Developmental and Comparative Immunology 24 (2000) 325342

www.elsevier.com/locate/devcompimm

Immunity, vaccination and the avian intestinal tract

W.I. Muir a,*, W.L. Bryden b, A.J. Husband a
a

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

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W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

number of characteristics, such as its polymeric

structure, the presence of the J chain and the secretory component, which distinguish it from
other immunoglobulins [2]. Our understanding of
these features and the function of avian SIgA has
been recently reviewed [3], and dierences exist in
both humoral and cell-mediated immunity (CMI)
between avian species [4].
A number of studies have demonstrated the secretion of intestinal SIgA in the chicken in response to antigen which is present at the
intestinal surface including Escherichia coli [5],
Salmonella typhimurium [6,7], Campylobacter
jejuni [8,9], and Eimeria tenella [10,11]. Immune
responses to antigen in the bursal lumen have
also been reported [12,13]. Gut microora is important for early stimulation and maturation of
the cellular compartment of the intestinal
immune system [14].
The avian intestinal immune system can mount
an eective antigen-specic SIgA antibody response to enteric pathogens, but it is a paradox
that there continues to be a lack of success with
the design and application of vaccines to control
intestinal pathogens. As the initial encounter
with intestinal antigen occurs across the mucosal
surface it is expected that oral administration of
antigen will induce an eective local intestinal
immune response, improving protection of the intestinal mucosa. However, the highly variable
immune response elicited following oral delivery
of non-replicating native antigen in mammals
[15,16] is also observed in chickens and continues
to be an obstacle in the design of vaccines for
oral delivery [9,17]. The digestive enzymes of the
gastrointestinal tract degrade much of the antigenic material before it reaches the immune system. The small amount of fully immunogenic
material which is then sampled by the gut-associated lymphoid tissues (GALT) tends to induce a
predominantly suppressor response, following its
presentation to the immune system via normal
villus epithelial cells [18,19].
Faced with few eective vaccines for the control of enteric diseases, the poultry industry has
adopted widespread use of antibiotics to contain
disease and maintain bird health. However, the
long-term use of these compounds has caused

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

W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

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

Noor et al. [8]

Ahmad and Sharma [28]
Stone et al. [30]
Reddy et al. [29]
Stone et al. [30]
Wakenell et al. [165]

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

Barbour and Newman [47]

Fatunmbi et al. [48]

Immune stimulating complexes (ISCOMs)

SC
IM
SC
O

MG(nr)
MG(nr)
Sh(nr)
NDV(l)

Q Ab
Q Pt
Q Pt; Q sxPt
Ab

McLaren et al. [51]

Sundquist et al. [52]
Charles et al. [23]
Rehmani and Spradbrow [53]

Naked DNA plasmids

P/M comb.
P comb.
P/M comb.

AI
AI
Chps

Q Pt
Q Pt
Q Pt

Fynan et al. [54]

Robinson et al. [55]
Vanrompay et al. [56]

O
O
O

St(l)
St(l)
St(l)

Q Pt; Q sxPt
Q Pt; Q sxPt
Q Mab; Q Pt

Curtiss and Hassan [57]

Hassan and Curtiss [58]
Hassan and Curtiss [59]

IBDV(rv)

Q Pt

Sheppard et al. [66]

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

Boyle and Coupar [166]

Binns and Boursnell [61]
Jieyuan and Spradbrow [167]
McMillen et al. [62]
Taylor et al. [63]

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.

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W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

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

Lillehoj et al. [76]

McGruder et al. [31]
McGruder et al. [74]
Kogut et al. [73]

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

Lowenthal et al. [79]

Lowenthal et al. [81]
Lillehoj and Choi [80]

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

Meinersmann and Porter [84]

Hoshi et al. [85]
Girard et al. [88]
Girard et al. [88]
Vervelde et al. [87]
Takada and Kida [86]

O
O

Cj(nr)
Cj(nr)

Q Pt
Ab

Khoury and Meinersmann [91]

Rice et al. [90]

SC
IV

Af(nr)
NDV (l)

2Pt
Ab

Richard et al. [27]

Neumann et al. [168]

O
Ip/O
IM

NDV(l)
T.t.
NDV(nr)

Ab
Q Ab
Q Ab

Rehmani and Spradbrow [53]

Muir et al. [17)
Hilgers et al. [95]

IM
SC

NDV(nr)
NDV(nr)

Q Ab
Q Pt

Hilgers et al. [95]

Katz et al. [94]

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

Rehmani and Spradbrow [53]

Rehmani and Spradbrow [53]
Fatunmbi et al. [48]
Rweyemamu et al. [96]
Richard et al. [27]

IM
IM

NDV(nr)
NDV(nr)

Q Ab
Q Ab

Hilgers et al. [95]

Hilgers et al. [99]

Vitamin nutrition
Vitamin E

In-ovo D18E

Q Ab
Q MAb
Dose dep.

Gore and Qureshi [33]

Haq et al. [106]
Friedman et al. [108]

Sr & Dose dep.

Dose dep.
Dose dep.
Ab; MAb

Davis and Sell [110]

Lessard et al. [111]
Sklan et al. [109]
Coskun et al. [112]

Bacterial enterotoxins
Cholera toxin

E. coli heat-labile toxin

Mycobacterial components
Lipopolysaccharide (LPS)
Muramyl-dipeptide (MDP)
Quil A
Amine containing compounds
Dimethyl dioctadecyl ammonium bromide (DDA)
Avridine

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

W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

delivery, the inclusion of novel adjuvants or

immunoregulators in the vaccine preparation and
manipulation of intestinal microora, and their
application to induce mucosal immune responses
in poultry are reviewed.
2.1. Route of immunization
Antigen can eciently access the GALT using
combined systemic and mucosal immunization
protocols (Table 1). The success of primary intraperitoneal vaccination followed by an oral booster immunization in generating anti-antigen SIgA
at the intestinal surface in chickens has been
described [9,17]. This immunization regimen
overcomes oral unresponsiveness to non-replicating antigens through antigen presentation across
the intestinal serosal surface. Further, birds
immunized in this manner with whole killed S.
typhimurium were partially protected from an
oral challenge of the same bacteria [7].
It is interesting to observe the frequency with
which immunizations designed to induce mucosal
immunity in poultry have been delivered parenterally, especially subcutaneous (SC) vaccination
(Tables 1 and 2). Certainly some of these delivery
systems, for example immune stimulating complexes (ISCOMs), eciently induce CMI, increasing opportunities to combat invading pathogens
[23]. Further, the SC delivery of non-replicating
antigen in adjuvant can elicit substantial levels of
systemic antibody [24,25] as well as intestinal
SIgA [9], where, for the latter, an oral booster
immunization may be required [17]. However,
the protection provided to the intestinal surface
of poultry by an SC-based immunization regimen
varies [23,24,26,27]. Nevertheless, the success of
some novel immunological techniques delivered
SC justies evaluation following direct delivery
to the mucosal surface.
The application of in ovo delivery of antigen
for enhancing immune status of chickens at
hatch, and improving resistance to pathogen
challenge, is well established [2830]. Embryonic
exposure to immune lymphokines isolated from
the culture supernatant of T cells derived from
hens immunized with Salmonella enteritidis
stimulated increased heterophil bactericidal ac-

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-

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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,

partly due to their large DNA genome which can

express substantial quantities of foreign DNA
[60,61]. Immunization with a recombinant fowlpox virus expressing Newcastle disease virus has
shown protective immunity against both pathogens [6163]. A recombinant of herpes virus
expressing the VP2 gene of infectious bursal disease virus (IBDV) protected immunized birds
from a challenge with virulent IBDV [64]. More
recently, adenovirus which, following oral delivery, induces systemic, local and distant mucosal
immunity [65] has been investigated as a vector
for use in poultry vaccines. An avian adenovirus
recombinant vector, expressing the VP2 gene of
IBDV, was successfully constructed [66] and
invoked an immune response which protected
chickens from an IBDV challenge. Recombinant
avian adenovirus may be delivered on poultry
food pellets [67], allowing immediate access to
the mucosal surfaces of the digestive tract. However, the ability of this delivery system to stimulate protective immunity is still to be ascertained.
The suitability of adenovirus vectors for the
delivery of cytokines to the avian immune system
is discussed in this special issue [68].
2.3. Adjuvants and immunomodulators
The inclusion of adjuvants and immunomodulators in a vaccine formula can improve the
quantity and possibly the quality of the immune
response. Some adjuvants which have been used
in parenteral vaccines have been evaluated in the
mucosal immune system, and other adjuvants
have been specically designed to upregulate
mucosal immune responses (Table 2).
The regulatory role of cytokines in directing
and ne-tuning immune responses makes them
ideal immunomodulators [69]. From murine studies there is evidence supporting the application
of cytokine therapy in mucosal immunity. For
example, delivery of recombinant vectors expressing either IL-5 [70], or IL-6 [71], induced substantial increases in IgA responses at the mucosa.
In chickens, the delivery of lymphokine cocktails,
generated from T cells of Salmonella enteritidis
immunized chickens, signicantly increased the
number and activity of heterophils [72], which,

W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

under a number of experimental conditions,

improved protection from challenge [31,7375].
Similarly, T cell lymphokine preparations stimulated in the presence of species of Eimeria
improved resistance of chicks to coccidiosis
[76,77]. Chicken myelomonocytic growth factor,
when delivered to chicks in a live recombinant
fowlpox viral vector, enhanced the number and
functional activity of circulating blood monocytes
[78], but the impact of this on disease resistance
is still to be evaluated. A number of recent studies have established the positive inuence of
chicken interferon-gamma (ChIFN-g) on IgY
antibody responses to antigen and improved bird
weight gain [79]. Reduced fecal oocyst output
and improved bird weight gain was observed in
chickens administered ChIFN-g and challenged
with Eimeria acervulina [80,81].
A number of bacterial enterotoxins have
demonstrated adjuvant properties. Cholera toxin
(CT), the enterotoxin produced by Vibrio cholera,
binds with very high anity to intestinal epithelial cells inducing very strong SIgA and serum
IgG antibody responses, and long-term immunological memory in mammals [82]. CT-B subunit,
which is less toxic than the complete toxin, maintains adjuvant eects, but does not induce memory cells [83]. Evaluation of CT as a mucosal
adjuvant in chickens has met with mixed success
[8486]. Identication of receptors on epithelial
cells of the chicken cecum which bind with CT
has recently been reported [87]. When delivered
with a recombinant Eimeria antigen, CT was an
eective mucosal adjuvant in chickens [88], especially evident when the antigen and CT were
conjugated [87]. Escherichia coli heat-labile toxin
(LT) functions as an adjuvant in a similar manner to CT [89]. A formalin-killed C. jejuni whole
cell vaccine was not improved when LT was
added to the orally delivered preparation [90].
Oral immunization, however, with LT-B subunit
fused to C. jejuni agellin, stimulated antibody
production and reduced levels of colonization
following challenge [91].
Dimethyl dioctadecyl ammonium bromide
(DDA) is a synthetic surface active quaternary
amine which has proven to be an eective adjuvant for the induction of CMI and humoral

331

immune responses [92]. The mechanisms of DDA

adjuvanticity are not entirely clear. DDA signicantly stimulated delayed type hypersensitivity
and, to a lesser degree, antibody titers, in a number of studies with viral antigens [93]. Studies
using DDA as an adjuvant with inactivated Newcastle disease virus delivered parenterally to
chickens, indicate its ability to induce antibody
production, CMI, high levels of protection in
challenge studies [93,94], and in inciting secondary immune responses in previously primed
chickens [95]. Avridine, which is structurally similar to DDA, acts as an adjuvant through upregulation of interferon production. Avridine gave
variable results when assessed as a mucosal adjuvant with Newcastle disease virus vaccine [53,96].
The polyanionic polymers, polyacrylic acids
(PAA), are high molecular weight strongly negatively charged compounds, which have shown
adjuvant eects for both humoral and CMI
[97,98]. When delivered to primed chickens, PAA
enhanced the secondary immune response to
inactivated Newcastle disease virus [95], but this
was not equal to the `gold standard' water-inmineral oil (W/O) emulsion. However, formation
of alkyl-PAA esters signicantly improved antibody responses compared to PAA alone, equaling that of the W/O emulsion [99].
The nutrient components of diets, including
protein, carbohydrate, lipids, vitamins and minerals, all modulate systemic and mucosal immune
function [100]. Nutrition also signicantly inuences the interaction between bacteria and the intestinal mucosa [101].
Following a report of the systemic and mucosal adjuvant activity of uoride [102], its immunomodulatory activity with oral immunization in
chickens was investigated [103], impacting most
notably on systemic serum IgY antibody, generating only low and variable titers of IgA antibody in bile and mucosal uids.
The fat soluble vitamins, vitamin A (retinol)
and vitamin E (a-tocopherol), are structurally
similar and have been utilized in W/O emulsions
[104]. In addition to adjuvant activity, both vitamins have other immunomodulatory eects [100].
The exact mechanisms of action require further
elucidation; however, they are most likely associ-

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W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

ated with antioxidative properties [105]. In recent

studies, vitamin E delivered into the embryo [33],
enhanced antigen-specic antibody titers and
increased the percentage and phagocytic activity
of macrophages. Chicks hatched from hens on a
vitamin E supplemented diet demonstrated a signicant increase in antibody titers [106]. Vitamin
E had no impact on the macrophage and lymphocyte population in the spleen [107], but higher
levels of supplementation had a positive inuence
on the percentage of CD4+CD8 thymocytes.
Evidence for the negative impact of excessive
vitamin E on immune function, and in particular
antibody production, has also been reported
[108].
Vitamin A is also intrinsic to the maintenance
of a competent immune system in a number of
animals [105] such that a deciency increases
host susceptibility to disease. Research undertaken using markedly dierent concentrations [109]
and sources [110] of vitamin A makes evaluation
of many publications dicult. Birds on diets supplemented with vitamin A had lower serum antibody titers, after vaccination and challenge with
Newcastle disease virus [110] but, after revaccination, supplementation improved antibody based
immunity. Following immunization with Newcastle disease virus vaccine, meat chickens on a vitamin A-decient diet had improved antibody titers
but impaired lymphocyte proliferation (determined by response to phytohemagglutinin
(PHA), a thymus-dependent response mediated
by T cells), whereas birds receiving high levels of
vitamin A maintained lymphocyte reactivity to
PHA. No eect of vitamin A on natural killer
cell activity or on the frequency of cells expressing MHC class II antigens was observed [111].
In contrast, similar vitamin A supplementation
of layer bird diets did not impact on T cell lymphocyte populations or antibody titers following
immunization to Newcastle disease virus, and no
detectable levels of maternal immunity were
identied in their progeny [112].
2.4. Immunological benets of microbes in the
intestinal tract
The gastrointestinal tract is an intriguingly

complex microenvironment, where close contact

is maintained between the host tissue and dietary substances, their components, and microorganisms, parasites and exogenous toxins
[113]. Within this milieu the immune system
distinguishes between self and non-self, acting
to eliminate threatening foreign substances.
However, the host will withstand the presence
of some resident microorganisms, which interact in a symbiotic manner [114]. The resident
microora contribute to host nutrition as well
as defence against enteric pathogens. In respect
of the latter, it is well appreciated that healthy
resident gut microbiota reduce the opportunity
for colonization by pathogenic bacteria
through competitive exclusion such that the
Nurmi principle, where young chicks are
inoculated with adult gut micoora [115] is
being examined for practical application
[116,117].
Lactobacilli have long been recognized for
their ability to stimulate the SIgA immune system, increasing resistance to disease [118,119] in
part through the release of low-molecular-weight
peptides which induce immune activation [120].
However, species of Lactobacillus vary in their
immunoregulatory properties [121,122] and in the
cytokine prole induced, which inuences the
ensuing immune response [123]. Given their symbiotic status and immunoregulatory attributes
Lactobacilli have been considered as antigen vectors suitable for parenteral priming and oral
delivery, invoking an intestinal immune response
to the inserted immunogen [122,124] while presenting no threat to the host (in comparison to
other live vectors such as Salmonella). These
organisms are found in the intestine of birds of
all ages, with host-specic attachment to the intestinal wall, making them ideal candidates for
safe vaccine vectors, which neatly complements
their innate ability to improve bird health and resistance to disease. This multiple pronged
approach to regulating the composition of intestinal microbes, bird immunity and health has
many benets and great potential, making it
worthy of further evaluation.

W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

3. Mechanisms of mucosal immune responses

3.1. Site of origin and tracking of IgA+ cells in
mammals
The limited success of the emerging vaccination strategies discussed in the preceding section in inducing mucosal immunity in poultry
may be, in part, due to our limited appreciation
of the operation of the avian intestinal immune
system. This is particularly striking when compared to the extensive knowledge available on
the mechanisms involved in inducing SIgA antibody in mammals.
In laboratory animals the origin of precursor
IgA B cells and their migratory route from
aector tissues to eector sites is well understood. The Peyer's patch (PP) is the main
source of precursors of IgA-producing cells
which reconstitute the intestinal and bronchial
lamina propria (LP) with IgA-containing cells in
B-cell depleted animals [125,126], whereas cells
isolated from the popliteal lymph nodes failed
to demonstrate similar repopulation characteristics. The thoracic duct is the main tracking
route through which plasma cells migrate to the
LP [127]. A common mucosal immune system,
where IgA-committed B cells originating from
GALT and bronchial-associated lymphoid tissues migrate to, and localize at, either of these
mucosal sites, has been identied [126,128].
However, regional preferences in tissue repopulation are evident, including preferential migration to tissues of origin [128].
In mammals secondary lymphoid tissues
within the intestinal tract such as the PP are
capable of sampling antigen which, subsequent
to its processing and presentation by antigen
presenting cells, initiate a local mucosal immune
response. These tissues are classied as inductive
or aector lymphoid sites, containing both B
and T cells. Following antigenic stimulation B
lymphocytes committed to IgA production leave
the PP and move through the interstitial spaces
into lymph channels which transport them to
the mesenteric lymph nodes (MLN). Here they
undergo further proliferation and maturation
before migrating via the eerent lymphatics of

333

the MLN into the thoracic duct lymph. From

here they enter the circulation which distributes
them to the mucosal eector tissue, the LP. As
mature plasma cells in the LP they secrete IgA
antibody specic for the antigen that elicited
their production. PP-derived T lymphocytes follow a similar migratory route to that of the
mucosal IgA-producing cells, resulting in a T
lymphocyte population of the LP and intraepithelial regions, which may be involved in CMI
or cytotoxic T lymphocyte function [129]. Both
the mature T and B cell populations present in
the intestinal tissue act to protect it from pathogens.

3.2. The role of T cells and cytokines in intestinal

immunity
The induction of B cells to IgA production is
dependent on T helper (Th) cells [130], which
provide two distinct signals one involving
direct cellcell contact and another mediated
through T cell cytokines [131]. CD4+ T cells
have been divided into two groups depending
on their typical cytokine prole [132]. T helper
(Th)1 cells, which are especially important for
CMI, but may also provide B cell help, preferentially produce IFN-g and interleukin (IL)-2. T
helper 2 (Th2) cells, which preferentially produce IL-4, IL-5, IL-6, and IL-10, provide B cell
help for all major isotypes, including IgA. In
vitro [133135] and in vivo studies [136] have
demonstrated the inuence of Th2 cytokines on
promoting IgA expression by surface positive
IgA B cells and their proliferation and maturation to IgA secreting plasma cells. Further, T
cells isolated from the mucosal lymphoid tissue
strongly favor Th2 type cytokine proles [137].
A comprehensive discussion on mucosal cytokines, their receptors and regulation of IgA production has recently been published [138]. The
key role of cytokines in regulating and ne-tuning immune responses makes them suitable vaccine adjuvants [136,139] and their application to
this end in avian mucosal immunology is presented in Table 2.

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W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

4. Features of avian GALT and the design of

intestinal vaccines
4.1. Site of origin of IgA+ cells in chickens
As previously described, the induction of an
IgA immune response in mammals is initiated
through eective delivery of antigen to PP. However, identication of an equivalent site/s, which
contains precursor IgA B cells and mucosal T
cells, has not been delineated in chickens. Studies
designed to clarify the location of this tissue are
crucial, especially in light of the anatomical and
organizational dierences between avian and
mammalian lymphoid tissues. Within the avian
GALT, the main areas of lymphoid aggregation
include the avian equivalent of the mammalian
PP, the bursa of Fabricius, cecal tonsils and
Meckel's diverticulum. Comprehensive descriptions of these tissues, their development and cellular components have been published [3,140,141]
and therefore only brief reference to their main
features is presented here. Payne [142] provided
an early overview of lymphoid tissue in the alimentary tract of poultry, which also included
lymphoid tissue of the pharynx, esophagus, proventriculus, gizzard, ceca, colon and cloaca.
Further, given the heterogeneity in the anatomy
and physiology of the digestive tract in birds
[143], diversity in their lymphoid structures [4] is
not unexpected.
Young chicks have approximately six PP; however, following age-associated involution only
one PP, the ileal lymphoid aggregate, is evident
[144,145]. The structure of the avian PP has been
described in detail and is similar to mammalian
PP [144146]. The bursa of Fabricius, which is a
primary lymphoid organ in chickens, is also an
inductive lymphoid tissue for intestinal immunity.
Typically, the cloacal orice draws antigen into
the bursal lumen [147] or, in older chickens
which have undergone bursal involution, into the
ceca and cecal tonsils [148]. The cecal tonsils are
a relatively immunologically mature secondary
lymphoid aggregation [149] similar to the PP
[150]. Smaller lymphoid aggregates are found in
the ceca [151], especially in the cecal apex. Only
rudimentary cecal tonsils are present at the time

of hatch, containing a majority of CD4+ T cells

[152]. By 6 weeks of age the number of B cells
(both IgM and IgA isotypes) is signicant, and
comparatively few T cells (mainly CD8+) are
present. The immunological signicance of the
cecal tonsils may be more relevant following
bursa involution [148]. The involvement of the
remnants of the yolk sac, Meckel's diverticulum,
as an inductive lymphoid site requires clarication [3]. Despite uncertainty of antigen access to
the lymphoid areas of the diverticulum [153,154],
both of these groups classied it as a part of the
GALT. However, it may play a role in the induction of immunological memory, rather than as a
site for the generation of antigen-specic antibody. Chickens do not have organized, encapsulated lymph nodes, but rather develop small
collections of mural lymphoid nodules within the
wall of the lymphatic vessels following antigenic
exposure [155].
The dierences in the location and structure of
chicken GALT compared to mammals highlights
the need for specic examination of the site/s of
origin of the precursor cells of IgA-producing
plasma cells in the intestinal mucosal tissue of
chickens. Using adoptive cell transfer, we have
investigated the ability of cells originating from
various intestinal lymphoid sites to repopulate
the duodenal and cecal tonsil LP in B-cell
depleted chickens [26]. Cells isolated from the
bursa of Fabricius and, less frequently, the intestinal lymphoid aggregate and cecal tonsil reconstituted B-cell decient birds with IgA secreting
plasma cells in the intestinal LP. These results
signify the importance of these lymphoid tissues
as sites which contain precursor cells of IgA+ B
cells, which localize in the intestine of the
chicken. Therefore, incorporation of techniques
that will ensure ecient antigen presentation to
these sites must be a priority in vaccine design
and intestinal immunization strategies. Interestingly, in another study [156], the Harderian gland
was found to contain surface IgA+ lymphocytes
which demonstrated signicant migration to the
cecal tonsils and, to a lesser extent, the bursa of
Fabricius. These observations may be the outcome of two possible scenarios which require
clarication. The common mucosal immune sys-

W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

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

mechanisms involved, which present additional

points for consideration in the design of ecacious poultry vaccines.
Only a few sites of the avian intestinal mucosa
possess specialized epithelial cells which can
sample antigen. The epithelium of the avian PP
is attened containing areas of M cells and very
few goblet cells [145]. The bursa of Fabricius
contains follicle associated epithelium (FAE)
which sample lumenal antigens, presenting them
to the underlying lymphoid tissue [161]. Cecal
tonsils and other lymphoid aggregates in the ceca
possess antigen sampling cells, which have been
classied as M cells [151], or alternatively M celllike cells [162]. The latter were relatively weak at
sampling antigen, which concurs with previous
reference [144] to the varying ability of the cecal
tonsils to sample antigen, depending on its site of
entry into the gastrointestinal tract. The infrequency of specialized antigen sampling cells in
avian GALT may be compensated for through
the increased frequency of lymphocyte inltration
into both epithelial cells and M cells [148], and
an increased ability of epithelial cells to sample
antigen [140].
The limited studies investigating antigen
uptake from the lumen in avian GALT are
inconclusive. Orally administered carbon was
identied in PP M cells and between the lymphocytes in the germinal centers [145]. This contrasts
with localization of carbon particles in the mononuclear phagocytes in the lymphoid tissue under
the PP epithelium in a study of similar design
[140]. Orally delivered carbon particles have been
observed in the germinal center of the cecal tonsils [144], whereas others [140,162] could not
identify the particles in either the epithelial cells
or M cell-like cells respectively. In contrast,
horseradish peroxidase reaction products were
located in the intercellular spaces, epithelial cells
and M cell-like cells of the cecal tonsils [162].
Carbon particles have been observed in the FAE
and medulla of the bursa of Fabricius [140].
Particles present in the cloaca are transported
into the bursal lumen and then localize throughout the medullary lymphoid tissue [161,163]. Following anal administration of carbon, [144] only
the cells of the bursa of Fabricius, not the cecal

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W.I. Muir et al. / Developmental and Comparative Immunology 24 (2000) 325342

tonsils or PP, sampled the particles. However,

after long-term administration of carbon it was
identiable in the macrophages of the cecal tonsils [147]. Similarly, anally administered ferritin
was eciently sampled by the FAE of the bursa
[161]. A strong immunoglobulin response was
generated following per anum administration of
a thymus-independent antigen but the response
to thymus-dependent antigens was variable
[12,13], and may be an age-dependent phenomena [164].
The foregoing discussion highlights some of
the unique anatomical structures and poorly
understood processes of antigen sampling and
immune response induction in the avian intestine.
This lack of information has resulted in an `ad
hoc' approach in the search for eective avian
mucosal vaccines (Tables 1 and 2). However,
more recently there is a trend to assess the ability
of new immunization strategies to maintain their
unique immunological functions in the avian
immune system, prior to their application. For
example, examination of the characteristics of intestinal uptake of microspheres in chickens [39]
identied a maximum microsphere size which
enables ecient sampling by the intestinal tissues.
In studies designed to determine the feasibility of
priming chickens parenterally for the induction
of intestinal SIgA an array of antigen delivery
routes were initially compared [9,17]. Following
identication of the most ecacious delivery
route, as determined by antigen-specic antibody
titers, this regimen was employed in vaccination/
challenge studies with the pathogen of interest
[7,9]. In a similar manner, identication of a
receptor for CT in the epithelium of the avian
cecum was undertaken before examining the ability of CT to act as a mucosal adjuvant in chickens [87]. Other studies applying microspheres
[9,26], the mucosal adjuvant CT [8486,88], and
other novel vaccination strategies in chickens, as
summarized in Tables 1 and 2, have utilized
emerging immunological technology in the
absence of empirical studies. This `hit and miss'
use of new vaccine techniques in chickens reiterates the benets in initially determining the sustainability of the immunization technology when
applied to birds. This will reduce the frustration

and expense of research with inappropriate avian

application of technology developed for mammals.

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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