Establishment of immune competence in the avian GALT

during the immediate post-hatch period

Enav Bar-Shira, David Sklan, Aharon Friedman*,†

Sections of Immunology and Nutrition, Department of Animal Science, Faculty of Agricultural, Food and Environmental Quality Sciences,

Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

Received 6 March 2002; revised 1 July 2002; accepted 22 August 2002

Abstract

Population dynamics of intestinal lymphocytes and the temporal development of lymphocyte functions were studied in broiler

chicks during the first 2 weeks post-hatch. This period is of major immunological importance as the chick is immediately exposed to

environmental antigens and pathogens. We show that the gut-associated lymphoid tissue contains functionally immature T and B

lymphocytes at hatch, and that function is attained during the first 2 weeks of life as demonstrated by mRNA expression of both ChIL-

2 and ChIFNg. Functional maturation occurred in two stages: the first—during the first week post-hatch, and the second during the

second week, which was also accompanied by an increase in lymphocyte population, as determined by expression of antigen receptor

genes. Evidence is presented to show that in the intestinal milieu cellular immune responses mature earlier, and are a prerequisite for

humoral responses. Hence, the lack of antibody response in young chicks is primarily due to immaturity of T lymphocytes.

q 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Avian; Gut-associated lymphoid tissue; CD3; Chicken IFNg; Chicken IL-2; Enteric; T lymphocyte; B lymphocyte

1. Introduction

Many antigens encountered by the immune system

enter the body through mucosal surfaces lining the

respiratory, digestive and genitourinary tracts. As a

result a unique and complex immune apparatus has

developed, the mucosa associated lymphoid tissue

(MALT), which provides the first line of defense

against these antigens. A major component of MALT is

the gut-associated lymphoid tissue—GALT [1].

In chickens, GALT is responsible for inducing immune

responses against bacterial, viral and parasitic enteral

(i.e. introduced via the digestive tract) antigens [2–6]

as well as responses to innocuous antigens [7]. The

avian GALT contains unique lymphoid structures such

as the bursa of Fabricius, caecal tonsils (CT) and

Meckel’s diverticulum as well as Peyer’s patches (PP),

intraepithelial lymphocytes (IEL) and scattered

immune cells residing in the intestinal lamina propria

[2]. As chickens do not have other peripheral

encapsulated lymph nodes, GALT lymphoid structures

serve as major secondary lymphoid organs [5,8–12]

0145-305X/03/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.

PII: S0 14 5 -3 05 X( 02 )0 0 07 6 -9

Developmental and Comparative Immunology 27 (2003) 147–157

www.elsevier.com/locate/devcompimm

† A. Friedman is incumbent of the Ron Barbaro Chain in

Veterinary Medicine.

E-mail address: fridman@agri.huji.ac.il (A. Friedman).

* Corresponding author. Tel: þ972-8-9489027; fax: þ972-8-

9489869.

Abbreviations: GALT, gut-associated lymphoid tissue; BSA,

bovine serum albumin; Hem, hemocyanin; CT, cecal tonsils; IEL,

intraepithelial lymphocytes.

emphasizing the importance of the avian GALT in

protection of birds.

Age-related changes in GALT of chickens have been

described. These include changes in composition of T and

B lymphocyte populations [13–15], innate cell popu-

lations [16] anatomical and morphological changes such

as enlargement of bursa and its involution in adulthood,

appearance of CT and PP [17,18] and maturation of the

response to enteral pathogens [2,17]. Furthermore, many

investigations have studied the cellular and functional

changes of the avian GALT due to pathogenic challenge

[2,19–21]. However, there is a little information

describing the normal development and immunological

function the avian GALT in the immediate post-hatch

period. This appears to be critical for the chick’s survival

immediately after hatch, for concomitant with foraging it

is exposed to adult type microflora. It is not known

whether the chick GALT is functionally mature at this

time and able to provide enteral protection in the absence

of maternal oral antibodies as is observed in mammals.

The present study was undertaken to investigate the

normal development and functional maturation of the

GALT in broiler chicks from hatch until 2 weeks of age.

This was achieved by following the intestinal

lymphocyte populations and determining immunologi-

cal function of these cells as manifested by cytokine

mRNA expression and antibody specific immune

responses generated following the enteral stimulation.

2. Material and methods

2.1. Chickens

Newly hatched, unvaccinated Ross broiler chicks

were obtained from a commercial hatchery (Kvuzat

Yavne, Yavne, Israel) and housed in an isolated,

disease-free, facility in battery brooders at 32 8C for

the first week post-hatch followed by 28 8C in the

second week in light controlled rooms with free access

to commercial starter feed (Matmor Feed Co., Ashdod,

Israel) and water. All procedures were approved by the

Animal Care and Welfare Committee of our Institute.

2.2. Preparation of intestinal slices

Chicks were euthanized 0 (2–4 h post-hatch), 1, 4,

6, 8 and 12 days post-hatch using CO2 (n ¼ 3 for each

time point). Intestines were removed and flushed with

cold PBS. Cross-sections (60–80 mg) from duode-

num (apex of pancreatic loop), jejunum (midsection

between tip of ascending duodenum and Meckle’s

diverticulum), ileum (midsection between Meckle’s

divertivulum and caeco–iliac junction), CT (caeco–

ileac junction) and colon (midsection between caeco–

ileac junction and cloaca) were collected and snap

frozen in liquid nitrogen. Slices were stored at

280 8C until analysis.

2.3. Oligonucleotide primer pair design

Specific oligonocleotide primer pairs were

designed and synthesized for b-actin, CD3gd, Bu-1,

chicken IL-2 (ChIL-2) and chicken IFNg (ChIFNg)

according to published chicken sequences (GeneBank

accession numbers L08165, AJ250458, X92865,

AF000631, U27465, respectively) as follows:

b-actin oligonucleotide primer pair (sense

50-CCTCTTCCAGCCATCTTTC-30; antisense 50-TC

ACAGAGGCGAGTAACTTCC-30), CD3gd oligonu-

cleotide primer pair (sense 50-CAGGGATTGTGGTC

GCAGAT-30; antisense 50-TACTGTCCATCATTCC

GCTCAC-30), Bu-1 oligonucleotide primer pair

(sense 50-GGTGTCCAGTGAAGGTGTG-30; anti-

sense 50-GATGCAAAGGATGGGTGTC-30), ChIL-2

oligonucleotide primer pair (sense 50-ATCTTTGGCT

GTATTTCGGT-30; antisense 50-GATTAGTTAGCC

ACGGGATA-30) and IFNg oligonucleotide primer

pair (sense 50GAACTGGACAGAGAGAAATGA-30;

antisense 50-TACTTTCATTGTTTGCCTGGTT-30).

The expected PCR product lengths were: 645 bp for

b-actin, 164 bp for CD3gd, 305 bp for Bu-1, 505 bp

for ChIL-2 and 477 bp for IFNg. Computer searches

and sequence alignments were performed by means of

software from Genetics Computer Group Inc. (Madi-

son, WI, USA).

2.4. Gene expression assays

Total RNA was extracted from tissue slices using

TRI reagent (Molecular Research Center, Cincinnati,

OH, USA), according to the protocol provided by the

manufacturer. Identical quantities of RNA were then

reverse transcribed into cDNA, and the expression

levels of b-actin (internal standard; present in all

tubes of the assay), CD3gd, Bu-1, ChIL-2 and

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157148

ChIFNg were determined by a semi-quantitative PCR

using the specific primers described earlier [22]. The

RT-PCR amplification was calibrated in order to

determine the optimal number of cycles that would

allow detection of the appropriate mRNA transcripts,

while keeping amplification for these genes in the log

phase (primer dropping method) [22]. PCR reactions

were performed using a programmable thermal

controller (MJ research INC. Waltham, MA, USA).

The number of cycles used for PCR reactions was 16

cycles for b-actin, 26 cycles for CD3gd, 28 cycles for

Bu-1, 32 cycles for ChIL-2 and 34 cycles for ChIFNg.

2.5. Immunizations and rectal antigen administration

Chicks were immunized orally with bovine serum

albumin (BSA; Sigma Chemical Co., Israel) or

rectally with hemocyanin (Hem; Sigma Chemical

Co, Israel) dissolved in sterile water

(5 mg/1 ml/chick/day and 1 mg/100 ml/chick/day,

respectively) for five consecutive days. The use of

two antigens was to insure that the absence of response

(Section 3) was not due to the type of antigen used.

Rectal immunization was performed by gently placing

a micropipette tip above the anal lips, and slowly

dripping the solution into the cloacal opening, thus

allowing the chick to voluntarily take up the solution

via retrograde peristalsis [23]. Oral immunization was

performed by gently placing a blunt-tipped feeding

needle above the tongue, and slowly dripping the

solution into the pharynx, thus allowing the chick to

voluntarily swallow the solution [7]. Positive control

chicks were immunized intramuscularly (IM) with

Hem or BSA emulsified in Freund’s complete

adjuvant (CFA). Briefly, antigen was dissolved in

sterile PBS (2 mg/ml) and the solution was then

emulsified in an equal volume of CFA. Each injection

(0.5 ml containing 500 mg antigen) was delivered into

four IM sites: left and right shank and breast muscles.

Blood samples of native and treated chicks were

obtained from jugular vein 1, 5 and 10 days after last

immunization.

2.6. Anti-Hem and anti-BSA antibody responses

Serum samples were collected from centrifuged

clotted blood and stored at 220 8C until used. Sera

from IM immunized chicks served as positive controls.

Sera obtained immediately after hatch or from

untreated 14 days old chicks served as negative

controls. Antigen specific antibody levels were

determined in serum pooled from six chicks per time

point by ELISA. Immunoplates (Nunc, Denmark) were

coated with antigen (50 mg/ml) dissolved in coating

buffer (100 ml 50 mM sodium carbonate/bicarbonate

buffer, pH 9.6) and held at 4 8C overnight. Plates were

washed three times with Wash Solution diluted 1:20 in

water (Kirkegaard and Perry, Gaithersburg, MD).

Blocking was performed with BSA (1% in phosphate

buffer, Kirkegaard and Perry) for anti-Hem antibody

responses, or with skim milk (Difco, Detroit, MI)

(0.5% in phosphate buffer) for anti-BSA antibody

responses. Block solution was added to each well and

then incubated at 37 8C for 2 h. Following washing,

test sera diluted 1:200 in block solution were added and

plates were incubated for 1 h at 37 8C and washed

again. Peroxidase labeled goat anti-chicken IgGHþL

(Kirkegaard and Perry), detecting antibody (diluted

1:2000 in block solution) was added, incubated 1 h at

37 8C and then washed. Following washing, bound

antibodies were detected by TMB (Kirkegaard and

Perry). Optical absorbance at 450 nm was determined

using a Spectra II ELISA reader (SLT, Salzburg,

Austria). The results are the average of duplicate

measurements and are expressed as absorbance units.

2.7. Statistics

Means of results are presented after factorial

analysis of variance as using the general linear models

procedures of SASw (SAS Institute, 1986) and

correlation between variables examined by linear

regression; significance was assessed at p , 0.05

unless otherwise stated.

3. Results

3.1. Population pattern of CD3 þ cells of the avian

gut is similar in all intestinal segments

The mRNA of the CD3gd antigen was used as a

marker to determine and follow gut colonization by

cells of both the innate and acquired immune systems

(Natural Killer Cells—NK and T lymphocytes,

respectively). The CD3gd antigen is expressed on

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157 149

avian T lymphocytes and is involved in signal

transduction pathways leading to T cell activation

[24] while avian NK cells express cytoplasmic CD3

[25]. Basal levels of CD3gd mRNA were observed in

all intestinal segments of newly hatched chicks

(Fig. 1). This primary colonization remained almost

unchanged during the first day post-hatch, while at 4

days post-hatch a dramatic increase in CD3 þ cells

was observed, which was significant at all sites

examined ( p , 0.05). Further increases in CD3 þ

cell population occurred after day 4, however, these

increases were of smaller magnitude. The dynamics of

seeding patterns were similar in all intestinal seg-

ments tested except for the colon, in which after day 4

post-hatch, cell frequency was lowest in this segment.

3.2. Functional development of immunity in the

intestine corresponds to T lymphocyte seeding pattern

To study the developmental and functional matu-

ration of the intestinal T cell population, ChIL-2

mRNA levels were measured. Levels of ChIL-2

mRNA increased in the hind gut and in the ileum

starting at day 4 post-hatch with a further major

increase observed during the second week post-hatch

(Fig. 2; p , 0.05). In the duodenum and jejunum there

were little changes in ChIL-2 mRNA levels until day

8 post-hatch after which profound increases in ChIL-2

mRNA levels were observed (Fig. 2; p , 0.05).

Correlations between the mRNA levels of CD3gd

and ChIL-2 were determined at the different intestinal

sites and the degree of correlation for both genes was

significant throughout the intestines ( p , 0.05). Thus

the dynamics of ChIL-2 mRNA expression in the

intestine correspond to the seeding pattern of T

lymphocytes. Moreover, the results suggest that

although T lymphocytes colonize the various parts

of the intestine at the same time the functional

maturation of the intestine is not uniform and is

biphasic. In the early maturational phase, which

occurs during the first week post-hatch, hind gut

maturation precedes that of the upper midgut. The

second maturational phase occurs towards the end of

the second week and leads to full maturation of the T

lymphocyte-dependent enteric immune system.

3.3. Effector cell functions corresponded to

development of functional immunity and CD3 þ

cell dynamics

Expression of ChIFNg mRNA levels was used

as a measure of effector cell functionality along

Fig. 1. CD3gd mRNA expression in intestinal segments from broiler chicks (D, duodenum; J, jejunum; I, ileum; C, caecal tonsils; C, colon) with

age. Results at each point are the averages of three individual chicks and are presented as mRNA ratio using b-actin as an internal standard.

SEM within groups was less than 5% of the values. The experiment shown was repeated three times with similar results.

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157150

the intestine. ChIFNg mRNA expression similar to

that of ChIL-2 demonstrated a biphasic functional

maturation (Fig. 3). The first maturational stage

occurred during the first week post-hatch and was

characterized by increases in intestinal ChIFNg

mRNA levels on day 4 post-hatch. Decreased

ChIFNg mRNA levels between days 4 and 6 post-

hatch, especially in the jejunum, ileum and colon,

Fig. 3. ChIFNg mRNA expression in intestinal segments from broiler chicks (D, duodenum; J, jejunum; I, ileum; C, caecal tonsils; C, colon)

with age. Results at each point are the averages of three individual chicks and are presented as mRNA ratio using b-actin as an internal standard.

SEM within groups was less than 4% of the values. The experiment shown was repeated three times with similar results.

Fig. 2. ChIL-2 mRNA expression in intestinal segments from broiler chicks (D, duodenum; J, jejunum; I, ileum; C, caecal tonsils; C, colon) with

age. Results at each point are the averages of three individual chicks and are presented as mRNA ratio using b-actin as an internal standard.

SEM within groups was less than 7% of the values. The experiment shown was repeated three times with similar results.

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157 151

were followed by a second maturational stage at the

beginning of second week that was characterized by

substantial increases in ChIFNg expression in all

intestinal segments. The relationship between

ChIFNg mRNA and CD3gd mRNA levels was

examined, and at all intestinal sites, with the

exception of the colon, significant correlations were

found ( p , 0.05). In the colon the relationship tended

towards significance ( p , 0.1). In addition, ChIFNg

mRNA levels were also correlated, throughout the

intestine, with ChIL-2 mRNA levels. The correlation

was significant ( p , 0.05) with the exception of the

CT, where the relationship tended towards signifi-

cance ( p , 0.1). Hence, the dynamics of ChIFNg

mRNA levels corresponded with those of intestinal T

cell colonization.

3.4. B lymphocyte population of the midgut precedes

that of the hind gut

mRNA expression of the Bu-1 antigen was used as

a marker to follow gut colonization by B lympho-

cytes. The Bu-1 antigen is expressed on B lympho-

cytes throughout their development [26]. Basal levels

of Bu-1 mRNA were detected along the intestine of

newly hatched chicks (Fig. 4). At hatch, Bu-1 mRNA

levels were higher in the CT compared to other parts

of the intestine. Increases in Bu-1 mRNA were

observed from 4 days post-hatch in the small

intestines. The primary colonization of the hind gut

remained unchanged until day 6 post-hatch where-

upon a marked increase in Bu-1 mRNA levels

occurred. These results imply that small intestine

colonization by B lymphocytes preceded that of the

hind gut. Bu-1 mRNA levels remained relatively

unchanged in all intestinal segments between 8 and 12

days post-hatch. These results suggest a steady flow of

B lymphocytes to the intestine after day 6. It is

interesting to note that despite the early seeding of B

lymphocytes in the colon, their frequency in that

segment was the lowest of all segments tested, similar

to that observed for T lymphocytes.

3.5. Development of antibody responses following

enteral immunization corresponds with gene

expression studies

To test whether seeding patterns and lymphocyte

cytokine gene expression were appropriate indicators

of the physiological status of GALT in the young

chick, we studied antibody specific responses to

enterally administered antigens. While oral protein

antigens induced potent systemic and gut-specific

antibody responses in adult chickens (Fig. 5 and [7]),

Fig. 4. Bu-1 mRNA expression in intestinal segments from broiler chicks (D, duodenum; J, jejunum; I, ileum; C, caecal tonsils; C, colon) with

age. Results at each point are the averages of three individual chicks and are presented as mRNA ratio using b-actin as an internal standard.

SEM within groups was less than 5% of the values. The experiment shown was repeated four times with similar results.

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157152

they did not induce responses in chickens orally

immunized prior to 10 days of age. To exclude the

possibility that lack of responses at young ages was

due to antigen or the route of administration, chicks

were immunized rectally with Hem at different time

points post-hatch and antibody levels were deter-

mined in sera (Fig. 6). Rectally administered Hem

induced immune responses in chicks immunized at

Fig. 5. Development of antibody responses to orally administered BSA. Chicks were orally immunized against BSA at different ages. In each

age group antibody responses to BSA were determined by ELISA 1, 5 and 10 days after the last feeding. The control groups include (a) sera

from 24 days old nainodotve chicks, (b) sera from 24 days old chicks immunized at 2 weeks of age with BSA–CFA, and (c) sera from 29 days

old chicks orally immunized with BSA at 2 weeks of age. Antibody responses of treated and control groups are presented as mean antibody

responses ^ SEM (n ¼ 6) at a serum dilution of 1:200. The experiment shown was repeated five times with similar results.

Fig. 6. Development of antibody responses to rectally administered Hem. Chicks were rectally immunized against hemocyanin (Hem) at

different ages. In each age group antibody responses to Hem were determined by ELISA 1, 5 and 10 days after the last immunization. The control

groups include (a) sera from 24 days old nainodotve chicks, (b) sera from 24 days old chicks immunized at 2 weeks of age with Hem–CFA, and

(c) sera from 29 days old chicks rectally immunized with Hem at 2 weeks of age. Antibody responses of treated and control groups are presented

as mean antibody responses ^ SEM (n ¼ 6) at a serum dilution of 1:200. The experiment shown was repeated four times with similar results.

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157 153

2 weeks of age, at levels comparable to those obtained

by IM immunization. However, when Hem was

administered to chicks between 0 and 4 days post-

hatch or 4 and 8 days post-hatch no antibody

responses were observed 5 and 10 days after last

rectal immunization. Antibody responses began to

appear only in chicks challenged between 8 and 12

days post-hatch. The first antibody response was

observed in this group 5 days after the last rectal

immunization. Ten days after the last immunization

all chicks in the group responded, however, only one

chick showed a response comparable in magnitude to

that of chicks immunized at 14 days of age. These

results are in agreement with the colonization and

cytokine gene expression data and demonstrate the

gradual maturation of the broiler GALT during the

first two weeks of life.

4. Discussion

Development of systemic immune competence in

the chicken is known to occur during the post-hatch

period [3,9,27], however, little is known about the

development of immune competence in the avian

GALT. The post-hatch period is of major immuno-

logical importance since the chick is immediately

exposed to environmental antigens (including patho-

gens), in the absence of additional post-hatch maternal

immunity such as that provided by colostrum and milk

in the mammal. To investigate the development of

immune competence in the avian GALT during the

immediate post-hatch period we studied population

dynamics of intestinal lymphocytes and the temporal

development of lymphocyte functions.

Our findings demonstrate that GALT maturation

occurs in two stages or waves: the primary wave

occurred during the first week post-hatch and a second

wave during the second week. The two maturational

stages were clearly demonstrated by mRNA

expression of both ChIL-2 and ChIFNg. The primary

stage was characterized by an increase in both

cytokine mRNA levels on day 4 post-hatch. The

increase in ChIL-2 expression was observed mainly in

the distal parts of the intestine which may imply that

this part of the intestine is more active immunologi-

cally during the first day following hatch. However,

this was not supported by the changes in ChIFNg, as

the observed increase in ChIFNg mRNA on day 4

post-hatch was of a similar magnitude in all intestinal

segments. A possible explanation for this discrepancy

could be that the increase of ChIL-2 mRNA reflects

activation of T lymphocytes alone, whereas the

increase in ChIFNg expression represents activation

of both T and NK cell populations; both have recently

been demonstrated to be constituents of the avian IEL

compartment [25]. Concomitant with the primary

activation and function, we observed a substantial

increase in CD3 þ cells in all intestinal segments.

The CD3 antigen is expressed on chicken T

lymphocytes [24]; however, as avian NK cells express

the cytoplasmic portion of this marker [28,29], the

increase in CD3 mRNA levels can be also attributed

to gut colonization by NK cells. The increase in the

CD3 þ cell population marks the beginning of the

second stage of GALT maturation and it is followed

by a second wave of activation as observed by

increased ChIL-2 and ChIFNg mRNA levels during

the second week of life.

A recent study in chicks has shown that three

successive waves of gd and ab T cells depart

sequentially from the thymus en route to the periphery

[30]. The second wave described in this study by

Dunon et al. coincides temporally with the increase in

CD3gd observed here. This could indicate the arrival

of thymus derived ab T lymphocytes to the intestine,

but needs to be demonstrated directly by monitoring T

cell trafficking. As ab T cells reside mainly in the

intestinal lamina propria [31–33], the second stage of

maturation might reflect the maturation of this

compartment. The time lapse between the cell

population period (day 3) and the extensive

expression of functional cytokines (day 6 and later)

suggests that the thymic migratory cells might need an

acclimation period before activation by gut borne

antigens. Thus, the extensive increase in ChIL-2 and

ChIFNg observed during the second week of life is an

expression of the maturation of the lamina propria

compartment. Our interpretation is supported by

studies of Lowenthal et al. who demonstrated that

lymphocytes obtained from 1 day old chicks, although

phenotypically mature, were functionally immature

and gained functional maturation gradually [34].

The events leading to GALT maturation can be

suggested to occur as follows: exposure to environ-

mental antigens together with feed, leads to initial

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157154

activation of lymphocytes and NK cells residing

mainly in the IEL compartment of the newly hatched

chicks. This activation is manifested by an increase of

ChIL-2 and ChIFNg mRNA levels observed on day 4

post-hatch. The arrival of new T lymphocytes

commencing day 3 post-hatch indicates the beginning

of a second maturational stage during which the

lamina propria compartment matures. GALT devel-

opment may involve maturation of both adaptive

immune cells (T lymphocytes) and innate immune

cells (NK cells). However, as peripheral innate cells

are functional at young age [35], we suggest that full

activation of NK cells is dependent on maturation of T

cells, and that most of the development is accounted

for by responsive T cells.

Our results show that GALT maturation also

involves changes in the B cell compartment. Age-

related differences in B cell population were pre-

viously described by Fagerland and Arp in bronchus

associated lymphoid tissue (BALT) of newly hatched

chicks [36]. These researchers showed that at hatch, B

cells are present in small numbers in BALT; however

significant population of B cells occurs at two weeks

of age. We demonstrate here that similar to BALT,

GALT is also populated by B cells at hatch, and in

contrast to BALT, population of GALT by B cells

begins as early as 4 days post-hatch and is further

increased over the first two weeks of age. The increase

in population size might either reflect influx of new B

lymphocytes or proliferation of residing cells; both

possibilities are not mutually exclusive. The earlier

colonization of GALT relative to that of BALT

suggests an earlier maturation of GALT and empha-

sizes the importance of GALT in the protection of the

young chicks during the first 2 weeks of life. Within

GALT, a substantial colonization of B cells in the

small intestine was observed on day 4 post-hatch,

while an increase of a similar magnitude was observed

in the hind gut only 2 days later. Non-uniform

distribution of B cells has also been described in the

intestines of dogs, demonstrating higher B cell

frequencies in the duodenum [37]. The cause for the

differences in B cell population dynamics is presently

unknown. As avian B lymphocytes, like their

mammalian counterparts, migrate in response to

chemokines [38], we propose that the differential

distribution and early colonization of B cells in the

proximal end of the intestine could be due to

chemokines secreted by epithelial cells in that region.

We suggest that the differential homing pattern of B

cells, during the immediate post-hatch period, is

associated with the maturational status of epithelial

cells lining the different regions of the intestine. The

potential role for gut epithelium in B cell colonization

is based on studies by Vervelde and Jeurissen who

showed the presence of B cells in the epithelium layer

of the avian digestive tract [39] and a recent

observation by Bowman et al. [40], who showed

that IgA-commited B cells are attracted to the

intestine by the intestinal epithelium derived chemo-

kine TECK. As the functional maturation of epithelial

cells, as expressed by brush border enzyme activities,

varies along the intestine [41], regional differences in

secretion of epithelial chemokines might also occur.

Further studies are needed to test whether these

regional differences in B cell colonization are

mediated by gut epithelial cells and whether they

affect antibody production in the various segments.

Higher Bu-1 expression in CT of newly hatched

chicks, compared to those in other parts of the

intestines, indicates that this region contains a larger B

cell population at hatch, and thus may be more active

in generation of antibody responses during the early

post-hatch period than other parts of the intestine.

This observation is in accord with previous reports

that have indicated the hind gut, specifically, the caeca

and CT as the main site of immune activity, at least in

terms of number and distribution of PP and lymphoid

follicles [2,17,42,43], and as the most active lymphoid

organ in the adult chick gut [2,11,14].

Functional maturation of B lymphocytes was

determined by their ability to initiate antibody specific

responses to innocuous antigens (BSA or Hem)

administered enterally. Piquer et al. have demon-

strated gradual maturation of intestinal B cell

responses in turkeys [44]. Exhibiting a similar pattern,

the data presented here demonstrate that maturation of

enteric induced antibody responses in young chickens

developed gradually maturing towards the end of the

second week of age. As both BSA and Hem are T-

dependent antigens capable of inducing enteral

responses in the absence of adjuvant [7,45], lack of

antibody responses during the first week post-hatch

might be attributed to immaturity of T lymphocytes

residing in the LP or CT and in terms of their inability

E. Bar-Shira et al. / Developmental and Comparative Immunology 27 (2003) 147–157 155

to provide help necessary for the induction of

antibody responses.

Absorptive and digestive capabilities of the chick

intestine develop during the first two weeks of life

[41]. Hence, lack of antibody responses in the young

hatchling could also be due to inability to process or to

absorb intact antigens at a young age. This possibility,

however, is unlikely for two reasons: first it has been

shown previously that while young chicks were

unable to absorb antigens administered orally, they

were capable of absorbing antigens administered via

the cloaca [10].The data presented here do not

indicate significant differences between the kinetics

of antibody response to antigens administered by

either route. Secondly we demonstrate that immune

cell activation as expressed by cytokine gene

expression is initiated as early as 4 days post-hatch

which indicates the ability of GALT to respond to gut

born antigens at an early age. Therefore, we favor the

idea that in the intestinal milieu, cellular responses

mature earlier and are a prerequisite for initiation of

humoral responses and that lack of antibody response

of young chicks is primarily due to immaturity of T

lymphocytes.

Acknowledgements

This study was supported by a grant from the

Israeli Ministry of Agriculture and Rural Develop-

ment.

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