TRANSMISSSON STUDIES ON AVIAN
LY’MPHOMATOSIS

Thesis for the Degree of M. S.
MICHIGAN STATE COLLEGE

George Edward Coffral
1950

This is to certify that the

thesis entitled

Transmission Studies on Avian Lymphomatosis

presented bg

George E. Cottral

has been accepted towards fulfillment
of the requirements for

M,S. degree in Animal Pathology

 

 

 

 

 

 

raw-aura! m. ~93 .T—sfit'

TRANSMISSION STUDIES ON AVIAN LYMPHOMAII‘OSIS

TRANSMISSION STUDIES ON AVIAN LYMPHQ-IATOSIS

13?
George Edward Qgttrel

A THESIS

Submitted to the Graduate School of Michigan State
College of Agriculture and Applied Science in
partial fulfilment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Pathology

1950

 

AC KNC‘WLED GLEN!

The writer wishes to express his sincere appreciation to
Dr. B. R. Burmster and Dr. Nelson F. Waters for their help
and cooperation in carrying out these studies. Gratitude is
also expressed to Mr. Barley Hinton, Dr. Frank Thorp, Jr.,
Dr. R. A. Runnells, Dr. A. M. Lucas, and Dr. E. F. Oakberg for
their helpful and encouraging suggestions during the course of
this study.

Special thanks are due to Mr. B. M. Larkins for his technical
assistance in the experimental work, and to Mrs. Hazel I. Garrison,
Mrs. Florence F. Hickok, and Miss Patricia A. Soavarda for

consultation on the preparation of the manuscript.

"3.. v:
£1.11", ~7‘Q

TABLE OF CONTENTS

Page
INTR®UCTI<M.........................l
HISTORY OF THE ETIQOGICAL CONCEPTS . . . . . . . . . . . . . 3
REVIEWOFTETRANSMISSIONSTUDIES.............. 10
THEPROBIEMOFEGGTRANSNHSSION............... 20
MATERIAhSLNDltETHGDS....................24
RESULTSANDDISCUSSION................... 36
SUMMYANDCONCLUSIONS...................45
BIBLIOGRAPHY........................ 47

Tms . O O O O O O O O O O O O O i. O O O O O O O O O O O O O 55

INTRODUCTION

Avian lymphomatosis is a fatal infectious neoplastic disease of
chickens caused by one or more virus-like agents. It is characterized
by the infiltration and multiplication of’lymphoid cells in the various
tissues of the bird's body. The invading cells ultimately become
neoplastic and result in the fonmation oi’tumors of the visceral organs
and other tissues, or may cause paralysis, blindness, or skeletal
changes.

Avian lymphomatosis has been divided into four types, according to
the tissues involved: visceral, neural, ocular, and ostecpetrotic lympho-
matosis. .All of these disease entities are included under the classifica-
tion, the avian leukosis complex, which also embraces three additional
neoplastic diseases, namely, erythroblastosis, granuloblastosis, and
myelocytomatosis. The scheme of nomenclature, as worked out by a
committee of poultry pathologists, is given in table 1 along with some

of the common names that have been applied to these diseases (Jungherr,

 

 

1941).
TABLE l.--Outline of nomenclature of the
avian leukosis complex
Eecommenaed nomenclature Common.names
The avian leukosis complex
Lymphomatosis:
Neural Fowl paralysis,8 marek's paralysis
Ocular Grey eye iriti
Visceral Lymphoc one, big liver disease
Oeteopetrotic marble bone, big bone disease
Erythroblastosis Fowl leukemia, blood disease
Granuloblastosis ” " " '

Hyelocytomatosis Leukochloroma, white tumors

 

Visceral and neural lymphomatosis are the two most important
diseases in this group; the others occur much less frequently. These
diseases are widespread throughout the world, and it has been estimated
that they cause an annual loss to the poultry industry of about 60 million
dollars in the United States alone (Hinton, 1949).

The plan of this thesis is to present the history of the various
etiological concepts of the avian leukosis complex, to review the litera-
ture on the transmission of avian lymphomatosis, and to present the results
of recent studies on the problem of egg transmission of avian lymphonatosis.

The problem has been approached from the viewpoint that the various
diseases embraced by the avian leukosis complex are separable and that at
least some of them are probably caused by separate, filtrable oneogenic
agents. The pathological changes and diagnostic aspects of these diseases
have been omitted purposely because they have been adequately covered in
other reports (Jungherr, 1948; Feldmn and Olson, 1948; Runnells, 1948).

For clarity, a standard nomenclature has been used throughout.

HISTORY OF‘TEE ETIOLOGICAL CONCEPTS

In 1896 Caparini in Italy described fowl leukemia, in 1905
Butterfield reported on WAleukemic lymphoid tumors othhe hen,” and in
1907 Marek in Hungary described "Multiple polyneuritis of fowl." From
these beginnings two independent groups of workers evolved-~one was the
fowl leukosis group, and the other was the fowl paralysis group. The
fowl leukosis group was composed of veterinary and comparative patholo-
gists and medical men who were interested in tumors and in.human leukemia.
They found in fowl leukosis a means of studying,experimenta11y, a
leukemic disease. The fowl paralysis group was composed of veterinary
pathologists, poultrymen and other investigators who were interested in
fowl paralysis prflmarily because of its economic importance to the

poultry industry.

The Original Etiological Concepts (1896 to 1929)

The fowl leukosis group.--The disease entities originally included

 

under the term.fow1 leukosis were: erythroblastosis, granuloblastosis,
myelocytomatosis, and visceral lymphomatosis. In Ellernann's (1921. 1925)
concept of fowl leukosis all of these diseases were caused by the same
etiological agent. The studies by this group of workers were highlighted
by the discovery of Ellermannand Bang, in 1908, that fowl leukosis could
be transmitted with a filtrable agent. .111 of the workers agreed that
there were several types of fowl leukosis, but they disagreed on the
nomenclature and the transmissibility of the various types. Some inves-
tigators reported an inability to transmit ”lymphatic" or 'extravascular
leukosis" and others were inclined to classify visceral lymphomatosis as

a separate disease (Andersen and Bang, 19283 Furth, 1929). Mhthews (1929)

-4-

believed that leukochloroma (myelocytomatosis) was also a separate
disease. Attempts to discover the natural mode of spread of fowl
leukosis failed. This was the status of fowl leukosis in 1929.
Ellermann's concept of the disease was beginning to be seriously
questioned.

The fowl paralysis group.--Originally fowl paralysis meant only
neural lymphomtosis; later, ocular and visceral lymphomatosis were also
included because of their frequent association with neural lymphomatoeis.
During the period 1907 to 1929 fowl paralysis spread throughout the major
poultry producing areas of the world. It spread like an infectious disease,
and the etiological agent was assumed to be a filtrable virus (Kaupp, 19213
Van der Walls and Uinkler-Junius, 1924). Doyle (1929) suggested that
fowl paralysis may be transmitted from dam to offspring through the egg.
Pappenheimer and his associates (1926) demonstrated that this disease
could be experimentally transmitted. The hmwledge of fowl paralysis in
1929 was summed up quite well by Pappenheimer and his associates (1929a,
1929b): ”Although the evidence, epidemiological and experimental, points
to an infective agent, we are totally ignorant as to its nature, and
as to the manner in which the disease is conveyed under natural condi-
tions.......... Our studies at least indicate that the agent which is
responsible for the pathological changes in the nervous system is not
exclusively neurotropic; it may in certain cases stimulate the prolifer-
ation of lymphoid cells in the viscera, and to a degree which makes them
take on the morphological character of a neoplasm."

During the period 1921 to 1929 there were numerous cements in the
literature that attempted to show a relation between fowl paralysis and

intestinal parasites (coccidia, tapeworms, etc.) Eventually this

- 5 -

erroneous supposition was dropped as more facts concerning the disease
became known.

The status of visceral_1ymphomatosis (lymphocytoma).-AViscera1

 

lymphomatosis was independently claimed by both the fowl leukosis and
fowl paralysis groups. This dual relationship of visceral lymphomatcsis
was the result of its confusing association with.the diseases studied

by each group. Ultimately, visceral lymphomatosis became the common
denominator that resulted in drawing the two groups closer together.
Pappenheimer and his associates (1929a, 1929b) were the first to notice
this overlapping. They saw that what Ellermann (1923) and his associates
were calling "lymphatic leukosis," was similar to their "visceral
lymphomata.” However, they were emphatic in their denials of an assoc-
iation of visceral lymphomatosis with leukemia.

In the fowl leukosis group the later tendency was to exclude
visceral lymphomatosis because it was thought to be a nonptransmissible
lymphoid tumor that was not accompanied by a true leukemic state.
Feldman.(l927) preferred to classify'this disease as lymphocytoma, defined

as a.malignant lymphoblastic neoplasm of chickens (Fumth, 1929).

Later Eti010gical Concepts (1929-1950)
The fowl leukosis and fowl paralysis groups maintained their sep-
arate identifies up to 1932. At that time the two groups were united;
however, many workers refused to join the union. The influence of the

original groups has persisted up to the present time.
Unitarian view.--In 1932 Johnson came to the conclusion that fowl

paralysis and fowl leucosis were all part of the same disease complex,

after observing that all types of’inwolvement resulted in.his transmission

trials. In the same year, Patterson, Wilcke, Hurray and Henderson (1932)
presented the results of their study of‘"so-called range paralysis of the
chicken," and they also concluded that these diseases were all part of
the same disease complex. Johnson (1932) summed up this viewpoint as
follows: ”From.this work it appears that the common cause of leucosis

and lymphomatosis of fowls is a filtrable agent, and that the predominat-
ing cell in the lesions is the hemocytoblast which is a trivalent cell
capable of differentiation into erythrocytes, granulocytes, and lympho-
cytes." Mamy*workers adopted this viewpoint. Jungherr (1948) has

termed this concept "the unitarian view."

Multitarian view.--Opposed to the unitarian view is the multitarian

 

view, which as presented by Furth (1933) tended to demonstrate that each
of the various diseases had its own etiological agent that performed
quite true to type when.maintained as a transmissible strain. Furth (1936)
was inclined to view the few discrepancies that occurred as contaminants
that were picked up in the course of serial passage. Feldman and Olson
(1933, 1934) had a somewhat similar viewpoint based primarily on the
pathological changes. They believed that lymphocytoma (visceral lympho-
matosis) was nondtransmissible, and that lymphocytoma, neural lymphomatcsis,
myelocytoma, and leukosis (myeloid and erythroleukosis) were all distinct,
unrelated diseases.

Division of visceral lymphomatosis.--Furth (1934b) and Stubbs (1937)
recognized “hepatolymphomatosis” (big-liver disease) as a separate
disease, but classified it as lymphoid leukosis along wdth.myeloid and
erythroleukcsis. Stubbs (1939) advocated placing the other visceral
lymphoid tumors (i.e., other than hepatolymphomatosis) in the fowl

paralysis category. Thus, a concept developed that there are two kinds

-7-

of visceral lymphomatosis: the visceral lymphomtosis associated with
neural and ocular lymphomatosis, and a visceral lymphomatosis or
lymphoid leukosis associated with erythroblastosis and granuloblastosis.
This was essentially a return to the concepts of the two original groups.

leukosis-sarcoma virus .--Oberling and Guerin (1933a, 1933b) pre-

 

sented evidence which suggested that fowl leukosis and sarcoma were
caused by the same agent . Their viewpoint was as follows: "The virus
of leukemia of fowls can give rise to sarcoma by affixing itself to
connective or histiocytic cells, and this histiotropism is most plainly
exhibited if, instead of making the transfers by intravenous injection
of leukemic blood, one nukes intramuscular or subcutaneous grafts of
leukemic material, which hinders the virus from exteriorizing its always
predominant hemotropism.‘ Other workers also reported the apparent
association of sarcoma and fowl leukosis or lymphomatosis (Troisier and
Sifferlen, 1935: Rothemeyer and Engelbreth-Holm (1933). However, some
workers felt that the association was simply a coincidence (Furth, 1934a),
and Stubbs and Forth (1935) could not duplicate Oberling's claim that
storage in glycerine modified the fowl leukosis agent, so that it would
produce sarcoma. Nevertheless, the virus tumors described by Rous and
his associates (1911-1914) had to be considered in subsequent studies
on leukosis and lymphomatosis.

Mixed and complex strains .--Furth (1934b) found that endotheliom

 

and myelomatosis were sometimes associated with lymphomatosis. This
brought the transmissible endothelioma described by Begg (1927) into the
already confused picture. Furth (1934b, 1936) postulated mixed and

complex strains. A mixed strain had several agents each one of which

- 3 -

produced a different type of neoplasia. A complex strain had a single
agent wdth multiple potentialities for producing different types of
neoplasia.

Osteopetrosis.--Jungherr and Landauer (1938) observed that osteo-

 

petrosis was frequently associated with lymphomatosis. In their concept
the agent of osteopetrosis was "non-dissociable from.the agent of
lymphomatosis."

Hemocytpblastosis.--Emmel (1934, 1937) thought that the basic

 

process in this group of diseases was a hemooytoblastosis, which resulted
from numerous non-specific factors, such as vitamin and mineral deficiencies
and bacterial toxins, particularly of thesalmonella group.

Chick disease virus.--In 1939 Blakemore reported on the discovery

 

of a filtrable agent in the tissues of birds with lymphomatosis. This
agent when inoculated into young birds produced an inflammatory response.
Blakemore advanced the theory that this acute disease in chicks was
caused by the same agent that was responsible for'lymphomatoeis in older
birds, and that the lymphomatous involvements represented the end result
or chronic stage offan acute infection. Glover (1940) and 1ater.Asplin
(1947a, 1947b, 19470) were also able to demonstrate this agent, which
Asplin has since termed ”'chick disease' virus." In his latest report
Asplin (1947c) made the following comment, "Experiments were carried out
in an effort to determine the validity of the theory that 'chick disease'
is an acute reaction_caused by the agent responsible for lymphomatosis.
The results, whilst not conclusive, present evidence that this is not so.”

The avian leukosis complex.--In 1940 a committee of'pathologists

 

Inst at the U. S. Regional Poultry Research Laboratory and suggested a

tentative classification in which this whole group of diseases was

- 9 -

collectively called “the avian leukosis complex.“ (Jungherr, 1941) The
classification was not intended to imply an etiological relationship of
the various diseases, but was suggested as a means of‘arriving at a
uniform terminology in the literature. In spite of the admonition, many
people construed the classification to mean acceptance of the unitarian
view.

Recent trends.--In the more recent literature the trend has been

 

away from.the unitarian viewpoint, especially in regard to separating
lymphomatosis from.erythroblastosis and granuloblastosis. myelocytomatosis
and sarcomatosis likewise have been set apart from the other diseases.
Leukosis has been thought of as meaning only erythroblastosis and
granuloblastosis. and lymphomatosis has been accepted generally as a
term.that includes neural, visceral, ocular and osteopetrotic forms.
Recently there has been an attempt to separate the various types of
lymphomatosis on an etiological basis, and the new evidence indicates

that this eventually may be possible (Davis and Doyle, 1947a, 1947b,

1947c, 19493'Burmester, 1947c).

The difficulty in the separation of these agents in the past may
have'been due to the use of heterogeneous populations cf'birds, in
which many agents may have been present in a latent fern, The use of
pedigreed inbred birds, whose strain and familial histories have been
followed, and the use of isolated quarters may enable investigators to
solve this problem of separation. Fortunately we have passed the day
when an experimenter will rely on the results obtained with a crate of

nondescript chickens from.the local market.

- 10 -

REVIEW'OF THE TRANSMISSION STUDIES ON AVIAN LYMPHDMATOSIS

Transmission of neural lymphomatosis.--Neural lymphomatosis has been

 

transmitted by inoculating birds with suspensions of nerve, ganglion,
spinal cord, and brain tissues taken from.birds with lesions of neural
lymphomatosis. The inoculations were made via the intraperitoneal,
intracranial, intraneural, subcutaneous, intramuscular, subdural, and
intravenous routes (Pappenheimer, et a1., 1926, 1929b; Baumann, 1936;
Jungherr, 1937; Beach, 1938; DeOme, 1943). Intravenous inoculation of
fresh or heparinized whole blood from cases of neural or ocular lympho-
matosis appears to be the method of'choice in transmitting neural.
lymphoontosis (Durant and MbDougle, 1937, 1939, 1945, 1947). ‘When the
transmission of neural lymphomatosis is attempted, the resulting inci-
dence is usually not very high, and other forms of the avian leukosis
complex are likely to be transmitted also, especially visceral lympho-
matosis. Transmissible strains of neural lymphomatosis have been
reported (Furth, 1934c, 1955). Freezing at -30° C and dnying from.the
frozen state inactivates inocula from such transmissible strains.

Transmission of neural lymphomatosis with a filtrable agent has
been claimed (Van der'fialle and'Winkler-Junius, 1924). The evidence to
support this assertion is quite insufficient, yet most workers agree
that it is very likely that neural lymphomatosis is caused by a filtrable
agent. The hypothetical agent is thought to be present in the circulat-
ing blood and in the involved nervous tissue.

The lesions found in peripheral nerves of paralyzed birds that
show typical symptoms of neural lymphomatosis may be either microscopic

foci of small lymphoid cells that have an inflammatory appearance, or

-11...

large areas of definite neoplastic cells. Birds that have been destroyed
‘within a few days after they first developed paralytic symptoms usually
had the inflammatory type of involvement; whereas, paralytic birds that
were allowed to live several weeks after the appearance of symptoms
usually had the neoplastic type of involvement. Microscopic lesions
suggestive of neural lymphomatosis have been.seen frequently in the
peripheral nerves, adrenal ganglia and other sympathetic ganglia in
birds that apparently were normal and showed no symptoms of nerve dis-
order during life.

Transmission of ocular 1ymphcmatosis.--Ocular lymphomatosis has

 

been observed in the chickens of the inoculated groups by many workers
(Johnson, 1932; Durant and McDougle, 1937, 1939, 1945). However, the
incidence of this disease was usually so low that a significant differ-
ence between the inoculated and control groups could not be demonstrated.
This has been a most disconcerting disease entity because of its rarity
and sporadic occurrence. However, a rather high incidence of the disease
has occasionally been reported in certain farm flocks.

Transmission of other types of lymphomatosis has resulted and tumor
strains have been developed from donors that had ocular lymphomatosis
(Brandly, et a1., 1942). .L series of transmission studies using whole
blood from.donors with ocular or neural lymphomatosis demonstrated that
lymphomatosis could be transmitted in this manner. 'lhen a large amount
of blood was transfused from such donors into chicks, a high incidence
of‘neural lymphomatosis resulted (Durant and McDougle, 1945). Successful
transmission of lymphomatosis, especially neural lymphomatosis, was
accomplished using whole blood from.normal chicks as the incculum. The

chicks were the offspring of birds with ocular lymphomatosis (Durant and

MCDougle, 1937, 1939).

- 12 -

Recently it has been demonstrated that a transplantable visceral
lymphomatous tumor strain will survive and grow when inoculated into
the anterior chamber of chicken eyes. In such cases the iris was
often involved and presented a gross and histological picture similar
to ocular lymphomatosis (Little, et a1., 1949, Burmester, 1949).

Transmission of visceral lymphomatosis.--Viscera1 lymphomatosis

 

has been transmitted by inoculating chicks with whole blood from.birds
with visceral, neural, and ocular lymphomatosis (Johnson, 1932; Furth

and Breedis, 19333 Brandly et a1., 1942). The use of cell-containing
inocula prepared from.blood or tumor tissue from birds with naturally
occurring visceral lymphomatosis led to the discovery that transplantable
lymphoid tmmor strains could be developed and carried in serial passage.
One of these strains produced visceral lymphomatosis, myelomatosis and
endothelioma (FUrth and Breedis, 1933). .A filtrable agent was demons
strated in this tumor strain. This evidence was not readily accepted
because in the other tumor strains that produced visceral lymphoid tumors
(lymphocytoma), filtrable agents could not be detected (Olson, 1941, 1944).
This led to the idea that lymphocytoma could be set apart from the other
diseases of the avian leukosis complex as a nonrtransmissible but
transplantable neoplasm» ‘Meanwhile, others contended that visceral
lymphomatosis was a part of the avian leukosis complex and that it could
betransmitted by inoculating whole blood into chicks (Brandly et a1. 1942).
Thus, transmissible strains of’lymphomatosis were developed and carried

in serial passage, using blood as an inoculum; later, other lymphoid tumor
strains were developed by inoculating into chicks tumor’cell suspensions
from naturally occurring cases of visceral lymphomatosis (Burmester and

Prickett, 1945).

- 13 -

The Olson lymphoid tumor, RPL 12 (Olson, 1941) was carried in
serial passage and studied at the U. S. Regional Poultry Research
Laboratory (Burmester and Cottral, 1947d). A filtrable agent that
produced lymphoid tumors and osteopetrosis was found in this tumor strain.
subsequently, this tumor strain was carried through six serial passages
using cell-free inocula. Because the filtrable agent was found in a
transplantable tumor strain and filtrable agents had not been demon-
strated in naturally occurring visceral tumors, it was not accepted as
the etiological agent of visceral lymphomatosis. It was believed that
there was a difference between the naturally occurring visceral lympho-
matosis and the filtrate induced lymphoid tumors. Recent studies have
shown that this objection is no longer valid (Burmester and Denington,
1947e; Burmester, 1947f).

In a recent study it was found that visceral lymphomatosis (and in
one group osteopetrosis) could be transmitted with cell-free inocula
prepared from.lymphoid tumors of donors with naturally occurring lympho-
mtosis (Burmester and Denington, 1947s). Four out of ten tumor bearing
donors tested gave positive results using cell-free inocula. The types
of’lymphomatosis in the four donors were as follows: two had gross
visceral and neural lymphomatosis; one had visceral lymphomatosis, only;
and the other had gross visceral and ocular lymphomatosis and micro-
scopic evidence of neural lymphomatosis. It was also found that lymphoid
tumor strains could be developed and carried in serial passage using cell
suspensions prepared from the tumors of some of these donors. Further-
more, transmission cf visceral lymphomatosis and osteopetrosis using cell-

free inocula could be demonstrated at different stages during the course

-14-

of serial passage of some of these and other tumor strains. Some of the
tumor strains were carried through several serial passages using cell-
free inocul a. One tumor strain was carried through seven serial
passages using cell-free inocula, but it did not produce typical vis-
ceral lymphomatosis, and hemangiomatosis was associated with it
(Burmester, 1947f).

Many workers have reported on the transmission of lymphomatosis
using whole blood or tumor cell suspensions taken from cases of the
naturally occurring disease. These cell-containing inocula have dual
potentialities and it is exceedingly difficult, if not impossible, to
demarcate strictly between these lymphoid tumors resulting from the
multiplication of transplanted tumor cells and lymphomatosis caused by
the etiological agent. Undoubtedly, many of the transmission studies
reported in the literature, where cell suspensions were used, should be
regarded as true transmissions and not transplantations. The difference
in the incubation periods may be one method of separation. In trans-
mission studies, using cell-free inocula and inbred susceptible birds,
the incubation period for visceral lymphomatosis varied from about 60
to 250 days, with an average of about 150 days. After serial passage
with cell-containing inocula the transplantable lymphoid tumor strains
had a much shorter incubation period, varying between 6 and 30 days.
Thus, when a cell-containing inoculum is used, those birds that die of
lymphomatosis after a long incubation period, are more likely to develop
lymphomatosis from the etiologic agent than from the multiplication of
the transplanted tumor cells but a strict differentiation cannot be made.
Recently, the relationship of the other forms of the avian leukosis

complex to visceral lymphomtcsis was reviewed and subjected to critical

- 15 -

study. The conclusion was reached that visceral lymphomatosis could

be readily transmitted, and that neural and ocular lymphomatosis were
not transmitted by the method employed by these workers. The incculum.
consisted of cell suspensions prepared from the tumorous organs of

birds wdth.viscera1 lymphomatosis (Davis and Doyle, 1947a, 1947b, 1947c,
1949). Other studies have borne out this conclusion. Further work
suggested that there was no marked concentration of the causative agent
or absence of it in any of the organs tested in the transmission studies.
The organs tested were the liver, heart, spleen, kidney, ovary, and
blood from a bird with naturally occurring visceral lymphomatosis (Davis
and Doyle, 1947a, 1949).

In other experiments, visceral lymphomatosis, and possibly neural
lymphomatosis, were transmitted to young chicks by inoculating them
intratracheally and intranasally with tracheal and nasal washings from
donors with neural and ocular lymphomatosis (Cottral and Burmester, 1948;
'Hinton, 1948, 1949). Filtrates of the respiratory washings were not
effective in producing lymphomatosis. In the same study, respiratory
washings rendered bacteriologically sterile with penicillin and strep-
tomycin were found to be effective in producing lymphomatosis when
inoculated intravenously. However, the same inoculum was ineffective
when inoculated via the respiratory route. Peroral administration of
tumor-cell suspensions appeared to be effective in transmitting visceral
lymphomatosis but not all workers have been successful in the transmission
of lymphomatosis when the oral and respiratory routes were used (Brewer
and Brownstein, 1946; Barber, 1948). Successful transmission has been

reported with fresh and desiccated feces (Jungherr, 1937).

- 15 -

Studies with tumor strains induced with cell-containing inocula.--
The transplantable tumor strains have been carried in serial passage
via the intraperitoneal, intravenous, intramuscular, or intraocular
rovtes by adaptation. Serial passage reduced the incubation period of
the tumor strain so that some of the strains produced tumors and
caused the death of the recipients in 6 to 30 days (Olson, 1944;
Burmester, 1945, 1947f). Dilute inocula containing as few as eight
tumor cells still resulted in successful transplantation. The thermal
death point of the tumor cells has been found to be between 45° and 50°C.
(Cottral and Burmester, 19483 Hinton, 1947, 1948). Chicks so days or
less of age were the most susceptible to the transplanted tumors.
However, the resistance of many of the older birds was overcome by
increasing the dosage of the incculum. Birds that developed trans-
planted tumors and survived were immune to further implants, but their
immunity appeared to be only against the cells, as many of'them.develcped
typical visceral lymphomatosis and died at a later date (Burmester, et a1.,
1946, 1947b, 19470). In some tumor strains a ”hemorrhagic disease" has
been seen in some of the inoculated birds. The lesions appeared to be
similar to those that have been described in studies with the Rous
agent (Duran-Reynals, 1940).

Several lymphoid tumor strains have been successfully transplanted
on the chorio-allantoic membrane of embryonated eggs, and the resulting
tumors have been carried in serial passage. When embryonated eggs were
inoculated with cell-free filtrates of visceral tumors no measurable
reaction took place in the embryos. Thus, the available embryonic time
prior to hatching is sufficient for transplanted tumor cells to form.a

tumor, but appears to be insufficient for the induction of tumors with

- 17 -

the filtrable agent. Another interpretation would be that other factors
necessary for tumor production with the cell-free agent may be lacking.

Transmission of osteopetrosis.--Ostecpetrosis has been a relatively

 

rare disease. The incidence was reported to be less than 0.05 percent.
In an endemic outbreak studied, the incidence was less than 2.0 percent
(Jungherr and Landauer, 1938). Osteopetrosis has usually occurred in
association with visceral lymphomatosis. 'Nhen.birds with spontaneous
osteopetrosis were used as donors the disease was transmitted to recip-
ient birds by using cell suspension prepared from.blood, bone marrow,

and visceral lymphomata. Four serial passages were made with this strain.
The agent survived desiccation up to 105 days.

A ”lymphomatosis-osteopetrosis" strain was developed from donors
that had neural and ocular lymphomatosis, but showed no evidence of
osteopetrosis (Brandly, Nelson, and Cottral, 1942). Osteopetrosis
occurred in six successive serial passages of this strain. Heparinized
whole blood was used as the inoculum.

Recently it was feund that osteopetrosis could be transmitted with
a filtrable agent (Burmester, 1947a; Burmester and Cottral, 1947d).

The filtrable agent was first demonstrated with cell-free inocula pre-
pared from.the Olson visceral tumor strain (RPL 12). Filtrable agents
producing osteopetrosis and visceral lymphomatosis were carried through
six serial passages using cell-free inocula prepared from.visceral tumors
and blood derived originally from.birds inoculated with strain RPL 12.

Osteopetrosis has been.transmitted with'a filtrable agent derived
from.an ovarian tumor of a bird with naturally occurring visceral and
neural lymphomatosis (Burmester and Denington, 1947e). Cell suspensions

prepared from this tumor also produced osteopetrosis. In other studies

- 18 -

osteopetrosis was produced with cell-free and cell-containing inocula
prepared from visceral tumors and blood derived from birds inoculated
with other lymphoid tumor'strains. In.many instances osteopetrosis has
been latent or ”masked” in donor birds and has been found in subsequent
passages. The incubation period for osteopetrosis was quite variable

in transmission studies. It ranged from.about 60 to 300 days. Centrif-
ugation studies indicated that it might be possible to effect a partial
separation of the agents of osteopetrosis and visceral lymphomatosis
(Burmester, 1947a). It has been suggested that these two diseases may
be caused by separate agents.

A condition resembling osteopetrosis was found in chicks that
received inoculations of bone marrow, both embryo and x-rayed adult
marrow (Gohs, 1934). Osteopetrosis was occasionally seen in some of
the complex strains of leukosis and lymphomatosis (Furth, 1936).
Osteopetrosis was found in groups of chickens that had been inoculated
with the Rous sarcoma (Pikovski and Doljanski, 1946). Recently a
disease resembling Osteopetrosis was observed in groups of chickens
that had been inoculated when embryos with the blood of mice having
mouse leukemia (Thiersch, 1944). In the same series of studies, osteo-
petrosis also occurred in groups that received chronic myeloid leukemic
blood from.man. These birds were also inoculated when they were embryos.
In this particular study none of the other forms of the avian leukosis
complex occurred.

mechanical transmission.--Insect and rodent vectors for the agents

 

of'ths avian leukosis complex have been postulated. It has also been
suggested that accidental transmission may take place during.surgical,

blood testing, and vaccinating operations, and by the use of certain

-19-

vaccines prepared from ombryonated eggs. However, none of these methods
of'transmission have been established.

Contact transmission.--The evidence indicates that naturally acquired

 

lymphomatosis can.be transmitted by contact of infected chicks with non-
infected chicks in the incubator and during the first few weeks of life
(Waters and Bywaters, 1949; Haters 1947a, 1947b). Contact transmission
after the first month of'life appears doubtfud. Chicks artifically
infected by inoculatiOn via the intraperitoneal route usually do not
transmit the disease to contact controls that are raised with them,
On the other hand, chicks inoculated via the intravenous, oral, and
respiratory routes in some instances appear to transmit the disease to
chicks in the contact control groups. This suggests that the route of
infection may play an important part in determining whether or not the
disease is spread by contact. However, an important question.not
answered by the available data is: do those exposed birds pick up the
etiological agent and become carriers not destined to die of the disease
themselves, but capable of passing the agent on.to the next generation?
The transmission studies using the oral and respiratory routes of
inoculation indicated that lymphomatosis can be transmitted via these
routes and that the agents appear to be present in the respiratory tract
of certain birds.

All of the evidence seems to favor the conclusion that erythro-
blastosis, granuloblastosis, and the Rous sarcoma are not transmitted

by contact.

- 20 -

THE PROBLEM OF‘EGG TRANSMISSION

The natural mode of transmission of avian lymphomatosis has not been
clarified. One of the main questions that needs to be answered is whether
or not avian lymphomatosis can.be transmitted from the dam or sire to
the offspring through.the egg. The second phase of this problem.is to
determine whether apparently normal birds, i.e., those without clinical
or gross evidence of the disease, can be carriers of the agent or agents
of lymphomatosis. With these points established, control measures for
the disease may be more easily sought. An answer to this whole problem
is of vital importance to the poultry industry.

Doyle (1929) was the first to suggest that avian lymphomatosis may
be transmitted through the egg. Since then, many other workers have pre-
sented observations and experimental evidence both for and against this
contention. The evidence favoring egg transmission of lymphomatosis
may‘be classified as follows: epiornithic observations, flock isolation
studies, family isolation studies, incubator exposure studies, histo-
logical studies, and transmission studies involving inoculation.

Epiornithological observations on the spread of‘lymphomatosis
indicated to some workers that the disease may have been introduced
into flocks by the purchase of hatching eggs from infected flocks (Doyle,
1929: marginson and mcGaughey, 19313‘Warrack and Dalling, 1932).

Flock isolation studies,such as the establishment of the U. 8.
Regional Poultry Research Laboratory flock, also suggested egg trans-
mission. In.this flock only eggs were introduced into previously
unused quarters and the first cases offlymphomatosis were found during

the second and subsequent months after the chicks hatched (Waters, 1945).

- 21 -

Family isolation studies were carried out in which only the eggs
of individual hens were placed in isolated incubators (Waters and
Prickett, 1944). Thus, each family group of chicks was hatched and
reared in an isolated pen. Gross manifestations of lymphomatosis were
later evident in the birds of certain family groups, but not in others.
This was further evidence of egg transmission.

The incubator exposure studies also indicated egg transmission
(Waters and Bywaters, 1949). These experiments were carried out with
the offspring of isolated dams. Control groups were hatched and reared
in isolated pens and their sibs, comprising the test groups, were hatched
and reared in incubators and pens with the offspring of non-isolated
highly I'contaminated" parent stock. Other test groups of sibs were
batched in isolation and then exposed to the non-isolated or ”contaminated"
stock at different ages following hatching. At the termination of the
experiments a significant difference in the incidence of gross lympho-
matosis was found between the groups exposed continually from.hatching
on to termination and the groups that were hatched in isolation and then
exposed from one day of age on to termination. The incubator exposed
groups had the highest incidence of lymphomatosis. These results were
interpreted to indicate that the offspring of the nonrisolated or
”contaminated” stock were being infected with lymphomatosis through the
egg and that they were capable of infecting the test group in the
incubator at hatching time to a greater degree than at any time there-
after. Thus, those birds hatched in isolation and exposed at one day
of age had a lower incidence of lymphomatosis because they missed the

incubator exposure.

- 22 -

Histological studies have given information relative to egg trans-
mission, especially dealing with a possible mechanism.of transmission
and the carrier-bird problem.(Lucas, 1946; Lucas, et al., 1949a, 1949b,
1949c, 1950; Oakberg and Lucas, 1949). If the agent of lymphomatosis
causes the formation of lymphoid areas in the viscera and nerves of'birds,
then there is histological evidence for the presence of the agent in
embryos, since such areas have been feund in some embryos.

Transmission studies that tended to indicate egg transmdssion were
conducted by Durant and McDougal (1937, 1939). They inoculated the
blood from.chicks that were the offspring of diseased parent stock
(ocular lymphomatosis) into other susceptible chicks. A higher inci-
dence of neural lymphomatosis was found in the inoculated chicks than
in'their uninoculated control sibs.

The evidence against egg transmission is very weak. Some workers
have noted a lower incidence of'lymphomatosis in heavily infected stock
when it was raised in isolation as compared to similar populations left
in the exposed environment (Johnson and‘flilson, 1937; may, Tittsler and
Goodner, 1925). Other investigators have been impressed by the low
incidence of lymphomatosis in the offspring of certain dams with ocular
lymphomatosis, and they have shown that the offspring of such dame do
not have any higher incidence of lymphomatosis than the offspring of
other apparently normal dams-(Madsen, 1937} Ball and Cole, 1946). Other
workers have reported the opposite effect when infected dams were used
(McClary and Upp, 1939).

Hutt and Cole (1947, using the results obtained with diallel and
reciprocal crosses between lines that had been bred for resistance or

susceptibility to lymphomatosis, concluded that the influence of'egg

-23-

transmission, if it does occur, is not nearly as great as are genetic
and environmental influences.

Recently, Cole (1949) made a study of several populations of birds,
dividing them into two main groups. These two groups were as follows:
(1) Chickens that were the offspring of birds that died of lymphomatosis
during the breeding season or shortly thereafter, and (2) chickens that
were the offspring of birds that survived throughout the breeding season.
There was no significant difference between the two groups of chickens
in respect to the incidence of lymphomatosis. This evidence was inter-
preted to indicate that if egg transmission of lymphomatosis does occur
it is not of great importance in determining the viability of the progeny.
These experiments were based on the premise that carrier birds are
destined to die of lymphomatosis within a relatively short period of

time--a conclusion that does not seem to be justified.

- 24 -

MATERIALS AND METHODS

In the present study conducted at the U. 8. Regional Poultry
Research.Laboratory only single-comb'fihite Leghorn chickens of'both
sexes were used. The experimental chickens were all from inbred line 15
of the laboratory flock. The history of this line, which has been raised
in isolation, has been described by Waters, et al. (1944, 1949). This
line oi‘chickens has had a relatively low incidence of lymphomatosis
when raised in isolated quarters, yet the birds were quite susceptible
to lymphomatosis, as proved by their performance when exposed in a
"contaminated" environment.

Fourteen hatches of inbred line 15 chickens were used in this study.
All of the line 15 chickens were hatched in isolated incubators away
from all other chickens. The chickens were brooded and raised in isolated
pens; thus, they had no direct contact with any other chickens. In all
of the experiments, except experiment No. l, and the isolated controls
for the other experiments, the following procedure fer'housing was used:
‘When the birds were about 60 days old the males from the various experi-
mental lots were removed and placed in a separate pen adjoining the pen of
females. This was done to lessen the danger of cannibalism. Later,
when the birds were about 100 days old, they were moved from their
brooding pens and placed in laying house pens, still maintaining the
separation of the sexes by placing them.in adjoining pens. The diet fed
to the birds was the standard laboratory poultry ration (Hinton, Lucas,
and Cottral, 1950). The experimental work was started in April, 1947,
and was terminated in.May, 1950. The experimental period for each group
of birds was set at 310 days, except in the case of two groups which were

terminated at 250 days. All birds that died during the course of'the

experimental period were subjected to a necropsy examination. At the
termination of the experimental period all surviving birds were killed
and examined. The diagnosis of lymphomatosis was based on the gross
necropsy findings.

Source and preparation of incculum.-~1n order to provide inocula

 

for these studies certain dams were selected as donors. The dams used
were either picked at random from.the exposed population or were selected
on the basis of the incidence of lymphomatosis in their sibs and progeny.
Eggs were saved from these selected dams and were then incubated. At the
appropriate time these embryos or hatched chicks were killed and their
tissues were aseptically removed. Fbr the most part liver tissue was
used in these studies.

Embryo and chick tissue.--In general the inocula were prepared as

 

follows: The liver tissue was weighed, then ground in a sterile test

tube grinder. Sterile 0.85 percent saline was added at the rate of

1 part tissue to 9 parts of saline. The cell suspension was then filtered
through sterile gauze to remove the large clumps of tissue. The inoculum
was then placed in rubber stoppered serum vials and held under refrigera-
tion until used. The inoculum was never held in storage longer than two
hours. Sterility tests were made at the time the inoculum was placed in
the vials.

'Hhen filtered inocula were prepared the same procedure was followed
except that the cell suspension was passed through a Seitz filter, using
a No. 8 pad, and N2 pressure. The filters were tested following filtra-
tion of the inoculum for their ability to hold back cultures of bacteria
in.broth medium. Usually a culture of micrococci or Serratia marcescens

was used fer this test. Thus, one could assume that no cells were able

- 26 -

to pass through to the filtrate and that the filter was in good work-
ing condition.

material from donor hens.--In one experiment the blood of‘adult

 

birds was used as an incculum. The blood was drawn from the wing vein
and was heparinized to prevent coagulation by using a solution of
heparin at the rate of 1 mg. per 7.5 m1. of blood.

The respiratory washings were collected from.the donor hens by
holding the bird with its head down and washing sterile saline through
its nasal passages. A syringe with a rubber tip was used for this purpose
and the fluid was injected into the choanae and caught as it came out the
external nares. Approximately 10 ml. of fludd was used to wash through
the nasal passages of each donor. Penicillin (100,000 units) and
streptomycin (35.0 mg.) were added to the respiratory washings to
inactivate the bacteria. The material was held in the refrigerator Ibr
two hours before it was used.

All inoculations were made intraperitoneally, unless otherwise
specified, and the amount used for this route was 0.25 ml. The birds
were inoculated at one day of age.

Experimental groups.--There were eight separate experiments, each

 

of which was composed of various lots or groups of chickens that received
different treatments. Each hatch used was divided so that each lot received

as nearly an equal family representation as possible.

Experiment No. l
Donors.--In this preliminary experiment 9 donor chicks were taken
from the incubator tray on the morning they hatched. These chicks were

selected at random, taking one chick from each of nine different inbred

- 27 -

lines (Table 2). The chicks were sacrificed and inocula were made as
follows:

Inoculum(A) was composed of the pooled liver tissue of all of the

 

Chicks 0

Inoculum (Bl_was the pooled yolk sacs of all of’the chicks.

 

Inoculum (01 was the pooled intestines and trachea of the chicks.

 

Recipients.-4The inocula were administered to the recipient line 15

 

chicks when they were one day old as follows: Lot No. 1.--l4 chicks,
inoculum (A) given intraperitoneally, 0.25 ml.: inoculum (0) given intra-
tracheally, 0.12 ml.

Lot No. 2.--l4 chicks, inoculum (B) given intraperitoneally, 0.25 ml.
inoculum (C) given intratracheally, 0.12 ml. Lots 1 and 2 were from a
single hatch and were raised together in an isolated pen.

Lot No. 3.--34 non-inoculated control chicks from.a single hatch,
hatched and raised in an isolated pen adjacent to the pen in which lots 1
and 2 were located.

Lot No. 4.--28 non-inoculated control chicks from.a separate hatch,
also raised as an isolated group.

This experiment was started on June 30, 1947, and terminated about

300 days later.

Experiment No. 2
2222:23--The donors for this experiment were 15 day old embryos,
18 day embryos and newly-hatched chicks--all were the offspring of 5
selected dams. These dams were selected because there was a fairly high
incidence of lymphomatosis among their offspring or their sibs. (See
table 3). All of the dams and sires of the donor'embryos lived for 600

days, at which time they were killed. None of'them showed any clinical

- 23 -

or gross evidence of lymphomatosis. No abnormalities were noted in
the donor embryos and chicks. In order to carry out this experiment,
it was necessary to set eggs from these selected dams at specified
intervals so that on a predetermined date there would be available,
embryos 15 and 18 days old and chicks just hatching. In each age
group two embryos (or chicks) were used from.aach of the five dams.
Thus, each inoculum was composed of the liver tissue of 10 embryos (or
chicks).

Recipients.--One hatch of line 15 chicks was divided into 5 experi-

 

mental ldzs, i.e., lots 5 to 9 inclusive. All inoculations were made
intraperitoneally.

Lot No. 5.*--l7 chicks were inoculated with liver tissue from 10
newly-hatched donor chicks.

Lot No. 6.--l4 chicks were inoculated with a cell-free filtrate of
the liver tissue of the 10 newly-hatched donor chicks.

Lot No. 7.--l7 chicks were inoculated with liver tissue from 10
embryos, 15 days old.

Lot No. 8.--l7 chicks were inoculated with liver tissue from.10
embryos, 18 days old.

Lot No. 9.--l7 chicks were used as uninoculated contact controls.
(All of the above groups of chickens, i.e., lots 5 to 9, inclusive, were
hatched and reared together in an isolated environment. lhen the birds
were about 90 days old all of the males were removed and placed in an

adjacent pen .

 

*Note: Lot numbers were assigned consecutively through all of the
experiments. Thus, experiment No. 1 contains lots 1 to 4, experiment
No. 2 contains lots 5 to 11, etc.

- 29 -

Lot No. 10r-20 chicks used as non-inoculated controls. This repre-

 

sented a single hatch and the birds were raised in complete isolation.

Lot No. ll.--25 chicks, likewise used as non-inoculated isolated

 

controls.
This experiment was started on April 5, 1948, and terminated about

500 days later.

Experiment No. 3
2222:23--The infermation on the donors for this experiment is given

in table 4. Liver tissue taken from.embryos 15 days old was used as

the inoculum. The embryos supplying inoculum for each experimental lot
were from only one dam. At the time the eggs were laid and the embryos
were used the dams and sires of the respective embryos were all clinic-
ally normal and apparently in good health. These dams and sires lived
in an exposed environment where they had ample opportunity to become
infected with lymphonatosis.

Recipients.--0ne hatch of line 15 chicks was divided into 10 lots

 

with as nearly as possible an equal family representation in each lot.
All inoculations were made intraperitoneally when the chicks were one
day old. The protocol fbr'this experiment was as follows:

Liver tissue inoculum

 

 

No. of birds (dam No.)
Lotto. I2 19 W
' " 13 16 J430H
" " l4 l7 J404N
" " 15 17 J716R
T ' 16 17 J313N
" " 17 17 J310R
" " 18 18 J2560
Filtrate of liver tissue
" " 19 18 J310R
" " 20 16 Amnionio fluid J313N
" " 21 18 Uninoculated contact controls

This experiment was started on Jan. 17, 1949 and terminated 220 days later.

- 50 -

Experiment No. 4

Eggeggs-dThis experiment was similar to experiment No. 3 except
that all of the dams and sires but two, lived in an isolated environment
where they had less chance of becoming infected with lymphomatosis. In
addition these isolated dams and sires were closely related to the recipient
chicks, because they came from the same inbred line. The information on
these dams is given in'tables 4 and 5. All were clinically healthy at
the time their eggs were used.

Recipients.--One hatch of line 15 chicks was divided into 12 experi-

 

‘mental lots, as in the previous experiment. All inoculations were made
intraperitoneally when the chicks were one day old. The protocol for

this experiment was as follows:

 

No. of birds Liver tissue inoculum
(dun NO 0)

Wife??? 15 fizsq

" ' 23 18 J709l

' T 24 18 I1414H2

" ' 25 18 Il406U2

“ ” 26 19 J1318K2

” ' 27 18 Il415B2

" ” 28 18 J131882

" ' 29 19 J150Dl2

" " 30 19 JlSOlMZ

” ' 31 19 JISlflI

" ' 52 17 J151832

' ‘ 33 19 Non-inoculated contact control

 

Lot No. 34.-4th composed of 34 chicks from a single hatch reared
in complete isolation. .All of the other birds in lots 22 to 35 lived
together as a population, except that the two sexes were separated as
in the previous experiment. This experiment was started.on Jan. 31,

1949, and terminated 220 days later.

Experiment No. 5

2222333--In this experiment 5 families were selected as donors
(i.e., 5 dams and the males to which they were mated, table 6). Only
high producing hens were used, and that was the main.basis for the
selection of the families. Eggs were set from these hens, so that on
a predetermined day there would be available, embryos and chicks from
these families as follows: embryos 1 day old, 8 days old, 15 days old,
and chicks one day old, 8 days old, 15 days old, 29 days old and 43 days
old. Two embryos or chicks from each respective age group and from
each of'the five families were sacrificed to prepare the inocula. Thus,
a pooled inoculum was prepared from the livers of the birds in each
age group. In addition, a filtered inoculum was prepared from the
livers of the one day old chicks. Part of this filtrate was placed in
a glass tube, sealed, and heated to 45°C for'30 minutes in a water bath.
This treatment furnished another incculum,

Recipients.--One hatch of line 15 chicks was divided into 11 experi-

 

mental lots. All inoculations were made intraperitoneally when the chicks

were one day old. The protocol for the experiment was as follows:
Pooled liver tissue

 

No. of birds inoculum.froms
Lot No. 35 22 Embryos 1 day old
' " 36 23 ' 8 days old
II N 37 25 It 15 I! I!
' “ 38 21 Chicks 1 day '
" " 39 26 " 8 days "
" " 4O 24 ' 15 " "
" " 41 25 " 29 ' "
" ' 42 22 ' 43 " '
" " 43 20 Filtrate of livers - 1 day old chicks
" ' 44 24 Heated ' ' ' 1 n ' '
‘ ' 45 23 Uninoculated contact controls

 

All birds lived together in a common isolated environment with the

two sexes in separate adjoining pens. The experiment was started march 21,
1949, and was terminated approximately 310 days later.

- 32 -
Experiment No. 6

Donors.--In this experiment a recheck was made on some of the donor
dams previously used in experiments No. 3 and 4. In addition, other
dams were tested. Eggs were set from the selected dams and when the
embryos were 18 days old they were sacrificed to furnish liver tissue
for the preparation of inocula. A pool of liver tissue from two sibling
embryos was used for each incculum. All of the donor hens and sires lived
in the exposed population and they were clinically normal at the time
the eggs were used. The information on the donors is given in tables 4
and 6.

- magenta-4 single hatch of line 15 chicks was divided into

 

11 experimental lots as in the previous experiments. The inoculations
were all made intraperitoneally when the chicks were 4 days old. This
experiment was initiated on May 2, 1949, and was terminated 310 days
later.

The protocol for this experiment was as follows:

Embryo inoculum

 

No. of birds (dam No .)
Lot No. 46 19 (Filtrate of liver) 37031
" ' 47 18 J70”
” a 48 13 J40“
u ' 49 18 J716R
" " so 18 (Amnionic fluid) J7me-
" " 51 18 (Filtrate of liver) J716R
w n 52 20 .1506?
" u 53 20 J430H
" T 54 18 J709D2
T ' 55 19 $5101!
" " 56 18 Uninoculated contact controls

 

All of the birds in this experiment were housed in a common isolated
environment and the management practices used were similar to those in
the revious experiments.

*In lot No. 50 the undiluted amnionic fluid from the J716R embryos was
used as the inoculum.

Experiment No. 7

M"'In this experiment the donor hens used were some of the
same ones that had been used in experiment No. 4. They were all housed
in the isolated environment. They were closely related to the recipient
chicks. From 2 to 5 embryos 18 days old were used to prepare each liver
tissm inoculum.

Recipients.--A single batch of line 15 chicks was divided into 6

 

experimental lots. The birds were inoculated when they were four days
old. The birds were housed together in a comon isolated environment as
in the other experiments. This experiment was started on May 16, 1949,
and was terminated 310 days later. The protocol for the experiment was
as follows:

Liver tissue inoculum

 

No. of birds (dam No.)
Lot No. 57 so 1141:5132
" " 58 22 J131832
" '_' 59 29 .11301112
" 3' so 24 J1314fl
: 3 s1 15 J1301MZ

62 23 Uninoculated contact controls

 

-34..

Experiment No. 8

Donors.--In experiment No. 8 an attempt was made to'determine what
effect the inoculation of blood and respiratory washings from.the dams,
themselves, would have on the recipient chicks. Seven of the hens that
had been previously tested by using their offspring were used in this
study. The collection and preparation of the blood and respiratory
washings have already been described. Two of the donor hens were from
the isolated environment and the others were from.the exposed environ-
ment. All of the hens were clinically normal when they were used as
dono rs .

Recipients.--A single hatch of line 15 chicks was divided into

 

10 experimental lots. The housing of the birds was similar to that of
previous experiments. The experiment was started on.May 50, 1949, and

was terminated 310 days later. The protocol for the experiment was as

 

follows:
No. of birds Inoculum and donor No.
Lot No. 63 19 Blood J716R
" “ 64 18 " J604N
" " 65 19 Resp. washings J70lO
" ' 66 20 " ' J709W
" " 67 17 Blood J7010
" ” 68 18 " J7oas
" ' 69 16 ' 1141382
" " 70 18 ” J4SOH
” ' 71 13 " 11414H2
" ' '72 19 Noninoculated contact controls

 

Lot No. 73 was composed of’3l birds from.a separate hatch that were
used as a non-contact control group. This group of birds was hatched in
isolation, but they subsequently had some degree of indirect exposure to

lymphomatosis in the brooder house in which they were housed. During

their early life, these birds had more potential exposure to naturally

occurring lymphomatosis than did any of the other groups of birds.

RESULTS AND DISCUSSION

The dmor dams used in these experiments were all clinically normal
at the time they were used as a source of eggs, blood, or respiratory
washings. Later, some of them.died of lymphomatosis, but most of them.
showed no clinical or gross evidence of the disease at the time of'autopsy.
Likewise, most of the sires were clinically normal. The donor embryos and
chicks did not show any gross abnormalities when they were sacrificed.

The results cf'the eight experiments are given in table 7. The
incidence of lymphomatosis in the various experimental lots is given in
cumulative percentages, broken down into intervals of 30 days. The column
marked "terminal" includes those birds that had lymphomtosis at the tim9
they were killed. Experiments 3 and 4 were terminated at 220 days. Thus,
the birds that survived to the end of the experiment, and showed lesions
of lymphomatosis upon necropsy examination, were placed in the 221-250 age
group. This was justified because observations have shown that it takes
about 30 days for the tumors to develop from the microscopic stage (grossly
invisible) to a.large tumor mass (Davis and Doyle, 1947b; Lucas, 1950).
Thus, the birds that had grossly evident tumors at 220 days would probably
have died before 250 days had elapsed.

Both.visceral and neural lymphomatosis occurred in the various experi-
mental and control lots. The incidence of neural lymphomatosis varied
considerably, but there was no significant difference in the incidence of
neural lymphomatosis between.the treated groups and the control groups.
Thus, the difference in the incidence between the various lots was mainly
due to visceral lymphomatosis. No cases of'ccular lymphomatosis were found.

In experiment No. 3, three cases of osteopetrosis occurred in combination

-37-

with visceral lymphomatosis during the 191 to 220 days age period (two
in lot 13 and one in lot 15). Two uncomplicated cases of osteopetrosis
were found in lot 23 of experiment No. 4 at the time of termination.

One case of osteopetrosis was observed in the 281-310 days ago period of
lot 37 in experiment No. 5. No cases of osteopetrosis were observed in
any of the control groups for these experiments or in any other line 15
control groups for other experiments. However, this line of inbred birds
has been found to be quite susceptible to osteopetrosis when inoculated
with tumor strain RPL 12 (Burmester and Cottral, 1947d) .0

Birds of both sexes were used and the sex ratio in the various lots
was balanced for the most part. Due to the small number of birds used
in each lot it was not possible to make an accurate test of the effect
of the two sexes. in armlysis of the entire group of birds led to the
observation that more males than females died of lymphomatosis during
the early age periods. However, more females than sales died later in
the experimental period. Thus, at the termination of the experiments the
females had a slightly higher incidence of lymphonatosis than the males.
This difference was not statistically significant. -.

The first procedure followed in making an analysis of the eight
experiments was to determine whether or not the birds in the various
lots of each experiment belonged to the same population in respect to
the incidence of lymphonatosis. This was accomplished by using the

chi-square test as in the two examples that follow:

Experiment No. l

 

 

 

 

No. in No. dying of Prdbability of dying Products
group lymphomatosis of lymphomatosis RX
14 9 0.6428 5.7852
14 5» 0.3571 1.7855
34 6 0.1765 j__ 1.0590
2 srx-Tb'sx 8.6297 - 6.4520
X " 5'5 " (.3226) (.6774) ' 9'956"
(with 2 D.F. at 1% level 1:2 = 9.210)
Experiment No. 2
No. in No. dying of Probability of dying Products
group lymphomatosis of'yymphomatosis RX
17 13 0.7647 9.9411
14 12 0.8571 10.2852
17 14 0.8235 11.5290
17 17 1.0000 17.0000
17 3 0.1765 0.5295

 

55 SK - 59

2 49.2848 - 42.4505

1 ' W57— ' 33-866“

(with 4 D.F. at 1% level 1:2

**Significant at the 1.0 percent level.

W

' 13 0277)

The results of the chi-square test for the various experiments

were as follows:

 

Inclusive Degrees of Valug of
Experiment No. lot Nos. freedom. X

1 1 " 3 2 90966 **

2 5~~ 9 4 33.866 *1

3 12 -21 9 42.801 to

4 22 -33 11 26.051 to

5 35 -45 10 13.409 H.S.
6 46 -56 10 35.560 so

7 57 '62 5 40915 N030
8 63 -72 9 21.578 s

** Significant at the 1.0 percent level
* N n I 5.0 II I!

11.8. Not significant

From these tests it can be seen that the various lots of birds
wdthin each of these experiments do not represent homogeneous popula-
tions with respect to the incidence of lymphomatosis. This suggests
that some of the treatments given to the birds within each experiment
were responsible for the rise in the incidence of lymphomatosis over
what the controls showed. Experiments 5 and 7 were the only ones that
failed to show a significant difference between the various experimental
lots.

Since the population of birds wdthin each experiment (i.e., except
5 and 7) was not a homogeneous one, the next logical test is to compare
each lot within an experiment to its respective control group. The contact
control was used for'this purpose in all cases except in experiment No. 1,
which did not have a contact control. In this experiment the isolated
control with the highest incidence of lymphomatosis was used. Therefore,
in all of the experiments except No. l, the experimental lots were tested
against the most comparable control group. Thus, the control group that

was used was made up of birds from the same hatch, the some families, and

was subjected to the same environment as the inoculated groups. This

comparison was made using the chi-square test as in the following

 

 

example:
Experiment No. 1
(Test of lot 1 with lot 3)
Treatment Take Fail Total
Lot—1 inoculated 9 5 14
' 3 control 6 28 34
15 33 48

_ _ 2
X2 . ((9: (28) (s) (5:) 24) (48> _ 7.986 ..

*t - significant at the 1.0 percent level.

 

The results of the chi-square test for each experimenters given in
table 8. The analysis was made at the 250 day point and at the termina-
tion point (310 days).

In experiment No. 1 there was no significant difference between the
two control groups or between the control lot 3 and the inoculated lot 2.
However, there was a significant difference between inoculated lot 1 and
control lot 3. Both lots 1 and 2 received inoculum.C, the pooled intes-
tines and trachea oi‘the donor chicks. However, lot 1 received the liver
inoculum and lot 2 received the yolk sac incculume Thus, the higher
incidence of lymphomatosis in lot 1 was attributed to the liver incculum.
For this reason the liver of the donor'chicks and embryos was chosen.as
the tissue to use as a source of inoculum for the experiments that
followed.

In experiment No. 2 there was no significant difference between the
three control groups. All of the inoculated groups had a high incidence

of lymphomatosis, varying from 76.5 to 100.0 percent. Thus, lymphomatosis

- 41 -

was transmitted by using liver tissue from.embryos 15 and 18 days old,
chicks one day old, and by using a cell-free filtrate of the chick liver
tissue.

The controls in experiment No. 3 had a rather high incidence of
lymphomatosis, 22.2 percent. This was the highest incidence for all of
the control groups at the 250 day point. Only two inoculated groups,
lots 12 and 13, had an incidence ol?lymphomatosis that was significantly
high.

The controls in experiment No. 4 had a low incidence of lympho-
matosis and the incidence in the inoculated groups was also correspond-
ingly lower than in the third experiment. Three inoculated groups (lots
23, 24, and 25) gave significant differences when compared to the controls.

Experiment No. 5 was designed to detemmine at what ages the embryos
and chicks would have the agent of lymphomatosis in their liver tissue.
However, when the experiment was terminated, none of the inoculated lots
differed significantly from.the controls.

In experiment No. 6, some of'the previously used donor hens were
retested. A filtrate prepared fronlthe livers of embryos of J709W pro-
duced a significant increase in lymphomatosis in the inoculated birds,
but the unfiltered preparation failed to give significant results. In
the previous test of this hen's embryos the results were also positive
(experimnt No. 4).

The embryos of hen.No. J716R gave positive results this time for
both the liver preparation and the amnionic fluid, but the filtrate
group did not show a significant increase in the incidence of lymphomatosis.
In the previous test (experiment No. 3) the results wdth the offspring of

this bird were not significant. Embryo J404A liver tissue gave significant

-42-

results. This hen was not previously tested. J430H, which gave posi-
tive results before, was negative in this test. J310R embryos were
negative in both tests (experiments Nos. 3 and 6).

In experiment No. 7 none of the inoculated lots had as high an
incidence as the control lot. The embryos of these hens had been
previously tested and they were negative in both tests. Unfortunately,
the hens, whose embryos had given significant results in experiment
No. 4, had not produced any fertile eggs by the time this experiment
was scheduled, so they were not represented in experiment No. 7.

The purpose of experiment No. 8 was to test the blood and respiratory
washings of some of the donor hens that had been previously used in the
other experiments. Hen No. J716R, which had given questionable results
in experiment 3, and significant results in experiment 6, gave significant
results this time. Hen J404N which also gave questionable results in
experiment 3, was positive in this test. Hen No. J7010, whose embryos
had shown significant results in experiment No. 3, gave only'questionable
results this time. The respiratory washings of this bird and those of
hen No. J7097.’ likewise produced questionable results. The results
obtained with the other donor hens tested in this experiment were not
significantly different from those of the control group.

A significantly high incidence of lymphomatosis was transmitted
to experimental birds by inoculating them with the liver tissue of
newly-hatched chicks and embryos 15 and 18 days old. The logical conclu-
sion was that the donor chicks and embryos were infected with the agent
of lymphomatosis. Thus, if the donor embryos and chicks were infected,
they must have acquired the infecting agent from their parents. Lympho-
matosis was transmitted by using the blood of some of these donor hens.

The respiratory washings of two of these hens gave only :qlestionable results.

-43..

At the time they were used, the donor hens and sires were all
clinically normal and most of them remained clinically normal or free of
gross evidence of lymphomatosis. (Some of them are still living.) There
did not appear to be any definite correlation between the infectivity
of the embryo or chick tissue or the dam's blood and the subsequent
development of gross lymphomatosis by the dam or sire. Thus, clinically
normal dams and sires may be carriers of the agent of lymphomatosis.

Some dcnor hens were checked several times, either by two tests
with their embryos or by using the dam's blood for the re-test. The
tests did not agree in all cases, however, one could not expect biological
material to give constant results. Furthermore, the infected hens may
be transmitting the disease to their offspring only at certain intervals
or cycles. Thus, every egg laid by a carrier hen would not be infected.
The work with pullcrum disease brought out the fact that Salmonella
pullorum could not be isolated at every attempt from carrier birds or
their offspring (Rettger and Stoneburn, 1909).

In this study the work was greatly handicapped by the presence of
naturally occurring lymphoratcsis in the birds that were used. Thus,
lymphomatosis was transmitted to some of the birds, but in others we
apparently were only adding to the infection that they already had;
therefore, the interpretation of the results depended upon the inoculated
lots developing an incidence of lymphomatosis that was significantly
higher than that observed in the control groups.

The concept of egg transmission of disease producing agents was
established many years ago, in fact, the work of Pasteur in 1870 proved
that pébrine, a protozoan disease of silkworms, caused by m bombycis,

was egg-borne. Since then it has been shown that nearly all types of

- 44 -

disease producing agents--protozoa, fungi, bacteria, rickettsia, and
viruses--have been proved to be transmitted through eggs or seeds.
This type of transmission takes place in certain helmdnths, arthropods,
birds, and plants (Cottral, 1949).

There are a great many difficulties in unequivocally proving that
a disease producing agent is transmitted through the egg. The ideal
situation would be one in which the etiological agent could be demone
strated in the dam, isolated from the interior of'the dam's eggs at
different stages of?incubation, isolated from the hatched offspring,
and, finally, in the ideal situation.the offspring would remain.carriers
of the agent or subsequently develop the disease. Not all of these
conditions have been satisfied in the study of lymphomatosis. However,
three of the most important steps have been accomplished, namely, demon-
stration that the agent is present in certain 15 and 18 day old embryos,

and in newly-hatched chicks, and in the blood of‘certain dams.

-45..

SUMMARY’AND'CONCLUSIONS

The history of the etiological concepts of the avian leukosis
complex is presented, and the literature dealing with the transmission
of avian lymphomatosis is reviewed. The problem of egg transmission
of avian lymphomatosis is discussed and new evidence is presented.

The problem.of egg transmission of avian lymphomatosis was studied
by attempting to transmit the disease to experimental birds by making
use of inocula prepared from.the tissues of newly-hatched chicks and
embryos l5 and 18 days old, and from the blood and respiratory washings
of certain selected donor hens. Eight experiments were conducted and
the results indicate that certain clinically normal dams and sires may
produce offspring infected with the agent of'lymphomatosis. Furthermore,
in two experimental groups lymphomatosis was produced with a filtrate
prepared from embryo and chick liver tissue. In another test, the
amnionic fluid from.eggs of a certain dam was infective.

The blood from.two donor-hens produced lymphomatosis when inocu-
lated into susceptible birds. The results obtained with respiratory
washings from donor'hens were not statistically significant.

The interpretation of the results is rendered difficult due to the
presence of the agent of naturally occurring lymphomatosis in the experi-
mental birds that were used. Thus, the disease produced by inoculation
was superimposed on the infection.already present in the birds. If stock
free of the agent of lymphomatosis could be found, studies of this kind
would be much easier to carry out and interpret.

Many experiments have been conducted which suggest that lymphoma-

tosis may be egg-borne, such as: epiornithic Observations, flock isolation

-45-

studies, incubator exposure studies, histological studies and trans-

mission studies. The results of these studies in conjunction with the
result of the present study justify the conclusion that avian lympho-

matosis is transmitted from carrier parents to the offspring through the

egg.

- 47 -

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- 54 -
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Regional Poultry Research Laboratory, East Lansing, Michigan, 1947.

Ninton, Berley. Ninth annual report. U. S. Bureau of Animal Industry.
Regional Poultry Research Laboratory, East Lansing, Michigan, 1948.

Winton, Berley. Tenth annual report. U. S. Bureau of.Anima1 Industry.
Regional Poultry Research Laboratory, East Lansing, Michigan, 1949.

'Winton, Berley, E. H. Lucas and G. E. Cottral. The effects of feeding
tomatoes on the incidence of lymphomatosis in chickens (in press).

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TABLE h.--History of parents of donor

(Experiments 3, 6, and 8)

embryos

 

 

 

Days lymphomatosis in sibs
Age at lived Age of dams
Dams and first after killed Number Percent
Sires use use or died Diagnosis of sibs lymphomatosis
Dam: J70lO 301 259 560 Cannibalism 16 56.25
Sires: K 315 177 h92 Normal
T 301 177 h78 "
V 301 53 35h Visceral lymph.
Dam: JLl3OH 309 365+ alive Normal 17 £11.18
Sire: Q 350 152 502 Neural lymph.
Dam: Jllolm 350 168 518 No gross lesions 15 116.67
Sires: P 350 177 527 Normal
V 336 129 M65 No gross lesions
Dam: J716R 282 365+ alive Normal 11 72.73
Sire: E 329 163 h92 "
Dam: J313N 309 365+ alive " 10 60.00
Sire: G 321 178 A99 " '
Dam: J310R 301 365+ alive " 1h 28.57
Sires: D 3L6 " " "
N 320 n H u
0 320 N n n
Dam: J2560 350 365+ alive " 12 25.00
Sire: D 350 177 527 Peritonitis
Dam: J726Q 29h 17 311 Reproductive 11 36.36
disorder
Sire: E 329 163 L92 Normal
Dam: J709W 316 365+ alive " 1h 50.00
Sire: E 329 163 1.92 "
Dam: JhOhA h96 365+ alive " 15 h6.67
SirethO3P h55 72 527 No gross lesions
V be 25 h65 Normal
A2 N40 73 513 "
Dam: J306P L20 365+ alive " 26 15.38
SileszJ3l6C M40 73 513 "
JBZOG h26 73 h99 "
J321L 14110 75 513 "
Dam; J709D2 392 365+ alive " 1h 50.00
smmm:J707H bl} 72 h85 "
J7151 h13 72 h85 "

TABLE 5.--History of parents of donor embryos
(Experiments h, 7, and 8)

 

 

 

Age at Days lived Age killed
Dams and Sires first use after use or died Diagnosis
Dam: 111111.112 711; 365+ alive Normal
Sire: R2 696 31 727 Bacterial infection
Dam: I1h06U2 71b 38 752 Visceral lymphomatosis
Sire: *
Dam: Ilhl382 7111 365+ alive Normal
Sire: *
Dam: J1318K2 23o 111b, 3711 Visceral and neural
lymphomatosis
Sires : T 230 1L1; 37h Norma 1
T2 230 21 251 Neural lymphomatosis
L 230 1hh 37h Visceral and neural
lymphomatosis
V 230 9 239 Neural lymphomatosis
Dam: J131882 203 136 339 Visceral lymphomatosis
Sires: **
Dam: J131882 259 365+ alive Normal
Sires: **
Dam: J1301Wé 231 365+ alive Normal
Sires: **
Dam: J1301M2 259 365+ alive Normal
Sires: **
Dam: J131hW 231 365+ alive Normal
Sires: Q2 2hh. 28h. 528 "
L2 2hb- 389 633 "
D2 216 175 391 No gross lesions
v2 230 215 L115 Neural lymphomatosis
Y2 2hb. 320 56h. Visceral lymphomatosis

* The sire used was IlhlhR2,

** The four sires used were J1318T, T2, L, and V.

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