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Department of Veterinary Anatomy and Pathology, Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006,
Australia
b
Department of Animal Science, Faculty of Veterinary Science, University of Sydney, Sydney, NSW 2006, Australia
Accepted 1 November 1999
Abstract
Defence of the intestinal mucosal surface from enteric pathogens is initially mediated by secretory IgA (SIgA). As
oral immunization of non-replicating antigen induces minimal SIgA antibody titers, novel immunization strategies
which selectively induce mucosal immune responses in mammals are now being assessed in chickens. The strategies
reviewed include the route of antigen delivery, the incorporation of antigenic components in delivery vehicles, the
inclusion of immunomodulators in the vaccine formula or in the diet, and manipulation of intestinal microora. The
dierences in anatomical organization and immunological mechanisms between birds and mammals must be
considered when manipulating avian intestinal immunity with the latest immunotechnologies developed for
mammals. Our knowledge of the function and functioning of the avian mucosal system is discussed. Progress in our
understanding of this system, the location of precursor IgA B cells and antigen sampling by these sites is not as
advanced as knowledge of the mammalian system, highlighting the need for ongoing research into the avian
application of novel vaccination strategies. 7 2000 Elsevier Science Ltd. All rights reserved.
Keywords: Chicken; Intestinal immunity; IgA antibody; Mucosal vaccines; Immunization; Immunomodulation; Antigen delivery;
Avian intestinal lymphoid aggregates
Abbreviations: ChINF-g, chicken interferon g; CMI, cellmediated immunity; CT, cholera toxin; DDA, dimethyl dioctadecyl ammonium bromide; DL-PG, DL-lactide-co-glycolide;
FAE, follicle-associated epithelium; GALT, gut-associated
lymphoid tissue; IBDV, infectious bursal disease virus;
ISCOM, immune stimulating complex; LP, lamina propria;
LT, E. coli heat-labile toxin; MLN, mesenteric lymph node;
M, microfold; PAA, polyacrylic acid; PHA, phytohemagglutinin; PP, Peyer's patches; SC, subcutaneous; SIgA, secretory
IgA; Th, T helper cell.
* Corresponding author. Tel.: +61-2-9351-7130; fax: +612-9351-7348.
E-mail address: w.muir@vetp.usyd.edu.au (W.I. Muir).
1. Introduction
Many pathogens establish contact with a potential host at mucosal surfaces. Mediation of
adaptive immune defence at these sites is initiated
by lymphocyte activation and the local secretion
of SIgA. One of the main functions of SIgA is
immune exclusion, where binding of SIgA to
antigen interferes with pathogen attachment and
colonization [1]. To function in the aggressive environment of mucosal surfaces SIgA possesses a
0145-305X/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 5 - 3 0 5 X ( 9 9 ) 0 0 0 8 1 - 6
326
public concern regarding the eect on environmental sustainability and antibiotic resistance in
human medicine. There is increasing consumer
pressure to reduce, if not eliminate, antibiotic use
in food producing animals. This coincides with
increased public expectation for the provision of
safe food, where the impact of perceived threats
to public health from Salmonella spp. and Campylobacter spp. have a strong inuence on consumer aversion to poultry products, in addition to
production losses [20]. The prevailing circumstances have placed increased pressure and
urgency on the need for safe and eective vaccines to control poultry pathogens and disease.
Therefore, there is a demand for development of
technologies which circumvent the diculties
encountered with non-replicating antigens,
enabling the induction of signicant SIgA and
improved defence at the intestinal surface.
The development of eective vaccines for oral
delivery in poultry has been hampered through,
rstly, a lack of techniques suitable for the chaperoning of antigenic substances to the intestinal
lymphoid tissues and, secondly, an inadequate
understanding of the structure and function of
the avian intestinal immune system. As discussed
in this review the past decade has seen signicant
advances in novel immunological technologies
and vaccination strategies in an attempt to overcome diculties with oral immunization. These
approaches have demonstrated great potential in
mammals [21,22] but they have been met with
only varying success in stimulating mucosal
immunity in chickens.
2. Induction of mucosal immune responses by
vaccination
While the preceding discussion highlights the
vaccination diculties encountered with presentation of native, non-replicating antigen across
the epithelial cells of the GALT, there are a number of novel strategies for circumventing these
obstacles. A recent review [3] discussed the application of some of these technologies to poultry.
An update on these strategies, including variations in the route and system of immunogen
327
Table 1
Route of antigen delivery and antigen delivery systems utilized in poultry for the induction of mucosal immunity
Delivery routea
Antigenb
Outcomec
Reference
Ip/O
P/O comb.
Ip/O
Ip/Ip
T.t.
KLH
St(nr)
Cj(nr)
Q Ab
2Ab
Q Pt
Q Pt
Muir et al.
Widders et
Muir et al.
Widders et
D16E
D18E
D18E
D18E
D18E
D18E
Cj(nr)
NDV(l)
NDV(nr)
NDV(rv)
AI(nr)
IBV(l)
Q Ab
Q Pt
Q Pt
Q Pt
Q Pt
2Ab
In-ovo D16E
Ip/O
Ip/O
Cj(nr)
Cj(nr)
T.t.
Q Ab
Pt
Ab
Noor [40]
Widders et al. [9]
Muir [26]
Liposomes
SC
SC
MG(nr)
AI(nr)
Q MAb, Q Pt
Q Ab
SC
IM
SC
O
MG(nr)
MG(nr)
Sh(nr)
NDV(l)
Q Ab
Q Pt
Q Pt; Q sxPt
Ab
P/M comb.
P comb.
P/M comb.
AI
AI
Chps
Q Pt
Q Pt
Q Pt
O
O
O
St(l)
St(l)
St(l)
Q Pt; Q sxPt
Q Pt; Q sxPt
Q Mab; Q Pt
IBDV(rv)
Q Pt
P comb.
WW
O
P comb.
P
AI(rv)
AI (rv)
FPV(l)
FPV and NDV(rv)
FPV and NDV(rv)
Q
Q
Q
Q
Q
Vaccine strategy
Route of delivery
Intraperitoneal
In ovo
Delivery systems
Non replicating
DL-PLG microspheres
Replicating
Bacterial
Salmonella typhimurium
Viral
Adenovirus
Fowlpox Virus
Ab
Pt
Pt
Pt
Pt
[17]
al. [9]
[7]
al. [9]
Ip, intraperitoneal; O, oral; D18E, day 18 embryonation; SC, subcutaneous; IM, intramuscular; M, mucosal; WW, wing web;
P, dierent parenteral immunization routes; comb, combinations of immunization.
b
T.t., tetanus toxoid; KLH, keyhole limpet hemocyanin; Cj, Campylobacter jejuni; NDV, Newcastle disease virus; MG, Mycoplasma gallisepticum; AI, Avian inuenza; IBV, Infectious bronchitis virus; Sh, Salmonella heidelberg; Chps, Chlamydia psittaci; St,
Salmonella typhimurium; IBDV, Infectious bursal disease virus; FPV, Fowl pox virus; (nr), non-replicating, (l), live; (rv) inserted in
a recombinant vector.
c
, no eect; Q, increase; 2, inconsistent; Ab, antibody; MAb, maternal antibody; Pt, protection; sxPt, serotype cross protection.
328
Table 2
Immunomodulatory techniques used in poultry for the induction of mucosal immunitya
Immunomodulator
Cytokines
Lymphokine cocktails
Recombinant chicken
Interferon-gamma
Delivery routeb
Antigenc
P
In-ovo D18E
Ip
Ip
Outcomed
Reference
Q
Q
Q
Q
Pt to E
Pt to S
Pt to S
sxPt to S
P comb.
Ip
IM
SRBC
Q Ab
Q Pt to E
Q Pt to E
O
O
O
O
It
IN
T.t.
IBDV(nr)
E(l)
E(nr)
E(nr)
NDV(nr)
Ab
Ab
Ab
Q Ab
Q Ab
Q Pt
O
O
Cj(nr)
Cj(nr)
Q Pt
Ab
SC
IV
Af(nr)
NDV (l)
2Pt
Ab
O
Ip/O
IM
NDV(l)
T.t.
NDV(nr)
Ab
Q Ab
Q Ab
IM
SC
NDV(nr)
NDV(nr)
Q Ab
Q Pt
Polyanionic polymers
Polyacrylic acids (PAA)
alkyl-PAA esters
O
O
SC
SC
SC
NDV(nr)
NDV(l)
NDV(nr)
NDV(nr)
Af(nr)
Ab
Q Ab
Q Ab
Q Pt
2Pt
IM
IM
NDV(nr)
NDV(nr)
Q Ab
Q Ab
Vitamin nutrition
Vitamin E
In-ovo D18E
Q Ab
Q MAb
Dose dep.
Bacterial enterotoxins
Cholera toxin
Vitamin A
NDV(l)
NDV(l)
b-casein
NDV(l)
Abbreviations as on Table 1.
IN, intranasal; It, intra-intestinal; IV, intravenous.
c
SRBC, sheep red blood cells; E, Eimeria spp,; Af, Aspergillus fumigatus.
d
S, Salmonella spp.; Sr, source; dep, dependent.
b
329
tivity and signicant protection from S. enteritidis challenge [31]. Enhanced non-specic
intestinal SIgA antibody titers have been
observed in young chicks following in ovo delivery of antigen [8] plus immunomodulators, such
as cholera toxin B subunit [32] and vitamin E
[33].
2.2. Antigen delivery systems
To ensure that antigenic material reaches the
mucosal epithelium in an immunogenic form and
that maximal uptake occurs across the epithelium, a number of novel antigen delivery strategies have been designed (Table 1).
2.2.1. Non-replicating delivery systems
One of the most promising non-replicating
delivery systems for oral administration of antigen is the biodegradable DL-lactide-co-glycolide
(DL-PLG) microsphere [34]. The dispersion of
antigen throughout the copolymer microsphere
protects it from gastric degradation, assists in its
selective uptake by the GALT (due to the hydrophobic exterior of the polymer) and regulates the
rate of antigen release. This delivery system can
induce antigen-specic responses at intestinal
[3436] and distant mucosal sites [37], in addition
to its codelivery with cytokine [38]. A few studies
have examined the ability of orally administered
microspheres in chaperoning the delivery of antigen to the avian intestinal immune system.
Uptake of microspheres by the avian intestine is
a size-dependent process, where microspheres
E2 mm are taken up by most areas of the intestine within 1 h of delivery [39]. However, the
immune response to antigen encapsulated in
microspheres has generated varying responses
[26] and may be an age-dependent process. It has
been shown that in ovo delivery of microspheres
induced signicant intestinal immunity in chicks
[32,40] while oral delivery in birds post-hatch did
not elicit an immune response [26].
Liposomes are another form of microencapsulation technology which can improve antigen
delivery. They can deliver antigen and cytokine
to mucosal sites [41,42] but their success varies,
depending on liposome size, chemical compo-
330
sition, surface charge and the incorporated antigen [4345]. Overall, administration of liposomal
preparations containing antigen to poultry has
induced eective immune responses [4648]. Shimizu et al. [49] have also demonstrated successful
liposomal encapsulation of egg-yolk-derived IgY
(IgG), and its improved resistance to gastric conditions compared to native IgY.
The matrix structure of ISCOMs is formed
naturally when antigen is mixed with cholesterol
and the saponin Quil A. The resistance of
ISCOMs to gastric conditions, in addition to
their selective uptake and processing by antigenpresenting cells, makes them suitable for oral and
parenteral delivery [50]. ISCOMs have been used
successfully in chickens for delivery of Mycoplasma gallisepticum immunogens [51,52]; however, local immunity was most evident following
parenteral delivery. Oral delivery of ISCOMs
containing Newcastle disease virus [53], did not
induce any eective immune response.
DNA vaccination, via the delivery of plasmid
DNA vectors containing antigen-encoding DNA
has, in preliminary studies in mice and chickens,
induced systemic and mucosal responses to inuenza virus [54,55] following parenteral and mucosal immunization. Protection was provided from
a subsequent challenge of inuenza virus. DNA
vaccination of turkeys for Chlamydia psittaci
demonstrated partial protection from a subsequent challenge [56]. However, despite these
promising results, there is a paucity of publications on the application of DNA vaccination
in poultry.
2.2.2. Replicating delivery systems
Developments in DNA technology have made
available attenuated viral or bacterial vectors for
delivery of their genes or, following recombination, gene inserts encoding the protective antigens
of other pathogens, to the mucosal surface. Attenuated mutants of Salmonella spp. are eective
as live attenuated vectors [57,58], stimulating signicant serotype cross-protection and protective
maternal immunity [59]. However, viruses have
been the preferred recombinant vectors for mucosal delivery. Recombinant herpes virus and fowlpox virus have been favored for use in poultry,
331
332
333
334
tem in chickens may be extensive with particularly strong links between the ocular and intestinal immune systems. Alternatively, as the
authors suggest, the Harderian gland may accumulate IgA+ lymphocytes which have recently
emigrated from the bursa of Fabricius, with a
governing role for their supply and relocation to
the intestinal immune system. It is evident that
further research is necessary to identify the tissues of residence of precursor IgA+ B cells, and
their preferred sites of localization in unimmunized and immunized chickens. Studies which
elucidate the tracking features of B and T cells
involved in the generation of intestinal IgA
immune responses in chickens are also urgently
required.
4.2. Antigen uptake in avian GALT
A number of mammalian studies have shown
that presentation of antigen from the intestinal
lumen into the PP lymphoid aggregate is a prerequisite in the stimulation of precursor IgA+ B
cells and the induction of immune responses in
the intestinal LP [128]. Secondly, antigen is
necessary for the permanent localization and proliferation of IgA secreting plasma cells in the LP
[157]. A fundamental requirement of vaccines is
the delivery of antigen in a form suitable for its
sampling by the intestinal lymphoid inductive
sites, and its processing and presentation to the
immune cells. Dierences in the features and
mechanisms of antigen sampling in the avian
GALT as compared with mammalian GALT,
may present a shortcoming in some recently
emerging immunization strategies when applied
to chickens.
Antigen present in the intestine of mammals is
typically taken into the GALT across the specialized microfold (M) cells of the numerous PP
[158]. Antigen can be presented to immune cells
directly by enterocytes [19], by enterocytes that
have developed an M cell phenotype [159], or it
can move either directly across or between enterocytes [160]. As the specialized epithelium
designed for antigen uptake in mammalian
GALT is not so frequently found in chickens
[140], there may be variation in the cell type and
335
336
5. Conclusion
The potential for novel immunization strategies
to induce intestinal immunity in mammals is well
established and oers exciting new opportunities
in other species. Many of these technologies,
which include the route of antigen administration, delivery systems for antigen and antigenic
components,
immunomodulatory
substances, and manipulation of the gut microora, have the potential to considerably improve
intestinal immune responses in poultry. However,
to gain full benet from these immunization procedures, further elucidation of the avian intestinal
immune system is essential. It is crucial to understand the immune mechanisms of the avian intestinal lymphoid sites for the specic local
activation of SIgA secretion. This includes clarication of the site of origin of the precursor cells
of IgA-producing cells, which preferentially home
to the intestinal mucosa, and features of intestinal uptake of antigen and antigen delivery vehicles. These advances must be paralleled with
studies verifying the activity of novel vaccine
technologies in inducing the desired response in
the avian intestinal immune system. Progress in
these areas will aid in dening research priorities
to the most promising emerging immunization
strategies for oral vaccination.
Acknowledgements
Research undertaken by W.I. Muir was nancially supported by the Australian Rural Industries Research and Development Corporation,
Chicken Meat and Egg Industry Councils.
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