development of maternal igg-free chick obtained from surgically bursectomized hen

Development of maternal IgG-free chickobtained from surgically bursectomized hen

Masahiro Yasudaa,e, Shuichi Furusawab, Haruo Matsudab,Yasuho Taura a, Toru Uranoc, Yuichi Yokomizod,

Shigeo Ekinoe *aDepartment of Veterinary Hospital, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan

bDepartment of Immunobiology, Faculty of Applied Biological Science, Hiroshima University, Hiroshima,

JapancLaboratory Animal Research Center, Kumamoto University School of Medicine, Kumamoto, Japan

dDepartment of Biological Product, National Institute of Animal Health, Ibaraki, JapaneDepartment of Anatomy, Kumamoto University School of Medicine, Kumamoto, Japan

Received 3 October 1997

Abstract

IgG-free eggs and chicks were developed, so as to study the role of maternal IgG in the

development of the immune system. Surgical bursectomy on the 18th day of incubationdeprived chickens of B cells and eliminated IgG synthesis. Bursectomized chickens areusually dead before sexual maturity under conventional conditions. When surgicallybursectomized chickens were housed in an isolated clean room and antibiotics were

administered to them, they could survive to sexual maturity. Finally, we succeeded inobtaining IgG-free fertilized eggs and maternal IgG-free chicks from surgicallybursectomized hens. The amount of yolk IgG in IgG-free eggs was one-ten thousandth less

than that in normal eggs. The level of IgM in the serum of maternal IgG-free chicksreached six times higher than that of normal chicks 5 days after hatching. # 1998 ElsevierScience Ltd. All rights reserved.

Keywords: Surgical bursectomy; Maternal IgG; Yolk IgG; B cell

Comparative Immunology, Microbiology

& Infectious Diseases 21 (1998) 191±200

0911-6044/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved.

PII: S0147 -9571 (98)00006 -X

C OMPARATIVE

I MMUNOLOGY

M ICROBIOLOGY &

I NFECTIOUS

D ISEASESPERGAMON

* Author for correspondence. Tel.: 81-96-373-5047; Fax: 81-96-373-5048; e-mail: ekino@

kaiju.medic.kumamoto-u.ac.jp

ReÂ sumeÂ

Des úufs et poussins sans IgG ont eÂ teÂ deÂ veloppeÂ s pour eÂ tudier le roÃ le des IgG

maternelles dans le deÂ veloppement du systeÁ me immunitaire. Une bursectomie chirurgicale,le 18eÁ me jour de l'incubation a priveÂ les poulets de cellules B et a supprimeÂ la syntheÁ sedes IgGs. Les poulets bursectomiseÂ s, eÂ leveÂ s dans des conditions conventionnelles, meurentavant d'avoir atteint leur maturiteÂ sexuelle. Quand les poulets chirurgicalement

bursectomiseÂ s ont eÂ teÂ eÂ leveÂ s dans une chambre isoleÂ e et propre et ont eÂ teÂ traiteÂ s par desantibiotiques, ils ont pu survivre jusqu'aÁ leur maturiteÂ sexuelle. Finalement, nous avonsreÂ ussi aÁ avoir des úufs feÂ condeÂ s dans IgG, et des poussins sans IgG maternelles aÁ partir

de poules bursectomiseÂ es. Le sac vitellin des úufs obtenus aÁ partir de poulesbursectomiseÂ es contenait dix mille fois moins d'IgG que les úufs obtenus aÁ partir depoules normales. Le niveau des IgM dans le seÂ rum des poussins sans IgG eÂ tait 6 fois plus

eÂ leveÂ que dans celui de poussins normaux 5 jours apreÁ s l'eclosion. # 1998 ElsevierScience Ltd. All rights reserved.

Mots-cleÂfs: Chirurgicale bursectomie; IgG maternelles; IgG du sac vitellin; Cellule B

1. Introduction

Maternal IgG in the yolk and milk is thought to protect the newborn animalsfrom infections [1±3]. In addition, our recent study suggests that maternal IgGbinds with bursal B cells and this binding is dependent on environmentalantigens from the gut [4]. It is hypothesized that maternal IgG in the yolkmodi®es B-cell di�erentiation and diversi®cation in the bursa of Fabricius. Toexamine this hypothesis, we developed maternal IgG-free chicks (MIgG±freechick).

Surgical bursectomy (SBx) before hatching depletes B cells in peripherallymphoid organs and eliminates IgG synthesis [5, 6]. The degree of B-cell depletionand IgG reduction by SBx is dependent on the stage of the chicken embryo [5].SBx on the 18th day of incubation is expected to cause the severest e�ect on B-celldevelopment in the periphery. Since a few B cells seed before the 18th day ofincubation, B-cell depletion is incomplete in occasional SBx-chickens [7]. Toexclude the incomplete SBx-chickens, the frequency of B cells and serum IgG inSBx-chickens must be examined before laying eggs. Severe depletion of B cellsoften causes infection and death in SBx-chickens, so that it is di�cult to keepSBx-chickens for a long term. We succeeded in keeping SBx-chickens for 18months which showed extremely low IgG in serum and laid IgG-free fertilizedeggs. MIgG-free chicks were hatched from IgG-free fertilized eggs.

M. Yasuda et al. / Comp. Immun. Microbiol. Infect. Dis. 21 (1998) 191±200192

2. Materials and methods

2.1. Chicken

Fertile eggs of H.B15 (B15) strain (Kumamoto University School of Medicine,Japan) were used. Eggs were incubated and hatched in our own facilities. Thechickens were housed together under conventional conditions and were allowedfree access to food and water.

2.2. Surgical bursectomy

On the 18th day of incubation, most of the chicken embryos were in theposition shown in Fig. 1(b). When the eggs were candled and rotated, the specialappearance of egg was found [Fig. 1(a)]. In the rather light concave part of theembryo, the head of the chicken embryo was located. The tail of embryo wasdistinguished as shown in Fig. 1(b). The rectangle was marked for drilling. Eggswere sterilized with tincture of iodine and surgical spirit before drilling. The eggshell was cut out with a dental drill without any damage of the shell membraneand the underlying chorioallantoic membrane. The shell, chorioallantoic andamniotic membrane were removed one at a time without hemorrhage. The tailcould be held ®rmly with the forceps. SBx was carried out basically according tothe method described by Aitken and Penhale [8]. SBx was performed with extremecare to avoid damage to the oviduct, so as to prevent stenosis of the oviduct.

2.3. Experimental design (Fig. 2)

SBx-chicks were housed together in an isolated room of a central air-conditioned building, in which ®ltrated air was supplied. The room was keptunder constant temperature (22228C) and humidity (50±70%). Antibiotics wereadministered to SBx-chickens with food and water. Ampicillin (Shionogi, Tokyo,Japan) was mixed with drinking water (2 g/liter of water). Chlortetracycline(Shionogi, Tokyo, Japan) was mixed with food (100 mg/1 kg of food). Serum IgMand IgG in SBx-chickens was examined with the enzyme-linked immunosorbentassay 11 weeks after hatching. Yolk IgG was examined after 8 months old.

2.4. Enzyme-linked immunosorbent assay (ELISA)

An ELISA for the detection of immunoglobulin in serum and yolk wasperformed as previously described [9, 10]. Brie¯y, 96-well ¯exible ¯at-bottomedpolyvinyl plates (Nunc, Roskile, Denmark) were coated with rabbit anti-chickenIg (Nordic Immuno. Lab., Tilburg, The Netherlands) or rabbit anti-chicken IgY(Jacson Immuno Lab. Inc., West Grove, PA). Serially 10- or 3-fold dilutedsamples (serum and yolk respectively) were added to precoated ELISA plates.After 1 h incubation at 378C, plates were washed in phosphate-bu�ered saline

M. Yasuda et al. / Comp. Immun. Microbiol. Infect. Dis. 21 (1998) 191±200 193

(PBS) with 0.01% Tween 20, and incubated with goat anti-chicken IgM or IgG

(Bethyl Inc., Montgomery, TX) for 1 h at 378C. Following washing, plates were

incubated with peroxidase-labeled rabbit anti-goat IgG (Bethyl Inc., Montgomery,

TX) for 1 h at 378C. After washing, binding of the peroxidase conjugate was

Fig. 1. Position of chick embryo in egg on the 18th day of embryonation. (a) Appearance of

embryonated egg. (b) The posture of chick embryo in egg. The head of chick embryo is located in the

rather light concave part. The backbone turns round through 2708 and reaches the tail.


detected by addition of o-phenylenediamine dihydrochloride (Sigma St Louis,MO) substrate solution. Absorbance was read at 492 nm with a microdilutionplate reader (BIO-RAD 450, Hercules, CA). The concentrations of serum IgM,IgG and yolk IgG were determined by plotting the OD values against a standardcurved formed by optional normal chicken serum or egg yolk. The ELISA datawere converted into mg/ml as previously described [11].

2.5. Preparation of cells and immuno¯uorescence

Bursa and spleen cells were obtained by teasing the minced organs throughstainless steel mesh. Spleen cells were further puri®ed on a Ficoll-Hypaque(Pharmacia, Uppsala, Sweden) gradient. For immuno¯uorescence, cells weresuspended in PBS supplemented with 10% foetal calf serum (FCS). Viable cellswere incubated with L22: anti-Bu1a, 2G11: anti-class II MHC, and HIS C12; anti-m as previously described [4]. After washing, a second incubation was conductedwith ¯uorescein isothiocyanate (FITC)-conjugated sheep anti-mouse IgG F(ab)2(Silenus Laboratories, Hawthorn, Australia). Relative immuno¯uorescenceintensities were determined by ¯ow cytometry using a FACScan (BectonDickinson, Mountain View, CA).

3. Results

3.1. Selection of IgG-free hens

SBx-hens in which serum IgG was less than 0.05 mg/ml were selected as IgG-free hens 11 weeks after hatching (Fig. 3). The mean amount of IgG in IgG-freehens was 0.014 mg/ml and 0.16% of that of normal chickens (Table 1). The meanamount of IgM in the IgG-free hens was 3% of that in normal chickens (Table 1).B cells (Bu1+ cells) were not detected in the peripheral blood of IgG-free hens.Typical FACS pro®les of Bu1+ cells in the peripheral blood of normal and SBx-hens are shown in Fig. 4. Histological examinations clari®ed that any Ig+ cellswere not found in spleen nor caecal tonsil of IgG-free cocks (data not shown).

Fig. 2. Experimental schedule for the development of IgG-free hens.


After sexual maturity (8 months old), yolk IgG in fertilized eggs derived fromIgG-free hens was 0.007% of that of normal hens (Table 1).

3.2. E�ects of maternal IgG on B-cell development

IgG-free fertilized eggs were hatched in a normal rotating incubator. Thosechicks hatched from IgG-free eggs were free of maternal IgG and abbreviated as

Fig. 3. Selection of IgG-free hens. Closed circles represent selected SBx-hens. Open circles represent

sacri®ced SBx-hens. Open triangles represent normal chickens. In selected SBx-hens, IgG was hardly

detected and IgM was lower than those in any normal chickens.

Table 1

Concentrations of serum IgM, IgG and yolk IgG in selected SBx-chickens

Ig class Normal (mg/ml) SBx-chicken (mg/ml)

IgM 5.729.3 0.220.4

(16) (9)

IgG 8.725.2 0.0120.01*

(16) (9)

Yolk IgG 27.128.0 0.00220.002*

(6) (6)

All values are mean2SD (number of chickens examined). Data of SBx-chickens were compared with

data of normal chickens. p value was examined by Student t-test (*p< 0.0005).


MIgG-free chicks. MIgG-free chicks started to develop serum IgM and IgGimmediately after hatching. Five days after hatching, serum IgG in MIgG-freechicks reached 0.04 mg/ml (Table 2). It was 1% of serum IgG in normal chicks.Interestingly, IgM in MIgG-free chicks was about six times higher than that innormal chicks. The deprivation of maternal IgG caused hyper IgM in serum at aneonatal stage. The frequency of Bu1+ and IgM+ cells in the bursa of Fabriciusand spleen of a MIgG-free chick was comparable to that of a normal chick(Table 3). Apparently, the development of B cells in the bursa and spleen was notin¯uenced by deprivation of maternal IgG, whereas the frequency of class II+

cells in the spleen was reduced in neonatal MIgG-free chicks. Typical FACSpro®les of MIgG-free and normal chicks were shown in Fig. 5.

Fig. 4. FACS pro®les of Bu1+ cells in peripheral blood of SBx hens 18 months after hatching. Normal

chickens contained 3% of Bu1+ cells in the peripheral blood. No Bu1+ cells were detected in the

peripheral blood of SBx hen.

Table 2

Concentrations of serum IgM and IgG in MIgG-free chicks at 5 days after hatching

Ig class Normal chick (mg/ml) MIgG-free chick (mg/ml)

IgM 0.620.3 3.521.7*

(7) (7)

IgG 3.020.3 0.0420.02**

(7) (7)

All values are mean2SD (number of chickens examined). Data of MIgG-free chicks were compared

with data of normal chicks. p value was examined by Student t-test (*p< 0.01, **p< 0.000001).


4. Discussion

IgG-free hens were obtained from chickens surgically bursectomized on the 18thday of incubation. SBx at this stage could not always cause the completesuppression of B-cell development. Sixty percent of SBx-chickens had hardlydetectable IgG in the serum and were selected as IgG-free hens (Fig. 3). In thoseIgG-free hens, serum IgM was also less than that in normal chickens and any Bcells were not detected in the peripheral blood (Figs. 3 and 4). When IgG-freecocks were histologically examined 11 weeks after hatching, any Ig+ cells were

Table 3

E�ect of maternal IgG on the frequency of the bursa and spleen

Bursa Spleen

mAb Normal MIgG-free Normal MIgG-free

Bu1 9522 9522 21212 1828

(7) (7) (6) (6)

Class II 7427 7524 5828 4328*

(7) (7) (7) (7)

IgM 9422 9522 16210 1528

(6) (6) (6) (7)

All values are mean2SD (number of chickens examined). Data of maternal IgG-free chicks were

compared with data of normal chicks. p value was examined by Student t-test (*p< 0.001).

Fig. 5. FACS pro®les of Bu1+, class II+ and IgM+ cells in spleen of MIgG-free chick and normal

chick at 1 week old. The pro®les of Bu1+ cells and IgM+ cells in spleen were not di�erent between

normal chicks and MIgG-free chicks. The frequency of highly class II+ cells in MIgG-free chicks was

clearly less than that in normal chicks.


found neither in the spleen nor in the caecal tonsil (data not shown). Takentogether, these results suggest that the successful SBx on the 18th day ofincubation causes the complete deprivation of B cells.

We could succeed on keeping IgG-free hens in the clean room for more thanone year. There would be chances of infections as it was not completely isolatedfrom conventional places. In spite of such conditions, SBx-hens could survive formore than one year. It is speculated that nonspeci®c defense mechanisms mayhave supported the survival of SBx-hens. In this aspect, the long-lived IgG-freechicken will be a good animal model for studying nonspeci®c defence mechanisms.

In MIgG-free chicks, serum IgM was six times higher than that in normalchicks (Table 2). Subba Rao et al. reported that IgG controlled the developmentof serum IgM [12]. We speculate that the de®cit of maternal IgG would removethe control of IgM level in serum. To identify this phenomenon, furtherinvestigations are required. On the other hand, the frequency of class II+ cells inthe spleen was statistically lower than that in normal chicks (Table 3). Thedevelopment of bright class II+ cells was suppressed in MIgG-free chicks (Fig. 5).It is known that antigen±antibody complexes strongly stimulate reticuloendothelialsystem [13±15]. From these facts, we speculate that antigen±MIgG complexes arestrong stimulants to the reticuloendothelial system and induce the activation ofclass II+ cells in the spleen.

Acknowledgements

This work is supported in part by a Grant-in-Aid (Bio Media Program) fromthe Japanese Ministry of Agriculture, Forestry and Fisheries, the Ministry ofEducation, and The Osaka Foundation for Promotion of Clinical Immunology.We are grateful to Dr S. Kawamura for critical reading of the manuscript.

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development of maternal igg-free chick obtained from surgically bursectomized hen

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