Fish & Shellfish Immunology 22 (2007) 57e67

www.elsevier.com/locate/fsi

Effects of administration of probiotic strains on GALT

of larval gilthead seabream: Immunohistochemical and
ultrastructural studies
Simona Picchietti a, Massimo Mazzini a, Anna Rita Taddei b, Raffaella Renna a,
Anna Maria Fausto a, Victoriano Mulero c, Oliana Carnevali d,
Alberto Cresci e, Luigi Abelli f,*
a
Dipartimento Scienze Ambientali, Università della Tuscia, 01100 Viterbo, Italy
b
Centro Interdipartimentale Microscopia Elettronica, Università della Tuscia, 01100 Viterbo, Italy
c
Department Cell Biology, University of Murcia, 30100 Murcia, Spain
d
Dipartimento Scienze del mare, Università Politecnica delle Marche, 60100 Ancona, Italy
e
Dipartimento Scienze Morfologiche e Biochimiche Comparate, Università di Camerino, Italy
f
Dipartimento Biologia, Sezione Anatomia Comparata, Università di Ferrara, Via Borsari 46, 44100 Ferrara, Italy
Received 12 December 2005; revised 16 January 2006; accepted 14 March 2006
Available online 28 March 2006

Abstract

Two bacterial strains Lactobacillus fructivorans (AS17B), isolated from adult seabream (Sparus aurata L.) gut, and Lactoba-
cillus plantarum (906), isolated from human faeces, were administered contemporaneously during seabream development using
Brachionus plicatilis and/or Artemia salina and dry feed as vectors.
Experimental group A received the probiotic strains already via rotifers from day 5 post-hatch (ph), whereas treatment of group
B began with Artemia feeding from day 27 ph. Fish were sampled at day 28 ph (group A and control) and day 99 ph (groups A, B
and control) for electron microscopy, histology and immunohistochemistry with the polyclonal antiserum ORa against homologous
serum Ig and the mAb G7 specific for seabream acidophilic granulocytes. In all groups, timing and pattern of differentiation of the
digestive tract did not differ. Furthermore, neither tissue damage nor manifest inflammation was provoked by probiotic adminis-
tration.
At day 28 ph, the developing GALT already housed mucosal leucocytes, including Igþ cells but no acidophilic granulocytes. No
differences were seen between experimental groups.
At day 99 ph, the density of Igþ cells (þ51%) and acidophilic granulocytes (þ284%) was significantly higher (p < 0.05) in
group A than in controls. Also group B had a higher density of Igþ cells (þ17%) and acidophilic granulocytes (þ130%) compared
with controls, although less pronounced. Light and electron microscopy observations detailed the occurrence of heterogeneous pop-
ulations of lymphocytes and granulocytes in the developing intestinal mucosa, and highlighted the net expansion of G7þ acidophilic
granulocytes (A þ536%, B þ292% vs. control) due to probiotic administration.

Abbreviations: GALT, gut-associated lymphoid tissue; IEL, intraepithelial lymphocyte; IHC, immunohistochemistry; IR, immunoreactive;
mAb, monoclonal antibody; MGG, May-Grünwald-Giemsa; Lp, lamina propria; PBS, phosphate-buffered saline.
* Corresponding author. Fax: þ39 0532 291715.
E-mail address: abl@unife.it (L. Abelli).

1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fsi.2006.03.009
58 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67

Evidence is provided that early feeding with probiotic-supplemented diet increased the number of Igþ cells and acidophilic
granulocytes in seabream gut and that the effects were more pronounced when administration started during gut metamorpho-
sis. These results point to a stimulatory effect of probiotics on the gut immune system that correlates with improvement of fry
survival.
Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Lactobacillus fructivorans; Lactobacillus plantarum; Probiotics; Sparus aurata; GALT; Larval immunity

1. Introduction

Bacterial diseases are considered to be a major cause of mortality [1] and economic loss in fish hatcheries. Either
treatment with chemotherapeutic agents or vaccination by bath and/or injection route is currently used to protect fish
against different bacterial diseases. The former method may alter the profile of a healthy gut microflora and lead to
selection of resistant strains [2] and causes environmental concerns, thus its use should be restricted, while vaccination
by injection is a stressful delivery method for fish.
The recent, growing interest in probiotic bacteria for their use in aquaculture has developed as a consequence of the
continuous search for alternative preventive strategies [3]. Administration of various microbial products has in some
instances been shown to enhance the survival of cultured fish when exposed to pathogens [4e6]. At least two hypoth-
eses to explain such findings have been proposed: (i) an inhibitory action of antimicrobial substances released by the
bacterium in question [7], (ii) an immune stimulating effect of the bacterial products [8].
Stimulatory effects, such as increased cytotoxic and phagocytic activities, were found after feeding gilthead seab-
ream (Sparus aurata L.) a diet supplemented with Lactobacillus delbrueckii ssp. lactis or Bacillus subtilis [9]. How-
ever, no data are available about dietary administration of probiotics during the larval stage, the most critical period in
the production, when mass mortalities are commonly caused by bacterial infections.
The two bacterial strains Lactobacillus fructivorans (AS17B), isolated from adult seabream intestinal
microflora [10], and Lactobacillus plantarum (906), isolated from human faeces [11], were administered using
Brachionus plicatilis, Artemia salina and dry feed as vectors. The present data on the effect of probiotics on
the gut-associated lymphoid tissue (GALT) of S. aurata have been taken from the same experiment that showed
that the bacterial strains produced desirable effects on seabream during development by significantly improving
survival of larvae and fry [10].

2. Materials and methods

2.1. Experimental design

For specific technical details and studies on efficiency of bacterial strain administration and gut colonization,
see [10].
Briefly, the two bacterial strains L. fructivorans (AS17B) and L. plantarum (906), both at a concentration of
1010 CFU g1 cell pellet, were mixed (80% and 20%, respectively, w/w) and then incubated under low aeration for
15 min with the live food (rotifers or Artemia) for allowing their uptake before administration to fish larvae. Further-
more, bacteria were added to dry food pellet (NippaiÒ, Tokio, Japan) sifting the mixture to obtain homogeneous
distribution. All treatments (live or dry feed) were made to obtain a final concentration of 105 bacteria ml1 sea water
in the larvae tanks.
Seabream larvae at day 1 post-hatch (ph) were distributed in 400 l tanks containing UV-treated, filtered (sand) sea
water. The water temperature in the tanks was gradually increased from 17  C (day 1 ph) to 18.5  C (day 5 ph). Three
experimental groups of 25,000 larvae (each in duplicate) were set up: A, B and control (150,000 larvae in total). Group
A received a mixture of both bacterial strains via rotifers from day 5 to day 26 ph and via Artemia from day 27 to day
56 ph. From day 57 to day 99 ph, the fry received the treatment in dry pelleted feed. Group B received identical
bacterial mixture only using Artemia as vector from day 27 to day 56 ph. From day 57 to day 99 ph, the treatment
was given in dry pelleted feed. No bacteria were administered to the control group.
 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67 59

2.2. Histology and immunohistochemistry

Fifteen specimens per group were collected at 28 and 99 days ph. Larvae and fry were fixed in Bouin’s liquid for 7 h
at 4  C and embedded in paraffin wax. Serial sections 7 mm thick were stained with haematoxylin and eosin or
May-Grünwald-Giemsa (MGG) for general histology.
Immunohistochemistry (IHC) was performed by ABC peroxidase with nickel enhancement as previously described
[12]. Serial sections 7 mm thick were incubated for 18 h at room temperature with the rabbit polyclonal antiserum ORa
(diluted 1:1500 in PBS 0.1 M, pH 7.3, containing 5% normal goat serum and 0.1% sodium azide) that recognizes gilt-
head seabream Ig H (81 kDa) and L (25 kDa) chains [13] or with the mouse mAb G7 (diluted 1:10 in PBS 0.1 M, pH
7.3 containing 5% normal horse serum and 0.1% sodium azide) specific for gilthead seabream acidophilic granulo-
cytes [14]. Pre-immune serum substituted the primary antibody in negative controls. Thereafter, sections were incu-
bated for 60 min with biotinylated goat anti-rabbit IgG serum or horse anti-mouse IgG serum (Vector Labs,
Burlingame, USA) diluted 1:1000 with PBS containing 0.1% sodium azide and 1% BSA, followed by incubation
for 60 min with avidinebiotinylated peroxidase complex (ABC, Vectastain Elite, Vector). Following rinses and stain-
ing (diaminobenzidine and nickel enhancement), sections were dehydrated, mounted and examined under bright-field
illumination.
In each specimen, multiple sets of consecutive sections were differentially immunostained with the antibodies
mentioned above and some were counterstained with eosin or MGG. Counts of immunoreactive cells and histologi-
cally stained cells (nucleated only, cell area ranging from 4 to 100 mm2) in 1 mm2 areas of posterior intestine (from
three non-consecutive sections per specimen) were performed by an observer unaware of treatments. Cell measure-
ments were obtained using a computer-assisted image analysis system which includes a Zeiss microscope equipped
with a colour video camera (Axio Cam MRC, Arese, Milano, Italy) and a software package (KS 300 and AxioVision).
The number of immunoreactive and histologically stained cells was then calculated by averaging the cell numbers
from all specimens. Measurements of cell diameter (major axis) were performed in five specimens and pooled. Data
analysis was performed using the GraphPad Prism 3.0 software statistical package. Mean and SEM were calculated
for each parameter. Two-way ANOVA was used to determine the effect of treatment. One-way analysis of variance
followed by Dunnett’s multiple comparison test were used to determine differences among groups. The level for
accepted statistical significance was p < 0.05.

2.3. Electron microscopy

Samples of larvae and fry (days 28 and 99 ph) were fixed with a mixture of 2.5% glutaraldehyde, 4% paraformal-
dehyde in 0.1 M, pH 7.2, cacodylate buffer [15], postfixed in 1% osmium tetroxide in cacodylate buffer then dehy-
drated in a graded ethanol series and embedded in Spurr resin (TAAB, England). Semithin and ultrathin sections
(60e80 nm) were obtained by means of a Reichert Ultracut ultramicrotome. Sections were stained with uranyl acetate
and lead citrate and examined with a Jeol JEM EX II transmission electron microscope at 120 kV.

3. Results

3.1. Histology and immunohistochemistry

Observations on the posterior intestine refer to seabream specimens at day 28 and day 99 ph. At day 28 ph, the
intestinal mucosa was folded and consisted of a simple epithelium and subepithelial connective tissue containing
a moderate number of leucocytes (Fig. 1a) both in control and group A. The major population of mucosal leucocytes
was represented by Ig lymphocyte-like cells. The polyclonal antiserum ORa immunostained a fraction of mucosal
leucocytes (size 5.1  0.5 mm, range 4.4e6.1 mm, N ¼ 100), whose density in control and group A (77  8 and
85  16 cells/mm2, respectively) was not significantly different. Cytoplasmic Ig was detected in ORa-immunoreactive
(-IR) cells (Fig. 1b). Neither acidophilic granulocytes, detectable after eosin or MGG stainings, nor G7-IR cells were
found at this age in the intestinal mucosa.
At day 99 ph, numerous lymphoid cells infiltrated the mucosal epithelium and lamina propria (Lp), together with
acidophilic granulocytes (Fig. 1c) and macrophages (Fig. 1d). A considerable presence of ORa-IR cells (Fig. 2a) that
had significantly smaller size (5.5  0.6 mm, range 4.5e6.5 mm, N ¼ 100) compared with macrophages (size
60 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67

Fig. 1. Histology and IHC of posterior intestine in day 28 ph larvae (a, b) and day 99 ph fry (c, d). (a) Semithin section of the folded mucosa
(control group) showing a moderate number of intraepithelial lymphocyte-like cells (arrows). (b) Pab ORa-IR cells (arrows) in the mucosa (control
group). (c) MGG staining showing lymphoid cells (arrows) and acidophilic granulocytes (arrowheads) in the mucosa epithelium and lamina prop-
ria (group A). (d) Semithin section showing lymphoid cells (arrows) and macrophages (asterisks) amongst leucocytes scattered in the intestinal
mucosa (group A). E ¼ epithelium, Lp ¼ lamina propria. Bars ¼ 10 mm in (a, b); 5 mm in (c, d).

14  1.6 mm, range 8.2e24.7 mm, N ¼ 50) containing phagocytosed material was detected. ORa-IR cells were distrib-
uted both intraepithelially and in the Lp (Fig. 2b) and had cytoplasmic Ig. Their density in group A was significantly
higher (þ51% and þ29%, respectively, p < 0.05) compared with controls and group B (Table 1). The density of Igþ
cells in group A was significantly higher (p < 0.05) at day 99 ph than at day 28 ph (Fig. 3).
Total acidophilic granulocytes were significantly more frequent in groups A and B (þ284% and þ130%, respec-
tively, p < 0.05) than in controls (Table 1). Furthermore, group A was significantly (p < 0.05) different from group B
(Table 1). The mAb G7 detected IR cells in the intestinal mucosa (Fig. 4a) and the density of these cells in groups A
and B largely exceeded (þ536% and þ292%, respectively, p < 0.05) that found in controls (Table 1). These cells had
a staining pattern as previously observed [14]. Counterstaining with MGG or eosin revealed that all of the intraepi-
thelial acidophilic granulocytes were G7þ (Fig. 4b). Otherwise, only a subpopulation of acidophilic granulocytes was
recognised by the mAb in the Lp (Fig. 4c). G7þ cells accounted for 23% (controls), 44% (group A) and 39% (group B)
of total acidophilic granulocytes.

3.2. Electron microscopy

Already at day 28 ph, leucocytes resided in the intestinal mucosa, still undergoing metamorphosis. Most leucocytes
had typical characteristics of lymphocytes, e.g. large nucleus and thin rim of cytoplasm. Lymphoid cells containing
more cytoplasm and free polysomes were observed in the subepithelial connective tissue (not shown).
At day 99 ph, the post-metamorphosis intestinal mucosa housed numerous lymphocytes characterized by hetero-
geneous shape (e.g. almost regular or elongated with short cytoplasmic processes), large nucleus and thin rim of
 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67 61

Fig. 2. IHC of Igþ lymphocytes in day 99 ph fry. (a) ORa-IR cells seed both mucosal epithelium and Lp (group A). (b) Intraepithelial and Lp Igþ
lymphocytes (arrows) are shown at higher magnification. E ¼ epithelium, Lp ¼ lamina propria. Bars ¼ 20 mm in (a); 10 mm in (b).

cytoplasm. Particularly, at least two types of intraepithelial lymphocytes (IEL), often contacting each other, were dis-
tinguished (Fig. 5a). A first cell type (IEL1) is characterized by heterochromatic nucleus and cytoplasm with short
processes and rich in ribosomes. A second type (IEL2) has often a rather regular shape, euchromatic nucleus with
marginal heterochromatin, and cytoplasm with large mitochondria. IELs are often in contact with large macrophages
with typical features of phagocytic cells (large lysosomes, multivesicular and residual bodies) (Fig. 5b) and

Table 1
Relative density (N cells/mm2) of intestinal leucocytes in day 99 ph fry fed with probiotic-supplemented diet
Control Group A Group B
G7þ granulocytes 53  10 390  80a 208  19a
Total acidophilic granulocytes 232  41 892  107a 534  73a,b
Igþ lymphocytes 94  11 142  9a 110  6b
Treatment of group A with the probiotic strains began via rotifers from day 5 ph, whereas treatment of group B began with Artemia feeding from day
27 ph. Numerical results were expressed as the mean  SEM and analysed by means of two-way ANOVA to determine the effect of treatment
(p < 0.001). One-way analysis of variance and Dunnett’s multiple comparison test were used to determine differences among groups.
a
Significantly different from control (p < 0.05).
b
Significantly different from group A (p < 0.05).
62 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67

200
Control

Ig-immunoreactive cells (N/mm2)

Group A a,b
150

100

50

0
day 28 ph day 99 ph

2
Fig. 3. Relative density (N cells/mm ) of Ig-immunoreactive cells in the posterior intestine of day 28 ph larvae and day 99 ph fry fed with
probiotic-supplemented diet (group A). Numerical results were expressed as the mean  SEM and analysed by means of two-way ANOVA to
determine the effect of treatment (p < 0.001). One-way analysis of variance and Dunnett’s multiple comparison test were used to determine
differences among groups. Significantly different from a: control and b: day 28 ph (p < 0.05).

granulocytes (Fig. 6a). Macrophages and lymphoid cells containing more cytoplasm and euchromatic nucleus with
marginal heterochromatin were found in the Lp (not shown).
At this age, two predominant typologies of granulocytes were found in the intestinal mucosa (Fig. 6). The first one
(Ga) is localized both intraepithelially (Fig. 6a) and in the Lp (Fig. 6b, c) and is characterized by rounded shape (cell
diameter 5.1  0.3 mm, range: 3.5e6.8 mm, N ¼ 100), endocytotic vesicles and cytoplasmic granules with an electron
dense paracrystalline core (Fig. 6c, d). The second one (Gb) is localized exclusively in the Lp (Fig. 6b), has elongated
shape (major axis of the cell: 8.0  0.4 mm, range: 6.0e10.9 mm; minor axis: 3.7  0.3 mm, range: 2.3e5.8 mm,
N ¼ 100) and contains a distinct granule type (irregular shape and homogeneous electron dense content) and autoly-
sosomes (Fig. 6b, e).

4. Discussion

In recent years, there has been great interest in the use of probiotic bacteria in aquaculture to improve resistance to
diseases and/or growth of farmed fish and water quality [16]. Most frequently, probiotics are associated with lactic
acid bacteria that can often produce bacteriocins and other compounds that may inhibit the growth of pathogenic bac-
teria [4]. Beneficial effects of microbial products on fish resistance to pathogen challenge were described but, in con-
trast to human studies, no reports have yet demonstrated any effects of probiotics on the fish intestinal immunity.
Furthermore, early probiotic administration has to be exploited since the larval forms of most fish are released in
the external environment at an early ontogenetic stage and are exposed to gastrointestinal microbiota-associated dis-
orders. In fact, they start feeding even though the digestive tract is not yet fully developed [17] and the immune system
is still incomplete [18,19].
The objective of our work has been the study of gut immune status, as characterised by the lymphocyte B (Igþ cells)
and granulocytic population of the intestinal epithelial layer and Lp, in an attempt to explain the observed beneficial
effect of probiotic administration [10]. Histology did not reveal any differences concerning timing and general pattern
of differentiation of the digestive tract in all experimental groups. Already at day 28 ph, leucocytes resided in the in-
testinal mucosa during gut metamorphosis, and no significant differences between control and group A were found. At
this stage, the ultrastructural analysis showed that the GALT mainly consisted of cells with lymphocyte-like cytology,
as described in other fish species [20]. The antiserum ORa recognised a fraction of mucosal leucocytes containing
cytoplasmic Ig. The contemporaneous lack of membrane Igþ cells and cells with typical ultrastructural features of
plasma cells (e.g. well developed RER cisternae) leads to consider these IR cells as putative pre-B cells. In fact, cy-
toplasmic Ig was found in pre-B cells in mammals and fish [21e23]. Furthermore, cell size (mean 5.1 mm) of Igþ cells
was significantly smaller compared with macrophages. It is worth noting that in other fish species intestinal
 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67 63

Fig. 4. IHC of intestinal acidophilic granulocytes in day 99 ph fry. (a) G7-IR granulocytes (arrows) scattered in the mucosa of the posterior
intestine (group A). (b) Double stained intraepithelial acidophilic granulocyte after mAb G7 immunostaining and eosin counterstaining (group
A). (c) Double staining with mAb G7 and MGG discriminates G7þ (arrowheads) and G7 acidophilic granulocytes (arrows) in the Lp (group
B). E ¼ epithelium, Lp ¼ lamina propria. Bars ¼ 10 mm in (a); 5 mm in (b, c).

Fig. 5. Electron microscopy of intestinal leucocytes in day 99 ph fry. (a) Two main typologies of lymphocytes, often contacting each other, are
found in the intestinal epithelium. IEL1 (arrow) is characterized by heterochromatic nucleus and cytoplasm with short processes and rich in
ribosomes. IEL2 (double arrow) has more regular shape, euchromatic nucleus and marginal heterochromatin. (b) IEL2 (double arrow) in contact
with a large macrophage (asterisk). E ¼ epithelium. Bars ¼ 0.5 mm in (a); 2 mm in (b).
64 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67

Fig. 6. Electron microscopy of intestinal granulocytes in day 99 ph fry. (a) The mucosal epithelium houses Ga granulocytes characterized by rounded
shape, endocytotic vesicles (arrow) and cytoplasmic granules. Ga is in contact with a IEL2 (double arrow). (b) The lamina propria contains Ga and Gb
granulocytes. The granules in Ga have electron dense paracrystalline core (shown at high magnification in (d)). Gb granulocytes have elongated shape,
a distinct granule type (shown at high magnification in (e)) and autolysosomes (arrows). (c) A Ga granulocyte in the lamina propria contains numerous
granules with complete (1), not complete (2) or small core (3) in the section. Bars ¼ 2 mm in (aec); 250 nm in (d); 200 nm in (e).

macrophages behave as Ig-binding cells [24] that can ingest antigens, and are proposed to have a role in antigen pre-
sentation [25,26]. The occurrence of Igþ cells in the larval intestine (controls included) suggests the early onset in
seabream of adaptive mechanisms of mucosal immunity, compared with ontogenetic data obtained in other teleosts
[27,28]. On the other hand, the absence at this age of acidophilic granulocytes suggests that some components of in-
nate mucosal immunity can appear later in seabream gut. Species differences are commonly observed in teleosts, thus
patterning of the immune system can reflect different evolutionary achievements as well as peculiar adaptations to
environment and antigens/pathogens.
 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67 65

At day 99 ph (post-metamorphosis stage), the intestinal mucosa was well differentiated with an evident organi-
zation of the subepithelial Lp. The abundant leucocytes mostly consisted of lymphocytes, macrophages and acido-
philic granulocytes localised both intraepithelially and in the Lp. As observed at the earlier stage, the dominant
population of ORa-IR cells had cytoplasmic Ig (mean size 5.5 mm). The density of Igþ cells in groups A and B
was higher compared with controls, the group A being significantly higher (p < 0.05) than group B. In addition,
the density of Igþ cells in group A was significantly higher (p < 0.05) at day 99 ph than at day 28 ph. These
data may indicate that early probiotic administration would affect intestinal immune cells, depending on the larval
age and/or the duration of the treatment. Whether such stimulation would derive from local or systemic effects
remains to be determined. In fact, oral administration of Lactobacillus spp. in trout stimulated antibody production
[29] in agreement with data obtained in humans [30].
At day 99 ph, the ultrastructural analysis revealed at least two types of lymphocytes, often contacting each other, in
the epithelial compartment. The existence of two IEL typologies was previously proposed in trout, where both cell
populations showed a significant cytotoxic activity [31]. Otherwise, neither specific nor non-specific cytotoxicity
was exhibited by IELs in carp [28]. Therefore, the functional spectrum of fish IELs is far from being understood
and the lack of specific T-cell markers in seabream greatly hampers comprehension of the effects of probiotic admin-
istration. As a matter of fact, the identity as T-cells of most IELs has been convincingly proposed in at least two teleost
species (carp and sea bass) [32,33]. Further studies are expected to evaluate the effects of probiotics on fish T-cells and
their relationships with B-cells.
Our ultrastructural data provided evidence that IELs are often in contact with large macrophages and granulocytes.
Although several, earlier studies indicated monocytes/macrophages as the major phagocyte populations in fish, recent
data in seabream have convincingly demonstrated that acidophilic granulocytes have a relevant role in the initiation
and support of the adaptive immune response [14]. Intraperitoneal injection of intact Vibrio anguillarum induced a rise
in the number of acidophilic granulocytes in the head kidney (main source), mobilization to the peritoneal cavity, and
capture and transport of antigens to the spleen [34,35]. Interestingly, these cells constitutively express MHC class II
mRNA [36] and produce IL-1b following bacterial infection [34].
In our study, at day 99 ph total acidophilic granulocytes significantly increased in groups A and B compared with
controls, group A being significantly higher than group B. According to their distribution and fine cytology two main
typologies of granulocytes (Ga and Gb) were described in the intestinal mucosa. Ga granulocytes are localized both
intraepithelially and in the Lp, and have the ultrastructural characteristics of G7-IR granulocytes previously described
[14,37]. Gb are localized in Lp only and contain a different granule type. IHC of the intestinal mucosa with the mAb
G7 and eosin/MGG counterstaining also discriminated two main subpopulations of acidophilic granulocytes (EþG7þ
and EþG7), corroborating ultrastructural data. According to their localization and staining pattern EþG7 granulo-
cytes may represent the gilthead seabream EGCs [38] which do not exhibit phagocytic activity in vivo.
Quantitative analysis demonstrated a net expansion of the G7þ cell population in groups treated with probiotic.
G7þ cells account for nearly all of the intraepithelial acidophilic granulocytes and for a subpopulation of acidophilic
granulocytes in the Lp. Our findings and previous ones altogether show that probiotic treatment (mainly protocol A)
particularly stimulated the G7þ subpopulation, shown to be phagocytic against Vibrio anguillarum [14,35]. These
results are apparently in keeping with recent data showing that administration to adult seabream of Lactobacillus
delbrueckii and Bacillus subtilis enhanced phagocytosis and cytotoxic activity [9]. Likewise, oral administration of
Lactobacillus rhamnosus stimulated respiratory burst in rainbow trout [29]. Increased phagocytic activity, resistance
to infection and host survival were consequent to administration of Lactococcus lactis [39] in turbot. These results in
fish agree with data gathered in mammals that lactobacilli increase phagocytic activity of peripheral leucocytes and
macrophages [40e42].
Although the recruitment of acidophilic granulocytes to a persistent inflammatory site is thought a generalized
response in teleosts [43], the integrity of the intestinal mucosa (including the epithelial brush border) and the lack
of edema and vasodilation, as well as the general absence of tissue(s) damage in fish treated in our study, altogether
indicate safety of probiotic administration. Indeed, in the same experiment the positive effects on seabream health
during development is demonstrated by significant improvement of larvae and fry survival. The effective strain col-
onization of seabream larval gut was monitored and confirmed, as well as the potential ability of Lactobacillus spp. to
affect gut microflora during development [10]. The present study dealt with analysis of selected leucocyte populations
for which some selective markers are available for this fish species. The data provide evidence of a significant effect of
early feeding with probiotic-supplemented diet on the number of Igþ cells and acidophilic granulocytes in seabream
66 S. Picchietti et al. / Fish & Shellfish Immunology 22 (2007) 57e67

GALT. Whether it would derive from enhanced proliferation, differentiation and/or recruitment of leucocyte sub-
populations will be addressed in future studies.

Acknowledgements

The authors wish to thank the staff of Panittica Pugliese, Torre Canne di Fasano (BR), Italy for helping in fish han-
dling, and Drs. Maria Rosaria Baldassini (Tuscia University) and Roberto Sulpizio (Ancona University) for technical
support. This study was supported by grants from the Italian Ministry for Agriculture (MIPAF 2002) to M.M., O.C.
and A.C.

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