Veterinary Immunology and Immunopathology 109 (2006) 209–217

www.elsevier.com/locate/vetimm

Immune responses after local administration of IgY loaded-PLGA

microspheres in gut-associated lymphoid tissue in pigs
Anne-Marie Torché a,b,*, Mireille Le Dimna b, Pascal Le Corre a, Alain Mesplède b,
Sébastien Le Gal b, Roland Cariolet c, Marie-Frédérique Le Potier b
a
Laboratoire de Pharmacie Galénique, Biopharmacie et Pharmacie Clinique, UPRES EA 3892,
Faculté des Sciences Pharmaceutiques et Biologiques, Université de Rennes I - 2, Avenue du Professeur Léon Bernard,
35043 Rennes cedex, France
b
Unité Virologie, Immunologie Porcine, UPRES EA 3892, Agence Française de Sécurité Sanitaire des Aliments,
Les Croix – B.P. 53, 22440 Ploufragan, France
c
Service de Production de Porcs Assainis et d’Expérimentation, Agence Française de Sécurité Sanitaire des Aliments,
Les Croix – B.P. 53, 22440 Ploufragan, France

Received 3 January 2005; received in revised form 25 July 2005; accepted 11 August 2005

Abstract

Oral vaccination of large animals using PLGA MS (poly(D,L-lactide-co-glycolide)microspheres) appeared to be more

challenging than immunization of mice. The purpose of this study was to deliver to GALT an immunogenic model protein (IgY),
free or encapsulated by spray-drying in PLGA MS, and to evaluate systemic immune response in SPF Large White pigs. Pigs
were surgically processed for local administration of IgY in three sets of experiments. In two sets of experiments, administration
was locally performed in temporary ligatured intestinal segments, in jejunal Peyer’s patches and in mesenteric lymph nodes. In
the third experiment, pigs received IgY via an intestinal cannula. Total IgY-specific antibodies were detected in the sera of pigs
after a single local immunization, but not in the sera of cannulated pigs. The study of IgG1 and IgG2 isotypes indicated that
PLGA MS are able to elicit a combined serum IgG2/G1 response with a predominance of IgG1 response when locally
administered. PLGA MS can be a potential oral delivery system for antigen but our results underlined the difficulty to immunize
large animals like pigs. Transposition of data between small and large animals appears to be complex and suggests that
physiological features need to be considered to increase intestinal availability of oral encapsulated vaccines.
# 2005 Elsevier B.V. All rights reserved.

Keywords: Pig; Gut-associated lymphoid tissue; Local administration; PLGA microspheres; Immune response

Abbreviations: GALT, gut-associated lymphoid tissue; PP, Peyer’s patches; PLGA MS, poly(D,L-lactide-co-glycolide)microspheres;
Specific Ig, total anti-IgY antibodies; IgY, avian immunoglobulin Y; IM, intramuscular; jPP, jejunal Peyer’s patches; jPP-E, administration
in jPP after enterectomy; jPP-IW, administration in jPP through intestinal wall; MLN, mesenteric lymph nodes; SPF, specified pathogen-free
* Corresponding author. Tel.: +33 2 23 23 49 50; fax: +33 2 23 23 48 46.
E-mail address: galenic@univ-rennes1.fr (A.-M. Torché).

0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetimm.2005.08.016
210 A.-M. Torché et al. / Veterinary Immunology and Immunopathology 109 (2006) 209–217
1. Introduction
Mucosal tissues of the gastrointestinal, respiratory
and urogenital tracts play a key role in the organism
since they are both, the gateway and the first line of
defence against invasion by microorganisms. Protec-
tion requires effective vaccination that stimulates
immune responses at the mucosal tissue (Brandtzaeg
et al., 1997). Thus, there is a great deal of interest in
the development of mucosal vaccine strategies (Chen,
2000). For this purpose, poly(D,L-lactide-co-glycoli-
de)microspheres (PLGA MS) designed for the
encapsulation of soluble antigens (Ag) are intensively
investigated.
Most of the studies focus on oral administration of
vaccines since: (i) the oral route appears to be easy to
handle in humans and animals; (ii) the gut-associated
lymphoid tissues (GALT) represent the largest
mucosal area. GALT is characterized by lymphoid
follicles, named Peyer’s patches (PP). Pig presents
isolated PP in the jejunum (jPP) and a continuous PP
in the terminal ileum (iPP) (Pabst et al., 1988). jPP
have a preferential function as sites for antigen
sampling, frequency of Ig secreting cells and
induction of intestinal immune response (Barman
et al., 1997; Pabst and Rothkotter, 1999; Bianchi et al.,
1999). PP are specifically covered by specialized
epithelial cells, M cells. These cells allow the transport
of Ag across the follicle-associated epithelium (FAE)
to the antigen-processing cells of the underlying dome
region with subsequent stimulation of B and T
lymphocytes (Gebert, 1997; Kraehenbuhl et al.,
1997; Neutra, 1999). Transcytosis activity has been
demonstrated for various polymeric nano- or micro-
particles (Jepson et al., 1993a; Ermark et al., 1995;
Smith et al., 1995) in many species including pig
(Torché et al., 2000). Different attempts were
performed to demonstrate the potentiality of PLGA
MS as vaccine adjuvant systems in order to induce
both systemic and local immune responses following
oral immunization. Experiments with antigens
entrapped in PLGA microspheres have demonstrated
that immunization boosts the systemic antibody
response, enhances mucosal IgA responses in mice
following oral or intragastric administration (Conway
et al., 2001; Jung et al., 2001; Fattal et al., 2002; Yeh
et al., 2002; Carcaboso et al., 2003) and is able of
priming cellular (cytotoxic T lymphocyte) immune
responses in vivo (Maloy et al., 1994; Partidos et al.,
1999).
Most vaccination experiments were successfully
performed on small animals like mice. Even if the
number of published data is smaller, it appeared
clearly more difficult to obtain the same results with
large animals. To date, these delivery systems remain
unsuccessful (Felder et al., 2000) or are largely untried
for oral vaccine delivery in large animals (Mutwiri
et al., 2002; Lin et al., 2003). The apparent
discrepancy between results from small and large
animals raises the problem of transposition and
suggests that additional barrier may impede the local
trafficking of MS throughout the GALT.
In order to challenge this assumption, we have
investigated the systemic immune response (total
immunoglobulins, IgA, IgG1 and IgG2 isotypes) after
administration of a model antigen (IgY) to pigs, as
solution or encapsulated in PLGA MS, at different
steps of Ag trafficking in the GALT. A surgical
experimental model was set up to locally deliver IgYat
different GALT locations, i.e. in intestinal lumen, in
mesenteric lymph nodes or within Peyer’s patches.
2. Materials and methods
2.1. Preparation of PLGA microspheres
containing IgY
Avian immunoglobulin Y (IgY 160 kD) was
isolated from chicken egg yolks by sodium precipita-
tion. Two milliliters of aqueous solution containing
IgY was emulsified by an ultrasonic probe with a 3%
(w/v) solution of PLGA dissolved in dichloromethan
(40 ml, RPE-ACS, Carlo Erba Reactifs) in a thermo-
stated beaker (2 8C) to produce a stable W/O
nanoemulsion. PLGA MS were subsequently formed
by spray-drying the nanoemulsion at a rate of 2 ml/
min under the following conditions: inlet air
temperature 50 8C; aspiration setting 100%; pump
control 15%; air flow rate 500 NL/h (Mini-Büchi 190).
Two formulations of PLGA MS (A and B) were
prepared by a spray-drying method. Formulation A
was prepared using an organic phase containing
Phusis1 75:25 poly(L-lactide-co-glycolide) and
0.05% (w/v) phosphatidylcholine. IgY was diluted
in PBS at a concentration of 4.5 mg/ml (w/v).
 A.-M. Torché et al. / Veterinary Immunology and Immunopathology 109 (2006) 209–217
Formulation B was prepared using Phusis1 75:25
poly(D,L-lactide-co-glycolide). The aqueous phase
contained IgY in PBS pH 7.4 at a concentration of
7.2 mg/ml (w/v) and 1% mannitol (w/v).
2.2. Characterization of PLGA microspheres
The protein content of PLGA MS was determined
by BCA assay (Pierce) after recovering IgY included
in PLGA MS by acetonitrile precipitation. Encapsula-
tion efficiency (%) was expressed by relating observed
IgY entrapment to theoretical IgY quantity used.
In vitro IgY release kinetics was performed and IgY
was measured (micro-BCA) in collected supernatants
after incubation of PLGA MS in PBS/azide (pH 7.4) at
37 8C.
PLGA MS size was determined by laser diffracto-
metry (Mastersizer S, Malvern Instruments) and
expressed as mean volume diameter (D 4.3 mm).
Structural integrity and immunogenicity of entrap-
ped IgY was assessed by 4–6% SDS–PAGE electro-
phoresis and Western blotting. The blot was incubated
with 1:1000 diluted alkaline phosphatase-conjugated
goat anti-IgY IgG (Jackson Immunoresearch).
2.3. Animals
Specified pathogen-free (SPF) Large White pigs
(n = 22) were used in this study. The pigs were housed
and reared in protected facilities and fed ad libitum.
All animal experiments described below have been
performed in the AFSSA Ploufragan facilities (regis-
tration No. B-22-745-1, authorization number: 22-25).
2.4. Immunization protocols
2.4.1. Surgical procedures
In the three sets of experiments, except pigs that
received intramuscular administration (IM), pigs were
locally administered IgY (free or encapsulated) or
negative control (BSA solution and blank micro-
spheres) after a surgical procedure.
Pigs were fasted 24 h before surgery. Each pig was
intravenously anaesthetized with tiletamine/zolaze-
pam/ketamine/xylazine. Then, the abdomen was
aseptically opened. After local injection of the antigen
as described below (experiments 1 and 2), muscle and
skin of the abdomen were closed using a nylon suture.
211
In experiment 3, IgY was administered through a
PVC/silicon cannula positioned by a surgical techni-
que described by Cuche and Malbert (1999). Cannula
was put into the duodenum and locally fixed with
sutures. The end of cannula was exteriorised through
an incision in the abdominal wall. The abdomen was
sutured and the cannula was fixed on the back of the
pig. Pigs recovered quickly, showing no clinical signs
and no difference in weight gain.
All pigs used in the study received an antibiotic
cocktail of penicillin–streptomycin for 5 days to avoid
infection.
2.4.2. Antigen preparation and administration
Soluble IgY was diluted in sterile PBS prior to
administration. PLGA MS, formulation A for experi-
ments 1 and 3 and formulation B for experiment 2,
were accurately weighted and suspended in sterile
PBS immediately before immunization. IgY, free or
encapsulated, was delivered as a single dose to pigs in
experiments 1 and 2. In experiment 3, pigs were
primed 3 weeks after the surgical procedure and then
boosted 28 days after the first administration. Soluble
BSA (7 mg) or blank MS were used as negative
control antigen.
2.4.3. Sample collection
Blood samples were weekly collected during the
time period of the assay (experiment 1: 56 days,
experiment 2: 61 days). Samples were centrifuged and
serum was collected and stored at 20 8C.
2.4.4. Experiments
IgY was delivered into different compartments of
the GALT. The three sets of experiments performed in
this study are summarized in Table 1.
Administration in jejunal Peyer’s patches (jPP)
were performed either directly through the intestinal
wall (jPP-IW) since PP were visible on the serosal
surface or after enterectomy (jPP-E). Administration
in mesenteric lymph nodes (MLN) was performed
directly in draining MLN. Administration in intestinal
lumen was performed directly via the cannula or in an
intestinal segment containing one or two isolated PP
ligatured and filled with 10 mg of IgY, either free or
encapsulated. The ligature was maintained for 15 min
in order to increase the time contact between IgY with
GALT.
212 A.-M. Torché et al. / Veterinary Immunology and Immunopathology 109 (2006) 209–217

Table 1
Synoptic of immunization experiments performed on SPF pigs
Route Number of pigs Age (weeks) IgY (mg/dose) Volume (ml) Antigen
Intramuscular 4 Solution Exp 1
3 1 2 Formulation A
7 Formulation B Exp 2
In situ
Mesenteric lymph node 2 4 7 1.5 Solution Exp 1
5 Formulation A
Jejunal Peyer’s patches—intestinal wall 4 4 7 1.5 Solution Exp 1
5 Formulation A
0 1.5 BSA solution
5 Blank MS
Jejunal Peyer’s patches—enterectomy 2 4 7 1.5 Solution Exp 1
5 Formulation A
Jejunal Peyer’s patches—intestinal wall 2 7 10 1.5 Solution Exp 2
5 Formulation B
Intestinal lumen
Jejunal segment 3 7 10 9 Solution Exp 2
Formulation B
Ileal segment Formulation B
Cannula 2 4 10 20 Solution Exp 3
4 Formulation A

2.5. Total Anti-IgY antibody detection
Anti-IgY antibodies (specific Ig) were measured by
conventional indirect ELISA. Microtitre plates (Nunc-
Immuno Plate1 FP96 Maxisorp) were coated with
100 ml per well of 0.5 mg/ml IgY in carbonate buffer
(pH 9.6) and incubated 1 h at 37 8C, then placed
overnight at +4 8C. Plates were washed with PBS
containing 0.1% Tween-20 (PBST) and blocked for
1 h at 37 8C with 200 ml PBST containing 2% (w/v)
non-fat dry milk (Bio-Rad). Serum samples and the
negative control (SPF pig serum) were diluted 1:100 in
PBS, and 100 ml of each sample were added to each
well. The positive control (hyperimmune pig serum)
was serially diluted from 1:1000 to 1,000,000. Plates
were incubated for 1 h at 37 8C and washed three
times with PBST. Hundred microliters of HRP-
conjugated polyclonal rabbit anti swine immunoglo-
bulins (P0164; Dako) diluted 1:1000 in blocking buffer
were added to each well. Plates were incubated for
30 min at 37 8C and washed three times with PBST.
Then 100 ml of chromogen (orthophenylenediamine
and hydrogen peroxide in citrate buffer (pH 5.0) were
added to each well and incubated at 37 8C for color
development. The reaction was stopped after 6 min by
addition of 50 ml of 1N H2SO4. Absorbance values (A)
for each sample were read at 490 nm with a plate reader
(Dynatech). ELISA was done in triplicate. Results are
expressed as positive percentage of hyperimmune
serum, diluted 10,000, calculated as follows:
Aexperimental serum Anegative control
 100
Ahyperimmune serum Anegative control
A positive threshold (25%) was established for
specific total Ig.
2.6. Anti-IgY isotypes detection
Antibody isotype analysis of pig sera was
performed using the same ELISA with some
modifications. Microtitre plates were coated with
100 ml per well of a 2.5 mg/ml IgY in carbonate–
bicarbonate buffer (pH 9.6). After identical steps of
washings, blocking and serum samples (dilution
1:100) incubation, monoclonal antibodies (Id-DLO,
Lelystadt), anti-swine IgA (mabaSw 27.9.1), anti-
swine IgG1 (mabaSw 23.4.91) and anti-swine IgG2
(mabaSw 34.1.1a) were added to the wells at the
dilution of 1:500 in PBST. The plates were incubated
for 1 h at 37 8C and washed three times with PBST.
 A.-M. Torché et al. / Veterinary Immunology and Immunopathology 109 (2006) 209–217 213

100 ml of HRP-conjugated polyclonal rabbit anti

mouse immunoglobulins (P0260; Dako) diluted 1:500
in blocking buffer was added to each well. The plates
were incubated for 30 min at 37 8C and washed three
times with PBST. After addition of the substrate as
previously described, the reaction was stopped after
3 min of incubation by the addition of 50 ml of 1N
H2SO4. ELISA was done in triplicate. Results are
expressed as described above. A positive threshold
(10%) was established for isotypes.

3. Results and discussion

3.1. Characterization of PLGA MS

The average diameter for formulation A and

formulation B was 4.2 mm and 5.6 mm, respectively,
with a narrow size distribution and a lack of aggregates
in the suspension. The encapsulation efficiency of IgY
for both formulations was near 90% (w/w).
After a small initial burst release after 1 h incubation
(6.1% for formulation A and 9.3% for formulation B), a
zero-order release was observed with an increase rate
above 5–7 days from 0.4 to 2.4%/day for formulation A
and from 1.0 to 4.9%/day for formulation B. The release
profiles obtained between the two formulations were
different since formulation B displayed substantially
higher release rate (Fig. 1). This may be explained by
the differences between the two formulations (i.e.
phosphatidylcholine and mannitol). It could be
suggested that IgY was differently localized in
formulations A and B and/or each formulation could
present distinct water uptake rates influencing the
release of IgY.
No additional bands indicating the presence of IgY
aggregates or fragments were observed on SDS–
PAGE gel and Western blot confirmed that the
immunogenicity of IgY was not altered during PLGA
MS fabrication (data not shown).
3.2. Induction of systemic IgY-specific antibody
responses
3.2.1. Induction of systemic IgY-specific antibody
responses after intramuscular administration
Following a single IM administration, free IgY
induced higher serum specific Ig response compared
Fig. 1. Cumulative in vitro release profiles of IgY from PLGA MS
formulations A (*) and B (&). Values are represented as mean
(standard deviation).
to encapsulated IgY (Fig. 2a). Moreover, formulation
B could not induce specific immune responses so
effectively than formulation A. This observation
suggests that the properties of PLGA MS influence
the induction of systemic immune response. A rapid
clearance of PLGA MS and/or difference in IgY
release, as we have seen between formulations A and
B, may explain this observation.
3.2.2. Induction of systemic IgY-specific antibody
responses after local administration
Oral vaccination with PLGA MS appears difficult
to be extrapolated from small laboratory animals to
large animals. Since we have met difficulties to induce
immune response with pigs assayed under a conven-
tional experimental procedure (i.e. oral gavage), we
have decided to focus on local delivery of PLGA MS
in the compartments where such particles could be
found after oral administration.
The Fig. 2b shows the immune response after
administration of IgY in jejunal Peyer’s patches
214 A.-M. Torché et al. / Veterinary Immunology and Immunopathology 109 (2006) 209–217

Fig. 2. Induction of total anti-IgY immunoglobulins in pigs immunized with free IgY (open symbol) or with PLGA MS (full symbol) containing
IgY. Experiment 1/formulation A (plain line). Experiment 2/formulation B (dashed line). One curve corresponds to one pig. (a) Intramuscular
administration (^ ^); (b) direct administration in jejunal PP through the intestinal wall (& &); (c) direct administration in jejunal PP through
the FAE after enterectomy (~ ~) and direct administration in MLN (* *); and (d) instillation in ligatured segment in intestinal lumen (~ ~
jejunum; * ileum).
 A.-M. Torché et al. / Veterinary Immunology and Immunopathology 109 (2006) 209–217
through the intestinal wall (jPP-IW) (experiments 1
and 2). Ig production was similar until about day 40.
Then, in experiment 1, Ig production still increased by
opposition to experiment 2. This discrepancy between
experiments 1 and 2 cannot only be attributed to the
use of two PLGA MS formulations since free IgY has
led to different Ig levels. The influence of pig age (4
and 7 weeks in experiments 1 and 2, respectively) may
explain such discrepancy and should be further
studied.
Negative controls administered in jPP-IW led to
positive percentage lower than the threshold
(BSA  18.4%, blank MS  11.4%).
Local administration was also performed by
enterectomy (jPP-E), a more invasive method.
Specific Ig response obtained with PLGA MS
(formulation A) was three-fold higher than that
obtained with free IgY (Fig. 2c) and was different,
almost two-fold, to the immune response induced after
administration through the intestinal wall (Fig. 2b). It
could be suggested that the depth within PP where IgY
was delivered was not the same by the two methods
since PLGA MS formulation used and pig age were
the same.
PLGA MS, in our size range, can be translocated to
mesenteric lymph nodes (MLN) and subsequently to
the spleen (Carr et al., 1996). MLN contain a large
population of lymphocytes and antigen-presenting
cells, allowing a good immune response to be
expected after local induction. Direct administration
of PLGA MS in MLN induced a stronger systemic
immune response, almost two-fold higher than free
IgY (Fig. 2c). Specific Ig production was sustained or
increased during all the period studied. The present
result illustrated the potential of PLGA MS as
adjuvant.
Serum immune responses following instillation
into ligatured segments of IgY, free or encapsulated,
are presented in Fig. 2d. PLGA MS appeared to elicit
relatively close specific Ig levels when instillated in
jejunal and ileal segments. Free IgY instilled in
intestinal lumen was able to induce serum Ig,
suggesting that the time contact with enzymatic
content of the intestine was too short for a complete
denaturation.
Since instillation of PLGA MS in intestinal
ligatured segments has elicited a systemic immune
response, we have investigated the local delivery of
215
PLGA MS in intestinal lumen in cannulated pigs
without any intestinal ligatures. Previously, formula-
tion A appeared to elicit better immune responses than
formulation B, and hence was employed in experiment
3. However, no specific Ig were detected, despite of
the boost administration at day 28.
The comparison of results from IgY administration
in a temporary ligatured intestinal segment and via a
cannula gave us interesting informations. PLGA MS
not only gives rise to significant antibody titres when
instilled in a ligatured segment but also indicate the
importance of residence time of PLGA MS for
efficient uptake in lymphoid follicles and induction of
immune response, since no Ig were dosed in
cannulated pigs. We could suggest that PLGA MS
were flushed by peristaltism and/or cleared by normal
mucus clearance in faeces. Results strongly suggested
that formulations, which can further increase the
residence time in the intestine and subsequently the
extent of particle uptake, are desirable.
3.2.3. Induction of systemic IgY-specific antibody
subclasses
IgA response was different according to the site of
Ag administration and the vehicle, free or encapsulated
(Fig. 3a). Intramuscular administration of PLGA MS
induced similar IgA level than with free IgY. Production
of serum IgA was greatly enhanced when PLGA MS
(formulation A) were used in MLN and in PP after
enterectomy compared to solution and was much higher
than after IM administration. After instillation in
ligatured intestinal segments, the slight IgA production
was higher when PLGA MS were used.
IgG1 appeared to be the main subclass in serum
responses whatever the administration sites (Fig. 3b);
moreover, specific IgG1 level remained stable after Ag
administration (mean of positive percentages from day
20 to day 56 (experiment 1) or to day 61 (experiment
2): CV < 15%). After IM administration, IgG1
production was similar with formulations A and B.
However, administration of PLGA MS seemed to have
no influence on IgG1 production in all cases studied.
After administration of PLGA MS in MLN and
jPP-E, it should be noticed that a high level of IgG2
was observed compared to free IgY and by opposition
to IM route (Fig. 3b). Instillation in ligatured segments
led to small IgG2 levels, which were higher when
PLGA MS were used.
216 A.-M. Torché et al. / Veterinary Immunology and Immunopathology 109 (2006) 209–217
Fig. 3. (a) Anti-IgY serum IgA in pigs immunized with IgY in PLGA
MS (filled bar) or in aqueous solution (open bar). Experiment 1 at days
0, 20, 36, 49 and 56 (black filled bar), experiment 2 at days 0, 20, 39, 53
and 61 (grey filled bar). (b) Anti-IgY serum IgG subclasses: IgG1
(open bar) and IgG2 (filled bar) in pigs immunized with IgY in PLGA
MS or in aqueous solution. IgG1was expressed as a mean (positive
percentage from day 20 to day 56 (experiment 1) or to day 61
(experiment 2)). Experiment 1 at days 0, 20, 36, 49 and 56 (black
filled bar), experiment 2 at days 0, 20, 39, 53 and 61 (grey filled bar).
These combined serum IgG1/IgG2 responses could
reflect Th1/Th2-like balance of immune responses.
However, such functions of porcine isotypes have not
been yet described in pig, only hypothesized (Piriou
et al., 2003; Crawley and Wilkie, 2003).
4. Conclusion
Despite of promising results obtained in mice, it
appears much more difficult to obtain similar results
with large animals like pigs. In this study, PLGA MS
were able to induce a systemic immune response after
intestinal administration (lumen–PP–MLN). How-
ever, the intensity of systemic total antibody level
was different according to the site of administration
highlighting the influence of diffusion barriers (i.e.
luminal < PP < MLN). The study of isotypes, IgA,
IgG1 and IgG2, indicated the influence of antigen
presentation to immunocompetent cells since isotype
production was increase when PLGA MS were locally
administered, i.e. intestinal lumen, PP and MLN.
Accordingly, the effectiveness of PLGA MS to deliver
antigen to PP depends on their ability to cross mucosal
barriers and on appropriate release kinetics of the
encapsulated antigen.
Acknowledgements
This work was financially supported by Direction
Generale de l’Alimentation (DGAl), Ministère de
l’Agriculture, de l’Alimentation, de la Pêche et des
Affaires rurales. We thank Dr C. Malbert (INRA Saint
Gilles, France) for providing cannula.
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