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Developmental and Comparative Immunology 26 (2002) 151±160 www.elsevier.com/locate/devcompimm The immune system of sea bass, Dicentrarchus labrax, reared in aquaculture G. Scapigliati a,*, N. Romano a, F. Buonocore a, S. Picchietti a, M.R. Baldassini a, D. Prugnoli a, A. Galice a, S. Meloni a, C.J. Secombes b, M. Mazzini a, L. Abelli c a Dipartimento di Scienze Ambientali, UniversitaÁ della Tuscia, Largo dell'UniversitaÁ, I-01100 Viterbo, Italy b Department of Zoology, Aberdeen University, Aberdeen AB24 TZN, UK c UniversitaÁ di Ferrara Ferrara, Italy Abstract The sea bass Dicentrarchus labrax is one the most important seawater ®sh species of south Europe and Mediterranean aquaculture, and studies on its immune system are important for both scienti®c and applied purposes. In this paper, we summarise the results obtained in studies of the immune system in this species, and present original data on cell-mediated acquired immune response. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fish; Dicentrarchus labrax; Sea bass; Immunology; Monoclonal antibody; Immunopuri®cation; Leucocytes 1. Introduction Teleost ®sh are the largest group of vertebrates (about 20,000 species), arising around 300 million years ago and sharing similar immune system organisation with other vertebrates [1]. This aspect includes the presence of functional lymphocytes [2±4], MHC [5], TCR [6], and cytokines [7]. In this respect, teleosts are interesting models to study the phylogeny of vertebrates immune system. Teleosts are also important in marine biotechnology and aquaculture, because many freshwater and marine species have been introduced into ®sh farms, and other species are being evaluated and developed for farming production. Several diseases can affect ®sh at all stages of their life cycle, and knowledge of the immune system is of major importance for their health * Corresponding author. Tel.: 139-0761-357-137; fax: 1390761-357-179. E-mail address: [email protected] (G. Scapigliati). control. In fact, this will allow the introduction of treatments such as vaccines and immunostimulants as alternatives to the use of drugs and antibiotics which raise a number of environmental concerns. The anatomical organisation of teleost lymphoid tissues includes the thymus, head kidney, spleen and mucosal associated lymphoid tissue [8] and cellular components display humoral and cellular immune reactions. These components include non-speci®c cell-mediated cytotoxicity (NCC) [9±11], microbial killing by macrophages [12,13], B-cell activities [14±16], and T-cell activities [17,18]. Despite the fact that our knowledge of the ®sh immune system is continuously increasing, the biology of cellular reaction is, at present, largely unknown mainly due to the lack of speci®c markers for leucocytes. In this respect, the sea bass is the only marine species for which B-cell and T-cell markers are available [19,20] and studies on its immune system can consequently have signi®cant value. Due to its importance for aquaculture, sea bass rearing is 0145-305X/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0145-305 X(01)00 057-X 152 G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160 continuously improving [21], with the main pathologies affecting this species in aquaculture being vibriosis [22], pasteurellosis [23] and virosis [24,25]. Thus, the development of proper vaccination strategies can be fundamental to limit the use of chemotherapeutic agents. In this respect, knowledge of the mucosal-associated system is important to understand the mode of action of vaccines and their protection against speci®c pathogens. Regarding the sea bass, experimental evidence has recently shown that gut-associated lymphoid tissue (GALT) of teleosts contains an elevated number of T-cells [26], the presence of which in the ®sh gut may represent the ®rst step in the evolution of an adaptive mucosal immune system [27,28]. In addition, other researchers have established in vitro cell culture conditions for sea bass leucocytes [29,30] that are fundamental for proliferation studies. In this review we summarise and update the progress of studies on the sea bass Dicentrarchus labrax immune system in aquaculture. 2. Methods 2.1. Immunopuri®cation of cells Sea bass were bred and reared in seawater at a local ®sh farm (La Rosa, Orbetello, Italy). Two-year old ®sh, 298 ^ 76 g in weight, were used in the experiments. All buffers and solutions used in handling ®sh cells were brought to 355 mOsm kg 21 with 2 M NaCl. Organs were removed and placed in cold Hank's balanced salts solution without Ca 21 and Mg 21 (HBSS). Cells were obtained by disrupting organs over a nylon mesh (100 mm) in HBSS. The resultant cell suspensions were resuspended at 1 £ 108 cells ml 21, washed with HBSS at 680 g, and then layered over discontinuous gradients of Percoll (Pharmacia AB, Uppsala, Sweden) diluted in RPMI to yield densities of 1.02 and 1.07 g cm23. Peripheral blood leucocytes (PBL) were obtained from heparinised blood. Whole blood from individual ®sh was washed twice in HBSS±heparin, resuspended in 8 ml of the same solution and loaded over Percoll gradients as described above. After centrifugation (30 min at 840 g) at 48C, cells at the interface between the two densities were collected and washed twice (10 min at 680 g) at 48C. After centrifugation, cell pellets were resuspended at 1 £ 10 8 cells ml 21 in the Mab DLT15 as culture supernatant for 45 min at 48C, or in the af®nitypuri®ed DLIg3 Mab at 20 mg/ml, then centrifuged and resuspended at 1 £ 10 9 cells ml 21 in HBSS. Three hundred microlitres of this cell suspension were incubated at 158C for 20 min with 60 ml of anti-mouse Ig labelled with magnetic Fe2O3 microparticles (Milteny biotec, Sunnyvale, CA, USA). Mab 1 cells were collected in HBSS by magnetic sorting following the manufacturer's instructions using MiniMacs columns. Puri®ed and released cells were counted and immediately monitored for their immunoreactivity, or employed for RNA extraction. 2.2. In vitro Ig production and cell proliferation The modi®ed ELISPOT assay was essentially as previously described [60]. Brie¯y, ®sh (n 4) were injected i.p. with 250 ml/®sh of PBS containing bacteria centrifuged from a 0.5 ml solution (ca. 2.5 £ 10 8 cells) of Vibrio anguillarum serotype 1 and serotype 2 vaccine (Microtek Europe, Brambles, UK) without adiuvant. Control ®sh (n 10) were injected i.p. with 250 ml of PBS. After 10 days, all immunised ®sh received an i.p. boost without adjuvant. Fifteen days later, ®sh were killed by anaesthetic overdose. Blood samples were obtained by caudal vein puncture using a syringe, and serum obtained from clotted blood by centrifuging at 2000 g for 10 min. Leucocytes from head kidney were adjusted to a concentration of 1 £ 10 7 cells/ml in RPMI containing 5% foetal calf serum (FCS, GIBCO) and 70 mg/ml heparin. One hundred microlitres of cell suspension were plated in a 96-well polystyrene plate coated with antigen (see below), and incubated at 258C for 48 h. Eighteen hours before plating cells, ELISA polystyrene plates (Nunc, Roskilde, Denmark), were coated with 100 ml of carbonate±bicarbonate buffer (50 mM pH 9.4) containing 1 ml of bacterin suspension. Alternatively, wells were adsorbed with dilution of ®sh sera in the same solution. After washing, the wells were processed for ELISA assay exactly as previously described [20] using DLIg3 as culture supernatant diluted 1:30 with DMEM. Each experimental point was in triplicate, and absorbance of each well was read at 492 nm with an automated plate reader. G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160 Controls were performed for serum samples by omitting serum or by substituting DLIg3 with DMEM, and for in vitro Ig production by adding 10 mg ml 21 of cycloheximide (Sigma) to block protein synthesis. Numerical values are expressed as the mean of different experiments ^ standard error of the mean (S.E.). Statistical analysis was performed using the Student ttest. For cell proliferation, head-kidney leucocytes or DLIg3-immunopuri®ed cells from the same animal (®sh immunised as above) were incubated for two days at 258C at 1 £ 10 6 cells/well in 100 ml of RPMI containing 5% FCS and, where necessary, with 2 ml of bacterin suspension. Cell proliferation was measured with a non-radioactive system (Celltiter 96, Promega) following manifacturer's instructions. Each experimental point was in triplicate. Proliferation was determined numerically by assuming 1 for the control. Results are expressed as the mean of two experiments ^ standard deviation (S.D.). 3. Innate immunity It is well established that teleost ®sh display innate responses against antigenic stimulants and pathogens [9,31±35]. In sea bass, at the morphological level, leucocytes from head-kidney were described as stromal cells, macrophages and lymphocyte-like cells [36]. Subsequently, the in vitro cytotoxic reaction of head-kidney, blood or peritoneal exudate leucocytes against tumour target cells was studied by transmission and scanning electron microscopy, and the effectors exhibited ultrastructural features of either monocytes or lymphocytes [37]. Nonspeci®c cell-mediated cytotoxicity was further studied morphologically, indicating that leucocytes were able to kill their targets by inducing necrosis and apoptosis in a similar way to mammalian cytotoxic cells [38]. In another work, spontaneous in vitro cytotoxic activity against tumour cell lines by unstimulated sea bass leucocytes was determined by trypan blue exclusion test and lactate dehydrogenase release assay, and high anti-tumour cell line activity of resident peritoneal leucocytes was found. Low activity was displayed by head-kidney and spleen cell populations whereas blood leucocytes revealed no signi®cant activity. Eosinophilic granule cells, 153 isolated from a peritoneal wash, appeared to be mostly responsible for the in vitro cytotoxic activity [39]. The phagocytic activity of head-kidney adherent cells following stimulation by bacterial (Aeromonas salmonicida) and fungal (Candida albicans) pathogenic agents was studied by light microscopy and by measuring production of reactive oxygen intermediates (ROI) [40]. In this work it was shown that the ratio of macrophages±pathogenic agents and the amplitude of the ROI response varied with the type of pathogenic agents, and that opsonisation by ®sh serum increased the macrophage ROI response. Phagocytic responses of macrophages were further studied [41] morphologically by analysing the in¯uence of leukocyte source, bacterial species, presence or absence of a bacterial wall, bacterial status (live or dead), and bacterial opsonisation. These studies showed that peritoneal macrophages from sea bass exhibited a greater capacity to engulf bacteria than did those isolated from blood which, in turn, had greater engulfment properties than those isolated from head-kidney. Parasites such as fungi can affect sea bass health in aquaculture, and although a speci®c humoral response against these organisms was recently reported [42], these results show that immunisation with S. dicentrarchi resulted mainly in the activation of the non-speci®c immune response as measured by lysozyme activity, enhanced phagocytosis by macrophages, and active production of ROI. Some dietrelated changes in non-speci®c immune responses have been studied [43], and describe the modulation of responses to substances such as alpha-tocopherol and dietary oxidised ®sh oil introduced in the food [44]. In the latter study, the non-speci®c immune factors assayed were plasma lysozyme and complement activities, natural haemolysis of sheep red blood cells, and chemiluminescence response of head-kidney phagocytes. 4. Humoral immunity and B-cells Teleost ®sh display a primary and a secondary humoral response upon antigen administration, although in contrast with mammals a shift in immunoglobulin (Ig) class is absent [35]. Dicentrarchus labrax is a teleost species susceptible to 154 G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160 many pathogens, the most studied pathology being vibriosis, a septicaemia caused by Vibrio anguillarum serotypes 01 and 02 and Pasteurella piscicida. With regard to Vibrio anguillarum, during experimental or `in ®eld' vaccination trials, it is usually used as a bacterin suspension administered intraperitoneally (i.p.), orally, by immersion, or by other means. However, in earlier studies [22], comparison of oral and i.p. vaccination of sea bass demonstrated the lack of ef®cacy of oral treatment, as measured by both antigen-speci®c Ig serum titres and bacteriostatic activity. However, passive immunisation using serum from orally vaccinated ®sh (2 months after vaccination) conferred weak protection against a challenge of virulent Vibrio. Subsequently, the relationships between the levels of total proteins, immunoglobulins and antibody activity in serum of sea bass broodstock, following one or two i.p. injections of heat-killed Vibrio anguillarum, were investigated [45]. Results from this work showed that Vibrio injection did not modify total serum protein levels, and that Ig production was signi®cantly higher in immunised animals. Furthermore, no signi®cant difference was found between males and females in antigen-speci®c antibody levels. With regard to the Gram-negative bacterium, Photobacterium damselae, previously classi®ed as Pasteurella piscicida, many studies are currently investigating the potential for development of effective vaccines. Fish were injected with live and heat-killed bacteria, and serum antibody activity examined by western blot analysis [23]. Great variation among the sera was evident with reference to the recognition of antigens in the high molecular weight group, and lipopolysaccharide and/or lipoprotein situated in the low molecular weight range appeared to be the most immunogenic material in the bacterial cell. Western blot analysis was also employed by others to assess the presence of Pasteurella antigens in organs of sea bass [46], showing also in this case a variability in the molecular weight of antigens recognised by immune sera. This reported variability in the recognition of diverse bacterial antigens was further studied by others [47]. In this work, different antigen preparations were administered i.p. and, interestingly, most of the toxic activity was carried by extracellular products, and not bound to the cell. To study humoral reactions of the sea bass, some monoclonal antibodies (Mabs) have been prepared against Ig and Ig-bearing cells [48]. All these monoclonal antibodies were prepared using Ig as the immunogen puri®ed with various biochemical methods [49±51]. Most of these Mabs recognised the heavy chain of Ig, whereas a few were obtained against the light chain [20,52,53]. Initially, the Mabs obtained were employed to set up an immunoenzyme assay to detect total and antigen-speci®c serum Ig [52], whereas the organ distribution and the immuno¯uorescence staining pattern of Ig-bearing cells were not studied. Subsequently, a Mab raised against the light chain of the Ig molecule was selected for its ability to recognise Ig in denatured and native form [20]. In this study, sea bass immunoglobulins were single-step puri®ed from the whole serum by af®nity chromatography on protein A-Sepharose and used as immunogen in mice. Among the positive hybridomas obtained, some clones were selected according to their ability to recognise either the immunoglobulin light chain (DLIg3) or the heavy chain (DLIg13 and DLIg14). Indirect immuno¯uorescence (IIF) and ¯ow cytometric analysis showed that DLIg3 stained 21% of PBL, 3% of thymocytes, 30% of splenocytes, 33% of head-kidney leucocytes, and 2% of gutassociated lymphoid tissue (GALT) [54]. DLIg13 and DLIg14 were unable to stain living cells, but recognised ®xed cells following ABC-immunoperoxidase staining of spleen, head-kidney and midgut. The Mab DLIg3 (IgG class) proved the most interesting since it works in all assay systems used, and was used to establish a sensitive ELISA assay (detection limit 1.2 ng/ml) to detect and quantify puri®ed and serum immunoglobulins. Finally [53], three anti-Ig Mabs were selected (WDI 1±3) on criteria based on ELISA, Western blot, IIF and ¯ow cytometric analysis. All Mabs were found to belong to the IgG class, and were effective in detecting antigen-speci®c antibody by ELISA. Under reducing conditions WDI 1 recognises the heavy chain and both WDI 2 (slightly) and WDI 3 (strongly) recognise the light chain. The average percentage of surface Ig-positive cells identi®ed by these Mab in PBL, head-kidney, spleen, thymus and gut were similar to that previously reported, thus con®rming the estimation of B-cells in sea bass organs. Mabs DLIg3 and WDI 1±3 were also employed in immunogold labelling, and showed speci®city for subpopulations of lymphoid cells G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160 155 Fig. 1. Proliferation of head-kidney leucocytes and immunopuri®ed cells. Bars represent absorbance mean value ^ SD of two experiments where unfractionated or DLIg3-puri®ed head-kidney leucocytes (10 6 per well) from Vibrio anguillarum-immunised ®sh were cultured with the same antigen (1 ml bacterin) added into the culture medium. Proliferation was measured as absorbance value at 570 nm, and proliferation index indicated as the ratio of absorbance value with respect to controls without bacterin. (B-cells, plasma cells and macrophages) in both PBL and lymphoid tissues [53,54]. A useful application of Mabs that are able to recognise Ig in its native conformation is for the immunopuri®cation of viable Igbearing cells. In this respect we employed the Mab DLIg3 for the immunopuri®cation of B-cells from head-kidney leucocytes by immunomagnetic sorting (Fig. 1). Percoll-enriched leucocytes from Vibrio anguillarum immunised ®sh were incubated with af®nity-puri®ed DLIg3 IgG, and subsequently with a secondary iron-labelled anti-mouse antibody. DLIg3-positive cells were retained in a column over a magnetic ®eld and recovered by removing the magnetic ®eld (see methods). DLIg3-puri®ed cells, when re-stimulated in vitro with the immunising antigen, displayed a strong proliferative response with respect to non-puri®ed leucocytes. By using polyclonal antisera and Mabs in ELISA, it is possible to monitor the ef®cacy of vaccination by measuring the production of antigen-speci®c Ig in sea bass biological ¯uids [44,53,55]. We also employed the Mab DLIg3 to investigate by ELISA the effects of non-pathogenic conditions in the content of total serum Ig in groups of ®sh at different ages, and farmed in different water oxygen concentrations [56]. The results showed that the immunoglobulin levels increased consistently with age and size, hyperoxygenation of sea water resulted in a two-fold increase of immunoglobulins, and that immunoglobulin levels from adult ®sh varied in relation to the spawning season. In recent years, the evaluation of antibodysecreting cells in ®sh has been conveniently achieved using the ELISPOT assay [57±59]. To further study the humoral immune response of sea bass we introduced a simpli®ed ELISPOT that would allow quantitative data on B-cell activities in vitro (production of antigen-speci®c Ig) without manual counting of the read out at a microscope, allowing manipulation of large number of samples and reducing considerably the assay time [60]. This method could be applied to monitor the presence of `memory' B-cells in the headkidney which secrete Ig speci®c for a certain antigen at a certain time during the life of the ®sh and, in this respect, we present interesting data on the conservation of B-cell memory after immersion vaccination (see below). Immersion vaccination is an effective and practical method for mass treatment of ®sh and most commercial bacterins are currently administered by this method, even though the exact mechanisms of antigen uptake and protection still remain unclear [61]. The role of humoral immunity in protection mechanisms after immersion vaccination has been controversial and potentially important roles for cell-mediated immunity or local immunity have been proposed. During trials with immersion vaccination, antibodies against pathogens are not detectable in the serum by ELISA and, even when antibodies are found, the titre does not always correlate with protection. To further investigate these observations, we employed both 156 G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160 Fig. 2. In vitro Ig production and serum antibody levels in ®sh immunised with Vibrio anguillarum. (a) In vitro production of anti-Vibrio Ig by head-kidney leucocytes from control ®sh or Vibrio anguillarum immunised ®sh. Bars represent the mean ^ SD from two different experiments. (b) Vibrio anguillarum-speci®c antibodies were quanti®ed in sera of the same animals using an indirect ELISA assay employing DLIg3. ELISA assay for serum Ig, and the in vitro Ig production assay by head-kidney leucocytes from 14 month-old ®sh which were vaccinated once by immersion in Vibrio anguillarum bacterin one year before. The results of these experiments (Fig. 2) clearly showed that with respect to controls, leucocytes from ®sh that received vaccine were able to produce speci®c Ig against Vibrio. These ®sh also had detectable titres of circulating anti-Vibrio Ig. Hence, it can be af®rmed that immersion vaccination induced B-cell memory in sea bass, and these ®sh could be potentially protected against subsequent pathogen exposure. 5. T-cells Studies on cell-mediated immune reactions have demonstrated that teleosts exhibit T-cell responses based on functional criteria like proliferation induced by T-cell mitogens [62], response in the mixedleucocyte reaction (MLR) [63], function as helper cells in antibody production against thymusdependent antigens [64], allograft rejection [65], and secretion of lymphokines [66]. Furthermore, as it was demonstrated in other teleosts, leucocytes from sea bass head-kidney can ef®ciently proliferate in response to mitogens which in mammals are speci®c for T-cells, such as concanavalin-A or phytohemoagglutinin [30,67]. However, due to the lack of speci®c Mabs, the involvement of T-cells in these responses has only been monitored indirectly, and their participation only presumed. In recent years, some work has been published on Mabs recognising T-cells. However, in the majority of cases these Mabs recognised antigens expressed by most PBL leucocytes, and/or by Ig-bearing cells, so they were not speci®c for T-cells [48]. The sea bass is, at the present moment, the only marine teleost species for which a putative speci®c G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160 anti-T-cell marker is available. The Mab DLT15, speci®c for thymocytes and peripheral T-cells, was obtained by immunising mice with paraformaldehyde-®xed thymocytes from sea bass juveniles [19]. This antibody (IgG class) is able to recognise its antigen(s) both in living cells and in tissue sections, and its use in IIF and cyto¯uorimetric analysis of leucocytes enriched over Percoll permitted the ®rst evaluation of T-cell populations in sea bass. DLT15 positive cells constitute 3% of PBL, 9% of splenocytes, 4% of head-kidney cells, 75% of thymocytes, 51% of GALT, and 60% of gill-associated lymphoid tissue [54]. In the view of oral delivery of antigens, gut-associated lymphoid tissue has been the subject of particular research, since it revealed a striking abundance of T-cells [26], and a remarkable precocity of their appearance during development [68]. DLT15 was used in immunocytochemistry to show for the ®rst time in a piscine system a T-cell activity in vivo, where muscle transplants were grafted in allogenic recipient ®sh [65]. The immunocytochemical analysis with DLT15 of rejected sea bass muscle allografts showed many positively staining cells in®ltrating the tissue. Another important use of DLT15 was to purify immunoreactive cells from sea bass organs, mainly from blood and gut-associated lymphoid tissue [69]. Puri®cation was performed by immuno-magnetic sorting of leucocyte fractions enriched by Percoll density gradient centrifugation, and the purity of DLT15-puri®ed cells was 90% for gut-associated lymphoid tissue, and 80% for blood leucocytes. DLT15-puri®ed cells from gut-associated lymphoid tissue were employed for RNA extraction and cDNA synthesis. In RT±PCR experiments using degenerate oligonucleotide primers corresponding to the peptide sequence MYWY and VYFCA of the trout T-cell receptor (TcRb) chain, a 203 bp product was ampli®ed. When sequenced and analysed, the cDNA was found to show 60% nucleotide identity to the trout TcRVb3. Elongation of DNA ampli®ed with MYWY and VYFCA primers by semi-nested 3 0 -RACE experiments gave ampli®ed products of 1000 bp. The sequence of these ampli®ed products was obtained and compared to database sequences, and the best scores were with different TcRb chains or TcR precursor molecules. The deduced amino acid sequence of one of these clones was compared with TcR constant regions sequences from cartilagineous 157 and bony ®sh. The highest similarity was observed with cod (51%). From these results we strongly argue that cells recognised by DLT15 are putative T lymphocytes. 6. Ontogenesis of the immune system The sea bass has been the subject of studies for the elucidation of ontogenesis of lymphoid cells and lymphoid organs. Initial studies addressed myelopoiesis in the thymus [70], showing morphologically the presence of intrathymic developing myeloid cells in the sea bass. The occurrence of an apoptotic process throughout thymic development suggested that thymocytes undergo selection processes in the thymic microenvironment [71]. Subsequently, other authors described the ontogeny of IgM-bearing cells using anti-Ig antibodies [72], showing their ®rst appearance 2 months after hatching. Studies on sea bass ontogenesis have greatly bene®ted from the use of the anti-B and anti-T cell Mabs described above. DLT15-antigenic determinants are expressed during development primarily in the thymus and gut, and subsequently in head-kidney and spleen, respectively [73]. DLT15 immunoreactivity was ®rst detected in thymocytes at day 30 ph (at 168C), 3 days after the ®rst appearance of lymphoid cells, shortly after in the epithelium of gut mucosa, and later in the head-kidney (day 35 ph) and spleen (day 44 ph) [67,73]. These ®ndings fall into the general scheme of teleost lymphopoiesis, but also show an early establishment of GALT. Throughout development, DLT15-immunoreactive cells became very numerous in the thymus, mainly localised in the cortical region and increased signi®cantly in the intestinal mucosa from day 44 ph onward, while they remained infrequent in the developing head-kidney and spleen. Numerous cells positive to DLT15 (51% of GALT) were found in the epithelium and lamina propria of the gut mucosa, and a gradient of such cells was present, increasing in concentration towards the anus [26]. This may suggest that, as in other ®sh species, the posterior gut may have a greater immunological relevance. The number of T cells found in the gut of sea bass largely exceeded that of Ig-bearing cells recognised by Mab DLIg3 [54]. The same observation was made in carp, where a large fraction 158 G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160 (50±70%) of intestinal leucocytes reacted with the MAb WCL38, thus representing presumptive Tcells, while Ig-bearing cells were a minor population (5±10%) [74]. These ®ndings suggest the predominance of T-cells in the GALT thoughout the digestive system. In the sea bass, IgM-bearing cells were ®rst detected by immunohistochemistry at day 38 ph in the head-kidney of fry reared at 16±208C. Earlier detection (day 18 ph) by FACS of small numbers of Ig-bearing cells in cell suspensions from whole larvae was not con®rmed by immunocytochemistry [72]. Low numbers of Ig-bearing cells were detected with anti-B cells Mabs (DLIg3, DLIg13, DLIg14)) at day 49 ph in the head-kidney, and were very infrequent in spleen and thymus of fry reared at 168C [67]. These ®ndings suggested that the immune system of the sea bass larvae is probably competent for antibody production around day 50 ph [72]. Field observations show that sea bass fry are highly sensitive to bacterial diseases during this period and that vaccination at this stage can provide good protection. Together, these ®ndings suggest that in sea bass the maturation of the humoral immune system takes place around the second month pf [48]. [10] 7. Conclusions [11] The sea bass has become one of the most important ®sh species reared in aquaculture, and much work has been done to investigate its immune system. Reagents are available to study the in vivo and in vitro immunobiology of B-cells and T-cells and cell culture conditions have been established. 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