us also with information on the expression of important immune genes during infection.

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2.2 Lectins Lectins in fish can be detected as natural precipitins or agglutinins. Fletcher (Fig. 2) showed, that lectins are probably important in neutralizing bacterial components (e.g. exotoxins) or in immobilizing micro-organisms and hence will facilitate phagocytosis (Fletcher, 1982). Fish lectins are not structurally related to Ig, but resemble plant or invertebrate agglutinins. Lectins have been found in coho salmon eggs (Yousif et al., 1995), trout serum (Hoover et al., 1998) and ayu mucus (Itami et al., 1993). A mannosebinding lectin, isolated from salmon serum, has been shown to opsonize a virulent Aeromonas salmonicida strain and lectin-coated bacteria can induce macrophages to kill them (Ottinger et al., 1999). Fish mucus lectins have been extensively studied by Suzuki's group (Fig. 3), revealing molecular diversity with wide range of sugar specificity and target binding spectrum (Suzuki et al., 2003; Tsusui et al., 2006). 2.3 Lysozyme This enzyme can be found in fish mucus, serum and eggs and is able to digest the peptidoglycan layer of bacterial cell walls. It was already published in the 1970s, that lysozyme is produced by macrophages and neutrophilic granulocytes (Murray and Fletcher, 1976). Ellis (Fig. 4) mentions in an important review, that lysozyme is bactericidal even for serious pathogens such as A. salmonicida and A. hydrophila (Ellis, 1999). Fish have been shown to possess two distinct lineages of lysozyme, chicken-type and goose-type, suggesting their phylogenetically ancient origin (Hikima et al., 2001). 2.4 C-reactive protein In teleost fish, C-reactive protein (CRP) is a serum component, which increases rapidly upon exposure to bacterial endotoxins (Ingram, 1980) or experimental infection with bacterial pathogens (Murai et al., 1990). Nakanishi and co-workers have shown, that CRP has lectin-like properties and can act as an opsonin to enhance phagocytosis or to activate the complement system after binding to the bacterium, Vibrio anguillarum (Nakanishi et al., 1991). 2.5 Complement There are a number of excellent studies describing, that most classes of fish possess a lytic complement system. In the early 1980s Nonaka and coworkers isolated C3 and C5 from rainbow trout plasma and C3 from lamprey plasma (Nonaka et al., 1981, 1984). Yano (Fig. 5) and his group have shown that C1-C9 are present in carp plasma (Yano, 1996; Nakao and Yano, 1998). Important comparative work has also been done in the laboratory of Lambris in the USA (Sunyer and Lambris, 1998; Holland and Lambris, 2002). They mention, that unlike homoiotherms, several species of fish have shown to possess multiple forms of complement components (C3 and factor B), that are structurally and functionally more diverse than those of higher vertebrates. Furthermore it is noteworthy to mention, that Woo (Fig. 6) has published, that the alternative pathway is the protective mechanism 3

against hemoflagellate parasites (Cryptobia) in naive fish (Woo, 1992). The classical pathway turned out to be important in acquired immunity after survival of parasitic infections or against bacteria, such as V. anguillarum (Boesen et al., 1999). Another example of the role of complement in the response against scuticociliate (Ciliophora) parasites in turbot will be given in the contribution from the group of Lamas in this special issue. It is tempting to speculate which complement pathway predominates in fish. Yano did suggest, that the alternative pathway in fish is more active than in mammals (Yano, 1996). However, this may also depend on the ambient temperature (season), age or condition of the animals. More recently also evidence was provided for the existence of a lectin complement pathway in fish (Fujita, 2002). These studies, taken together, suggest, that all mammalian complement factors (C1-C9, B, D) are present in teleost blood and that all known pathways are operating in fish (Boshra et al., 2006; Nakao et al., 2011). For further information on complement in fish see review on immunity to infection with piscine flagellates by Woo and Ardelli in this special issue. 2.6 Inflammation We know already for a long time from histopathological studies, that inflammatory responses occur during bacterial, viral, fungal, protozoan and metazoan parasitic infections in fish (Roberts, 1978). According to Finn and Nielsen (1971) acute inflammation responses in bony fish are comparable with those in mammals. Granulocyte infiltration appears 12-24 h after injection of bacteria or Freund's complete adjuvant in rainbow trout. The infiltrating cells (granulocytes and macrophages) increase in numbers till day 2-4. Work by the group of Secombes (Fig. 7) shows, that the macrophages are stimulated to secrete interleukin-1 (IL-1) and eicosanoids, which attract and activate other leukocytes, including lymphocytes (Secombes et al., 1999). These observations can be regarded as an example of the interaction between the innate and the acquired immune system in fish. 2.7 Phagocytic cells Several studies have described, that macrophages and neutrophilic granulocytes are the principal phagocytic cells in fish (Secombes and Fletcher, 1992; Verburg-Van Kemenade et al. 1994). Upon stimulation these cells phagocytose antigenic material and/or exert cytotoxic activity. The killing of intracellular or extracellular pathogens is based upon the release of a number of reactive oxygen species and nitric oxide (NO) (Campos-Perez et al., 2000). The Canadian group of Belosevic (Fig. 8) and the group of Savelkoul / Wiegertjes (Fig. 9) have shown, that macrophages play a key role in the defense against blood parasites in cyprinid fish (Joerink et al. 2004). In one of these experiments carp were infected with Trypanoplasma borreli or Trypanosoma carassii and the activation state of the head kidney leukocytes was determined. The results showed, that T. borelli-infected carp were more prone to increase nitrite (marker for NO release) by classically activated macrophages while T. carassii-infected animals were more prone to increase arginase activity by alternatively activated macrophages (Joerink et al., 2006). This is a clear indication for macrophage polarization in immune responses against parasites. Another interesting study showed, that the NO response of carp 4

macrophages was influenced by genetic differences in the fish host. It was suggested, that transferrin polymorphism could influence the NO release in T. borreli-infected animals (Jurecka et al., 2009). For the role of phagocytes in innate immunity to intracellular bacteria (e.g. Mycobacterium marinum) see also the contribution by the group of Belosevic in this special issue. Phagocytosis of antigenic material by macrophages is not only an activity of the non-specific innate defense system but can also be an initial step in the specific adaptive immune response. As in mammals, we are probably dealing with subpopulations of mononuclear phagocytes, which differ in function. In this respect it is interesting to mention that macrophages from immune fish are more active in phagocytosis than those from control animals. This is probably due to opsonization of antigens by antibodies or due to metabolic activation of the macrophages (Griffin, 1983). Elegant studies by Rombout (Fig. 10) and Verburg-Van Kemenade (Fig. 11) and their teams have shown, that most macrophages from the hindgut of carp are binding purified immunoglobulin (Ig), which is an indication for Fc receptors on the surface of these cells (Koumans-Van Diepen et al., 1994). This is another example of cooperation between the innate immune system (phagocytes) and the acquired immune system (Ig molecules). A new paradigm on phagocytes emerged by the finding of phagocytic B-cells in teleost, suggesting that phagocytosis is a leukocyte function with ancient origin, and B-cell phagocytosis plays a significant protective role especially in lower vertebrates like teleost (Sunyer, 2012). 2.8 Non-specific cytotoxic cells Excellent experiments by the group of Evans (Fig. 12) did reveal the presence of non-specific cytotoxic cells (NCC) in channel catfish. (Graves et al., 1984; Evans and Jaso-Friedmann, 1992). The monocyte-like NCC show a clear lytic activity against certain transformed mammalian cell lines in vitro. Today NCC have been shown in the blood, spleen and head kidney of several teleosts. They are probably involved in killing protozoan parasites and virus infected cells. In addition to NCC distinct natural killer (NK)-like cells have been isolated and cloned from catfish blood (Shen et al., 2004). These NK-like cells are killing allogeneic targets, but are negative for markers that define neutrophils, monocytes and NCC. 2.9 Anti-viral defenses Interferon (IFN) is a cytokine, which is produced by different cell types in response to viral infections. IFN increases the resistance of host cells to different viruses by inducing the expression of proteins, which inhibit the translation of viral mRNA. IFN in teleosts is species specific, e.g. IFN produced by rainbow trout does not protect cyprinid cells in vitro. De Kinkelin and coworkers have shown, that in vivo synthesis of IFN peaks 2-3 days after viral infection and usually precedes the virus neutralizing effects of circulating antibodies, which appear 1 or 2 weeks later (De Kinkelin et al., 1977). In an extensive study on the phylogeny of cytokines, Secombes (Fig. 7) describes, that type I and type II IFN can be distinguished in rainbow trout based upon acid stability (pH 2) and relative temperature resistance (60 °C) (Secombes, 1991). Today IFN activity has been demonstrated in a large number of fish species (Robertsen, 1999, 2006). The 5

group of Leong (Fig. 13) showed, that IFN-induced proteins, e.g. Mx protein, confer antiviral resistance upon uninfected cells and can be used as marker for IFN activity in salmonid fish (Trobridge et al., 1997, Nygaard et al., 2000). For additional information on innate immune responses to viral infections in salmonids see paper by Collet in this special issue. For immunity to betanodaviruses in marine fish see also the contribution from the group of Chen in this special issue. 2.10 Cell cooperation and cytokines The acquired immune response in fish shows the expected characteristics of specificity and memory. At the start of the humoral (Ig) response it takes some time before the first specific antibodies appear in the circulation. This lag phase is needed for antigen processing and cell co-operation between distinct leucocyte populations (accessory cells, B and T cells). Accessory cells (monocytes and macrophages) process different antigens and present the processed antigenic determinants in association with MHC class II molecules to lymphocytes. We know from mammalian studies that the T cell receptor (TCR) on the membrane of a T helper (Th) cell is important for the recognition of the antigenic determinant. It took a long time before the presence of the TCR on lymphoid cells of fish could be proven. The group of Charlemagne (Fig. 14) was the 1st to provide evidence for TCR genes in rainbow trout (Partula et al., 1996). During the interaction between macrophages and Th cells other molecules such as CD3 and CD4 are also essential as co-receptors. Activated macrophages will secrete interleukin-1 (IL1), which is essential for the induction of the response by activating Th cells. Th cells regulate the proliferation and differentiation of B cells into Ig secreting plasma cells by producing IL-2 and other interleukins. Most of these B, Th and accessory cell functions have been verified by using monoclonal antibodies and functional in vitro tests for channel catfish in the group of Miller (Fig. 15) (Miller et al., 1985, 1987) or for carp in the group of Avtalion (Fig. 16) (Caspi and Avtalion, 1984). The occurrence of Th1 and Th2-like responses during parasitic sea lice infection in Atlantic salmon will be discussed in the contribution by Fast in this special issue. Surprisingly, the apparently old and conserved IL-system exhibits a low degree of homology among vertebrate species when its ligands are compared at the level of amino acid sequences (approximately 30% homology between the human and teleost forms of IL-1ß and TNF- ). On the other hand, the secondary and tertiary structure of the IL-1 molecule appears quite conserved: Secombes (Fig. 7) and co-workers have shown that the trout IL-1 sequence can be superimposed on the human crystal structure for IL-1ß (Secombes et al., 1998). It would appear that in an evolutionary context the conservation of three-dimensional structure is more important for cytokine function than its primary sequence. In recent years a variety of cytokine sequences was elucidated for several fish species. Fibroblast growth factor (FGF) and some CC and CXC chemokines have been cloned from a number of fish species (Secombes et al., 1999; Alejo and Tafalla, 2011). Several isoforms of the anti-inflammatory cytokine TGF-ß are described for fish and of the pro-inflammatory cytokines IL-1ß and TNF- ß sequences are published (Secombes et al., 1999). The 1st teleost sequence for IL-1ß was published for rainbow trout in 1999 (Zou et al., 1999) followed in 2000 by the IL-1ß sequence for common carp (Fujiki et al., 2000). A second IL-1ß sequence was found for rainbow trout (Pleguezuelos et al., 2000) 6

and for carp in the following year (Engelsma et al., 2001). An explanation for the existence of 2 related but distinct forms may be the tetraploidization event that occurred (independently) in some species during evolution. In addition to the IL-1ß sequences also the IL-1 receptors type I (Holland et al., 2000) and type II (Sangrador-Vegas et al., 2000) have been published for rainbow trout. Elegant three-dimensional models of IL-1ß and IL-1 receptor type I from trout and sea bass were predicted by comparison with those available from humans or mice (Scapigliati et al., 2004, Fig. 17). The multiple forms of IL-1ß and the presence of both types of receptors indicate that the complexity of the IL-1 system in teleosts is similar to that in mammals. Functional aspects of TNF- action in fish were demonstrated so far using human recombinant TNF- in rainbow trout macrophage (Knight et al., 1998) and assaying for hepatocyte serum amyloid A expression (Jørgensen et al., 2000, Fig. 18). TNF- sequences have been published for Japanese flounder in 2000 by the group of Aoki (Fig. 19) (Hirono et al., 2000). While most teleost cytokine sequences are now becoming available, functional information on cytokines in neuro-endocrine communication in teleosts is still limited. IL-1ß is the beststudied teleost cytokine up till now, with considerable importance and potency in the communication between the neuroendocrine system and the immune system (for reviews see Weyts et al., 1999; Verburg-Van Kemenade et al., 2009). 2.11 Major histocompatibility complex (MHC) During the last 25 years an impressive amount of information became available on class I and class II loci in bony fish. Using the polymerase chain reaction (PCR) Kurosawa and colleagues were able to demonstrate that the genome of carp contains nucleotide sequences showing considerable homology with MHC class I and II sequences in man and mice (Hashimoto at al., 1990). Subsequently, classical class I, class II and/or 2-microglobulin genes have been found in carp (Stet et al., 1993), rainbow trout (Hansen et al., 1999), medaka (Naruse et al., 2000) and Atlantic salmon (Grimholt et al., 2002). An important observation in all bony fish studied is the fact that - in contrast to the situation in mammals - the class I loci are not linked to class II loci, but found on different linkage groups. In other words: an MHC does not exist as a gene complex in the original definition in fish. Several suggestions have been made to explain the absence of linkage between these class I and class II genes (Kuroda et al., 2002). For example, duplication of parts of a chromosome bearing the MHC could have taken place followed by translocation and subsequent loss of certain loci or class II loci were translocated from a prototype MHC to other chromosomes in the ancestor of fish. Stet, Dixon (Fig. 20) and their colleagues suggest that unlinked classical class I and II genes could have an evolutionary advantage (Stet et al., 2003). The offspring in mammals are endowed with only 4 possible MHC genotypes (haplotypes) per pair. Local populations will usually show only a small number of MHC haplotypes. This limited diversity could provide a risk when environmental circumstances are changing or new diseases arise. This risk could only be counteracted by investing a relative large amount of resources (care, energy) in a relative small number of offspring. Fish, on the other hand, usually have high numbers of offspring; up to thousands or even millions. Mortality in fishes can be over 80% in early life, which can be due to predation or diseases. Fish with unlinked MH genes have the ability to endow their offspring with high numbers of genotypes, which will increase the chance that at least some individuals will survive. 7

2.12 Genetic aspects of disease resistance A classic case of selection for disease resistance in carp is described by Kirpichnikov (Fig. 21) (Kirpichnikov et al., 1993). In the period between 1960 and 1990 they made crosses between 3 local fish stocks and managed to select for fast growth and resistance to dropsy (caused by Rhabdovirus carpio). Several other examples of genetic differences in disease resistance in fish have been described (Chevassus and Dorson, 1990; Houghton et al., 1991; Wiegertjes et al., 1993; Jeney et al., 2009; Dixon et al., 2009), but well defined genetic markers are still scarce. For Dorson see Figure 1. In a study with captive-bred chinouk salmon (Oncorhynchus tshawytscha) it was shown that outbred and/or heterozygous (MHC genes) animals were usually more resistant to V. anguillarum, IHN virus and a parasite causing whirling disease than inbred or homozygous fish (Arkush et al., 2002). Only a few studies have yet addressed the functional aspects of MHC molecules in fish. An important study in Atlantic salmon discovered, that there is a significant association between resistance to infectious salmon anaemia virus as well as A. salmonicida and MHC gene polymorphism (Grimholt et al., 2003). Another elegant study in carp shows, that resistance against koi herpesvirus (KHV) is influenced by MHC class II B genes (Rakus et al., 2009). For further information on the biology and host responses to herpesviruses in carp see contribution from the groups of Aoki (Fig. 19) and Steinhagen (Fig. 22) in this special issue.All these studies underline the importance of genetic variation – in particular MHC polymorphism - in a population of fish. Rather peculiar are the observations in Atlantic cod (Gadus morhua), showing that these animals have relatively high numbers of MHC class I genes, but class II and CD4 genes are missing (Pilstrøm et al., 2005; Star et al., 2011). Nevertheless cod is not exceptionally susceptible to disease. This indicates, that their immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. For Pilstrøm see figure 7. Utilizing genomic sequences and genetic linkage maps, quantitative trait loci (QTL) approaches in recent years achieved breeding of some disease-resistant fish families, as evidenced by the pioneer studies of Okamoto's group (Ozaki et al, 2001; Fujii et al, 2007). 3. Ontogeny and tolerance In contrast to higher vertebrates, most fish species are free-living organisms at an early life stage. In other words: the need for an effective defense system against a variety of aquatic pathogens is evident. It is widely accepted, that acquired immunity is not fully functional at hatching. Consequently, during a certain time period fish larvae are mainly depending on their innate immune system (Zapata et al., 2006). Maternal Ig, complement factor 3, lysozyme, lectins, pentraxins and protein inhibitors are present in the eggs of various fish species (Magnadóttir et al, 2005). Moreover, it was suggested recently, that maternal transfer of immunity can be affected by environmental factors experienced by brood fish, such as pathogens or nutrition (Zhang et al, 2013). This means, that manipulation of maternal immunity (e.g. by vaccines, imunostimulants) could be used to 8

enhance the survival rate of fish larvae in the future (Vadstein, 1997). As far as the embryo itself is concerned, several studies show, that blood cell formation and expression of complement and acute phase protein genes starts already before hatching (Huttenhuis et al, 2006a). Just before and soon after hatching myeloid cells (macrophage precursors) can be found in different hematopoietic tissues of carp and zebrafish, followed a few days later by the first lymphocytes in the thymus and in the intestine (Danilova et al, 2004; Huttenhuis et al, 2006b, 2006c). B cells appear 1-2 weeks later in lymphoid organs, such as the kidney and the spleen of carp (Huttenhuis et al. 2005), but also in the pancreas of zebrafish (Danilova and Steiner, 2002). It takes several more weeks before typical B cells appear in the intestine and gills of carp (Huttenhuis et al, 2006c). From these studies the general picture arises, that lymphoid cell populations develop in a certain order. Functional studies are supporting this idea. It was shown by the group of Manning (Fig. 23), that carp are capable of mounting an allograft response as early as 16 days posthatching and a faster secondary response at 1 month old to an allograft from the same donor (Botham et al., 1980). In rainbow trout immunized at day 21 post-hatch with soluble human gamma globulin (HGG) or killed A. salmonicida bacteria a serum antibody response was observed to the bacteria, but not to HGG (Manning et al., 1982). Furthermore, when tested for their memory response by 2nd immunization 8 weeks later, the fish injected with HGG in their primary immunization remained unresponsive to HGG, although normal fish of the same age were able to respond These results with trout agree with findings of Van Muiswinkel et al. (1985) showing, that carp aged 4 weeks are unable to mount an antibody response to sheep red blood cells (SRBC). When these fish were reimmunized 3 months later they still failed to respond, although animals which received their 1st injection of SRBC at the same age showed normal anti-SRBC reactivity. These observations in rainbow trout and carp provide evidence for tolerance induction in young fish, which should be taken into account when vaccinating fish at an early age. 4. Memory and vaccination 4.1 Memory An important feature of the immune system is the capacity to develop immunological memory. A first contact with an antigen usually induces relatively shortlived effector cells (activated Th, Treg, cytotoxic T cells or plasma cells). However, there are also long-lived memory cells among the progeny of the original non-primed lymphocytes. These memory cells retain the capacity to be stimulated by the same antigen. The development of immunological memory was often examined indirectly by monitoring the secondary response. In the case of positive memory this response will be faster and more vigorous than the primary response. The height of the secondary response is dependent on the amount of priming antigen. Early work from the group of Van Muiswinkel (Fig. 24) shows, that a relative low priming dose is usually optimal for memory development in carp (Rijkers et al., 1980a; Lamers et al., 1985). In carp the ratio between secondary and primary antibody responses never reached the high levels as in mammals. Ardelli and Woo (Fig. 6) described rapid increases in complement fixing antibody titres after challenge of brook charr with the parasite, Cryptobia salmositica. 9

The parasites were lysed when they were incubated with immune plasma and complement, which confirms that complement-fixing antibodies can play an important role in protection (Ardelli and Woo, 1997). The existence of immunological memory in rainbow trout has been in further demonstrated in vitro by the group of Secombes. Separated T and B cells from trout, previously injected with A. salmonicida (causative agent of furunculosis), appeared to proliferate in response to various antigen preparations of A. salmonicida. (Marsden et al., 1995). All primed cell populations demonstrated enhanced responses to these antigens in vitro. This provides evidence for the existence of T and B cell memory in vaccinated individuals. Elegant in vitro studies by Kaattari (Fig. 25) and coworkers showed that the B precursor cell frequency in rainbow trout immunized with the hapten-carrier TNP-KLH increased about 15 fold (Arkoosh and Kaattari, 1991). The same authors also showed that there was no evidence for antibody affinity maturation during the primary or secondary response against this T-dependent antigen. Several differences between the secondary responses of mammals and teleosts have been found. One distinction, which can be made, is that the ratio between the secondary and the primary response is much higher in mammals than in teleosts (which can be expressed as “memory factor” or MF). The MF in mice, injected with Salmonella flagellar antigen, is for example 100 (Nossal et al., 1965), whereas in carp injected with A. hydrophila the maximum MF was 6 (Lamers et al., 1985). During the secondary response the dominant Ig isotype in mammals is IgG. Isotype switching (from IgM to IgG) is triggered in mammals during the immune response, in contrast to teleosts where this phenomenon has not been demonstrated. Thank’s to the excellent work on fish Ig by the research groups of Clem / Miller (Fig. 15), of Warr (Fig. 26) and of Jørgensen (Fig. 18) we know today, that teleost fish are producing both IgM and IgD (Wilson et al., 1997; Hordvik et al., 1999; Stenvik and Jørgensen, 2000). Recently, spectacular findings by groups in the USA, Japan, the Netherlands and India provide evidence for a existence of a new Ig class called IgT in trout (Hansen et al., 2005) or IgZ in zebrafish (Danilova et al., 2005) and carp (Ryo et al., 2010). Evidence is provided, that both IgZ and IgT play a role in mucosal responses against parasites and other pathogens (Ryo et al., 2010; Zhang et al., 2010). For a review on the biology and mucosal immunity to myxozoan parasites see the contribution from the groups of Sunyer and Bartholomew in this special issue. This paper underlines the important role of IgT in mucosal responses. In teleosts B-cel memory is probably due to an increase in the antigen-sensitive precursor pool without any of the accompanying characteristics observed in mammals (such as a switch in isotype). In mammals (based on work with rats) on the contrary, an increase in both the precursor pools and clone sizes after initial antigen priming have been observed (Kaattari, 1992) (Fig. 25). Affinity maturation as result of somatic mutation has not been found in teleosts, but recent studies in trout showed that antibodies (Abs) from late immune serum show a greater degree of polymerization than did Abs retrieved earlier in the response from the same animals (Ye et al., 2010). Moreover, a high degree of polymerization of the tetrameric IgM molecule was associated with a higher affinity of the molecule and a longer half-live. It will be important to see if this phenomenon can also be observed in other fish or even in other vertebrates. 4.2 Vaccination 10

Fish farming has grown significantly during the last 50 years. Fish like ayu, carp, catfish, tilapia and salmonids are often kept at high population densities. This increases the risk for dramatic disease outbreaks. Although antibiotics can be used for the treatment of bacterial diseases, this clearly has some drawbacks. Repeated use can induce drug resistance in microorganisms or suppress the immune system of fish (Rijkers et al., 1980b). Moreover, harmful residues may be present in fish sold for human consumption. Hence, it is not surprising that there is a high interest in protecting fish by vaccination. Early vaccination attempts against bacterial diseases by Snieszko, Schäperclaus, Duff and Goncharov in the period between 1938 and 1951 have been described in a previous paper (Van Muiswinkel, 2008). In the 1960s the groups of Klontz (Fig. 1) and Fryer were studying oral vaccination with positive results when rainbow trout were vaccinated against “enteric redmouth” (caused by Yersinia ruckeri) (Ross and Klontz, 1965) and negative results for coho salmon vaccinated against “furunculosis” (caused by A. salmonicida) (Spence et al., 1965). In addition to the usual injection method new procedures for bath or immersion methods have been developed. These impose less stress on fish and are almost as effective as injection. In 1979-1980 a salmon disease called “Hitra disease” was spreading along the Norwegian coast causing serious losses in the fish farming industry. Egidius (Fig. 27) and coworkers were able to determine the causative agent (Vibrio salmonicida sp. nov.) (Egidius et al, 1981) and to develop an effective bath vaccination method together with the research group of Jørgensen (Fig. 18) in Tromsø (Egidius and Andersen, 1979; Egidius et al., 1986; Holm and Jørgensen, 1987). In warmwater fish, such as Indian carps (Catla catla, Labeo rohita, Cirrhunas mrigala) pathogenic strains of Aeromonas hydrophila can cause serious problems. The group of Karunasagar (Fig. 24) has shown, that immunization using a haemolysin-negative strain of this bacterium is possible (Karunasagar et al., 1991). Recently a recombinant protein from the outer membrane of A. hydrophila was isolated, which offers new possibilities for vaccination on large scale (Maiti et al., 2009). A comparison of different application routes (injection, immersion, spray, oral) was made by Johnson and Amend (Fig. 28) in the 1980s using V. anguillarum and Y. ruckeri bacterins in salmonids. Injection and immersion gave usually the best protection upon challenge (Johnson and Amend, 1983a, 1983b). Oral vaccination usually evokes only minimal immune responses in the host. It is not easy to explain this phenomenon. In a classic study by Rombout (Fig. 10) and his team it was shown that the 2nd gut segment plays a key role in antigen transport and antigen processing by macrophages (Rombout and Van Den Berg, 1989). Numerous lymphoid cells are also present in this part of the gut. These cells probably play a role in local (mucosal) responses. Repeated oral administration of bacterial antigen resulted in antibodies in skin mucus and bile, but not in serum (Rombout et al., 1989b). It is expected that encapsulation of vaccines is needed to prevent digestion in the 1st part of the gut and to ensure that the essential antigenic determinants reach the 2nd gut segment in a non degraded and immuno-stimulatory form (Joosten et al., 1995). A detailed description of the cells types, molecules and responses involved in intestinal immunity in fish can be found in a thorough review by Rombout et al. (2011). A successful oral vaccine against Edwardsiella ictaluri in channel catfish was developed in a cooperative study by the groups of Plumb (Fig. 29) and Paterson (Fig. 30). Interestingly, they 11

observed protection of vaccinated animals in the absence of agglutinating serum Ab (Plumb et al., 1994). This could mean, that in this case a mucosal response was more important for protection than a systemic response. Another example of mucosal defenses is the antibody response of channel catfish to the ectoparasite, Ichthyophtirius multifiliis (causing white spot disease). The group of Dickerson has performed elegant studies showing, that catfish antibodies bind to a specific coat protein (“immobilization antigen”) forcing the parasite to exit the fish skin prematurely (Clark and Dickerson, 1997). In later studies they also discovered, that successful immunization of catfish was possible when these “immobilization antigens” were used as vaccine (Wang et al., 2002). For more information on immunity to I. multifiliis see review by Dickerson and Findly in this special issue. In the 1980s several licensed bacterial vaccines were developed, but viral vaccines were scarce. Probably the 1st commercial anti-virus vaccine (produced by Bioveta, Czech Republic) was an inactivated vaccine against spring viraemia of carp (SVC) caused by Rhabdovirus carpio was developed by Tesar ik (Fig. 31) and Macura in 1981. In the last 10-20 years, various vectors have been used to produce large quantities of antigens by recombinant DNA technology. In aquaculture, research on recombinant vaccines has focused mainly on viral diseases, because traditional production of viruses in cell culture systems is relatively expensive. Glycoproteins of viruses causing viral haemorrhagic septicaemia (VHS) and infectious haematopoietic necrosis (IHN) in rainbow trout elicit protective antibodies Lorenzen and Olesen, 1997). Studies by Lorenzen (Fig. 32), Boudinot (Fig. 9) and colleagues have shown, that genetic immunization using DNA is feasible. They showed, that intramuscular injection of plasmid DNA containing genes encoding glycoproteins or nucleocaspid protein in rainbow trout, protected against challenge by VHSV and IHNV (Boudinot et al., 1998; Lorenzen et al., 2002). This protection is probably not only due to the induction of a long-term specific immune response, but also due to an early, nonspecific antiviral protection mediated by interferon. Work by the group of Leong (Fig. 13) has shown, that DNA vaccines encoding viral glycoproteins induced nonspecific immunity and high levels of Mx protein in the kidneys and liver of injected fish (Trobridge et al. 1997; Kim at al. 2002). For a review on the role of different Ig classes and T cell populations in acquired immunity after vaccination against infectious pancreatic necrosis (IPN) virus in salmon see contribution from the group of Evensen in this special issue. 4.3 Immunostimulation Many vaccines based upon recombinant antigens or killed pathogens are not very effective as such. In most cases the use of adjuvants or immunostimulants is necessary to increase vaccine efficacy. In addition to traditional adjuvants (e.g. mineral oils) a new generation of adjuvants has been developed such as ligands for Toll receptors or cytokines (Tafalla et al., 2013). The application of immunostimulants as such (without a vaccine) is also an interesting application. Substances such as ß-glucans and certain plant extracts can be incorporated in food and may directly activate the innate defence system. Work from the group of Dinakaran Michael (Fig. 33) has shown, that the addition of medicinal plant (e.g. Eclipta alba, Nyctanthes arbortristis) extracts to fish feed is 12

effective in stimulating innate immunity (lysozyme, alternate complement activity) and protection against virulent A. hydrophila in tilapia (Christibapita et al., 2007; Kirubakaran et al., 2010). Moreover, the oral application of various immunostimulants is a promising approach for protecting young fish as long as their acquired immune system is not fully developed (Vadstein, 1997; Bricknell and Dalmo, 2005; Magnadottir et al., 2006). Another new approach is the use of probiotics in aquaculture. According to this method live bacterial or yeast strains are administered to fish in order to promote their health. Different probiotics can elevate phagocytic, lysozyme, complement, respiratory burst activity or influence cytokine production (Nayak, 2010). At present the various factors (e.g. dose, duration, species) affecting the effect of probiotics are under investigation in several laboratories.

5. Experimental fish models There is a high need for well defined inbred lines of fish as experimental animals for studies on the interaction between pathogens and the host immune system. The production of inbred lines by conventional full-sib mating is a time consuming process. For most species this will take between 10 and 30 years. An alternative is the use of fish reproducing under natural conditions by spontaneous gynogenesis, such as the Gibel or Ginbuna (Carassius auratus gibelio) (Yamashita et al., 1993). Using this animal model the group of Nakanishi (Fig. 34) together with colleagues from Japan, Germany and Chili was able to produce monoclonal antibodies against CD4 and CD8 surface molecules, which enabled them to distinguish between cytotoxic T cells (CD8+/CD4-), helper T cells (CD8-/CD4+) and CD4/CD8 double positive early T cells (Toda et al., 2011; Takizawa et al., 2011). These new tools will allow further research on the characterization and function of T cell subsets in fish (Nakanishi et al., 2011). For further information on the antiviral functions of CD8+ cytotoxic T cells in teleost fish see the review by Fischer et al. in this special issue. It is also possible to produce gynogenetic fish in the laboratory. The last approach is based on the production of diploids by the use of inactivated sperm and suppression of the 2nd meiotic division by cold-shocking of the eggs. This technique has been successful for a number of fish species, including trout (Purdom, 1969) and carp (Komen et al., 1991). These relatively large fish offer the advantage, that they yield sufficient numbers of immune competent cells for functional in vitro tests, cell sorting and subsequent transcriptome analyses (Henkel et al., 2012). For an overview of the available clones in fish see the excellent review by Komen and Thorgaard (2007). Another important publication which stimulated research in the production of homozygous gynogenetic fish came from the laboratory of Streisinger (Streisinger et al., 1981). This paper has led to the production of homozygous and heterozygous clonal lines of zebrafish (Danio rerio) and has given rise to the zebrafish as the animal model for research on embryonic development of vertebrates. Today a wealth of genetic and genomic information on zebrafish is available. Its genome is currently sequenced and gene microarrays and insertional mutants are available. Several laboratories have developed bacterial and viral disease models with the zebrafish to study the immune response to infection (Sullivan and Kim, 2008; Meijer and Spaink, 2011). For example, 13

the pathogenesis and inflammatory response to the bacterium, Edwardsiella tarda, was investigated (Pressley et al., 2005) and the susceptibility to Flavobacterium columnare was increased when a protein in the Toll-like receptor (TLR) signaling pathway was suppressed (Chang and Nie, 2008). Recent publications have shown, that a whole range of different TLRs can be found in fish (Palti, 2011). For a review on innate immunity to intracellular bacteria in zebrafish see also the contribution from the group of Belosevic in this special issue. One of the Chinese groups, who have been very active in the study of innate immune receptors in zebrafish and other fish species is the laboratory of Shao (Fig. 35). They have been studying the genomic organization of TLRs and the possible functions (Zhu et al., 2013). For more information on TLRs and their recognition of fish pathogens see also the review by Wiegertjes and co-workers in this special issue. The results of these studies will not only strengthen our knowledge of the mechanisms behind innate and acquired immunity, but also provide important clues for the development of new vaccines and prophylactic measures against diseases in economically important fish. 6. Books and meetings There has been a series of international meetings focusing on fish immunology and/or vaccination in the 1980s and 1990s. In most cases proceedings were published, which provide us with an insight in how our field was developing over the years. The International Association of Biological Standardization (IABS) held a symposium entitled “Fish Biologics: Serodiagnostics and Vaccines” at the National Fish Health Research Laboratory, Leetown (WV), USA, in 1981 with Anderson and his staff as local organizers. Knowledge of fish diseases and their prevention through the use of biologics (diagnosis, vaccines) was highly needed, but also data on basic fish immunology were required as background (Anderson and Hennessen, 1981). The group of Van Muiswinkel organized in the same year a conference on “Immunology and Immunization of Fish” at Wageningen University (NL) as special meeting of the International Society of Developmental and Comparative Immunology (ISDCI). The aim of this meeting was to bring together comparative immunologists with more applied scientists orientated towards aquaculture and toxicology (Van Muiswinkel and Cooper, 1982). In 1983 a successful symposium on “Fish Immunology” was organized at the university of Plymouth (UK) by the Fisheries Society of the British Isles. The local organization was in the hands of Margaret Manning (Fig. 23), Mary Tatner and their staff. Both innate immunity (transferrin, lectins, lysozyme) and acquired immunity (cell populations, responses, memory, tolerance), but also stimulating or suppressive factors (vaccines, adjuvants, pollution) were discussed and described in the proceedings (Manning and Tatner, 1985). Two years after the Plymouth meeting another international conference on “Fish Immunology” took place at the Sandy Hook Laboratory, NJ, USA. Again data on the fish immune system and more applied subjects such as immunosuppression by pesticides and heavy metals, immunity to bacterial, viral and parasitic diseases were discussed (Stolen et al., 1986). The main organizer of this meeting, Joanne Stolen, was also active in publishing books and organizing other meetings, such as a workshop on “Modulators of Fish Immune Responses” at Breckenridge (CO, USA) in 1993 (Stolen et 14

al., 1994b). She was the driving force behind a special series of books on “Techniques in Fish Immunology” providing a detailed description (including manuals) of an impressive number of techniques adapted for work with different fish (volumes 1-3) (Fig. 36) or shellfish species (volume 4) (Stolen et al., 1990, 1992, 1994a, 1995). Also in the 1990s two important IABS meetings on “Fish Vaccinology” took place in Norway. The 1st one was at Oslo in 1996 (organizers Gudding, Lillehaug, Midtlyng and Brown) and the 2nd one at Bergen in 2003 (principal organizer Midtlyng, Fig. 37). The proceedings of these 2 meetings provide an almost complete coverage of fish vaccinology and include such areas such as the immune system of fish and crustaceans, methods for vaccine delivery, bacterial, viral and parasitic antigens, antigen production, recombinant and DNA vaccines, adjuvant technology, regulatory affairs, environmental effects and vaccination strategies (Gudding et al., 1997, Midtlyng, 2005). An excellent textbook on “Fish Vaccination” edited by Ellis (Ellis, 1988) (Fig. 38) should be mentioned in this historic context. This book gives an overview of the general and practical aspects of fish vaccination and provides the reader with detailed information on vaccination against specific bacterial, viral and parasitic diseases as known in those days. One of the best general textbooks on the fish immune system was published by Iwama and Nakanishi (Fig. 35) as editors (Iwama and Nakanishi, 1996). This book contains extensive chapters on the cellular and humoral components of innate and acquired responses, ontogeny of the immune system, environmental factors and stress, infection and disease. There are several other organizations or societies, who have organized stimulating international meetings and/or workshops. The presentations were published as abstract or as full paper in special issues of international journals. The International Society of Developmental and Comparative Immunology (ISDCI), founded in 1976, organized every 3 years a meeting from 1980 onwards (www.isdci.org ). The International Society of Fish and Shellfish Immunology (ISFSI) was initially started in 1990 as the Nordic “Projektstyregruppen for Fiskeimmunologi” with the aim to coordinate fish immunology projects and to reduce diseases in aquaculture. Pilström (Fig. 7) was elected as 1st president of this group in 1993. This organization has now fully developed into an international society with meetings every 2-3 years (www.isfsi.org ). Since 1989 the Fish Health Section of the Asian Fisheries Society (AFS) organizes a symposium on “Diseases in Asian Aquaculture (DAA)” every 3 years (www.fhs-afs.net). During these DAA meetings there are one or more sessions on immunity in fish and shellfish. At the Papanin Institute for Biology of Inland Waters (IBIW), Russian Academy of Sciences, Borok, Yaroslavl (RU), every 3-5 years an international meeting on “Problems of Immunology, Pathology and Fish Health Protection” is organized by Mikryakov and colleagues (www.ibiw.ru ). Two more workshops turned out to be of high value for people, who are interested in the interaction between fish defenses and diseases: a) The Eastern Fish Health Workshop, organized annually since 1976 by Cipriano and the staff of the National Fish Health Laboratory, Leetown (WV), USA (www.lsc.usgs.gov ). b) The Fish Immunology and Vaccination Workshop, organized annually since 1998 at the Wageningen University (NL) by Wiegertjes (Fig. 9) and colleagues (www.wageningenur.nl/en and search for Cell Biology & Immunology Group).

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7. Conclusions During the last 50 years considerable progress has been made in describing and understanding the immune system of fish. Antigenic stimulation in fish evokes responses, which are in many respects comparable with those in warm-blooded vertebrates. An effective innate immune system is present and acquired immune responses show the expected characteristics of specificity and memory. There are clear influences of genetic factors, but also environmental factors such as temperature and stress play an important role. Our knowledge of the immune system of fish can be used for evaluation of the health status of fish under different conditions (water quality, pollution), but can also be used for vaccination and breeding for disease resistance in aquaculture. 8. Acknowledgments The authors wish to thank the following institutes for providing them with information or manuscripts: Library National Science Center, US Geological Survey, Leetown (WV, USA); Library Wageningen University-WUR (NL). The following people have been very helpful in collecting data or pictures: R. Cipriano (USA), E. Egberts (NL), C.H.J. Lamers (NL), V.R. Mikryakov (RU), V. Zlabek (CZ). Moreover, G.F. Wiegertjes (NL) and the referees are acknowledged for their valuable comments.

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Legends to the figures Fig. 1. From left to right George W. (Bill) Klontz (1929-2000), Michel Dorson and Trevor T.P. Evelyn at the symposium “Fish biologics: serodiagnostics and vaccines”, Leetown (WV), USA, 1981. Bill Klontz received his doctor of veterinary medicine degree from Washington State University, Seattle (WA, USA) in 1963. He was first employed by the US Fish & Wildlife Service and the Texas A&M University. From 1972 onwards he joined the University of Idaho as professor at the Fisheries department till his retirement in 1994. He developed the concept of fish health management, which concentrates on the importance of the environment of the fish in the disease response. His worldwide contributions were recognized by the American Fisheries Society, who awarded him the Snieszko Distinguished Service Award in 1994. Michel Dorson received his doctor of Microbiology degree from the Université Paris 7, Paris (France) in 1985. He joined the Institut National de la Recherche Agronomique (INRA) after finishing his MSc degree in Biology (1967). He was director of the Fish Pathology Laboratory (INRA) from 1981-1985 and director of the Experimental Fish Facilities (INRA) at Jouy-en-Josas (France) from 1986 till his retirement in 2004. He is an internationally recognized expert on anti-viral responses and vaccination of rainbow trout. Trevor Trust received his doctor of Microbiology-Biochemistry degree from the University of British Columbia (BC, Canada) in 1963. He was research scientist and head of the Fish Health and Parasitology Section, Biological Sciences Branch, Pacific Biological Station, Nanaimo (BC, Canada) from 1964-1997. Now he is scientist emeritus of the same institute in Nanaimo. He was president of the Fish Health Section of the American Fisheries Society (FHS/AFS) in 1983 and honoured by the FHS/AFS with the Snieszko Distinguished Service Award in 1992. He wrote > 85 scientific papers on bacterial diseases (e.g. Renibacterium salmoninarum) and vaccination of salmonids. Photographs in this review were made by the 1st author unless otherwise mentioned. Fig. 2. Thelma C. Fletcher obtained her PhD degree on mucus of fish at the University of Aberdeen (Scotland, UK) in 1968. Her publication on the immunoglobulins in the serum and mucus of plaice (Fletcher and Grant, 1969) was first in describing the presence of IgM-like molecules in this fish species. Fletcher wrote more than 100 papers in total and edited at least 6 books. Today she is honorary research fellow at the School of Biological Sciences, University of Aberdeen. Picture was taken during the “Fish Biologics: serodiagnostics and vaccines” symposium, Leetown (WV), USA, 1981. Fig. 3. Yuzuru Suzuki earned his PhD degree on inflammation in fish from the University of Tokyo (Japan) in 1976. His early research was in fish physiology with special focus on mucus lectins of eel. After his appointment as full professor at the Fisheries Laboratory, University of Tokyo, in 2000, studies by his group on fish mucus lectins have developed further utilizing Fugu (tiger puffer fish) as model and this approach was facilitated quite much by the available genome information. His new laboratory was called the “Fugu Breeding Institute (FBI)”, which has produced more than 30 high impact papers on the diversity of fish mucus lectins, identification of adaptive immune factors, and genome-based Fugu breeding. He has retired as "FBI Director" in 2013, and started his new investigation activities on conservation of the natural environment, while enjoying bird watching, his lifelong hobby. The picture was taken at the ISDCI Congress, 2003 (St. Andrews, Scotland, UK), provided by Y. Suzuki and published with his permission. 31

Fig. 4. Anthony E. (Tony) Ellis (1947-2010) received his PhD degree from the University of Aberdeen (Scotland, UK) in 1973. He has spent his whole career at the Marine Laboratory, Aberdeen, where he has been the head of the Immunology/Vaccines group, which has been closely integrated with the Scottish Fish Immunology Centre (founded 2001). He has published over 200 refereed papers on pathogenic mechanisms, virulence factors and fish immunology and edited 3 books. His review on innate host defence mechanisms (Ellis, 2001) belongs to the best cited papers of our field. He and Mary Tatner were the founding editors of the journal “Fish and Shellfish Immunology”. As a gesture of respect for Tony Ellis and his work, he was awarded an Honorary Professorship at the University of Aberdeen and the new fish health centre of the Marine Laboratory was called “The Ellis Building” in 2010. This picture was taken at the occasion of the PhD defence by Nuno M.S. dos Santos at Wageningen University (NL) in 2000. Fig. 5. Tomoki Yano finished successfully his PhD project on the molecular structure of kitol at the Division of Agriculture, Kyushu University (Japan) in 1968. In the period from 1968-1989 he was working as assistant/associate professor at the department of Fisheries, Kyushu University. He was appointed as full professor at the same department in 1989 till his retirement in 2004. From 2004 till 2009 he was professor at the Fukuoka Woman’s Junior College. He is well known for his work on the complement system of carp (at least 39 papers), humoral factors of the innate system (12 papers) and immunostimulants in fish (8 papers). In addition he published several reviews or book chapters of high quality. This picture was obtained from T. Yano and is published with permission. Fig. 6. Patrick T.K. Woo lecturing during the 4th Nordic Symposium on Fish Immunology at Hirtshals (DK) in 1998. He received his PhD degree from the University of Guelph (ON, Canada) in 1968. In the period between 1968 and 1972 he was MRC postdoctoral fellow and Ballard fellow in the Ontario Veterinary College, and was also FAO or IDRC fellow on trypanomiasis research in East Africa (e.g. Uganda) and West Africa (e.g. Nigeria). From 19721974 he was appointed as associated faculty at the department of Veterinary Microbiology & Immunology, University of Guelph. Subsequently he was active as assistant/associate professor at the department of Integrative Biology, University of Guelph from 1974-1985. In 1985 he was promoted to professor at the same department till his retirement in 2006. He was also appointed as director of the Axelrod Institute of Ichthyology, University of Guelph, in 2003. As recognition for his scientific achievements he received the Robert A. Wardle Award and Medal from the Canadian Society of Zoologists in 1995. He published more than 230 papers on African trypanomiasis and haemoflagellates (Cryptobia) in fish. Moreover, he is the editor of a wellknown book series, “Fish Diseases and Disorders” (CABI Publishing, Oxon, UK). Fig. 7. Christopher J. (Chris) Secombes (left) and Lars Pilström (right) (1938-2009) during a EU Erasmus Programme meeting at Uppsala (Sweden) in 1997. Chris Secombes did his PhD work in a project on developmental immunology of trout and carp and on the evolution of antigen-trapping mechanisms coordinated by the University of Hull (UK, Margaret Manning) and the Marine Laboratory, Aberdeen (Scotland, UK, Tony Ellis). He obtained his PhD degree from the University of Hull in 1981. After a post-doc period at Wageningen University (NL) he moved to the University of Aberdeen in 1982 and became professor of Zoology in 1997. Over the years he has been head of the department of Zoology and head of the School of Biological Sciences in Aberdeen. In 2001 he became the head of the Scottish Fish Immunology Research Centre and from 2003-2006 he was President of the 32

International Society of Developmental and Comparative Immunology. In 2004 he was appointed to the Established Chair of Zoology at Aberdeen. He has written over 300 (!) scientific publications and is editor of the journal “Fish and Shellfish Immunology” since 1995. In 2007 he was elected Fellow of the Royal Society of Edinburgh (RSE) and was also awarded the RSE Alexander Ninian Bruce Prize for “his outstanding contribution to our understanding of the immune system of fish, particularly salmonids”. Lars Pilström obtained his PhD degree in chemistry and biology from Uppsala University, Uppsala (Sweden) in 1970. From 1970 till 1972 he was assistant in Animal Physiology at the same university. After a short period in the pharmaceutical industry, he became university lecturer in Animal Physiology, Uppsala University, in 1978. His appointment as professor in Molecular Immunology at the department of Cell & Molecular Biology, Biomedical Centre, Uppsala Universty, followed in 2000. In the years 2002-2005 he was involved in projects of the Norwegian Research Council. His research group is famous for their work on the immune sytem of Atlantic cod (Gadus morhua), in particular immunoglobulin genes, humoral responses and MHC genes (Magnadóttir et al., 2001; Pilström et al., 2005). He was elected in 1993 as the 1st president of the Nordic Society for Fish Immunology (at present: International Society for Fish and Shellfish Immunology). Fig. 8. Miodrag (Mike) Belosevic received his PhD degree from the Institute of Parasitology, McGill University (Montreal, Canada) in 1985. From 1985-1988 he received post-doctoral training at the McGill Centre for Tropical Diseases, Montreal General Hospital, and at the Department of Immunology, Walter Reed Army Institute of Research, Washington (DC, USA). In the period between 1988 and 1997 he went through the ranks of assistant / associate professor at the departments of Zoology (Faculty of Science), Immunology (Faculty of Medicine), Medical Microbiology and Infectious Diseases (Faculty of Medicine), University of Alberta, Edmonton (Alberta, Canada). From 1997 till present he was appointed as professor at the department of Biological Sciences, and cross-appointed in the departments of Medical Microbiology & Immunology, Civil and Environmental Engineering (2000), Agricultural Food and Nutritional Sciences (2006) and School of Public Health (2008) of the University of Alberta. In 2008 he was appointed as distinguished university professor of Biological Sciences and School of Public Health. His primary research interest is comparative molecular immunology and his main research goals are: 1) cytokine regulation of macrophage hematopoiesis and antimicrobial mechanisms in mammals and fish; 2) mechanisms of cellular immune responses to protozoan parasites; 3) detection and inactivation of chemical contaminants, pathogens and prions in environmental samples. He has published >250 refereed publications and received many important awards, including the Wardle Medal of the Canadian Society of Zoologists (2002), Rudolph Hering Medal, American Association of Civil Engineers (2002), Kilam Mentor Award, Killam Trust (2004), Alberta Centennial Medal, Province of Alberta (2005), University Cup, University of Alberta (2006), Elected Fellow of the Royal Society of Canada (2008) and Clark P. Read Mentor Award, American Society of Parasitologists (2009), Fry Medal, Canadian Society of Zoologists (2013), Queen Elisabeth II Diamond Jubilee Medal (2013) and Doctor of Science (honoris causa), University of Waterloo. The picture was obtained from M. Belosevic and is used with permission. Fig. 9. Geert F. Wiegertjes (left) and Pierre Boudinot (right) during the Fish Immunology Workshop at Wageningen (the Netherlands) in 2010. Geert Wiegertjes received his PhD degree in Immunology from Wageningen University 33

(WUR, NL) in 1995. In 1989 he joined the Cell Biology & Immunology group of WUR as fellow and later was promoted to assistant and associate professor. In 2013 he was appointed as “personal professor” at the same department. He is well known for his work on immunogenetics of disease resistance in carp, in particular the interaction between the host and blood flagellates (Trypanoplasma borreli). Moreover, he was the main organizer of the annual Fish Immunology / Vaccination Workshop at Wageningen during the last 14 years. He was elected as Education Secretary of the International Society of Developmental & Comparative Immunology (ISDCI) in 2012. Pierre Boudinot is team leader of the Infection and Immunity of Fish group belonging to the laboratory of Molecular Virology and Immunology, Institut National de la Recherche Agronomique (INRA), Jouy-en-Josas (France). He received his PhD degree in 1995 from Université Paris 7, on lambda light chains of mouse Igs, with Pierre André Cazenave at the Institut Pasteur. His group has performed excellent studies on the defence mechanisms elicited in fish (rainbow trout, zebra fish) by viral pathogens, e.g. functional characterization of RNA and DNA virus-induced responses. He also developed analysis of immune repertoires (TCR, Igs and gene families of innate immunity) in fish. Fig. 10. Jan H.W.M. Rombout obtained his PhD in Zoology (Entero-endocrine cells of cyprinid fish) from Wageningen University (WUR, NL) in 1980. Starting in 1973 he went through the ranks of assistant and associate professor of the Developmental Biology and Cell Biology & Immunology groups (WUR) till his retirement in 2011. From 2007 onwards he was appointed as adjunct professor at the University of Nordland, Bodø (Norway) till present. Jan Rombout is well known for his extensive work on mucosal immunology and developmental biology of fish and vaccination of shrimp. He was awarded with the honorable membership of the International Society of Fish Immunology in 2010, because of his outstanding scientific contributions to this field. The picture was made by Willem J.A. Valen around 1980 and is used with permission. Fig. 11. Bertha M.L. (Lidy) Verburg-van Kemenade at work in 2011. She received her PhD degree in Animal Physiology (Neuroendocrinology) from the Radboud University, Nijmegen (NL) in 1987. In the same year she joined the Cell Biology & Immunology group of Wageningen University (WUR, NL) as assistant professor and started a successful research line on the interaction between the neuroendocrine system and the immune system in fish (for reviews see Weyts et al., 1999; Verburg-van Kemenade et al., 2009). She is an internationally recognized expert on the effect of stress in fish. She was board member of the Section Animal Physiology of the Netherlands Organisation for Scientific Research (NWO) and of the Royal Dutch Society of Zoology (KNDV). Since 2006 she is a board member of the European Society of Comparative Endocrinology (ESCE). Fig. 12. Donald L. (Don) Evans got his PhD degree from the University of Arkansas in 1971 and did a post-doctoral research fellowship (1971-1973) at the University of Texas M.D. Anderson Hospital and Tumor Institute, Houston (TX, USA). At present he is professor of Immunology at the department of Infectious Diseases, University of Georgia, College of Veterinary Medicine, Athens (GA, USA). His “Don Evans Lab” is famous for their work on non-specific cytotoxic cells in catfish, tilapia and zebrafish. The picture was obtained from D.L. Evans and is published with permission.

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Fig. 13. Jo-Ann C. Leong earned her PhD in microbiology (reverse transcriptase and retroviruses) from the University of California (San Francisco Medical School) in 1971. After postdoctoral training in biochemistry at the University of California she joined the department of Microbiology, Oregon State University (OSU), Corvallis (OR, USA) in 1975 and was appointed chair of this department in 1996. She has received numerous recognitions, including the OSU Alumni Association Distinguished Professor Award in 1991 and the OSU Distinguished Professor Award in 1993. She and her group at OSU have studied the viral diseases of Pacific salmon and, using genetic engineering techniques, developed vaccines (e.g. against Infectious Hematopoietic Necrosis Virus) to protect young fish. In 2001 she became director of the Hawaii Institute of Marine Biology and professor at the School of Ocean and Earth Sciences, University of Hawaii, Kaneohe (HI, USA). After her arrival in Hawaii she has become intrigued by the devastating diseases in corals (Van Oppen et al. 2009) and tropical fish and expanded her research into these research arenas. This picture was obtained from J-A. C. Leong and is published with permission. Fig. 14. Jacques Charlemagne (1939-2002) lecturing during a Comparative Immunology meeting at Masserberg (former DDR, present Germany) organized by Herwart Ambrosius in 1982. Charlemagne did his PhD work in the 1960s at the laboratory for Animal Biology, Université Paris 6, Paris (France) with famous embryologist, Charles Houillon, as supervisor. At that time he was focusing on graft rejection in urodele amphibians (Pleurodeles waltlii and axolotl). It turned out, that these animals reject grafts in a rather chronic way. In later studies, when he was working as professor in the Comparative Immunology group, CNRS, Pierre & Marie Curie University (Paris), it became clear, that urodeles are also poor antibody producers upon immunization. In order to explain these phenomena his team developed a series of molecular and gene cloning approaches. They found, that these animals express many MHC molecules of the class I type, but that their class II were poorly polymorphic. This special feature is shared by Atlantic cod (Pilström et al., 2005) and makes them rather unique experimental models (Star et al., 2011). In 1993 the Paris group was able to clone TCR beta genes in the axolotl. This was the 1st time, that TCR genes were shown in a cold-blooded vertebrate. Not much later they were also the 1st to publish the structure and diversity of the TCR alpha chain in fish (rainbow trout) (Partula et al., 1996). This picture was obtained from Cor H.J. Lamers and is published with permission. Fig. 15. Norman W. Miller received his PhD degree in Biology from the University of Delaware, Newark (DE, USA) in 1980. He joined the Department of Microbiology (headed by L.W. Clem), University of Mississippi Medical Center, Jackson (MS, USA) as postdoctoral research associate (1980-1985). Subsequently he went through the ranks of instructor (1985-1987), assistant professor (1987-1992), associate professor (1992-1998) and was appointed as professor at the same department in 1998. From 2009 onwards he is active as professor emeritus. He served as secretary-treasurer for the International Society for Developmental and Comparative Immunology (ISDCI) from 2000 till 2009. His publications on cell cooperation during the immune response (Miller et al., 1985), the effect of temperature on different cell populations (Miller and Clem, 1984) have certainly played an important role in the success of the “Jackson catfish lab”. Moreover, together with his colleagues, Melanie Wilson and Eva Bengtén, he developed long term lymphoid cell lines (Miller et al., 1994, Shen et al, 2004), which are widely used today. This picture was obtained from N.W. Miller and is used with permission. 35

Fig. 16. Ramy R. Avtalion received his degree of Doctor of Veterinary Medicine (1961) in France and completed post-doctoral studies at the Pasteur Institute of Paris (1963). From 1963 till 1964 he was the head of the section Anaerobic Vaccine Preparation, Kimron Veterinary Institute, Bet Dagan, Israel. He joined the department of Life Sciences, Bar-Ilan University, Ramat Gan, Israel, in 1964. Subsequently he was appointed as lecturer (1964-1969), assistant/associate professor (1969-1981) and professor (1981-2002). He was awarded the honourable certificate of appreciation of the Israel Immunology Society in 2005.At present he is still active as professor emeritus. He founded the Laboratory of Fish Immunology and Genetics in 1967. This laboratory has been very active in different fields of comparative immunology, genetics and tissue regeneration. Well known are his studies on the effect of temperature on the immune response in fish (Avtalion, 1981), the development of gynogenetic lines and clones of tilapia (Don and Avtalion, 1988) and the role of natural antibodies in protection against fish pathogens (Sinyakov et al., 2002). This picture was obtained from R.R. Avtalion in 2010 and is used with permission. Fig.17. Giuseppe Scapigliati visiting an emperor penguin rookery at the Cape Washington (Ross Sea, Antarctica) in 2010. He received his Doctoral degree in Biological Sciences (PhDequivalent) from the University of Siena, Siena (Italy) in 1980. Moreover, he received a PhD degree on “Cellular and molecular studies of the immune system of sea bass (Dicentrarchus labrax L.)” from the University of Aberdeen, Aberdeen (Scotland, UK) in 2005. From 19831991 he was post-doctoral researcher in the immunopharmacology group of a human vaccines company, Sclavo SpA (Siena, IT). From 1991-1998 he was appointed as lecturer in Zoology at the University of Tuscia, Viterbo (IT) and from 1998 to 2011 as associate professor of Animal Biology and Animal Biotechnology at the Faculty of Sciences, University of Tuscia, Viterbo (IT). He is currently full Professor of Animal Biotechnology and Zoology at the same University. He has published extensively on fish immunobiology with particular emphasis on the marine species, Dicentrarchus labrax, the first fish species where T cells have been directly investigated with specific cell markers. Most of experimental work focussed on functional immunology of control and immunized fish, and recently he participated in a consortium on the coelacanth fish genome cloning (Nature doi:10.1038/nature12027). Scientific interests also include Antarctic fish immunobiology and he participated in several expeditions of the Ross Sea Italian base. In 2010 he organized the 1st congress of the European Organisation for Fish Immunology at Viterbo (IT) and became the 1st president of this organisation for the years 2010-2013. The picture was obtained from G. Scapigliati and is used with permission. Fig.18. Trond Ø. Jørgensen got his PhD degree on “Tumour immunology / idiotype specificity and network regulation” from the University of Tromsø (Norway) in 1982. He received postdoctoral training at the department of Immunology, University of Tromsø from 1982 till 1984. Between 1984 and 1989 he was active as senior research scientist at the department of Marine Biotechnology, University of Tromsø. In 1989 followed his appoinment as professor at the department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø. From 2007 onwards he also became director of the MabCent-SFI, a centre for researchbased innovation on marine bioactives and drug discovery (hosted by the University of Tromsø). He and his group have published extensively (> 100 papers) in the field of immunology and biotechnology. Important for the field of fish immunology and vaccination have been his publications on disease agents, such as Vibrio salmonicida, V. anguillarum and Aeromonas salmonicida in Atlantic salmon and the description of the peculiar immune system of Atlantic 36

cod. Picture was obtained from University of Tromsø and is used with permission. Fig. 19. Takashi Aoki obtained his PhD degree in Agriculture from the University Tokyo (Japan) in 1973. After a postdoctoral period (1973) at the department of Fisheries (Tokyo University) he was appointed as assistant professor at the department of Microbiology, School of Medicine, Keio University (Tokyo, Japan) in 1974. Subsequently, he became associate professor of the Laboratory of Aquatic Pathomicrobiology, Department of Fisheries, Faculty of Agriculture, Miyazaki University (Miyazaki, Japan) from 1975 to 1990 and later professor at the Laboratory of Applied Genetics and Biochemistry, Department of Biological Resources, Faculty of Agriculture, Miyazaki University. In 1994, he became professor at the Tokyo University of Fisheries (now Tokyo University of Marine Science and Technology). He retired in 2007 and was appointed Professor Emeritus and also Distinguished Professor of the Laboratory of Genome Science, Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology from 2007 to 2012. He is currently a Visiting Professor at the Consolidated Research Institute for Advanced Science and Medical Care (ASMeW), Waseda University, Tokyo. He has published extensively (> 470 papers!) on fish and shellfish diseases and immunology and on cloning and characterization of immune-related genes. Moreover, he and his group have studied virulence and drug resistance genes of fish pathogenic bacteria, vaccines for aquaculture and molecular diagnostic techniques. He has been president of the Japanese Society of Fish Pathology, vice president of the Japanese Society of Fisheries Science and board member of several other relevant organisations. He received several important awards, including the Japanese Society of Fish Pathology Prize (1998) and the Japanese Society of Fisheries Science Prize (2007). The picture was obtained from T. Aoki and is used with permission. Fig. 20. René J.M. Stet (1954-2007) and Brian Dixon analyzing data at Wageningen University (NL) in 1994. René Stet got his PhD degree (graft versus host disease in rats) from the University of Groningen Medical School (NL) in 1986. With the support of a UK Royal Society Fellowship he went to the Marine Laboratory (Aberdeen, Scotland, UK) for a postdoctoral study on the genetic basis of disease susceptibility in fish (1987-1988). In 1988 he moved to the Cell Biology & Immunology group of Wageningen University, first as assistant and later as associate professor. It was here where he performed most of his innovative work on carp immunology over the following years (1988-2005). He was awarded visiting scholarships, twice to visit the Parham laboratory at Stanford University (CA, USA) in 1996 and 2001. In 2003 he visited the Dixon laboratory at the University of Waterloo (ON, Canada). In 2005 he moved again to Aberdeen to take up a full professorship at the Scottish Fish Immunology Research Centre, where he initiated studies using histocompatibility genes to understand population dynamics of fish and developed further his research on immune response genes in salmonid fish. He served the International Society of Developmental and Comparative Immunology (ISDCI) as Secretary Education (19972003) and as Vice-President for Europe and Africa from 2003 till his unexpected death in 2007. As no other he understood the importance of a molecular approach to prove the existence of a fish equivalent to the mammalian major histocompatibility complex (MHC). For an excellent review on the impact of René Stet’s work see Dixon, 2008. Brian Dixon received his PhD from Dalhousie University, Halifax (NS, Canada) in 1993. He held a Natural Science and Engineering Research Council of Canada postdoctoral fellowship in Wageningen (NL) from 1994 to 1995 and a Medical Research Council of Canada postdoctoral 37

fellowship at Stanford (CA, USA) from 1996 to 1998. He was appointed as a professor in the department of Biology at the University of Windsor, Windsor (ON, CA) in 1998 and then in 2000 he was appointed as professor at the department of Biology, University of Waterloo, Waterloo (ON, CA). In 2011 followed his appointment as Canada Research Chair in Fish and Environmental Immunology at the same university. The research of his laboratory is directed towards characterizing the fish immune system. His research program has focused on the Major Histocompatibility Complex (MHC) receptors and their function. One of the applications of this approach is the use of MHC polymorphism in population studies of fish (e.g. walleye, yellow perch). Other important subjects are fish lymphokines (e.g. CK-1) and the effect of toxic chemicals on the immune system of aquatic animals. Fig. 21. Valentin S. Kirpichnikov (1908-1991) completed his advanced studies at the Institute of Experimental Biology (1932-1941) and the All-Union Research Institute of Pond Fish Culture, Rybnoe, Moscow Province (Russia) (1933-1937), where he founded the world’s 1st laboratory dedicated to the genetic improvement of commercial fish species. After serving in the Soviet Army during World War II (1941-1945), he joined the Institute for Zoology, Russian Academy of Sciences, Leningrad (1946-1948). As a classic geneticist he opposed the false genetic doctrines of Lysenko, which caused hard times for him in the period from 1948 till 1968. It was not until 1966, that the degree of Doctor of Sciences was granted to him. From 1969 till 1991 he was able to continue his productive research at the All-Union Research Institute, Rybnoe, and the Institute for Cytology, Russian Academy of Sciences, in Leningrad (now St. Petersburg). He published over 200 scientific papers, including 2 monographs (e.g. Kirpichnikov, 1981), and edited at least 7 proceedings. During his later years he received the Medal-in-Memorial of N.I. Vavilov and was elected as honorary member of the All-Union Society of Geneticists and Selectionists USSR, Russian Academy of Sciences and the International Association for Genetics in Aquaculture (IAGA). The picture was obtained from the journal “Aquaculture” (Elsevier, Amsterdam) and is used with permission. Fig. 22. Dieter Steinhagen at his office in 2009. He received his Dr. rer. nat. degree (PhD equivalent) in “Fish Diseases & Fish Culture” from the University of Hannover, Hannover (Germany) in 1985. Moreover, he passed the state exam for higher education (“Habilitation”) in 1995. From 1980 onwards he has been working at the Fish Disease Research Unit, Institute for Parasitology, School of Veterinary Medicine, University of Hannover. In 2005 he was appointed as professor (“apl. Prof.”) and became Head of the Fish Disease Research Unit. He and his group have published extensively on the interaction between the immune system of fish (carp, tilapia) and parasites (e.g. Goussia carpelli,Trypanoplasma borreli). In recent years they have also performed excellent work on herpesviruses and the bacterium, Aeromonas hydrophila (effect on intestinal and skin mucus). They also played an active role in several international cooperative research and training projects (both EU and non-EU). Fig. 23. Margaret J. Manning in discussion with colleagues during an international symposium on "Immunology and Immunization of Fish", Wageningen (NL), 1981. She graduated on a PhD thesis entitled “The thymus and its relationship to growth and development” from the university of Bristol (UK) in 1956. Following a period as assistant in Zoology at the university of Glasgow (Scotland, UK), she was appointed to the University of Hull (UK) as senior lecturer in Zoology. In 1982 she moved to the University of Plymouth (formerly Plymouth Polytechnic, Devon, UK) as professor at the Department of Biological Sciences. She is well known for her publications on 38

the immune system of amphibians and fish, in particular the role of the thymus during development. She is co-author of one of the 1st books on “Comparative Immunobiology”, which contains an excellent chapter on cartilaginous and bony fishes (Manning and Turner, 1976). Moreover, she organized several international meetings and is editor of at least 2 books in our field. Fig 24. Picture shows university staff and participants during a training course of “Techniques in Fish Immunology” at the Karnataka Veterinary, Animal & Fisheries Sciences University (KVAFSU, formerly University of Agricultural Sciences, Bangalore), College of Fisheries, Mangalore (India) in 1993. This course was organized by Iddya Karunasagar (1st row, 3rd from left) and Indrani Karunasagar (1st row, 2nd from right). Willem B. Van Muiswinkel (1st row, 3rd from right) was an invited lecturer at this course. Indrani Karunasagar received her MSc degree in Medical Microbiology from Kasturba medical College, Manipal (India), in 1975 and PhD in Microbiology in 1985. From 1975 to 1981, she was a lecturer in KMC Manipal and later Senior Resident in JIPMER, Pondicherry, 1982-2000, assistant professor till 1992 and then as associate professor of Fishery Microbiology at the University of Agricultural Sciences, College of Fisheries, Mangalore. In 2000 she was appointed as professor and head of the department of Microbiology, KVAFSU. At present she is also Director of the UNESO Centre for Marine Biotechnology (MIRCEN), Mangalore. She and her husband, Iddya Karunasagar (today working as Senior Fishery Officer for FAO, Rome, Italy), have been active in the area of aquaculture and marine biotechnology over many years and published > 250 papers on viral and bacterial diseases affecting aquaculture facilities, molecular diagnostics of fish and public health related pathogens, marine toxins and toxic algae, antibiotic resistance and residues, vaccines and immunostimulants for fish and shrimps. She received the Outstanding Woman Scientist Award (ICAR, Govt. of India) in 1998, the National Woman Bioscientist Award (Dept. of Biotechnology, Govt. of India) for her lifetime achievements in 2003, the M.S. Swaminathan Award, Professional Fisheries Graduate Forum, Mumbai (India) and the highest award of the Indian Council of Agricultural Research, the “Rafi Ahmed Kidwai Award” for her research contributions to Fisheries, in 2006. She was elected Fellow of the National Academy of Agricultural Sciences (India) in 2011. Willem B. Van Muiswinkel obtained his PhD degree in Immunology from the Erasmus University, Rotterdam (EUR, the Netherlands) in 1975. In the period from 1969-1975 he was assistant professor at the department of Cell Biology, EUR and from 1975-1985 he worked as assistant / associate professor in Cell Biology & Immunology at the Wageningen University (formerly Agricultural University), Wageningen (WUR, NL). He was appointed as professor in Cell Biology & Immunology at WUR in 1985. He and his staff have build up the 1st research group working in the field of “Fish Immunology & Vaccination” in the Netherlands. They organized several international meetings and workshops, including the 6th Congress of the Int. Soc. Dev. Comp. Immunology at Wageningen in 1994. He has been the Coordinator of the EU Erasmus Student Exchange Programme in “Fish Immunology & Diseases” from 1981-2001. Since his retirement in 2003 he has a special interest in the history of fish immunology and the nature of Texel island (NL). Fig. 25. Stephen L. (Steve) Kaattari at the Eastern Fish Health Workshop, Leetown (WV, USA) in 2001. He received his PhD in Microbiology at the University of California, Davis (CA, USA) in 1979. Steve Kaattari was research associate of the department Microbiology & Immunology, Oregon Health Sciences University, Portland (OR, USA) from 1979 till 1982. In the period 39

between 1982 and 1993 he joined the department of Microbiology, Oregon State University (OSU), Corvallis (OR, USA) first as assistant / associate professor and from 1992 onwards as full professor. In 1993 he was appointed as professor in Environmental Sciences at the Virginia Institute of Marine Sciences (VIMS), College of William & Mary, Gloucester Point (VA, USA). In 2002 he became the Chair of the department Environmental & Aquatic Animal Health at the VIMS. He received several honors, including the Oregon State University Service Award in 1989, the James and Mildred Oldfield Team Award (OSU) in 1993, CSX professor of Marine Science in 2001. His laboratory has performed excellent studies on the differentiation of longlived plasma cells and plasmablasts and their role in generating immunological memory (Zwollo et al. 2005; Kaattari et al., 2005), B cell affinity transduction leading to the structural / functional modifications of the antibody molecule (Ye et al., 2010) and the development of antibody-based sensors for the detection of pollutants in estuarine waters (Bromage et al. 2007; Spier et al., 2007). Fig. 26. Gregory W. (Greg) Warr removing the thymus from a rainbow trout at the National Fish Health Laboratory (NFHL), Leetown (WV, USA) in 1981. He graduated from King’s College in the University of Cambridge (BA and MA degrees) and his PhD degree (1973) is from the University of London (England, UK). After postdoctoral study at the University of Edinburgh (Scotland, UK) from 1973-1974 and at the Walter & Eliza Hall Institute, Melbourne (Australia) from 1974-1977 he joined the Basic Research Program (Cell Biology & Biochemistry Unit), NCI Frederick Cancer Research Center, Frederick (MD, USA) as a scientist in 1977. In 1980 he was appointed associate professor in the Biochemistry Department of MUSC (the Medical University of South Carolina, Charleston, SC, USA). In 1986 he was promoted to full professor of Biochemistry & Molecular Biology and in 2003 he was appointed as Associate Director for Research at the Marine Biomedicine & Environmental Sciences Center of MUSC. He is an emeritus professor of MUSC and since 2008 has been a Program Director in the Molecular and Cellular Biosciences Division of the National Science Foundation (NSF), Arlington (VA, USA). In 2008 he was elected Fellow of the American Association for the Advancement of Science (AAAS) based on his contributions to research, teaching and as Editor-in-Chief of the journal Developmental & Comparative Immunology (1993-2009). He has edited 3 books and published over 200 papers primarily in the field of evolutionary immunobiology (where his interests included marine mammals, birds, fish, and marine invertebrates) and marine ecogenomics. The picture was taken by Monte Stucky (NFHL) and is used with permission. Fig. 27. Emmy C. Egidius (1929-1989) during the Immunology & Immunization of Fish conference at Wageningen (NL) in 1981. She obtained her PhD degree in marine bacteriology from the University of Oslo (Norway) in 1956. Subsequently she worked some years at the University of Bergen (NO) on the infectious disease, Gaffkemia, in lobsters and with Leo Pharmaceutical Products, Copenhagen (DK), on new antibiotics and bacterial enzymes. In 1970 she joined the Institute of Marine Research at Bergen (NO), where she became the leading scientist in marine bacteriology and fish pathology. In 1979-1980 a new disease, called “Hitradisease”, broke out in Norwegian salmon culture. Her team at Bergen, together with colleagues from the University of Tromsø (NO), was able to prove that the disease was of bacterial origin and was caused by a new species, Vibrio salmonicida. In the 1980s this work was carried further and an effective vaccine was developed to combat “Hitra-disease”. She took an active part in national and international organizations, especially the International Council for the Exploration of the Seas (ICES). At the time of her death she was a member of the ICES Advisory Committee 40

on Marine Pollution and chairman of the Working Group on Pathology and Diseases of Marine Organisms. In 1987 she was elected President of the European Association of Fish Pathologists (EAFP) and in 1988 she was appointed as associate professor at the University of Bergen. Fig. 28. Donald F. (Don) Amend obtained his PhD degree (Pathophysiology of IHN virus disease in rainbow trout) from the University of Washington, Seattle (WA, USA), in 1973. From 1965-1976 he was active as research microbiologist for the US Fish & Wildlife Service, Western Fish Disease Laboratory, Seattle (WA, USA). In this period he developed methods to control infectious diseases of salmon. Many of these new methods are still in use today. In 1976 he was appointed as Director of Research for Tavolek Laboratories, Inc. (a Johnson & Johnson company), Redmond (WA, USA). He and his colleagues developed vaccines and pharmaceuticals for the aquaculture industry. They introduced the 1st vaccines to control vibriosis and enteric redmouth disease (ERM). In the period 1981-1983 he joined the University of California, Davis (CA, USA) as associate professor in a joint appointment between Veterinary School of Medicine and School of Agriculture-Aquaculture. In Davis he taught fish pathology and conducted research on fish diseases. From 1983-1998 he has been General Manager and CEO for the Southern Southeast Regional Aquaculture Alaska, inc. (SSRAA), Ketchikan (AK, USA). The SSRAA developed long term and short term strategic plans and was responsible for implementing them. SSRAA oversaw the production of over 100 million salmon fry and smolts each year. Picture (taken in the late 1970s) was obtained from D.F. Amend and is used with permission. Fig. 29. John A. Plumb lecturing during the 35th Eastern Fish Health Workshop at Shepherdstown (WV, USA) in 2010. He got his PhD degree in Zoology-Fisheries from the Auburn University, Auburn (AL, USA) in 1972. He joined the Auburn faculty in 1969 and began his research programme on fish diseases, which has helped improve the health and production of channel catfish and other warmwater fish species. His research focused on ways to vaccinate fish against infectious diseases and examined the environmental influence on disease outbreaks. He has written >100 scientific publications and authored 5 books (e.g. Plumb, 1994). Plumb and his team developed the 1st licenced oral vaccine against enteric septicemia of catfish (Edwardsiella ictaluri) (Plumb and Vinitnantharat, 1993). In 1982 he received the Distinguished Service Award from the Catfish Farmers of America and in 1990 the Snieszko Distinguished Service Award from the Fish Health Section of the American Fisheries Society. In 2013 he was honored with the “Friend of the Eastern Fish Health Workshop” award. He is Professor Emeritus at the department of Fisheries and Allied Aquacultures of Auburn University since 1998. Picture was obtained from R. Cipriano and is used with permission. Fig. 30. William D. (Bill) Paterson received his PhD in Microbiology from Oregon State University, Corvallis (OR, USA) in 1972. He is well known for his pioneering work in the field of vaccines for aquaculture. In the period between 1972 and 1984 he investigated the diseases and defence mechanisms of cold blooded vertebrates and invertebrates for Fisheries and Oceans Canada, Halifax (NS, Canada) and Connaught Laboratories Ltd, Willowdale (ON, Canada). From 1984 till 1993 he became Co-founder, President and Research Director of Aqua Health Ltd., Charlottetown (PEI, Canada). This company developed, produced and sold vaccines for prevention of diseases in aquaculture. They were the 1st to develop a vaccine to prevent disease (Gaffkemia) in invertebrates (Keith et al., 1988). Moreover, they were also active in the field of other veterinary biologicals and as a testing laboratory. From 1993 onwards he became the 41

owner-operator of Paterson Applied Technologies, Tottenham (ON, Canada). This company provided consulting services on fish health and aquaculture. Photograph was obtained from W.D. Paterson in 2010 and is used with permission. Fig. 31. Jan F. Tesar ik received his PhD degree from the University of Veterinary Sciences, Brno (Czech Republic) in 1970. From 1958-1961 he worked as assistant at the department of Physiology, University of Veterinary Sciences, Brno. In the period between 1961 and 1991 he was appointed as researcher at the Research Institute of Fish Culture and Hydrology (VURH) in Vod any (Czech Republic) and from 1991-1996 as the head of “Fishery Ostrava”. During his entire career he focused on the diagnosis and treatment of fish diseases. In the 1970s his team developed cell culture techniques enabling them to identify Rhabdovirus carpio as the causative agent of Spring Viraemia of Carp (SVC). Under his supervision the 1st commercial anti-viral vaccine (against SVC) was developed in the 1980s (Tesar ik and Macura, 1981). The picture was taken around 1970 and obtained from V. Žlábek (VURH) and is used with permission. Fig. 32. Niels Lorenzen received his PhD degree from the University of Copenhagen (Denmark) in 1991. The title of his thesis was “Viral haemorrhagic septicemia virus in rainbow trout: immunological investigations in relation to vaccine development”. At present he is the head of the Fish Health Research group at the department of Animal Sciences, Aarhus University, Århus (DK). In the period 2005-2011 he has been the coordinator of a major EC supported collaborative fish & shellfish immunology research project known as IMAQUANIM (Improved Immunity of Aquacultured Animals). His main interests are in fish rhabdoviruses: innate and adaptive protective immune mechanisms, fish vaccinology in general and DNA vaccines in particular (against the salmonid rhabdoviruses VHSV and IHNV). The picture was obtained from G.F. Wiegertjes and is used with permission. Fig. 33. R. Dinakaran Michael earned his PhD in Immunology from the Madurai Kamaraj University, Madurai (India) in 1986. In the period between 1985 and 2003 he was appointed as Professor and Head of the department of Zoology, The American College, Madurai (India). He published extensively on the immunostimulatory effect of medicinal plant extracts/fractions, immunomodulation by social stress (crowding, sex ratio), the effects of pollution (chromium, cadmium) and vaccination (against Aeromonas hydrophila) in fish (e.g. tilapia, Indian carps). Couple of his marine macroalgal derived immunostimulant products have been filed for patents. From 2003 till 2010 he was active as Director of the Centre for Fish Immunology, department of Zoology & Biotechnology, Lady Doak College, Madurai (India). From Jan 2011 till present he is the Director, School of Life Sciences, Vels University, Chennai (India) and is currently working with striped snakehead and Asian seabass. He widely travelled on teaching and research assignments, was the recipient of state and national Best Teacher Awards and was selected as Consultant (for Fish Immunology) by the Food & Agriculture Organisation (FAO) of the United Nations, Rome (IT) in 2003. Picture was obtained from R. Dinakaran Michael and is published with permission. Fig. 34. Teruyuki Nakanishi at the ISDCI Congress at Prague (CZ) in 2009. He obtained his PhD degree from the Faculty of Fisheries, University of Hokkaido (Japan) in 1983. After a postdoctoral year at Plymouth University (Plymouth, UK) he became chief of the Immunology Section, Fish Pathology Division, National Institute of Aquaculture, Mie (Japan). In 1999 he was appointed as professor at the department of Veterinary Medicine, Nihon University (Japan). He 42

has written more than 130 papers on subjects such as cytotoxic T cells, MHC genes, cytokines, hematopoietic stem cells and fish vaccines. He is editor of a “classic” fish immunology textbook (Iwama and Nakanishi, 1996). Fig. 35. Jian-zhong Shao obtained his PhD degree in Biomedical Engineering from the Zhejiang Universitity, Hangzhou (China) in 1998. In the period between 1987 and 2000 he worked as lecturer and associate professor at the College of Life Sciences, Zhejiang University. In 2000 he was appointed as professor in Life Sciences at the same university. Since 1998 he is also acting as Director of the Institute for Cell Biotechnology of Zhejiang University, and Director of the Key Laboratory for Cell and Gene Engineering of Zhejiang Province, China. In 2003 he worked as visiting scientist at the Medical School of Harvard University, Boston (MA, USA). At present he is Vice Dean of the College of Life Sciences, Zhejiang University, Executive Director of the Chinese Society for Cell Biology, Council Member of the Chinese Society for Immunology, and Council Member of the Chinese Society for Oceanology & Limnology. He is also a member of the American Association of Immunologists (AAI), and the American Society for Biochemistry and Molecular Biology (ASBMB). His laboratory has 2 main research lines: a) Stem cell regulation and engineering with a focus on regenerative medicine; b) Molecular and cellular basis of immune signaling network control mechanisms using zebrafish as animal model. Picture was obtained from J. Shao and is used with permission. Fig. 36. Cover of the handbook on “Techniques in Fish Immunology”, Vol. 3, by J.S. Stolen, T.C. Fletcher, A.F. Rowley, D.P. Anderson, S.L. Kaattari, J.T. Zelikoff, and S.A. Smith (Eds.) (Stolen et al., 1994a). Fig. 37. Paul J. Midtlyng received his Doctoral degree (Evaluation of furunculosis vaccines in Atlantic salmon) from the Norwegian College of Veterinary Medicine, Oslo (Norway) in 1998. From 1995-2009 he was first head of department and later head of research & development, VESO Vet Research, Oslo / Namsos (Norway). In the period from 2009 to 2011 he was appointed as head of the Laboratory Animal Unit, Norwegian School of Veterinary Science (NSVS), Oslo. From 2011 onwards he is part-time associate professor of the Centre for Epidemiology & Biostatistics, NSVS, and regulatory affairs manager for Novartis Aqua Norway, Oslo. He has served on Norwegian Research Council program committees for fish health, and in the period between 1997 and 2007 he has been Council member and General Secretary for the European Association of Fish Pathologists (EAFP). He has published extensively on fish vaccination and other means of controlling bacterial and viral diseases in fish. He is also editor / co-editor of at least 4 books. The picture was obtained from P.J. Midtlyng and is used with permission. Fig. 38. Cover of the book on “Fish Vaccination” by A.E. Ellis (Ed.) (Ellis, 1988).

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Abstract This review describes the history of research on immunity to infectious diseases of fish in the period between 1965 and today. Special attention is paid to those studies, which are dealing with the interaction between immune system and invading pathogens in bony fish. Moreover, additional biographic information will be provided of people involved. In the 1960s and 1970s the focus of most studies was on humoral (Ig, B-cell) responses. Thorough studies on specific cellular (T-cell) responses and innate immunity (lectins, lysozyme, interferon, phagocytic cells) became available later. In the period between 1980 and today an overwhelming amount of data on regulation (e.g. cell cooperation, cytokines) and cell surface receptors (e.g. T-cell receptor; MHC) was published. It became also clear, that innate responses were often interacting with the acquired immune responses. Fish turned out to be vertebrates like all others with a sophisticated immune system showing specificity and memory. These basic data on the immune system could be applied in vaccination or in selection of disease resistant fish. Successful vaccines against bacterial diseases became available in the 1970s and 1980s. Effective anti-viral vaccines appeared from the 1980s onwards. There is no doubt, that Fish Immunology has become a flourishing science by the end of the 20th century and has contributed to our understanding of fish diseases as well as the success of aquaculture.

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Paper DCI 2023 Highlights • We describe the interaction between the immune system and pathogens in fish. • Vaccination and selection for disease resistance contributed to success in aquaculture. • Data on important books and meetings in this field are discussed. • Unique pictures and short biographies are published of 42 researchers involved.

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