Download The immune system of sea bass, Dicentrarchus labrax, reared in

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 ˆ 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 ˆ 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. Leucocyte subpopulations can be puri®ed and induced to proliferate
in response to T-cell and B-cell mitogens. Finally,
some DNA probes for genes encoding important
molecules of the immune system will soon be
released. Taken together, this scenario suggests that
the sea bass may become a reference marine ®sh
model for basic and applied research in aquaculture
and biotechnology.
References
[1] Secombes CJ, Van Groningen JJM, Egberts E. Separation of
lymphocyte subpopulations in carp Cyprinus carpio L.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[12]
[13]
[14]
[15]
[16]
[17]
by monoclonal antibodies: immunohistochemical studies.
Immunology 1983;48:165±75.
Yamaguchi K, Kodama H, Miyoshi M, Nishi J, Mukamoto M,
Baba T. Inhibition of cytotoxic activity of carp lymphocytes
(Cyprinus carpio) by anti-thymocyte monoclonal antibodies.
Vet Immunol Immunopathol 1996;51:211±21.
Passer BJ, Chen CH, Miller N, Cooper MD. Identi®cation of a
T lineage antigen in the cat®sh. Dev Comp Immunol
1996;20:441±50.
Nishimura H, Ikemoto M, Kawai K, Kusuda R. Crossreactivity of anti-yellowtail thymic lymphocyte monoclonal
antibody (YeT-2) with lymphocytes from other ®sh species.
Arch Histol Cytol 1997;60:113±9.
Greenlee AR, Ristow SS. A monoclonal antibody which binds
a carbohydrate moiety broadly expressed on rainbow trout
leucocytes. Fish Shell®sh Immunol 1993;3:317±30.
Partula S, De Guerra A, Fellah JS, Charlemagne J. Structure
diversity of the T cell antigen receptor b-chain in a teleost ®sh.
J Immunol 1995;155:699±706.
Secombes CJ, Hardie LJ, Daniels G. Cytokines in ®sh: an
update. Fish Shell®sh Immunol 1996;6:291±304.
Zapata A, ChibaÁ A, Varas A. Cell and tissues of the immune
system of ®sh. In: Iwama Q, Nakanishi T, editors. The ®sh
immune system: organism, pathogen and environment. San
Diego: Academic Press, 1996. p. 1±62.
Evans DL, Harris DT, Jaso-Friedmann L. Function associated
molecules on nonspeci®c cytotoxic cells: role in calcium
signalling, redirected lysis, and modulation of cytotoxicity.
Dev Comp Immunol 1992;16:383±94.
Stuge TB, Miller NW, Clem LW. Channel cat®sh cytotoxic
effector cells from peripheral blood and pronephroi are
different. Fish Shell®sh Immunol 1995;5:469±71.
Fischer U, Ototake M, Nakanishi T. In vitro cell-mediated
cytotoxicity against allogeneic erythrocytes in ginbuna
crucian carp and gold®sh using a non-radioactive assay. Dev
Comp Immunol 1998;22:195±206.
Jang SJ, Hardie LJ, Secombes CJ. Elevation of rainbow trout
Oncorhynchus mykiss macrophage respiratory burst activity
with macrophage-derived supernatants. J Leukocyte Biol
1995;57:943±7.
Solem ST, JoÈrgensen JB, Robertsen B. Stimulation of respiratory burst and phagocytic activity in atlantic salmon (Salmo
salar L.) macrophages by lipopolysaccharide. Fish Shell®sh
Immunol 1995;5:475±91.
Rijkers GT, Frederix-Wolters EMH, Van Muiswinkel WB.
The immune system of cyprinid ®sh. Kinetics and temperature
dependence of antibody-producing cells in carp (Cyprinus
carpio). Immunology 1980;41:91±7.
SaÂnchez C, Alvarez A, Castillo A, Zapata A, Villena A,
DomIÂnguez J. Two different subpopulations of Ig-bearing
cells in lymphoid organs of rainbow trout. Dev Comp
Immunol 1995;19:79±86.
Boesen HT, Pedersen K, Koch C, Larsen JL. Immune
response of rainbow trout (Oncorhynchus mykiss) to antigenic
preparations from Vibrio anguillarum serogroup O1. Fish
Shell®sh Immunol 1997;7:543±53.
Feng JS, Woo PTK. Cell-mediated immune response and
G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
T-like cells in thymectomized Oncorhynchus mykiss
(Walbaum) infected with or vaccinated against the pathogenic
haemo¯agellate Cryptobia salmositica Katz, 1951. Parasitol
Res 1996;82:604±11.
Marsden MJ, Vaughan LM, Foster TJ, Secombes CJ. A live
(Delta aroA) Aeromonas salmonicida vaccine for forunculosis
responses in rainbow trout (Oncorhynchus mykiss). Infection
Immunity 1996;64:3863±9.
Scapigliati G, Mazzini M, Mastrolia L, Romano N, Abelli L.
Production and characterisation of a monoclonal antibody
against the thymocytes of the sea bass Dicentrarchus labrax
L. (Teleostea, Percicthydae). Fish Shell®sh Immunol 1995;
5:393±405.
Scapigliati G, Romano N, Picchietti S, Mazzini M, Mastrolia
L, Scalia D, Abelli L. Monoclonal antibodies against sea bass
Dicentrarchus labrax (L) immunoglobulins: immunolocalisation of immunoglobulin-bearing cells and applicability in
immunoassays. Fish Shell®sh Immunol 1996;6:383±401.
Nehr O, Blancheton IP, Alliot E. Development of an intensive
culture system for sea bass (Dicentrarchus labrax) larvae in
sea enclosures. Aquaculture 1996;142:43±58.
Dec C, Angelidis P, Baudin-Laurencin F. Effects of oral
vaccination against vibriosis in turbot, Scophthalmus maximus
L., and sea bass Dicentrarchus labrax L. J Fish Dis
1990;13:369±76.
Bakopoulos V, Volpatti D, Adams A, Galeotti M, Richards R.
Qualitative differences in the immune response of rabbit,
mouse and sea bass, Dicentrarchus labrax, L., to Photobacterium damsela subsp piscicida, the causative agent of
®sh Pasteurellosis. Fish Shell®sh Immunol 1997;7:161±74.
Sideris DC. Cloning, expression and puri®cation of the coat
protein of encephalitis virus (DIEV) infecting Dicentrarchus
labrax. Biochem Mol Biol Int 1997;42:409±17.
Skliris GP, Richards RH. Induction of nodavirus disease in sea
bass, Dicentrarchus labrax, using different infection models.
Virus Res 1999;63:85±93.
Abelli L, Picchietti S, Romano N, Mastrolia L, Scapigliati G.
Immunohistochemistry of gut-associated lymphoid tissue of
the sea bass Dicentrarchus labrax (L.). Fish Shell®sh
Immunol. 1997;7:235±46.
Litman GW. Sharks and the origins of vertebrate immunity.
Sci Am 1996;275:67±71.
Matsunaga T. Did the ®rst adaptive immunity evolve in jawed
®sh?. Cytogenetics Cell Genetics 1998;80:138±41.
MunÄoz J, Esteban MA, Meseguer J. In vitro culture
requirements of sea bass (Dicentrarchus labrax L.) blood
cells: differential adhesion and phase contrast microscopic
study. Fish Shell®sh Immunol 1999;9:417±28.
Galeotti M, Volpatti D, Rusvai M. Mitogen induced in vitro
stimulation of lymphoid cells from organs of sea bass
(Dicentrarchus labrax L.). Fish Shell®sh Immunol 1999;
9:227±32.
Ingram G. Substances involved in the natural resistance of ®sh
to infection. A review. J Fish Biol 1980;16:23±60.
Fletcher TC. Non-speci®c defence mechanisms of ®sh. Dev
Comp Immunol 1982(suppl 2):123±32.
Ellis AE. The immunology of teleosts. In: Roberts RJ, editor.
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
159
Fish pathology, 2nd ed. London: Bailliere Tindal, 1989. p.
135±53.
Manning MJ. Fishes. In: Turner RJ, editor. Immunology: a
comparative approach. Chichester: John Wiley & Sons Ltd,
1994. p. 69±100.
Van Muiswinkel WB. The piscine immune system: innate and
acquired immunity. In: Woo PTK, editor. Fish diseases and
disorders. Volume 1: protozoan and metazoan infections.
Canada: Department of Zoology, University of Guelph,
1995. p. 729±49.
Meseguer J, Esteban MA, Agulleiro B. Stromal cells,
macrophages and lymphoid cells in the head-kidney of sea
bass (Dicentrarchus labrax L.). An ultrastructural study.
Arch Histol Cytol 1991;54:299±309.
Mulero V, Esteban MA, MunÄoz J, Meseguer J. Non-speci®c
cytotoxic response against tumor target cells mediated by
leucocytes from seawater teleosts, Sparus aurata and
Dicentrarchus labrax: an ultrastructural study. Arch Histol
Cytol 1994;57:351±8.
Meseguer J, Esteban MA, Mulero V. The nonspeci®c
cell-mediated cytotoxicity in the seawater teleosts (Sparus
aurata and Dicentrarchus labrax): ultrastructural study of
target cell death mechanisms. Anat Rec 1996;244:499±505.
Cammarata M, Vazzana M, Cervello M, Arizza V, Parrinello
N. Spontaneous cytotoxic activity of eosinophilic granule
cells separated from the normal peritoneal cavity of
Dicentrarchus labrax. Fish Shell®sh Immunol 2000;10:143±
54.
Bennani N, Schmid-Alliana A, Lafaurie M. Evaluation of
phagocytic activity in a teleost ®sh, Dicentrarchus labrax.
Fish Shell®sh Immunol 1995;5:237±46.
Esteban MA, Meseguer J. Factors in¯uencing phagocytic
response of macrophages from the sea bass (Dicentrarchus
labrax L): an ultrastructural and quantitative study. Anat
Record 1997;248:533±41.
MunÄoz J, Sitja-Bobadilla A, Alvarez-Pellitero P. Cellular and
humoral immune response of European sea bass (Dicentrarchus labrax L.) (Teleostei: Serranidae) immunized with
Sphaerospora dicentrarchi (Myxosporea: Bivalvulida). Parasitology 2000;120:465±77.
SitjaÁ-Bobadilla A, PeÂrez-SaÂnchez J. Diet related changes in
non-speci®c immune response of European sea bass (Dicentrarchus labrax L.). Fish Shell®sh Immunol 1999;9:637±40.
Obach A, Quentel C, Laurencin FB. Effects of alpha-tocopherol
and dietary oxidized ®sh oil on the immune response of sea bass
Dicentrarchus labrax. Dis aquatic org 1993;15:175±85.
Coeurdacier JL, Pepin JF, Fauvel C, Legall P, Bourmaud AF,
Romestand B. Alterations in total protein, IgM and speci®c
antibody activity of male and female sea bass (Dicentrarchus
labrax L, 1758) sera following injection with killed Vibrio
anguillarum. Fish Shell®sh Immunol 1997;7:151±60.
Pretti C, Milone MTA, Cognetti Varriale AM. Fish
pasteurellosis: sensitivity of Western blotting analysis on the
internal organs of experimentally infected sea bass (Dicentrarchus labrax). Bull Eur Ass Fish Pathol 1999;19:120±2.
Mazzolini E, Fabris A, Ceschia G, Vismara D, Magni A,
Amadei A, Passera A, Danielis L, Giorgetti G. Pathogenic
160
[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
G. Scapigliati et al. / Developmental and Comparative Immunology 26 (2002) 151±160
variability of Pasteurella piscicida during in vitro cultivation
as a preliminary study for vaccine production. J of App
IchthyolÐZeit Ang Ichthyol 1998;14:265±8.
Scapigliati G, Romano N, Abelli L. Monoclonal antibodies in
teleost ®sh immunology: identi®cation, ontogeny and activity
of T- and B-lymphocytes. Aquaculture 1999;172:3±28.
EsteÂvez J, Leiro J, Santamarina MT, Dominguez J, Ubeira
FM. Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in
immunoassays. Vet Immunol Immunopathol 1994;41:353±
66.
Bourmaud CAF, Romestand B, Bouix G. Isolation and partial
characterization of IgM-like sea bass (Dicentrarchus labrax L.
1758) immunoglobulins. Aquaculture 1995;132:53±58.
Palenzuela O, SitjaÁ-Bobadilla A, Alvarez-Pellitero P. Isolation
and partial characterization of serum immunoglobulins from
sea bass (Dicentrarchus labrax L.) and gilthead sea bream
(Sparus aurata L.). Fish Shell®sh Immunol 1996;6:81±94.
Romestand B, Breuil G, Bourmaud CAF, Coeurdacier JL,
Bouix G. Development and characterisation of monoclonal
antibodies against sea bass immunoglobulins Dicentrarchus
labrax Linnaeus, 1758. Fish Shell®sh Immunol 1995;5:347±
57.
Dos Santos NMS, Taverne N, Taverne-Thiele AJ, De Sousa
M, Rombout JHWM. Characterisation of monoclonal
antibodies speci®c for sea bass (Dicentrarchus labrax L)
IgM indicates the existence of B cell subpopulations. Fish
Shell®sh Immunol 1997;7:175±91.
Romano N, Abelli L, Mastrolia L, Scapigliati G. Immunocytochemical detection and cytomorphology of lymphocyte
subpopulations in a teleost ®sh Dicentrarchus labrax (L).
Cell Tissue Res 1997;289:163±71.
Vigneulle M, Baudin-Laurencin F. Uptake of Vibrio
anguillarum bacterin in the posterior intestine of rainbow
trout Oncorhynchus mykiss, sea bass Dicentrarchus labrax
and turbot Scophthalmus maximus after oral administration
or anal intubation. Dis Aquatic Org 1991;11:85±92.
Scapigliati G, Scalia D, Marras A, Meloni S, Mazzini M.
Immunoglobulin levels in the teleost sea bass Dicentrarchus
labrax (L.) in relation to age, season, and water oxygenation.
Aquaculture 1999;174:207±12.
Joosten PHM, Tiemersma EW, Threels A, Dhieux-Caumartin
C, Rombout JHWM. Oral vaccination of ®sh against Vibrio
anguillarum using alginate microparticles. Fish Shell®sh
Immunol 1997;7:471±85.
Bandin I, Dopazo CP, Vanmuiswinkel WB, Wiegertjes GF.
Quantitation of antibody secreting cells in high and low
antibody responder inbred carp (Cyprinus carpio L.) strains.
Fish Shell®sh Immunol 1997;7:487±501.
Davidson GA, Lin SH, Secombes CJ, Ellis AE. Detection of
speci®c and costitutive antibody secreting cells in the gills,
head-kidney and peripheral blood leucocytes of dab (Limanda
limanda). Vet Immunol Immunopathol 1997(58):363±74.
Meloni S, Scapigliati G. Evaluation of immunoglobulins
produced in vitro by head-kidney leucocytes of sea bass
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
[73]
[74]
Dicentrarchus labrax by immunoenzymatic assay. Fish
Shell®sh Immunol 2000;10:95±99.
Nakanishi T, Ototake M. Antigen uptake and immune
responses after immersion vaccination. Dev Biol Stand
1997;90:59±68.
Sizemore RC, Miller NW, Cuchens MA, Lobb CJ, Clem LW.
Phylogeny of lymphocyte heterogeneity: the cellular
requirements for in vitro mitogenic responses of channel
cat®sh leucocytes. J Immunol 1984;133:2920±4.
Miller NW, Sizemore RC, Clem LW. Phylogeny of
lymphocyte heterogeneity: the cellular requirements for in
vitro antibody responses of channel cat®sh leucocytes.
J Immunol 1985;134:2884±8.
Miller NW, Van Ginkel JE, Ellsaesser F, Clem LW.
Phylogeny of lymphocyte heterogeneity: identi®cation and
separation of functionally distinct subpopulations of channel
cat®sh lymphocytes with monoclonal antibodies. Dev Comp
Immunol 1987;11:739±48.
Abelli L, Baldassini MR, Mastrolia L, Scapigliati G.
Immunodetection of lymphocyte subpopulations involved in
allograft rejection in a teleost, Dicentrarchus labrax (L.). Cell
Immunol 1999;191:152±60.
Graham S, Secombes CJ. Cellular requirements for
lymphokine secretion by rainbow trout, Salmo gairdneri,
leucocytes. Dev Comp Immunol 1990;5:75±83.
Volpatti D, Rusvai M, D' Angelo L, Manetti M, Galeotti M.
Evaluation of sea bass (Dicentrarchus labrax) lymphocytes
proliferation after in vitro stimulation with mitogens. Boll
Soc Ital Patol Ittica 1996;8:22±26.
Picchietti S, Terribili FR, Mastrolia L, Scapigliati G, Abelli L.
Expression of lymphocyte antigenic determinants in
developing GALT of the sea bass Dicentrarchus labrax (L.).
Anat Embryol 1997;196:457±63.
Scapigliati G, Romano G, Abelli L, Meloni S, Ficca AG,
Buonocore F, Bird S, Secombes CJ. Immunopuri®cation of
T-cells from sea bass Dicentrarchus labrax (L.). Fish Shell®sh
Immunol 2000;10:329±41.
Aviles Trigueros M, Quesada QA. Myelopoiesis in the thymus
of the sea bass, Dicentrarchus labrax L. (Teleost). Anat
Record 1995;242:83±90.
Abelli L, Baldassini MR, Meschini R, Mastrolia L. Apoptosis
of thymocytes in developing sea bass Dicentrarchus labrax
(L.). Fish Shell®sh Immunol. 1998;8:13±24.
Breuil G, Vassiloglou B, Pepin JF, Romestand B. Ontogeny of
IgM-bearing cells and changes in the immunoglobulin M-like
protein level (IgM) during larval stages in sea bass (Dicentrarchus labrax). Fish Shell®sh Immunol 1997;7:29±43.
Abelli L, Picchietti S, Romano N, Mastrolia L, Scapigliati G.
Immunocytochemical detection of a thymocyte antigenic
determinant in developing lymphoid organs of sea bass
Dicentrarchus labrax (L.). Fish Shell®sh Immunol 1996;
6:493±505.
Rombout JHWM, Joosten PHM, Engelsma MY, Vos N,
Taverne N, Taverne-Thiele A. Indication of a distinct putative
T cell population in mucosal tissue of carp. Dev Comp
Immunol 1998;22:63±77.