Suppression of IgA synthesis in chickens

Veterinary Immunology and Immunopathology, 7 (1984) 305--313 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands

305

S U P P R E S S I O N OF IgA S Y N T H E S I S IN C H I C K E N S

B.K. T I W A R Y and M.C. GOEL Immunology Section, D e p a r t m e n t of V e t e r i n a r y Microbiology, Haryana A g r i c u l t u r a l University, H i s s a r 125004, India (Accepted 12 M a r c h

1984)

ABSTRACT Tiwary, B.K. and Goel, M.C., 1984. S u p p r e s s i o n of IgA synthesis in chickens. Vet. Immunol. Immunopathol., 7: 305-313. Four d i f f e r e n t p r o t o c o l s were tested for the induction of IgA d e f i c i e n c y in chickens: (I) i n o c u l a t i o n of anti-~ i n t r a - p e r i t o n e a l l y (i.p.) on alternate days after h a t c h i n g up to a p e r i o d of three weeks; (II) b u r s e c t o m y within 6 h and at 24 h a f t e r hatching; (III) in-ovo injection of anti-~ on the 17th day of embryo n a t i o n f o l l o w e d by b u r s e c t o m y at 24 h of h a t c h and a single injection of anti-~ i.p. on the day of hatching; (IV) as in III above but b u r s e c t o m y within 6 h of hatch, f o l l o w e d by three further injections of anti-~ on days 3, i0 and 34 after hatching. T r e a t m e n t (I) p r o d u c e d temporary d y s g a m m a g l o b u l i n e m i a during the p e r i o d of treatment. B u r s e c t o m y at 24 h of hatch rendered 75% of the chicks IgA d e f i c i e n t up to four weeks of age. Early b u r s e c t o m y within 6 h of h a t c h r e s u l t e d in s u b s t a n t i a l i m p r o v e m e n t of IgA suppression. Such chicks, when tested at 4, 6 and i0 weeks of age, were found to be 81, 72 and 58.3% IgA deficient, respectively. With treatment (III) all the t r e a t e d birds were IgA deficient at four w e e k s of age. However, as the b i r d s g r e w older, IgA a p p e a r e d in the serum so that at the age of 12 weeks only 27.3% were deficient. T r e a t m e n t (IV) c o m p l e t e l y s u p p r e s s e d the IgA system of 13 out of 14 chickens. These chickens lacked both serum and secretory IgA as well as I g A - c o n t a i n i n g cells in their intestinal mucosa. Both IgG and IgM c o n t i n u e d to be produced. INTRODUCTION The a v a i l a b i l i t y of an I g A - d e f i c i e n t model will be of much h e l p in understanding the m e c h a n i s m of m u c o s a l im~unity s p e c i f i c a l l y in c h i c k e n s and absorption of various b i o m o l e c u l e s

from the mucosa,

in general. P r e v i o u s studies have

d e m o n s t r a t e d that removal of b u r s a sufficiently early results in IgA d e f i c i e n c y in some of the c h i c k e n s Leslie and Martin, selective

(Kinkade and Cooper,

1974; Ewert and Eidson,

(Kinkade and Cooper,

1973; Martin and Leslie,

1973;

1977). This d e f i c i e n c y is either not

1973) or is transitory

(Martin and Leslie,

1973).

Invariably, b u r s e c t o m y a f t e r h a t c h i n g leaves a lage n u m b e r of c h i c k e n s with an intact IgA system, but when it is c o m b i n e d with thymectomy at one day of age, IgA synthesis is c o m p l e t e l y s u p p r e s s e d

(Perey and Bienenstock,

1973) whilst

IgG and IgM synthesis is not a p p r e c i a b l y affected. This procedure, however, also c o m p r o m i s e s the c e l l - m e d i a t e d r e s p o n s e which is c o n t r o l l e d by the thymus. K i n k a d e and C o o p e r

(1973) showed that cells c o m m i t t e d to IgA synthesis arise

from cells that f o r m e r l y made IgM by a genetic switchover m e c h a n i s m h a v i n g the

0165-2427/84/$03.00

© 1984 Elsevier Science Publishers B.V.

306 f o l l o w i n g sequence, Leslie

IgM ÷ I g G ÷ I g A . On the b a s i s of this model, M a r t i n and

(1974) a t t e m p t e d to suppress s p e c i f i c a l l y IgA f o r m a t i o n in c h i c k e n s

by embryonic a d m i n i s t r a t i o n of a n t i - ~ a n t i b o d y in c o n j u n c t i o n with n e o n a t a l b u r s e c t o m y and r e p e a t e d a d m i n i s t r a t i o n of anti-~ after hatching, but were unable to a c h i e v e total suppression.

It seems that as yet there is no simple and

s t r a i g h t f o r w a r d m e t h o d for raising s e l e c t i v e l y I g A - d e f i c i e n t chickens Leslie, p e r s o n a l c o m m u n i c a t i o n ) .

(G.A.

In the p r e s e n t i n v e s t i g a t i o n we r e - e x a m i n e d

the effect of v a r i o u s t r e a t m e n t s viz.,

i n o c u l a t i o n of anti-~, b u r s e c t o m y

c a r r i e d out at 6 h or 24 h after h a t c h i n g and a c o m b i n a t i o n of both with the aim of d e v e l o p i n g a p r o t o c o l for s e l e c t i v e l y s u p p r e s s i n g IgA synthesis in chickens. The efforts seem to have succeeded.

M A T E R I A L S AND M E T H O D S Chickens F e r t i l e W h i t e L e g h o r n eggs were used. W h e n required,

intravenous injections

of 17-day-old embryos were c a r r i e d out through a w i n d o w cut over a b l o o d vessel in the c h o r i o a l l a n t o i c membrane. The shell w i n d o w was sealed with c e l l o p h a n e tape. S u r g i c a l b u r s e c t o m y was p e r f o r m e d either within 6 or at 24 h of hatch.

R a b b i t IgG a n t i - c h i c k e n alpha c h a i n The r a b b i t a n t i - c h i c k e n IgA and anti-~ earlier by Goel et al.

(RACA) were the same as p r e p a r e d

(1980) and C h h a b r a and Goel

(1980). The R A C A was p r e c i p -

itated twice w i t h a m m o n i u m sulphate at 45% saturation and the p r e c i p i t a t e d i s s o l v e d in 0.i M p h o s p h a t e buffer, p H 7.5, and d i a l y s e d a g a i n s t the same buffer. The s a l t - p r e c i p i t a t e d anti-~ g l o b u l i n was c h r o m a t o g r a p h e d on a DEAES e p h a d e x G-50 column. T h e first p e a k e l u t e d from the column was c o n c e n t r a t e d by p r e s s u r e dialysis. For in ovo i n o c u l a t i o n it was m i l l i p o r e f i l t e r e d and stored at -20 ° . S u b s e q u e n t l y it was r e f e r r e d to as rabbit a n t i - c h i c k e n - ~ IgG

(RACA/IgG). It c o n t a i n e d

14 m g p r o t e i n per ml.

M o n i t o r i n g of IgA d e f i c i e n c y Gel d i f f u s i o n

(GD) and i m m u n o e l e c t r o p h o r e s i s

(IE) of serum a g a i n s t R A C A for

d e t e c t i n g IgA in c i r c u l a t i o n were c o n d u c t e d as d e s c r i b e d earlier 1980). Intestinal w a s h i n g s et al.

(Goel et al.,

(IW) w e r e c o l l e c t e d b y the m e t h o d of B i e n e n s t o c k

(1972). The IW were c e n t r i f u g e d at 12000 g for 60 min and l y o p h i l i s e d in

a v a c u u m drier

(high v a c u u m drier, Edwards). The l y o p h i l i s e d IW were reconsti-

tuted in 0.4 ml of 0.05 M veronal buffer, pH 8.6, a n d t e s t e d for the p r e s e n c e of IgA by GD and IE a g a i n s t RACA.

Indirect immunofluorescence

s t a i n i n g for I g A - c o n t a i n i n g cells

C h i c k e n intestine was c o l l e c t e d and w a s h e d with c h i l l e d p h o s p h a t e b u f f e r e d

307 saline

(PBS),

pH

7.4,

cut and snap frozen. according

and a p o r t i o n Cryostat

to the m e t h o d

each of duodenum,

sections

of Y a m a m o t o

et al.

incubation

with R A C A or R A C A / I g G

tions w e r e

s t a i n e d w i t h goat a n t i - r a b b i t

cyanate

labelled

Netherlands). 9.0)

antibody

The

and e x a m i n e d

Microscope

Experimental

Federal

as s u m m a r i s e d

after

in T a b l e

hatching

followed

i.p.

I, were

tested

of a n t i - ~

Laboratories,

in b u f f e r e d

The

glycerine

(Leitz U n i v e r s a l

in chickens,

(pH

Widefield

for the

(III)

in-ovo

injection

by b u r s e c t o m y

further

and 34 after hatching.

Chickens

which

on a l t e r n a t e

(II) b u r s e c t o m y

within

and a single

6 h and at

injection

of anti-ci on days

treatment

according

only for serum IgA and those a c c o r d i n g IgA as well as for I g A - c o n t a i n i n g

cells

in days

17th day of em-

III above but b u r s e c t o m y

injections

received

protocols

of IgA d e f i c i e n c y (i.p.)

of a n t i - ~ on the

(IV) as in

by three

secretory

induction

at 24 h of hatch

followed

I-III w e r e m o n i t o r e d

four d i f f e r e n t

intraperitoneally

on the day of hatching;

6 h of hatch,

serum and

by

the sec-

of Germany).

up to a p e r i o d of three weeks;

24 h after hatching;

anti-~

stained

washing,

fluorescein-isothio-

Immunological

and m o u n t e d

microscope

Republic

section was

After

immunoglobulin

(Nordic

of IgA s y n t h e s i s

(I) i n o c u l a t i o n

bryonation

Each

were cut and stained

design

For the s u p p r e s s i o n

chickens:

1:8

were washed,

with a f l u o r e s c e n c e

Orthoplan,

(1977).

at 37 ° for one hour.

diluted

sections

j e j u n u m and ileum were

of 5 U m t h i c k n e s s

of

within

3, I0

to p r o t o c o l s

to p r o t o c o l

IV for

in the intestinal

mucosa.

Immunoglobulin

levels

IgG and IgA levels diffusion paration

(RID)

in sera of c h i c k e n s

as d e s c r i b e d

of i ~ n u n o g l o b u l i n

given p r e v i o u s l y of d e t e c t i o n

(Chhabra

earlier

(Chhabra

standards and Goel,

by RID for both

were d e t e r m i n e d and Goel,

and s p e c i f i c i t y 1980;

Goel

by radial

1980,

immuno-

1981).

The p r e -

of their a n t i s e r a

et al.,

1980).

The

has been

lower

limit

IgG and IgA was 20 Dg/ml.

RESULTS Evaluation Since

of IgA s u p p r e s s i o n

the m a i n

for p r e p a r i n g

objective

cols of treatment,

ginning given

RACA.

in sera of 8 - d a y - o l d from one w e e k of age.

in T a b l e

II.

investigation

model,

as s u m m a r i s e d

IgA o n l y by GD test a g a i n s t appeared

of the

an I g A - d e f i c i e n t

in T a b l e Using

birds. Results

was to s t a n d a r d i s e

the chickens

subjected

I, w e r e m o n i t o r e d

this m e t h o d

Subsequently, obtained

a method

to various

proto-

initially

for serum

it was found that

IgA first

all m o n i t o r i n g

with d i f f e r e n t

was done be-

protocols

are

308

5 I

I

I

+

+

+

+

+

I

I

-t-

+

~ o 0

•~

4J

~

o

o" 0

0

r~

kt o

O

-

~

,~

,.~ 0

3

~ o •~ ~ ~

O 4J O

4J

-,-I ~

0

.~ 0

E 0

d I

i

.~

.,4

~

U I •,-i .IJ

U I .,4 .P

-~>,-i

,4-1 tM .,-t

c,I

O

"k

I

I

I

~

.iJ 0

.H 0

0

,.q

c~

~

~

II

II

II

II

-~

Z

H ~J ,.q

~

m

•tJ ¢1

~ ¢1 m

E

0 4J

U

~

÷

,

309

Table

II

Time

c o u r s e of s u p p r e s s i o n of IgA s y n t h e s i s

Treatment given protocol

Status of cNickens

Batch A Batch

D p D P

9/12

8/11

4/12

3/13

3/12

3/11

8/12

10/13

9/11 2/11

8/11 3/11

-

7/12 5/12

-

III

m

13/13

8/13

-

4/ii

3/11

P D P

0/13 13/14 1/14

5/13

IV

13/14 1/14

7/11 6/6 0/6

8/11 3/3 0/3

II

B

Not t e s t e d Numerator Denominator = Batch A = Batch B Note:

Protocol

Age of c h i c k e n s

in c h i c k e n

4

6

8

in weeks

i0

12

24

-

3/3 o/3

N u m b e r of b i r d s d e f i c i e n t (D)/Positive (P) for IgA N u m b e r of b i r d s t e s t e d B u r s e c t o m y w i t h i n 24 h of h a t c h i n g Bursectomy within 6 h of h a t c h i n g The IgA l e v e l s in t r e a t e d b i r d s u n d e r p r o t o c o l I were d e p r e s s e d but not s u p p r e s s e d .

I

In ovo a d m i n i s t r a t i o n of R A C A / I g G

f o l l o w e d by p o s t - h a t c h i n g

treatment with

R A C A up to three w e e k s of age f a i l e d to s u p p r e s s the s y n t h e s i s of IgA. The t r e a t m e n t did,

however,

d e l a y the a p p e a r a n c e of IgA;

serum of t r e a t e d c h i c k s even up to two weeks of age. the

r e t u r n e d to c o n t r o l

Protocol

Once the t r e a t m e n t was d i s c o n t i n u e d

50 Pg and

the IgA levels

levels.

II

B a t c h A.

This g r o u p c o m p r i s e d c h i c k e n s b u r s e c t o m i s e d at 24 h of hatching.

four w e e k s of age,

75% of the c h i c k s were

12 w e e k s of age, 23.3,

B a t c h B.

the p e r c e n t a g e

for serum IgA so that at 8,

of c h i c k e n s d e f i c i e n t

in IgA were

72.7,

10 and 33.3

respectively.

Bursectomy within

6 h of h a t c h r e s u l t e d in s u b s t a n t i a l

IgA s u p p r e s s i o n as c o m p a r e d w i t h b u r s e c t o m y at 24 h of hatch. case also,

At

f o u n d d e f i c i e n t for serum IgA. W i t h

the lapse of time m o r e b i r d s b e c a m e p o s i t i v e

the d e f i c i e n c y

i0 w e e k s of age, tively.

in the

level of IgA in

serum of t r e a t e d a n d c o n t r o l c h i c k s at three w e e k s of age was

106 ~Ig/ml, r e s p e c t i v e l y .

and

it was not d e t e c t e d The m e a n

81,

in some birds was t r a n s i t o r y .

i m p r o v e m e n t of

However,

in this

W h e n t e s t e d at 4, 6 and

72 a n d 58.8% of the c h i c k e n s were IgA d e f i c i e n t ,

respec-

810

Protocol

III

In this g r o u p b u r s e c t o m y t i o n of R A C A / I g G

age all the t r e a t e d g r e w older,

chickens

IgA a p p e a r e d

and 12 weeks of age, 36.5,

a n d 27.3,

initially even

at 24 h of h a t c h was c o m b i n e d

in ovo and a n o t h e r

b u t at I0 weeks

Thus

Protocol

of d e f i c i e n t treatment

for m a x i m a l

chicks

treated

6 h of h a t c h and four i n j e c t i o n s treatments

sis so t h a t at f o u r weeks of age was no r e v e r s i o n Thereafter, (SIgA)

and

IgA-containing

The l y o p h i l i s e d

To a c h i e v e intestinal

for a longer

cell

when

indicating

sensitive

was e x a m i n e d

stained

cells.

When

viewed

cryostat

at m a g n i f i c a t i o n s

lacked

their

the s u r v i v i n g

secretory

chickens

the l o n g e s t p e r i o d t e s t e d

a typical

IW f r o m eight birds the a b s e n c e

assessment

IgA b a n d

treated

re-

(Table

II)

to p r o t o -

of SIgA.

of IgA s y s t e m suppression,

by i n d i r e c t - i m m u n o f l u o r e s c e n c e

sections

in IE. No p r e c i -

according

from intestines

chicken

for I g A - c o n t a i n i n g

of normal

the I g A - c o n t a i n i n g

birds w e r e

cells w e r e

seen

although

the cell c o n t o u r

was n o t a l w a y s regular.

the l a m i n a p r o p r i a

of I g A - d e f i c i e n t

birds was l a c k i n g

in

cells.

sera c o l l e c t e d

from IgA-deficient

birds were also tested

of IgG and IgM by GD t e s t b u t o n l y the f o r m e r was quantified. quantitated

because

ing r e s u l t s

in n o r m a l

been r e p o r t e d

sera.

up to the 8th w e e k of

for m o n i t o r i n g

However,

of IgA synthe-

IgA in t h e i r

IW f r o m n o r m a l b i r d s g a v e a single p r e c i p i -

of 175 X or more,

in the c o r i u m of the villus, On the other hand,

status.

by b u r s e c -

0, 3, i0 and 34

suppression

of these c h i c k e n s sacrificed

R A C A in GD and p r o d u c e d

a more

mucosa

IgA-containing

were

u p to 24 weeks of age,

b a n d was o b s e r v e d

col IV were tested,

1974)

in IgA was

It b e c a m e a p p a r -

followed

of R A C A on days

13 out of 14 c h i c k e n s

and r e c o n s t i t u t e d

tation band against

The

to 61.5,

of IgA synthesis.

in ovo,

there was a m a r k e d

of IgA status

some c h i c k e n s

IgA d e f i c i e n t

pitation

6 h alone.

suppression

At 6, i0

III was e f f e c t i v e

of birds d e f i c i e n t

with RACA/IgG

W i t h these

IgA

birds was r e d u c e d

within

and p r o l o n g e d

post-hatch.

mained

birds.

along w i t h R A C A i n o c u l a t i o n

tomy within

age.

However,

deficient

of

IV

This group comprised

There

serum.

under protocol

with bursectomy

that early bursectomy

p e r i o d was n e c e s s a r y

as the chicks

of age the p e r c e n t a g e

lower than t h a t o b t a i n e d

ent t h e r e f o r e

IgA in their

lacked

the p e r c e n t a g e

injec-

At 4 w e e k s

in the serum of p r e v i o u s l y

respectively.

w i t h a single

of R A C A on the day of hatch.

it is a l r e a d y

to be d e c r e a s e d

or r e m a i n

well

or s u p r a - n o r m a l

unaffected

established

that bursectomy

levels of IgM,

whereas

(Perey a n d Bienenstock,

(Blythman

and White,

for the p r e s e n c e

Only

1977).

1973;

IgG was after hatch-

IgG levels Leslie

Therefore,

have

and Martin, chickens

311

producing

IgG could not be d e f i c i e n t

b o t h of these

immunoglohulins,

ful in s e l e c t i v e l y serum

IgG levels

controls

suppressing

were

(3.73 + 0.61

indicating

IgA s y n t h e s i s

of I g A - d e f i c i e n t

mg/ml)

2.25 + 0.32

in IgM. T h e birds

thereby

were found p o s i t i v e

that p r o t o c o l

in chickens.

birds at 4 and 7 weeks

significantly

lower

(p < 0.05)

IV was

However, of age

for

success-

the mean

(1.77 + 0.31

than those

and

of u n t r e a t e d

mg/ml).

and 3.32 + 0.44

DISCUSSION The r a t i o n a l e hatching, mice.

for the

inoculation

as in our first protocol,

Some

success

in s u p p r e s s i o n

achieved

in m i c e

(Manning,

However,

in our hands

this

o n l y delay the a p p e a r a n c e

1972;

of K i n k a d e This

two w e e k s

of age,

It became

remains

1973).

Bursectomy

may result

by v e r y h i g h levels al.,

1969;

previously

Kincade

et al.,

IgM,

IgA s u p p r e s s i o n

(Leslie and M a r t i n

investigation

IgA in some c h i c k e n s Even

inoculation

hatch

White

(1977)

inoculation The

0,3,

within

i0 and 34 p o s t - h a t c h .

inoculated

within

intravenously

1973;

results

Martin

IgG and a b s e n t 1977).

Ewert and E i d s o n

its a p p e a r a n c e

In c h i c k e n s

IgA

(Cooper

Delayed

1977).

and hatch,

characterized et

bursectomy

of c h i c k e n s

to escape

During

the

the s y n t h e s i s

in the m a j o r i t y

17th day of e m b r y o n a t i o n

bursectomised

of others.

within

6 h of

but a larger n u m b e r

of c h i c k e n s

was

of B l y t h m a n

and

6 h of hatch does not a p p r e c i a b l y early b u r s e c t o m y

alter

with RACA

(IV). was i n o c u l a t i o n and i n o c u l a t i o n

of the l i m i t a t i o n

embryos,

with RACA/IgG

in

w i t h R A C A on days

on v o l u m e

it was e s s e n t i a l

of

and on the day of

w i t h the o b s e r v a t i o n

of t r e a t m e n t s

Because

in suppres-

and Leslie,

16 days of e m b r y o n a t i o n

and White,

6 h of h a t c h i n g

into

different

the source of

or dysgamlmaglobulinemia

led us to combine

protocol

combination

bursectomy

bursectomy

1974;

similar

of anti- H

significantly

a large n u m b e r

combined

of chickens,

in our final

successful

embryos,

This,

that b u r s e c t o m y

IgG and IgM levels

between

this reversion.

was s o m e w h a t

IgA deficient.

injections

at 24 h of h a t c h did suppress

on the

and could

is in a g r e e m e n t

of both IgM and IgG until

allows

but o n l y d e l a y e d

of R A C A / I g G

the p a t t e r n

rendered

hand,

bursectomy

could n o t p r e v e n t

hatch,

surgical

and Cooper,

Blythman

on the o t h e r

1973).

will not be suppressed.

low or absent

after hatching,

present

that

stage,

1973;

with multiple levels

results

This

with

has been

et al.,

that as long as the bursa,

agammaglubulinemia,

of serum

Murgita

the levels were not

(Kinkade

at an e a r l i e r

in c o m p l e t e

(1973)

IgA s y n t h e s i s

IgA d e v e l o p m e n t

1973;

for some time.

in subnormal

apparent

intact,

It has b e e n r e p o r t e d sion of serum

et al.,

of w o r k e r s

with this a p p r o a c h

did not yield d e s i r a b l e

et al.

b u t by four weeks

from the controls. B lymphocytes,

Lawton

resulted

in ovo and after

was b a s e d on the e x p e r i e n c e

treatment

without

antibody

of IgA s y n t h e s i s

of IgA in serum

w i t h the e x p e r i e n c e bursectomy.

of specific

which

can be

that m a x i m u m

anti-d

the

312 antibody should be given in least volume; RACA/IgG was used for this purpose. As there was no such limitation on the volume which could be given to chicks, only RACA was used. In case it was necessary, RACA could be inoculated in larger doses or more frequently. However, if available in sufficient quantity, RACA/IgG can very well be used for both purposes. This treatment resulted in complete suppression of the IgA system in chickens. The birds continued to produce both IgG and IgM, though the IgG levels of treated birds were lower than those of the controls. Martin and Leslie (1974) used a similar approach to suppress specifically IgA formation, but were unable to achieve total suppression. They inoculated DeKalb 161 White Leghorn chickens, with 4.5 mg of anti-~

on day 18 of embryonation, performed bursectomy within 18 h of hatching

and gave three more injections of a n t i - ~ on days 0, 4, and 8 after hatch. This treatment delayed the appearance of detectable IgA until day 21, and subsequent concentrations of IgA were about 25 and 10% of the control values at 49 and 71 days of age, respectively. It is rather difficult to explain the discrepancy between the two studies. The two most probable reasons would seem to be earlier bursectomy and inoculation of a n t i - ~ for a longer period in the present study. Martin and Leslie(1974) thought that their inability to achieve total IgA suppression was perhaps due to precursor cells not yet expressing IgA surface determinants which had migrated from the bursa before bursectomy. Probably this migration takes place quite early after hatching, and was prevented by early bursectomy in the present study. It can be concluded that early bursectomy, as close as possible to hatching followed by prolonged inoculation of anti-d after hatch

(about a month), in the particular strain of WLH chickens used in the

present study, resulted in total and selective suppression of IgA synthesis. The availability of this IgA-deficient chicken model will help in investigating the role of SIgA in local immunity in chickens in particular, and in prevention of uptake of antigens from mucosal surfaces in general.

ACKNOWLEDGEMENTS Thanks are given to Dr. S. Prasad for providing necessary facilities and to Dr. B.K. Sinha for quantitation of IgG. The techmical assistance of Mr. R.S. Yadav is acknowledged.

REFERENCES Bienenstock, J., Perey, D.Y.E., Gauldie, J. and Underdown, B.J., 1972. Chicken immunoglobulin resembling gamma A. J. Immunol., 109: 403-406. Blythman, H.E. and White, R.G., 1977. Effect of bursectomy on germinal centre and in~nunoglobulin production in chickens. Immunology, 33: 671-677. Chhabra, P.C. and Goel, M.C., 1980. Normal profile of immunoglobulins in sera and tracheal washings of chicken. Res. Vet. Sci., 29: 148-152. Chhabra, P.C. and Goel, M.C., 1981. Immunological response of chickens to Mycoplasma gallisepticum infection. Avian Dis., 25: 279-293.

313

Cooper, M.D., Cain, W.A., Van Alten, P.J. and Good, R.A., 1969. Development and function of the immunoglobulin producing system, i. Effect of bursectomy at different stages of development on germinal centres, plasma ceils, immunoglobulins and antibody production. Int. Arch. Allergy, 35: 242. Ewert, D.L. and Eidson, C.S., i977. Effect of bursectomy and depletion of immunoglobulin A on antibody production and resistance to respiratory challenge after local or systemic vaccination of chickens with Newcastle Disease Virus. Infect. Immun., 18: 146-150. Goel, M.C., Chhabra, P.C. and Sharma, V.K., 1980. Preparation of chicken immunoglobulins and their specific antisera. Indian J. Anim. Sci., 50: 983-988. Kincade, P.W. and Cooper, M.D., 1973. Immunoglobulin A: site and sequence of expression in developing chicks. Science, 179: 398-400. Kincade, P.W., Self, K.S. and Cooper, M.D., 1973. Survival and function of bursa-derived cells in bursectomised chickens. Cell. Immunol., 9: 93-102. Lawton, A.R., Asofsky, R.M., Davie, J.M. and Hylton, M.B., 1973. Suppression of immunoglobulin synthesis in mice: age dependence of anti-~ suppression and effect of anti-y1, anti-y2 and anti-~. Fed. Proc., 32: 1012. Leslie, G.A. and Martin, L.N., 1974. The secretory immunoglobulin system of fowl. IV. Serum and salivary immunoglobulins in normal, agammaglobulinemic and dysgammaglobulinemic chickens. Int. Arch. Allergy, 46: 834-841. Manning, D.D., 1972. Induction of temporary IgA deficiency in mice injected with heterologous anti-immunoglobulin heavy chain antisera. J. Immunol., 109: 1152-1155. Martin, L.N. and Leslie, G.A., 1973. Ontogeny of IgA, IgY and IgM in normal and neonatally bursectomized chickens. Proc. Soc. exp. Biol. Med., 143: 241-243. Martin, L.N. and Leslie, G.A., 1974. IgM-forming cells as the immediate precursor of IgA-producing cells during ontogeny of the immunoglobulin-producing system of the chicken. J. Immunol., 113: 120-123. Murgita, R.A., Mattioli, C.A. and Tomasi, T.B., Jr., 1973. Production of a runting syndrome and selective yA deficiency in mice by the administration of anti-heavy chain antisera. J. Exp. Med., 138: 209-228. Perey, D.Y.E. and Bienenstock, J., 1973. Effects of bursectomy and thymectomy on ontogeny of fowl IgA, IgG and IgM. J. Immunol., iii: 633-637. Yamamoto, H., Watanabe, H. and Mikami, T., 1977. Distribution of immunoglobulin and secretory component containing cells in chickens. Am.J.Vet.Res., 38: 1227-1230.