The Effect of Aging on Immune Competence in the Chicken:
Antibody-Mediated Immunity
FRED McCORKLE and BRUCE GLICK
Poultry Science Department,
Mississippi State University,
Mississippi State, Missouri 39762
(Received for publication March 12, 1979)
INTRODUCTION
We have demonstrated that cell-mediated
immunity in the chicken is influenced by aging
and sex (McCorkle et al., 1979). In this manuscript we describe an age related decline in
antibody response to a thymic dependent
antigen, sheep red blood cells, but not to a
thymic independent antigen, Brucella abortus
(Toivanen et al., 1972). Also, the plaque
forming cell response of female spleens was
influenced less by age than were spleens from
males.
heart puncture 7 days after injection.
Plaque Forming Cell (PFC) Assay. An
agarless monolayer hemolytic plaque technique
(Cunningham and Szenberg, 1968) as modified
for the chicken system (Mueller et al., 1971)
was employed. Seven days following an iv
injection of one ml of a 5% SRBC suspension,
spleens were removed, cells expressed, and
antibody producing cells counted by the plaque
forming assay. The results were expressed as the
number of PFC/10
spleen lymphocytes.
Between 6 and 10 males and females were used
at each age in the PFC assay.
MATERIALS AND METHODS
Hemagglutinin Assay. The SRBC antibody
titers were determined by the microtiter
procedure of Wegman and Smithies (1966).
Each bird's serum sample was titrated individually. The titers were expressed as the log base 2
of the reciprocal of the highest dilutions giving
a visible agglutination.
Animals. A closed flock of New Hampshire
chickens (an American breed) developed by L.
J. Dreesen on the poultry research farm at
Mississippi State University was used. The
chickens were kept in Oakes batteries until 6
weeks of age when they were moved to floor
pens with wood shavings. Food and water were
available ad libitum.
Antigens. The antigens were sheep red blood
cells (SRBC), obtained from the Department of
Animal Science and Brucella abortus (BA) (Difco Laboratories, Detroit, MI). The SRBC were
stored in Alsever's solution and were washed 3
times in physiological saline (.9% NaCl) before
intravenous (iv) injection.
Immunizations.
All immunizations were
made iv either with 1.0 ml of a 5% suspension
of SRBC or with .1 ml of undiluted BA. Birds
at each age were injected one time and bled by
Brucella abortus Agglutinin Assay. The
procedure for determining BA antibody titers
was that of Steven Lerman, Department of
Pathology, New York University Medical
School. Each well of a microtiter plate (ji plate)
was charged with .1 ml of diluent (75 ml of pH
7.2 phosphate buffered saline and .1 ml of 35%
BSA). The initial well then received .1 ml serum
which was serially diluted (1:2, 1:4, . . .)
followed by the addition of .1 ml of a 1:10
dilution of BA. Control wells with diluent plus
serum and diluent plus antigen were included.
The microtiter plates were incubated at room
669
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ABSTRACT The plaque forming cell (PFC) response of males exhibited comparable peaks at 1
and 12 months of age and significantly lower values at 3, 6, 18, and 24 months of age. Female PFC
responses peaked at 1 month, declined significantly by 3 months, and returned to a high level by 6
months. PFC response of females, unlike that of males, did not decline subsequent to 12 months of
age. There were no significant sex differences in antibody titers to the thymic dependent antigen,
sheep red blood cells (SRBC), or the thymic independent antigen, Brucella abortus (BA). Peak
hemagglutinin values of SRBC occurred between 3 and 6 months of age and declined thereafter.
However, titers to BA peaked at 3 months and remained high thereafter.
1980 Poultry Science 59:669-672
McGORKLE AND GLICK
670
TABLE 1. The plaque forming cells/106 spleen lymphocytes with age of male and female chickens
Month
No.
birds
Males
1
3
6
12
18
24
7
8
8
10
7
9
12,038 a ±
5,531 c ±
6,773 b ±
9,403 a b ±
5,926 c ±
4,322 c ±
907
848
848
758
907
799
No.
birds
Females
6
8
10
9
7
10
12,866 a
6,668 c
9,910 a b
8,168 b
9,371 a b
10,123 a b
±979
+ 848
± 758
± 799
±907
±758
a,b,c Means with different superscripts within a column are significantly different (P < .01).
RESULTS AND DISCUSSION
The number of PFC in males was significantly higher at 1 month than at 3 and 6 months of
age (Table 1). At 12 months of age the male
PFC response was not significantly different
from 1 month values but declined significantly
by 18 and 24 months of age. In females the
number of PFC was significantly higher at 1
than at 3 months of age, and then increased by
18 and 24 months to levels that were not
significantly different from 1 month old
females.
The antibody titers to SRBC and BA were
significantly influenced by age but not sex
(Tables 2 and 3). The total SRBC agglutinin
titers increased significantly from 1 to 6 months
of age and then declined by 12 and 24 months
to levels that were not significantly different
from 1-month-old chickens (Table 2). While the
IgG antibody was not influenced by age, the
IgM antibody showed the same pattern as the
total SRBC agglutinin. The total antibody titer
and IgM antibody for BA increased significantly
between 1 and 3 months and remained high
throughout the ensuing 32 months (Table 3).
The decline in antibody production to a
T-cell dependent antigen, SRBC, in the chicken
followed the same pattern described in other
vertebrates (Makinodan et al, 1976; Segre and
Segre, 1976a, b ; and Albright and Makinodan,
1977). The difference between the PFC response and SRBC titers of females might reflect
TABLE 2. The total, ME-resistant (IgG), and ME-sensitive (IgM) antibody titers (Log2)
to sheep-red-blood cells (SRBC) with age in chickens
1
3
6
12
24
month
month
month
month
month
No.
birds
Total
15
19
13
14
19
6.83 c **
8.59 a b
9.57 a
8.85abc
7.13bc
± .3
± .4
±.6
± .5
± .4
ME-resistant
IgG
ME-sensitive
IgM
3.73 a *
4.45 a
5.15 a
4.95 a
3.74 a
3.10 b **
4.50 a
4.42 a
3.90 a b
3.38 b
±
±
±
±
±
.22
.26
.34
.34
.26
± .31
± .37
± .48
± .47
± .37
' ' Means ± SE with different superscripts in the same column are significantly different (*P < .05; **P <
.01).
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temperature for 24 hours in moist chambers.
Each bird's serum sample was titrated individually. The titers were expressed as the log base 2
of the reciprocal of the highest dilutions giving
a visible agglutination.
2-Mercaptoethanol (ME) Sensitive and Resistant Antibody. Using the techniques of Delhanty and Solomon (1966), equal volumes of
serum and .2M ME in phosphate buffered saline
(pH 7.5) were mixed and incubated at 37C for
30 min. Agglutination tests were conducted for
SRBC serum and BA serum. The titers were
recorded as ME-resistant antibody (IgG) and the
differences from the total titer were recorded
as ME sensitive antibody (IgM).
Statistics. The data were analyzed by analysis
of variance (Steele and Torrie, 1960) and all
means were separated for significance by
Student-Newman-Kuels' test (Newman, 1939).
RESEARCH NOTE
671
TABLE 3. The total, ME-resistant (IgG), and MEsensitive (IgM) antibody titers (Log2) to
Brucella abortus (BA) with age in chickens
No.
birds
Total
1
3
6
9
12
18
24
32
20
10
20
21
21
18
13
16
7.7C**
12.5 a
12.2 a
ll.O* 15
12.0 a
10.3 b
12.1 a
12.l a
a
±.4
+ .5
± .4
+.4
± .3
±.4
+.5
± .6
ME-resistant
IgG
ME-sensitive
IgM
2.0 C **±.3
2 . 6 b c + .5
4.4 a
± .3
4 . 1 a b +.3
ab
4.3
+ .3
3.7abc±.3
2.7abc±.4
3 . 6 a b c ± .6
5.5 d ** ± .3
9.9*
± .5
7 . 8 b c ± .4
bcd
6.9
± .4
7 . 8 b c + .4
6.7cd ±.4
9 . 4 a b +.5
8 . 6 a b c ± .5
' ' c ' Means ± SE in the same column with different superscripts are significantly different (*P < .05; **P <
.01).
t h e greater sensitivity of t h e P F assay t o d e t e c t
levels of IgM. T h e ability of female, b u t n o t
male, spleens t o m o u n t a PFC response at 18
a n d 2 4 m o n t h s of age equal t o t h e early p e a k
splenic response suggests t h a t t h e B-cells of
aged females m a y b e m o r e efficient IgM producers t h a n are t h e B-cells of aged males.
Since t h e BA a n t i b o d y titers remained high
after peaking, o n e might assume t h a t B-cell
function declines less with age t h a n does t h e
function of T-helper cells. These d a t a are
c o n t r a r y t o Callard et al. ( 1 9 7 7 ) w h o d e m o n strated a loss of B-cell function w i t h age in
C B A / H mice i m m u n i z e d t o t h e t h y m i c - i n d e p e n d e n t antigen p n e u m o c o c c a l polysaccharide
t y p e III, b u t t h e y agree with o t h e r research
with mice which indicate n o change in B-cell
response t o a T-cell i n d e p e n d e n t antigen (Price
and M a k i n o d a n , 1 9 7 2 ; Makinodan and Adler,
1 9 7 5 ; and Haferkamp et al, 1 9 6 6 ) .
ACKNOWLEDGMENTS
We t h a n k Karen A n d e r s o n for t y p i n g t h e
manuscript. This is j o u r n a l article n u m b e r 4 1 9 0
for t h e Mississippi Agricultural and F o r e s t r y
E x p e r i m e n t Station.
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[