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THE EFFECT OF NEGHATAL THYMECTOMY
(3&3 Xci-RRAEEATEQN OF THE
CHECKEN GK THE PERCENT OF

ELOQ-D TYMPHGQXTTES

Tim-sh: {on time Emma a? M“ 5’.
MECREGAN STKFE UHETERSET

Inguna Siiavs Fauser
‘5 9-69

 

LIBRAR y 1'?
Michigan Sm: ;

1'" University 3
l ’ .

THESIS

_ 1‘34”...“ ”w"!

 

 

 

THE EFFECT OF NEONATAL THYMECTOMY AND
X-IRRADIATION OF THE CHICKEN ON THE

PERCENT OF BLOOD LYMPHOCYTES

BY

Inguna Silavs Fauser

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1969

ACKNOWLEDGMENTS

The author wishes to express her sincere appreciation
to Dr. Virginia H. Mallmann for her continued guidance and
assistance. Sincere appreciation is also expressed to my
colleague Dr. Dibakar Panigrahi with whom thymectomies were
jointly performed as part of our integrated research project.
Appreciation is also expressed to Dr. John L. Gill for
consultation regarding the statistical analyses employed.

To.Mrs. Betty Leiby for her clerical and laboratory
assistance wherever needed, a special thank-you is extended.

I am particularly indebted to my parents and aunt,
Dr. Anna Silaus for their continued interest in my pursuit

of education and to my husband for his daily encouragement.

*****

I wish to acknowledge the financial support of the
American Thoracic Society and the National Tuberculosis
Association, the Agricultural Experiment Station, and the

Department of Microbiology and Public Health.

*****

ii

TABLE OF

ACKNOWLEDGMENTS . . . . .
LIST OF TABLES . . . . . .
LIST OF FIGURES . . . . .
INTRODUCTION . . . . . . .
LITERATURE REVIEW . . . .
MATERIALS AND METHODS . .

Chickens . . . . . .

Surgical Procedures

X-Irradiation . . .
Differential Counts

Total Leukocyte Counts

Necropsy . . . . . .
StatisticaltAnalysis
Infection . . . . .
RESULTS . . . . . . . . .
DISCUSSION . . . . . . . .
SUMMARY . . . . . . . . .
LITERATURE CITED . . . . .

APPENDIX . . . . . . . . .

CONTENTS

Page

. . . . . . . . . . . V
. . . . . . . . . . . . . l
. . . . . . . . . . . . . 3
O O O O O O O O O O C O 9
. . . . . . . . . . . . . 9
. . . . . . . . . . . . . 9
O O O O O O O O O 0 O O O 9

iii

10.

LIST OF TABLES

The number of lymphocytes/100 leukocytes
14 days after hatching . . . . . . . . . . .

The number of lymphocytes/100 leukocytes
38 days after hatching . . . . . . . . . . .

Aging effect: number of lymphocytes/300
leukocytes in normal, sham operated, and
thymectomized chickens at 14 and 38 days
post hatching . . . . . . . . . . . . . . . .

Two-way analyses of the variance of lympho-
cytes/100 leukocytes 38 days after hatching--
three replicate scores . . . . . . . . . . .

Leukocytes/mm3 in eXperimental chickens l4
and 38 days after hatching . . . . . . . . .

The analyses of leukocytes/mm3 at 14 and 38
days after hatching and differences between
14 and 38 days . . . . . . . . . . . . . . .

The number of lymphocytes/100 leukocytes in
experimental chickens 5% months of age . . .

The analyses of the number of lymphocytes/100
leukocytes in eXperimental chickens 5% months
of age (four—way analysis of variance, three

scores) . . . . . . . . . . . . . . . . . . .

Four-way analysis of variance--three scores
combined from Table 8 . . . . . . . . . . .

The significance of treatments in the per—
centage of lymphocytes due to thymectomy,
X—irradiation, infection, and tuberculin
testing . . . . . . . . . . . . . . . . . . .

iv

Page

17

18

19

2O

22

23

26

27

28

33

LIST OF FIGURES

Figure Page

1. Percent mortality of 3 day old thymec-
tomized and normal chickens following
X-irradiation . o o o o o o o o o o o o o o o 15

2. Percent mortality of 3 day old thymec—
tomized and normal chickens following
800 R X-irradiation . . . . . . . . . . . . . l6

3. .Continuity of thymus cells with cells of
the thyroid gland (Hematoxylin and Eosin
stain) . . . . . . . . . . . . . . . . . . . . 24

4. Representation of the three-way interaction
between cross classified thymectomy,
X-irradiation, and infection . . . . . . . . . 29

5. Representation of the two-way interaction
between cross classified thymectomy and
x-irradiation I O O O O O O O O O O O O O O C 31-

6. Representation of the two-way interaction
between cross classified infection and
tuberculin testing . . . . . . . . . . . . . . 32

INTRODUCTION

The chicken is a model in which the immunologic
aspects of immediate hypersensitivity and delayed hypersen-
sitivity can be studied singly. Removal of the bursa of
Fabricius, one of the central lymphoid organs, prevents the
development of the chicken's capacity to respond to primary
antigenic stimulation by production of humoral antibodies.
Removal of the thymus, the other central lymphoid organ,
prevents the capacity to develop delayed hypersensitivity
of the tuberculin type.

The following is a description of the techniques
employed to thymectomize one day old chickens. The LD50/35
(lethal dose to 50 percent of the chickens 35 days after
treatment) for three day old chickens was determined to
investigate if there is a need to follow thymectomy with
X—irradiation.

Because the lymphocyte is the cell type postulated
to be under thymic influence, differential blood counts were
performed to determine the effect of X-irradiation and/or
thymectomy on the percent of lymphocytes in the peripheral
blood. It was postulated that a prediction could be made by
the percent of lymphocytes as to the effectiveness of

thymectomy prior to necropsy.

Total white blood cell counts were performed to
demonstrate the effect of X—irradiation in lowering the
total number of leukocytes/mm3.

The effect that an infection with Mycobacterium
avium and tuberculin testing had on the percent of lympho-
cytes in chickens belonging to the following four treatment
groups: thymectomized, thymectomized—X-irradiated, normal,

and X—irradiated was determined.

LITERATURE REVIEW

In man and many animals, the thymus is primarily
responsible for the maturation of humoral and cellular immu-
nity. The study toward an understanding of the immune mech-
anism has received great impetus with the discovery that
morphologically compartmentalized and separable immune sys—
tems exist in chickens. The report by Glick in 1956 as
summarized in 1964 that the bursa of Fabricius functioned in
the development of humoral immunity was an indication of the
morphologic separability of the central lymphoid tissue of
the chicken. The bursectomized chicken cannot respond to

t al., 1966). That

primary antigenic stimulation (Jankovic
the bursa of Fabricius and the thymus are two separable
immunologic systems, functionally as well as morphologically,

t EL-: 1962 and confirmed eXperi-

was postulated by Warner

mentally by Cooper t al. in 1966.

In the chicken the development of the cell system,
which includes the plasma cells and the large lymphocytes in
the red pulp of the spleen, and Peyer's patches, is largely

t go I 19667

bursa-dependent (COOper _£.§1,, 1965; Cooper
Woods _§.gl., 1965). Relatively few bursa-dependent lympho-
cytes are found in the peripheral circulation (Lucas at al.,

1961). The thymus is responsible for the development of the

small lymphocytes of the circulation, either indirectly or
directly. A humoral factor of thymic origin may affect
cells in the peripheral lymphoid tissue or thymocytes
(lymphocytes of the thymus) may migrate to the peripheral
lymphoid tissue (Larsson, 1966; Nossal, 1964; Toro, 1967;
Owen _£__1., 1969). The small lymphocytes are believed to
be the mediators of delayed hypersensitivity, homograft
rejection, and possibly for the graft versus host reaction.
The small, thymus dependent lymphocytes found in the spleen
are thought to be confined to the white pulp in the peri-
arteriole zone. The white pulp develops shortly after
hatching; the red pulp develops 4—5 weeks post hatching
(COOper gt _l., 1965).

Functionally, the two systems are not completely
independent. The thymus-dependent system may be necessary
for the recognition of a substance as foreign which precedes
the inititation of an immunologically specific response by
either the thymus or bursa dependent systems (Peterson,
1965).

The thymus in the Chicken is reported to be the
first lymphoid organ to function in the embryo. Lymphocyto-
poiesis begins sooner in the thymus than in any other lym-
phoid organ. Thymic lymphocytes are well develOped prior
to hatching, but bursal lymphocytes are not (Peterson gt al.,

1965).

COOper et_§1, (1966) recommended x—irradiation
together with thymectomy in the newly hatched period to
establish an experimental model in which the thymus depen-
dent system is nonfunctional. The purpose of X—irradiation
after thymectomy was to destroy any thymus dependent periph-
eral lymphoid tissue present at hatching. The use of X—
irradiation is a two-fold disadvantage. First, an LDSO is
used to insure sufficient irradiation of the surviving
chickens and therefore, half of all X-irradiated chickens
are lost due to X-irradiation. The cost is slight but
thymectomies are tedious and delicate. The second disadvan—
tage is that another variable in the experimental model is
introduced.

The assumption under which X—irradiation is used is
the following: Lymphoid and myeloid tissues are most easily
damaged so that a dose of radiation can be found which
destroys every cell in these tissues without irreparable
damage to the rest of the body (Dresser _Eng1., 1959).
Stearner gt 21. (1951) reported that the amount of tissue
destruction by X—irradiation is dependent on dose as well
as upon the length of time during which the dose is admin-
istered. The manner in which X-irradiation destroys cells
is not known (Stearner _£_§1,, 1956).

There are several reports that total body X-irradia-
tion of the chicken destroys lymphocytes (Lucas t 1., 1957;

t 31., 1948; COOper t al., 1966). The dose and age

Murray

at which X-irradiation was administered differed in all the
reports and the results varied with the age and dose.

The rapid destruction of lymphocytes in the periph-
eral blood, internal tissues and organs by total body X-
irradiation in the 3 week old chicken has been described
most extensively by Murray §£_§1,, 1948. The lymphocytes
throughout the body were severely affected by X-irradiation.
A reduction of 50 percent in the total white blood cell
count occurred within the first hour after X—irradiation,
and was due to the sudden destruction of lymphocytes. The
bone marrow was among the first tissues to show extensive
damage. Destruction of almost all lymphocytes of the cortex
and medulla of the thymus was followed by regeneration of
lymphocytes, but never more than a third of normal. Damage
to the liver included reduction of lymphatic areas followed
by partial restoration. Many lymphocytes of the lymphatic
tissue of the lamina propria were destroyed, but damage to
the lymphatic tissues was not as extensive as in the spleen
or bone marrow. It was emphasized that in 3 week old chick—
ens the small lymphocytes are more susceptible to damage by
total body X-irradiation than large lymphocytes, although
destruction occurs to both.

A reduction in the percent of lymphocytes in the
peripheral blood of thymectomized chickens has been reported
by several workers (Warner _t._1., 1964; Jankovic et_gl,
1964; Isakovic gg_gl,, 1964; Jaffee, 1966). None of these

workers made a comparison of the decrease in lymphocytes in

thymectomized and in thymectomized X—irradiated chickens.
Cooper _£H_1. (1966) reported a decrease in the numbers of
small lymphocytes in thymectomized X-irradiated chickens but
in their earlier work had found no reduction in thymectomized
chickens. Whether thymectomy only causes a significant
depletion in the percent of lymphocytes in the peripheral
circulation or whether thymectomy must be followed by X-
irradiation should be resolved. Because an LD50 irradiation
dose is used, one half of the chickens die. Omission of the
irradiation would reduce the number of chickens to be thy-
mectomized necessary to provide for a statistically signif-
icant number to be used in subsequent studies (Pearson g£_§l,,
1951). Besides this practical consideration, the additional
variability in results due to the added experimental condi-
tion of X—irradiation would be avoided.

Determination of the role of the lymphocyte in the
delayed hypersensitivity reaction has potential diagnostic
importance in the identification of antigens to which an
animal or individual is hypersensitive (Benezra $5.31., 1967).
The lymphocyte has been postulated as being the cell type
either mediating or effecting the delayed hypersensitivity
reaction (Mills, 1966; Kay §£_§1,, 1963). McFarland §£_§1,
(1966) have demonstrated that in a mixed leukocyte reaction,
the lymphocytes interact with macrophages, cell debri and
lymphoblasts. The reaction is by the uropod, suggesting that

stimulatory material may be acquired through the appendage.

In_vitro studies to determine the specificity of the delayed

immune response have been reported (George t 1., 1962;

Thor, 1967; David t 1., 1964). In_vitro, lymphocytes from

 

individuals hypersensitive to tuberculin increase in blast
formation when cultured with Purified Protein Derivative
(PPD) (Gell, 1967). Migration in capillary tubes of peri-
toneal exudate cells, consisting of macrophages and lympho—
cytes, from guinea pigs with delayed hypersensitivity to PPD,
ovalbumin and diphtheria toxoid is markedly inhibited only
by the antigen to which the animal had been sensitized
(David, 1964). Lymphocytes from sensitive guinea pigs
release a factor, the migration inhibition factor (M.I.F.)
which inhibits the migration of monocytes in_yi§£g_(Bloom
._£.gl., 1966). This factor inhibits the migration of mono—
cytes from sensitive or normal guinea pigs, the latter

t al.,

acting as an in vitro passive sensitization (Bloom
1966). If in fact the migration inhibition phenomenon of
ig_vitro studies is a manifestation of the cellular hyper—
sensitivity residing in the sensitive animal, the peritoneal
exudate cells from sensitized thymectomized chickens would
not be capable of displaying migration inhibition when
exposed to the antigen used in sensitizing. The chicken
model provides the opportunity to study whether the origin
of the capacity to develop delayed hypersensitivity as
detected in skin testing and by ig_vitro studies depends on

a continued thymic function.

MATERIALS AND METHODS

Chickens

Fertile eggs from single comb white Leghorns were
incubated at dry bulb 99°-100° F, wet bulb 850-860 F in an
International incubator (automatic rotation, controlled

humidity).

Surgical Procedures

Thymectomy of chickens was performed one day after
hatching. After anesthetization by 0.05-0.08 cc Combuthal
(R)* administered intraperitoneally, a dorsal incision
approximately 4—6 cm long was made on the neck to expose the
lobes of the thymus. Each lobe and the surrounding connec—
tive tissue and fat deposits were removed by blunt dissec-
tion. Sham thymectomies were performed in the same manner,
removing only fat deposits and connective tissue. The
incisions were closed with clamps. Aseptic technique was

used and no antibiotics were administered.

X-Irradiation
Three days after hatching, irradiation was adminis-

tered with the General Electric Maxitron 300 X-ray machine.

 

*
Diamond Laboratories, Inc., Des Moines, Iowa.

10

The conditions of irradiation were: 220 PKV, 20 ma with
0.25 mm cu + 1.0 mm Al. added (with hvl of approximately
0.25 mm cu) at a dose rate of 6.4 R/min. A total dose of
800 R was given in 125 min. in air. The target distance was

31 inches square. Under these conditions the LD was

50/35
calculated to be 800 R. To place the chickens individually
during irradiation, four cardboard boxes were constructed to
hold 100 chickens, 25 chickens per box, one chicken per com-
partment. The four boxes were arranged in‘a square and
rotated during irradiation in relation to each other as well
as about their own axis to insure an even distribution of
exposure to X-ray. In so doing, the calculated variation in
the dose each chicken received was less than 2 percent
(Mostosky, 1966).

In the experiments conducted to determine the LD50/35
the conditions of irradiation were altered in the amount of

total dose administered. The doses used were 650 R, 750 R,

and 850 R. The above dosage was calculated from the results.

Differential Counts

Samples of blood were collected by puncture of the
marginal wing vein. All samples were collected in the
morning. Blood films were stained with Wright's stain.

At least three differential counts per sample per

t al., 1961). All slides were

chicken were made (Lucas
scanned from right to left, left to right, and at several

levels to diminish errors due to different distributions of

11

the cells on the slide because of size and/or density. The
leukocytes were classified as lymphocytes, monocytes, hetero—
phils, basophils, or eosinophils (Lucas e£_§l,, 1961). If
the number of leukocytes on two slides was less than 300, as
in the slides from irradiated chickens at 14 days post hatch—

ing, no differential counts could be recorded.

Total Leukocyte Counts*

Total leukocyte counts were made of the chickens at
14 and 38 days of age to determine the effect of irradiation
on the total white blood cell counts. A free flowing drop
of blood from the wing vein was drawn to the 0.5 mark on a
standard white blood cell diluting pipette and diluted imme—
diately to the 11 mark with Reese-Ecker stain as a diluent.
Each pipette was shaken for a minimum of 30 seconds, after
which a sample was transferred to a hemocytometer and total
white blood cell counts made. The number of white blood

cells per cubic mm was calculated.

Necropsy

After completion of all studies, the chickens were
necrOpsied. The thymectomized chickens were examined
grossly from the cranium to the thyroid gland for remnants
of thymic tissue. Serial sections of the thyroid gland were

examined microscopically for remnants of lymphoid tissue.

 

*These determinations were made cooperatively with a
fellow graduate student, Hugh T. Fauser.

12

When thymectomies were considered complete by gross examina-
tion, but remnants of lymphoid tissue were found on micro-
scopic examination of the thyroid gland or fat deposits in
the vicinity of the thymus gland locations, the results
Obtained from such chickens were included in the thymecto-
mized group in the studies of the blood after a preliminary
examination of the results indicated that somewhat less than

complete removal was effective.

Statistiggligpalysis

All data was analyzed by the basic analysis of vari-
ance technique for cross classified data. Where supplemen-
tary testing was indicated orthogonal contrasts (Li, 1964)
were used. Because the number of variables under study
either exceeded 2, or analysis was further complicated by
unequal sample sizes and sub-sampling (Ruble gt al., 1968),
the MSU computer was used.* Data was tested for variance

homogeneity with Cochrans test (Kirk, 1968)°

Infection

At 2% months of age chickens from each of the four
treatment groups (thymectomized, thymectomized irradiated,
normal, and irradiated) were inoculated intradermally with
5 mg wet weight of viable fl; aging. Twelve weeks after

infection, blood samples were collected for differential

 

*

Use of the Michigan State University computing
facilities was made possible through support, in part,
from the National Science Foundation.

13

counts from the infected and control chickens. All chickens
were then skin tested intradermally in the wattle with 0.1
cc of mammalian tuberculin (Panigrahi, 1969). Three days
after skin testing, blood samples were taken from all

chickens for differential counts.

RESULTS

The percent mortality due to 850 R, 750 R, and 650 R
for 3 day old thymectomized and normal chickens is presented
in Figure 1. The irradiation at 850 R produced a 79 percent
mortality, and 750 R and 650 R caused a mortality rate below
50 percent.

Figure 2 represents the percent mortality and range
of variation of the mortality rate for three separate groups
of chickens subjected to 800 R, the LD50/35.

The effect of thymectomy, sham thymectomy, and
X—irradiation on the number of lymphocytes/100 leukocytes
14 and 38 days after hatching is shown in Tables 1 and 2.
Table 3 indicates that the mean difference of the three com-
bined scores within each group between 14 and 38 days after
hatching was the same. Surgical manipulation did not affect
the aging effect as reflected by the number of lymphocytes/
300 leukocytes. The analysis of this data is presented in
Table 4. For each of the three replicate scores, surgery
produced a significant reduction of the number of lympho-
cytes/100 leukocytes. Supplementary testing indicated the
reduction was found only in the thymectomized group. X—irra-
diation had no significant effect upon lymphocytes/100 leuko-

cytes. No significant interaction between thymectomy and

14

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Table l. The number of lymphocytes/100 leukocytes 14 days
after hatching, three counts/chicken

 

 

Non—X-Irradiated

 

 

Chicken # # Lymphocytes/100 Leukocytes
N—135 63,63,60
N-l34 63,50,64
N-108 81,67,73
N-123 62,65,74
N-llO 68,56,60
Normal N-107 44,55,47
N-109 48,56,60
N—115 44,58,59
N-ll8 51,59,59
N-113 66,66,62
N-ll6 56,71,67
S-69 57,56,69
S-64 63,60,50
S—68 46,59,67
S—6l 59,70,73
Sham S-66 55,72,72
Thymectomized S-75 79,63,59
S-73 73,74,64
S—60 51,52,58
S-62 48,48,41
Tx—37 35,40,38
Tx-130 37,36,36
Tx-39 30,42,39
Tx-34 59,55,53
Tx-4l 46,52,55
Tx-40 25,24,28
Thymectomized 1&442 40,39,43
Tx-31 45,43,48
Tx-36 30,38,31
Tx-33 54,56,53
Tx—131 28,39,39
Tx-38 28,39,38
Tx-43 48,36,32

 

18

 

 

 

 

 

Table 2. The number of lymphocytes/100 leukocytes 38 days
after hatching, 3 counts/chicken
Non-X-Irradiated X-Irradiated
No. No.
Chicken Lymphocytes/100 Chicken Lymphocytes/100
No. Leukocytes No. Leukocytes
N-l35 52,58,54 x-119 42.50.52
N-l34 21,29,16 X—86 52,51,56
N-108 63,54,69 x-99 40,39,56
N-123 27,33,32 X-85 61,56,55
H N-llO 59,66,72 x-79 39,32,47
g N-107 32,32,36 X—lOO 52,55,48
u N-109 72,81,74 X-80 65,64,67
g N-115 58,62,78 x-94 43,49,39
N-118 41,42,55 x-101 41,42,46
N-113 51,56,60 X-84 55,56,49
N-ll6 56,55,63 X—87 55,45,48
N-105 28,29,29 x-102 41,33,33
x-121 48,51,55
x-93 32,43,37
X-78 69,67,64
S—69 41,61,59 Sx-57 71,67,58
g S-64 23,34,42 Sx-59 55,56,59
.2 S-68 53,48,49 Sx—45 82,81,76
e S-61 46,46,41 Sx-54 58,63,60
33 S-66 66,55,61 Sx-Sl 55,58,51
ET” s-75 56,67,62
E s-73 61,61,61
g: S-60 49,42,45
e S—62 31,41,47
Tx-37 19,24,23 Tx-X-18 22,40,36
Tx-130 17.29.29 Tx—X-24 20,19,24
Tx-39 14,08,05 Tx—X—l3 27,20,24
3 Tx-34 42,43,43 Tx-X-126 04,14,08
.2 Tx-4l 25,23,28 Tx-X-128 29,42,45
a Tx-4O 15,14,17 Tx-X-125 29,26,28
3 Tx—42 33,31,31 Tx-X-5 23,22,23
o Tx-3l 17,29,22 Tx-X—17 25,28,21
2 Tx—36 19,31,35 Tx-X—3 23,30,32
E* Tx-33 38,33,36 Tx-X-9 13,11,23
B Tx—131 12,12,17 Tx-X-129 39,29,30
Tx—38 49,48,33 Tx—X-27 26,33,36
Tx-43 22,38,38

 

19

Table 3. Aging effect: number of lymphocytes/300 leuko-
cytes in normal, sham operated, and thymectomized
chickens at 14 and 38 days post hatching

 

 

 

 

 

 

Normal (N) Sham Operated (S) Thymectomized (Tx)
Chicken 14 38 (Chicken 14 38 Chicken 14 38
No. Days Days No. Days Days No. Days Days
N—118 169 138 S-73 211 183 Tx-33 163 107
N-113 194 167 S-69 182 161 Tx-34 167 128
N-107 146 100 S-62 137 119 Tx-3l 136 68
N-llS 161 198 S-66 199 182 Tx—37 113 66
N-123 201 92 S-64 173 99 Tx-40 77 46
N—135 186 164 S-60 161 136 Tx-42 122 95
N-109 164 227 S-68 172 150 Tx-130 109 75
N-llO 184 197 S—75 201 185 Tx-4l 153 76
N-l34 177 66 S—61 202 133 Tx-43 116 98
N-116 194 174 Tx-39 111 27
N-108 221 186 Tx-36 99 85
N-105 131 86 Tx-13l 106 41

Tx-38 105 130

 

One way analysis of variance for unequal sample size:

 

 

H :u . = u . = u .
0 differenceN differences differenceTX
N :u = u = u
l d N d S d Tx

Source d.f. 8.8. M88. F_
Among 2 428.619 214.310 0.177
Within 31_ 37461.499 1208.435
Total 33 37890.118 At 0.05 level not sig.

With addition transformation F = 0.177

(2.31)

20

 

 

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21

X—irradiation occurred. The simple correlation between
score 1 and 2, score 2 and 3, score 1 and 3 are: 0.91992,
0.92522, and 0.89022, respectively.

The number of leukocytes/mm3 of blood for each of
the four groups of chickens, thymectomized, thymectomized
X-irradiated, X-irradiated and normal at 14 and 38 days
after hatching is presented in Table 5. Table 6 presents
the analyses of the effects of X-irradiation and thymectomy
in the chicken on the leukocytes/mm3 of blood.

The two way analysis of the leukocytes/mm3 at 14
days after hatching indicated a significant reduction in the
leukocyte/mm3 due to X-irradiation but no significant effect
due to thymectomy. At 38 days after hatching the affect of
X-irradiation was no longer significant (d==0.05) and thymec—
tomy produced no significant effect. The last analysis in
Table 6 indicated that the difference in the effect due to
X—irradiation at 14 and 35 days after hatching is statisti-
cally significant. No interaction between thymectomy and
X-irradiation was detected in any of the analyses presented
in Table 6.

Figure 3 shows the lack of separability of the
thymus and the thyroid gland.

Data were collected at 5% months after hatching to
determine if thymectomy or X-irradiation caused a prolonged
effect upon the number of lymphocytes/100 leukocytes. In
addition, data to determine the effects of infection with

'M. avium and tuberculin testing were collected. The data

22

Leukocytes/mm3 in experimental chickens l4 and 38 days
after hatching

Table 5.

 

 

Non-Irradiated Irradiated

 

 

 

 

Chicken # 14 Days 38 Days Chicken # 14 Days 38 Days
Normal
N-645 22,208 27,712 X-665 8,000 23,488
N-667 20,032 28,416 X-666 4,800 32,512
N-524 27,712 26,432 X—667 7,488 11,712
N-526 20,224 28,288 X-668 14,400 18,688
N-527 13,056 5,952 X-670 7,488 14,912
N-529 13,504 11,072 X-672 9,600 17,600
N-531 11,520 12,800 X-674 6,400 19,712
N-532 30,400 27,328 X-675 8,512 13,312
N-533 26,688 17,472 X-676 3,200 20,800
N-705 40,512 42,688 X-677 4,800 18,688
N-706 40,000 17,088 X-679 13,312 16,512
N-707 15,424 18,112 X-685 1,088 16,000
N—708 57,088 16,000 X—686 5,888 7,424
N-710 26,688 24,000 X-687 5,312 34,688
N-711 29,312 36,800 X-688 18,112 19,712
N-712 14,400 49,088 X-689 3,712 9,600
N-713 24,000 35,200 X-690 14,400 30,912
N-715 13,888 25,600 X-691 3,712 11,712
N-7l8 14,400 37,888 X-693 8,512 58,688
N—719 32,000 37,312 X-696 4,800 19,200
N-720 29,888 20,288 X-698 9,088 9,600
N-723 19,712 22,400 X-699 10,112 22,400
N-724 11,200 9,088 X-7OO 8,512 14,912
X-701 7,488 18,688
X-702 12,800 19,200
X-703 11,712 21,888
Thymectomized
Tx-640 5,888 27,712 TX-X-601 8,000 25,600
Tx-641 26,688 9,088 Tx-X-602 20,800 10,848
Tx-648 40,000 41,600 Tx-X-603 12,800 26,112
Tx-649 30,400 16,512 Tx-X-604 16,000 14,400
Tx-X-605 9,088 25,600
Tx-X—606 11,200 13,824
Tx-X-608 6,912 17,600
Tx-X-609 17,088 12,288
Tx—X-610 13,312 22,912
Tx-X-612 5,312 24,512
Tx-X-615 16,512 32,000
Tx-X-618 8,000 18,688
Tx—X—622 12,224 14,400
Tx—X-623 18,688 19,712
Tx-X-624 6,400 12,800
Tx-X-625 14,400 29,888
Tx—X-626 6,912 28,288
Tx—X-627 9,600 13,824
Tx-X-629 6,912 22,912
Tx-X-630 4,288 6,400
Tx-X-632 3,200 2,688
Tx-X-635 9,600 16,512
Tx-X-636 10,624 10,688
Tx-X-639 10,112 14,400

 

23

 

 

 

 

 

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24

 

 

 

 

Figure 3. Continuity of thymus cells (darker staining area)
with cells of the thyroid gland (Hematoxylin and
Eosin stain).

25

is presented in Table 7. Table 8 shows the four way analy-
sis of variance for unequal sample sizes performed on the
data of Table 7. In the analysis for each of the three
scores, there were significant interactions (d==0.05). The
following interactions existed: (1) two three-way interac-
tions: among thymectomy, X—irradiation and infection; and
among irradiation, infection, and tuberculin testing. Two-
way interactions between thymectomy and infection, thymec-
tomy and X-irradiation, and infection and tuberculin testing
were found. The existence of these interactions makes the
tests of significance listed in Table 8 for the main effects,
namely thymectomy, X-irradiation, infection, and tuberculin
testing, invalid.

To facilitate the interpretation of the nature of
interactions, the three scores from Table 8 were combined.
The simple correlations between scores 1 and 2, scores 2
and 3, and scores 1 and 3 are: 0.91332, 0.88231, 0.83351,
respectively. The four-way analysis of variance for unequal
sample sizes with the average of the three scores taken per
sample is presented in Table 9. The dominant interactions
were ABC, CD, and AB: (1) thymectomy, with X-irradiation
with infection, (2) infection with tuberculin testing, and
(3) thymectomy with X—irradiation, respectively. The three-
way interaction as graphed in Figure 4 was characterized by
a different response in thymectomized and normal chickens,
depending upon whether or not they had been X-irradiated;

furthermore, this interaction between thymectomy and

26

 

 

 

 

 

Table 7. The number of lymphocytes/100 leukocytes in eXperimental chickens 5%
months of age
Non-X-Irradiated X-Irradiated
# Lymphocyte/100 # Lymphocyte/100
Chicken # Leukocytes Chicken # Leukocytes
Noninfected--Sampled Before Tuberculin Testing
N—920 55.57.54 X-893 85.90.92
N-924 52,52,42 X-912 76.80.62
Normal N-926 67,55,76 X—914 90.90.87
N-929 80.66.84 X-915 86,85,86
N-935 86.83.78
Tx-835 48.44.56 Tx-X-847 43.41.38
. Tx-836 68.69.63 Tx-X-848 81.73.58
Thy"e°t°mlzed Tx-837 57.60.68 Tx-X—859 47.59.47
Tx-842 36.27.31 Tx-X-864 29.38.36
Noninfected--Sampled After Tuberculin Testing
N-920 66.69.64 X-893 88.79.81
N-924 55.47.50 X-912 80.75.82
Normal N-926 77.60.72 X-914 84.92.86
N-929 62.55.51 X—915 83.81.86
N-935 67.77.79
Tx-835 40.40.50 Tx-X-847 35.43.52
. Tx-836 59.70.67 Tx-X—848 49.56.55
ThymeCtomlzed Tx-837 44.51.72 Tx-X-859 48.49.50
Tx-842 45.41.45 Tx-X-864 36.31.25
Infected-~Sampled Before Tuberculin Testing
N-922 91.94.94 X-892 60.63.65
N-931 96.93.89 X-897 90.90.86
Normal N-934 73.84.88 X-898 91.93.88
N-937 86.91.92 X-899 84.87.90
N-938 94.88.86 X-902 83.76.88
X-908 87.86.78
X-909 91.80.79
Tx-831 72,77,67 Tx-X-853 74.72.76
Tx-833 75.81.87 Tx-X-855 82.80.85
Thymectomized Tx-843 79.76.89 Tx-X-857 70.71.74
Tx—X-861 74.65.56
Tx—X-876 78.77.75
Infected--Sampled After Tuberculin Testing
N-922 46.67.79 X-892 87.81.76
N-931 66.56.58 X-897 90.85.83
Normal N-934 66.73.63 X-898 85.88.94
N-937 88.80.87 X-899 86.82.79
N-938 84.69.80 X-902 82.84.76
X-908 79.84.81
X-909 83.87.91
Tx-83l 65.67.51 Tx—X-853 64.70.65
Tx-833 40.52.58 Tx—X—855 73.72.75
Thymectomized Tx-843 43.40.56 Tx-X-857 71.71.57
Tx-X-861 58.56.55
Tx-X-876 65.50.48

 

 

 

 

 

27

 

 

 

 

Table 8. The analysis of the number of lymphocytes/100 leukocytes in experimental
chickens 5% months of age (four-way analysis of variance, three scores)
3 Degrees of
Source Sum of Squares Freedom Mean Square F Significance
Score 1
A 7909.76558 1 7909.76558 69.8921 0.0005
B 821.28579 1 821.28579 7.2570 0.009
C 2743.54915 1 2743.54915 24.2425 0.0005
D 1299.48815 1 1299.48815 11.4825 0.001
AB 335.44329 1 355.44329 2.9640 0.090
AC 693.57627 1 693.57627 6.1286 0.016*
AD 220.94239 l 220.94239 1.9523 0.168
BC 0.00131 1 0.00131 0.0000 0.997
BD 331.71162 1 331.71162 2.9311 0.092
CD 358.64904 l 358.64904 3.1691 0.080
ABC 649.29101 1 649.29101 5.7372 0.020*
ABD 13.82974 1 13.82974 0.1222 0.728
BCD 356.95230 l 356.95230 3.1541 0.081
ACD 17.99532 1 17.99532 0.1590 0.692
ABCD 1.84069 1 1.84069 0.0163 0.899
Error 6563.92619 58 113.17114
Total 22380.55405 73
Score 2
A 6323.35876 1 6323.35876 59.9295 0.0005
B 803.46046 1 803.46046 7.6148 0.008
C 2914.35746 1 2914.35746 27.6208 0.0005
D 1189.55090 1 1189.55090 11.2739 0.001
AB 645.93034 1 645.93034 6.1218 0.016*
AC 363.33582 1 363.33582 3.4435 0.069
AD 83.71257 1 83.71257 0.7934 0.377
BC 205.98249 1 205.98249 1.9522 0.168
BD 200.86511 1 200.86511 1.9037 0.173
CD 436.19497 1 436.19497 4.1340 0.047*
ABC 555.13712 1 555.13712 5.2613 0.025*
ABD 42.68259 1 42.68259 0.4045 0.527
BCD 714.53395 1 714.53395 6.7720 0.012*
ACD 49.79341 1 49.79341 0.4719 0.495
ABCD 1.69171 1 1.69171 0.0160 0.900
Error 6119.77143 58 105.51330
Total 20963.09459 73
Score 3
A 6251.63939 1 6251.63939 49.6535 0.0005
B 36.88711 1 36.88711 0.2930 0.590
C 2656.80314 1 2656.80314 21.1016 0.0005
D 728.79115 1 728.79115 5.7884 0.019
AB 1076.60888 1 1076.60888 8.5509 0.005*
AC 305.30301 l 305.30301 2.4249 0.125
AD 82.01149 1 82.01149 0.6514 0.423
BC 52.84539 1 52.84539 0.4197 0.520
BD 287.54473 1 287.54473 2.2838 0.136
CD 917.64330 1 917.64330 7.2884 0.009*
ABC 792.26990 l 792.26990 6.2926 0.015*
ABD 48.48346 1 48.48346 0.3851 0.537
BCD 210.18776 l 210.18776 1.6694 0.201
ACD 246.53196 1 246.53196 1.9581 0.167
ABCD 5.28346 1 5.28346 0.0420 0.838
Error 7302.59714 58 125.90530
Total 21105.36486 73
aA = not thymectomized vs. thymectomized; B = not X-irradiated vs. irradiated;

C = not infected vs. infected; and D = before tuberculin testing vs. after
tuberculin testing.

28

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90"
4 B2
80“
f3
70"
353 65 B1
0.
8 60
H 0
0
50“ 52
. CD.
47 l
49 73
40 T 5: i § ; 5 fi-
30 40 A250 60 70 A1 80 90
Group Mean
90‘?
83 B2
80" 80 B1
5 70
m 9
z 6
g 65
o 60"
u
(D
50”
C2D.
4O : % : €74 :%2 4
30 40 50 60 A2 70 80A1 90
Group Mean
Figure 4. Representation of the three-way interaction

between cross classified thymectomy. X-Irradiation,
and infection. A1 = Normal. A2 = Thymectomized,

B = Not X-Irradiated, B2 = X-Irradiated, C1 = Not
Infected. C2.= Infected, D. = Summation Before and

,After Tuberculin Testing.

30

X—irradiation occurred only in the noninfected group. The
two—way interaction between thymectomy and X-irradiation did
not occur in the infected group of chickens. The interac-
tion between thymectomy and X-irradiation as graphed in
Figure 5 is such that X-irradiation resulted in an increase
in the percent of lymphocytes in normal chickens but when
coupled with thymectomy was followed by no detectable effect
as compared with thymectomy. The manner in which infection
and tuberculin testing interacted as shown in Figure 6 is
the following: in infected chickens, tuberculin testing
caused a lowering in the percent of lymphocytes, in nonin—
fected chickens, tuberculin testing was not accompanied by
such a response. In Table 9. as in Table 8. the signifi-
cance of the four main treatments as given is invalid.
Because the significance of thymectomy, X-irradia—
tion, infection, and tuberculin testing could not be taken
directly from the four-way analyses of variance of either
Table 8 or 9. orthogonal contrasts were made to test the
effects of these four main treatments. The results obtained
are in Table 10. The effects of thymectomy, X-irradiation,
infection, and tuberculin testing were tested singly within
different treatment combinations of the effects not being
tested. For example. D/Cl represents the following contrast
comparison: the percent of lymphocytes prior to tuberculin
testing of all chickens within the noninfected group. with
the percent of lymphocytes after tuberculin testing within

the noninfected group. There was no significant difference.

 

31

 

90T
83 B
80" 2
g .. 72 B1
a, 70
z
53;
o 60“ 59
H
<9 5
50” C.D.
4o . . 5.8. . 7?. .
30 4o 50 A2 60 70 Al 80 90

Figure 5.

Group Mean

Representation of the two-way interaction between
cross classified thymectomy and X-irradiation.

A1 = Normal, A2 = Thymectomized. Bl = Not X-Irra-
diated, B = X-Irradiated. C.D. = Summation of
Infected and Noninfected Chickens Before and
After Tuberculin Testing.

 

32

 

90‘r
80» 81 D1
g L 71 132
g '70
g 63
<3 60‘~ 61
a...
0
50“
A.B.
62 76
40 5 5 ‘1 He : z 1
30 40 50 60 Cl 70 C2 80 90
Group Mean
Figure 6. Representation of the two—way interaction between

cross classified infection and tuberculin testing.
C1 = Noninfected Chickens. C2 = Infected Chickens,
D1 = Before Tuberculin Testing. D2 = After Tuber—
culin Testing. A.B. = Summation of Normal and
Thymectomized Chickens Whether Irradiated or Not.

 

33

 

 

Table 10. The significance of treatments in the percentage
of lymphocytes due to thymectomy, X-irradiation,
infection, and tuberculin testing

Contrasta Significance

D/Cl . . . . . . . . . . . . . . . . . . n.s.

*

D/C2 . . . . . . . . . . . . . . . . . .

*

A/Cl . . . . . . . . . . . . . . . . . .

*

A/C2 . . . . . . . . . . . . . . . . . .

B/C2 . . . . . . . . . . . . . . . . . . n.s.

*

A/Blcl OOOOOOOOOOOOOOOOOO

*

A/BZCl 00.0.0000000000000

*

B/Alcl . . . . . . . . . . . . . . . . . .

*

B/A2Cl . . . . . . . . . . . . . . . . . .

*

C/AlBlDl

*

C/AlBlD2 . . . . . . . . . . . . . . . . . .

*

C/AZBlDl . . . . . . . . . . . . . . . . . .

C/A2B1D2 . . . . . . . . . . . . . . . . . . n.S.

*

C/A2B2Dl . . . . . . . . . . . . . . . . . .

*

C/AZBZDZ . . . . . . . . . . . . . . . . . .

aAl = no thymectomy, A2 = thymectomy; B = no X—irradiation.

B2 = X-irradiation; C1 = no infection, C2 = infection; and
D = before tuberculin testing. D2 = after tuberculin
testing.

*Significant (a==0.01); n.s. = not significant.

34

The second contrast D/CZ shows that tuberculin testing had a
significant effect in decreasing the percent of lymphocytes
in infected chickens. Tuberculin testing had a significant
effect only in infected chickens.

The effect of thymectomy was tested within four
levels of the other treatment combinations. within nonin-
fected, within infected. within nonAX-irradiated noninfected,
and within X-irradiated noninfected. In all of these con-
trasts thymectomy produced a significant effect upon the
percent of lymphocytes/100 leukocytes. The data shows the
effect to be a decrease.

The significance of X-irradiation was tested within
the infected chickens. within nonthymectomized noninfected
chickens, and within thymectomized noninfected chickens. In
chickens not infected with M, avium. there was no significant
effect on the percentage of lymphocytes due to X-irradiation.
In both nonthymectomized and thymectomized chickens infected
with M, gyigm,ZX-irradiation had a significant detectable
effect upon the percent of lymphocytes. The data shows the
effect to be an increase in the percent of lymphocytes.

The significance of infection in affecting the per-
centage of lymphocytes also depends upon the combination of
other treatments within which the effect of infection were
being tested. Infection with.M, 31133 had a significant
effect on normal, nonirradiated chickens in increasing the
percent of lymphocytes both before and after tuberculin

testing. The thymectomized nonirradiated chickens had a

35

significant increase only before tuberculin testing but
none after tuberculin testing. Infection had a significant
effect in increasing the percent of lymphocytes in.thymec—
tomized X-irradiated chickens before and after tuberculin
testing.

X-irradiation. infection, and tuberculin testing
show significant effects upon the percent of lymphocytes
depending upon the combination of other treatments. Thy-
mectomy was associated with a significant decrease in the
percent of lymphocytes in all of the meaningful contrasts
tested.

The results obtained from the reactions to tubercu—

lin are given in the Appendix.

DISCUSSION

The thymus is the first lymphoid organ to function
in the chicken embryo and the thymus dependent white pulp
of the spleen develops shortly after hatching; therefore,
X-irradiation after thymectomy has been recommended to
destroy any peripheral thymus dependent lymphocytes. The
LD50/35 was established for 3 day old chickens to determine
whether the percent of lymphocytes in the peripheral blood
was reduced more by X-irradiation and thymectomy than by
thymectomy only; neonatal thymectomy in the chicken is
followed by a depletion in the percent of lymphocytes in
the peripheral blood.

The results of differential blood counts 14 days
after hatching indicated that thymectomy has a statistically
significant effect in lowering the percent of lymphocytes.
No comparisons could be made with the X—irradiated chickens
because of a marked anemia in this group at 14 days after
hatching and there were not sufficient leukocytes on two
blood smear slides for a differential count.

A comparison of the mean difference in the percent
of lymphocytes between 14 and 38 days among thymectomized,

sham-thymectomized and normal chickens showed all groups to

36

37

be equal. Therefore, the aging or maturation effect was not
affected by thymectomy.

At 38 days after hatching, thymectomized chickens
and thymectomized X-irradiated chickens had a decrease in
the percent of lymphocytes. There was no difference between
thymectomized and thymectomized X—irradiated chickens in the
extent of the decrease. Thymectomy with X-irradiation did
not cause any greater' decrease 10f the lymphocytes in the
peripheral blood than thymectomy only. The conclusion drawn
is that X-irradiation is not required after thymectomy to
reduce the percent of lymphocytes in the peripheral blood.

The X-irradiation was not without effect. but this
effect was not detected as having long term infuence on the
lymphocyte alone or as persistent as the effect of thymec-
tomy. Fourteen days after hatching. the total leukocyte
count from X-irradiated chickens was significantly lower
than in nonirradiated chickens. This explains why there
were insufficient leukocytes to perform differential blood
counts 14 days after hatching. Significant to note is that
there was no decrease in the total leukocyte count in the
thymectomized group of chickens.

There was recovery with time of the lowered leuko-
cyte count of the blood due to X-irradiation. By 38 days
after hatching no effect on the total white blood cell count
could be detected due to X-irradiation or to thymectomy. A
comparison of the total leukocyte count made between 14 and

38 days after hatching indicated that a difference occurred

38

only in the X—irradiated group. In the X-irradiated group,
recovery from the effects of X-irradiation was complete by
38 days after hatching or 35 days after X-irradiation. It
was concluded that the thymus was not required for this
recovery.

Thirty-eight days after hatching was chosen as the
time to collect blood samples in these studies because the
death rate due to X-irradiation reached its plateau by this
time. and chickens in the group surviving X-irradiation were
to be used in future studies. By 38 days after hatching the
spleen which is a lymphogenic organ in the mature chicken
has released the heterophils stored there during embryona-
tion and is functioning in its adult capacity. If the
effects of thymectomy in decreasing the percent of lympho—
cytes were to be counteracted by production of lymphocytes
by the spleen. these should have been detected. No such
counteracting effects were detected. At this time chickens
are still considered immunologically incompetent (Wolfe gt
.gl., 1949). Differential blood counts showed a persistent
decrease in lymphocytes in the thymectomized chickens at
38 days post hatching. The total leukocyte counts confirmed
that X-irradiation decreased the numbers of all leukocytes
and that recovery occurred by 35 days post X-irradiation.

No such recovery occurred from the effects of thymectomy as
detected by differential blood counts.

The results showed that thymectomy in chickens per-

formed 24 hours post hatching was followed by a decrease in

39

the percent of lymphocytes in the peripheral blood. This
decrease was persistent through 38 days after thymectomy.
X-irradiation is followed by a significant decrease in all
white blood cells as measured 11 days after irradiation but
this decrease did not persist.

The data indicate that if thymectomy in the chicken
was performed early, at least within 24 hours after hatching.
X-irradiation was not necessary to cause a significant
decrease in the percent of lymphocytes. So far as known,
the chicken does not have lymphatics through which lympho-
cytes travel. Furthermore, leukocytes are not found in
the peripheral circulation of the embryo. All leukocytes
are released into the blood stream within 24 hours after
hatching, and the lymphocytes are released after 24 hours
after hatching (Lucas gt al., 1961). This emphasizes the
importance of performing thymectomy early enough to prevent
the peripheralization of thymocytes into the circulation.

No attempts were made to classify the lymphocytes
according to size. Szenberg and Shortman (1966) reported
that lymphocytes represent a mixture of cells with different
biological functions. Cell p0pu1ations from the thymus and
bursa differ in density and function, but this difference
exists between cells of the same size and morphology. Also.
the large bursa dependent lymphocytes are rarely found in
the peripheral blood (Lucas §E_gl,. 1961).

Variability in determinations of total white blood

cell counts depending upon the methods used has been reported

4O

(Denington §£_§l,. 1955). The choice of Reese-Eckers stain
as a diluent was made because it has been reported to have
the lowest coefficient of variability. The use of a diluent
with the lowest coefficient of variability increased the
statistical power of the analysis.'

The variability in the reported normal ranges of
differential blood counts in the chicken is greater than in
mammals (Lucas _t.gl,, 1961). This variability exists among
chickens and within one chicken. A diurnal rhythm has also
been reported (Glick §£_§l,, 1960; Khussar. 1966); therefore,
all blood samples were collected in the morning. To deter-
mine how much variabiltiy occurred during the counting
process, three separate blood counts were performed. The
simple correlations between the three scores were considered
to be high enough to indicate that subsequent differences
detected between the eXperimental groups of chickens were
indicative of a true difference and were not due to errors
in counting.

Chickens which contained remnants of thymus tissue
adhering to the thyroid gland at necropsy (incomplete-
thymectomy) were included in the studies for two reasons.
First, when the differential blood counts of complete-
thymectomy chickens were compared with those of incomplete—
thymectomy chickens, regardless of X-irradiation. no differ—
ences were detected. Therefore, all results should be

reviewed in terms of the significance of the removal of:

substantial thymic tissue rather than the persistence of

41

small remnants. The second consideration for including
incomplete-thymectomy chickens in the tabulation of results
is that cells of the thymus invade the thyroid. In fowl,
cells of the thymus lie in contact with the thyroid. In
many cases, lymphoid area cells of the thyroid are directly
continuous with the cells of the thymus, Figure 3. The
membrane separating the two glands has disappeared (Payne
_£__1.. 1952). This strongly indicates that complete
thymectomy, in the sense of complete removal of all thymic
tissue can be obtained with certainty only if the thyroid
gland is also removed.

Having shown that neonatal thymectomy in the chicken
is followed by a significant decrease in the percent of
lymphocytes of the peripheral blood at 38 days post hatching,
a study was conducted to determine whether in 5% month old
thymectomized chickens the percent of lymphocytes was still
lower than in normal chickens. In addition, the effect on
the percent of lymphocytes of X-irradiation, infection with
M, gyigm at 2% months of age and tuberculin testing on 5%
month old chickens was determined. Interactions between
various combinations of the four main conditions were found.
Each of the conditions tested showed significant effects
upon the percent of lymphocytes, but this significance was
restricted to certain combinations within the other condi-
tions. To facilitate the interpretation of the interactions.

the three replicate scores of differential blood counts were

42

averaged. A three way interaction between thymectomy, X-
irradiation and infection was found: (a) as illustrated in
Figure 4 in the noninfected group there was an increase in
percent of lymphocytes in the nonthymectomized group due to
X-irradiation, (b) in the thymectomized group there was a
decrease due to the effects of X-irradiation, (c) the
effects of thymectomy upon the percent of lymphocytes was

a decrease. In the infected group: (a) the effect of thy”
mectomy upon the percent of lymphocytes was a decrease, (b)
the effect of X—irradiation was measured as an increase in
both normal and thymectomized groups. (c) all infected
chickens showed a higher percent of lymphocytes than their
uninfected controls. The prominent two-way interaction
(lack of a parallel response) between thymectomy and X—
irradiation as illustrated in Figure 5 was due to a greater
increase in the percent of lymphocytes in normal irradiated
chickens than in thymectomized irradiated chickens. The
two—way interaction between infection and tuberculin testing
as shown in Figure 6, indicates that tuberculin testing
alters the percent of lymphocytes in the infected chickens
only. The decrease in the percent of lymphocytes in in-
fected chickens after tuberculin testing may be due to a
total increase in some other leukocyte type; this could
produce an apparent decrease in percent of lymphocytes, or

it may be a real decrease in percent of lymphocytes.

43

Orthogonal contrasts were constructed, restricting
each of the four main conditions within combinations of the
other conditions to test what significance the conditions of
thymectomy, X-irradiation. infection. and tuberculin testing
had upon the percent of lymphocytes in the peripheral blood
of chickens. Caution must be used in evaluating the signif-
icance of these analyses because the data was not orthogonal
(independent). To take this into consideration, an alpha
level of 0.01 was set, but the degree of confidence at which
the hypothesis could be accepted or rejected is more closely
representative of an alpha level of 0.05.

Having determined in Table 9 and illustrated in
Figure 6 that an interaction exists between tuberculin
testinganuiinfection. the test for the significance of the
effect of tuberculin testing was made separately for the
noninfected and infected groups. Tuberculin testing in non-
infected chickens regardless of thymectomy or X-irradiation
caused no detectable change in the percent of lymphocytes.
By contrast, tuberculin testing decreased the percent of
lymphocytes in all chickens infected with.M, avium.

Using this same procedure. it was shown that X-
irradiation appeared to have no discernible effect upon the
chickens subsequently infected. However, in noninfected
X-irradiated chickens there was a significant effect due to
X-irradiation in both normal and thymectomized groups. In

normal chickens the effect the X-irradiation was an increase

44

in the percent of lymphocytes. but in thymectomized chickens,
X—irradiation was followed by a decrease in the percent of
lymphocytes.

This increase in the percent of lymphocytes in
normal chickens following X-irradiation is a strong source
of the interaction as well as an indication that the long
termed effects of X—irradiation are not predicted by the
initial sequence of events immediately following irradiation.
After the destruction of leukocytes and the initial recovery
to normal values. X-irradiated chickens appear to have been
stimulated, presumably as a result of X-irradiation, to
produce or maintain a higher percentage of lymphocytes than
normal. To achieve a reduction in the percent of lympho-
cytes in thymectomized chickens. by coupling X-irradiation
with thymectomy would not be justified, because X-irradiated
chickens cannot function as proper controls.

Infection with M, avium increased the percent of
lymphocytes in normal, thymectomized, X-irradiated and
thymectomized X-irradiated chickens with one exception. The
percent of lymphocytes in thymectomized chickens after tuber-
culin testing is the same in both infected and noninfected
chickens. Prior to tuberculin testing. the infected thymec—
tomized group did show a higher percent of lymphocytes than
the noninfected thymectomized group.

Thymectomy was always followed by a decline in the
percent of lymphocytes in both subsequently infected and non-

infected chickens. Whether or not X—irradiation was

45

administered, there was a persistent decrease in the percent
of lymphocytes in all noninfected chickens.

To achieve a reduction in the percent of lymphocytes
of the peripheral circulation of the chicken, neonatal
thymectomy is indicated. The decrease effected by thymec-
tomy is persistent through 5% months of age. The need of
coupling X—irradiation with thymectomy is highly question-
able and in fact contradictory because the longer term
effect cflf X-irradiation was an increase in the percent of
blood lymphocytes at 5% months of age. Furthermore, X-irra-
diation following thymectomy did cause a greater decrease in
the percent of lymphocytes at 38 days after hatching than
did only thymectomy.

Because the lymphocyte has been credited as the cell
type which mediates the delayed hypersensitivity reaction.
the thymectomized chicken affords a unique biological model
in which the controlling mechanism of delayed hypersensitiv—
ity can be studied in 2139 as well as in_yi££9, The focus
of the research reported here was to deve10p the technique
of thymectomy and to determine its effects upon the percent
of lymphocytes in the peripheral blood.

The kind of lymphocyte. and their function or bio-
logic capacities has not been determined. Lischner §£_§1,
(1967) have reported in a human an analogous situation with
the thymectomized chicken. They report that lymphocytes in

congenital absence of the thymus fail to participate in a

46

host versus graft rejection and to develop tuberculin sen-
sitivity. Moreover. the passive transfer for these capac—
ities from immunologically competent cells was unsuccessful.

In the research reported here. what appears to be an
increase or decrease in the percent of lymphocytes, may be
the result of an unequal decrease or increase in leukocytes
other than the lymphocyte; the increase or decrease reported
here must be viewed as relative to their controls. Because
the thrombocytes in the chicken are stained differently from
the leukocytes so as to be differentiated from them when
counting in a hemocytometer for total leukocyte counts.

a simultaneous differential count is not possible. The
morphology of the leukocytes is distinct from that of the
thrombocytes but individual differences between them are
lost. A technique in which total leukocyte counts and dif-
ferential counts could be performed simultaneously would
answer the question whether the increases and decreases in
percent of lymphocytes are relative or absolute.

Of primary interest was the determination that neo-
natal thymectomy in the chicken is followed by a statisti-
cally significant decrease in the percent of lymphocytes
up to 5% months of age. X—irradiation is not necessary to
effect a further decrease. The additional conditions of
infection with.M, avium and tuberculin testing were included
as a pilot study to determine whether thymectomized and
normal chickens responded in parallel fashion. There are

strong indications that the effects of tuberculin testing

47

are dependent upon infection rather than upon thymectomy.
On the other hand the influence of infection upon the per-
cent of lymphocytes within thymectomized chickens only was
the same after tuberculin testing as in noninfected thymec—
tomized chickens. These are the first investigations into
the possible functions of the lymphocyte during infection
and tuberculin testing and must be further studied for a

meaningful interpretation.

SUMMARY

The decrease in the percent of lymphocytes following
thymectomy one day post hatching and X-irradiation three
days post hatching was the same as the decrease in chickens
thymectomized one day post hatching. These observations
were made in chickens 38 days and 5% months of age. The
necessity of coupling X-irradiation with neonatal thymectomy
to destroy thymocytes (thymic lymphocytes) which may have
peripheralized prior to hatching was studied by comparing
the percent of lymphocytes of X-irradiated normal and thy—
mectomized chickens with non-X-irradiated normal and thymec-
tomized chickens. It was found that complete thymectomized
chickens could not be distinguished from incomplete—thymec-
tomized chickens by percent of lymphocytes in differential
counts regardless of X—irradiation.

The long term effects of X-irradiation and thymec—
tomy were determined in 5% month old chickens infected with
.M. avium at 2% months before and after tuberculin testing at
5% months of age. There were statistically significant in-
teractions and only the most prominent were diagrammed. A
three-way interaction between thymectomy, X-irradiation, and
infection was found. Infection with M, gyigg increased the

percent of lymphocytes in all chickens. In noninfected

48

49

chickens, X-irradiation caused a significant increase in

the percent of lymphocytes, but when coupled with thymectomy
caused a decrease in the percent of lymphocytes. In in-
fected chickens, X-irradiation regardless of thymectomy was
followed by increases in the percent of lymphocytes. Regard-
less of infection or tuberculin testing, X—irradiation in—
creased the percent of lymphocytes in normal chickens more
than in thymectomized chickens. There was a decrease in the
percent of lymphocytes in infected chickens only after tuber-
culin testing.

Thymectomized chickens showed a persistent decrease
in the percent of lymphocytes at 5% months of age. The use
of X-irradiation after thymectomy is not recommended because
of the interactions with thymectomy at 5% months of age and
because no greater' decrease in the percent of lymphocytes

than that due to thymectomy only was found.

LITERATURE CITED

10.

LITERATURE CITED

Benezra. D., I. Gery, and A. M. Davies. 1967. Trans-
formation of rabbit lymphocytes by specific antigens.
Proc. Soc. EXptl. Biol. Med. 125:1305-1308.

Bloom, B. R.. and B. Bennett. 1966. Mechanisms of a
reaction ig vitro associated with delayed hypersen-
sitivity. Science 153:80-83.

Clark. W. G. B., C. B. Williams, and J. M. Yoffery.
1966. Thoracic duct lymphocytes following thymec-
tomy in the guinea pig. J. Lab. Clin. Med. 61:
439-446.

COOper, M. D., R. D. A. Peterson, and R. A. Good. 1965.
Delineation of the thymic and bursal lymphoid systems
in the chicken. Nature 205:143-146.

Cooper. M. D., R. D. A. Peterson, M. A. South. and R. A.
Good. 1966. The functions of the thymus and the
bursa systems in the chicken. J. EXptl. Med. 123:
75-102.

David. J. R.. S. Al-Askari, H. S. Lawrence. and L.
Thomas. 1964. I. Delayed hypersensitivity i3
vitro. J. Immunol. 23:264-273.

David, J. R.. H. S. Lawrence, and L. Thomas. 1964.
II. Effects of sensitive cells on normal exudate
cells in the presence of Ag. J. Immunol. 93:274-278.

David, J. R.. H. S. Lawrnece, and L. Thomas. 1964.
III. The specificity of hapten-protein conjugates
in the inhibition of cell migration. J. Immunol.
23:279-282.

Denington. E. M.. and A. M. Lucas. 1955. Blood tech-
niques for chickens. Poult. Sci. 34:360-368.

Dresser, D. W., and N. A..Mitchison. 1959. Cellular
basis for the immunological memory, pp. 227-242.
;g_Ciba Foundation Symposia cellular aspects of
immunity, B. H. Waksman (ed.). J. A. Churchill.
London.

50

ll.

l2.

13.

14.

15.

16.

17.

18.

19.

20.

21.

51

Ford, C. E., H. S. Micklem, E. P. Evans. J. G. Gray, and
D. A. Ogden. 1966. Studies with point-body irradi—
ated mice injected with chromosome-marked bone marrow
and subjected to antigenic stimulation. Ann. N. Y.
Acad. Sci. 122:283-296.

Gell, F. A. 1967. The association of increased
spontaneous lymphocytes transformation ig_vitro with
clinical manifestations of drug hypersensitivity.

J. Immunol. 28:778-785.

George, M.. and J. H. Vaughan. 1962. ;g_vitro cell
migration as a model for delayed hypersensitivity.
Proc. Soc. Exp. Biol. 111:514—521.

Glick, B. 1960. Leukocyte count variation in young
chicks during an 18-hour period. J. Appl. Physio.
15:965.

Glick, B. 1964. The bursa of Fabricus and the devel-
opment of immunologic competence. p. 343-358. .;Q
R. A. Good and A. E. Gabrielson (ed.). The thymus
in immunobiology. Hoeber-Harper. New York.

Gowens, G. L., and D. D. McGregor. 1965. The immuno-
logical activities of lymphocytes. Prog. Allergy
2;1-78.

Isakovic, K., and B. D. Jankovié. 1964. Role of the
thymus and the bursa of Fabricius in immune reac-
tions in chickens. II. Cellular changes in
lymphoid tissues of thymectomized, bursectiomized
and normal chickens in the course of first antibody
response (to human erythrocytes). Internat. Arch.
(Allergy Appl. Immunol. 24:296-310.

Jaffe, W. P. 1966. The role of the bursa of Fabricius
and the thymus in chickens. W. Poult. Sci. J. 22:
25-30.

Jankovié, B. D., and K. Isakovic. 1966. Antibody
production in bursectomized chickens given repeated
injections of antigen. Nature 211:202-203.

Kay, K., and W. O. Rieke. 1963. Tuberculin hypersensi-
tivity: Studies with radioactive antigen and mono-
nuclear cells. Science 139:487-489.

Kirk. R. E. 1968. Experimental design: Procedures for
the behavioral sciences, p. 62. Brooks/Cole Publish—
ing Co., Belmont, California.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

52

Khussar. Y. P. 1966. Circadian rhythm of thymic lympho-
cyte mitotic activity under normal conditions and in
acute radiation sickness. Arkh. Anat. Gestol.
Embriol. 5;:109-112.

Konda. S., and T. N. Harris. 1966. .Effect of appendec-
tomy and thymectomy with X-irradiation. on the pro-
duction of antibodies to two protein antigens in
young rabbits. J. Immunol. 21:805-814.

Larsson, B. 1966. Venous output of H3-thymidine
labelled lymphocytes for the thymus. Acta. Pathol.
Microbiol. Scand. 68:622-624.

Li, J. C. R. 1964. Statistical inference, vol. I,
p. 437. .Edwards Brothers Inc., Ann Arbor. Michigan.

Lischner, H. W., H. H. Punnett, and AMM. D. DiGeorge.
1967. Lymphocytes in congenital absence of the
thymus. Nature 214:580-582.

Lucas. A. M.. and E. M. Denington. 1957. Effects of
total body X-ray irradiation on the blood of female
single comb white leghorn chickens. Poult. Sci. 81:
1290—1310.

Lucas, A. M.. and C. Jamorz. 1961. Atlas of avian
hematology. U.S.D.A. Agriculture monOgraph 25,
Washington, D.C.

McFarland. W., and D. H. Heilman. 1966. Comparison of
lymphocyte transformation and intradermal reactions.
Am. Rev. Resp. Dis. 235742-748.

McFarland, W., D. H. Heilman. and J. F. Moorhead. 1966.
Functional anatomy of the lymphocyte in immunologi-
cal reactions ig_vitro. J. EXptl. Med. 124:851-858.

Mills, J. A. 1966. The immunological significance of
ag. induced lymphocyte transformation ig_vitro.
J. Immunol. 91:239-247.

Mostosky, U. V. 1966. M.S.U. Vet. Clinic, unpublished
data.

Murray, R.. M. Pierce. and L. O. Jacobson. 1948. The
histological effects of X-ray on chickens with
special reference to the peripheral blood and
hemOpoietic organs. AECO 2303. 1-69.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

53

Nossal, G. J. V. 1964. Studies on the rate of seeding
of lymphocytes from the intact guinea pig thymus.
Ann. N.Y. Acad. Sci. 120:171-181.

Owen, J. J. T., and M. A. Ritter. 1969. Tissue inter—
action in the development of thymus lymphocytes.
J. EXptl. Med. 129:431-437.

Panigrahi, D. 1969. To be published in Ph.D. thesis,
Department of Pathology, Michigan State Univesity.

Payne, F.. and W. R. Breneman. 1952. Lymphoid areas in
endocrine glands of fowl. Poult. Sci. 31:155-165.

Pearson. E.S., and H. 0. Hartley. 1951. Power function
for analysis of variance tests derived from the non-
central F. distribution. Biometrica 3§_part 1 and 2.

Peterson. R. D. A., M. D..C00per, and R. A. Good. 1965.
The pathogenesis of immunologic deficiency diseases.
Am. J. Med. 38:579—604.

Peterson, R. D. A., and R. A. Good. 1965. Morphologic
and developmental differences between the cells of
the chicken thymus and bursa of Fabricius. Blood J.
Hematol. g§;269-280.

Pribnow, J. F.. and M. S. Silverman. 1967. Studies on ’
the radiosensitive phase of the primary antibody
response in rabbits. J. Immunol. 28:225-229.

Ruble. W. L., and C. Cress. 1968. Analysis of covari-
ance and analysis of variance with unequal frequen-
cies permitted in the cells (L. S. Routine), M.S.U.
Ag. Exptl. Station Stat. Descrip. No. 18. M.S.U.,
East Lansing, Michigan.

Stearner, S. P. 1951. Effect of variation in dosage
rate of roentgen rays. Am. J. Roentgenology Radium
Therapyl§§:265-27l.

Stearner. S. P., and E. J. B. Christian. 1951. The
effect of overall time of exposure upon survival
of young chicks following roentgen irradiation.
Am. J. Roentgenology Radium Therapy 65:272-276.

Stearner, S. P.. M. Sanderson. E. J. B. Christian, and
A. M. Brues. 1956. Initial radiation syndrome in
the adult chicken. Am. J. Physiol. 184:134-140.

46.

47.

48.

49.

50.

51.

52.

53.

54

Szenberg, A., and K. Shortman. 1966. Fractionation of
fowl blood lymphocytes by their buoyant density:
Localization of cells active in graft versus host
reactions. Ann. N.Y. Acad. Sci. 122:310-326.

Thor, D. E. 1967. Delayed hypersensitivity in man:
A correlate ig_vitro and transfer by an RNA extract.
Science 157:1567-1569.

Toro. I., and I. Olah. 1967. Penetration of thymocytes
into the blood circulation. J. Ultrastructure Res.
11:439-541.

Warner. N. L. 1967. The immunological role of the
avian thymus and bursa of Fabricius. Folia Biologica
(Prague) l3:1-17.

Warner, N. L., A. Szenberg, and F. M. Burnet. 1962.
The immunological role of different lymphoid organs
in the chicken. I. Dissociation of immunological
responsiveness. Aust. J. Exptl. Biol. Med. Sci.
49:378-388.

Warner, N. L., and A. Szenberg. 1964. Immunologic
studies on hormonally bursectomized and surgically
thymectomized chickens: Dissociation of immunologic
responsiveness. p. 395-413. ;g_R. A. Good and A. E.
Gabrielson (ed.). The thymus in immunobiology.
Hoeber-Harper, New York.

Wolfe, H. R.. and E. Dilks. 1949. Precipitation produc-
tion in chickens. IV. A comparison of the antibody
response of eight avian species. J. Immunol. 61:
251-257.

Woods, R.. and J. Linn. 1965. The transport of cells
from the bursa of Fabricius to the spleen and thymus.
Acta. Path. Microbiol. Scand. 64:470-475.

APPENDIX

REACTIONS ELICITED BY TUBERCULIN

OF M. avium INFECTED CHICKENS

Normal
X-irradiated
Thymectomized

Thymectomized
X-irradiated

APPENDIX

Positive

(%)
82
86

100

82_

55

Negative
(%)

9

0

AT 48 HOURS

Questionable

(%)
9
14

0

18