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mass

This is to certify that the

thesis entitled

PATHOGENESIS 0F OSTEOPETROSIS IN CHICIGINS

presented by

vance LaVerne Sanger

has been accepted towards fulfillment
of the requirements for

_P.tLJL_ degree in My

 

Pathology
/' . ‘7
flc 2%,.550
Majorjprofessor

Date November 18, 1963

0-169

LIBRARY

MiChRSdu State
University

 

ABSTRACT
PATEDGENESIS OF OSTEOPBTROSIS IN CHICKENS
By Vance L. Sanger

The experiment was designed to determine the earliest date at
which lesions of experimentally produced osteopetrosis appear and to
follow the progressive development of the lesions grossly and micro-
scopically. One hundred, day-old, Regional Poultry Laboratory (RPL),
line 15 I chicks were inoculated intraperitoneally with 0.2 ml. of
RPL strain 12, preparation L 29, lymphomatosis virus. Twelve control
chickens of the same strain and hatch were used. All chickens were
identified by wing band number. The numbers were randomized and a
group of experimental birds and their corresponding controls were
killed each week starting on day h3 after inoculation. The decision
for killing the first group on day h3 was based on preliminary work
which indicated that this was about as early as lesions could be
expected.

Laboratory procedures included radiographs of the long bones of the
legs, hematocrits, hemoglobin levels, differential leucocyte counts, serum
alkaline phosphatase measurements and gross and microscopic descriptions
of lesions in both soft and hard tissue.

Well-developed gross lesions were present on day h3. The lesions
nearly always appeared first at the center of the tibial diaphysis at

the Junction of the posterior edge of the fibula and the tibia. From

Vance L. Sanger
here the lesion spread around the circumference of the bone as well as
longitudinally, giving the bones a fusiform appearance. The enis were
never affected.

Microscopicslly, the lesions appeared to develop subperiosteally
first and appeared at the endosteum only after the subperiosteal lesion
was well developed. Or else if the subperiosteal and endosteal lesions
started simultaneously the subperiosteal lesion developed at a more
rapid rate. The osteoblastic layer of the periosteum was hyperplastic,
sometimes measuring 20 to 30 cells in thickness. Spongy bone was formed
at a rapid rate. Haversian systems did not form. Instead, the areas
that simulated Haversian canals remained much larger than normal and
. developed into irregular, large spaces,sometimes Joining others nearby.
These large spaces contained fibrous tissue, one or more capillaries,
and were lined by a row of osteoblasts. Bony spicules developed which
reached from the surface of the original, normal, cortical bone to the
periosteum. Both the spicules and spaces were reoriented from a plane
parallel with the longitudinal axis of the long bone to a perpendicular
plane. The lesions grew simultaneously in depth on the bone surface, in
length along the shaft and in circumference around the shaft.

By a means which could not be determined, a subperiosteal lesion
gradually penetrated the original, cortical bone and reached the
endosteum and from this point, spongy bone spicules developed in the
marrow cavity, gradually reducing its lumen. Or else the lesion
started at the point of deepest development at the time the periosteum
and endosteum were in close apposition. It then grew both endosteally

-and subperiosteally at the same time from that point rather than that

Vance L. Sanger
the lesion penetrated and replaced original, normal, cortical bone.

Osteoclastic activity was not part of the lesion. The lesion was
progressive and proliferative in nature and not degenerative. The
characteristics of malignancy were not evident in the osseous lesion.

No evidence of invasion of nearby tissue nor metastasis to soft tissue
was encountered. '

Early lesions were visible grossly and microscopically before they
were visible on radiographs. The bones from some chickens with osteo-
petrosis were easily broken.

Soft tissue lesions included a proliferation of the intima of
some arteries and veins to the point the lumens were partially or com-
pletely closed. Early bile duct and pancreatic carcinomas were present,
and were probably a result of the action of the lymphomatosis virus.
Some spleens contained amyloid deposits. Focal necrosis was present in
some livers. This was attributed to a contaminant virus described by
other workers that was carried in the inoculum.

Hematological studies were limited in significance. Serum alkaline

phosphatase measurements were inconclusive.

PNTHOGENESIS OF OSTEOPETROSIS IN CHICKENS

By

vance Laverne Sanger

A.THESIS

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

DOCTOR OF PHILOSOPHY
Department of Veterinary Pathology

1963

8

3907?
MM”

ACKNOWLEDGEMENTS

Appreciation is expressed to my co-advisers, Dr. C. C. Morrill,
Chairman of the Department of Veterinary Pathology, Michigan State Uni-
versity and Dr. B. R. Burmester of the United States Regional Poultry
Research Laboratory for their helpful suggestions in planning my graduate
program and for their guidance in this research.

Appreciation is expressed to Dr. Roy M. Kottman, Director, and Dr.
w. E. Krauss, Associate Director of the Ohio Agricultural Experiment
Station, for granting the sabbatical leave which made it possible to
pursue this program of graduate study and research.

Appreciation is expressed to Mr. B. Hinton, Director of the United
States Regional Poultry Research Laboratory, East Lansing, Michigan, for
his cooperation and use of the facilities, chickens and supplies and to
Dr. T. N. Fredrickson for his suggestions and cooperation in this research.

Appreciation is expressed to Dr. U. V. Mostosky, radiologist in the
Veterinary Clinic, for taking the radiographs.

Appreciation is expressed to Dr. w. D. Pounden, Chairman, Department
of Veterinary Science, Ohio Agricultural Experiment Station, for his help
and guidance, to Dr. C. R. Weaver, Experiment Station Statistician, for
his help with the statistical analyses, and to the other members of the
Department of Veterinary Pathology, Michigan State University, for their
interest and help.

Finally, appreciation is expressed to the laboratory technicians at
Michigan State, the Regional Poultry Research Laboratory and the Ohio
Agricultural Experiment Station for their assistance in preparing the

tissues and other research material for my use.

11

I.

II.

III.

TABLE OF

I m0 wCT I ON C C O O O O O O 0

REVIEW OF LITERATURE . . . . .

A.
B.
C.
D.
E.
F.
G.
H.

I.

Osteoperiastitis . . . . .
Osteomyelosclerosis . . .
Osteitis Deformans . . . .
Osteopetrosis . . . . . .
Alkaline Phosphatase . . .
GALVirus ........
Thyroid . . . . . . . . .
Nephroblastoma . . . . . .

Endothelioma e e s e e e e

MATERIALS AND METHODS . . . .

A.
B.
C.
D.

E.

Inoculum.........
Chicks . . . . . . . . . .
Laboratory Procedures . .
Necropsy.........

Histopathologic Technic .

REUL'IB O O O O O O O O O O O

A.
B.

C.

Clinical Observations . .
Gross Pathology . . . . .
Microscopic Pathology . .
(a) General Observations

(b) Nam]. Bone e e e e e

CONTENTS

e
C
e
e
e
e
e
e
e
C
e
e
s
G) -] 0‘ U) U)

. . . . . . . . . . . . . l6
. . . . . . . . . . . . . 18
. . . . . . . . . . . . . 18
. . . . . . . . . . . . . l9

. . . . . . . . . . . . . 20
. . . . . . . . . . . . . 2O
. . . . . . . . . . . . . 20
. . . . . . . . . . . . . 21
. . . . . . . . . . . . . 22
. . . . . . . . . . . . . 23
. . . . . ... . . . . . . 2S
. . . . . . . . . . . . . 25
. . . . . . . . . . . . . 25
. . . . . . . . . . . . . 32
. . . . . . . . . . . . . 32
. . . . . . . . . . . . . 3h

iii

V.

D.
E.

F.

(e)
(d)
(e)
(f)
(s)
(h)
(1)
(J)
(k)
(1)
(m)
(n)

Osteopetrotic Lesions

Muscle

Vascular'Lesions

Liver e e e e e

Spleen . .
Lung . . .
Testicle .
Thyroid . .
Thymus . .
Parathyroid

PIDCI‘Q” e

Other Tissues

Radiological Changes

Hematological Studies

Serum Alkaline Phosphatase

DISCIJSS I ON 0 O O O O

A.

B.

C.
D.
E.
F.

G.

Clinical s e e e

Bone e e e e e e

(a)

(b) Inclusion Bodies

Fractures .

Intimal Growths

Liver and Pancreas

TestLCIC e e e e e

Thyroid

MUGCI. s e s s e s

iv

F5“

37
A9
52
52

57

8‘

63
63
63
65
65
67
72

7:.
7h
75
79
80

81
81
82
83

 

I. it I'll!» all I

 

H. Spleen........
I.'lhymus........
J. Parathyroid . . . . .
K. Hematology......
L. Serum Alkaline Phosphatase
M. Work Planned for Future
SUMMARY ...........

LISTOFRIF'ERBNCE......

86

8

Table

I.
II.
III.
IV.

V.

LIST OF TABLES

ADBIYIII Of bentocritfl s e e e s e s e e e s

Analysis of hemoglobin levels . . . . . . . .

Analysis of differential leucocyte counts . .

Analysis of serum alkaline phosphatase tests

Summary of all lesions

vi

71
71

Figure

3U)”

\J'I

\0 CD —4 G\

10
11
12
13
1h

15

16
17

18

19

LIST 01“ FIGURE

Osteopetrosis in h9-day-old live chicken . . . . . . . .
Osteopetrosis in l83-day-old live chicken . . . . . . .
Gross appearance of tibia affected with osteopetrosis .
Cross section of tibia affected with osteopetrosis . . .

Low-power photomicrograph of osteopetrotic lesion
in longitudinal and CTOIB section e e s s e s s s s s e

Radiograph of double fracture of tibia . . . . . . . . .
Gross appearance of atrophic spleens . . . . . . . . . .
Early lesion showing A fundamental changes . . . . . . .
Six separate osteopetrotic lesions on the same bone . .
NormalHaversiansystem ................
Normal periosteum and beginning of Haversian system . .
Normal periosteum with single row of osteoblasts . . . .
Spongy bone and large lacunae in a lesion . . . . . . .
Transition between normal cortical bone and spongy bone.

Irregular size and shape of Reversian spaces in the

lesion e e e e e e e e e e e e e e e e e e e e e e s s e
Congestion in subperiosteal normal bone . . . . . . . .

Extent of lesion both in depth and in growth around

the bone e e e e s e e e e e e e e e e e e e e e e e e e

Rapid growth of spongy bone and formation of excess

03t901d e e e e e e s s e e e e e s e e s e e s e s e e

Orientation of bone spicules and Hiversian spaces in
”sun ray burst" appearance e e e e e e e e e e e e e s s

Gatedblafltic hyperplasia s s s e e e e e e e e e e e e e

vii

33
33
35
35
36
36
38
38
39
39

’41
h1

h2

“3

“3
“5

26
27

28
29
30

31

32
33
3h
35
36
37
38
39
ho
hl
1&2
h3
hh

Osteoblasts in mitosis in a lesion . . . . . . . . . .
Vacuolation of cytoplasm in osteoblasts . . . . . . .
Inclusion body in an osteoblast . . . . . . . . . . .
Inclusion body in an osteoblast . . . . . . . . . . .

Inclusion body in an osteoblast and a degenerating
erythrocyte nearby e s e s e e e e e e e e s e e e s e

Erythrocyte nucleus undergoing karyorrhexis . . . . .

Point of transition from margin of a lesion to normal
periosteum and bOUQ s e e e e e s e s e e e s e e s s

Hyperplasia of periosteum above normal bone . . . . .
Hyaline cartilage in 3 1.310n s s e e e e e e e e-e e
Endosteal changes in 3 ICBIOD e e s e e e e e s e e e

Endosteal changes in a well-developed subperiosteal

lesion . . . . . . . . . . . . . . . . . . . . . . . .
Atrophy of skeletal muscle . . . . . . . . . . . . . .
Lumen of small artery closed by intimal growth . . . .
Lumens of 2 arteries nearly closed by intimal growth .
Intimal growth in a small renal vessel . . . . . . . .
Intimal growth in an artery . . . . . . . . . . . . .
Internal elastic membrane in an artery . . . . . . . .
Three intimal growths in l intrahepatic vein . . . . .
Higher magnification of figure 38 . . . . . . . . . .
Intimal growth penetrating the tunica adventitia . . .
Focal necrosis in liver from GAL virus . . . . . . . .
Small focus of bile-duct carcinoma . . . . . . . . . .
Higher magnification of figure 1&2 . . . . . . . . . .

Invasion of hepatic parenchyma by bile-duct epithelium

viii

’45
1.6
146

.147

w
us

1&8

SO
SO

51

51

53

~53

5h
5%
SS
55
56
56
58
58
59

59
61

Figure Page
us AUYIOid in the Cpleen e s e e e s e s e e s e e s e e s e 61

us Higher magnification or figure “5 e e e e e s s s e e s e 62
h? TUbUlar atrOfihy 1n teBtACIG e e e e e e e e e e e e e e e 62
h8 Early malignant growth in pancreas . . . . . . . . . . . 6h

h9 Radiograph of tibias and femurs of bones which were
positive grossly but could not be seen on radiograph . . 66

SO Radiograph of control chichen for comparison with
figure R9 e e e s s e e e s e‘e e e s s e s e s e e e e e 66

51 Earliest osteopetrotic lesion found by radiograph . . . . 68

52 Radiograph of extensive osteopetrotic lesions on the
femurs, tibias and metarsals of a 60-day-old chicken . . 69

53 Radiograph of control chicken for comparison with
figure 52 e e s e e e e s e e e e e e e e e e e e e e e e 70

1.x

I. INTRODUCTION

Avian osteopetrosis is a disease which usually affects the long
bones but occasionally may affect every bone in the body. Initially,
in a long bone, a focal lesion appears on the surface of the midshaft of
the diaphysis and gradually grows both longitudinally and in circumfer-
ence until the entire bone may be affected with the exception of the
articular surfaces.

The lesions usually become clinically visible in the second or
third month of life. In uncomplicated cases, the long bones become
markedly enlarged with roughened, irregular, convex surfaces. Gaits are
affected and the chickens become anemic. In less severe cases, the
lesions may escape notice until the birds are dressed for market.
Generally males are affected more often than females.

Osteopetrosis is included in the leukosis complex because it can
be reproduced by the leukosis virus and many times it is accompanied
by some other form of the disease. Two other forms of leukosis which
frequently accompany osteopetrosis are lymphomatosis and erythroblastosis.
When either of these is present, it, rather than osteopetrosis, is
usually responsible for the death of the bird.

Osteopetrosis can be experimentally transmitted and numerous
reports of such experiments have been published. No report, however,
has shown the earliest age at which lesions appear in chicks inoculated
at 1 day of age with the virus and no report has described the pro-
gressive deve10pment of lesions from the earliest moment of change to
well-developed bone changes. It seemed desirable to attempt to answer

1

these 2 points in question which would contribute to the knowledge of
the disease. Thus the objectives of this study were: (1) to determine
the earliest date at which lesions were present either on radiographs of
the long bones of the legs, by gross examination or by microscopic study
of representative sections of several bones, and (2) to describe both
the gross and microscopic development of the lesions up to a well-

developed definitive point.

II. REVIBV OP LITERATURE

A. Osteoperiositis

Besnoit and Robin (1922) described a condition in a year-old male
chicken and published gross photographs of the long bones which had the
typical appearance of osteopetrosis. The chicken was thin and emaciated
and walked haltingly and with difficulty. He was negative to the subcu-
taneous and intradermal tuberculin skin tests. At necropsy the viscera
and Joints were negative. The muscles were atrophied and the diaphyses
of the long bones were hypertrophic and fusiferm in shape. The diameter
of the affected bones was about 3 times greater than normal. 0n cross
section the modullary canal was reduced in size. The bony wall was
thickened and the bone had a homoganous appearance.

Microscopically the Haversian canals were enlarged; the lamellae
were small and completely disorganized; the cells presumably osteocytes
were round or elongated and connective tissue was present. New blood
vessels were forming.

They stated that the condition appeared specifically as inflammation
and diagnosed the bone lesion as osteoperiostitis. They implanted a
small piece of bone subcutaneously in a rabbit and after 3 months
recovered tubercle bacilli from the rabbit. They concluded that this
was primary tuberculosis of the bone, but stated that the lesions were
not typical for those of tuberculosis.

Bell and Auger (192A) reported a condition in a chicken which
probably today would be called osteopetrosis. The long bones of the legs
and wings were enlarged and spindle-shaped. Microscopically, the perios-

teum was thickened and the osteogenic layer was hyperplastic. Beneath
3

this was a layer of newly formed bone which had been deposited on the
original, normal, compact bone. This same kind of newly formed, spongy
bone partially filled the medullary canal. There were no visceral
lesions. They raised the question as to whether this was a chronic
avisceral form of tuberculosis with only scattered tubercle bacilli
present without the formation of liquified or caseous pus. They con-
cluded that the condition they described was primary in the bones, and
therefore was apneumonic and avisceral and not related to tuberculosis.

Pugh (1927) described a condition which he called osteoperiositis
that probably was osteopetrosis. He described the disease in 7 of hh
affected chickens, all from the same flock. The disease appeared in
the sixth to seventh month of life and was not limited to l breed.
Mostly males were affected. There was enlargement of the long bones
of the legs and wings with thickening in the middle of the diaphyses and
obliteration of the moduliary cavity. The new bone was softer than
normal bone; yellow in color and cut easily with a knife. The lesions
gradually progressed toward the bone ends but the ends were not enlarged.
The total weight of the bone was increased. The periosteum was thickened.
Microscopically, the process was one of osteitis and periosititis with ‘
laying down of new osseous tissue. There were no other signs of ill
health.

Carpentier (1931) reported a single case of a condition which was
probably osteopetrosis. A male chicken, 3 months of age, was noticed to
be lame and less well developed than others of the same age. He was
killed at 5 months of age; his weight was lhOO Gm. Upon examination

the long bones of the legs and wings were enlarged and spindle-shaped

with the greatest enlargement in the centers of the diaphyses tapering
to the ends. The ends were unaffected. The subperiosteal regions were
affected with the new growth of osseous tissue which was harder than
normal. The bones were also heavier than normal bones from chickens of
the same age. The diseased tibia weighed 39 Gm. compared to 12 Gm. for
a normal bone. The medullary canal was normal. There were no visceral
lesions.

He originally thought this was tuberculosis of the bone but tuber-
culosis was eliminated after studying the flock history and killing the
100 remaining chickens in the flock for examination.

Bayon (l93h) described 2 unrelated cases of a bone disease in
chickens.

"One, a cross-bred cockerel, had thick bones of the legs. Microscopic
examination of the bones revealed a condition analogous to Paget's
disease in man and which in fowls has been previously described by Pugh
(1927). The inner layers of the internal surfaces of the bones are
eroded and carried away and the bony substance so obtained is deposited
on the outer surface of the skeleton, thus rendering the bones abnormally
thick and deformed. This perverted destruction and reconstruction are
clearly evident in the bones of another case.”

He did not know the cause. The appearance of the affected metatarsal
bones in the photograph was indistinguishable from the condition recog-
nized today as osteopetrosis.

Patsy (1935) reported on several bone malformations l of which may
have been osteopetrosis. Several hens developed a progressive thickening
of the long bones of the legs and wings and sternum. walking was diffi-
cult and affected birds remained small. Cross sections of the long bones
revealed a complete absence of the medullary canal. Microscopically,

there was evidence of both bone formation and.bone destruction. The

medullary canal was filled with bone spicules between which were canals

and spaces. Osteoblasts, multinucleoted giant cells and fibrous tissue
filled the spaces. On the surface of the bones, osteogenesis was taking
place and the surface had a roughened appearance. Parathyroids were
hypertrophic. There were no other lesions.

Brocket (1935, a) found a hypertrophic osteopathy in a bird which
had been killed and inspected for market. All long bones were affected
but not the skull or vertebrae or pelvis. The bones were large in the
center and tapered toward the ends. The tibias were most seriously
affected. The new bone appeared spongy. The medullary cavity seemed
slightly larger when compared to a normal bone but the bone size was
due to an increase in bone substance. The new bone was hard and difficult.
to saw. The bony surface was abnormal and had transverse lines. The
viscera and skin were normal.

He reasoned that this condition started with an acute general
infection, probably of respiratory origin, and spread to the bones, with
a possible endocrine predisposition which resulted in hyperossification.
He listed 7 possible diagnoses: pneumo-hypertrophic osteitis,
acromegaly, Paget's disease, tuberculosis, gigantism, osteooarcoma and
vitamin deficiency. In a later communication he concluded this was the

same condition described by Pugh (Brecket, 1935, a).

B. Osteomyelosclerosis

Seifried and Sassenhoff (who) described osteomyeloscleroois in
chickens. This was a disease of the spongiosa and not the compacta.
The spongy bone eventually filled the marrow spaces but the cortex re-
mained unchanged. Bones of the body skeleton were affected but the

bones of the extremities remained unchanged. This condition was not like

osteopetrosis as it appears today.

Seifried (19M) described a disease of the bones and of the blood
forming organs of chickens which did not resemble any other disease in
domestic animals including the marble-bone disease described by Jungherr
and Landauer. However, it did appear to be similar to the group of
diseases in humans known as osteomyelosclerotic blood diseases.

Szfics (1952) described a condition which he called osteomyelo-
sclerosis. This disease was different from osteopetrosis but some
features were similar. The spongy substance of the epiphyses and marrow
cavities gradually became more compact. In advanced cases the entire
marrow cavity was filled with bone substance. There was no evidence of
leukosis in the experimental birds and the author did not mention any
roughening of the periosteal surfaces of the affected bones. The disease
appeared at about 1 year of age; the cause was not determined.

Theiss (l9hh) described a condition in 62 chickens which he called
”osteomyelosklerose”. The number of affected chickens increased as they
grew older. He did not describe the condition except to quote other
authors. A gross photograph of the bone reveals a condition indistin-

guishable from what is known today as osteopetrosis.

C. Osteitis Deformans

Venkataraman (1936) reported that a hen and a cock developed diffuse
thickening of long bones and anemia and emaciation. The medullary cavity
. of long bones contained dense compact bone resembling ivory. No cause

was found. He called the condition osteitis deformans.

D. Osteopetrosis

Karshner (1926) was the first to use the term ”osteopetrosis" for
a human disease called ”marble bone” or Albers-Schoenberg disease. In
this hereditary disease he found an increase in thickness and density of
the cortical portion of the osseous system. He felt that "osteopetrosis"
suggested fragility as well as hardness, limestone rather than marble.

Jungherr and Landauer (1938) were the first to transmit the disease
experimentally. In the early stages, the metatarsi developed an
anterior convexity and thickening and irregularity of the surfaces.
Articular surfaces were unaffected. Affected bones were more resistant
to fracture than normal bones.

MicroscOpically, in the early stages, medullary fibrosis, increased
osteoclasia and degeneration of old bone were found. Trabeculae showed
stippling with a fine basophilic dust-like material.

Newly formed bone consisted of many irregular-sized blood spaces
which contained a dense, central, fibrous material, osteoblasts and a
few osteoclasts. There was a reduction in the marrow cavity in affected
bones. Later, Jungherr (1959) stated that in comparison with other forms
of leukosis, the occurrence of osteopetrosis was rare. In the initial
phase of the disease he found sequestration, granular degeneration of
old trabeculae, marrow fibrosis and increased osteoclasia. These changes
were accompanied by new large-celled, vascular, fibrous bone tissue.
Later, new bone gradually replaced the original spongiosa and compacts
while osteoclastic activity regressed. In the arrested phase, new bone

lamellae appeared condensed, hypercalcified and subdivided by numerous,

thick, irregular, cement lines. Secondary anemia was common because of
the progressive reduction of hemopoietic tissue.

Brandly (l9hl) injected embryonated eggs with whole blood and
washed erythrocytes. In 7 to 10 days, the liver, spleen and diaphyses of
the long bones of the legs and wings were enlarged. Microscopically,
bone changes were similar to those seen in osteopetrosis. Brandly,
Nelson and Cottral (l9hl) produced lymphomatosis and osteopetrosis with
strain 3 of their agent. Osteopetrosis occurred in 9 percent of ino-
culated birds. Some of these lesions were visible grossly. Others
were found microscopically. More males than females were affected.
Brandly, Nelson and Cottral (l9h2) obtained their strain 3 inoculum
from‘White Leghorn hens suffering from ocular lymphenatosis. Strain 3
inoculum caused visceral, neural and ocular lymphomatosis and osteopetro-
sis. One chicken inoculated at 21 days of age had clinical osteopetrosis
57 days later. Another chicken developed osteopetrosis 300 days after
inoculation. Osteopetrosis was found in 9 percent of inJected birds.

The sex ratio of 25 affected birds was 17 males and 8 females. There was
hyperpyrexia of affected bones as well as a marked enlargement in the
diameter of the long bones. A few skeletal bones were affected. Micro-
scopically, there was increased cellular activity of the periosteal
region, blood spaces were enlarged and irregular; leukemic blood changes
were not found in the osteopetrotic birds although they frequently were
anemic. The medullary cavity was obliterated in some bones. Gross

examination of the parathyroid glands in osteopetrotic birds revealed

no changes.

10

Duran-Reynals (191:2) injected chickens with Rous sarcoma virus
and produced osteopetrosis. The gross and microscopic appearance of
the lesions was the same as that described by Jungherr and Landauer.

The parathyroid glands were not enlarged. Osteopetrosis did not develop
in ducks injected with Rous sarcoma virus. Bieley (l9h3) found osteo-
petrosis in a chicken. The long bones were enlarged and irregular in
outline. The vertebrae and skull were unaffected and there were no

soft tissue lesions. Moynihan (l9h3) reported osteopetrosis in 7
chickens, 1 male and 6 females. The chickens were anemic and oligo-
cythemia was a constant feature. Leukemia was not observed. All bones
except the skull and vertebrae were affected. Lesions were characterised
by hypercalcification resulting in partial or complete obliteration of
the marrow cavity. Bone surfaces were rough and irregular and diseased
bones were several grams heavier than bones from healthy chickens of the
same age. . ‘

Thiersch (l9hh) inoculated blood from human patients suffering from
myeloid leukemia into ll-day-old chick embryos. Only a small percentage
of the injected chicks survived; of 12 chickens hatched from the inocu-
lated embryos, 5 developed osteopetrosis. Similar lesions were obtained
by injecting day-old chicks. OsteOpetrosis could be induced with blood
from 3 different patients with chronic myeloid leukemia. Injection of
blood from human subjects with chronic lymphoid leukemia did not induce
osteopetrosis.

Coles and Bronkhorst (l9h6) observed osteopetrosis occurring
equally in both sexes. Affected bones were exceptionally hard and did

not fracture easily. The ends of the bones were unaffected. They

11

reported that susceptibility to osteopetrosis was an inherited recessive
character.

Burmester, Prickett and fielding (19h6) inoculated 2-3-dayeold-chicks
with cell-free preparations of RPL 12 lymphoid tumor. By 10 weeks of
age, clinical manifestations of osteopetrosis were seen and by 6 months
of age, kl percent had osteopetrosis.

Affected bones were irregular in outline, warmer than usual and hard
to the touch. The pathological changes were confined to the diaphysis.
The narrow cavity was reduced in diameter by deposition of spongy bone.
The periosteum was hypertrophic. Microscopically there was hyperplasia
of the periosteum and an increase in its thickness with formation of new
and abnormal cancellous bone beneath the periosteum. Numerous irregularly
placed marrow cavities contained hyporplastic tissue which appeared to be
bone marrow. The normal diaphyseal architecture of bone was completely
altered.

Burmester and Cottral (19%?) stated that there was some evidence to
suggest that osteopetrosis and visceral lymphomatosis were produced by
different agents and that either might remain latent in recipients and
become overt in subsequent passages.

Burmester (19%?) found that RPL strains 18 and 21 produced osteo-
petrosis as well as visceral tumors.

In summarising the work at the Regional Poultry Research Laboratory,
Hinton (1951) reported that visceral lymphomatosis and osteopetrosis had
been transmitted with cell-free filtrates which indicated that these forms
of leukosis were caused by a filterable virus-like agent or agents. He
suggested that osteopetrosis may have a causative agent separate from the

one causing visceral lymphomatosis.

12

Pearce and Brown (191:8) and Pearce (19h8, 1950a, 1950b) described
osteopetrosis in an inbred group of rabbits. The condition was an
inherited, lethal characteristic that somewhat resembled.Albors-Shoenborg
disease in man. Bone lesions, as well as several other anomalies, were
present at birth. The bones were hard but brittle. Spontaneous fractures
were rare.

Radiographs revealed clubbing of the ends of ribs and long bones,
shortening of the long bones, increased bone density, reduction of the
marrow cavity and scanty marrow.

The periosteal surfaces of the bones appeared normal but there was
size reduction. The bones felt tough and fibrous when cut with a knife.
Osteocytes were numerous, many were pyknotic and a variation in
size and staining reaction was noticed. Groups of 2 to 6 osteocytes were
crowded together and formed small cellular feel. When compared to nor-

mal bone, osteoclasts were reduced in number in some areas and more
numerous in other fields.

Fibrous tissue was prominent in the marrow cavity among the
trabeculae and extended into the cortex and reached the periosteum in
some places. Small cysts were frequently seen in the fibrous tissue.
There was actual failure of development of marrow cavity and marrow
tissue.

The primary parathyroid glands were enlarged and aberrant glands
were found along the cervical area. This was in contrast to small glands
and only a few aberrant glands in controls. Microscopically more cysts
were found in the glands of the affected rabbits than in those of

controls.

13

HemOpoietic centers were far more numerous in the livers of
affected rabbits than in those of the controls.

Davakula (1953) reported that ”in comparison with the frequency
of the occurrence of other forms of the avian leukosis complex, osteo-
petrotic lymphomatosis was rare.” ”Clinically, observation and palpa-
tion of long bones, as the metatarsi, may reveal abnormal convexities
or irregular thickenings of the affected regions. Gross alterations of
the long bones were observed in various degrees depending on the stage
of development of the disease. Hematological and radiological examina-
tion may also confirm the evidence of avian leukosis complex.”

In a discussion of the classification of the different pathological
expressions of leukosis, Campbell (l95h) suggested that osteopetrosis
should be removed from the leukosis complex and set apart as a distinct
pathological entity.

Over a 2-year period in the examination of hh59 chickens for
diagnostic purposes, Jordan (1956) found 2 birds with osteopetrosis.

Holmes (1958a, l958d, 1959‘, 19590. 1961c, 1961f, 19638) Published
a series of papers in which he described the results of his experimental
work on osteopetrosis in chickens and turkeys and observations he made
as a result of the work. He transmitted osteopetrosis by the use of
whole blood, unfiltered and filtered plasma and bone marrow. The earliest
lesion was detected by palpation at 35 days of age. Lesions developed
later in turkeys than in chickens. The earliest radiological detection
of osteopetrotic lesions was at 37 days following inoculation at 1 day
of age. Embryos which were inoculated and allowed to hatch developed
osteopetrosis. Lesions were characterised initially by subperiosteal
hyperemia followed by the formation of new subperiosteal bone. The
earliest changes occurred on the diaphyses of the long bones. The entire

skeleton was sometimes affected, including the skull and vertebrae.

Bones grew wider and denser than normal. The subperiosteal lesions

1%

‘were later followed by endosteal lesions with narrowing and obliteration of
the marrow cavity. Initially the radiological appearance of very early
lesions was that of periostitis. This periosteal reaction was the
characteristic response of long-bone periosteum to any stimulus and may
appear in a number of conditions. These primary changes in osteopetrotic
lesions were constructive and not destructive and therefore osteopetrosis
should not be considered a neoplastic disease. He further suggested
that osteopetrosis may be caused by a virus separate from the leukosis
virus; therefore this disease should be considered as a separate entity
from the leukosis complex and avian neoplasia. Cortisone injections did
not influence the development of the disease in young chicks. Osteopetrosis
resembled infantile, cortical hyperostosis (Caffey's syndrome).

In testing strains of leukosis virus used for research which were
obtained from places other than the U. 8. Regional Poultry' Research
Laboratory, Burmester,‘Walter, Gross and routes (1959) found that cell-
free preparations of myeloblastosis strain A caused osteopetrosis but
similar preparations of erythroblastosis strain R did not. Burmester,
Gross, waiter and Fontes (1959) also found that osteopetrosis was
common in 5 of the 7 preparations of RPL 12 virus given parenterally
which they tested. It did not occur in chickens inoculated with oral
washings, fecal extracts or embryo extracts of naturally infected hens.

Gross, Burmester and waiter (1959) reported that in the early stages
of uncomplicated osteopetrosis, diseased birds had an anemia, unthrifty
appearance.

IMany suffered from leg fractures and all long bones could be

crushed easily at necropsy.

15

Microscopically the first visible alteration was an increase in
size and number of the cells in the deep layer of the periosteum,
endosteum and Haversian canals. This proliferation progressed rapidly,
and assumed neoplastic features so evident by the partial lysis of the
compact bone. New cartilaginous tissue was laid down in the lytic spaces
and mineralization followed. This produced bone with an imperfect
lamellar system.

A markedly atrophic and fibrous spleen frequently accompanied
osteopetrosis and occasionally a cirrhotic liver. Osteopetrosis was
considered a neoplastic disease. However, a question regarding malig-
nancy was raised because the proliferative process eventually subsided.

Ressang (1960), in a discussion of the avian leukosis complex,
reported that leukosis had been recognised in Indonesia since 1928 but
was not considered to be of economic importance. The visceral, ocular
and neural forms had been diagnosed but osteopetrosis, erythroblastosis,
granuloblastosis and myelocytomatosis had not been diagnosed.

Darcel (1960) stated that osteopetrosis was a non-neoplastic bone
malformation.

Campbell (1961) found that inoculation of day-old chicks with blood
from a florid case of osteopetrosis produced well-developed lesions in
lZ-lh weeks, especially in males. Affected bones had a thickened periosteum
and were highly vascular.

The initial lesion on x-ray was an endosteal bone proliferation fol-
lowed by progressive laminar deposition of periosteal bone. The medul-
lary cavity was eventually obliterated by new bone with an abnormal

architecture due to enormously enlarged and elongated Haversian canals

16

extending centripetally toward the shaft center. These vascular spaces
contained no marrow but had a scanty fibro-cellular tissue. Anemia

followed which stimulated an extramedullary myelopoiesis in the liver

and kidneys.

He further stated that osteopetrosis was not a neoplastic condition
but rather an osteopathy characterised by hyperplasia. It was associated
with a virus. It had not occurred in conjunction with lymphoid and mye-
loid leukosis and spontaneous development was unknown. No experimentally
induced osteopetrosis cases developed any leukosis. It cannot be said
that the leukoses and osteopetrosis had a common etiology.

DeVolt (1961) reported that osteopetrosis was being encountered

with increasing frequency in turkeys.

H. Alkaline-Phosphatase

Changus (1957) analysed osteoblasts for alkaline-phosphatase activity
in both malignant and nonmalignant diseases. He found that in all bone
lesions from patients with fibrous dysplasia of bone there was high
alkaline-phosphatase activity in the osteoblasts along the margins of
bone trabeculae and in almost all those cells distant to the bone
trabeculae that have been called fibroblasts.

The periosteum removed from tibias not directly involved with
specific disease had abundant alkaline-phosphatase activity in the inner
layer adjacent to the bone. Histochemically, these cells are interpreted
as osteoblasts. The outer layer of the periosteum had little or no
alkaline-phosphatase activity, and the cells composing this layer were

interpreted as histochemical fibroblasts.

17

In 5 cases of fibrous dysplasia of bone the serum alkaline-
phosphatase levels were within normal range and varied from 2.5 to 3.7
Bodansky units. The inner layer of the periosteum was rich in alkaline-
phsophatase activity but the outer layer was poor in or devoid of this
activity.

Osteogenic sarcoma of the bone was high in histochemical alkaline-
phosphatase activity while fibrosarcomas of the bone were devoid of this
activity.

The data presented here demonstrated that the osteoclasts, giant
cells and macrophages all had acid-phosphatase activity and no alkaline-
phosphatase activity. This indicated that the osteoclasts, like other
reactive giant cells, were derived from epitheloid cells or macrophages
and not from pro-existing osteoblasts or fibroblasts. The findings also
indicated that alkaline-phosphatase does not participate in decalcifica-
tion of the bone but that acid-phosphatase does.

Fibrous dysplasia is an exaggerated hyperplasia or response of
osteoblasts to some unknown stimulus.

Bell'gt’gl, (1959) found an increase in plasma alkaline-phosphatase
in laying hens suffering from cage layer fatigue. The bones were exten-
sively decalcified and flexible. Histologically, there was marked
osteoclastic activity and thinning of the compact and spongy bone.

Siller (1959) described an osteogenic sarcoma from the proximal end
of the right tibia of a 7 year old hen. Mitotic figures were present
and nucleoli of the osteoblasts were large and prominent in the primary
tumor. There was invasion of muscle and metastasis to the kidney and

lungs. The metastatic tumors resembled the primary tumor and were

18

unencapsulated and infiltrative. Calcification was present and mitotic
figures were numerous. The plasma alkaline-phosphatase measured 360
units compared to 25 to 120 units for a normal non-laying hen.

Bell (1960) found that the plasmas of immature birds (5 to 6 weeks
old) of both sexes had alkaline-phosphatase levels up to 10 times those
found in non-laying adults. In the hen, the average level of plasma
ensymic activity increased by about 50 percent when laying began. At
this time, also, wide fluctuations became apparent. He attributed the
rise to increased osteoblastic activity.

Hurwits and Griminger (1961) found enlarged parathyroids, elevated
plasma alkaline-phosphatase and loss of bone material in calcium-

deficient hens.

r- lax-.1222

Sharpless and Jungherr (1961) reported that a Callus adeno-like (GAL)
virus was isolated from a lymphomatous liver which produced areas of
necrosis in the livers of experimentally infected chicks. The presence
of this virus is transmissible lymphomatous virus preparations might
explain earlier reports of a primary necrotising agent in transmissible
avian lymphomatosis.

In his studies on inflammation, Menkin (l9h8) reported that a sub-
stance called necrosin was released by damaged cells and caused thrombosis
of small local lymphatics and cloudy swelling, vacuolation and granularity

Of cytoplasm of liver cells.

6- Thmid
(Sable (l95h) described atrophy and degenerative changes in the

thyl‘oid glands in dogs infected with Trypanosoma crusi. He found that

l9

”grossly the thyroid glands were normal in size, configuration and con-
sistency. Microscopically, however, the follicles were small and con-
tained little or no colloid. The epithelium was uniformly cuboidal and
showed no tendency to become columnar or to form proliferative papillae.
Many of the follicles contained spherical agranulocytic cells with
eccentric nuclei. There was no evidence of acute inflammatory process,
fibrosis of either capsule or stroma. The changes were diffuse rather
than focal.

He believed these changes were caused by thyrotoxic substances but he

did not know the site of origin.

H. Nephroblastoma

Walter, Burmester and Cunningham (1962) reported that the HA1
strain A (myeloblastosis) virus produced nephroblastomas. These tumors,
in turn, were passed and both cell suspensions and cell-free filtrates
produced tumors as well as forms of leukosis including granuloblastosis,
lymphomatosis and osteopetrosis.

These avian renal tumors should possibly be considered an additional
member of the group of diseases known collectively as the avian leukosis
complex.

I. Endothelioma

Furth described endothelomas in chickens injected with his strain 2
leukosis virus. Tumor also varied from microscopic to 7 cm. They
developed in 2132 through proliferation of preformed endothelium. How-
ever, many times the endothelial, mesenchymal or mesothelial origin could
not be determined. Necrosis and giant cells usually occurred in the tumors.
Many tumors formed cavities and channels in which hemocytoblasts and erythro-
blasts and erythrocytes were present. These blood cells originated from
detached, swollen, endothelial cells. Many tumors were accompanied by
hematomas. Cells composing the tumors were sarcoma-like or multinuclear

giant cells. Some tumos were invasive.

III. MATERIALS AND METHODS

A. Inoculum

The inoculum was preparation L 29 of the RPL 12 lymphomatosis virus
maintained at the U. S. Regional Poultry Research Laboratory. The dosage
was 0.2 ml. of a S-percent extract not further diluted, given intra-
peritoneally. The extract was prepared from the liver of a chicken
suffering from erythroblastosis. Preparation of the virus was begun
by homogenizing a S-percent suspension of liver in Sims' solution in a
Waring blender for 20 minutes. This preparation was centrifuged for 30
minutes at 20,000 revolutions per minute under refrigeration. The super-
natant was removed and again centrifuged for 30 minutes at the same speed
under refrigeration. It was then sealed in vials and stored at -70

Fahrenheit until used.

8.22.122

The chicks were hatched at the Regional Poultry Research.Laboratory
from line 15 I Leghorns maintained at this laboratory and were inoculated
when they were 8 hours old. Only cockerels were used and each chicken
was wingAbanded at the time of inoculation. One hundred chickens were
given virus and placed in an isolation room in a battery for the first h
weeks; then they were transferred to a separate room and placed on the
floor which was formed by wire stretched over 2 X h" boards placed on
edge. Feed was placed in metal trough feeders. Water dripped continuously
from a faucet into an overflow cup. Ventilation was by window from the

outside.

21

Thirty-six control chicks from the same batch were housed similarly
but protected from exposure to the virus or to inoculated chicks.
The numbers were randomized, and 15 experimental and 3 control

chickens were killed each week starting at h3 days of age.

C. Laboratory Procedure

Just prior to killing each bird, 2 milliliters (ml.) of blood were
drawn from the wing vein or by cardiac puncture. One ml. was deposited
in a clean, dry test tube and allowed to clot so that serum could be
obtained.

The remainder was placed in a tube containing 0.1 ml. of a lO-percent
solution of the anticoagulant, ethylenediamintetraacetic acid (BDTA),
which had been oven dried.

Blood smears were made in duplicate, air dried and stained with
Wright's stain.

For the hematocrit measurement, a plain capillary tube, 75 milli-
meters (mm.) long and 1.3 to 1.5 mm. in diameter was filled and one end
plugged with a vinyl plastic putty. The tube was placed in a micro-
capillary centrifuge and spun for 5 minutes at 11,500 revolutions per
minute (RPM). The packed cell volume was read in percent of the total
column from a micro-hematocrit reading device.

For hemoglobin determination the acid-hematin method of Banlowski, as
described by Fredrickson (1963) was followed. Two m1. of 0.h percent
aqueous ammonium hydroxide was placed in a colorimeter tube. Two-tenths
ml. of fresh whole bleod measured in a Sahli pipette was added and
thoroughly mixed. After 5 minutes, 3 ml. of tenth normal hydrochloric
acid was added, mixed and allowed to stand 20 minutes. This solution was

then read in a Bausch and Lomb colorimeter which had been standardized

22

by a hemoglobin sample prepared the same way from dog‘s blood and
compared with the reagent blank and hematin standard.

The clotted blood samples were centrifuged immediately upon return-
ing to the laboratory from the necrOpsy room where the chickens were
bled.

The serum alkaline-phosphatase level was measured on the fresh serum
as soon as it could be separated from the coagulated blood sample. This
enzyme is not very stable; therefore the test was completed as quickly as
possible in order to avoid any loss of enzyme activity. The enzyme was
measured in Sigma units according to the method described by the Sigma
Company (1960).

Radiographs were made of the legs of 97 of the 112 birds in the
experiment. The other 15 either died at an early age or at a time that
was not convenient for taking the radiograph. The skin and feathers
covering the femurs, tibias and fibulas was removed. The flesh was left
intact. The legs of each pair were placed on their lateral surfaces
with the femurs directed toward the top of the film so that the left leg
facing the viewer in the radiograph was the right leg of the bird.

Kodak, non-screen, ready-pack X-ray film was used. The focal
distance was 36 inches and the exposure was 15 milliamperes per second

(MAS) in hh-kilivolt peak (KVP).

D. Necropsy

Relatively complete necropsies were done on all chickens. Brain,
thyroid, thymus, lung, heart, proventriculus, duodenum, pancreas, liver,
spleen, kidney, adrenal, gonad, cranium, and all long bones of the right

wings and right legs were saved in 10 percent neutral formalin. Bones of

23

the left leg and pieces of liver, kidney, spleen, adrenal, duodenum and
pancreas were saved in Zenker's fixative. Liver, kidney and adrenal were

also fiked in Carnoy's fixative.

E. Histopathologic Techniques

For routine sections all tissues were embedded in paraffin, sec-
tioned at 6 microns and stained with hematoxylin and eosin. Special
stains included Masson's trichrome, Van Gieson, Perls' iron stain,
periodic acid Schiff (PAS), Hansen-Bock stain for bone, Weil-Weigert's
elastic stain, and crystal violet for amyloid. Histologic techniques
described by Lillie (195h) and the manual of the Armed Forces Institute
of Pathology (1957) were followed.

Bone segments were prepared by sawing cross-sections from the
approximate centers of the shafts of long bones and longitudinal sec-
tions from the proximal third of the tibia and femur in a line to
approximately divide the bone into two equal parts. The longitudinal
sections of the proximal ends of the femurs were cut on a medic-lateral
plane Just anterior to the head. The posterior half was used for
sectioning. The tibial sections were sawed on the antero-posterior
plane Just medial to the fibula. The lateral half was always saved for
sectioning and in most instances the stained section included part of
the fibula.

A cross section of both frontal bones was taken at the level of the
center of the cerebral hemispheres.

The proximal end of the humerus was sawed on a dorse-ventral plane
and the anterior half saved for sectioning. Occasionally cross-sections
of the radius and ulna were taken from the middle thirds of the

diaphyses.

2h

A Jeweler's saw with 52 teeth per inch was used for sawing the
bone sections.

Bones were demineralized in "Decal*".

 

’Scientific Products, Evanston, Illinois.

IV. RESULTS

A. Clinical Observations

Chickens suffering from osteopetrosis were stunted in growth and
had a pale appearance around the head and wattles and the feathers were
rough and ungroomed (Figures 1 and 2). Affected birds walked with a
limp or a stilted gait and lifted the feet higher than usual when walk-
ing. Some birds sat down most of the time unless disturbed.

Usually the first visible sign of osteopetrosis occurred as a
swelling on the metatarsi with gradual enlargement and development of
a convex curvature on the anterior surfaces. Palpation of the other
long bones of the legs and wings revealed increased size and roughened
surfaces. These changes were accompanied by a decrease in the thickness
and size of the muscles covering these bones. Affected bones were warmer
to the touch than those of normal chickens.

At necropsy, when the lengths of the femurs and tibias of the
stunted birds were compared with bones from the same age controls, the
bones from the sick birds were shorter than controls but this was
considered to be a result of reduced growth rather than the effect of

the disease on the individual bones.

B. Gross Pathology

As a result of preliminary work on osteopetrosis at the U. 3.
Regional Poultry Research Laboratory (Fredrickson and Sanger, 1961) it
was decided to kill the first group of chickens at h3 days of age.

Fifteen experimental and 3 control birds were killed initially and each

25

26

 

Figure l. Porty-nine-day-eld chickens. Chicken on the left was
injected with lymphomatesis virus at 1 day of age and has well-
developed osteopetrotic lesions. Compare with figure 2.

27

 

Figure 2.

Chickens 183 days of age. The chicken on the left was
inJected with lymphomatous virus at 1 day of age and has extremely
large, osteopetrotic lesions.

28

week after that until the entire group was gone. Statistically it was
found that 3 controls were adequate for each group of 15 experimental
birds.

Sixty birds had gross lesions of osteopetrosis. In nearly every
instance, the earliest appearance of a gross lesion was on the center of
the diaphysis of the tibia at the posterior edge of the fibula. The
lesion increased in depth at this point and at the same time advanced
rapidly from this point posteriorly around the shaft and toward each end
of the bone giving the bone a fusiform appearance (Figure 3). The prog-
ress of the lesion anteriorly from the fibula was much less rapid. In
advanced lesions the two edges of the lesion eventually met on the
anterior surface of the tibia and stopped at the extremities of the
shaft so that the ends of the bones were never affected. Occasionally
a lesion started first in either the proximal or distal third of the
shaft, and on a few bones 2 separate foci were visible at the same time.

On the femur, the first lesion nearly always appeared on the proximal
third of the diaphysis on the antero-medial border. From this point it
progressed around the circumference of the shaft and also developed
distally.

The lesion appeared as a distinct, pale, yellowish focus in contrast
to the grayish-white to translucent appearance of the normal bone. Except
in the very earliest lesion, it was always elevated above the level of
the adjacent bone surface. The periosteum was thickened and the surface
of the lesion was less firm to the touch than the normal bone. A knife
easily cut through the new osseous tissue until the original compact
bone was encountered. If a piece of the new growth was removed and

pressed between the fingers, it gave the impression of a piece of firm

29

W

 

Figure 3. Upper tibia is from a Sh-day-old chicken inoculated
at 1 day of age. Notice the enlargement of the diaphysis. The
lower tibia is from a control bird of the same age.

3O

cheese or caseous material which did not crumble easily, and it had a
distinct pale, yellowish color.

Cross section of an affected bone revealed an eccentric appearance
of the cortical bone, the affected wall being noticeably thickened while
the opposite wall was thin and normal in appearance with the medullary
canal appearing to be situated near one edge of the bone rather than
in the center (Figure h).

If the lesion had not completely surrounded the bone, the edges of
the newly forming bone tapered to a fine line which blended with the
normal periosteum and bone. If it had progressed completely around the
shaft, the entire cortical wall was thickened and yellowish in color
but one side was always thicker than the other. In the more advanced
lesions, the medullary canal was reduced to a small opening. In this
experiment, in these early stages of development of the lesion, the
medullary canal was always visible but in older, more advanced lesions
the medullary canal may be completely obliterated.

On the longitudinal section of bone cut through an advancing lesion,
the new growth could be followed from where it tapered from a notice-
able elevation to a fine line that merged imperceptibly with the un-
affected bone covered by normal periosteum (Figure 5). The contrast in
color between the lesion and normal bone was quite apparent on the cut
surfaces. The newly formed bone could be broken and crumbled away from
the normal bone by slight pressure with the fingernail or a metal
instrument.

The long bones of the wings were affected in some chickens and in
1 the sternum had osteopetrotic lesions. Gross lesions were visible in

the bones of 11 of 15 birds that were killed on day h3.

31

   

METRIC I 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure h. Tibias from 60-dey-old chickens. Notice
the large osteopetrotic lesion, most prominent on
the posterior surface of the tibia on the riat,
from a chicken that was inJectod at 1 day of age.
Control on left.

32

Many birds, when they were examined, had fractures of long bones
which apparently had occurred spontaneously or were unknowingly caused
when they were caught for selection of the birds to be killed each week.
At necropsy, many bones were broken from manipulation during dissection
(Figure 6).

The spleens in some chickens were atrophic while in others there
were no changes. The color and consistency were unaffected. The dimen-
sions of atrophic spleens from 2 experimental birds and 1 control of the
same age were compared. The atrophic spleens each measured 8xhx3 and
9x5x5 millimeters compared to 17xllxlO millimeters for the control
(Figure 7).

Many of the experimental birds also had erythroblastosis and,
early in the experiment, deaths occurred rapidly for awhile because of
this condition. In birds with erythroblastosis the livers were enlarged,
dark red to mottled and sometimes they were covered by a yellowish,

fibrinous pseudomembrane. Ascites was also present.

C. Microscopic Pathology

These findings are summarized in Table 5, page 73.

(a) General Observations

Seventybthree chickens had osteopetrosis. Thirteen of these had
microscopic lesions which would have been missed if the diagnosis had
been based only on the gross observation. The earliest and smallest
lesions that were found microscopically presented h distinct character-
istics which distinguished the lesion from normal bone. So far as
could be determined they all appeared simultaneously. These changes

included, (1) a deep basophilic staining of the new osseous tissue

33

 

   

Figure 5. Osteopotrotic lesion on longitudinal and cross section of
tibia. Notice how the lesion diminishes in height and merges imper-
ceptibly with the normal periostou and bone (1). Nematoxylin and
Bosin. x 7.

I“ V 1
/.

Figure 6. Radiograph showing double fracture of right
tibia with displacement of one fracture. Fractures
were co-on in experimental birds.

3h

(2) an increase in size and irregularity of the newly forming Haversian
canals, (3) an increase in number and change in position of the lacunae
and, (h) a fibrous appearance to the new bone (Figure 8). A fifth
characteristic, present in most lesions but absent from a few, was a
remarkable increase in the number of layers of osteoblasts in the
periosteum.

The early lesions were always located under the periosteum. No
endosteal lesions were found where there were not already subperiosteal
changes and no endosteal changes occurred until subperiosteal changes
were well advanced. The lesions were progressive and proliferative in
nature and not degenerative.

In most instances a single focus of osteopetrosis was present on the
bone. In a few, 2 or 3 separate foci were found and on 1 bone, 6 small,
separate and unrelated foci were found. All of these were subperiosteal
and 1 reached from the endosteum to the periosteum (Figure 9). Occasion-
ally a well-developed osteopetrotic lesion was present on 1 side of the
diaphysis and on the opposite side a new, very small lesion was found

which bad Just started.

(b) m P...
Normal compact bone was eosinophilic, dense, and homogenous. It had
a distinct and uniform structure formed by many Haversian systems.
Mature osteocytes lying in the lacunae were deeply basophilic and
shrunken from fixation. The lacunae appeared as small, oval to fusiform,
open spaces regularly spaces in concentric circles around a small, cir-
cular Haversian canal (Figure 10). The periosteum contained 2 layers.

The inner layer adJacent to the bone surface consisted of a single row

35

 

meme ’1 2

Will IH HH HH

Figaro 7. Two atrophic spleens on the rifit from chickens with
osteopetrosis compared to spleen from same age control chicken.
Atrophy of the spleen was a canon finding in chickens with
osteopetrosis. All chickens were 5h days of age.

 

 

 

 

 

 

Figure 8. Early lesion which depicts the is fundamental changes in
an osteopetrotic lesion: (1) basophilic staining of new bone,

(2) large irregular Reversion canals, (3) increase in number and
size of lacunae, and (h) fibrillar structure of new bone. Normal
bone is to the left in the picture. Hematoxylin and Rosin. x 219.

 

36

. _ - q -
. . ,, .
-- ‘Clx 'e‘F/If "A’i.

 

Figure 9. Six separate, subperiosteal, osteopetrotic foci on
the humerus. Indostsal changes have occurred at one place.
Nematoxylin and Bosin. x 18.

 

Figure 10. An [aversion system in nornl bone with a small
round Reversion canal and concentric lamellar rings surround-
ing it. Nematoxylin and tools. x 5&7.

37

of osteoblasts. The outer layer was composed of fibrous tissue whose
fibers ran in parallel lines with thin elongated nuclei lying along the
course of the fibers. The fibers lay in the same plane as the longitu-
dinal axis of the bone. If a new Haversian canal was forming on the
surface, the osteoblasts appeared to be increased in number at this

point (Figures 11 and 12).

(c) Osteopetrotic Lesions

All osteopetrotic lesions were essentially the same with a few
exceptions which will be mentioned. A single description will be made
of the lesion in which the progressive development of the microscopic
changes will be described starting with an early lesion and progressing
to the well-developed stages of the disease.

The newly forming, spongy bone was deeply basophilic. There were
no Haversian systems present because of complete disorganization of bone
structure. Osteocytes were large and eosinophilic. Lacunae were large,
irregular in shape and were present in countless numbers (Figure 13).
Many lay adJacent to others and often several opened into adJoining
lacunae forming a large open space. The bone tissue appeared as ir-
regular fine fibers with no systematic arrangement. In early lesions
adJacent to normal compact bone, these fine, basophilic lines lay between
Haversian systems and penetrated to deeper parts of the cortical wall
(Figure 1%). Baversian canals were greatly increased in size and the
shape varied from round to irregular geometric designs (Figure 15).

The spaces were separated only by narrow spicules of spongy, fibrous-
appearing bone (Figure 15). They were either filled with osteoblasts or

contained only a single row around the periphery of the canal, in which

38

 
      
 

\\_\

  

.|_
.‘ \b

\
\

  

‘ _
\
a-

A

Figure 11. Poriostoum and early developsnt of Reversion
canal in normal bone. Open spaces are fintisu artefacts.
lsmatoulin and Rosin. x 957.

.——e; . .’.//J .

 

 

Figure 12. Normal periosteum with a single row of osteo-
blasts on the bone surface (1). Longitudinal section of
laversian canal in normal bone below. Clear space is an
artofact. losstoxylin and losin. x 501.

 

Figure 13. Spongy bone and large, irregular spaces in osteo-
petrotic lesion. Notice the large, irregular lacunae which
are so numerous that they touch and sometimes coalesce. no
large spaces are filled with fibrous tissue. Compare with
figures 10 and 11. Nematoxylin and Rosin. x 51.0.

 

- . , . ' " .
‘ '7 .' ,". L". . ‘ .'

Figaro 1h. Gradual transition between normal compact bone
and basophilic, spongy bone at the margin of an osteopetrotic
lesion (1). Nematoxylin and losin. x H.

hO

case they contained increased amounts of a fibrous network of tissue
containing a few fibroblasts or modified osteoblasts. There were 1 to
several capillaries in each canal. Occasionally a lesion was found in
which there was congestion of all capillaries but this was unusual and
the same condition occurred in normal bone (Figure 16). Therefore this
was considered physiological and not a pathological entity. There was
never any hemorrhage or necrosis.

As the lesion progressed, it develOped peripherally around the
circumference of the bone surface, extended itself toward each end of
the bone and at the same time increased rapidly in depth by formation
of spongy bone at the surface (Figure 17).

At the margin of the lesion, the newly forming Haversian canals
were at once abnormal in shape and size and in the center of the pro-
gressive lesion, the canals were altered to a greater degree. Instead
of forming a small circular canal, the appearance changed to that of a
large, irregular space which sometimes opened into adJoining spaces
forming even larger, open, irregular spaces. Between these large spaces
were long spicules of fibrous, highly cellular, spongy, new bone which
reached to the surface of the lesion under the periosteum. They were
capped by a thick layer of osteoid tissue. The fibrous strands and
cytoplasm of these highly active osteoblasts covering the tips of the
spicules were forced into a convex, saucer-shaped cap because of the
extremely rapid growth at these points (Figure 18).

The direction of the canals also changed from running parallel with
the longitudinal axis of the bone to a position at right angles to the
long axis. In the well-developed lesions this gave the effect of rays

of light radiating out from a central focus (Figure 19).

hl

 

Figure 15. Notice the ruarkablo irregularity in size
and shape of the Eversian spaces in an osteopetrotic

lesion. Compare with figures 10 and 11. Nematoxylin
and losin. x 55.

 

Figure 16. Congestion of capillaries in subperiosteal,
usual bone from control chicken. his condition was found
in only a few experimental and control birds and was not
considered significant. [astonlin and Rosin. x 125.

 

The fibula is completely surrounded by a lesion.

The progressive growth of the lesion both in depth
lsmatoxylin and losin. x 16.

and in extension around the circumference of the shaft is clearly

demonstrated.

um 17o

33

 

Fistula. nerepidgrcI-thei’thesponahonois
depicted by the lerge eeount of osteoid tissue end
ceepressien of the estoohlssts st the ups of the bone
spicules (l). lenetoxylin end lssin. x 219.

 

Figure 19. Orientstien of the bone spicules end specos
st right engles to the longitudinsl exie or the bone.
lustexylin end Resin. 2 Mt.

u

The periosteum was 20 to 30 cells in depth over many of these
lesions and was composed of hypertrophic, basophilic osteoblasts
(Figure 20). They were fusiform to stellate in shape and cytoplasm
streamed from the cells in long fine strands and bundles intermingling
with the cytoplasmic processes from adjacent cells. Occasional mitotic
figures were found among the cells (Figure 21). Many cells had vacuoles
in the cytoplasm so that the nuclei were forced to one edge of the cell
(Figure 22). Some osteoblasts contained small,round, homogenous, baso-
philic bodies in the cytoplasm (Figures 23, 2h, and 25). These small
bodies usually were surrounded by a halo and l to 3 were occasionally
seen in the cytoplasm of a cell. They resembled cytoplasmic inclusion
bodies described in some mammalian diseases. Degenerating erythrocytes
were also present but their staining reaction was much more eosinophilic
and they contained nuclear remnants (Figures 25 and 26).

An occasional lesion was found which contained an unusual number of
capillaries at the surface of the newly forming bone, but this was only
an incidental finding and was not a consistent part of the lesion.

The highly cellular layer of the periosteum was covered with the
dense fibrous layer as seen in a normal periosteum. At the margin of
the developing lesion the osteoblasts gradually diminished in number
until they finally faded out to a normal, single row of cells. The
basophilic characteristic of the spongy bone also gradually gave way to
normal osteoid and compact bone. The transition from the abnormal
osteopetrotic lesion to normal adJacent tissue was so gradual that it
almost defied pinpointing the exact place where the lesion ended and

normal tissue began (Figure 27). Occasionally the spongy bone ended but

 

Figure 20. A wide layer of hypertrophie osteoblasts at the
surface of a lesion. Compare with figure 12. leestoxylin
“a Bull. 1 70h.

 

Figure 21. Osteoblasts undergoing mitosis. Mitotic figures
were seldom seen, even in these very active lesions. Bona-
toxylin and losin. x 985.

#6

 

lemstemylin and losin. 1 £85.

Vacuolation of the cytoplasm of osteoblasts

in e subperiosteal lesion.

118‘" 22a

 

Largo, basophilic inclusion body surrounded
nema-

by a hole in the eyteplsse of an osteoblast (l).

toxylin and Iosin. x 121.

Figure 23o

 

Figure 215. Large, basophilic inclusion body lying free
among osteoblasts in the periosteum (l). lematexylin
and Resin. 1 1221.

 

Figure 25. Small, basophilic inclusion body (1) in the
cytoplasm of an osteoblast. A degenerating erythrocyte
containing a pyknetic nucleus is nearby (2). lemotoxylin
and looin. x 1221.

 

Figure 26. Degenerating erythrocyte with haryorrhexis (1).
here is a distinct difference between the inclusion bodies
and degenerating erythrocytes. nematoxylin and Rosin. x 1221.

 

Figure 27. Gradual transition from the margin of a
lesion to normal periosteum and bone (1). The transi-
tion is so gradual that the point of demarcation narrows
to the vanishing point. Compare with fimre 5. neme-
toxylin and Basin. 2 #00.

1‘9

the hyperplastic periosteum continued beyond the edge of the lesion
(Figure 28).

In a few bones, where the lesions had been present for a considerable
time, islands of hyaline cartilage had formed. This was not a usual part
of the osteopetrotic process, however (Figure 29).

In the advanced lesions, there was a gradual transition between
original, normal, dense, cortical bone and the osteopetrotic process
(Figure lh). Osteoclasts usually were not found in the lesions. There
was no necrosis and no transition to cartilage followed by remodeling of
bone. Sometimes the lesion extended from the endosteum to the periosteum.
If the endosteal surface was affected long, narrow spicules of spongy
bone formed and projected into the marrow tissue (Figures 30 and 31).

This caused a marked irregularity in the outline of the medullary canal
and probably explained the manner in which the canal was eventually
obliterated in advanced lesions. The marrow was sometimes more fibrous
than usual; in other instances it had not changed from its usual cellular
character.

There were no giant cells in the osteopetrotic lesion and no

metastasis to soft tissue was observed.

(d) Muscle

Muscle fibers and bundles in the area of these lesions were under-
going atrophy or were entirely gone and had been replaced by fibrous
connective tissue (Figure 32). Therewwere no hemorrhages, no necrosis and

no inflammatory cells around the atrophic muscles.

50

;.

9:4" 0
¥' '

' ?

a .
{Va .

;
a,
s

x . .
g. _..... .
72—32.,

_‘

\

 

the hyperplastic periosteum

lesions

In some

28.
(1) continued beyond the edge of the essoeus lesion (2).

nematexylin end lesin. x 128.

P1

 

thiswes

lswever,
lemotoxylin and Resin. x H.

In a few lesion? extensive development of
l .
uncommon and was not part of the usual development of

hyaline cartilage occurred

the osteopetrotic lesion.

Figure 29.

 

Figure 30. The endosteum and bone marrow were affected
only if there was extensive development of the subperiosteal
lesion. Fibrosis of the marrow tissue may develop after
this. no new endosteal bone is spongy in nature. noma-
toxylin and Rosin. x 55.

 

Figure 31. Marked development of osteopetrotic lesion
on the tibia with some involvement of the endosteum.
llotice the small lesion on the fibula. Compare with
£183" 3°e Hentoxylin “a. “no I 12o5o

52

(e) Vascular Legion

Either arteries or veins or both in the legs, lungs, kidneys, liver
and around the adrenal glands contained lesions which partially or
wholly obliterated the lumen (Figures 33 and 3h). The lesion was formed
by the proliferation and growth of the loose, reticular, intimal layer of
the vessels (Figure 35). The fibers and cells were somewhat increased
in number and in size and the direction of growth was toward the lumen
which gradually reduced its size (Figure 36). In the arteries, the growth
was sharply delineated at the internal elastic membrane (Figures 33,36,
and 37). In some vessels, there were several foci of growths developing
at different locations at the same time (Figure 38) and sometimes the
adjacent foci had develOped to the point they were touching each other
(Figures 38 and 39). The growths were covered on the free surface by
endothelium; neither inflammatory cells nor thrombosis were present, and
in only 2 instances had these growths penetrated the outer structures of
the vessel wall (Figure ho). In the liver, some small vessels were
entirely closed. In 1 liver, 1 focus was found where a similar kind of
tissue was originating from the deep face of Glisson's capsule and pene-
trating the liver parenchyma. No mitotic figures were found in any

lesion and other characteristics of malignancy were absent.

(1‘) L142

Some livers contained 2 other lesions unrelated to the 1 Just
described. These were fecal necrosis and bile-duct carcinoma. The
necrotic foci were variable in size and not limited to 1 part of a
lobule. Some foci were quite small and were located within 1 lobule mid-

way between the periphery and central vein. Others were larger and

53

..w

r....,...\_
.

 

Degeneration and atrophy of skeletal muscles

Figure 32.

over the lesions followed by fibrous replacement is charac-

lematonylim and Rosin. x 156.

teristic of this disease.

I 2“. .3. “\N‘Vewk
W/un DA . WW.“ NW ,‘;a! PA.
. Mafia ea
..., ...... “Y

..
a”
d

34$.
. a

        

Kn.»
, ... . ....
a ......

 

                 

  

     

”k. )1. . . H...
......) .. ATM...
; . .. . . . . ..

p

f 13

.
:1“
A.”
v-

F???
5
a

"i
‘t- 5,
”‘33. .

a
.'&-

Lumen of a small artery completely closed by
Notice that the growth stops at the internal
lemotoxylin and Rosin. x 391.

intimal growth.
elastic membrane.

 

Figure 3h. The lumens of two arteries nearly closed by
the intimal growths. lematoxylin and Resin. 1 55.

 

Figure 35. Intimal growth in a vein in the kidney. Cells
and fibers are undergoing hyperplasia but mitotic figures are
not evident and invasion of parenchyma has not occurred.
lie-toxylin and Resin. 2 5&7.

 

Figaro 36. Intimal growth in an artery. The direction
ofgrowthweotowardthelumenwhichgraduallyrodueedits
sine. lematoxylin and Resin. 1 19h.

 

Figure 37. higher magnification of figure 36, showing the
sharp line of demarcation at the internal elastic membrane
which separates the intimal growth from the tunica media.
Hematoxylin and losin. x 1182.

56

 

Figure 38. Large hepatic vein with three separate
intimal foci, two of which are touching. nematoxylin
‘m Mine 1 “a

 

Figure 39. Higher magnification of figure 38. lone-
toxylin and Resin. 1 125.

57

involved parts of 2 adjacent lobules. The hepatic cells had undergone
necrosis and remained only as a pale eosinophilic mass (Figure hl).
Sinusoids usually were distinguishable and separated parts of the
necrotic liver cords from each other. They contained nuclear remnants,
cellular debris and erythrocytes. In many instances, erythrocytes were
intact even in the centers of the necrotic area. If erythroblasts were
present they were even more resistant to destruction. This suggests that
either the erythrocytes and erythroblasts moved into the sinusoids after
the liver cells died or else they were not susceptible to the causative
agent. Hemorrhage was quite marked around the periphery of the necrotic
foci.

The bile-duct carcinomas appeared as dense basophilic unencapsulated
growths of epithelial cells in the liver parenchyma. A few abortive
ducts were forming but most cells were growing in an unorganized arrange-
ment (Figures h2 and h3). Mitotic figures were numerous and invasion of

liver parenchyma was apparent (Figure an).

(g) Spleen

The atrophic spleens did not contain lesions which could account
for the atrophy except that they were nearly completely devoid of all
lymphocytes and germinal centers. The reticular framework and cells
seemed to be increased but this was only from being condensed rather than
a real increase. Erythrocytes were present in sinusoids and small
arterioles were open.

One significant lesion which was present in many spleens was amyloid.
This material was eosinophilic, smooth, homogenous, and foci of it were

variable in size. The amyloid was distributed throughout the sections

 

Figure to. One of 2 instances in which the intimal growth
penetrated the tunica adventita. nematoxylin and Rosin. x 5157.

m 4135303015. W... >
(Ey'x’fiiti‘fi as?" ..

 

e2... ,
. ..

k. ”. ~‘e' . .

~, {... s",I-o-a..‘ 8 st- _. s. ‘ _

r" : . 5‘.» .‘»'.J"...~.‘. . ~ Im'e " '

V ' ,"b;

Figure hl. Focal necrosis surrounded by huerrhoge in the
liver. his was probably caused by the GAL virus. Hema-
toxylin and losin. x 206.

 

Figure 32. A small focus of bile duct carcinoma with invasion
and destruction of hepatic cells. Abortive ducts are forming
at one point (1). nematoxylin and Rosin. x 181.

 

of.

Figure ha. Higher magnification of figure #2 showing bile
duct formation and mitotic figures (1). Hematoxylin and
“line I 816o

60.

and sometimes formed a thick collar around a small arteriole but at other
times was not associated with a vessel. Nuclei and cellular remnants

were trapped in spaces in some of the amyloid deposits (Figures its and 1+6).

(h) 2225,

The lung in 1 chicken contained a large area of necrosis, cellular
proliferation and much fibrin. Large, nonseptate, branching mycelia were
found throughout the lesion. These mycelia resembled those of the
Phycomycetes and were probably Mucor. This fungus apparently was the
cause of the lesion. There were no other respiratory changes except

impaction of all capillaries and vessels in some lungs with erythroblasts.

(i) Testicle

The testicle from several chickens had a lesion which was similar
in all instances. This consisted of tubular strephy with the epithelial
cells lying unattached in the lumen (Figure h7). They were shrunken
and formed a cast although the individual cells were still visible. This
atrophy occurred usually at l pole or occasionally along 1 edge. It never
affected the entire gland. There was never any evidence of inflammation,
hemorrhage or trauma to account for the atrophy. In the older lesions,
fibrous tissue had grown in from the capsule and filled in the spaces

around the atrophic tubules. None of these changes were visible grossly.

(J) Thyroid

The thyroid gland in several chickens had undergone degenerative
changes characterized by loss of colloid, partial collapse of follicles
and some desquamation of epithelial cells. The cytoplasm of the cells

consisted of fine granular debris or interlacing strands of granular

261;. ‘0’} are!“

2";~f,§‘=~"7 -- ; '

:69 fifi’flgi‘i‘éfl‘ifiiga g... . . .
3' ‘35:? ""3 ‘
s_ \‘\g ’d .1" .1. \ ‘

  
  

  
    
 

3‘.
eo

 
   

3‘?

.2” Mb lo’i’a

Figure u. Invasion of hepatic parenchy- by bile duct epithe-
lium (l). Mitotic figures at 2. lsmatoxylin and eosin. x 595.

 
  
 

.- t“? ‘7 mug
"gig?“ data?”
we ‘ F

.K’ ,

  

Figure #5. Ixtensive amyloid deposits in the spleen. lemo-
toxylin and Rosin. x 532.

 

Figure 156. Higher magnification of amyloid around a
small vessel in the spleen. nematoxylin and losin.
X 1mo

 

Figure #7. Tubular atrophy in the testicle. Ipithe-
lium in atrophic tubules is detached and forms a cast
in the lumen. Fibrous tissue is penetrating the area
from the tunic. Hematoxylin and losin. x H.

63

debris. They had the appearance of liver cells filled with glycogen. A
few of the nuclei were pyknotic and some had been displaced to the margin
of the cell. Some follicles had lost part of the epithelium which had

undergone lysis. There was no inflammation or necrosis.

(k) Thymus

The thymus gland occasionally varied in structure but degenerative
changes were not present. The usual arrangement in a thymus gland
consisted of a dense border of large, deeply basophilic lymphocytes with
the center of the gland being open, lightly stained and containing small
lymphocytes and Hassel's corpuscles.. Occasionally this arrangement was
changed and the entire gland contained the small lymphocytes with
Hassel's corpuscles distributed throughout the section. The dense
border was entirely absent except for small foci of these large, darker
cells.

(1) Parathyroid

Parathyroid glands were unaffected except for l which contained a
cystic space.

(m) Pancreas

Several pancreatic glands contained areas of hyperplasia with no
acinar formation. These islands of hyperplastic cells had displaced the
normal architecture or had surrounded some acini which were present in
the area. In 1 gland, the pancreatic cells were large, had vesicular
nuclei, were invading the surrounding normal gland structures and
occasional mitotic figures were seen. This was considered an early

manifestation of malignant growth (Figure h8).

 

Figure lt8. Pancreas from 50-day-old chicken. The large,
vesicular nuclei, mitotic figures and lack of organisation
were considered evidence of early malignancy. nematon'lin
“a. “line 1 632o

6S

In 1 pancreas, many acinar cells were undergoing hydropic degenera-
tion, acinar walls were obliterated or indistinct and the tissue took
only a light stain. Islands of normal acini were scattered throughout
the section. There was a slight increase in fibrous tissue in the areas

of degenerative changes.

(n) Other Tissues
No changes were found in the brain, heart, proventriculus, duodenum

or adrenal gland from any chicken in this experiment.

D. Radiological Changes

The entire length of both legs and occasionally a humerus were
exposed on the radiograph. Occasionally because of the position of the
leg, the fibula happened to lie in the exact plane of the outer surface
of the cortex of the tibial diaphysis. This caused the cortical line
to appear slightly more dense or blurred in a few instances.

Osteopetrotic lesions were diagnosed in h6 chickens by radiograph
compared to 60 at necropsy and 73 microscopically.

With the exception of 1 chicken, no radiographs were made before
day h3, the time at which the first group was killed. The single chicken
died on day h2, and microscopically osteopetrotic lesions were found on
the tibia and femur, but no lesion was seen on the radiograph. Five
other chickens which were killed on day h3 had grossly visible lesions of
osteopetrosis but no changes were detected on radiographs. These findings
indicate that osteopetrotic lesions can be seen grossly before they can be
detected by radiograph (Figures R9 and 50).

The early and advanced lesions presented the same appearance except

for size. The lesion appeared as an area of increased density with slight

66

Figure #9. Radiograph of the tibias and femurs from a
h3-day-old experimental chicken in which osteopetrotic
lesions were visible grossly but could not be seen on
the radiograph. Compare with the same aacontrol,
figure 50.

 

Figure 50. Radiograph of tibias and femurs from con-
trol bird, #3 days of age. Compare with figure It9.

67

thickening of the cortical wall followed by marked elevation above the
level of the surrounding normal bone as the lesion progressed. The new
osseous tissue develOped under the periosteum and on the surface of the
original cortical bone (Figures 51 and 52). There was no evidence of

rarefaction or bone destruction in the developing lesions.

E. Hematological Studies

Seventy-six chickens were available for hematocrit and hemoglobin
studies. They were placed in l of 5 groups according to their leukosis
status. Group 1 was the control. Group 2 was given the virus but did not
develop osteopetrosis or erythroblastosis; therefore, group 2 was compar-
able to the control group. Group 3 had osteopetrosis; group h had erythro-
blastosis, and group 5 had both forms of the disease.

According to the analysis of variance the hematocrit differences
among the groups were highly significant. Groups 3, h and 5 had lower
hematocrit readings than group 1. Group 2 was not different from group 1.
In other words, the variation among these groups was sufficiently greater
than the normal variation within groups that these differences would be
considered due to the disease and not differences within the population.

The mean for each group was obtained by addition of all values and
dividing by the number of observations (2 3%).

Standard deviation was obtained by substitution of approximate

 

numbers into the following formula: f'fifi' Z‘X-if—

where n - sample size, x - each observation and i’- the mean. After
standard deviations were calculated the analysis of variance was carried
out (Snedecor, l956; Huntsberger, 1958). These data are summarized in

Table I o

 

 

Figure 51. larlieot lesion that was detected by radio-
giiaph. The lesion is on the right tibia of a Sk-day-old
c cken.

69

 

Figure 52. Radiograph shoving marked osteo-
petrotic lesions on the femurs, tibias and
metatarsi of a 60-day-old, experimental
chicken. Compare with figure 53.

70

 

Figure 53. Radiograph of tibias and femurs of 60-day-old,
control chicken.

71

TABLE I. Analysis of the hematocrit readings for 6h
experimental and 12 control chickens.

 

 

 

Group Number of Birds Mean Standard Deviation
l 12 28.6 1.7
2 12 28.2 3.3
3 25 2h.2 3.h
h 2 26.7 3.5
S 25 2h.7 h.h

 

The variation in hemoglobin levels among the groups was not signi-

ficant. These data are summarized in Table 2.

TABLE II. Analysis of the hemoglobin levels for 6%
experimental and 12 control chickens.

 

 

Group Number of Birds Mean Standard Deviation
1 12 22.2 h.2
2 12 22.2 h.2
3 25 22.9 3.0
h 2 248.48 505
5 25 23.2 3.6

 

Seventy-three blood smears were satisfactory for making differential
leucocyte counts. These chickens were grouped in the same 5 categories as
for the other blood studies and were numbered in order 9, 10, ll, 12 and
13 in the same order for the 5 conditions as given above.

An analysis was performed using a square-root transformation of all
counts except lymphocytes to see if there were any significant differ-
ences among the groups (Snedecor, 1956). According to the test, there was
a significant difference in the monocyte counts of the 3 infected groups,
numbered ll, 12 and 13, when compared to the 2 noninfected groups numbered
9 and 10. There also was a difference in the basophil count in the 2

birds in group 13. These data are summarized in Table 3.

TABLE III.

Analysis of the differential leucocyte counts
for 61 experimental and 12 control chickens.

 

 

 

 

No.

Group Birds Lymphocytes Monocytes Heterophils Eosinophils Basophils
9 12 85.9 0.83 h.16 3.88 0.19
10 12 81.0 1.56 10.36 2.10 0.0h
11 2h 7h.9 h.08 9.2h 3.h5 0.26
12 2 70.5 3.92 15.60 3.31 5.h7
13 23 80.2 2.52 10.11 1.36 0.73
All 73 79.3 2.h6 8.88 2.52 0.37
Significant at NoSo oos NoSo NoSo oos

 

N.S. - not significant.

F. §grum_Alkaline Phosphatase

Fifteen serum samples from experimental birds with no osteOpetrosis
(group 7) and h9 samples from birds with osteopetrosis (group 8) were
compared with the serums from the 12 control chickens (group 6). There
was no significant difference in the serum alkaline-phosphatase levels

among the 3 groups. These data are illustrated in Table h (see discussion,

 

 

 

p0 85).

TABLE IV. Analysis of the results of the serum
alkaline phosphatase levels from controls
and chickens with osteopetrosis.

Group Number of Birds Mean Standard Deviation
6 l2 1h.l 3.7
7 15 13.9 5.h

8 A9 13.8 1.2

 

73

TABLE V. Summary of lesions that were found in each
of the 8 groups of chickens. These groups
also include those that died during the half
week prior to or following the day on which
each group was killed.

 

 

Group 1, h3 Group 2, 50 Group 2, 57 Group h, 63

 

Lesion days of age days of age days of age days of age
.Amyloid + + + +
Bile duct carci-

noma - - + +
Erythroblastosis + + + +
Fractures + + + +
Hepatic necrosis + + + +

Inclusion bodies

in osteoblasts - + + -
Intimal growths - + + +
Mycosis, pulmo-

nary + - - -
Osteopetrosis + + + +

Pancreatic carci-

Parathyroid cyst - + - -
Splenic atrophy + + + +

Testicular degen-
eration + + - -

Thyroid degenera-
tion . + O I-

 

+ . Present in 1 or more chickens

- a Not seen

Vo DISCUSSION

A. Clinical

The chickens in figures 1 and 2 were from 2 other experiments on
osteopetrosis. Birds from this experiment had not developed lesions
large enough to be photographed before being killed. Therefore, these
other h chickens were substituted in order to illustrate 2 different
degrees of bone involvement in 2 different ages of birds as well as to
show the massive size which the lesions will attain if birds are allowed
to live long enough. The contrast between controls and experimental
birds also emphasized the difference in growth rates, appearance and
feather condition. In figure 1, the birds were h9 days of age. They
were from the same hatch and had the same care, feed and type of housing.
The control on the right weighed 1350 Gms. and the experimental bird
weighed 1080 Gms. The circumference of the left metatarsus of the control
measured h.0 cm. compared to 5.6 cm. for the experimental bird.

The birds in figure 2 were 183 days of age. They were from the same
hatch and had always been penned together. The control on the right
weighed 2520 Gms. and the experimental bird weighed 1&85 Gms. The cir-
cumference of the left metatarsus of the control measured h.7 cm. compared
to 10 cm. for the experimental bird. All measurements were made at the
center of the diaphysis.

The stilted gait, limping, and preference for sitting down most of

the time suggested that the birds were uncomfortable and probably

7h

75
experienced pain when moving about. However, pressure on the bony lesions
did not illicit a response, which fact indicated that the bones were not
sensitive. The latter is in agreement with the observation of Jungherr
and Landauer (1938).

B o BOne

 

In speaking of hard-tissue (bone, cartilage and teeth) lesions the
fact should be borne in mind that the primary changes must first occur
in the cells and tissues forming the periosteum and marrow and filling
the Baversian canals, and that reactions of the bone tissue proper are
always secondary (Weinmann and Sicher, 1955). Osteoblasts are very
sensitive to the action of bacteria and viruses. The viruses of hog
cholera, swine influenza, canine distemper and avian encephalomyelitis
cause hypoplasia, atrophy and even death of the osteoblast (Monlux, 1961).
On the other hand, the severity of the irritant (living or non-living) may
not be great enough to cause death of the cells. There may be a minimal
response or the cells may be stimulated to reproduce themselves at a rapid
rate, in which case the response is called hyperplasia.

In all instances in this study, the earliest microscopic lesions
were located under the periosteum and never under the endosteum, and
there was never a lesion deep in the cortical bone that did not stem
directly from the periosteal lesion immediately above it. No endosteal
lesion, marrow involvement or change in the original compact bone was seen
without accompanying well advanced subperiosteal lesions. The conclusion
here, then, was that the osteopetrotic lesion was basically a function of
the infected osteoblasts and the changes in the bone tissue and structure

were secondary. This is supported by‘Weinmann and Sicher (1955).

76

In other words, the fundamental change occurred in the osteoblast
at the time it was parasitized. The change, however, was not visible by
the methods used in this study. This fundamental change in the osteoblast
caused it to elaborate tissue which was different in several respects from
normal bone. These changes were characterized by a difference in stain-
ing reaction and structure of the tissue. Eventually, osteoblasts increas-
ed in number and size as seen in many of the lesions.

In radiographic studies, Holmes found the earliest lesions under the
periosteum, but Bell and Campbell (1961) reported their earliest radio-
graphic lesions were endosteal. Jungherr and Landauer (1938) stated that
in the earliest stages, medullary fibrosis, increased osteoclasia and de-
generation of old bone were present. These observations were not support-
ed in this study. Osteoclasts were notably absent from the lesions. How-
ever, they were present around the trabeculae and endosteal surfaces of
nearly all bones indicating that they were present in the normal location
but were not part of the developing osteopetrotic lesion. This is merely
to say that the osteoblastic activity at this stage in the progressive
develOpment of the osteopetrotic lesion was more important than the
osteoclastic activity. Medullary fibrosis did not occur until the lesions
were well advanced.

It was estimated from the well-developed lesions in a few of these
chickens that lesions were present probably by day 30, and microscopically
they would have been seen several days before this.

The study of this series of lesions commencing with the smallest and
progressing to the largest suggested the following step-by-step development
of the lesion. Initially a few osteoblasts were infected by the virus.

The time at which this infection occurred was not apparent; but reasoning

77

from the advanced state of some lesions found in the first birds killed
on day h3, the infection could have occurred anytime from 1 to 25 days of
age. The irritant (virus) apparently stimulated the osteoblasts but did
not kill them.

Under the influence of the irritant (virus) the osteoblasts increased
in activity and immediately elaborated large, coarse fibrils that were
deeply basophilic in contrast to the eosinophilic character of the adja-
cent, compact bone. Simultaneously, the superficial indentations that
were destined to become Haversian canals in the compact cortical bone
were not closed or reduced in size by the laying down of lamellar bone;
consequently, they appeared in the new, basophilic, fibrillar matrix as
large, open, round to irregular spaces with little resemblance to normal
Haversian canals. Immediately thereafter, a noticeable increase in
cellularity occurred in the new bone, and osteocytes and lacunae were
often so numerous and crowded that adjacent lacunae coalesced and formed
larger, open spaces containing several osteocytes. This rapidly forming
new bone had a noticeable fibrillar appearance. Finally there was a
marked increase in the number of layers of osteoblasts in the proximal
layer of the periosteum. These are the 5 characteristics mentioned in
the results which distinguished a very early osteopetrotic lesion from
normal bone. Because of the extremely rapid formation of osteoid matrix,
the trabeculae became oriented to the deep face of the periosteum and
appeared as long columns of very cellular, fibrous bone capped at the
periosteal surface by a thick layer of osteoid tissue. The large simulat-
ed Haversian spaces were formed by the columnar trabeculae and 2 structures
together, radiating at right angles to the longitudinal axis of the bone,

gave the appearance of the "sun ray burst" described previously and

I: It'Il-lllllll.fil 1' ill ..

 

78

below. There was no resemblance of this new bone to the regular, system-
atic organization of normal, compact bone. As the lesion continued to
grow, its development was characterized only by an exaggeration of the
above steps and not by any change in form or structure. The lesion
spread both around the bone and longitudinally toward each end. In a
rapidly growing lesion, new spongy bone formed at a rate greater than 1
mm. per week (Sanger, £5.3l., 1963).

It was concluded from this study that the changes which were describ-
ed in the osteopetrotic lesions developed initially at the time the new
bone was formed and did not occur later as a result of remodeling or
penetration of the original normal bone by the osteopetrotic process.
Osteoclasts were extremely scarce in these lesions and bone remodeling
depends upon osteoclastic activity. Since they were very scarce, it
seemed reasonable to conclude that whatever changes were seen occurred
as the new bone was formed. This is not to suggest that osteoclastic
activity might not be present in later stages of the disease.

The course and development of the lesion which has Just been
described is the usual response of the periosteum to an irritant.

"The bundles of fibrils in the matrix of immature bone are coarse and
irregularly arranged. The osteocytes are numerous, but irregular in
shape and arrangement, and have only a few processes. This immature,
primitive or coarse fibrillar bone is always arranged in trabeculae
and is, therefore, always spongy bone" (Weinmann and Sicher, 1955).
"Fibrous bone, in repair, neoplastic, inflammatory and irritative
(or reactive) processes is laid down as a rapidly manufacturable
material. When the stimulus to fibrous bone production is intense,
the trabeculae begin to show uniform orientation, typical examples
being the trabeculae in the sun ray burst of an osteogenic sarcoma"
(Frost, 1960).

This description of the "sun ray burst" appearance of the newly forming

trabeculae is the appearance of the trabeculae and enlarged irregular

79

Haversian-like spaces in all of the well-developed osteopetrotic lesions in
this study. It, also, is the appearance of inflammatory reactions of
bones in humans as described and illustrated by'Weinmann and Sicher
(1955, pp. 351, 355).

Early writers described the condition that is now recognized as
osteopetrosis as an inflammatory condition (Bell and Anger, l92h),
(Besnoit and Robin, 1922), (Bracket, 1935), Pugh, 1927) before it was
associated with the leukosis complex. Burmester gt_gl. (19h6), Monlux
(1961) and Moulton (1%1) described the lesion as one of hyperplasia. Darcel
(1960) and Campbell (1961) also considered it a non-neoplastic condition.
Neither the cell characteristics nor the nature of the lesion conform
to the criteria which are used for the recognition of malignancy
(Anderson, 1961).

(a) Fractures

The reason for the many fractures was not apparent. Some of the
fractures occurred either spontaneously or when the chickens were handled
and were not discovered until the birds were necropsied. Other fractures
occurred during manipulation at necrOpsy in spite of careful efforts to
avoid breaking the bones. In some chickens, the fracture was complete
with displacement of broken ends. In others the fracture appeared only
as a line across the diaphysis with no displacement. On the radiograph
no attempt at repair was visible even on the ones with no displacement.
Some chickens had fractures but there was no evidence of osteopetrosis
on the bones and sections that were studied either grossly, histopathologi-
cally or by radiograph. However, osteopetrosis may have been present on A
some bone that was not examined; therefore, it cannot be concluded that

fractures occurred in the absence of osteopetrosis. Controls did not

I I “la. I I.) {Ii 1.! I _ I

 

 

 

 

80

have any fractures. Radiographs of fractured bones did not reveal any
decreased density in the cortical bone when compared to controls of the
same age. Cross, 53 21. (1959) also reported that the bones broke or
could be crushed easily. Jungherr and Landauer (1938) and Coles and
Bronkhorst (19h6) reported that the bones were more resistant to fracture

than normal.

(b) Inclusion Bodies

The small objects which were present in the cytoplasm of osteoblasts
had the characteristics of inclusion bodies. Sharpless and Jungherr (1961)
described intranuclear inclusion in liver cells from chickens infected
with the GAL virus. It may be that these bodies were related to the
presence of this virus in these chickens. It has also been reported that
so-called inclusion bodies are nonspecific such as the "hyaline-droplet"
degeneration described by Smith and Jones (1957). It may be that these
small bodies were only products of degeneration. If they were degenera-
tive products, they were different from degenerating erythrocytes which
had a distinct eosinophilic color and contained nuclear remnants. To
attribute them to degenerative changes is not entirely consistent because
the osteopetrotic lesions were not degenerative in nature and osteoblasts
were not undergoing recognizable degeneration, either those with or

without inclusions in the cytoplasm.

C. Intimal Growths
The fibrillar growths which developed in the vessels of the liver,

kidney, lung, limbs and in the large artery adJacent to the adrenal gland

81

did not present any evidence of malignancy. The direction of growth was
consistently toward the lumen, the line of least resistance. In only 2
instances, was the growth penetrating the media of the vessel wall. In

1 liver, a similar tissue of fibrllar type appeared to be originating
from the deep face of Glisson's capsule. The growth did not cause
thrombosis of vessels or necrosis even though some vessels were virtually
closed. Collateral circulation must have been sufficient to meet the
needs of the tissues, at least in some organs. The intimal growths
described here did not form channels, did not contain blood cells or
giant cells and were not accompanied by necrosis like the endotheliomas
described by Furth (l93h). Jungherr and Landauer (1938) reported that
portal cirrhosis occurred and also lymphoid infiltration and hyaline
degenerative changes in the vascular walls. Gross ethal. (1959) found an
occasional cirrhotic liver. The lesions which these authors described did

not accompany the blood vessel growths reported in this study.

D. ElZEEMEEE Pancreas

The focal necrosis was attributed to the GAL virus described by
Sharpless and Jungherr (1961) which was an inapparent contaminant carried
in the RPL 12 inoculum. There were no intranuclear inclusion bodies,
however. The early bile-duct carcinomas and pancreatic adenomas were
probably caused by the lymphomatosis virus much like those in the kidney

described by Burmester gt 9;. (1959) and Walter £13 11;. (1962).

E. Testicle
The reason for testicular atrophy was not apparent unless this was
a function of the GAL virus or of the intimal vascular growths. The

atrophy was more than a transitory lesion because in several testicles,

82

fibrous tissue was growing into the gland from the deep face of the tunic
and filling the spaces vacated by the tubules. In no instance, however,
was there atrophy of the entire gland. Holmes (1961) found infantile
testicles in some experimental birds. This condition wasnot part of

the lesion described here. Bell and Campbell (1961) reported that the
gonads were retarded in development.

Tubular atrophy may also have been primary as a result of ischemia
caused by the narrowing of vascular lumens. This, in turn, was then
followed by connective tissue growth, or the connective tissue growth
could have been primary, being caused by the same thing that stimulated
the intimal vascular growths. The crowding from the connective tissue
would then, in turn, cause tubular atrophy from pressure as well as

interference with blood supply.

Fe 1352mm Gland

The degenerative changes in the thyroid glands resembled those
described by Gobel (l95h) which he thought were caused by thyrotoxins.
However, he could not identify the source of the toxins. The photo-
micrographs in his paper showed degenerative changes in glands which
appeared similar to those described in this work. Follicles were partial-
ly collapsed; colloid was not present or only partially filled the fellicle.
Epithelial cells were undergoing degeneration and many were lying free in
the lumens. In thse avian thyroids, these cells were considered to be
desquamated epithelial cells but Goble (1951:) stated that the ones he saw
were spherical agranulocytes; this was interpreted to mean they were
macrophages although Goble may not have intended to imply this. There was

no inflammation and no fibroplasia like that seen in the degenerative

 

ill I. I} i 'ialll I! II

 

83

testicular lesion. It may be that this lesbn is a reaction to the GAL

virus but less severe than that in the liver.

Ge MUBCIQ

 

Muscular atrophy and degeneration probably occurred as a result of
2 conditions acting either singly or in combination. The reduced blood
supply brought about by closure of the lumens of the vessels from intimal
proliferation would reduce both nourishment and oxygen to the muscles.
This would cause atrophy, and continued loss of nutrition would eventually
lead to degeneration. The second cause could be attributed to pressure
atrophy and degeneration from the increase in bone size from osteopetrosis.
This gradual increase in bone size would apply pressure to the muscles
gradually reducing their blood supply as well as restricting movement.
There would also be incomplete elimination of waste products which, in
turn, would aggravate the condition, or both of these conditions could be
acting together which did happen in some chickens. These degenerative
muscular changes would also account in part for the affected gait and

reluctance of the birds to stand.

H e S 21900
Splenic atrophy was a common finding in birds'with osteopetrosis;

however, it did not occur in every one. The reason for the decrease in
size stemmed from acomplete absence of white pulp in the spleen. The
other elements appeared normal. There seemed to be a slight increase in
the reticular framework but this was more apparent than real because of
the concentration of reticulum due to reduced size. Splenic atrophy was
reported by Gross (1959), Holmes (1961), and Bell and Campbell (1961.

Jungherr and Landauer (1938) reported hyperplasia of the Malpighian

corpuscles.

I. Thymus
Absence of lesions in this gland was in agreement with the findings

of Bell and Campbell (1961).

J. Parathyroid

The histological appearance of these glands was no different from
the controls. The large cystic space in the single gland was not con-
sidered significant. Jungherr and Landauer (1938) found significant
changes in only 2 glands. One was lymphocytic infiltration associated
with generalized lymphomatosis and the second was a small cell hyper-
plasia associated with secondary osteoporotic lesions. Krook and Barrett
(1962) listed 3 morphological ways to evaluate the functional capacity of
the parathyroid glands: (a) by weight or volume of the glands; (b) by
conventional histological examination of the glands; and (c) by
quantative morphological analysis of the glands. They also quoted Bger
and Van Lessen and Engfeldt that estimating functional activity based on
histological examination was rather inaccurate. Krook and Barrett (1962)
further stated that the histological picture of the parathyroid glands of
most species is often heterogeneous with transitional cell types. The
glands from these chickens were studied only by histological examination.
It might be that additional studies would have revealed significant

changes.

K. Hematology
The 3 groups of chickens which were suffering from osteopetrosis

or erythroblastosis or both diseases had much lower hematocrit readings
than the controls or the unaffected experimental birds. However, the

circulating erythrocytes contained the normal amount of hemoglobin. The

85

explanation for the anemic appearance of affected birds in this experiment
lies, then, in a deficiency of erythrocytes rather than in reduced con-
centrations of hemogldbin in the cells.

According to Table 1 published by Bell and Campbell (1961) there
was very little change in the hematocrit readings between affected and
unaffected birds. They also stated that the red cell counts were unchanged.
They found a deficiency of white cells typical of a myelophthisic anemia
(granulocytes originating in the bone marrow). They did not report any
measurement of hemoglobin levels.

Jungherr and Landauer (1938) reported there was a decrease in hemo-
globin in affected birds when compared to controls. They concluded that
blood from affected chickens was essentially aleukemic but showed evidence
of a mild, nonspecific anemia.

The 3 groups of diseased birds, divided on the basis of the
manifestations described above, had a significant increase in monocytes
when compared to the 2 groups of uninfected birds. The reason for this
was not apparent.

The 2 chickens which had only erythroblastosis had an increase in
basophils but the reason for this was not known.

Total leukocyte counts were not made; therefore, it is not possible
to say whether the increased monocyte and basophil counts were relative

or absolute increases.

L. Serum Alkaline Phogphatase
In the osteopetrotic lesions, the osteoblasts were hypetrophic
and hyperplastic and new, spongy bone was formed at a rapid rate. Accord-

ing to thk 33 31., 195k the rapid formation of normal bone, or new bone

86

in repair, or calcified and uncalcified pathological bone causes an
increase in the level of the enzyme, phosphatase, in the blood. Rapid
destruction of bone also causes an increase in the level of the enzyme.
Numerous reports in the literature show that chickens have elevated
phosphatase levels under varying conditions (Bell 33 21., 1959; Bell,
1960; Siller, 1959, and Hurwitz and Griminger, 1961). Therefore, it was
anticipated that the serum enzyme level would be increased noticeably in
these chickens which were suffering from osteopetrosis. Hewever, compari-
son of the enzyme levels between affected and unaffected birds showed
that there was no difference between the 2 groups in this experiment. This
was unexpected and the question was raised whether there might have been
some error in the test. Correspondence with the Sigma Company, whose

test was used, did not clarify the matter. But their offer to test serums
from other osteopetrotic and normal birds was accepted.

Their test showed that on serums from h osteopetrotic birds, the
enzyme levels were approximately twice as high as on controls of the same
age. Therefore, it can be stated that serum alkaline phosphatase levels
are elevated in some chickens with osteopetrosis. Review of the techniques
and procedures revealed that the serums from osteopetrotic birds should
have been diluted before testing. It may also be that the spectrophoto-
meter had been improperly calibrated or was not working properly. This

part of the experiment needs to be repeated.

M. 19.15.13. Planned 221:. m

One of the objectives of this eXperiment was to determine the
earliest date at which osteopetrotic lesions appeared on the bones of
chickens which had been given the RPL 12 virus at 1 day of age. This

objective was not fully satisfied, the reason being that the time for

87

killing the first birds was not set early enough. In retrospect it can
be seen that a large number of birds would have been needed, and the first
group should have been killed not later than the third week of the experi-
ment. An experiment will be designed to answer this question by using a
larger number of birds and killing the first group at a younger age.

New spongy bone is formed at a remarkably rapid rate in this disease.
It would be desirable to know how fast this bone is formed. An experiment
has been planned which will permit accurate measurements of the rate at
which the lesion grows.

No references were found in the literature in which the density of
the bony substance of these lesions had been measured. A measurement
of the degree of mineralization of the various lesions would contribute
to the knowledge of the disease. An experiment has been designed to
provide this information.

For several years, chickens have been maintained at the Ohio Agri-
cultural Bxperiment Station (OAES) which carry the causative agent of
osteopetrosis. A comparative study of the RPL 12 agent and the OAES agent
in producing osteopetrosis has been planned.

Because of the inconsistent results of the serum alkaline-
phosphatase test between birds of this experiment and other birds

with osteopetrosis, plans have been made to repeat this work.

VI. SUMMARY

Osteopetrosis was experimentally produced by injecting day-old
chicks with lymphomatosis virus; clinically, affected chickens were
ragged in appearance, stunted in growth and the comb and wattles were
pale.

The first group of chickens was killed at h3 days of age. Several
had well-developed lesions. The lesions nearly always appeared first at
the center of the diaphysis of the tibia at the posterior border of the
fibula where it touched the tibia. The color of the lesion was pale
yellow compared to a grayishdwhite appearance of normal bone. The
lesion gradually spread around the shaft, toward each end and grew in
depth at the center tapering toward each end, giving the affected bone
a fusiform appearance.

Microscopically the lesion was typical of an inflammatory periosteal
reaction. Osteoblasts were hypertrophic and hyperplastic. The periosteum
was 20 to 30 cells thick in some places. Bhrersian systems did not form.
Instead a basophilic, fibrous, cellular, spongy bone formed at a rapid
rate on the surface of the original cortical bone. Beversian canals
developed as large, open, irregular spaces filled with fibrous tissue.
Bone spicules formed rapidly, and both the spicules and spaces were
oriented at right angles to the longitudinal axis of the bone. On cross

section, the lesion presented a ”sun ray burst” appearance.

88

89

Osteoclastic activity was not a part of the lesion. The process
was proliferative and progressive. Necrosis and degeneration were not
present. The characteristics usually associated with malignancy were
not evident in the osseous lesions.

Many livers had focal necrosis which was attributed to the GAL virus
carried in the inoculum. Some spleens were atrophic and largely devoid
of lymphocytes and germinal centers. Many spleens contained amyloid
deposits. Many arteries and veins had proliferative lesions formed by
rapid growth of the fine, reticular, intimal layer. These growths
completely occluded the lumens of some vessels. They were not considered
malignant. The testicles had small areas of tubular and epithelial
atrophy.

In the liver, foci of neoplastic bile-duct epithelium were present,
and in the pancreas, there were neoplastic foci of acinar epithelium.

The hematological studies were limited in significance. The serum

alkaline-phosphatase tests were inconclusive.

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