MAREK’S DISEASE: WRUS- HOST CELL ' ‘
RELATIONSHIPS IN ViTRO AND EN VWO, AND
BIGLDGiCAL MARKERS FOR CLONED
PREPARATIONS OF THE VIRUS AND A
HERPESVIRUS 0F TURKEYS

Thesis for the Degree of ‘Ph. D.
#1::chsz STATE umvsasaw
HARVEY GRAHAM PURCHASE

1970 '

 

VHF“? HLIBRARY $9}

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This is to certify that the

thesis entitled
MAREK'S DISEASE: VIRUS- -HOST CELL RELATIONSHIPS
IN VITRO AND _I__N VIVO, AND BIOLOGICAL MARKERS
FOR CLONED —PREPARAT|0NS OF THE VIRUS
AND A HERPESVIRUS 0F TURKEYS

presented by
HARVEY GRAHAM PURCHASE

has been accepted towards fulfillment
of the requirements for

PH.D. degreein MICROBIOLOGY 5 PUBLIC
HEALTH

 

 

 

/« «s 27%“--- A,

Major professor

Date November 19 , I 970

0-7639

 

 

ABSTRACT
MAREK'S DISEASE: VIRUS-HOST CELL RELATIONSHIPS
IN VITRO AND IN VIVO, AND BIOLOGICAL MARKERS

" FOR CLONED—PRERARATIONS OF THE VIRUS
AND A HERPESVIRUS OF TURKEYS

BY

Harvey Graham Purchase

A specific, indirect fluorescent antibody test was
developed for the detection of Marek's disease (MD) virus induced
antigen in cell culture and antibody in the serum of chickens.
The test has limited application for the detection of
antigen. It is 10 to more than 320 times more sensitive
than the agar gel precipitation test for the detection of
antibody. Maternal antibody could be detected in the sera
of 1 to 11 day old chicks from MD exposed dams after anti-
body was no longer detectable by the precipitation test.
Actively acquired antibody in contact exposed chickens was
detected earlier than by the precipitation test. Some sera
that had a high titer of antibody in the fluorescent antibody
test did not produce precipitation in agar. Eight isolates
of MD virus could not be distinguished from one another by
the indirect fluorescent antibody test which indicated that
either the strains were antigentically identical or con-

tained a common antigen or contaminant.

Harvey Graham Purchase

Cloned preparations of MD virus differed from one
another in pathogenicity for chickens, antigenicity and
effect on cell cultures. These three properties were
independent of one another. All avian cells tested were
susceptible both to the MD virus and to the herpesvirus of
turkeys.

Antigens induced by MD virus were detected in the
feather follicle epithelium, the lung, bursa of Fabricius,
thymus, spleen and caecal tonsil of MD infected birds by
both the direct fluorescent antibody and agar gel precipita—
tion tests. However the antigens could not be detected in
the tumors themselves by either test, although MD was
readily transmitted with intact tumor cells. There was a
direct relationship between the presence of antigen and the
cells undergoing degeneration and necrosis. Intranuclear
inclusion bodies were in the feather follicle epithelium
where similar necrobiotic changes were taking place.
Filtrable virus was found only in skin extracts and originated
from the feather follicle epithelial cells where virus
maturation and envelopement were completed. This epithelium
is probably the portal of exit of the virus from the host

and accounts for the highly infectious nature of MD.

MAREK'S DISEASE: VIRUS-HOST CELL RELATIONSHIPS

IN_VITRO AND IN_VIVO, AND BIOLOGICAL MARKERS

 

FOR CLONED PREPARATIONS OF THE VIRUS

AND A HERPESVIRUS OF TURKEYS

BY

Harvey Graham Purchase

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1970

ACKNOWLEDGEMENTS

I wish to express my sincere thanks to Dr. Charles
H. Cunningham, Professor of Microbiology and to Dr. Ben R.
Burmester, Director of the U. S. Regional Poultry Laboratory
and Professor of Microbiology, for their encouragement and
assistance in the research work and preparation of this
thesis. I would also like to express my appreciation to
Dr. Philipp Gerhardt, Chairman and Dr. Delbert E. Schoenhard,
Assistant Chairman of the Department of Microbiology and
Public Health for aiding my efforts and to all at the Regional
Poultry Research Laboratory for rendering verbal and moral
assistance and valuable constructive criticism.

I acknowledge the help of Mr. H. Burgoyne in per-
forming precipitating antibody tests as referred to in one
of the papers. For technical help, I wish to express my
gratitude to Mrs. Cheryl Hunt, Mrs. Audre Gudanowski and

Miss Phyllis Frank.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS . . . . . . . . .
TABLE OF CONTENTS . . . . . . . . .
LIST OF TABLES . . . . . . . . . .

LIST OF ABBREVIATIONS. . . . . . . .
INTRODUCTION. . . . . . . . . . .

LITERATURE REVIEW . . . . . . . . .

Incidence and Distribution of Marek's Disease

Etiologic Agent . . . . . . . .
Strains of MD Virus . . . . . . .
Host Range in Vivo and in Vitro . . .

 

Gross Pathology . . . . . . . .
Microscopic Pathology . . . . . .
Pathogenesis . . . . . . . .

Specimens of Choice for Virus Isolation

Isolation, Cultivation and Identification Methods

Serology. . . . . . . . . . .
Epizootiology . . . . . . . . .
Control . . . . . . . . . . .

MATERIALS AND METHODS. . . . . . . .

RESULTS AND DETAILED DISCUSSION . . . .

Article I.
Marek's Disease. I.

Eight Isolates. . . . . . . .

Article II.
Marek's Disease:

iii

Immunofluorescence in the Study of
Detection of Antigen in

Cell Culture and an Antigenic Comparison of

Immunofluorescence in the Study of
Detection of Antibody

Page
ii

iii

vi

30

40

Article III. Virus-specific Immunofluorescent
and Precipitin Antigens and Cell-free Virus
in the Tissues of Birds Infected with Marek's
Disease . . . . . . . . . . . .

Article IV. Pathogenicity and Antigenicity of
Clones from Strains of Marek's Disease Virus

and the Herpesvirus of Turkeys . . . . .

Article V. Responses of Cell Cultures from

Various Avian Species to Marek's Disease Virus

and the Herpesvirus of Turkeys . . . . .
GENERAL DISCUSSION AND CONCLUSIONS . . . . . .

Techniques for Detection of Antigen and Antibody

in Vitro and in Vivo . . . . .
Differences Between Strains of MDV and HVT . .
Growth of Clones of Virus in Cultures of Various

Types. . . . . . . . .
Virus- Host Cell Relationships in Vitro. . . .
Virus- -Host Cell Relationships in_Vivo . . . .
The Etiology of MD . . . . . . . . . .

SUMMARY...............

LITERATURE CITED 0 O O O O O O O O O O 0

iv

Page

48

60

94
121
121
123
124
124
125
126
128

132

LIST OF TABLES

TABLE Page
1. Index of Materials and Methods . . . . . . 27
2. Index of Results. . . . . . . . . . . 28

AGP

B14

Cal-l
CAM
CEF
CK

Conn A

Cornell S

CR64

DEF
DFA
DK
DNA
FA

FAPP

LIST OF ABBREVIATIONS

agar gel precipitation

P.M. Biggs' 14th strain of MDV which
is of classical type from the HPRS

California one isolate is an acute
strain of MDV from R. Bankowski,

University of California, Davis,
California

C1

chorioallantoic membrane

chick embryo fibroblasts

chick kidney

Connecticut A isolate is a classical
MDV from R. Luginbuhl, University
of Connecticut, Storrs, Connecticut

Cornell Line of chickens susceptible
to MD and bred by R. Cole, Cornell
University, Ithaca, New York.

an acute isolate of MDV passed in
chickens by W. Staples, Cobb Breed-
ing Corporation, Connecticut in
1964 and isolated in cell culture
by H. G. Purchase

duck embryo fibroblasts

direct fluorescent antibody

duck kidney

deoxyribonucleic acid

fluorescent antibody

filtered air positive pressure

vi

FCSO

GEF

HPRS

HPRS l6
HPRS 17
HPRS 18
HPRS 20
IBA

IF

IFA
ILTV

JM

JM19,JM30,JM31,
JM32,JM34,JM35,JM36

JMHP

JMN

JMP

an acute isolate of MDV from field
case 50 from Port Huron, Michigan
by H. G. Purchase

goose embryo fibroblasts

high passage MDV i.e. the JM strain
of MDV which had been passed over
40 times in CEF and DEF = JMHP

Houghton Poultry Research Station,
Houghton, Huntingdon, England

 

I
acute
classical
acute isolates of MDV made by
H. G. Purchase while on
acute J secondment to the HPRS

infectious bursal agent
immunofluorescent

indirect fluorescent antibody
infectious laryngotracheitis virus

an acute isolate of MDV from M.
Sevoian7University of Massachusetts,
Amherst, Massachusetts

different cloned preparation of the
JM strain of MDV

HMD

a line of chickens bred for resistance
to JM MD by three generations of
selection by R. Cole, Cornell
University, Ithaca, New York

a line of chickens bred for suscept-
ibility to JM MD by three genera-
tions of selection by R. Cole,
Cornell University, Ithaca, New
York

vii

JQEF

LMD

MD
MDV

MSD

nm
PBS
PhEF
PiEF

RPRL line 6

RPRL line 7

RPL

RPRL

RPL 39

RSV

TEF

USDA

Japanese quail embryo fibroblasts

low passage MDV, i.e. MDV which had
been passed less than 17 times in
CEF and DEF. Included are GA,
RPL39 and cloned preparation of
JM MDV.

Marek's disease
Marek's disease virus

Merck, Sharpe and Dohme one violate
of MDV from T. Maag, Rahway, New
Jersey

nanometers

phosphate buffered saline

pheasant embryo fibroblasts

pigeon embryo fibroblasts

an RPRL inbred line of chickens
resistant to MD and lymphoid
leukosis tumor development but not
resistant to virus infection

an RPRL inbred line of chickens
susceptible to MD and resistant
to subgroup A lymphoid leukosis
virus infection. Bred by L. B.
Crittenden and N. F. Waters

Regional Poultry Laboratory

Regional Poultry Research Laboratory
= RPL

RPL 39th identification, an acute
isolate of MDV from a flock in
Georgia, by H. G. Purchase

Rous sarcoma virus

turkey embryo fibroblasts

United States Department of
Agriculture

viii

INTRODUCTION

Marek's disease (MD) is a neoplastic disease of the
hymphoid system of chickens of great economic importance.
Infiltration and proliferation of lymphoid cells occurs in
the nerves and visceral organs. The etiologic agent can
be readily transmitted from affected birds to genetically
susceptible chicks using whole blood or intact tumor cells.
The agent is highly cell associated and treatments which
destroy the viability of the cells also eliminate infec-
tivity. However, in nature, MD is highly infectious and
nearly all mature flocks are infected.

MD virus (MDV) produces characteristic cytopathic
effects in avianepithelioidcu'fibroblastic cell cultures.
When MDV is propagated in cell culture, antigens are pro-
duced which react in the agar gel precipitation (AGP) test
with sera from birds that have been exposed to MDV. The
AGP test can be used to detect antibody in sera from
experimental birds and field flocks. As described in this
thesis, the indirect fluorescent antibody (IFA) test was
developed as an alternative method for detecting MDV anti-
gens and antibody (Purchase, 1969, Article I; Chen and

Purchase, 1970; Nazerian and Purchase, 1970; Purchase and

Burgoyne, 1970, Article II). This IFA test, however, failed
to reveal comparative serologic differences between iso-
lates of MDV.

At this stage in the understanding of MD, the author
formally proposed that, since different isolates of MDV
vary in their biological activity, distinguishable herpes—
viruses could be isolated both from clones derived from
a single inoculum and from clones derived from different
inocula. As reported in this thesis (Purchase et_al.,
1970a, Article IV; Purchase et_al., 1970b, Article V) cloned
preparatiors of MDV differed in pathogenicity, antigenicity
and cytOpathic effect in different cell types.

Concurrently, the author applied the direct
fluorescent antibody (DFA) test to the detection of antigen
in 1129. Antigen was found, among other locations, in the
epithelial cells lining the feather follicle (Purchase,
1970a, Article III). This was the only location in the
living host in which a significant amount of virus matured
to its fully infectious form. Enveloped infectious
virions, and both precipitating and immunofluorescent
antigens could be found at this site, which is the portal
of exit of MDV from the chicken. The discovery of this
"missing link" in the life cycle of MDV infection esta-

blished the herpesvirus as the etiologic agent of MD.

In the thesis below the important historical and
current literature on MD is reviewed with emphasis on
work that is relevant to the thesis research. The results
of preliminary research are presented in the form of three
published papers (Articles I, II, and III). The experi-
mental efforts to substantiate the aforementioned formal
proposal are described in two pre-publication manuscripts
(Articles IV and V). Finally, the composite findings are

discussed and drawn together as generalized conclusions.

LITERATURE REVIEW

Marek's disease (MD) is a common lymphoproliferative

disease of chickens which affects the peripheral nerves

and visceral organs. The syndrome was first described

by Marek (1907) who, thinking it to be an inflammatory
disease, called it "polyneuritis". More recent studies
(Payne and Biggs, 1967) indicated that "Marek's disease

is characterized by a neOplastic-like proliferation of
lymphoid cells in the nerves and in other organs, notably

the ovary".

Incidence and Distribution of MD

 

Marek's disease is of immense economic significance
to the poultry industry and causes losses among chickens
throughout the world, particularly in areas of intensive
poultry production (Churchill and Biggs, 1967; Biggs
gt_al., 1968a; Nazerian et_al., 1968; Solomon et_al., 1968).
In the United States, annual losses attributable to
"leukosis" (most of which now is considered to be MD) are
estimated to be in excess of 150 million dollars (Calnek,
1967). Whereas the number of chickens slaughtered and

inspected by the United States Department of Agriculture

(USDA) did not change significantly between 1961 and 1968,

the number condemned from "leukosis” per 10 thousand inspected
increased from 10 to nearly 200 (Goldstein, 1968). In 1968
leukosis replaced airsacculitis as the single most important
cause of condemnation in young chickens inspected by the

USDA. Marek's disease is not confined to young broilers.
Severe losses have occurred in started pullets and in

layers (Purchase et_§1., 1966; Purchase et_gl., 1969) where

it has been confused with lymphoid leukosis (Purchase, 1965;

Burmester and Witter, 1966; Siccardi and Burmester, 1970).

Etiologic Agent

 

Since 1926, attempts to transmit MD have met with
varying degrees of success (Pappenheimer et_al., 1926;
Pappenheimer et_al., 1929; Blakemore, 1939; Durant and
McDougle, 1939; Durant and McDougle, 1945). An important
contribution to the understanding of MD came from Biggs
and Payne (1963 and 1967), who succeeded in serially trans-
mitting the HPRS-B14 strain of MD virus (MDV). Purchase
and Biggs (1967) then isolated and characterized four
"acute" and one "classical“ strains of MDV. The discovery
in England (Churchill and Biggs, 1967) and in the U.S.A.
(Solomon gt_al., 1968; Nazerian et_al., 1968) of a herpes-
virus closely associated with MD was a significant break?
through. A large amount of data subsequently was accumulated,

which circumstantially linked the virus to MD (Biggs et a1.,

1968; Witter §E_al., 1969a). The discovery of the site

of maturation of the virus (Calnek et_al., 1970a; Nazeran
and Witter, 1970; Purchase, 1970a, Article III) and trans—
mission of the disease with cellvfree virus preparations
has left little doubt that the herpesvirus is the cause

of MD.

MDV (Burmester et_al., 1969; Witter et_31., 1969a)
belongs to the cytomegalovirus or B group of cell-associated
herpesviruses (Lee e£_31., 1969). Enveloped virions
from the feather follicles are 200-400 nm in diameter
(Calnek gt_al., 1970a; Nazerian and Witter, 1970). The
capsid has icosahedral symmetry with 162 hollow cylindrical
capsomeres approximately 85-100 nm in diameter (Ahmed and
Schidlovsky, 1968; Epstein et_al., 1968; Nazerian et_al.,
1968; Calnek gt_al., 1970a). Complete particles have a
centrally located, dense nucleoid approximately 65 nm in
diameter. Virions are rarely observed in tumors
(Schidlovsky et_al., 1969; Calnek et_al., 1970b). However,
naked particles are sometimes in the nuclei of cells in
the bursa of Fabricius (Calnek et_al,, 1970b) and occasion-
ally in the nuclei of Schwann cells (Ubertini and Calnek,
1970). Complete, enveloped particles are frequently present
in the nuclei and in cytOplasmic inclusions in cells of
the feather follicle epithelium (Calnek, 1970a; Nazerian

and Witter, 1970).

The deoxyribonucleic acid (DNA) of MD herpesvirus
contains 56 to 57 moles percent of guanine and cytosine
(Lee et_al,, 1969). The composition of the DNA and its
lack of infectiousness in cell—free preparations of cell
cultures also suggests that the virus belongs to the

herpesvirus group B (Melnick et a1., 1964; Wilner, 1969).

Strains of MD

 

Many different strains of MD have been described

such as JM (Sevoian et a1., 1962; Witter and Burmester.
1967), C1 (Bankowski et a1., 1969), GA (Eidson and Schmittle,
1968), Conn A (Chomiak et a1., 1967), CR 64, RPL 39, FC 50,
MSDl
Biggs and Payne, 1967), HPRS l6, HPRS 17, HPRS 18, HPRS 19,

(Purchase, 1969), HPRS B14 (Biggs and Payne, 1963;

and HPRS 20 (Biggs et_al,, 1965; Purchase and Biggs, 1967).
Herpesviruses have been identified in most of these strains

and they do not appear to differ from one another serologically
as measured by the AGP or IFA tests (Chubb and Churchill,

1968; Purchase, 1969). They do, however, differ greatly in

the pathologic manifestations they induce in chickens. Some
induce mainly visceral lesions, whereas others affect pre-

dominantly the nerves (Purchase and Biggs, 1967).

Host Range in Vivo and in Vitro

 

Chickens are the natural hosts but infection has been
artificially transmitted to turkeys (Sevoian et a1., 1963b;

Witter et a1., 1970a), pheasants (Harris, 1939; Johnson,

1941) and quail (Kenzy and Cho, 1969; Witter, 1970). How-
ever MDV could not be re-isolated from turkeys with MD
tumors (Witter gt_al., 1970a). Lesions similar to those
described for MD have been reported as occurring naturally
in the duck (Cottral and Winton, 1953), goose, canary,
budgerigar and swan (Wight, 1963). In one eXperiment
sparrows were apparently refractory (Kenzy and Cho, 1969).
Natural antibody to MDV could not be detected in pigeons,
starlings, yellow hammer, sparrows, and pheasants but MDV could
be re-isolated from ducks after artificial infection
(Baxendale, 1969). The few attempts that have been made
to infectmammals have been unsuccessful (Churchill, 1968;
Calnek, 1970; Purchase, 1970c).

The MDV propagates well in chick kidney (CK) cells
(Churchill and Biggs, 1967; Churchill, 1968) and duck
embryo fibroblasts (DEF) (Solomon et_al., 1968) and under
certain circumstances in chicken embryo fibroblasts (CEF)
where it may (Vindel, 1964; Kottaridis gt_al., 1968; Nazerian,
1968; Nazerian, 1970) or may not produce cytopathic effects
(Witter §E_al., 1968a). Procedures for assaying MDV in CK
and DEF have been thoroughly studied (Churchill, 1968;
Calnek and Madin, 1969; Witter gt_al,, 1969b). The MDV
has also been reported to grow in pheasant embryo fibroblast
cells (Baxendale, 1969). A thorough attempt to pr0pagate
this virus in a wide variety of mammalian cells was unsuccess-

ful (Calnek et a1., 1969).

Gross Pathology

 

The mildest or "classical" form of MD is character—
ised by paralysis of one or more of the extremities of 12
to 14 week old chickens. The motor function of any nerve
may be affected so the symptoms vary. Incoordination is
followed by paralysis of the leg which may result in a
characteristic attitude in which one leg is stretched for-
ward and the other backward. The wings, tail, neck or eye-
lids may droop and birds sometimes have respiratory diffi-
culty. Morbidity and mortality are usually low and birds
that die or are killed have enlarged gray nerves. In the
most acute form, the disease may affect birds as early as
6 to 8 weeks of age and the only antemortem signs may be
depression and anorexia. Morbidity and mortality may
exceed 50% of the flock and birds that die have lymphoid
tumors which affect most commonly the gonad, liver, lung
and skin. The disease may also occur in older birds.

All degrees of severity of the disease may be
encountered in the laboratory and in the field. The gross
pathologic picture, morbidity, mortality and incubation
period are all influenced by the particular isolate of
the MD agent, the degree of exposure, the age at the time
of exposure and the genetic constitution of the host.
Examples of "classical" isolates are HPRS B14 (Biggs and
Payne, 1963; Biggs and Payne, 1967), HPRS 17 (Purchase

and Biggs, 1967) and Conn A (Chomiak et a1., 1967) and

10

examples of "acute" isolates are HPRS l6, HPRS l8, HPRS l9,
HPRS 20 (Purchase and Biggs, 1967) and GA (Eidson and
Schmittle, 1968). Examples of genetically susceptible
chickens are Cornell S and JMP lines (Cole, 1968), Regional
Poultry Research Laboratory (RPRL) Line 7 (Crittenden, 1968)
and Houghton Poultry Research Station (HPRS) Rhode Island
Reds (Biggs and Payne, 1967; Purchase and Biggs, 1967).
Examples of relatively resistant chickens are RPRL Line 6
(Crittenden, 1968), HPRS Brown Leghorns (Biggs and Payne,
1967; Purchase and Biggs, 1967) and the JMN line (Cole,
1968). Susceptibility to MD declines with age (Sevoian

and Chamberlain, 1963; Biggs and Payne, 1967). Females

are more susceptible to both the classical and acute forms
of MD than males (Biggs and Payne, 1967; Purchase and Biggs,

1967).

Microscopic Pathology

The most characteristic lesions of MD occur in the
nerves where there is an infiltration with lymphoid cells.
Initially, the lesion resembles neuritis but later the
size of the lesion and anaplasia of the cells are character-
istic of a neoplastic-like process. Lesions of type I
(Wight, 1962) or A type (Payne and Biggs, 1967) consist of
small, medium and large lymphocytes, a few plasma cells
and large dark staining cells which are thought to be
degenerating lymphoblasts and are referred to as "MD cells".

In lesions that appear neoplastic, large lymphocytes may

ll

predominate but there is still considerable pleomorphism.
Edema, myelin degeneration, and Schwann cell proliferation
may also occur. Type II (Wight, 1962), B type (Payne and
Biggs, 1967) or edematous type lesions contrast with the
type I, A type, or proliferative:lesionsdescribed above.
Theycxxnnrin older birds, or in long standing cases, where
the majority of the cellular infiltration is replaced by
edema and there may only be a few small lymphocytes and
plasma cells. Payne and Biggs (1967) described another,

C type, or mild lesion in which there was only a light
infiltration by plasma cells and small lymphocytes. Lesions
of this type occur in clinically normal older birds.

In the brain, the lesion is a nonpurulent encephalitis
with perivascular cuffing with lymphocytes and occasional,
small areas of gliosis and endotheliosis. In the eye, lesions
are often demonstrable only by histologic examination. The
most constant change is a mononuclear infiltration of the
iris but infiltration of the eye muscles has also been
found.

The lymphoid tumors of the visceral organs and skin
are all composed of masses of pleomorphic lymphocytes, much
like those in the A type neural lesions. In the liver,
lesions consisting of collections of lymphoid cells begin
around the portal tracts. The lesions then enlarge and
eventually large areas of the liver are replaced by lymphoid

tissue. In the heart and muscles, lymphoid infiltration

12

may be extensive causing degeneration and necrosis of the
muscle fibers. Skin lesions begin as enlarged lymphoid
foci in the subcutis but they progress until all layers

beneath the stratum basale are infiltrated with masses of

 

lymphocytes.

Changes in the bursa of Fabricius and thymus are
either degenerative or proliferative. Degenerative changes
in the bursa consist of cortical and medullary atrophy,
necrosis and cyst formation, or replacement of the cortex
and medulla of the follicles with reticular tissue. In the
thymus, there may be a complete absence of the cortex and
a lack of thymocytes in the medulla and Hassel's corpuscles
may become necrobiotic. Proliferative changes in the bursa
consist of an interfollicular lymphoid infiltration with
characteristic pleomorphic lymphoid cells. Eventually the
cells may penetrate the follicles and obliterate them. A
similar lymphoid proliferation occurs in some cases in the
thymus.

Alterations are present in the epithelium lining
the feather follicles. There are no changes in the basilar
layer but degenerative changes commence in the intermediate
and transitional layers. Cells filled with a clear vacuole,
which either displaces the nucleus into a crescent on one
side of the cell or which surrounds the nucleus, are more
frequently present in infected birds than in uninoculated

controls. Many of the nuclei in the transition layer may

13

contain characteristic Cowdry type A inclusions resembling
those produced by MDV in cell culture. Sometimes the
inclusion bodies are unusually dense and contracted, reflect—
ing the variable amounts of cell degeneration. The cytoplasm
of the cells in this layer is slightly eosinophilic and
granular. In the outer layers, in the position of the

stratum corneum, the nuclei of the cells are either basophilic

 

and about the size of inclusion bodies or have degenerated
completely and are not visible. Instead of becoming flattened,
the cytoplasm of the cells is filled with indistinct, highly
eosinophilic granules. The most superficial layers disinte-
grate into fragments and finely granular material. These
alterations are largely confined to the ephithelium in the
deeper two-thirds of the follicle and are present in only

a small proportion of follicles of an infected bird.

Pathogenesis

 

There is a difference of opinion as to whether MD
is primarily an inflammatory process or a true neoplasm.
Necrobiotic lesions have been described in the bursa
(Purchase and Biggs, 1967; Jakowski et;al,, 1969; Purchase,
1970a), thymus (Purchase and Biggs, 1967), hematopoietic
organs (Jakowski §£;alf, 1970) and the feather follicle
epithelium (Calnek et_al., 1970b; Nazerian and Witter, 1970;
Purchase, 1970a). Herpes virions have been reported in the

nerves of birds with MD (Ubertini and Calnek, 1970). It is

14

possible that the virus also causes some destructive changes
in the nerves which can not be observed histopathologically
but which account for the predilection for nerve tissue of
the infiltrating lymphocytes.

The destructive nature of the disease is exemplified
in the immunologic deficiencies in diseased birds (Purchase

et a1., 1968) and their inability to develOp immunity to

 

coccidiosis (Biggs et_al., 1968b; Kenzy et_al., 1970). These
observations agree with Marek's early interpretations and
those of Campbell (1956) and Wight (1962) who consider the
disease to be primarily inflammatory.

Several early workers considered the basic response
to be neoplastic with the inflammatory and degenerative
changes being secondary (Pappenheimer et_al., 1926; Furth,
1935). Payne and Biggs (1967) came to the same conclusions
as follows:

Undoubtedly, the pathological changes in birds
which died with A type nerve lesions and visceral
lymphoid tumors fulfill many criteria of neoplasia,
such as l.) progressive proliferation, 2.) qualitative
differences from, and excessive increases over, lymphoid
hyperplasia produced by many infections of the fowl,

3.) multifocal and diffuse origin, and 4.) possible
abnormal cells . . . . On this basis, we believe Marek's
disease should be classified as a neoplastic condition.

Wight and Siller (1965), while comparing birds with
MD to those in which they had induced an experimental,
allergic encephalomyelitis, and Vindel (1965) from histo-
patholigic studies, speculated that MD might be an autoimmune

disease. Support for the involvement of an autoimmune

15

phenomenon comes from observations that birds with MD have
increased levels of circulating globulin (Howard et a1.,
1967; Ringen and Akhtar, 1968; Samadieh et a1., 1969), that
there is a remission of clinical signs following treatment
with cortisone or 6-mercaptopurine, both immunosuppresive

agents (Foster and Moll, 1968), or stress from Mycoplasma

 

gallisepticum infection (Katzen et a1., 1969). However,

 

in spite of earlier references to the contrary (Payne and
Biggs, 1967), bursectomy does not reduce the incidence of
the disease in experimentally infected chickens (Payne and
Rennie, 1970a). This contrasts with the dramatic effect
of bursectomy on lymphoid leukosis (Peterson et a1., 1964;
Peterson et_al., 1966). It seems possible that the MDV
may initiate the primary change, but subsequent pathologic
manifestations may have an immunologic basis.

Payne and Biggs (1967) considered the earliest
microscopically visible changes to involve cells of the
lymphoid series. However, Sevoian and Chamberlain (1964)
thought the nerve changes resulted from proliferation of
neurilemmal cells followed by differentiation to lymphoid
cells. These observations do not resolve whether MD is

primarily a disease of the nervous or lymphoid systems.

Specimens of Choice for Virus Isolation

 

Virus can be readily isolated from diseased chickens
and from many normal appearing chickens once infection is

established in a flock (Witter et al., 1970b). Infection

l6

persists in some birds for long periods, possibly for the
remainder of their lives (Witter, 1970b). Although congenital
infection has been reported (Sevoian, 1968a) it probably
occurs very infrequently and is of no practical importance
since embryos and chicks from infected chickens are free

of virus (Solomon eE_al., 1970). Tumor cells, kidney cells
and leukocytes from the spleen or peripheral blood are the
specimens of choice for isolation of the virus (Witter
gt_al., 1969b). Since MDV is highly cell—associated, whole
cells must be used as inoculum. Storage of specimens for
virus isolation should be under conditions which preserve
the viability of the cells, i.e., addition of dimethyl-
sulfoxide, slow freezing and storage at -l96°C (Spencer and
Calnek, 1967). Recently, a method has been described so
that cell-free virus can be extracted from cell cultures in
reasonable quantity and lyophilized (Calnek gt_al,, 1970).
If repeatable, this technique offers many advantages for
the storage of MDV.

Contaminated dust and dander, oral and nasal wash-
ings, feces and litter are infectious even after prolonged
storage but they are not good sources of the virus (Kenzy
and Biggs, 1967; Witter and Burmester, 1967; Witter gt_al.,
1968b; Beasley et_al., 1970). Enveloped and filterable
virus has been recovered from the feather follicles which
may be the only place where complete virus is produced in

infected birds. Virus from this source is probably

l7

responsible for the infectivity of dander, litter, and
poultry house dust (Calnek et_al., 1970a; Nazerian and
Witter, 1970a). Viral preparations from feather follicles
or feather tips remain infectious even after dessication
and storage at room temperature.

Isolation, Cultivation, and
Identificatibn Methdds

 

 

The presence of MDV may be established by the inocula-
tion of susceptible chicks, cell cultures, and embryonating
chicken eggs with specimens suspected of containing MDV.
Pathogenic strains of MDV produce symptoms and lesions in
genetically susceptible chicks, such as line 7 or Cornell S,
18 to 21 days after chicks are inoculated at one day of
age. Gross or microscopic lesions in the nerves and/or
viscera, virus isolation in cell culture, specific antigen
in the feather follicles, or the presence of antibody in
serum are all suitable criteria of infection. Experiments
should be of at least 10 weeks duration for maximum sensi-
tivity (Biggs and Payne, 1967; Payne and Biggs, 1967;
Purchase and Biggs, 1967; Witter et;gl., 1969a; Witter
et al., 1969b). Birds for these bioassays should be kept
in strict isolation to prevent cross contamination.

The MDV produces characteristic cytopathic changes
in DEF and CK cell cultures (Churchill and Biggs, 1967;
Solomon et_al., 1968). Cytopathic areas which are produced

in 6 to 14 days consist of rounded and fusiform, refractile

18

cells and polykaryocytes which have Cowdry type A, DNA
containing intranuclear inclusions. The most sensitive test
for virus is the direct cultivation of kidney cells from
test chickens. Virus present in the kidneys produces a
characteristic cytopathic effect in the cultures. Inocula—
tion of CK cultures from uninfected chicks with tumor,
spleen, kidney, buffy coat cells or whole blood from test
chickens is also suitable. Cell culture is from 10 to more
than 1000 fold less sensitive than chickens for isolation
of virus (Witter gt_gl., 1969b).

The virus produces pocks on the chorioallantoic
membrane (CAM) of embryonating chicken eggs inoculated
via the yolk sac or CAM at 4 to 6 or 10 to 11 days of
incubation, respectively. If viable, competent lymphocytes
are in the inoculum administered by the CAM route, the
response is complicated by the graft-versus-host reaction
which produces non-specific pocks. This confusion is
reduced when the yolk sac route is used (Bfilow, 1968;
Bfilow, 1969).

Identification of the virus may be based on both
the in yiyg_response and cell culture changes. Present
or past MDV infection in a bird is confirmed by virus
isolation, demonstration of antibody, or by examination
of the feather follicles for immunofluorescent or pre-
cipitating antigen or for herpes virions by electron

microsc0py. In cell culture, the characteristic cytopathic

19

response may be prevented by inhibitors of synthesis of

viral DNA. Also, infected cells contain characteristic
inclusions and both nuclear and cytoplasmic immunofluorescent
antigen. Naked and occasionally enveloped herpes virions

are in the nucleus of infected cells and sometimes in the
cytoplasm. The DNA extracted from the virus has a high
guanine—cytosine content similar to that of cytomegaloviruses

(Lee et a1., 1969).

Serology

Antibody can be demonstrated in the sera of infected
or recovered birds by the AGP (Chubb and Churchill, 1968),
IFA (Purchase, 1969; Purchase and Burgoyne, 1970) or passive
hemagglutination tests (Eidson and Schmittle, 1969). Antigen
for the AGP test can be produced in CK or DEF cultures.
Cultures with confluent cytopathic areas induced by low
passage MDV are harvested in a small quantity of cultural
fluid and the cells are disrupted. This extract is placed
in a well in an agar layer containing 8% NaCl (Okazaki
gt_gl,, 1970b) and the antibody (serum) in an adjacent well.
After a suitable incubation period, as many as six different
lines of precipitates may form between a serum with anti-
body and MDV antigen. The major line has been referred to
as the A line and the others have been lettered alphabetically

(Churchill et a1., 1969a).

20

Antigen for the IFA test is usually produced in
infected CK cells on coverslips. After fixation of the
cells in acetone, the diluted chicken serum is added. The
serum and cells are allowed to react then the excess serum
is washed off. The cells are then stained with fluorescein
conjugated anti-chicken globulin and examined with a
fluorescence microscope. If antibody is present, the antigens
in both the nucleus and cytoplasm of the infected cells
fluoresce (Purchase, 1969, Article I).

In the passive hemagglutination test, antigen
similar to that of the AGP test is added to tanned chicken
erythrocytes, incubated for a period and then thoroughly
washed. The erythrocytes are mixed with dilutions of
serum. If serum diluted 1/16 or greater causes agglutina-
tion of the erythrocytes, the test is considered positive
for antibody (Eidson and Schmittle, 1969).

Agglutinins have been described in chickens with
MD but their specificity is in question (Zacharia and
Sevoian, 1969; Payne and Rennie, 1970b; Zacharia and
Sevoian, 1970).

The AGP test is most widely used for detection of
antibody to MDV and for distinguishing between the virulent
and attenuated MDV (Churchill e£;al,, 1969a; Purchase
§E_al., 1970a). The IFA test is used to detect antibody
to MDV and to distinguish between antibody to MDV and to

HVT (Witter et al., 1970b).

21

Most birds and almost all flocks of chickens have
antibody to MDV by the time they reach sexual maturity
whether or not the flock has apparent losses from MD. Thus,
inapparent infection is common. The presence of antibody
is only an indication of past infection and is of no value

in determining the cause of death.

Epizootiology

 

Although ovarian transmission of MDV has been
described (Sevoian, 1968), considerable evidence has
accumulated against this route of infection (Rispens gt_al.,
1969; Solomon et a1., 1970; Witter, 1970a). The observation
that large groups of commercial chickens reared in isolation
have no evidence of MDV infection indicates that if egg
transmission occurs at all it is a rare phenomenon (Drury
e£_al,, 1969; Rispens e£_al., 1969; Witter, 1970a).

The natural environment of chickens may be contam-
inated with MDV. Virus has been detected in saliva (Kenzy
and Biggs, 1967; Witter and Burmester, 1967), feces (Witter
and Burmester, 1967) and dander (Beasley eE_al., 1970) from
infected chickens. Air (Sevoian gE_al., 1963a), litter
and droppings (Witter eE_al., 1968b) from cages containing
infected birds, and even air from such a cage after the
chickens have been removed (Colwell and Schmittle, 1968) are
infectious. Also certain beatles, Alphitobius diaperinus,

mechanically transmit infection (Eidson et a1., 1966).

22

The natural route by which chickens become infected
has not been determined although infection via the respira-
tory tract appears to be the most logical. In a detailed
epizootiological study in broiler flocks conducted recently
in Georgia infection could be detected in chicks one to two
weeks after they had been placed in a contaminated poultry
house (Witter et al., 1970b). Once established, the
incidence of infection increased rapidly until at eight weeks,
MDV could be isolated from almost all birds and there was a
parallel increase in microscopic lesions. The presence of
maternal antibody in hatched chicks does not prevent
infection but it may slightly delay the subsequent develop-
ment of lesions (Chubb and Churchill, 1969). Virus and
antibody may persist for at least 18 months but the levels
may fluctuate. Although nearly all chicks become infected
before they reach sexual maturity, clinical disease is the
exception rather than the rule. The existence of viral
infection in the absence of clinical or pathological
evidence of disease has been well documented (Chubb and
Churchill, 1868; Witter gt_al., 1969a). The conditions
necessary for the development of the disease are poorly
understood.

Approximately one week after infection, chickens
begin to shed MDV (Kinzy and Biggs, 1967) into the environ-
ment in the form of enveloped virus associated with, or

originating from, the feather follicle (Calnek and Hitchner,

23

1969; Calnek et_al., 1970a; Nazerian and Witter, 1970;
Purchase, 1970a, Article III). Desquamated cells and
feather sheath cells are abundant components of chicken
house dust and dander which adheres to all objects within
the house or which enter or leave the house. Excretion of
virus in this volatile form provides a means for airborne

transmission.

Control

Conventional methods for controlling infectious
diseases have been largely unsuccessful in reducing the
incidence of MD (Chute et_al., 1964). Some success has
been obtained in controlled environment houses which employ
biologically filtered air under positive pressure (FAPP)
with strict environmental sanitation, and rearing in iso-
lation (Drury et_al., 1969).

Different genetic stocks of chickens are highly
variable in their susceptibility to MD (Hutt and Cole,
1947; Biggs and Payne, 1963; Purchase and Biggs, 1967;
Crittenden, 1968; Sevoian, 1968b). The mode of inheritance
is complex but resistance appears to be dominant (Cole, 1970).
There is no correlation between genetic resistance to MD
and the genetic control of various production traits or
genetic resistance to lymphoid leukosis. However, there
is a significant relationship between the susceptibility

of chickens to experimental inoculation and to natural

24

exposure (Biggs et_al., 1968c). Also, sufficient genetic
heterogeneity is present in most commercial chickens for
a worth-while selective breeding program directed towards
resistance to MD. Rapid progress can be made in this
direction in a few generations (Biggs gt;§l., 1968c; Cole,
1968).

Maternal antibody passed from a hen to her off-
spring delays the onset and reduces the incidence of disease
in chicks challenged at one day of age by a natural route,
but it has little effect when birds are challenged by
intra-abdominal inoculation (Chubb and Churchill, 1969).
The protective effect is lost by the time the birds are
three weeks of age. Hyperimmunization of dams with a
virulent strain of MD has conferred some degree of immunity
on the progeny (Eidson et_al,, 1968). Since field exposure
may not occur until after birds are three weeks old, this
procedure does not appear to have any practical application.

Adaptive transfer of resistance has been claimed
(Feldbush and Maag, 1969) but undoubtedly MDV was trans-
mitted with the immune spleen cells. There was no way of
assessing what portion of the resistance was due to the
immune competence of the cells themselves.

The development of live virus vaccines against MD
has been reviewed (Purchase §E_al., 1970b). There have
been at least four strains of virus used for this purpose:
namely two attenuated and one apathogenic MDV and an

apathogenic HVT.

25

An attenuated MDV vaccine has been developed
(Churchill gt_al,, 1969a and 1969b). When a strain of
"acute" MDV was passed serially in CK cell culture it
produced larger cytopathic areas (macroplaques) than those
produced by the parent strain and it was nonpathogenic for
chickens. This strain had also lost the "A" precipitating
antigen present in cultures infected with low passage MDV.
Chicks inoculated with the attenuated strain were protected
against the disease when subsequently challenged with a
virulent strain of virus. The vaccine is also effective
in the field (Biggs §E_gl,, 1970).

A nonpathogenic herpesvirus, HVT, isolated from a
group of turkeys (Witter et;gl,, 1970), had some antigens
in common with MDV. In CK or DEF cultures, it produced
macroplaques which were distinguishable from those produced
by the attenuated high passage and the virulent low passage
strains of MDV. In addition, antibody to HVT could be
distinguished from antibody to MDV by the IFA test. The
HVT protected chicks from MD when they were subsequently
challenged with virulent MDV. The vaccine virus only rarely
spread from vaccinated to unvaccinated birds in contact
with them (Okazaki et;al,, 1970). This vaccine is also

highly effective in field trials (Purchase et al., 1970c).

26

A strain of MDV which had undergone very few passages
in CEF and produced no detectable cytopathic effects,
reduced the incidence of MD in challenged chickens (Kottaridis
and Luginbuhl, 1969). Some of the vaccinated birds died
of MD and the designs of the experiments were not adequate
to determine whether this was due to the pathogenicity of
the vaccine or to its partial ineffectiveness as an immunizing
agent.

A naturally avirulent strain of MDV isolated from
a normal flock was also able to produce protection (Rispens
gt_al., 1969). The virus had undergone approximately 20
passages in cell culture. A virus of this description would
probably be serologically and virologically indistinguishable
from the virulent virus and would be expected to spread

rapidly from bird to bird.

MATERIALS AND METHODS

The details of materials and methods are presented in
each of the five articles. A convenient reference index is
given in Table 1.

TABLE l.--Index of Materials and Methods.

 

lit .

 

 

Item Article Page
MATERIALS

Chickens for in vivo studies . . . . . I 558

Embryos for CEIl culture . . . . . . IV 4

V 2

Embryos for CAM inoculation . . . . . V 2

Goose embryo fibroblast cell line . . . V 3

HeLa cell line. . . . . . . . . . V 3
Viruses

MDV . . . I 558

MDV passed many times in cell culture . IV 4

HVT . . . . . . . . . . . . I V 4

ILTV . . . . . . . . . . . . I 558

RSV pseudotypes . . . . . . . . I 558

IBA . . . . . . . III 1898
METHODS
Procedures for preparation of reagents
Cell cultures from various avian

species . . . . . . . V 3
Virus- infected cell cultures for IFA
test . . . . . . . . . . . I 558
II 118
Tissue sections for DFA test . . . III 1899
Cell culture antigens for AGP test . . II 118
Tissue antigens for AGP test . . . . III 1899
Antigens from cloned viruses for AGP
test . . . . . . . . . . . IV 7
Hyperimmune antisera. . . . . . . I 558
III 1898
Antisera to 8 isolates Of MD . . . . I 558
Antisera to cloned preparations of MD
and HVT . . . . . . IV. 8
Fluorescein- conjugated globulin for DFA
test . . . . . . . . . III 1898

Procedures for performing tests
Testing fluorescein- -conjugated anti-

chicken globulin . . . . . . . I 558
Staining, IFA test . . . . . . . I 558
II 118
Staining, DFA test . . . . . . . III 1899
Micro AGP test. . . . . II 118
Modified micro AGP test with plastic .
templates. . . . . . . . . IV 8
Cloning of MDV and HVT . . . . . . IV 5
Testing pathogenicity of viruses. . . IV 7
CAM inoculation . . . . . . . . V 4
Assay of filtrable virus . . . . . III 1899

 

27

RESULTS AND DETAILED DISCUSSIONS

 

 

Experimental results and detailed discussions are
presented in each of the five articles. A convenient refer—
ence index of results is given in Table 2.

TABLE 2.--Index of Results.
Item Article Page
IMMUNOFLUORESCENCE IN THE STUDY OF MAREK'S
DISEASE. I. DETECTION OF ANTIGEN IN CELL
CULTURE AND AN ANTIGENIC COMPARISON OF
EIGHT ISOLATES . . . . . . . . . . . I 559
Development of antigen . . . . 559
Staining of MD antigen in duck and chick
embry fibroblasts . . . . . . . . 559
Staining of heterologous antigens . . . 559
Controls within the indirect FA test . . 559
Comparison of sensitivity of FA and TC
tests . . . . . . . . . 559
Detection of MD herpesvirus in field
samples . . . . . . . . . . 561
Antigenic relationship between isolates
Of MD 0 O I O O O I O O O O O 561
Agreement between observers . . . . . 563
Heat stability of MD antigen . . . . . 563
IMMUNOFLUORESCENCE IN THE STUDY OF MAREK'S
DISEASE: DETECTION OF ANTIBODY . . . . . II 120
Agreement between results of FA and AGP
tests 0 O O I I O O O O 120
Comparative titration of serums . . . . 121
Detection of maternal and acquired anti-
bOdy O O I I O O I O O O O O 121

28

29

TABLE 2.--Continued.

 

Item Article Page

 

VIRUS-SPECIFIC IMMUNOFLUORESCENT AND
PRECIPITIN ANTIGENS AND CELL-FREE VIRUS
IN THE TISSUES OF BIRDS INFECTED WITH

MAREK’S DISEASE . . . . . . . . . . . III 1899
Distribution of IF antigen. . . . . . 1899
Demonstration of specificity of staining . 1901
Distribution of precipitin antigen . . . 1901
The presence of filtrable virus . . . . 1901

PATHOGENICITY AND ANTIGENICITY OF CLONES FROM
STRAINS OF MAREK'S DISEASE VIRUS AND THE

HERPESVIRUS OF TURKEYS . . . . . . . . IV 9
Filtration and cloning of viruses . . . 9
Morphology of plaques in CK cells . . . 9
Pathogenicity of cloned viruses . . . . 10
Antigenic analysis by the agar gel

precipitin test . . . . ll
Antigen analysis by the indirect fluores-
cent antibody test. . . . . . . . l4

RESPONSES OF CELL CULTURES FROM VARIOUS AVIAN
SPECIES T0 MAREK'S DISEASE VIRUS AND THE

HERPESVIRUS OF TURKEYS . . . . . . . . V 5
Morphology of uninfected cells . . . 5
Cytopathic changes produced by different

viruses . . . . . . . . . . 5
Cytopathic changes in different cell
types . . . . . . . . . . . 6

Comparative sensitivity of cells of

different types and CAMs to clones of

virus . . . . . . . . . . . . 7
Immunofluorescent antigen in infected

cultures . . . . . . . . . . . 3

 

ARTICLE I

IMMUNOFLUORESCENCE IN THE STUDE OF MAREK'S
DISEASE. I. DETECTION OF ANTIGEN IN CELL
CULTURE AND AN ANTIGENIC COMPARISON OF

EIGHT ISOLATES

BY

H. G. Purchase

Reprinted from J. Virol. 2:557-565, 1969.

30

JOURNAL or VIROLOGY, June 1969, p. 557-565
Copyright © 1969 American Society for Microbiology

Vol. 3. No. 6
Printed in U.S.A.

Immunofluorescence in the Study of Marek’s Disease

I. Detection of Antigen in Cell Culture and an Antigenic Comparison

of Eight Isolates1
H. G. PURCHASE

Regional Poultry Research Laboratory, Agricultural Research Service, US. Department of Agriculture,

East Lansing, Michigan 48823

Received for publication 29 January 1969

The indirect fluorescent-antibody (FA) test was applied to the detection of Marek’s
disease (MD) antigen in cell culture and antibody in the serum of birds. For the
detection of antigen, sera were obtained from birds hyperimmunized with the
JM strain of MD. MD antigen could be detected in the nucleus and in the
cytoplasm of duck and chick embryo fibroblasts and in those of chick kidney cells
infected with material known to contain the MD virus. Uninoculated cultures
of chicken cells were always free of MD antigen. When chick kidney cells were in-
fected with a stock cellular preparation of MD virus, infected cells could be detected
after 24 hr with the FA test. At this time no cytopathological areas were seen by con-
ventional light microscopy. By 7 days after infection, the same number of infected
areas were detected by both methods, and the fluorescent areas coincided with the
cytopathological areas. This indicates that the fluorescent areas and the areas with
cytopathology are caused by the same agent. A straight-line relationship between
the dilution of inoculum and the number of fluorescent or morphological foci ob-
tained indicates that one infectious unit produced one fluorescent or morphological
focus. In addition, this time sequence study confirmed the cell association of the
virus and demonstrated the cell-to-cell spread of infection. Cell cultures inoculated
with eight different isolates of MD were tested in all combinations with sera pre-
pared against the same isolates. The antigens were indistinguishable from one
another, indicating that either the strains are antigenically identical or there is a
common antigen or contaminant in all of them so that they stained equally well.
The FA test can detect MD antigen before cytopathological areas develop in cell
culture; however, the small size of the area usually examined precludes its use in
initial isolations in which only a small number of infectious units are present in the
inoculum. MD-infected cells contain a heat-stable antigen similar to that found in

herpes simplex-infected cells.

 

A wealth of circumstantial evidence has ac-
cumulated which incriminates a highly cell-associ-
ated herpesvirus as the etiological agent of
Marek’s disease (MD) (4, 12, 15, 18). The agent
produces characteristic cytopathic changes in cul-
tures of chick kidney cells, duck embryo fibro-
blasts, and, under special conditions, in chick
embryo fibroblasts (11). Although some attempts
to obtain cell-free virus have been successful,
most efl‘orts have failed. It has been impossible to
obtain suflicient cell-free virus to perform neu-
tralization tests for the identification of the virus
or the detection of antibody in sera of birds.

1 Preliminary results were reported at the 105th annual meeting
of the American Veterinary Medical Association in Boston. Mass...
July 1968.

557

Recently an antigen has been detected in infected-
cell cultures which produces a line of precipitation
in an agar-gel when diffused against serum from
infected or recovered birds (3). This antigen can
be used in the agar-gel precipitin test for the
detection of antibody in birds which have been
exposed to MD.

Kottaridis and Luginbuhl (10) have described
a direct fluorescent-antibody (FA) test for MD
antigen in cell culture. They used sera obtained
from rabbits which had been hyperimmunized
with extracts of infectious blood. This paper
describes the application of the indirect FA test
to the detection of MD antigen in cell culture and
antibody in the serum of birds.

PURCHASED BI
& '. hm. OF Acqil‘lWT'F‘
\ P0?“ 0??“ r _.“ _ lfL‘“

 

 

558

MATERIALS AND NIETHODS

Chickens and eggs. The inbred lines maintained at
the Regional Poultry Research Laboratory were used
throughout. Line 7 chickens and those produced by
the cross between line 15 males and line 7 females are
highly susceptible to MD, line 15 and line 151 are
intermediate, and line 6 chickens are resistant to MD
(5). Except where otherwise indicated the parent
lines are maintained in conventional chicken houses
and the flock is known to harbor MD viruses. Pro-
geny from these chickens reared in modified Horsfall-
Bauer isolators are usually free of all signs of infec-
tion.

Sources of viruses. The origin of the JM isolate of
MD has been described (17). MSD 1 was obtained
from T. Maag, Merck & Co., Inc, Rahway, N.J.;
GA from S. Schmittle, University of Georgia (8);
CONN A from R. Luginbuhl, University of Con-
necticut (2); CR 64 from W. Staples, Cobb Breeding
Corp., Connecticut; Cl from R. Bankowski, Univer-
sity of California (1); RPL 39 from a field outbreak
of MD in Georgia; and FC 50 from an outbreak of
MD in Michigan. These isolates had been passaged
40, 1, l9, 0, 5, 1, 2, and 1 times, respectively, in
chickens from the Regional Poultry Research Labora-
tory.

Two strains of laryngotracheitis virus were kindly
supplied by R. Luginbuhl. Bryan’s high titer strain of
Rous sarcoma virus with Rous-associated virus: as
helper [BH-RSV (RAVI)] and BH-RSV (RAvg) were
obtained from P. K. Vogt (9).

Antigen. Kidney cultures were prepared as de-
scribed by Churchill and Biggs (4) from a bird which
had been inoculated with MD-infected blood and
which had clinical signs and gross lesions of MD.
When cytopathological areas appeared (4, 19), the
cells were trypsinized and plated on fresh confluent
monolayers of kidney cells prepared from uninfected
birds. This procedure was repeated until an extensive
cytopathological effect (CPE) was obtained, where-
upon the cells were trypsinized and frozen in dimethyl
sulfoxide and stored in liquid nitrogen (7).

Primary chick kidney cultures were prepared and
grown on cover slips (11 by 22 mm) placed in 60—mm
plastic disposable petri dishes and they were infected
with various sources of MD virus. At intervals or
when a clearly visible CPE was present, cover slips
were removed, rinsed in phosphate-buffered saline
(PBS), and fixed by immersion in acetone at 4 C for
2 min. The cover slips were dried under an air blower
and stored at 4 C until used within the next few days.

Primary chick embryo and duck embryo fibroblast
cultures were prepared as described previously (15,
16). Chick embryo fibroblasts infected with the JM
strain of MD were obtained from K. Nazerian.

Antisera. Line 6 chickens were reared in modified
Horsfall-Bauer isolators. At 8 weeks of age, a blood
sample was obtained to confirm the absence of anti-
body. They were then inoculated intraperitoneally
with 0.5 ml of fresh whole blood obtained from a
chicken with clinical and gross signs of MD. Simul-
taneously, 0.5 ml of complete Freund’s adjuvant was
inoculated into the breast muscle. Birds were bled 2

PURCHASE

 

J. VIROL.

weeks later for the cross-fluorescence studies in
Table 1. In other instances in which a high titer of
antibody was required, the above procedure was
repeated 3 times at 2-week intervals and birds were
exsanguinated 2 weeks after the last inoculation. All
sera were heat-inactivated at 56 C for 30 min and
clarified by centrifugation at approximately 1,000 X g
for 5 min before dilution and application in the FA
test.

fluorescein-conjugated antichicken globulin. Con-
jugates from various commercial sources were tested
by the following procedure. Spleen sections from
freshly killed, 6- to 10—week-old chicks from any
source available were cut at a 6 rim-thickness on a
cryostat (Lab-tek; B. C. Ames C0,, Waltham, Mass).
They were immediately fixed in acetone at -— 10 C for
2 min and then air dried. After moistening with PBS.
the cover slips were flooded with twofold dilutions of
various commercial fluorescein-labelled antichicken
globulins and allowed to react for 30 min at room
temperature and then washed in PBS for 15 min. The
sections were then permanently mounted (6) and
examined under a Leitz fluorescence microscope with
a BC 12 excitor filter and an CG 1 barrier filter. Dilu-
tions of the conjugate which stained the globulin-
producing cells in the spleen with the least amount of
nonspecific fluorescence of the surrounding tissue
were selected for use in the indirect FA test. Conju-
gates from various commercial and laboratory
sources were found to dilfer greatly in quality. The
same dilution of the best conjugate was found to give
the most satisfactory results in the indirect test by
using MD virus-infected cell cultures as antigen and
reacting this antigen with globulin from recovered
serum and then with the antiglobulin as described
below.

Staining procedure. Cover slips were divided into 1
to 4 areas with water-repellent ink and were attached
horizontally to the top of rubber stoppers with adhe-
sive tape. They were flooded with PBS for a few
seconds, and then dilutions of serum were placed on
the dilferent areas of the cover slip. The stoppers were
carefully placed around the periphery of a plastic
beaker so that the cover slips pointed toward the
center. The tightly covered beaker contained sutlicient
PBS, which was stirred continuously on a magnetic
stirrer to keep the atmosphere humidified. After incu-
bation at room temperature for 30 min, they were
submerged in PBS and rinsed by gentle stirring for 15
min. The PBS was removed from the beaker with a
vacuum device, and the cover slips were covered with
an appropriate dilution of fluorescein-labelled anti-
chicken gamma globulin and allowed to react for 30
minutes. The cover slips were again submerged for 15
min in PBS, removed from the stoppers, dipped in
distilled water, and mounted on glass slides in 90%
glycerol and 1 (70 PBS, in Elvanol (13) or in Uni-
mount (6).

Terminology. Cytopathological areas observed
under conventional light microscopy are referred to
as morphological foci, whereas those observed after
FA staining are referred to as fluorescent foci.

VOL. 3, 1969

RESULTS

Development of antigen. Monolayers of chick
kidney cells on cover slips were infected with a
stock preparation of JM-infected chick kidney
cells. At 1, 3, 5, and 7 days after infection, cover
slips were removed, fixed, and stained with the
indirect FA technique in which sera prepared
against the JM isolate were used, and they were
examined under the fluorescence microscope. Un-
infected cultures were similarly treated.

On the lst day after infection, with conventional
light microscopy, many rounded refractile cells
could be seen attached to the monolayer, but they
could not be recognized as morphological foci.
Upon FA staining, however, some of the cells
fluoresced very brightly. Many of them were
spherical (Fig. 1), others had thin processes ex-
tending from them, and a few were flattened and
resembled the surrounding kidney cells which
were normal in shape but contained antigen (Fig.
2). There were many groups of cells which were
morphologically indistinguishable from the sur-
rounding cells but contained brightly staining
antigens (Fig. 3 and 5). These areas could not
have been recognized as cytopathological areas
by conventional light microscopy.

By 5 and 7 days postinoculation, progressively
more morphological foci were visible than at 3
days, and, on close examination, some larger
refractile cells could be seen. Some fluorescent
foci consisted mainly of rounded refractile cells
(Fig. 6), and others contained one or more
polykaryocytes (Fig. 7). Among different foci,
the proportion of rounded cells to polykaryocytes
varied. By the 7th day postinoculation, nearly all
the fluorescent foci also contained cells with
cytopathology (i.e., they coincided with morpho-
logical foci).

Both cytoplasmic and nuclear staining was ob-
served, and it was not possible to determine which
antigen appeared first. The staining in the nucleus
was usually difluse (Fig. 2 and 3) and did not
obscure the unstained nucleolus. There was usu-
ally a nonstaining halo around the brightly stained
“intranuclear inclusion” (Fig. 3 and 9). A difl‘use
staining was most common in the cytoplasm,
although some cells also contained brightly
staining, irregular granules (Fig. 9). The rounded
cells in the center of the focus stained the most
brightly and the intensity decreased centrifugally
(Fig. 6).

At no time during these experiments did unin-
fected chick kidney cultures stain (Fig. 8).

In another experiment similar to that described
above. chick kidney cultures were prepared di-
rectly from JM-infected birds showing clinical
signs of MD. Antigen was first detected on the

IMMUNOFLUORESCENCE AND MAREK’S DISEASE

559

2nd day after preparation of the cultures. The
development of staining and cytopathology pro-
gressed as described above.

Staining of MD antigen in duck and chick
embryo fibroblasts. Antigen in duck embryo fibro-
blasts infected with the J M isolate of MD stained
brightly. There was a difl‘ use nuclear antigen and a
diffuse and irregularly granular cytoplasmic anti-
gen. In addition, many cells in both the infected
and normal duck embryo fibroblast cultures con-
tained small, uniform, spherical cytoplasmic gran-
ules which tended to obscure the specific stain in
the infected cultures.

Chick embryo fibroblasts infected with MD
contained a diffuse nuclear and cytoplasmic anti-
gen, and granular cytoplasmic antigen could be
easily detected in infected cells (Fig. 4).

Staining of heterologous antigens. Chick kidney
cultures on cover slips were infected with two
strains of infectious laryngotracheitis virus, BH-
RSV (RAVI), BH-RSV (RAVZ), and JM isolate
of MD, and chick embryo fibroblasts on cover
slips were infected with BH-RSV (RAVI) and
BH-RSV (RAVZ). They were fixed and stained in
the indirect FA test with a JM antiserum. There
was no fluorescent staining in any of these cul-
tures, except in those infected with the J M isolate
of MD which showed bright specific fluorescence.

Controls within the indirect FA test. When
saline or serum from uninfected birds replaced
the anti-MD serum in the first step of the indirect
test, no staining was obtained

The specificity of the indirect test was also
examined by absorbing a positive serum with MD
antigen. Antigen was prepared from JM-infected
duck embryo fibroblasts and chick kidney cells by
a method similar to that described by Chubb and
Churchill (3). A similar batch of antigen was
prepared from normal duck embryo fibroblasts
and chick kidney cells. A 1:10 dilution of a posi-
tive serum was added to an equal quantity of MD
cell antigen, normal cell antigen, and saline in
separate tubes. The tubes were incubated at room
temperature with intermittent agitation for 2 hr
and centrifuged at 3,000 x g for 30 min; the
supernatant fluid was used in the indirect FA test
to stain positive JM antigen grown on cover slips.
The brightness of staining was scored from 0 to 4
plus. Both MD-cell antigens absorbed out the
MD antibody from the serum, and only a 1 plus
staining was obtained, whereas the normal cell
antigens and the saline left the antibody which
gave a 4 plus fluorescence of the MD antigen.

Comparison of sensitivity of FA and TC tests
for antigen. The supernatant medium from con-
fluent chick kidney cultures growing on cover slips
was replaced with 5 ml of 1/3 log dilutions of a
stock of JM-infected kidney cells. After 1, 3, 5,

 

 

no. 1—6

560

 

VOL. 3, 1969

and 7 days of incubation, cover slips were re-
moved and stained by the indirect FA technique
with antisera prepared against the JM isolate.
Replicate plates without cover slips were exam—
ined with an inverted conventional light micro-
scope for cytopathological areas (morphological
foci). The numbers of foci per 100 mm2 of surface
area as observed by each method were plotted
(Fig. 11).

’All infected viable cells could be detected by
the FA technique on the day after infecting the
culture. However, they were easier to count on
the 3rd day after infection when the fluorescent
foci were larger. It was not until the 7th day that
most fluorescent foci had developed cytopathol-
ogy which could be seen under the fluorescence
microscope as a rounding and retracting of cells
and as the presence of polykaryocytes. At this
time, there were individual, rounded, fluorescent
cells attached to the monolayer between the large
fluorescent foci. They were similar to the cells
seen at 1 day after infection and were probably
cells which had been washed off the foci and were
initiating secondary foci.

Morphological foci were first detectable on the
3rd day after infection when they consisted of
small~groups of six or more rounded, refractile
cells, often with adjacent, fusiform, refractile
cells. The foci increased in size and numbers until
they reached a maximum at about the 7th day
(Fig. 12).

There is a linear relationship between the
fluorescent foci and the dilution of inoculum and a
similar relationship between the morphological
foci and the dilution of inoculum (Fig. 11). The
lines for the fluorescent foci and for the mor-
phological foci seen at 3, 5, and 7 days after
infection are parallel.

Detection of MD herpesvirus in field samples.
Blood samples were obtained from different field
flocks and used to inoculate replicate plates of
chick kidney cells. One plate contained a cover
slip which was removed between the 4th and 7th
day after inoculation and stained by the indirect

IMMUNOFLUORESCENCE AND MAREK’S DISEASE

561

FA test with JM antiserum. The other plate was
examined for morphological foci between the
10th and let day postinoculation. MD herpes-
virus was detected in 21 (72.4%) of the 29 sam-
ples. Of these, 7 (33.3%) produced morphological
and fluorescent foci, whereas 14 (66.6%) pro-
duced only morphological foci; no fluorescent
foci were detected on the cover slip. There were no
samples which produced only fluorescent foci,
and eight samples (27.6%) were negative by
both tests.

In order to increase the number of infectious
units per culture, and thus increase the likelihood
of a focus occurring on a cover slip, cultures were
passaged to fresh confluent monolayers of kidney
cells 7 days after inoculation with blood. An
additional 43 field samples were examined by this
method. Only 15 (34.9%) contained MD herpes-
virus. Of these, eight (53.3%) produced mor-
phological and fluorescent foci, whereas seven
(46.7%) produced only morphological foci.
Twenty—seven samples (62.8%) were negative by
both tests, and one sample (2.5%) had fluorescent
foci on the cover slip but no morphological foci
on the petri dish. In this instance, a replicate
sample of blood was inoculated into line 15 X 7
chickens and lesions of MD were produced. None
of the 10 samples obtained from isolated control
birds and examined by this method produced
either morphological or fluorescent foci in cell
culture. '

Antigenic relationship between isolates of MD.
Antigen and antibody for the indirect FA test were
prepared from the same inoculum source. Set um
from each immunized bird was used in the in-
direct FA test on each of the antigens. The
brightness of fluorescence was scored from 0 to
4 plus by two observers, and the average score
for each group of antisera was referred to as the
staining index (Table 1).

Birds inoculated with the GA isolate failed to
produce antibody and died of MD shortly after
being bled for antibody. The staining index of
the antibody against homologous antigen was

 

FIG. 1. Chick kidney monolayer one day after inoculation with a stock of MD-infected chick kidney cells.

Single, spherical cell stains. Ca. X 380.

FIG. 2. Chick kidney monolayer one day after inoculation with a stock of [VD-infected CK cells. Single, flattened

cell with an intranuclear inclusion stains. Ca. X 380.

FIG. 3. Chick kidney monolayer one day after inoculation with a stock of IUD-injected CK cells. A group of
flattened cells with difluse and granular cytoplasmic antigen and intranuclear inclusions. Ca. X 380.

FIG. 4. Chick embryo fibroblast cultures infected with JM strain of MD virus. The diffuse nuclear antigen and
the irregular cytoplasmic granules stain brightly. Ca. X 250.

FIG. 5. Chick kidney monolayer at 1 day after inoculation with a stock of AID-infected CK cells (saute mono-
layer as Fig. 1—3). Three fluorescentfoci can be clearly seen. Ca. X 130.

FIG. 6. Chick kidney monolayer 5 days after infection with a stock of MD-in/ected CK cells. A fluorescent
(and morphological) focus composed of rounded cells. Ca. X 130.

 

 

 

 

FIG. 7-10
562

 

VOL. 3, 1969

IMMUNOFLUORESCENCE AND MAREK’S DISEASE

563

TABLE 1. Antigenic relationship among eight isolates of MD

 

 

 

 

 

 

 

Antibodsioproduced Chigkef’rfs Antigens 9| 3:“ 1 fit;
lmgmup IM 315m GA CONN A CRM C. I RPL39 cho i i

l

I
JM ‘ 4 3.0"" 2.0 2.8 1.3 1.5 2.3 2.0 2.1 I 2.1 i 1.9
MSDi 3 1.5 0.5 1.8 0.7 1.0 1.0 0.8 1.2 1.1 1.6
GA 2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 I 2.0
CONN A 4 1.5 1.3 1.9 1.3 1.0 1.8 1.5 1.4 1.5 , 1.6
CR64 4 3.8 2.8 3.3 3.3 2.0 3.5 3.6 2.8 3.1 l 1.3
CI 4 2.0 2.1 1.8 2.3 1.4 1.6 2.1 1.6 1.9 I 1.7
RPL39 3 2.3 2.3 2.5 2.2 1.8 1.8 2.2 1.5 2.1 i 1.7
FCSO 4 1.4 1.8 1.9 1.6 1.4 1.4 1.4 1.0 1.5 l 1.5

 

 

 

 

 

 

 

 

 

a Mean staining index of antibody, i.e. mean of the scores for one group of antisera against all eight anti-
gens. Sera from eight control birds had a mean index Of 0.0.
5 Mean brightness of antigen, i.e. mean of the scores for all groups of antisera against one antigen.

Control antigen had a mean brightness of 0.0.

‘ Values in boldface are average scores (staining indexes) for each group of antisera; the brightness
of fluorescence was scored from 0 to 4 plus by two observers.

higher than the mean staining index against all
antigens in two instances (JM and RPL 39), but
was lower than the mean staining index in the
others. The mean brightness of the antigen was
similar in each instance.

Agreement between observers. In the above
test, two observers examined each preparation
and scored the brightness of fluorescence from 0
to 4 plus, independently (Table 2). Of the 224
Observations, 143 (63.8%) were in full agreement,
79 (35.2%) deviated by a score of 1 plus and 2
(0.9%) deviated by a score of 2 plus. There was
full agreement among the 64 observations with
sera from control birds and among the 28 ob—
servations with control antigen. There was a
98.4% agreement among positives and negatives.

Heat stability of MD antigen. Two cover slips
on which J M-infected chick kidney cells had been
grown were fixed as described above. One was
placed in distilled water and boiled for 90 min.
They were then stained in the indirect FA test.

There was bright nuclear and cytoplasmic stain-
ing in the unboiled cover slip (Fig. 9). After being
boiled for 90 min, the cells shrank slightly but
there was no decrease in the intensity of staining

(Fig. 10).
DISCUSSION

When chick kidney cells were infected with a
stock cellular preparation of the JM isolate,

fluorescent cells could be detected after 24 hr,
but at this time no morphological foci were seen.
By 7 days after infection, the same number Of
infected areas were detected by both methods and
the fluorescent foci coincided with the mor-
phological foci. A straight-line relationship be-
tween the dilution of inoculum and the number
of fluorescent or cytopathic areas obtained indi-
cates that one infectious unit produced one
fluorescent or morphological focus. Thus the anti-
gens detected in cell culture by the indirect FA
test are induced by the virus which causes the
characteristic cytopathology.

The indirect FA test as described here is highly
specific since MD antisera did not stain cultures
infected with other poultry pathogens. In addition
the antibody could be absorbed from the serum
by MD-infected chick or duck cells but not by
uninfected cells. Control uninfected cultures de-
veloped neither cytopathic areas nor fluorescent-
staining foci.

Antigen could be detected in both the nucleus
and the cytoplasm of duck and chicken cells and
its morphology and distribution were similar to
those described for herpes simplex although no
small nuclear granules were seen (14).

The origin of the FA-staining cytoplasmic
granules in duck embryo fibroblast cultures is

 

FIG. 7. Chick kidney monolayer 7 days after infection with a stock of MD-infected CK cells. A fluorescent (and
morphological) focus composed mainly of polykaryocytes. Ca. X 130.

FIG. 8. Uninfected chick kidney monolayer fixed and stained at the same time as that in Fig. 7. Note areas of
rounded cpithelioid cells at top of picture which do not stain. The fluorescing artifact can be easily distinguished

from MD antigen. Ca. X 130.

FIG. 9. Chick kidney monolayer with a large proportion of MD-virus-infected cells. Arrows show diffuse and

irregularly granular nuclear staining. Ca. X 320.

FIG. 10. Chick kidney monolayer identical to that in Fig. 9 but boiled for 90 min in distilled water. Ca. X 320.

 

564

 
  
   
  
    

300 Count: / Morph F4
I00 sq m.

Day I A A

Day 3 V V

Day 5 I D

IOO - Dar 7 0 °

FOCUS COUNTS PER IOO 50 mm.

 

 

 
 

o -| -2
onunous IN Loam
FIG. 11. Numbers of foci detected morphologically

and by FA in cultures at various times after inocula-
tion with dilutions of MD-infected cells.

 

E |OO r-
E
0
U') 50
8 v
k 30 -
Lu
CL
*\
2
D
O
9 IO ~
U5 Caunts / Morph. FA.
3 IOO sq mm
8 Day I A A
L‘ 5 5 Day 3 v v
Day 5 . D
3 r Day 7 o o
I t 1 1 1
O 1 3 5 7

 

DAYS AFTER INOCULATION

FIG. 12. Numbers of foci detected morphologically
and by FA in cultures at various times after inocula-
tion.

unknown, but they could be easily distinguished
from the MD antigen by their morphology and
distribution. Since they were present in all cultures
examined, duck embryo fibroblasts were only
rarely used for antigen.

PURCHASE

J. VIROL.

TABLE 2. Number of sera examined by each observer
which ltad the brightness indicated

 

l

 

 

 

Score 2
Scorel i——_ ._. --- . . _ _-
0“ I 1 I 2 3 I 4
0 5,. , 3 l
l 2 i 24 ‘ l4
2 , 9 24 17 l
3 ll , 34 ' 6
4 2 l 17 i 7

 

“ Brightness of fluorescence was scored from 0
to 4 plus.

b The 64 observations with sera from control
birds and 28 observations with control antigen
were in full agreement and have been omitted from
this table. Values in boldface are in agreement.

MD herpesvirus-infected cells possess an anti-
gen which is not destroyed by boiling for 90 min.
In this respect MD herpesvirus is similar to herpes
simplex (14).

Cell culture antigens appeared about 3 days
before the CPE could be detected, but eventually
nearly all the fluorescent foci developed into
morphological foci. In this respect, the FA test is
as sensitive as that depending on morphological
foci for detecting infectious virus; however, it
suflers the disadvantage that only a small area
(in this case 100 mm”) is usually examined. Mor-
phological foci may occur on the petri dish and
not on the cover slip. This possibility accounts for
the observations in which morphological foci but
not fluorescent foci were detected. In an attempt
to increase the sensitivity of the FA test, I pas-
saged cultures to fresh kidney cells. This increased
the efliciency of recovery from 33.3 to 53.3%.
Larger cover slips could be used, but the time
required to scan them for fluorescent foci is much
greater than is required to examine a petri dish
for morphological foci. This precludes the use of
the FA test in initial isolations in which only a
small number of infectious units are present in
the inocula.

Approximately the same number of fluorescent
foci were detected at 1 day after infection of
chick kidney cultures as were detected at 7 days
after infection. Since all cultures were maintained
under liquid media, this indicates that there was
very little, if any, spread of virus through the
media. The slight increase in the number of foci
at 5 and 7 days after infection is probably due to
secondary foci originating from infected cells
which had drifted loose from cytopathological
areas. These individualcells were clearly visible
when stained with FA.

The fluorescence on day 1 probably reflects the

 

VOL. 3, I969

presence of infected cells in the inoculum. It is
possible that the foci originated from division of
the infected cells added to the culture rather than
by infection of surrounding cells. This is unlikely
since infected cells do not propagate in continuous
culture and slough ofl‘ from the monolayer and
die as the culture gets older. Also the morphology
of the foci produced is characteristic of the recipi-
ent monolayer and not of the donor cells (19).
In mature foci, there was a gradation of staining
from very bright staining in the center of a focus
to just detectable staining at the periphery. If the
entire focus had originated by division of an
infected cell, one would expect a focus of cells
containing approximately the same amount of
antigen as was seen at 1 day after infection (Fig.
3 and 5). The gradation of staining from the
center outward indicates that the infection is
spreading from cell to cell in a centrifugal direc-
tion (Fig. 6). These findings confirm the highly
cell-associated nature of this virus and demon-
strate that infection is transmitted from infected
to adjacent cells. Convincing proof of cell to cell
transmission awaits studies on the mechanism of
focus formation.

Of the eight isolates studied in these experi-
ments, seven could not be distinguished from one
another by the indirect FA test. No conclusions
an be drawn with regard to the eighth isolate
since the chickens that were inoculated did not
produce antibody to this isolate. This may have
been because the chickens succumbed to MD
before they could produce antibody. In another
experiment (unpublished data) chickens produced
antibody to this isolate and it stained J M antigen
well. These results indicate that either the eight
isolates are antigenically identical or that there is
a common antigen or contaminant in all stocks of
the isolates.

The indirect FA test can be considered to be
fairly objective. Both observers had considerable
experience with the indirect FA test and the
observations were made independently; there was
excellent agreement between them.

ACKNO WLEDG MENTS
The author wishes to acknowledge the skilled technical assis-
tance of C. A. Hunt and P. A. Frank.
ADDENDUM IN PROOF

Recent electron microscopic studies have demon-
strated that all cells containing antigen demonstrable

IMMUNOFLUORESCENCE AND MAREK’S DISEASE

565

by the FA test also contain herpesvirus and that cells
which do not contain antigen do not contain herpes-
virus particles (K. Nazerian and H. G. Purchase,
manuscript in preparation).

LITERATURE CITED

l. Bankowski, R. A., T. Mikami, and J. E. Moulton. 1968.
Characterization of the C1 strain of Marek's disease, p.
153-154. Program of the 105th Annual Meeting of the
American Veterinary Medical Association, 1968.

2. Chomiak, T. W., R. E. Luginbuhl, C. F. Helmboldt, and S. D.
Kottaridis. I967. Marek’s disease. I. Propagation of the
Connecticut A (Conn—A) isolate in chicks. Avian Dis.
11:646—653.

3. Chubb, R. C., and A. E. Churchill. 1968. Precipitating anti-
bodies associated with Marek‘s disease. Vet. Rec. 83:4—7.

4. Churchill, A. E... and P. M. Biggs. 1967. Agent of Marek's
disease in tissue culture. Nature (London) 215:528—530.

5. Crittenden, L. B. 1968. Avian tumor viruses: prospects for
control. World’s Poultry Sci. J. 24:18-36.

6. Culling, C. F. A. 1967. Pennanent mounting method for
fluorescent antibody preparations. Nature (London) 214:
1140.

7. Dougherty, R. M. 1962. Use of dimethyl sulphoxide for pres-
ervation of tissue culture cells by freezing. Nature (London)
193:550-552.

8. Eidson, C., and S. C. Schmittle. 1968. Studies on acute
Marek’s disease. I. Characteristics of isolate GA in chickens.
Avian Dis. 12:467—476.

9. Ishizaki, K, and P. K. Vogt. I966. Immunological relation-
ships among envelope antigens of avian tumor viruses.
Virology 30:375—387.

10. Kottaridis, S., and R. E. Luginbuhl. 1968. Marek's disease.
Ill. Immunofluorescent studies. Avian Dis. 12:383—394.

11. Nazerian, K. 1968. Electron microsc0py of a herpesvirus
isolated from Marek’s disease in duck and chicken embryo
fibroblast cultures. p. 222-223. Proceedings of the Electron
Microscope Society of America 26th Annual Meeting.

12. Nazerian, K., J. J. Solomon, R. L. Witter, and B. R. Bur-
mester. 1968. Studies on the etiology of Marek’s disease. 11.
Finding of a herpesvirus in cell culture. Proc. Soc. Exp.
Biol. Med. [27:177—182.

13. Rodriguez, J., and F. Deinhardt. 1960. Preparation of a
semipermanent mounting medium for fluorescent-antibody
studies. Virology 12:316—317.

l4. Roizman, 8., S. 8. Spring, and P. R. Roanc, Jr. 1967. Cellular
compartmentalimtion of herpesvirus antigens during viral
replication. J. Virol. 1:181—192.

15. Solomon, J. J., R. L. Witter, K. Nazerian, and B. R. Bur-
mester. 1968. Studies on the etiology of Marek’s disease. I.
Propagation of the agent in cell culture. Proc. Soc. Exp.
Biol. Med. 127:173-177.

16. Witter, R. L., G. H. Burgoyne, and J. J. Solomon. 1968.
Preliminary studies on cell cultures infected with the
Marek’s disease agent. Avian Dis. 12:169-185.

17. Witter, R. L, and B. R. Burmester. 1967. Transmission of
Marek’s disease with oral washings and feces from infected
chickens. Proc. Soc. Exp. Biol. Med. [24:59-62.

18. Witter, R. L., G. H. Burgoyne, and J. J. Solomon. 1968.
Evidence for a herpesvirus as an etiologic agent of Marek’s
disease. Avian Dis. 13:171-184.

19. Witter, R. L, J. J. Solomon, and G. H. Burgoyne. 1969. Cell
culture techniques for primary isolation of Marek’s disease-
associated herpesvirus. Avian Dis. 13:101—118.

Article II

IMMUNOFLUORESCENCE IN THE STUDY OF MAREK'S DISEASE:

DETECTION OF ANTIBODY

BY

H. Graham Purchase and G. H. Burgoyne

Reprinted from Am. J. Vet. Res. gizll7-123, 1970.

40

Reprinted from the AflERICAN JOURNAL OF VETERINARY RESEARCH, Vol. 81. No. 1. PI“: 117—123

 

 

 

Immunofluorescence in the Study of
Marek’s Disease: Detection of Antibody

H. Graham Purchase, B.V.Sc., M.R.C.V.S., M.S.,

and G. H. Burgoyne, M.S.

 

 

SUMMARY

Indirect fluorescent antibody (FA) and agar gel precipitin (AGP) tests
were used to detect antibody in serums of chickens which were exposed to
Marek’s disease (MD) virus. There was a 92% agreement between the
results of the FA and those of the AGP tests on 418 serums from various sources.
By the FA test, maternal antibody in young chickens from Ian-exposed dams
was detected after antibody was no longer detectable by the AGP test, and
acquired antibody in contact-exposed chickens was detected earlier than by
the AGP test. The FA test was 10 to more than 320 times more sensitive than
the AGP test; also, some serums which had a high titer of antibody demon-
strable by the FA test did not produce precipitation in the AGP test.

Because of the highly cell-associated
nature of the herpesvirus which is impli-
cated as the cause of MD, it has not been
possible to obtain sufficient cell-free virus
for serologic studies.1v"~4'6 However,
Chubb and Churchill2 described an AGP
test which can be used to detect antibody
to the MD virus in the serums of affected
chickens or of chickens in the recovery
stage. The purposes in the present re-
port are to describe the application of
the indirect FA test to the detection of
MD antibody in the serums of affected
and recovered chickens and to compare

 

Received tor publication April 10, 1969.

From the Regional Poultry Research Laboratory, Poul-
try Research Branch, Animal Husbandry Research Divi-
don, Agricultural Research Service, U. S. Department of
Agriculture, East Lansing, Mich. 48823.

'I‘heauthorsthsnkMrs. C. A. Huntfortechnieal assis-
tance and Dr. R. L. Witter for asdstenee in the prepara-
tion of this manmcript.

January. I970

Purchased by the U. S. Department

the FA test results with the AGP test
results. The application of the FA test
to the detection of antigen in cell culture
has been previously described.5

Materials and Methods

The chickens and embryos used were of
lines 6, 7, 15, and 151 and of 15 x 7 cross
kept at this laboratory.“ All chickens were
necropsied at the termination of the experi-
ments, and when necessary the diagnosis
was confirmed histopathologically. The vi-
ruses, JM, GA, and CR64 strains of MD,
have been described previously.‘

Antigen for the indirect FA test was made
as follows: Chicken kidney (CK) cells from
J M virus-infected chickens with clinical
and gross lesions of MD were cultured by
the usual procedure.” When cytopatho-
logic alterations developed, the cells were

I”

of Agricultum for oiiicial use

 

trypsinized and placed on additional CK
cell cultures prepared from J M-afiected
chickens. This was repeated 2 to 5 times
until there were more than 20 cytopatho-
logic areas per 100 sq. m. of the petri
dish surface.

The cells were then passaged on CK cells
prepared as described, but grown on 11-
by 22-mm. coverslips in plastic petri dishes.
When the cytopathologic changes were well
developed, the coverslips were removed,
rinsed in phosphate-buffered saline solution
(PBS), fixed in acetone at 4C. for 2 min-
utes, and air dried under a blower. The
coverslips were stored dry in a petri dish
at —70 C. and used over several months,
during which time they did not lose their
antigenicity. Before the coverslips were
used, they were divided into 5 areas with
waterproof ink. This division allowed 4
serum samples to be tested and the 5th
area to be used as a “handle” by which
the coverslip could be attached with ad-
hesive tape to a rubber stopper.

All serums were heated to 56C. for 30
minutes and were centrifuged at 1,000 g for
5 minutes before they were used. A 1/ 20
dilution of each serum in PBS was made in
a 96—cup plastic tray.‘ The diluted serum
was stirred and transferred to a section of
a marked coverslip with a capillary tube.
The staining procedure for the FA test was
the same as that previously described.“ The
brightness of staining in the FA test was
scored negative, 1+, 2+, 3+, and 4+
(brightest). Titers are expressed as recip-
meals of the highest dilutions of serums
which gave fluorescence.

Precipitating antigen was prepared from
cultured CK cells from JM virus-infected
chickens as described by Chubb and Chur-
chill,a except that the antigen used in the
present study originally contained 4 x 10'
cells/ ml. Antigen was prepared in a similar
manner from JM virus-infected duck em-
bryo fibroblasts (DEF) .' The technique of
double diffusion in agar gel containing 8%
sodium chloride was similar to that de-
scribed by Woernle,’ except that 1% agar
was used. Titers are expressed as recip-
rocals of the highest dilutions of serums
which gave a line of precipitation.

Experiment 1.—Serums from chickens
given difierent treatments were used to
determine whether a correlation existed

 

‘ Joseph E. Frankle Company, Philadelphia, Pa.

 

between results of FA and AGP tests and
previous exposure of chickens to MD virus.
Eleven chickens were inoculated intra-ab-
dominally with J M, GA, or CR64 strains
of MD virus when they were 1 or 42 days
of age. Blood samples were collected and
the chickens were killed 6 weeks later, at
which time 7 of 11 chickens (63.7%) had
lesions of MD. Another group of 10 chick-
ens was kept in direct contact from 1 day
of age with the J M virus-inoculated chick-
ens. Blood samples were collected, and
chickens were killed at 6 weeks of age.
All 10 chickens had lesions of MD. Two
chickens were inoculated intra-abdominally
with 0.2 ml. of normal blood when they
were 1 day old. Blood samples were col-
lected and chickens were killed 6 weeks
later. Lesions of m) were not detected in
either chicken. Serum was prepared from
blood collected from 7 noninoculated chick-
ens which were reared in Horsfall-Bauer
isolators adjacent to the inoculated chickens
and from 20 specific-pathogen-free chickens
more than 6 months old that were kept in
plastic isolators during their entire lifetime.
None of the control chickens had lesions
of MD at necropsy.

Experiment 2.—Serums from chickens
given different treatments were used for
comparative titration in FA and AGP tests
(Table 2). Serums 1, 2, 3, 10, ll, 13, and
14 were from 6—week-old line 15 x 7 chick-
ens inoculated when they were 1 day old
with blood from JM virus-infected chickens,
and serums 15, 16, 17, and 18 were from
similar noninoculated controls. Serums 7,
8, 9, and 12 were from 6-month-old line
15 x 7 chickens which survived contact ex-
posure to J M virus-inoculated chickens and
serums 19, 20, 21, and 22 were from line 7
chickens reared under specific-pathogen-
free conditions. Serums 4, 5, and 6 were
obtained from 8-week-old line 6 chickens
from which blood samples were collected 2
weeks after they were given a series of 3
inoculations with blood from J M virus-
infected chickens as described previously“
and serums 23, 24, 25, and 26 were from
similar noninoculated controls.

Experiment 3.—Serums from chicken
flocks in the field were used to compare FA
and AGP tests. Blood samples were collected
on the processing line directly from hearts
of 100 broilers, 8 to 9 weeks old, from 8
different farms, each of which held 3,800

Am. J. Vet. Res., Vol. 3|. No. I

TABLE l—Agreement Between Results of Indirect Fluorescent Antibody (FA) and Agar Gel
Precipitin (AGP) Tests on Certain Serums

 

 

 

 

 

 

 

 

Agreeme Disagreements
. Poe. to M Neg. to n Pos. to PA Neg. to n
Expen- test and test and test and test and
ment No. of no to pos. to neg. to neg. to pos. to
No. chickens Marek's disease virus AGP test AGP test AGP test AGP test
1 11 Inoculated with MD virus 9 1 0 1
10 Exposed by contact to no
virus-inoculated chickens 10 0 0 0
2 Inoculated with normal
blood 0 2 0 0
27 Kept in isolation
(nonexposed) 0 27 0 0
2 10 Inoculated with MD virus
infected blood 7 0 3 0
4 Exposed by contact to In)
virus-inoculated thickens 4 0 0 0
12 Kept in isolation 0 0 0
3 100 Exposed to up under field
conditions 32 59 4 5
74 Field isolated 0 74 0 0
4 24 Chickens Imder 3 weeks of
8 4 12 0
24 Inoculated with up virus 20 3 1 0
40 Exposed by contact to MD
virus-inoculated chickens 24 10 5 1
so Kept in isolation
(nonexposed) 0 so 0 0
Total 418 114 272 25 7
(Per curt) (92.3) (6.0) (1.7)

 

‘ The test-positive reactions indicate maternal antibody. Fee. = positive; Neg. = negative.

to 6,600 chickens. Total percentage of chick-
ens condemned because of “leukosis” ranged
from 0.5% to 32.3% of the chickens pro-
cessed, and approximately 50% of the blood

samples came from these chickens. In ad-
dition, 74 serums were prepared from blood
collected from 5‘ isolated breeder flocks
which were selected because they did not

TABLE 2—Comparison of Titers of Certain Serums by the Indirect Fluorescent Antibody (FA)
and Agar Gel Precipitin (AGP) Tests with Chicken Kidney and Duck Embryo Fibroblast Antigens

 

 

 

 

 

 

 

 

(Experiment 2)
PA titer‘ Acr- titer‘ rA/AGP"
Serum

No. or: our on use or: oar

1 640 640 82 32 20 20

2 640 80 8 8 80 10

8 320 320 8 8 40 40

4 320 320 2 4 160 80

5 320 320 Neg. Neg. >320 >320

6 320 320 Neg. Neg. >320 >320

7 320 160 4 4 80 40

8 320 160 2 2 160 80

9 320 160 1 2 320 80

10 320 80 16 4 20 20

11 mo 80 4 4 80 80

12 320 so 2 2 160 40

13 Id) 80 4 8 40 10

14 160 80 Neg. Neg. >160 >80

Av. titer of
serums 1—14 342.9 205.7 5.9 5.6

15-26 Neg. Neg. Neg. Neg. N .A. N.A.

 

Titers are expressed as the redprocal oI the highest dilution of serum which produced fluorescence or

. ” The relative sensitivity at the u
’ter by the Aer titer.
N.A. = Not amlimble.

January. I970

test over the we test is indicated by dividing the ra

ll9

 

AGP

16

FA

AGP

 

AGP PA AGP FA AGP FA AGP FA

FA

Age (wk.) of chickens at time blood samples were collected

AGP

FA
2+
2+
2+

AGP

Neg.
Neg.
Neg.

FA

Neg.
Neg.
Neg.

AGP

1%
FA
3 + Neg.
Neg. Neg.
1 + Neg.

AGP

0‘.
FA
4+
2+

 

 

TABLE 3—Detection of Maternal and Acquired Antibody by Indirect Fluorescent Antibody (FA) and Agar Gel Precipitin’ (AGP) Tests (Experiment 4)

Group

 

 

Inoculated

.....

Contact

0/ 8
0/8
0/8

0/8
0/8
0/8

0/8 0/8
N.D.
N.D.

0/8
0/8

0/8 0/8
N.D.
N.D.

0/8
0/8

”‘ Blood samples were collected when chickens were 1

0/8 0/8
0/8 N.D.
N.D.

N.D.

0/8 0/8
0/8 N.D.
N.D.

N.D.

0/8 0/8 0/8 0/8
0/8 N.D. 0/8 N.D.
N.D. N.D. N.D.

N.D.

0/8 0/8
N .D.
N.D.

N.D.
N.D.

0/8

N .D.
N.D.
: not determined.

6/8
N.D.
N.D.

N .D.

N.D.
N.D.
N.D.

N.D.
N.D.

N.D.
Brightness of fluorescence in the FA test was scored from Neg. to 4+; results of the AGP test are scored as + or Neg; numerator = No. of samples test-positive; denominator

‘ The AC? test was performed in duplicate with chicken kidney and duck embryo fibroblast antigen with identical results.
total No. samples tested;

day old; these are not the same chickens from which subsequent blood samples were collected.

 

Control 2
Control 3

Control 1

have history of losses from m) and because
none of the serums reacted in the AGP test.

Experiment 4.—To study the pathogen-
esis of MD, 55 line 15 x 7 chicks were di-
vided among 4 Horsfall-Bauer isolators as
follows. Ten chicks were placed in the
lst isolator and were inoculated intra-ab»
dominally with a dilution of JM virus-
infected blood which contained approxi-
mately 100 minimal infectious units of virus
per dose, and 15 noninoculated chicks were
placed in the same isolator to be exposed
by contact. Three groups of 10 chickens
each were kept in the other 3 isolators and
were used as noninoculated controls. Sam-
ples of blood were collected from chicks at
11 days and 2, 4, 5, 6, 7, 8, 12, and 16 weeks
after they hatched. Consecutive serum sam-
ples from 8 of the exposed and 24 of the
control chickens were tested serologically
(Table 3). In addition, a random sample
of 8 chicks from the same hatch and source
as those placed in isolators were exsan-
guinated when they were 1 day old and
serum prepared from the blood was also
tested.

Results

Agreement Between Results of FA and
AGP Tests—There was 92% agreement
between the results of the FA and the
AGP tests on a total of 418 serums (Ta-
ble 1). Of these serums, 199 were from
exposed chickens, and there was 87%
agreement between the tests on them.
All 193 controls were free of antibody by
both tests. There were 25 serums in
which results of the FA test were positive
and results of the AGP test were negative
and 7 serums where the reverse occurred.
A large proportion of the discrepancies
were in experiment 4 (Table 3) where
the FA test detected maternal antibody
in ll-day-old chicks and the AGP test
did not. Similarly, in the chickens ex-
posed to MD infection by contact, the FA
test detected acquired antibody before
it was detected by the AGP test. The 3
serums in experiment 2 that reacted in
the FA test, but not in the AGP test, will
be described later.

None of the control serums from chick-
ens which had been maintained under
different degrees of isolation reacted in
either the FA test or the AGP test.

Am. J. Vet. Res., Vol. 3|. No. I

 

 

A nonspecific patchy fluorescent stain-
ing of the nuclear membrane of all cells
in the monolayer occurred with 8 of 12
serums from the line 7 specific-pathogen-
free chickens in experiment 1. In con-
trast, the serums from 2 chickens inocu-
lated with normal blood did not react
in either the FA test or the AGP test.

Comparative Titration of Serums.—
Serums from 14 chickens which had been
exposed to MD virus and 12 nonexposed
chickens were titrated by both FA and
AGP tests to determine which of the tests
was the more sensitive and to determine
whether there was a relationship between
the titers obtained with the 2 tests. Ti-
trations were performed with both CK
and DEF antigens to determine whether
there were qualitative or quantitative
diflerences between the antigens.

The serums were ranked according to
the FA titers (Table 2) . The serum with
the highest titer in the FA tests also had
the highest titer in the AGP test, but
beyond this there was very little rela-
tionship between the titers of individual
serums. Serum titers in the FA test were
from 10 to more than 320 times higher
than those obtained in the AGP test, indi-
cating that less antibody can be detected
in serum by the FA test than by the AGP
test. Three samples which had a titer
of between 80 and 320 in the FA test did
not produce a precipitin line in the AGP
test.

With both the FA and AGP tests, there
was very little difference between the
titers of the different serums obtained
with the CK and DEF antigens, indicating
that there was probably no qualitative
difference between the antigens. In the
FA test, however, serum titers were higher
when measured with CK antigen than
when measured with DEF antigen, and
this is reflected in the average titer of
the test-positive serums which was 1/343
with CK antigen and 1/206 with DEF
antigen. There was no quantitative dif-
ference between the precipitin antigens.

Serums from 12 normal nonexposed
chickens were test-negative in both the
FA and AGP tests.

January. I970

Detection of Maternal and Acquired
Antibody.—Maternal antibody was de-
tected in all chickens at 1 day of age
(Table 3) by both tests. At 11 days of
age, maternal antibody was demon-
strated in 12 of 16 chickens (75%) by
the FA test, but antibody was not de-
tected by the AGP test. The staining of
serums from 11-day~old chickens was not
as bright as that obtained with serums
from l-day-old chickens. By 3 weeks of
age, chickens did not have antibody;
however, at 4 weeks of age, all 3 inocu-
lated chickens and 2 of 5 contact-exposed
chickens had antibody demonstrable by
the FA test, but antibody was detected
in only 2 of the inoculated chickens by
the AGP test. At 5 weeks, all the inocu-
lated chickens had antibody detectable
by both tests; however, 3 of the contact-
exposed chickens were test-positive by
the FA test only. From the 6th week on,
antibody was demonstrated in exposed
chickens by both tests, except for 1
chicken which was test-negative by the
FA test on the 6th week. The brightness
of staining in the FA test seemed to in-
crease with time. None of the control
chickens had antibody detectable by
either method after 3 weeks of age.

All inoculated chickens and 8 of 14
contact-exposed chickens (57%) either
died of the disease or had lesions at nec-
r0psy. Of the 30 control chickens, 1 had
a minor microscopic lesion indistinguish-
able from that of MD; however, other
lesions were not seen in this chicken and
antibody could not be demonstrated in
its serum. Lesions of MD were not de-
tected in any of the other 29 control
chickens.

Discussion

Agreement Between Tests—Results of
the FA and the ACP tests were in close
agreement; however, in most samples,
antibody was detected by the FA test and
not by the AGP test rather than vice
versa. This was especially evident when
there were low levels of antibody in the
serum just before maternal antibody was

l2l

 

 

 

 

lost and early in the development of ac-
quired antibody. In these circumstances,
the FA test seemed to detect antibody
which did not react in the AGP test.
There was no relationship between the
titers obtained by the 2 tests; thus, some
serums had high titers in the FA test but
had negative results in the AGP test. The
discrepancies between the 2 tests indi-
cate that diflerent spectrums of antigen-
antibody reactions were being observed
in the 2 tests. The FA test would be ex-
pected to be more sensitive than the AGP
test, since it would detect both the anti-
gen-antibody combinations that produce
a visible precipitate in addition to other
antibody-antigen combinations which did
not form a lattice large enough to pro-
duce a grossly detectable precipitate in
the AGP test. These factors explain the
instances in which the FA test seemed
more sensitive than the AGP test.

There were very few instances in which
a precipitin line was observed in the AGP
test and there was no staining in the FA
test. These discrepancies could have
been due to subjective differences in the
reading of the tests, particularly the FA
test.

The specificity of these reactions is
supported by the following observations.

Only young chickens from dams which
were known to be exposed to MD virus
and older chickens with lesions of MD or
known to be exposed to the MD virus had
antibody. Isolated controls always re-
mained free of antibody which could be
detected by either the FA or the AGP test
after they were 3 weeks old. Thus, even
though the 2 tests detect antibodies di-
rected against diflerent spectrums of
antigens, both are specific for MD virus-
induced antibody. In addition, serums
which had positive results in the indirect
FA test stained only the cytopathologic
areas in infected CK cultures. This pro-
vides evidence that the agent which in-
duces the cytopathologic effect in cell
culture is the same as that which pro-
duces MD in chickens. Since the cyto-
pathologic features are typical of those
induced by a herpesvirus and a herpes-
virus has been seen in similar infected

|22

cultures)?“6 this supports the view that
this herpesvirus is the etiologic agent
of MD.

The nonspecific nuclear staining ob-
tained with 8 serums from the line 7
specific-pathogen-free flock and the non-
specific precipitin lines obtained from 2
serums from the line 7 and 2 serums from
the line 151 specific-pathogen-free flocks
cannot be explained. Since the serum
from chickens inoculated with normal
blood did not react in either test, it was
unlikely that isoantigens were involved.

Sensitivity of the Tests.—The serum
titers obtained with the FA test were 10
to 320 times higher than those obtained
in the AGP test. Even when serums are
used in the FA test at a 1/20 dilution,
the test is usually more sensitive than
the ACP test with nondiluted serums.

Comparison of Antigens of Chicken
Kidney- and Duck Embryo Fibroblast-
0rigin.—Although noninfected DEF cul-
tures contained granules which stained
in the FA test, the fluorescence of these
granules could be distinguished by their
morphologic features and distribution
from the fluorescence obtained in MD
virus-infected cells. However, DEF anti-
gen was not as sensitive as CK antigen,
probably because there was a more cir-
cumscribed cytopathologic efl'ect in the
CK cultures than in the DEF cultures, and
this afforded a better contrast in staining
between the antigen-containing cells and
the background of noninfected cells.
Also, the fluorescence of “normal” gran-
ules in DEF cultures may have affected
the interpretation of the endpoint. Thus,
CK antigen was considered superior to
DEF antigen for the FA test.

There was very little difference be-
tween the serum titers obtained with the
2 antigens in the ACP test. This is also
reflected in the results of the pathogen-
esis study (experiment 4) where there
was a full agreement between positives
and negatives obtained with the 2 anti-
gens. However, antigen pools vary in
quality, and only single pools of CK or
DEF antigen were compared.

Pathogenesis of M arek’s Disease—Ma-

Am. J. Vet. Res.. Vol. 3|. No. |

 

 

 

 

 

 

ternal antibody was detected by both
tests in chickens shortly after they
hatched, but it was detected only by
the FA test at 11 days after chickens
hatched. Chubb and Churchill2 were
able to detect maternal antibody in 12
of 18 2-week-old chickens in one experi-
ment and 3 of 21 in another experiment.
The failure to detect antibody by the
AGP test in 11-day-old chickens in the
present experiments may be due to the
line of chickens used or to a difference
in the antigen used. Antibody could be
detected in chickens 4 weeks after they
were inoculated at 1 day of age. Some
chickens in contact with inoculated
chickens developed antibody which could
be detected by the FA test at the same
time as the inoculated chickens, but most
chickens did not produce precipitating
antibody until they were 6 weeks old.
Chickens which acquired antibody con-
tinued to have antibody in their serums
until they were at least 16 weeks old.

Advantages of the FA Test Over the
AGP Test.—In our experience, the FA
antigen is easier to prepare than the AGP
antigen for the following reasons. In the
FA test, the brightness of staining of in-
dividual cytopathologic areas is a mea-
sure of the amount of antibody in the
serum. Thus, the brightness of staining
is relatively independent of the number
of cytopathologic areas on a coverslip.
Also, coverslips can be fixed at any time
after cytopathologic features appear
when there are cells containing large
amounts of antigen. In the AGP test, in
contrast, the amount of precipitating
antigen is highly dependent on both the
amount of cytopathologic features in a
culture and the stage in the cycle of the
virus when the antigen is harvested.
Thus, the conditions necessary for pre-

' g antigen must be closely controlled,
and each batch of antigen should be stan-
dardized. In the FA test, the cytopatho-
logic areas can be examined morpho-
logically, and contaminating agents which

January. I970

do not produce the characteristic cyto-
pathologic features can be detected.
These agents could conceivably produce
false-positive reactions in the AGP test.

The FA test requires more manipula-
tions than the AGP test, since serums
must be centrifuged and diluted before
use; however, the tests are almost com-
parable in ease of performance. The FA
test is considerably more sensitive than
the AGP test, but where serums contain
very low levels of antibody, reading of the
results is more subjective and requires
more experience in the former than in
the latter. At certain stages in the
pathogenesis of MD, antibody can be de-
tected by the FA test but not by the AGP
test.

The indirect FA test for antibody is a
readily performed, sensitive test which
is highly specific for MD virus-induced
antibody. It should be considered in im-
munologic, epizootiologic, and patho-
genicity studies of MD, both in the lab-
oratory and in the field.

References

l. Biggs, P. M.: Marek’s Disease—Current
State of Knowledge. Current Topics in Micro-
biol. & Immunol., 43, (1968): 92-125.

2. Chubb, R. C., and Churchill, A. E.: Pre-
cipitating Antibodies Associated with Marek’s
Disease. Vet. Rec., 83, (1968): 4-7.

3. Churchill, A. E., and Biggs, P. M.: Agent
of Marek’s Disease in Tissue Culture. Nature,
215, (1967): 528—530.

4. Nazerian, K., Solomon, J. J., Witter,
R. L., and Burmester, B. R.: Studies on the
Etiology of Marek’s Disease. II. Finding of a
Herpesvirus in Cell Culture. Proc. Soc. Exptl.
Biol. & Med., 127, (1968): 177-182.

5. Purchase. H. G.: Immunofluorescence in
the Study of Marek’s Disease. 1. Detection of
Antigen in Cell Culture and an Antigenic Com-
parison of 8 Isolates. J. Virol., 3, (1969): 557—
565.

6. Solomon, J. J., Witter, R. L., Nazerian,
K., and Burmester, B. R Studies on the
Etiology of Marek’s Disease. I. Propagation
of the Agent in Cell Culture. Proc. Soc. Exptl.
Biol. & Med, 127, (1968): 173-177.

7. Woemle, H.: Diagnose der infektiosen
Bronchitis der Hiihner mit Hilfe der Prtizipi-
tationsreaktion im festen Agarmedium. Mo-
natsh. Tierheilk., 11, (1959): 154-167.

I23

 

Article III

VIRUS-SPECIFIC IMMUNOFLUORESCENT AND PRECIPITIN

ANTIGENS AND CELL-FREE VIRUS IN THE TISSUES OF

BIRDS INFECTED WITH MAREK'S DISEASE

BY

H. G. Purchase

Reprinted from Cancer Res. 0:1898-1908, 1970.

48

[CANCER RESEARCH 30, 1898-1908, June 1970]

Virus-specific Immunofluorescent and Precipitin Antigens and
Cell-free Virus in the Tissues of Birds Infected with Marek’s Disease

H. G. Purchase

United States Department of Agriculture, Poultry Research Branch,
Regional Poultry Research Laboratory, East Lansing, Michigan 48823

SUMMARY

A variety of organs from chickens inoculated with Marek’s
disease virus and from control uninoculated chickens were
examined for immunofluorescent antigen with the direct
fluorescent antibody test with Marek’s disease hyperimmune
chicken globulin, for precipitin antigen with selected sera in
the agar gel microprecipitin test, and for filtrable virus
infectious for chick kidney cell cultures. A parallel histo-
pathological examination of the organs was also made.

Immunofluorescent antigen was found in superficial cells of
the epithelium of the feather follicles, lungs, follicles of the
bursa of Fabricius, thymus, spleen, and cecal tonsil. It was
not present in tumors of any organ. Precipitin antigen, which
may have been identical to immunofluorescent antigen, was
detected in all these organs except the cecal tonsil. Filtrable
infectious virus was recovered from extracts of skin, but not
from extracts of lungs, bursas, or thymuses. Histopathologi-
cal examination revealed a close association between the
antigens in these organs and cells undergoing degeneration
and necrosis. There were intranuclear inclusion bodies in the
cells of the epithelium of the feather follicles where similar
necrobiotic changes were taking place.

INTRODUCTION

MD1 is a lymphoproliferative disease which is considered to
be caused by a highly cell-associated Group B herpesvirus (3,
17, 24, 30). la chick kidney or duck embryo fibroblast
cultures, the virus produces a characteristic area of syncytial
cytopathology (7, 24, 31) in which herpes virions and
virus-induced antigens are detectable by the indirect fluores-
cent antibody test (l6, 18, 20). Heavily infected cultures
also contain MD-specific antigens demonstrable by the agar
gel precipitin test (6, 21).

Kottaridis and Luginbuhl (12) described an antigen in bone
marrow smears which reacted in the indirect fluorescent
antibody test with rabbit antiserum. However, no antigen
was reported in other organs. Chubb and Churchill (6) were

 

1The abbreviations used are: MD, Marek’s disease; 11“, immuno-
fluorescent.
Received December 16, 1969; accepted February 25, 1970.

1898

Animal Husbandry Research Division, Agricultural Research Service,

unable to demonstrate antigen which would react in the agar
gel precipitin test in homogenates of MD tumors.

The objective of the present studies was to examine various
organs with and without MD tumors for the presence of IF
and precipitin antigens specific for MD and to correlate the
presence of antigen with histopathological changes. While the
work was in progress, Spencer and Calnek (25), Calnek and
Hitchner (5), and von Biilow and Payne (27) reported
finding IF antigen in various organs, including the bursa of
Fabricius, thymus, and kidneys. Calnek and Hitchner (5)
demonstrated antigen in the feather follicle epithelium and
Calnek et al. (4) recovered infectious cell-free virus from the
feather shaft. Nazerian and Witter (19) demonstrated
ultrastructural changes, including intranuclear inclusion
bodies and enveloped virus feather follicle epithelium. The
present communication confirms some of the above findings
and extends the histopathological and immunological
observations.

MATERIALS AND METHODS

Source of Chicken. Inbred lines 6 and 7 and the cross
between line 15 males and line 7 females maintained at the
Regional Poultry Research Laboratory were used throughout
these experiments (20).

Source of Viruses. The JM and RPL 39 isolates of MD have
been described (20). The RPL 39 isolate produces many
more visceral lesions than the JM isolate (H. G. Purchase,
unpublished data). Stocks of blood from birds with clinical
signs and gross lesions of MD or cultured duck embryo
fibroblasts with characteristic cytopathological areas were
preserved with 10% dirnethyl sulfoxide and stored in liquid
nitrogen (10). Infectious bursa] agent (2), as a homogenate
infected bursa, was obtained from Dr. Roland Winterfield,
Purdue University, Lafayette, 1nd.

Antisera. Groups of 6- to 8-week-old line 6 chickens were
inoculated intraabdominally 3 times at 2-week intervals with
0.5 ml of heparinized whole blood from birds with clinical and
gross signs of MD and simultaneously inoculated i.m. with
Freund’s complete adjuvant (0.5 ml) (20). The birds were
exsanguinated 2 weeks after the last inoculation, and serums
prepared from the blood were tested for antibody by the
indirect fluorescent antibody and agar gel precipitin tests.
Both chick kidney and duck embryo fibroblast antigens were
used in the agar gel precipitin test (6, 21).

Preparation of fluorescein-conjugated Globulin. The

CANCER RESEARCH VOL. 30

Purchased by

AC A__o- .-

the u. S. Department

globulin fraction of selected serums was precipitated 3 times
with 18, 14, and 14% sodium sulfate, as described by Wier
(28). The final precipitate was resuspended in borate buffer,
pH 7.4, to one-half the original serum volume. It was then
passed through a Sephadex 625 column (Pharmacia Fine
Chemicals Inc., Piscataway, N. J.) equilibrated with phos-
phate-buffered saline, pH 7.2 (FTA hemagglutination buffer,
Baltimore Biological Laboratories, Baltimore, Md.). The total
bed volume of the column was at least 3 times the volume
of the globulin. A solution of Blue Dextran 2000 (0.1% w/v)
(Pharmacia) was placed on the column ahead of the globulin
fraction to indicate when the void volume had been eluted.
A volume of eluant was collected equal to 20% more than
the volume of globulin placed on the column. The protein
content was determined by the method of Lowry et al. (14),
with bovine serum albumin as a standard. Immediately prior
to conjugation, the pH of the solution was adjusted to 9.0
with 0.1 N sodium hydroxide and fluorescein isothiocyanate
adsorbed onto Celite (Calbiochem, Los Angeles, Calif.) was
added at a rate of 0.5 mg/mg protein. Conjugation was
allowed to proceed for 30 min at 46, during which time the
reaction mixture was agitated continuously and maintained
at a pH of 9.0 by adding sodium hydroxide. The mixture
was centrifuged at 1500 X g for 3 min and the supernatant
was placed on a Sephadex G-25 column equilibrated with
phosphate-buffered saline. Once again a volume of eluant
was collected equal to 20% more than the volume of
conjugate placed on the column. Aliquots (1 ml) sealed in
glass vials and stored frozen were thawed when needed and
diluted in phosphate-buffered saline to a concentration
determined by experience with that conjugate (1:80 or
1:160 for the conjugate used for most of the work reported
here). Merthiolate (Eli Lilly and Co., Indianapolis, Ind.) was
added to a final concentration of 1:10,000, w:v, and the
solution was stored at 4° and used over a period of 2 to 3
months.

Preparation and Staining of Tissue Sections. Sections of
various organs from freshly killed birds were embedded in
OCT matrix, frozen, and cut at 6 [.1 thickness on a cryostat
(Ames Lab-Tek, Westmont, 11].), fixed immediately in
acetone at 4° for approximately 2 min, and then rapidly air
dried. They were stained, mounted in Elvanol (22), and
examined as described previously (20), except that for direct
fluorescent antibody staining only 1 incubation at room
temperature with the dilution of conjugated serum was
required. Whenever possible, duplicate samples of tissue were
fixed in formol sublimate, dehydrated, embedded in wax,
sectioned, and stained with hematoxylin and eosin.

Assay for Filtrable Virus. Portions of the blocks used for
frozen sections and stored in the cryostat were removed and
thawed and the excess embedding matrix was removed. A
10% extract prepared in cold phosphate-buffered saline by
mincing with scissors was sonicated for 30 sec with the small
probe of a Bronwill Biosonik oscillator (Will Scientific, Inc.,
Rochester, N. Y.) at 70% of maximal intensity. The extract
was centrifuged at 1500 X g for 5 min, and the supernatant
fluid was removed. The supernatant, after addition of 2
drops of a 3-times-washed 24-hr culture of Serratia
marcessens, was then passed through a 0.4511 filter (Swinnex 25

JUNE 1970

Antigen and Virus Localization in Marek’s Disease

unit, Millipore Filter Corp., Bedford, Mass), pretreated with
bovine fetal serum (26). The first and last few drops of
filtrate were placed in tryptose phosphate broth and
incubated aerobically at 37° for 3 days, during which time
bacterial growth was not detected. Duplicate 0.2-ml samples
of the filtrate were assayed for virus on chick kidney cells
(31).

Agar Gel Precipitin Test. Organs were treated as described
above, except that a more concentrated (25%) extract was
prepared. Some samples were homogenized with a
TenBroeck grinder, in place of sonic extraction. The micro-
precipitin test was performed as previously described, with
agar containing 8% NaCl (6).

Experimental Design. In the lst experiment, 15 3-week-old
chickens were inoculated intraabdominally with 0.2 ml of
the stock of JM MD blood, and another group of 15 birds
was inoculated by the same route with 1 X 107 JM
MD-infected duck embryo fibroblast culture cells. A 3rd
group of 15 birds was maintained uninoculated to serve as
controls. The 3 groups were placed in separate adjacent
Horsfall-Bauer isolators. Portions of liver, spleen, kidney,
adrenal, gonad, bone marrow, lung, bursa of Fabricius,
thymus, cecal tonsil, pancreas, proventriculus, brain, brachial,
sciatic and celiac plexuses, and skin from which the feathers
had been plucked were removed for examination from I bird
from each group at 5, 9, l4, I9, 23, 28, 35, and 42 days
postinoculation and the survivors were discarded at 42 days
postinoculation.

In the 2nd experiment, 15 l-day-old chicks were inoculated
intraabdominally with JM MD blood. A 2nd group of 15
chicks was similarly inoculated with RPL 39 MD blood and
a 3rd group of 9 chicks served as uninoculated controls.
Birds (2 or 3 from each group) were killed and portions of
the skin, lung, bursa of Fabricius, and thymus were removed
for examination at 5, 8, 12, 15, and 19 days postinocula-
tion. Survivors were discarded at 19 days postinoculation.

In the 3rd experiment, 1 dr0p of a 1:10 dilution of stock
infectious bursal agent virus was placed in each eye of 16
3-week-old chicks. A 2nd similar group was inoculated
intraabdominally with RPL 39 blood and a 3rd group served
as uninoculated controls. Pieces of the lung, bursa of
Fabricius, and thymus of 4 of each exposed and control
group were removed for examination at 1, 2, 4, and 8 days
postinoculation.

RESULTS

Distribution of IF antigen. IF antigen was first detected in
the feather follicle, lung, and bursa (Table l) at 5 days after
inoculation of loday-old or 3-week-old chicks. Antigen
occurred sporadically thereafter in these organs and in the
thymus, spleen, and cecal tonsils until 42 days postinocula-
tion, which was the longest time tested. Many of the birds
inoculated with either the JM or RPL39 strains of MD virus
had antigen detectable in several different organs.

Initially, IF antigen was diffusely distributed in the
cytoplasm of a few round cells in the affected organ (Fig. 5)
but in more advanced cases, some cells contained brightly
staining granules of irregular size and shape (Fig. 6).

1899

H. G. Purchase

Occasionally, the nuclei of cells were filled with a homo-
geneous IF antigen which stained very brightly.

Antigen was most commonly seen in cells in the superficial
layers of the corneous portion of the feather follicle epitheli-
um which lies adjacent to the coreum produced by the
growing feather (Fig. 1). It varied from a small amount of
diffuse cytoplasmic antigen in a few individual cells to large
amounts of diffuse and granular cytoplasmic and diffuse
nuclear antigen in a band of cells about 4 deep extending
from the basal end of the follicle to the interfollicular skin.
It usually appeared to be most concentrated in the deeper
2/3 of the follicle wall, and it never extended to the
interfollicular skin. When sections of skin including follicles
were stained with hematoxylin and eosin, alterations were
seen in the epithelium lining the feather follicles in 4 birds
(Table I and Fig. 2). All 4 birds had IF antigen in their
follicles. No changes could be seen in the basilar layer;
however, there were degenerative changes in cells in the
intermediate and transitional layers [The basal, intermediate,
and transitional layers together form the stratum germina-
tivum (15)]. Cells filled with a clear vacuole which either
displaced the nuclei into a crescent on 1 side of the cell or
surrounded the nucleus were more frequently seen in
infected birds than in uninoculated controls. In I bird with
IF antigen, many of the nuclei in the transitional layer
contained characteristic inclusion bodies (Fig. 2) resembling
those produced by MD virus in cell culture (31). Sometimes
the inclusion bodies were unusually dense and contracted,
reflecting the variable amounts of cell degeneration. The
cytoplasm of cells in this layer was slightly eosinophilic and

Table 1

Fluorescent antibody and hematoxylin and eosin staining of tissues
from MD-infected and control birds.

 

Fluorescent antibody Hematoxylin and eosin

 

 

Inoculated Control Inoculated Control

Feather follicle 19/29‘1 0/9 4/1 8” 0/8

Lung l2/53 0/20 5/48 0/17
Bursa 8/53 0/20 24/48“ 0/17
Thymus 7/50 0/19 5/48“ OH 7
Spleen 2/23 0/11 0/18 0/8
Cecal tonsil 1/19 0/9 1/16 0/7
Nerve 0/20 0/9 28/48 0/17
Brain 0/7 0/2 10/18 0/8
Gonad 0/19 0/9 9/18 0/7
Liver 0/11 0/4 1/18 0/8
Kidney 0/11 0/4 4/l 8 0/8
Pancreas 0/7 0/2 0/l 7 0/8
Proventriculus 0/7 0/2 2/18 0/8
Total positive 23/59 0/21 37/48 0/17

 

“Number positive over number examined. Birds were examined at
various times between 5 and 42 days postinoculation.

bLymphocytic infiltrations in the subcutis. 4/18 had vacuolization of
nucleus and cytoplasm and disruption of cells of the feather follicle
epithelium. Of these, 1 also had intranuclear inclusion bodies.

Clncludes atrophic and necrotic lesions in addition to the lymphocytic
infiltrations or tumors which are characteristic lesions of MD in other
organs.

I900

granular. In the outer layer, in the position of the stratum
corneum, the nuclei of the cells were either basophilic and
about the size of inclusion bodies or had degenerated
completely and were not visible. The cytoplasm of the cells
was filled with indistinct highly eosinophilic granules. The
most superficial layers were disintegrating into fragments and
finely granular material. In follicles from which the feathers
were not removed, the feather sheaths and the feathers
themselves appeared normal. Even in preparations in which
many of the feather follicles showed the above changes,
there were some follicles which appeared normal. Since, in
the few follicles sectioned longitudinally, the alterations were
largely confined to the epithelium in the deeper 2/3 of the
follicle, the level at which the follicles were sectioned
transversely may have determined whether alterations were
seen. In 4 birds, there were large accumulations of lymphoid
cells in the nerves and in the connective tissue of the
subcutis. There was no IF antigen in these areas; however. 2
of these had IF antigen in their feather follicles.

IF antigen also occurred in the lungs in cells located
between the epithelial linings of adjacent air capillaries and
sometimes in the epithelial cells themselves (Fig. 3).
Occasionally, fluid in the intermediate cavities and lumens of
tertiary bronchi also stained with the conjugate. Most of the
lungs appeared normal in histological sections. In some there
were diffuse infiltrations of lymphoid cells between the
epithelial linings of the air capillaries (Fig. 4). In the lungs of
5 of the birds examined, there were large areas of
pleomorphic lymphoid cells characteristic of MD lymphoid
tumors, but none of these contained IF antigen.

The earliest signs of antigen in the bursa of Fabricius, 5
days after inoculation of day-old chicks, consisted of
scattered individual cells in the medulla of the follicles (Fig.
5) which increased until the whole medulla stained very
brightly (Figs. 6 and 7). Granular cytoplasmic antigen in
cells also scattered through the cortex and sometimes
between the follicles (Fig. 6) could be distinguished from the
occasional granules of fluorescent precipitate in the serum by
their morphology and plane of focus (Fig. 13). After
hematoxylin and eosin staining, the bursas with few
scattered cells containing IF antigen had follicles which were
uneven in size and in some the medulla was atrophied. There
was usually some evidence of necrosis, i.e., pyknosis and
karyorrhexis. Many birds with atrophic follicles without signs
of necrosis contained no cells with IF antigen. In 3 birds
where the medulla of some follicles contained masses of
antigen, there was massive necrosis with lysis and karyor-
rhexis of the lymphoid cells (Fig. 8). In the bursas of birds
which had recovered from the necrotic process, there were
often cysts containing cellular debris and in some cases the
medullas were filled with reticular cells and fibrous tissue
(Fig. 9). Occasionally, in older birds, there were large groups
of heterOphils in the cortex of, or between, the follicles. IF
antigen was not detected in the bursas with these regenera-
tive changes, although antigen was often present in other
organs of the same bird. Bursas with massive extrafollicular
infiltration of pleomorphic lymphocytes (tumors) (Fig. 11)
and follicular atrophy did not contain IF antigen.

In the thymus, IF antigen was usually confined to isolated

CANCER RESEARCH VOL. 30

cells in the medulla. In some birds, particularly those with
advanced regenerative changes in the bursa, there were large
groups of cells in the thymus which contained antigen (Fig.
10). After hematoxylin and eosin staining, the thymuses
were severely atrophied (Figs. 15 and I6) and groups of cells
in the medulla exhibited necrosis and karyorrhexis (Figs. 17
and 18).

In the spleens and cecal tonsils, IF antigen was present in a
variable number of round cells. In 1 bird, the cecal tonsil
and surrounding musculature of the intestine were tumorous,
but there was no IF antigen in this organ.

Massive infiltrations of pleomorphic lymphoid cells occur-
red in the nerves, gonad, liver, kidney, or proventriculus. No
IF antigen was detected in any of these tumors.

Cells containing granules which stained very brightly (Fig.
12) were seen in the bone marrow smears of both normal
and infected birds. This staining was not considered specific.

Demonstration of Specificity of Staining. Two criteria were
used in the initial evaluation of the specificity of the
staining. Firstly, specific staining was very much brighter
than the background staining obtained in other areas of the
section and in sections of tissues from other birds. Secondly,
specific staining was absent from the tissues of control birds.
Staining was observed in bone marrow and skin (epidermis)
of both infected and control birds. Further evidence that the
staining in the epidermis was nonspecific was that it could
be eliminated without affecting the staining in the feather
follicles by further dilution of the conjugate; however, this
procedure did not reduce staining of the bone marrow cells.
Similar results were obtained by von Biilow and Payne (27).
This nonspecific staining was omitted from the tables of
results.

Serums from 3 different chickens hyperimmunized with
blood from birds infected with the JM strain of MD and
conjugated with fluorescein isothiocyanate stained similar
areas in the bursa and lung equally well, whereas fluorescein-
conjugated serum from a bird which was hyperimmunized with
normal duck embryo fibroblast cultures and did not contain

Antigen and Virus Localization in Marek ’s Disease

MD antibody detectable by the indirect fluorescent antibody
or agar gel precipitin tests did not stain this antigen.
Fluorescein-conjugated anti-rabbit globulin and anti-human
globulin and rhodamine-conjugated human albumin did not
stain IF antigen in similar sections.

The staining of IF antigen could be completely inhibited
by pretreatment of the tissue section with unconjugated
serum before staining with the conjugated serum. When
conjugated serum was doubly absorbed with MD chick
kidney precipitin antigen, it no longer stained tissue culture
antigen and the intensity of staining of IF antigen in the
bursa was considerably reduced. Control chick kidney
antigen prepared in a manner similar to the above precipitin
antigen did not alter the staining ability of the serum.

Sections of the bursa from one 16-week-old bird with a
grossly visible lymphoid leukosis tumor follicle induced by a
Subgroup A virus (RPL 12) and from ten 3-week-old birds
between 2 and 8 days after inoculation with infectious
bursal agent, showing characteristic degenerative and necrotic
changes, were free of IF antigen.

Distribution of Precipitin Antigen. Homogenates of various
organs (Table 2) were examined for precipitin antigen with
the fluorescein-conjugated MD hyperimmune serum used in
the above IF studies (but undiluted), serum from a bird
which had recovered from JM MD, sera from 3 birds which
had been inoculated at 1 day of age with MD-infected duck
embryo fibroblasts, sera from 2 adult birds which had been
challenged at 10 weeks of age by intraabdominal inoculation
of blood from a bird infected with MD, and sera from 4
naturally exposed birds from a commercial flock. A common
precipitin line was obtained with all these sera, but not with
serum from a 16-week-old bird reared in isolation. When
tested beside MD duck embryo fibroblast and chick kidney
antigens, there was a continuation between the precipitin
line obtained with the bursa and skin antigens and the
strongest line obtained with the cell culture antigens, which
was probably the A antigen line (9) (Fig. 14).

The Presence of Filtrable Virus. Six samples of skin, lung,

Table 2

Distribution of precipitin antigen among various
organs which had been previously examined

 

 

for [F antigen
Organ No. of IF antigen No. of organs with
homogenate Source birds examined status precipitin antigen
Skin MD inoculated 3 Positive 1
MD inoculated 7 Negative 2
Control 7 Negative 0
Lung MD inoculated 10 Positive 2
MD inoculated 26 Negative 0
Control 9 Negative 0
Bursa MD inoculated 12 Positive 3
MD inoculated 52 Negative 0
Control 18 Negative 0
Thymus MD inoculated 8 Positive 2
MD inoculated 28 Negative 0
Control 9 Negative 0
Spleen MD inoculated 2 Positive 2
Cecal tonsil MD inoculated 1 Positive 0

 

JUNE 1970

I901

H. G. Purchase

thymus, and bursa which contained IF antigen were
examined for filtrable virus. None of the lung, thymus, or
bursa samples contained virus demonstrable by the proce-
dures used; however, 4 skin specimens yielded 10, 7, l, and
1 focus-forming units/0.2 ml extract, respectively. When
these cultures were stained with the fluorescein-conjugated
chicken globulin by a modified procedure (16), IF antigen
was detected in the cytopathological areas. The distribution
of antigen was identical to that previously described (20).

DISCUSSION

Three different types of virus-host cell interactions oc-
curred in vivo. Firstly, there were processes in which neither
antigen nor infectious cell-free virus could be demonstrated.
Thus, tumors of the visceral organs and nerve lesions did not
contain IF antigen, precipitin antigen, or infectious cell-free
virus (8) and, except in rare instances (K. Nazerian, unpub-
lished, and Ref. 23), have not been reported to have virus
particles. Since small numbers of intact tumor cells will
induce MD when inoculated into susceptible disease-free
chickens (31), the viral genome must be present within these
cells.

Secondly, antigen may be present, but not infectious
virus, and a cytolytic process may occur. Both IF and agar
gel precipitin antigens were detected in the bursa of
Fabricius, where they were associated with necrosis of the
medullas of the follicles. The IF antigen and the cytolytic
process were described by Spencer and Calnek (25) and
Calnek and Hitchner (5). A degenerative process was also
seen in the thymuses which contained IF antigen. Thus it
appears that production of antigen, particularly in these
organs, is associated with a cytolytic process in the cells. The
IF antigen in the lung could have been infection from
inhaled material from other infected birds in the same
isolator. Many of the birds with IF antigen in their lungs
also had antigen in other organs, suggesting that the antigen
was being produced at several sites simultaneously. No
infectious cell-free virus was found in the bursa or lung,
indicating that replication of the viral genome and produc-
tion of antigen occurred without formation of complete
infectious virions, although incomplete virions may have
been produced at these sites. The same relationship probably
existed in other organs which contained IF antigen, but
which were not examined for infectious virus.

Lastly, in the feather follicle epithelium, both antigen and
infectious cell-free virus were detected. The virus was infec-
tious even after storage at -—15° for 2 months. Similar results
have been obtained by Calnek et al. (4). Both Calnek et al.
(4) and Nazerian and Witter (l9) detected virus in the base of the
feather shaft, but did not examine skin homogenates. In
both instances the origin of the virus was most likely the
feather follicle epithelium. This is the only place where
infectious cell-free virus has been detected in vivo. Cells in
this location are continuously being sloughed off in normal
birds. In infected birds, an additional degenerative process is
involved, and the cells fragment and release their contents.
These cells have been found to contain large numbers of
enveloped virions (K. Nazerian, unpublished).

1902

Instances have been reported in which cultured cells were
found to be infectious when inoculated in vivo and yet there
was no evidence of cytopathology in the cultures (13, 29,
30). This situation was similar to the lst type of virus-host
cell interaction described above. Usually, however, infected
susceptible cells in culture contained IF antigen, underwent
degeneration, and eventually became part of a cytopathic
area. Very few complete herpes virions were seen by electron
microscopy in these cultures and only small amounts of virus
can be recovered from them (16, 18). Thus, there is a
parallel between this situation and that which exists in the
bursa of Fabricius of infected birds, i.e., the 2nd virus-host
cell interaction described above.

The techniques used were not accurate enough to identify
with certainty each cell containing antigen; however, it
appeared that antigen did occur in lymphocytes in the bursa,
thymus, and lung, and only in the latter organ may some of
the epithelioid cells themselves have been involved. In the
feather follicles the interrelationships were unique, and the
only cells affected were the keratinizing cells of the stratified
squamous epithelium. No IF antigen was detected in any of
the kidneys, gonads, or nerves examined, whereas Spencer
and Calnek (25) and Calnek and Hitchner (5) have observed
fluorescence in these organs. On the other hand, they were
unable to observe fluorescence in the lungs, whereas this was
a common site of antigen detection in the present studies.
These discrepancies could have been due to differences in
the strains of chicken, in the virus used, or in the specificity
of the conjugates. Satisfactory frozen sections of feather
follicles were difficult to obtain in this laboratory. This may
have been responsible for the lower proportion of feather
follicles with [F antigen in this work than was reported by
Calnek and Hitchner (5), and it could account for the 2
instances in which precipitin antigen was detected, but no IF
antigen was detected.

Nuclear inclusion bodies in the superficial layers of the
epithelium of the feather follicle were observed by Nazerian
and Witter (19), who also found enveloped particles in
cytoplasmic inclusion bodies. However, they were not
described by Calnek and Hitchner (5).

Both the IF and agar gel precipitin antigens in cell culture
and in vivo are virus-induced and appear specific for MD
virus, since they are found only in cells or birds infected
with MD virus and not in cells or birds infected with other
agents, such as lymphoid leukosis or infectious bursal agent.
Antigens produced by the JM and RPL 39 strains of virus
were indistinguishable. It was not possible to determine
whether the same antigen was being detected by the fluores-
cent antibody and agar gel precipitin tests in vivo and in cell
culture. Since the organ extracts and cell culture extracts
gave a “line of identity” in the agar gel precipitin test, they
must both contain at least 1 antigen in common. Since the
line of identity between these antigens was obtained in the
agar gel precipitin test with the fluorescein-tagged antiserum,
it was likely that a similar common antigen was being
detected by both the fluorescent antibody and agar gel
precipitin tests. Here, as in the test for antibody (21), it
appears as if the fluorescent antibody test is more sensitive
than the precipitin test.

CANCER RESEARCH VOL. 30

IF and precipitin antigens were detected in the feather
follicles or skin extracts as early as 5 days and as late as 42
days postinoculation, which was the longest time tested. It is
probable that virus is also produced throughout most of this
period, since the cycle of replication of the virus is com-
pleted in these cells and since birds have been shown to be
infectious for most of this time (11). The infected feather
follicle cells are probably eased out of the follicle as the
feather grows and they form part of the dander which is
infectious (1) and which has also been shown to contain
infectious cell-free virus (1. N. Beasley, personal communica-
tion). Inasmuch as large amounts of dander are produced by
chickens, this is the most likely source of virus for dis-
semination to the surroundings.

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173—177, 1968.

Spencer, 1. L., and Calnek, B. W. Marek’s Disease: Application of
Immunofluorescence for Detection of Antigen and Antibody.
Am. 1. Vet. Res., 31: 345—358, 1969.

Ver, B. A., Melnick, J. L., and Wallis, C. Efficient Filtration and
Sizing of Viruses with Membrane Filters. J. Virol., 2: 21—25, 1
1968. .

von Bitlow, V., and Payne, L. N. Direkter fluoreszierendcr
Antikorper-test (FA-test) bei der Marek‘schen Krankheit. Zentr.
Veterinaermed., in press, 1969.

Weir, D. M. (ed.), Handbook of Experimental Immunology. pp.
7—8. Philadelphia: F. A. Davis Company, 1967.

Witter, R. L., Burgoyne, G. H., and Solomon, 1. 1. Preliminary
Studies on Cell Cultures Infected with Marek’s Disease Agent.
Avian Diseases, 12: 169—185, 1968.

Witter, R. L., Burgoyne, G. H., and Solomon, 1. 1. Evidence for
a Herpesvirus as an Etiological Agent of Marek‘s Disease. Avian
Diseases, 13: 171—184, 1969.

Witter, R. L., Solomon, 1. 1., and Burgoyne, G. H. Cell Culture
Techniques for Primary Isolation of Marek's Disease-associated
Herpesvirus. Avian Diseases, 13: 101—118, 1969.

1903

H. G. Purchase

Fig. 1. IF antigen (small filled arrow) in the superficial layers of the epithelium of the feather follicle. The antigen stains bright green,
whereas the feather (open arrow) reflected blue light of lower intensity. Large and small filled arrows, positions of the basilar and corneous
layers, respectively, in Figs. 1 and 2. Fluorescent antibody stain, X 120.

Fig. 2. Inclusion bodies (open arrow) in the nuclei of cells in the transitional layer of the epithelium lining the feather follicle 19 days after
inoculation of MD virus into a 3-week-old chick. The epithelium is convex instead of concave because the follicle collapsed after the feather was
removed. It is unusually deep because of the oblique angle of the section. The basal layer (large arrow) and intermediate layers in this section
are not affected, but the transitional (open arrow) and corneous layer (small arrow) show necrobiosis. H & E, X 300.

Fig. 3. IF antigen in cells between the air capillaries in the lung in a 28-day-old chicken inoculated with MD virus at 1 day of age. Both
diffuse bright fluorescence throughout the whole cell, and granular cytoplasmic fluorescence are visible. For orientation, compare with Fig. 4.
Fluorescent antibody stain, X 300.

Fig. 4. Cellular infiltration in the lung of a l9-day-old bird which also contained IF antigen. pb, tertiary bronchus or parabronchus; a.
atrium; i, intermediate space; c, air capillaries. H & E, X 300.

Fig. 5. Immunofluorescent antigen in the medulla of a follicle of the bursa of Fabricius 9 days after inoculation of MD virus into a
3-week-old chicken. The cytoplasm of many small round cells and 1 large round cell stain brightly. Fluorescent antibody stain, X 480.

Fig. 6. Large amounts of IF antigen in the medulla of a follicle in the bursa of Fabricius of a bird the same age and treatment as that in Fig.
5. Granular cytoplasmic antigen is present in many cells in the cortex. This bursa also had precipitin antigen. Fluorescent antibody stain, X 400.

Fig. 7. IF antigen in the medulla of another follicle from the bursa of the same bird as Fig. 6. Arrow, periphery of the cortex of the follicle.
Fluorescent antibody stain, X 120.

Fig. 8. Necrosis of the cells of the medulla of a follicle from the bursa of the same bird as Figs. 6 and 7. H & E, X 120.

Fig. 9. Replacement of the medullas of follicles of the bursa of Fabricius by reticular cells in a chicken 42 days after inoculation at 3 weeks
of age with MD virus. This bursa did not contain IF or precipitin antigen, although IF antigen was detected in the lung, thymus (same bird as
Fig. 10), and feather follicle. H & E, X 120.

Fig. 10. IF antigen in the thymus 42 days after inoculation at 3 weeks of age with MD virus. For orientation and location of antigen
compare with Fig. 18. This thymus also contained precipitin antigen. Fluorescent antibody stain, X 300.

Fig. 11. Massive interfollicular infiltration of the bursa of Fabricius with pleomorphic lymphocytes 19 days after inoculation at 1 day of age
with the RPL 39 strain of MD virus. This bursa did not contain IF antigen. H & E, X 120.

Fig. 12. Nonspecific staining of granules in cells of a bone marrow smear. Fluorescent antibody stain, X 300.

Fig. 13. Bursa of Fabricius from an uninfected bird stained with the fluorescein-conjugated chicken globulin used throughout these studies.
The granules which are precipitated from the serum can be distinguished from specific stain by their morphology and by the plane at which
they come into focus. Fluorescent antibody stain, X 120.

Fig. 14. Agar gel precipitin test. Well 1 contains antigen prepared from normal skin, Well 2 and 5 contain chick kidney cell culture precipitin
antigen, Wells 3 and 6 contain duck embryo fibroblast culture precipitin antigen, and Well 4 contains antigen prepared from the skin of an
infected bird with IF antigen in the feather follicles. The center well contains the fluorescein-conjugated chicken globulin, which was also used
in the fluorescent antibody tests. One line, probably the A antigen line, is common between the different antigens. X 2.

Fig. 15. A normal thymus from a bird 56 days old. H & E, X 30.

Fig. 16. Severe atrophy of the thymus of a chicken 42 days after inoculation with MD virus at 1 day of age. This thymus contained IF
antigen, but not precipitin antigen. H & E, X 30.

Fig. 17. Higher magnification of the medulla of the same thymus as in Fig. 15. h, Hassall’s corpuscle. H & E, X 300.

Fig. 18. Higher magnification of the same thymus as in Fig. 16. Note the Hassall’s corpuscle (h) and the area of degenerating cells (arrow).
which is thought to correspond with the areas which contain IF antigen, as in Fig. 10. H & E, X 300.

1904 CANCER RESEARCH VOL. 30

Antigen and Virus Localization in Marek’s Disease

 

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1908 CANCER RESEARCH VOL. 30

Article IV

PATHOGENICITY AND ANTIGENICITY OF CLONES FROM

STRAINS OF MAREK'S DISEASE VIRUS AND THE HERPES-

VIRUS OF TURKEYS

BY

H. G. Purchase, B. R. Burmester and C. H. Cunningham

Accepted by the Journal of Infection and Immunity.

60

IN 306

PATHOGENICITY AND ANTIGENICITY OF CLONES
FROM STRAINS 0F MAREK'S DISEASE VIRUS AND THE
HERPES VIRUS OF TURKEYSa

H. G. Purchaseb’c, B. R. Burmesterb’c, and C. H. Cunninghamc

Journal Article No. 5l85 of the Michigan Agricultural Experiment Station

 

a This report is a portion of a thesis submitted by the senior author

in partial fulfillment of the requirements for the Ph.D. degree.

b USDA Poultry Research Branch, Animal Science Research Division,

ARS Regional Poultry Research Laboratory, East Lansing, Michigan #8823

c Department of Microbiology and Public Health, Michigan State

University, East Lansing, Michigan #8823

ABSTRACT

Virus was extracted by filtration from chicken embryo fibroblast
cultures infected with the JM, high passage JM(JMHP), GA, and RPL39
strains of Marek's disease virus (MDV) and from the herpesvirus of
turkeys (HVT) and purified by cloning. The plaques produced by clones
of HVT, JMHP and other MDV differed in morphology from one another.
Clones of MDV varied greatly in pathogenicity for chickens but JMHP
and HVT were non-pathOQenic. Two pathogenic clones of JM virus and a
clone of JMHP virus lacked the A precipitin antigen present in all
other clones tested. All clones had at least one B antigen in common.
HVT and MDV clones with and without the A precipitin antigen could be
distinguished from each other by the indirect fluorescent antibody

test. Changes in virus-host cell relationships, loss of pathogenicity

and loss of the A antigens were independent events.

INTRODUCTION

Marek's disease herpesvirus (MDV) antigen and antibody
have been detected by the agar gel precipitin test (A, l6) and
by both the direct and indirect fluorescent antibody tests
(17, I8, 20, 22). Initial examination of several laboratory and
field isolates of MDV failed to reveal any antigenic differences
between them. However, there was no indication of the purity of
the strains used, and a common contaminant could have accounted
for the serologic cross-reactions.

An antigenic change has been described in MDV that had been
passaged many times in chicken kidney (CK) cell culture (7).
During the 20th to 30th passages in cell culture, the virus lost
an antigen which was usually found in the supernatant fluids of cdltures
infected with the original strain. Futhermore, the subcultured virus
had become apathogenic for chickens, its growth characteristics in
cell culture had become altered, and larger plaques containing
larger syncytia occurred in a shorter time than with the original
virus. A similar attenuation and morphologic change of the cytopatho-
logic areas (plaques) occurred on passage of the JM strain of MDV (13).

Hitter, 5;,21, (25) described a non-pathogenic herpesvirus
isolated from turkeys (HVT). The HVT produced larger plaques than
MDV and was antigenically similar, but could be distinguished from it by
the indirect fluorescent antibody test. Vaccination with HVT protected

chickens against subsequent challenge with MDV (IS). A similar herpes-

3
virus isolated from turkeys (ID) has given similar protection
(D.P. Anderson. unpublished data).

Variants of herpes simplex virus could be distinguished from
one another by the cytopathic effects they produced (9, IA, 2]),
the distribution and intensity of fluorescent staining in cell
culture (ll). and the size of the packs produced on chorioallantoic
membranes of chicken embryos (l2). Similar differences in cytopathic
effect were described for variants of B virus (Herpesvirus simiae),
and there was a relationship between plaque size and virulence for
rabbits (2). This virus could be distinguished from herpes simplex
by the direct or indirect fluorescent anyibody tests (l). Passage
of pseudorabies virus in cell culture altered the size of plaques
produced and decreased the pathogenicity for rabbits (23). From the
above, it appeared likely ;that variants of MDV might exist and that
characteristics used to detect the variants might be useful in
distinguishing MDV from HVT.

The present paper describes the purification by cloning of
three strains of MDV and one of HVT and their pathogenicity in
chickens and antigenic differences by the indirect fluorescent antibody
and agar gel precipitin tests. A description of the plaques produced
by the clones and of host range studies will be presented in another

communication.

MATERIALS AND METHODS

Source of Viruses. Blood or tumor cells from 3 to 6 week-old
chicks infected at one day of age with the JM, RPL39 and GA strains
of MDV were used (l7). A strain of JM virus (JMHP) which had been
passaged more than #5 times in CEF and duck embryo fibroblast (DEF)
culture was kindly supplied by Dr. K. Nazerian (l3). The FCl26
isolate of HVT used in these studies has been described (25).

Chickens and Embryos. Line l5 x 7 chickens (8) and line I900
embryos were from the Single Comb White Leghorn flocks reared at
the Regional Poultry Research Laboratory. Line 1900 are commercial
chickens of C/O phenotype reared under pathogen free conditions.
They are free of lymphoid leukosis viruses and MDV (H. A. Stone,
unpublished data). Embryonated duck eggs were obtained from
Truslow Farms Inc., Chestertown, Maryland 2l620.

Cell Cultures. Primary CEF and DEF cultures were prepared from
l0 and IA day old embryos respectively as previously described (26)
and grown in medium Flo and I99 (Grand Island Biological Co., Grand
Island, N.Y.) with 5%.calf serum (Colorado Serum Co., Denver, Colorado).
The CK cells were prepared from birds under A weeks of age and were
cultured in Eagle's basal medium with 9% bovine fetal serum (Grand

Island Biological Co.) (26).

Propagation and Cloning Procedures. Secondary DEF cultures
were inoculated with blood or tumor cells from birds infected with
the JM, RPL39 and GA strains of MDV. The cells were passaged l to
3 times until extensive cytopathologic effects had developed.
Because CEF cultures were shown to produce more cell-free virus than
DEF cultures (13) the viruses were adapted to grow in CEFs. They
were first propagated in a mixture of equal numbers of CEFs and
DEFs in culture. When extensive cytopathologic effects had
developed, usually 2 to 6 passages. the cells were passed onto
pure CEF cultures. At each subsequent passage on CEF cultures an
attempt was made to extract filterable virus as described below.

The JMHP and HVT produced cytopathologic changes in CEFs
and filterable virus was extracted from them.

Filtration and clonigg. The growth medium on heavily infected
CEF cultures was replaced with antibiotic free medium 2h hours
before extraction. When nearly all the cells in the cultures had
become morphologically altered and there were large numbers of rounded
cells floating in the medium, but before holes appeared in the
monolayer, the cells were scraped off the plate into the supernatant
medium. This su5pension was kept on ice and sonicated with the Bronwell
Biosonic oscillator (Hill Scientific Inc.. Rochester. N.Y.) using
the small needle probe at 70% of maximal intensity for ID seconds. a
procedure which will disrupt over 99% of the cells. The extract was
then centrifuged at approximately 2000 x g for 5 minutes. The supernatant

fluid was decanted into a fresh tube and a few dr0ps of a turbid,

Cs

3 times washed Zh-hour old culture of Seratia marcessens was added.
The fluid was then fi‘tered through a 0.h5 um membrane filter
(Swinnex, Millipore Corporation, Bedford, Mass.) which had been
pretreated with bovine fetal serum (2“).

The integrity of the filter was ascertained by bacteriological
sterility tests of the first few drops after the void volume of the
filter had been expressed and the last few dr0ps after filtration.

The filtrate, l ml/plate, was used to inoculate CEF, DEF and CK cultures
grown in 60 mm plastic petri dishes (Falcon Plastics, Los Angeles,
California). Growth medium was added to the cultures four hours

after inoculation and it was changed every 2 days thereafter.

Cultures were examined daily for cytOpathologic changes.

When plaques consisting of about ID or more rounded
refractile cells had appeared, the cultures were overlaid with growth
medium containing l%.agar. Holes, approximately 2 mm in diameter,
were cut with a sterile cork borer through the agar above the plaques.
These areas and a few surrounding cells were removed from the petri
dish with a few draps of 0.05%.trypsin and placed in 0.5 ml of calf
serum until they were used to inoculate DEF cultures. These clones
were prOpagated in DEF cultures until a sufficient stock was obtained,
usually 2 to 3 passages, at which time the cells were preserved in

liquid nitrogen.

Pathogenicity of clones. Dilutions of virus-infected cells
containing l,000 plaque forming units (PFU) per 0.2 ml were
inoculated intravabdominally into one-day-old line l5 x 7 chicks
which were then reared in modified, stainless steel Horsfall-Bauer
isolators. All chickens dying during the experiment were
necrOpsied. All survivors were exsanguinated and necropsied at
l0 weeks of age. When a diagnosis could not be made on gross
examination, portions of the left and right brachial and sciatic
plexuses, the celiac nerve and a gonad were removed for
histOpathologic examination.

Fluorescent antibodyiand,ggar gel precipitin tests. The
proceddres used for the indirect fluorescent antibody test have
been described (l7). Cell monolayers infected with each clone were
prepared by inoculating confluent CK cultures on ll x 22 mm coverslips
in petri dishes with approximately IOO PFU per coverslip. The
coverslips were removed and fixed when early cytopathologic
changes were visible, i.e., at l to 3 days after inoculation.

Reagents for the agar gel precipitin tests were prepared from
the stocks of cloned viruses after two additional passages in DEF
cultures (20). When there were advanced cytopathologic changes
the supernatant fluid and cells were harvested. The supernatant
medium was centrifuged at l500 x g for ID minutes to remove most
of the cells and then concentrated approximately 50 times by
precipitation twice with 80% and then 60% saturated ammonium
sulfate. The final precipitate, resuspended in distilled water, is

referred to as supernatant reagent.

Cells were scraped off the petri dish and su5pended in phOSphate
buffer saline, pH 7.“, at a concentration of approximately 2xl07 cells
per ml. They were placed on ice and sonicated for 30 seconds with the
small probe of a Biosonic oscillator at 70% of maximum intensity.

This material is referred to as cell reagent. Both the supernatant
and cell reagents were stored at -20°C until just before use.

Agar gel precipitin tests were performed as previously described
(I6, 20) with the following modifications. A 25x75 mm glass slide was
painted with a thin layer of a 0.9% agar containing 0.09% glycerine and
dried rapidly on a warm (70-900) surface. A second slide was placed
above the coated slide and separated from it by 2 layers of electrical
tape and agar (0.9%, l%.or 2%.in phOSphate buffered 8% NaCl solution)
was poured between them. After the agar had solidified, the uncoated slide
was removed and a plexiglas template (Bolab Incorporated, Reading, Mass.)
with a flat polished lower surface lightly coated with silicone
grease was placed on the agar. The holes in the template were then
filled with 50 um antigen or serum. Incubation was at room temperature
for 72 hours.

Sera used in both the indirect fluorescent antibody and agar gel
precipitin tests were from the Survivors of the pathogenicity tests.

The hyperimmune sera were the same as those used previously (l7).

RESULTS

Filtration and cloning of viruses. 0f the 50 attempts to
filter the viruses, only l8 (36%) were successful (Table l). The
JMHP and HVT were filtrable in h of 5 (80%) attempts whereas the low
passage JM, RPL39 and GA strains yielded filtrable virus in lh of #5
(32%) attempts. There was less virus (l.3-lh.7PFU/ml) produced by the
low passage strains than by the JMHP (56.5PFU/ml) and HVT (250PFu/ml),
0f the 8 filtration experiments performed with MDV which had been
passed between A and lo times in cell culture none were successful.
However, Sporadic successes were obtained with viruses between the llth
and l8th passages.

None of the uninfected control cells had cytOpathic changes or
yielded filtrable virus.

Morphology of plaques in CK cells. The appearance of plaques
produced in CK cultures by all the clones except JMHP and HVT was
similar to that previously described (5, 6, 7, 26). Infected cells
became rounded and highly refractile and were sometimes multilayered.
In each plaque the number of Spherical refractile cells increased with
time so that it resembled a "bunch of grapes”. However, the plaques

rarely exceed l mm in diameter even in cultures lh days postinoculation.

to

The plaques produced by JMHP were similar to those described
(7, l3). They appeared l to 2 days earlier, were larger than those
produced by low passage virus, and contained larger spheroidal,
highly refractile syncytia. Cells lysed and/or became detached so
that plaques developed a hole in the center. This occurred more
frequently with plaques induced by JMHP than with those induced by
low passage virus.

Plaques produced by HVT could be detected as early as 2 to 3
days after inoculation (25). After l0 days, the plaques were l.5 to
2 mm and occasionally 3 mm in diameter. In their earlier stages of
development, plaques consisted of a few polygonal refractile cells.
Later the cells in the center were lysed and cells to the periphery
became more rounded and refractile. Syncytia spread out over the
surface of the petri dish and were flattened. The syncytia and the
rounded cells were less refractile than those of the JMHP or low
passage viruses.

Pathogenicity of cloned viruses.

In order to compare the pathogenicity of different clones from
a single strain of MDV, the 7 clones from the JM strain were selected.
In addition, single clones from the JMHP, GA, RPL39 and HVT strains
were also used. The different clones derived from the JM strain killed
80% (JM l9) to 0% (JMHP) of the chickens and induced from 90% to 0%
lesions respectively (Table 2). The clones of the GA and RPL39 strains

produced an intermediate level of mortality and proportion of lesions.

However, these strains produced many more visceral lesions than any
of the clones of JM virus. In addition to tumors of the gonad,

over half the tumors produced by the GA and RPL39 strains occurred
in the liver, lung, kidney, heart and muscle, whereas those produced
by the clones by the JM strain occurred almost exclusively in the
gonad. The median latent period to death was not directly related
to the degree of mortality or gross or microsc0pic lesions. The

HVT was nonpathogenic in these tests.

Antigenic analysis by the agar_gel precipitin test.

Antigen preparations were the supernatant and the cell reagents
from each clone and from uninfected cells, calf serum and tryptose
phosphate broth. They were tested in the agar gel precipitin test
against sera from two birds inoculated with each clone, against
selected hyperimmune sera and against sera from uninoculated control
birds of the same source and age. Reagents from uninfected cells,
calf serum and tryptose phosphate broth did not react with any of the
sera and the sera from uninoculated birds did not react with any
antigen. clone reagents tested against positive sera produced as
many as 6 different precipitin lines. The strongest line of
precipitation produced by most supernatant reagents represented an

t £1 (7) as antigen A. This

antigen referred to by Churchill
antigen was not present in high passage viruses and was also absent
from the JMHP supernatant and cell reagents. In addition this line

of precipitation did not occur with JM30 and JM3I but was present

l2

in HVT reagents. All clones including the HVT appeared to have at
least one other antigen in common, the 8 antigen which was probably
similar to that described by Churchill (7). Although there were
often multiple lines of precipitation in this region no one line
could be consistently identified, so they were referred to as 8
antigen lines to distinguish them from the A antigen lines.

All supernatant reagents produced strong A antigen lines.
However, using selected sera the B and other antigens could also be
detected in most of these reagents. The cell reagents were more
variable. Some (e.g. JHl9 and GA) had A, B and additional antigens
whereas others (e.g. JHBA and JHBS) lacked the A antigen.

In order to confirm and extend the above observations the
following reagents and sera were selected for use in subsequent
tests:

I) The A antigens (supernatant reagents from JHI9 and GA) gave
strong A lines and very weak 8 and other lines.

2) The 8 antigen (JMHP cell reagent) gave no A precipitin line
with any antiserum.

3) The A8 antigens (cell reagents from RPL39, GA and JM3Z)
gave multiple strong precipitin lines.

A) The A antisera (sera from birds infected with JHl9 and JH32)
gave strong A lines when reacted with an A antigen and weak or no

8 lines when reacted with a 8 antigen.

l3

5) The B antisera (sera from birds infected with JM30 and JM3I)
gave no reaction with A antigen, strong reactions with B antigens
and were prepared against clones which lacked the A antigen.

All clones were examined for the presence of the A and B antigens.
RPL39, JMl9, GA, JM32, JM3h had A antigens and JMHP, JM30 and JM3l
lacked these antigens (Figures 1 and 2). Sometimes the A antigen
produced two lines of precipitation both of which were absent from
the clones lacking the A antigen (Figure l). The HVT had one antigen
in common with the other clones but another was absent or differed
(Figures l, 7 and 8). All clones had at least one B antigen in common
(Figures 3 and h). All MDV clones which had A antigen were
serologically indistinguishable.

In order to confirm that JM3O and JM3l lacked the A antigen,
sera from birds inoculated with these clones were examined for antibody
to the A antigen (Figures 5 and 6). None of the serums had antibody
to the A antigen.

The HVT supernatant and cell reagents were examined in more
detail for identity of their antigens with the A and B antigens of
the MDV. In both supernatant and cell preparations, one of the two
A antigens gave a line of identity and the second a reaction of partial
identity as there was a spur on the HVT antigen side (Figures 7 and 8).
The HVT cell reagent (Figure 7) had three antigens at least one of
which was in common with one of the B antigens of MDV. The supernatant
preparation reacted much more weakly with the B antiserum and only

one precipitin line could be identified (Figure 8).

J“

The A antigen appeared to be of lower molecular weight than the
precipitating globulin in the antiserum since the line was concave
towards the serum well. In addition, it formed as equally strong
line in a supporting medium containing 2% or 0.5% agar indicating
that it diffused readily through these matrices. The 8 antigen had
a molecular weight similar to that of the precipitating antibody since
it usually produced a straight line. It reacted more strongly in
0.5% agar than in 2% agar.

The A antigen line was nearly always closer to the serum well
than the other lines.

Antigenic analysis by the indirect fluorescent antibodyAtggt,
Uninfected cells and cells infected with each clone were analyzed
by the indirect fluorescent antibody test With the same sera as were
used above. Uninfected control cells did not become stained with
any serum and control sera did not stain any of the cell preparations.
A good cross reaction was obtained between cells infected with all MDV
clones and sera from chickens which survived inoculation with the
clones. When JM30, JM3l, and JMHP infected cells were reacted with
homologous antisera the rounded, refractile cells in the plaques
stained intensely whereas the surrounding flattened cells adjacent to
them stained very poorly or not at all (Figure 9). When cells infected
with other clones were treated with homologous antisera which stained
antigen intensely, the rounded cells in the plaques were surrounded by
morphologically normal cells whose cytoplasm contained a bright diffuse

or very finely granular stain (Figure l0). The number of flattened

cells staining in this way varied in different plaques and cell preparations

is

because of the different stages of devel0pment of the plaques. The
flattened cells could not be easily identified using sera which

stained the rounded cells less brightly. When MDV-infected cells

were treated with antiserum to HVT, the morphologically altered cells
stained weakly and it was not possible to distinguish between the MDV
clones. When HVT-infected cells were treated with homologous antiserum,
antigen could be demonstrated in both the nucleus and the cytOplasm

in the morphologically altered cells and in a broad band of morpho-
loQically normal cells surrounding them. There was frequently a per-
inuclear ring of brightly stained coarse sphericad particles and/or

a diffuse, very fine granular antigen-containing particles. When similar
cells were reacted with antiserum to any of the MDV clones only the
nuclear antigen stained, and then at a much lower intensity than

with homologous antiserum. When the nuclear staining was particularly
bright, a faint diffuse cytoplasmic antigen could also be detected

in the morphologically altered cells but usually not in the broad

band of cells surrounding them in which the nuclei were very prominent.
Thus, antibody against MDV could be readily distinguished from

antibody against HVT.

l6
DISCUSSION AND CONCLUSIONS

Infectious cell-free virus could be obtained inconsistently
from low passage MDV-infected CEF cultures. Although there were
minor variations in culture conditions, in stage of development of
cytOpathologic alterations at the time of harvest and in the method
of harvest, none of them could be directly linked to the success or
failure of a particular filtration experiment. It appeared to be
necessary to have optimal conditions so that there was a maximum
yield of infectious virus to offset the inefficiency of the
extraction and filtration procedures. As reported previously (l3, 25)
the JMHP and HVT generally yielded virus in larger amounts than the
low passage strains.

Extracted virus was filtered through a 0.h5 um filter in order
to remove clumps of virus and to increase the probability that each
cytOpathologic area would develop from a single infectious particle.
The average pore diameter was only slightly greater than the diameter
of an enveloped virion. MDV is entirely cell associated (6), thus
contamination of a clone by released virus is highly unlikely though
there is a danger from floating infected cells. To reduce the danger
clones of low passage MDV were selected as soon after infection as
possible from plates containing ll plaques or less. The cloning
procedure was not repeated because additional passages of MDV in cell
culture would have further modified the virus clones. The clones

prepared from JM virus were different from one another indicating that

17
a selection had taken place. Thus it is likely that in most instances
the procedure was sufficiently exact to ensure isolation of the
progeny of a single infectious particle.

The cloning procedure for JMHP and HVT were less rigorous
since clones were selected from plates with a larger number of plaques.
However conclusions derived from the comparison of these viruses with
the low passage MDV are not as dependent on success of the cloning

procedure.

18

Some of the characteristics of the original strains were present
in the cloned preparations. Thus, the RPL39 and GA clones produced
a large prOportion and wide distribution of visceral tumors
characteristic of these strains whereas the pathogenic JM clones
produced a low incidence of visceral tumors almost exclusively of the
gonad. The JMHP and HVT clones remained non-pathOgenic.

The pathogenicity of different clones of the JM strain of MDV
varied. The most pathogenic was JMl9 which induced lesions in 90%
of the birds and the least pathogenic was the JMHP which did not
induce any lesions. There are two possible explanations for this
variation, namely that the original stock of JM virus was a mixture
of many genetically different viruses which varied in pathogenicity
or that a change of pathogenicity occurred during passage of the virus.
Strains that vary widely in pathogenicity have been observed (l9),
and it is quite possible that unpurified stocks would contain more
than one virus. The variable responses obtained with different
unpurified stocks of the same strain and the variation in pathogenicity
with passage from chicken to chicken which has been well documented
(3) could be explained by an alteration in the relative amounts of
the more pathogenic and less pathogenic strains in a given stock.
However, loss of pathogenicity may also occur during passage in cell
culture. The contribution of cell culture passage to the lack of

pathogenicity of some of the clones can not be estimated.

'9

The viruses studied could be divided into three groups on the
basis of their antigenic properties. Firstly, there are those with
the A and other antigens namely JMl9, JM32, JM3h, JM35, JM36
RPL39 and GA. These clones were serologically indistinguishable.
Secondly, there are those which lacked the A antigen such as clones
JM30, JMBI, and JMHP. It is not known whether viruses of this group
occur naturally or whether they are products of cell culture passage.
Thirdly, HVT has an antigen in common with at least one component
of the A antigen of the first group of MDV's. Because of the close
proximity of the precipitin lines produced by the two components of
the A antigen they often appeared as a single line. The HVT had at
least one 8 component in common with the MDV. It is possible that
reagents could be developed which would distinguish between HVT and
the other viruses in the agar gel precipitin tests.

JMHP produced cytopathologic changes in a shorter time than the
low-passage clones of MDV, and the plaques were of different morhpology.
It is unlikely that virus similar to JMHP could have been present
in the original stock since it would easily have been recOQnized and
since none of the low-passage clones produced plaques of this type.
Thus the change in morphologic appearance and rate of growth of the

plaques can be attributed to passage in cell culture.

20

Loss of pathogenicity during passage in cell culture or innate
lack of pathogenicity are unrelated to the presence or absence of the
A antigen. Thus relatively pathogenic clones with (JMl9) and without
(JM3l) the A antigen and relatively non-pathogenic clones of the same
strain with (JM32) and without (JMHP) the A antigens have been
isolated. Similarly the change in appearance of the plaques and
increase in rate of growth characteristic of JMHP are not related to
loss of the A antigen since JM30 and JM3l were morphologically
indistinguishable from the other low passage clones and yet lacked
the A antigen. Thus the above observations indicate that although
passage in cell culture may be responsible for loss of pathogenicity,
loss of the A antigen and change in virus-host cell relationship,
these three events are independent of one another. Also, only in
the case of the change in virus-host cell relationship can the
alteration be attributed entirely to passage in cell culture.

Differences between viruses with and without the A antigen could
also be observed using the indirect fluorescent antibody test.
However, it was not possible to differentiate between these viruses
consistently because of variations in the stages of development of
the plaques in different preparationsand in the amount of antibody
in the sera. In plaques produced by clones with the A antigen, a
diffuse antigen in the cytoplasm of infected morphologically normal

cells surrounding the plaques stained with homologous antiserum.

21
Cells in close proximity to plaques induced by viruses without the
A antigen did not stain with their homologous antisera and in these
plaques only the rounded refractile cells stained. Since the virus
infection spreads centrifugally by cell to cell contact, the cells
around the periphery of a plaque would be the most recently infected.
Therefore, it appears that the A antigen is produced earlier in the
cycle of infection than the other antigens, an observation confirmed
by sequential harvesting of antigen for the agar gel precipitin test
(Okazaki and Purchase, unpublished observation).

As has been reported elsewhere (IS, 25) HVT can be distinguished
from MDV by the intensity and distribution of staining antigen in
HVT and MDV infected cells. The pattern of staining was consistent
for the sera prepared against all clones of MDV studied. A similar
difference in the appearance of nuclear and cytoplasmic antigen of
Herpesvirus hominis types I and 2-infected cells stained in the direct
fluorescent antibody test was reported by Nahmias gtugl (ll). They

could use the test to distinguish between the two strains of virus.

iz

Figure

Figure

Figure

Figure

Figure

and

The A antigen of different clones of MDV and HVT. The
center well contains the A antiserum (against JH32) and
wells l to 6 supernatant reagents from RPL39, JHIS, GA,
JMBI, 4:419, HVT. (1% Agar).

The A antigen of different clones of MDV. The center
well contains the A antiserum (against JH32) and wells

1 to 6 supernatant reagents from JMlS, JH30, JM32,

JHBA, JMBS. JM36. (l%.Agar)

The 8 antigen of different clones of MDV and HVT. The
center well contains the B antiserum (against JHBO) and
wells l to 6 cell reagents from RPL39, JMl9, GA, JH3l,
JHl9, HVT. (0.5%,Agar).

The 8 antigen of different clones of MDV and HVT. The
center well contains the B antiserum (against JMBO) and
wells l to 6 cell reagents from RPL39, JMHP, HVT, GA,
JH32, HVT. (0.5% Agar).

The absence of anti-A antibody among birds inoculated
with JM30 and JM3l. The center well contains the AB
antigen (RPL 39 cell reagent), wells l, 3, and 5 A anti-
serum (against JHBZ) and wells 2, h, and 6 sera from birds

inoculated with JM30 (Figure 5) and JH3l (Figure 6).

23
Figure 7

and 8.

Figure 9.

Figure l0.

Relationship between HVT antigens and MDV antigens. The
center well contains HVT cell reagent (Figure 7) and HVT
supernatant reagent (Figure 8), well I A antigen (GA
supernatant reagent), well 2 A antiserum (against JNl9),
well 3 B antiserum (against JH30), well A 8 antigen
(JMHP cell reagent) well 5 B antiserum from a different
bird than well 3 (against JH30) and well 6 A antiserum
(against JM32). (0.5%.Agar).

Antigen prepared from clone JH30 reacted in the indirect
fluorescent antibody test with homologous antiserum.
Note only the rounded cells stain. x300.

Antigen prepared from clone JM35 reacted in the indirect
fluorescent antibody test with serum against RPL39. Note
the cells of normal morphology surrounding the rounded
refractile cells have a diffuse of finely granular

cytoplasmic antigen. X300.

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REFERENCES

Benda, R. I966. Attempt to differentiate 8 virus from herpes simplex
virus by the fluorescent antibody technique. Acta Virol.
l0: 3&8-353.

Benda, R. and J. Cinatl. I966. Isolation of two plaque variants
from the prototype strain of B virus (herpes simiae). Acta
Virol. l0: I78.

Biggs, P. H. and L. N. Payne. I967. Studies on Marek's disease
I. Experimental transmission. J. Nat. Cancer Inst. 39: 237-280.

Chubb, R. C. and A. E. Churchill. l968. Precipitating antibodies
associated with Marek's disease. Vet. Record 83: h-7.

Churchill, A. E. l968. Herpes-type virus isolated in cell culture
from tumors of chickens with Marek's disease. I. Studies in
cell culture. J. Natl. Cancer Inst. kl: 939-950.

Churchill, A. E. and P. H. Biggs. I967. Agent of Marek's disease in
tissue culture. Nature 2l5: 528-530.

Churchill, A. E., R. E. Chubb and W. Baxendale. l969. The attenuation,
with loss of oncogenicity, of the herpes-type virus of Marek's
disease (strain HPRS-l6) on passage in cell culture. J. Gen.
Virology h: 557-56H.

Crittenden, L. B. 1968. Avian tumor viruses: Prospects for control.

World“s Poultry Science Journal 2“: l8-36.

ll.

12.

I3.

Hoggan, M. D. and B. Roizman. I959. The isolation and properties of
a variant of herpes simplex producing multinucleated giant cells
in monolayer cultures in the presence of antibody. Amer. J. Hyg.
70:208-2l9.

Kawamura, H., D. J. King and D. P. Anderson. l969. A herpesvirus
isolated from kidney cell culture of normal turkeys. Avian
Diseases l3:853-863.

Nahmias, A. J., W. T. Chiang, 1. Del Buono and A. Duffey. l969.
Typing of herpesvirus hominis strains by a direct immunofluorescent
technique. Proc. Soc. Expt. Biol. Med. l32:386-390.

Nahmias, A. J., W. R. Dowdle, Z. M. Naib, A. Highsmith, R. W. Harwell,
and W. E. Josey. l968. Relation of pock size on chorioallantoic
membrane to antigenic type of herpesvirus hominis. Proc. Soc.
Exptl. Biol. Med. l27:1022-l028.

Nazerian, K. I970. Attenuation of Marek's disease virus and study of
its properties in two different cell cultures. J. Natl. Cancer Inst.
(In press).

Nii, 5., J. KamahOra. I963. Pathological changes induced by herpes
simplex virus in the chorioallantoic membranes of hatching eggs.
Biken Journal 6:205-2hl.

Okazaki, W., H. G. Purchase and B. R. Burmester. 1970. Protection
against Marek's disease by vaccination with a herpesvirus of turkeys

(HVT). Avian Diseases lhlzhl3-h29.

l6.

l7.

l8.

20.

2|.

22.

23.

2h.

Okazaki, W., H. G. Purchase and L. Noll. I970. Effect of different
conditions on precipitation in agar between Marek's disease
antigen and antibody. Avian Diseases (In press).

Purchase, H. G. l969. Immunofluorescence in the study of Marek's
disease. 1. Detection of antigen in cell culture and an antigenic
comparison of eight isolates. J. Virology 3: 557-563.

Purchase, H. G. I970. Virus-specific immunofluorescent and precipitin
antigen and cell-free virus in the tissues of birds infected with
Marek's disease. Cancer Research (In press).

Purchase, H. G. and P. H. Biggs. I967. Characterization of five
isolates of Marek's disease. Res. Vet. Sci. 8: th-th.

Purchase, H. G. and G. H. Burgoyne. I970. Immunofluorescence in
the study of Marek's disease: Detection of antibody. Am. J.
Vet. Res. 31: ll7-l23.

Roizman. B. and L. Aurelian. I965. Abortive infection of canine cells
by herpes simplex virus I. Characterization of viral progeny from
co-operative infection with mutants differing in capacity to
multiply in canine cells. J. Mol. Biol. ll: 528-558.

Spencer, J. L. and B. W. Calnek. I970. Marek's disease: Application
of immunofluorescence for detection of antigen and antibody.

Am. J. Vet. Res. 3|: 345-358.

Svobodova-Somogyiova, J. l968. Pseudorabies virus with lost virulence
for rabbits after intramuscular inoculation, obtained from
persistently infected cultures. Acta Virol. l2: 285.

Ver, 8. A., J. L. Melnick and C. Wallis. l968. Efficient filtration
and sizing of viruses with membrane filters. J. Virology

2: 21-25.

25. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne. I970.
Isolation from turkeys of a cell-associated herpesvirus
antigenically related to Marek's disease virus. Am. J. Vet. Res.
31:525'533.

26. Witter, R. L., J. J. Solomon and G. H. Burgoyne. l969. Cell culture
techniques for primary isolation of Marek's disease-associated

herpesvirus. Avian Disease l3:l0l-ll8.

 

 

 

Article V

RESPONSES OF CELL CULTURES FROM VARIOUS AVIAN

SPECIES TO MAREK'S DISEASE VIRUS AND THE

HERPESVIRUS OF TURKEYS

BY

H. G. Purchase, B. R. Burmester and C. H. Cunningham

Submitted to the American Journal of Veterinary Research.

94

RESPONSES OF CELL CULTURES FROM VARIOUS
AVIAN SPECIES TO MAREK'S DISEASE
VIRUS AND THE HERPESVIRUS OF TURKEYS

H. G. Purchase, 8. R. Burmester, and C. H. Cunningham

Received for publication

From the Poultry Research Branch, Animal Science Research
Division, ARS Regional Poultry Research Laboratory, East
Lansing, Michigan #8823

And the Department of Microbiology, Michigan State University,
East Lansing, Michigan #8823

Journal Article No. EZAA of the Michigan Agricultural Experiment
Station.

Portion of a Ph.D thesis submitted by the senior author to
Michigan State University

SUMMARY

Cells were cultured from chicken, duck, bobwhite, and Japanese quail,
turkey, pheasant, pigeon, and goose embryos. These avian cell cultures,
HeLa cells and the chorioallantoic membranes of chicken embryos were tested
for susceptibility to cloned preparations of Marek's disease virus recently
isolated in cell culture (LMD), to N0 virus passed many times in cell culture
(HMD) and to the herpesvirus of turkeys (HVT). Avian cells were susceptible
to all of the viruses but the cultures varied considerably in degree of
susceptibility and in cytOpathological response. Morphological changes
produced by LMD, HMD, and HVT were characteristic for each virus but could
best be differentiated in chick kidney cultures. Chick embryo fibroblasts
were much less susceptible than duck embryo fibroblasts to LMD, equally
susceptible to HMD and much more susceptible to HVT. HeLa cells were
not susceptible to any of the viruses. Cytoplasmic virus-induced antigen
was detected by the indirect fluorescent antibody test in HVT-infected cells
of all types with HVT antiserum but not with LMD or HMD antiserum. Nuclear

antigens fluoresced with all antisera.

INTRODUCTION

Marek's disease (MD) viruses which range in pathogenicity from
”acute” to mild or "classical" have been isolated from various sources
in different lines of susceptible chickens and in cell cultures (6,l2,l3,
2l,22,2h). Chick kidney (CK) (l,h,6,12,20,24) and duck embryo fibroblast
(DEF) (2l,2h) cultures are most commonly used for primary isolation and
passage of MD viruses although isolation and propagation in chick embryo
fibroblasts (CEF) has been reported (l3,lh). The MD virus grew in pheasant
cell cultures and was re-isolated from ducks after experimental inocula-
tion (3). Antibody could not be demonstrated in pigeons (Columba palambus),
starlings (Stuonus vulgaris), Sparrows (EEEEEL domesticus), yellow hammer
(Emberoza citorinella), pheasants (Phasianus colchicus) and jackdaws
(Corvus monedula) (3). The MD virus did not propagate in cultures of
mammalian origin (5). The herpesvirus of turkeys (HVT) produced plaques
in DEF, CEF, CK, turkey embryo fibroblasts (TEF) and turkey kidney cell
cultures (TK) (23).

The studies described herein were initiated to determine whether
cell cultures derived from various avian species were susceptible to MD
virus and if changes produced could be used as markers to differentiate between
viruses, particularly those differing in pathogenicity. It appeared important
to be able to differentiate between the pathogenic MD viruses (l8), the non-
pathogenic MD virus which had been serially passed many times in cell culture
(HMD) (l5) and HVT since the last two could be used as vaccines to prevent MD
(7,8,l6,l9). Previously cloned MD viruses and HVT, which had been shown to

differ greatly in pathogenicity, were employed (l8).

MATERIALS AND METHODS
Source of viruses.

The origin of 7 cloned preparations of JM, h of RPL 39, and 7 of GA
viruses collectively referred to as low passage MD (LMD) have been described
(18). They had been passaged l0-l7 times during cloning. Three cloned
preparations of a strain of JM virus which had been passed over #0 times
in cell culture prior to cloning (54 passages in all) and h of the HVT
strain FCl26 (lS,l8,23) were also employed. Viruses were propagated in DEF
and CEF and virus-infected DEF stocks containing IOZ dimethyl sulfoxide and
l5% calf serum were stored in liquid nitrogen (ll).

The cloned viruses, in order of decreasing pathogenicity, were JMl9,
GA, JHBI, JM30, JMBS, RPL39, JHBQ, JM36, and JM32. The HMO and HVT were non-
pathogenic (l8). Two preparations of JM virus (JM30 and JM3l) and the HMD
lacked antigens identified as “A” antigens whereas all other viruses produced
these antigens (l8). The antigens and pathogenicity of 3 cloned preparations
of RPL39, 6 of GA and A of HVT were not tested.
Chickens and embryos

Line l5 x 7 chickens and line I900a and line 6 embryos were from the

Single Comb White Leghorn flocks (Gallus gallus vardomesticus) reared at the

 

Regional Poultry Research Laboratory (9). Embryonated eggs used for cell
culture were obtained as follows: bobwhite (Colinus virginianus) and Japanese
quail (Coturnix coturnix japonica), turkey (Mgleagris gallopavo), pheasant
(Phaseanus colchicus) and duck (Anas platyrhynos ggg_domesticus) eggs were
from Truslow Farms Inc., Chestertown, Maryland, and pigeon (Columba lixig)'
eggs were from Dr. A. M. Lucas, U.S.D.A, Avian Anatomy Project, East Lansing,

Michigan.

 

a Stone, H. A., unpublished data l970.

Cell cultures.

The preparation and propagation of CEF, DEF, and CK cultures has
been described (2h). Bobwhite quail (BQEF), Japanese quail (JQEF),
turkey (TEF), pheasant (PhEF), and pigeon (PiEF) embryo fibroblast
cultures were prepared from embryos at the midpoint of their incubation
period with a procedure similar to that used for DEFs. Duck kidney
(DK) cultures were prepared from 2 to A week old ducks in a similar manner
to CK cultures.

The established line of goose (Anser anser var domesticus) embryo
fibroblasts (GEF) was obtained from Dr. L. B. Crittenden, ARS, Beltsville,
Maryland and was received frozen. Immediately prior to use an ampule was
thawed rapidly, and propagated in a similar manner to CEFs except that it
was passaged at 7 to lh day intervals. HeLa cells obtained from Microbiological
Associates, Inc., Bethesda, Maryland were cultured in medium Fl0 and I99 with
h% calf serum (2h) and were passaged at 3 to A day intervals.

Primary fibroblasts were plated at l x l07 cells on each l60mm plastic
petri dishb, Assays were performed on secondary cultures seeded with l x l06
cells (5 x l0S for GEF) in 60mm plastic dishes. Primary kidney cultures,
seeded at 5 to 8 x l06 cells per 60mm plastic petri dish, were used for all
assays.

Susceptibility of cells to clones of virus.

The susceptibility of CEFs and DEFs was tested with all viruses, however,
the susceptibility of all other cell types was screened using the 7 cloned
preparations of JM and only one each of GA, RPL 39, HMD and HVT. The culture
medium from plates with a confluent monolayer was removed and 0.5m] of each

tenfold dilution of inoculum was added to each culture. After incubation at

 

b Falcon Plastics, Oxnard, California

h

37°C for one hour the cultures were washed with phosphate buffered saline
(PBS) pH7.h and fresh medium was added. The culture medium was changed
every other day thereafter. Cultures were examined daily and cytopathic
areas were counted when they were easily visible but before the cell mono-
layers began to degenerate.

CytOpathic changes were examined in greater detail in preparations stained
with hematoxylin and eosin (2) and the presence of virus-induced antigen was
demonstrated by the indirect fluorescent antibody test (17). Cultures on
ll x 22mm glass coverslips in plastic petri dishes were infected with
tenfold dilutions of virus. Coverslips with clearly visible discrete
cytopathic areas were removed from the petri dishes. One set of coverslips
was fixed in Zenkers acetic acid and stained with hematoxylin and eosin (2)
and another set was rinsed in PBS, fixed in acetone, and stained by the indirect
fluorescent antibody technique with antisera specific for some of the cloned
viruses (l7,l8).

Chorioallantoic membrane inoculation

Embryos from line 6 were inoculated with 0.2ml of tenfold dilutions of
inoculum on the l0th day of incubation using the false air cell technique (l0).
Embryos were killed on the l8th day by placing them in a refrigerator for 2 to

l8 hours after which time the chorioallantoic membranes were removed and examined.

RESULTS
Morphology of uninfected cells.

Cells from decapitated embryos were morphOIOgically fibroblastic and
so are referred to as embryo fibroblasts. The DEF were broad cells (Figs.
7a,b,and c), the BOEF, JQEF, and PhEF were intermediate and CEF (Figs. 2a,
b,and c), PiEF and GEF (Fig. 3a) were long and thin. The PiEF monolayers
contained many large vacuolated syncytia (Fig. 9a). The GEF cell line grew
better in sparse cultures and as they became confluent the cells overlapped
one another haphazardly (Fig. 3a and b).

The DK and CK cells were epithelioid in nature. The DK cells formed
an even monolayer of flattened polygonal cells (Fig. ha) whereas the CK
cells formed islands of flattened polygonal epithelioid cells separated by
cords of fibroblastic cells (Figs. 5a, b,and c). The HeLa cells were
characteristically epithelioid.

Cytopathic changes produced by different viruses.

Observations were made of unstained preparations and cells stained with
haematoxylin and eosin and the direct fluorescent antibody procedure.

In general, cytopathic areas produced by JMl9, JM30, JM3l, and JM36 were
more easily recognized than those produced by other LMD viruses but could not be
individually differentiated so they are described together (Table I). Altered
areas were small, developed slowly and contained few small syncytia and rarely
was there a hole in the center of them. Cells making up the areas were clustered
close together and tended to pile up.

Cytopathic areas produced by HMD and HVT appeared early and developed
rapidly and at times looked similar (Figs 2b and c). HMD produced larger, more
rounded and refractile syncytia than HVT (Figs 5b,6b,7b,8b). As reported (l5)
holes in the center of cytopathic areas seemed to develop largely as a result

of detachment from the monolayer. The syncytia produced by HVT were angular or

stellate and vacuolated and holes in the center of cytOpathic areas looked
as if they had formed by lysis of the cells (Figs 5c,6c,7c).

EosinOphilic Cowdrey type A intranuclear inclusion bodies were in plaques
produced by all clones of virus in all cell types (Figs. la,b, and c).
Inclusion bodies were never found in cells not involved in a cytopathic area.
Cytopathic changes in different cellgtypes.

Cell types are described in increasing order of their tendency to develop
syncytia (Table I). Changes in embryo fibroblasts, i.e., BQEF, CEF, GEF,
JQEF, PhEF, and TEF consisted mainly of small, spherical, highly refractile
cells which tended to become multilayered and some detached from the mono-
layer (Figs 2a,b,c,3a,and b). Interspersed polygonal and fusiform cells
were not obvious. Cytopathic areas on BQEF and JQEF tended to have fatter
fusiform cells and resembled those in DEF.

In DK cells all viruses produced cytOpathic areas with clearly defined,
biconcave edges (Figs ha and b). The diametrically opposite edges merged
gradually and irregularly into the surrounding monolayer and often contained
fusiform refractile cells. There were occasional small syncytia and holes in
the center of cytOpathic areas, particularly in those produced by HVT (Fig lb).

In CK cells cytopathic areas consisted mostly of rounded cells and small
syncytia (5a,b,c,6a,b,and c). The LMD produced groups of tightly packed very
refractile small spherical cells which formed a small grape-like cluster
(Figs. 5a and 6a). In cells infected with HMD there were thick, large,
stellate and round refractile syncytia around which were grouped fusiform and
refractile cells (Figs. 5b and 6b). Cells had little tendency to pile up and
a hole frequently developed in the center of the cytopathic area. The HVT
produced thin syncytia with many vacuoles and some rounded or polygonal cells,
however all affected cells had less of a tendency to become spherical and

refractile than those infected with the other viruses (Figs. 5c and 6c).

Holes invariably developed in the center of the cytopathic areas, probably
more due to lysis than detachment of cells. The monolayer around the holes
retracted and was thicker than on the remainder of the petri dish (Fig. 6c).
The cells immediately surrounding the holes were generally polygonal and vacuo-
lated.

Alterations in DEF cultures took longer to develop but were larger in
diameter than those in other embryo fibroblast or CK cells. They consisted
of many fusiform refractile cells which piled up. There were few small
syncytia in cytopathic areas produced by LMD (Fig 7a and 8a). However, HMD
produced characteristic flat brown almost hemispherical syncytia with the
numerous nuclei arranged in a circle (Fig. 7b and 8b) and which frequently
detached to form large black Spheres (Fig 8b). Usually HVT produced large
stellate syncytia which were thinner in the early stages than those produced
by HMD (Fig. 7c and 8c). They were often attached to the petri dish and to
one another by long pseudopods (Fig 7c).

In PiEF, LMD did not produce cytopathic effects however occasional
immunofluorescent antigen was detected in some cells. HMD produced rare large
flat syncytia which could be distinguished from those in uninfected cells
because they contained very few vacuoles and had intra-nuclear inclusion bodies
in stained preparations (Fig. la). HVT produced enormous thin syncytia with
very few vacuoles occupying large areas of the cell culture (Figs. 9b and 9c).
They frequently had thick central areas near where detachment and retraction
of pseudopods from the petri dish had occurred.

Comparative sensitivity of cells of different types and CAMS to clones of
2.11.9.2-

The susceptibility of all other cells was related to the susceptibility of

DEF since the stocks of virus were prepared in DEFS. The relative ability
of a virus to produce cytopathic effects in CEF and DEF is expressed as the
ratio of the titer of the virus on CEF to the titer on DEF (Table 2). The
viruses fall into 2 groups, namely, LMD viruses such as JM, RPL 39 and GA
which gave ratios from 0.08 to 0.lh and HMO and HVT which gave ratios of
3.0 and 2l.0 (See also Fig l2)

In a search for biological markers which would differentiate between the
viruses, the susceptibility of different cell types to the MD viruses was
screened in one or two experiments with each cell type and with CAMS. The
viruses did not differ greatly from one another in their ability to produce
cytopathic effects on each of the cell types or on CAMS so the data for
these viruses have been pooled (Fig l2). Except in a few instances, titers on
different cell types did not vary more than 20 fold. In particular the MD
viruses produced very few cytopathic areas in PiEF or PhEF and the HVT was
impotent in DK cells. However in each case these cells were not more susceptible
than DEF to the other viruses. The susceptibility of CEF to HVT has been reported
above.

Immunofluorescent antigensin infected cultures

Cultures infected with LMD and HMO viruses were treated with antiserums
prepared against these viruses. An area resembling an inclusion body,
excluding the nucleolus, stained in the nucleus and fine diffuse and coarse
granular particles stained in the cytoplasm of cells involved in the cyto-
pathic areas (Fig. 10a). Sometimes staining was observed in cells of normal
appearance surrounding the cytopathic areas.

Staining was observed in both the nucleus and the cytoplasm of cells infected
with HVT and treated with homologous antiserum (Figs. l0c and llc). However,

when such cells were treated with serum against LMD or HMD the nuclei stained

much more intensely than the cytoplasm (Figs. l0b and llb) and the

cytoplasm stained diffusely and did not contain the large number of brightly
staining coarse granules seen with homologous antiserum. As reported
previously (l9,23), the difference in staining between antibody to MD
viruses and antibody to HVT was most outstanding in HVT-infected CK cells.

HeLa cell cultures were the only cells tested which were not susceptible
to any of the viruses used. Inoculated cultures contained scattered areas of
fluorescent debris (Fig lla) which were considered to be remnants of degenerating
cells from the inoculum.

If significant amounts of cell-free virus had been produced by some of
the viruses in particular cell types, secondary small foci of infection should
have been seen. Areas which were not cytopathically involved but contained
antigen were not observed.

Uninfected cells did not stain with any anti-serum or negative sera
except DEFs which, as previously described (l7), contained easily recognisable

granules which stained with both positive and negative sera.

DISCUSSION

When using a cellular inoculum to infect test cells of different types,
it is possible that the inoculum cells themselves would proliferate and
produce cytopathic areas. The presence of cytOpathic areas would be
interpreted as susceptibility of the test cells. In the studies reported
here. growth and morphological change of the inoculum cells were not
responsible for the cytopathic areas observed for the following reasons. Very
few cells from the inoculum grew without a feeder layer and those that did soon
became cytopathically involved, detached and died. Thus once cells were infected
they either did not divide or divided so infrequently that they were unable to
maintain themselves. HeLa cells are not susceptible to MD virus infection (5).
When HeLa cells were inoculated with a large dose of a cellular inoculum and
stained by indirect fluorescent antibody procedure only cellular debris stained
(Fig Ila). This debris was probably the remnants of the inoculum cells which
died rather than forming cytOpathic areas. In Susceptible cells the appearance
of the cytopathic areas was characteristic of the recipient culture and not of the
culture in which the virus stocks were prepared, in this case DEF.

The titer of each clone of virus when assayed on a particular cell
type was a measure of the relative susceptibility of the cell type to the virus.
The titer depended not only on the transfer of infectivity from the inoculum
to the recipient cells but also on whether the recipient cells became altered
to form clearly distinguishable plaques. Whether recipient cells became altered
may have been affected by a large number of factors other than the source species
of the cell. For example, extraneous infectious agents could enhance or inhibit
the herpesvirus infection or its cytOpathic effect. Only the CEF were known
to have come from birds extensively tested for and free from known extraneous

viruses. There was a large amount of vacuolization and syncytial formation in

the PiEF which could have been due to an infectious agent in these cultures
which, in turn, could have inhibited the herpes virus infection. Other
factors might act synergistically. Among them are the physiological condition
of the cells, the different media in which they were cultured and the time at
which they were infected or examined. 0n the other hand, extraneous agents
could not have been solely responsible for the cytopathic effects since specific
virus-induced antigens and intranuclear inclusions were present in the cyto-
pathic areas in all cell types. The characteristic cytOpathic areas did not
occur in uninfected cultures.

The approximately twenty fold increase in susceptibility of CEF to HVT
was demonstrated several times (Table 2 and Fig. l) with similar results.
Thus CEF had an advantage in sensitivity for detecting this virus. However
it was much more difficult to distinguish between viruses by morphology of
the cytOpathic areas than when using DEF or CK cultures. Also CEF cultures
were not suitable for primary isolation of LMD and in agreement with
previous results (15) , this virus will only grow in CEFS after several passages
in CK or DEF cultures. CEFs are less susceptible to virus passed ll to l7 times in
cell culture (LMD) than to virus passed 5h times (HMD) or to HVT. A brief
examination of the susceptibility of other cell types did not reveal any that
would be significantly more sensitive to the MD viruses than DEFS or CKS. Also
there was no indication that there were cell types other than CEFs in which
adequate sensitivity to one virus and resistance to another could be used as a
biological marker to differentiate between viruses. Thus none of the cell types
examined offered any advantages over CK cultures for distinguishing between
LMD, HMD, and HVT. However when working exclusively with HVT and possibly also

HMD, CEF cultures were more sensitive.

Since the cultures were maintained under liquid medium cell-free virus
would have produced secondary foci of infection by the time the originally
infected cells had become morphologically altered. The absence of secondary
foci in monolayers stained by the indirect fluorescent antibody technique
until after infected cells floated free from the cytopathic areas (h-S days)
was consistent with the hypothesis that none of these cultures released
large amounts of infectious virus into the supernatant cultural medium.

This is in agreement with the previous observations that the viruses are
highly cell-associated (6,7,23).

Using the procedures described it was possible to demonstrate that
all avian cells tested and CAMS of chicken embryos were susceptible to
the herpesviruses used, however, HeLa cells were not susceptible to these
viruses. From this and similar work by Calnek e£_§1.(5) it can be concluded

that most mammalian cells are not susceptible to MD viruses.

REFERENCES

Ahmed, M. and G. Schidlovsky, 1968. Electron microscopic localization
of herpesvirus-type particles in Marek's disease. J. Virol. 2:
lhh3-lh57.

Armed Forces Institute of Pathology, 1960. Manual of Histologic and
Special staining techniques 2nd ed., McGraw-Hill Book Co. Inc.,

New York. N.Y. Helen K. Steward. Ed.

Baxendale, W., l969. Preliminary observations on Marek's disease in
ducks and other avian species. Vet. Rec. 85: 3hl-3h2.

Calnek, B. W., and S. H. Madin, l969. Characteristics of ig_xi££g
infection of chicken kidney cell cultures with a herpesvirus from
Marek's disease. Am. J. Vet. Res., 30: l389-lh02.

Calnek, B. W., S. H. Madin, and A. J. Kniazeff, l969. Susceptibility
of cultured mammalian cells to infection with a herpesvirus from
Marek's disease and T-virus from reticuloendotheliosis of chickens.
Am. J. Vet. Res. 30: l403-lhl2.

Churchill, A. E. and P. M. Biggs, l967. Agent of Marek's disease in
tissue culture.. Nature 2l5: 528-530.

Churchill, A. E., R. C. Chubb, and W. Baxendale, l969. The attenuation,
with loss of oncogenicity, of the herpes-type virus of Marek's
disease (Strain HPRS-l6) on passage in cell culture. J. Gen. Virol.
h: 557-56h.

Churchill, A. E., L. N. Payne, and R. C. Chubb, l969. Immunization against
Marek's disease using a live attenuated virus. Nature 221: 7hh—7h7.

Crittenden, L. 8., I968. Avian tumor viruses: Prospects for control.

World's Poul. Sci. J. 2h. l8-36, l968.

l0.

ll.

12.

I3.

lh.

Cunningham, C. H., 1966. Laboratory guide of virology 6th Ed., Burgess
Publishing Co., l86 pages.

Dougherty, R. M., l962. Use of dimethyl sulfoxide for preservation of
tissue culture cells by freezing. Nature 193: 550-552.

Eidson, C. S., D. J. Richey, and S. C. Schmittle, l969. Studies on acute
Marek's disease. XI. Propagation of the GA isolate of Marek's disease
in tissue culture. Avian Dis. l3: 636-653.

Kottaridis, S. D., R. E. Luginbuhl, T. N. Fredrickson, l968. Marek's
Disease. II. Propagation of the Connecticut-A Isolate in Cell
Cultures. Avian Dis. l2: 246-258.

Nazerian, K., l968. Electron microscopy of a herpesvirus isolated from
Marek's disease in duck and chicken embryo fibroblast cultures.

Proc. Electron Micro. Soc. America. 26th Ann. Meeting, 222-223.

Nazerian, K., I970. Attenuation of Marek's disease virus and study of
its properties in two different cell cultures. J. Nat. Cancer
Inst. #4: l257-l267.

Okazaki, W., H. G. Purchase, and B. R. Burmester, 1970. Protection
against Marek's disease by vaccination with a herpesvirus of turkeys.
Avian Dis. lk: hl3-h29.

Purchase, H. G., l969. Immunofluorescence in the Study of Marek's disease.
I. Detection of antigen in cell culture and an antigenic comparison
of eight isolates. J. Virol. 3: 557-563.

Purchase, H. G., C. H. Cunningham, and B. R. Burmester, I970. Pathogenicity
and antigenicity of clones from different strains of Marek's disease
virus and the herpesvirus of turkeys. J. Infection and Immunity.

In press.

20.

2l.

22.

23.

2h.

Purchase, H. G., R. L. Witter, W. Okazaki, and B. R. Burmester, I970.
Vaccination against Marek's disease. Perspectives in Virol. 7:
In press. Ed. M. Pollard. Academic Press Inc., New York, N.Y.

Sharma, J. M., S. G. Kenzy, and A. Rissberger, l969. Propagation and
behavior in chicken kidney cultures of the agent associated with
classical Marek‘s disease. J. Nat. Cancer Inst., 43: 907-916.

Solomon, J. J., R. L. Witter, K. Nazerian, and B. R. Burmester, l968.
Studies on the Etiology of Marek's disease I. Propagation of the
agent in cell culture. Proc. Soc. Exp. Biol. Med., l27: l73-l77.

Witter, R. L., G. H. Burgoyne, and J. J. Solomon, l969. Evidence for
a Herpesvirus as an etiological agent of Marek's disease. Avian
Dis. l3: l7l-l8h,

Witter, R. L., K. Nazerian, H. G. Purchase, and G. H. Burgoyne, I970.
Isolation from turkeys of a cell-associated herpesvirus antigenically
related to Marek's disease virus. Am. J. Vet. Res., 3l: 525-538.

Witter, R. L., J. J. Solomon, and G. H. Burgoyne, l969. Cell culture
techniques for primary isolation of Marek's disease-associated

Herpesvirus. Avian Dis. l3: l0l-ll8.

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Legends for Fiqures
Fig. l. Cowdrey type A intranuclear inclusion bodies: (a) A single large
syncytium produced by HMD in PiEF. HSE x #80. (b) A syncytium with
vacuoles, a hole in the center of the cytOpathic area and several adjoining
cells with inclusion bodies in HVT infected DK cells. H8E x #80. (c) A
syncytium and individual cells with inclusion bodies in GEF infected with
HVT. HeE x 525.
Fig. 2. Cytopathic areas in CEF induced by: (a) LMD, (b) HMD and (c) HVT.
Viewed unstained with an inverted microscope cytopathic areas consist mainly
of spherical, highly refractile cells which are sometimes multilayered. x30.
Fig. 3. Cytopathic area induced by HMD in GEF: (a) Unstained x30. (b) Two
syncytia beside the cytopathic area consisting of dark fusiform and spherical
cells overlapping in a haphazard fashion. NEE x l20.
Fig. #. Cytopathic areas in DK cells produced by HMD: (a) Two unstained areas
with characteristic biconcave appearance. x30. (b) One cytOpathic area stained
with homologous antiserum by indirect fluorescent antibody. x300.
Fig. 5 s 6. Cytopathic areas in CK cells infected with: (a) LMD, (b) HMD and
(c) HVT. For detailed description see text. Fig. 5 Unstained. x30. Fig. 6.
HSE x120.
Fig. 7 a 8. Cytopathic areas in DEFS infected with: (a) LMD, (b) HMD and
(c) HVT. For detailed description see text. Fig. 7, unstained x30. Fig. 8,
HSE x90.
Fig. 9. (a) Uninfected PiEF. A vacuolated syncytium can be seen beside a
hole in the monolayer. NEE x90. (b) PiEF infected with HVT. Enormous
syncytia with very few vacuoles are visible. HSE x90. (c) PiEF infected with

HVT and stained with homologous serum by indirect fluorescent antibody. x300.

Fig. l0. GEF stained by indirect fluorescent antibody: (a) Infected with LMD
and stained with homologous antiserum. There is both nuclear and diffuse
cytoplasmic staining. x300. (b) Infected with HVT and stained with LMD
antiserum. The nuclear staining predominates. x300. (c) Infected with HVT
and stained with homOIOgous antiserum. Clusters of granules are stained in

the cytoplasm. x300.

Fig. ll. (a) HeLa cells infected with a very high dose of LMD and stained

by indirect fluorescent antibody with homOIOgous antiserum. Some fluorescent
debris is staining. x300. (b) DK cells infected with HVT and stained by
indirect fluorescent antibody with LMD antiserum. Many nuclei are staining

in the cells bordering the hole in the cell culture. xl20. (c) DK cells
infected with HVT and stained by indirect fluorescent antibody with homologous
antiserum. The nuclei do not stain and appear like shadows but there is gran-
ular and diffuse stain in the cytoplasm. x300.

Fig. 12. Relative susceptibility of cell cultures of various types and
chorio-allantoic membranes (CAM) of chicken embryos to cloned preparations of
LMD, HMD and HVT. The susceptibility of duck embryo fibroblasts (DEF) is 1.0.
Embryo fibroblasts were from bobwhite quail (BQEF), chicken (CEF), goose (GEF),
Japanese quail (JQEF), pheasant (PhEF), pigeon (PiEF) and turkey (TEF). Kidney
cultures were prepared from chickens (CK) and ducks (0K). HeLa cells were not
susceptible to any of the viruses. Relative susceptibility of CEF to HVT - l8,

of PhEF to LMD ' 0.002, and of PiEF to LMD and HMD I <0.00l.

RELATIVE SUSCEPT/B/L/TY (DEF: /.0/-)

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GENERAL DISCUSSION AND CONCLUSIONS

Techniques for Detection of Antigen
and Antibody in Vitro and in Vivo

 

 

A specific IFA test for the detection of MD antigen
in cell culture has been described (Purchase, 1969, Article I).
There was a direct relationship between the number of fluores—
cent foci and the dilution of the inoculum used to infect
the cells indicating a one hit phenomenon. Thus, one infec-
tious unit of virus produced one fluorescent focus or one
morphological focus (cytopathic area) in cell culture.
Approximately the same number of fluorescent foci were
detected at one day after infection of CK cultures as were
detected at seven days after infection. The peak in the
number of morpholoqical foci was only reached at seven days
post-infection. The IFA test has limited application in
virus isolation studies because of the small area of the
culture which can be examined and the time and effort required
to perform the test. When examined by this test, all eight
isolates of MD were antigentically identical. An alter-
native explanation was that there was a common antigen or

contaminant in all stocks of the isolates.

121

122

The IFA and the AGP tests were compared in detail
(Purchase and Burgoyne, 1970, Article II). There was a 92%
agreement between the results of the IFA and those of the
AGP tests on 418 serums from various sources. The IFA test
was 10 to 320 times more sensitive than the AGP test; some
serums which had a high titer of antibody demonstrable
by the IFA test did not produce precipitation in agar. This
observation has been confirmed by Witter (1970). Thus, even
though the two tests detect antibody which reacts in differ-
ent ways, both are specific for MDV—induced antibody. The
IFA test would be expected to be more sensitive than the AGP
test since it would detect the antigen-antibody combinations
that produce a precipitate in addition to other antigen—
antibody combinations which did not form a lattice large
enough to produce a grossly visible precipitate.

A method is described for preparing fluorescein
conjugated anti-MDV chicken globulin for use in the direct
fluorescent antibody (DFA) test (Purchase, 1970a, Article
III). This globulin was used to detect MDV-induced antigen
in the feather follicles, lung, bursa of Fabricius, thymus,
spleen, and cecal tonsil of MD—infected birds. Precipitat-
ing antigen was also demonstrated in most of these sites.
Filtrable virus, however, was only found in the skin, and

presumably originated from the feather follicle epithelium.

123

Differences Between Strains of
MDV and HVT

 

 

Even though eight isolates of virus previously
examined by the IFA test were found to be identical, cloned
preparations from these isolates differed greatly in their
pathogenicity. Two pathogenic JM viruses and the HMD virus
lacked antigens identified as the A antigens in the agar
gel precipitin test. The HMD produced larger plaques in
a shorter time than the other MD viruses. Either there
was more than one virus in the original stocks or the virus
became altered during cloning. Unfortunately, because of
its highly cell-associated nature, ll to 17 passages in cell
culture were required to clone purify the virus and changes
may have occurred in the virus during passage. The change
in morphological appearance of the cytopathic areas must
have occurred during passage in cell culture since large
cytopathic areas would most certainly have been present in
the early passages. Since the absence of an antigen or
lack of pathogenicity of a small proportion of virus in the
original stock would not have been detected, it is not
possible to determine whether the differences in antigens
and pathogenicity were due to passage in culture or were
present in the initial stocks of virus.

The HVT produced large cytopathic areas and was
non-pathogenic for chickens. This virus and MDV shared

two A antigens and at least one B antigen. These viruses

124

could not be distinguished from one another by the AGP
test. However, MDV and HVT could be distinguished from one
another, as could antibody to each Virus, by the intra-
cellular location of antigen stained by the IFA test.

Growth of Clones of Virus in
Cultures of Various Types

 

 

Bobwhite quail, chicken, duck, goose, Japanese
quail, pheasant, pigeon, and turkey embryo fibroblasts,
chick and duck kidney cells were all susceptible to LMD
virus, HMD virus and the HVT. The cytopathic areas pro-
duced in cells of different types varied greatly. Clones
of these three types of virus could best be distinguished
from one another by the cytopathic changes they produced
in CK cultures. The LMD virus did not grow as well in
CEF as in DEF however the reverse was true for HMD and HVT

(Purchase et al., 1970b, Article V).

Virus—Host Cell Relationships in Vitro

 

The MDV is highly cell-associated and spreads from
cell to cell by direct contact. The areas containing
antigen which become the cytopathic areas or plaques
enlarge peripherally by infection of neighboring cells.
Even though cultures are maintained under liquid medium,
secondary fluorescent foci of infection are not initiated
until rounded cells float free from the initial cytopathic
areas and settle on uninfected cells. This process was

well demonstrated by the IFA test (Purchase, 1969, Article

125

I). On clone isolation or passage in cell culture, MDVs
become altered in pathogenicity, in the appearance of
cytopathic areas and presence or absence of the A antigen.
It appears that change in any one of these three properties

may occur independently of change in any other.

Virus-Host Cell Relationships in Vivo

 

The loss of maternal anitbody in chicks and the sub-
sequent acquisition of antibody after infection were
adequately demonstrated using the IFA test (Purchase and
Burgoyne, 1970, Article II). However, by the IFA test,
maternal antibody in chicks from MD-exposed dams was
detected after antibody was no longer detectable in the AGP
test. Also, acquired antibody in contact-exposed chickens
was detected earlier than by the AGP test. The sera of
some older birds had a high titer of antibody demonstrable
by the IFA test but did not produce precipitation in agar.
In addition to being more sensitive, the IFA test will
detect antibody of a type different than that by the pre-
cipitation test.

Three different types of virus-host cell inter-
actions occurred in_yiyg, Firstly, there was a situation
where neither antigen nor infectious cell-free virus could
be demonstrated. This occurred in tumors of the visceral
organs and nerve lesions. Secondly, antigen was present

but not infectious virus and a cytolytic process occurred.

126

This is exemplified by changes which occur in the bursa

of Fabricius of infected chickens. Naked viral particles
have been observed in the nuclei of cells undergoing this
type of degeneration (Calnek, et_31., 1970c). Lastly, in
the feather follicle epithelium, both antigen and infectious
cell-free virus can be detected. The virus was infectious
even after storage under conditions in which cells would
not retain their viability. Degenerative and necrobiotic
changes are continuously occurring in the superficial
layers of the stratified squamous epithelium and intra-
nuclear inclusion bodies could be seen in these cells.
Furthermore, enveloped virions were observed in the nucleus
and in cytoplasmic inclusions (Nazerian and Witter, 1970).
This is the only location in which virus maturation and
envelopement is completed. It is probably the portal of

exit of MDV from infected birds.

The Etiology of MD

 

At the time that the IFA test was developed, there
was still some uncertainity that the herpes-virus was the
cause of MD. Demonstration of a reaction between sera from
diseased birds and the cytopathic areas which contained
inclusion bodies and virions (Nizerian, 1968; Nazerian
gj;al,, 1968) added evidence that the herpesvirus was the
cause of MD. Demonstration by Calnek and Hitchner (1969)
of immunofluorescent antigen in the epithelium of the

feather follicles led to the discovery of enveIOped virus

127

infections for cell cultures and chickens at this site
(Calnek et_al., 1970a; Nazerian and Witter, 1970). Work
reported in this dissertation confirmed these findings.
Demonstration of the maturation and envelopement of
herpesvirus in the feather follicle epithelium and the
reproduction of the disease with virus from this site
removed the last shadows of doubt about the etiology in
agent of MD. A herpesvirus has now been firmly established
to be the cause of MD. This is the first time that a
herpesvirus has been definitely incriminated as the cause

of a neoplastic disease.

S UMMARY

1. The IFA test was applied to the detection of
MDV antigen in cell culture and MDV antibody in the serum
of birds.

2. Nuclear and cytoplasmic antigen could be
detected as early as 24 hours after infection at which time
no cytopathic changes could be detected by conventional
light microscopy. By seven days after infection, the same
number of infected areas were detected by both methods.

3. There was a linear relationship between the
concentration of inoculum and the number of fluorescent
foci or cytopathic areas indicating that one infections
unit produced one fluorescent focus or one cytopathic
area.

4. The time sequence study confirmed the cell
association of the virus and that there was cell-to-cell
Spread of infection.

5. When examined by the IFA test, eight isolates
of MD were indistinguishable indicating that either the
strains were antigentically identical or there was a

common antigen or contaminant in all stocks.

128

129

6. Cells infected with MDV contain a heat stable
antigen similar to that found in herpes simplex-infected
cells.

7. There was 92% agreement between the results of
the IFA and AGP tests for antibody.

8. By the IFA test, maternal antibody in young
chickens from MD exposed dams was detected after antibody
was no longer detectable by the AGP test and acquired anti-
body in contact exposed chickens was detected earlier than
by the AGP test.

9. The IFA test was 10 to more than 320 times
more sensitive than the AGP test. Some serums with high
titers of immunofluorescent antibody did not produce pre-
cipitation in agar gel.

10. Using the DFA and micro AGP tests, antigen
was found in superficial cells of the feather follicle
epithelium, lung, follicles of the bursa;of Fabricius,
thymus, spleen and cecal tonsil.

ll. Filtrable, infectious virus was recovered from
extracts of skin but not from extracts of lung, thymus or
bursa of MD-infected chickens.

12. Histopathologic examination revealed a close
association between antigens in these organs and cells
undergoing degeneration and necrosis.

13. There were intranuclear inclusion bodies in

cells of the feather follicle epithelium.

130

14. Different cloned preparations of virus varied
greatly in pathogenicity.

15. Some viruses had lost the A precipitin antigen
during passage in cell culture.

16. HMD produced larger plaques in a shorter time
than other MDVs.

17. Loss of pathogenicity, loss of the A antigen,
and change in virus-host cell interrelationships are inde-
pendent events.

18. The MDV and HVT shared at least two A antigens
and one B antigen. These two viruses could not be readily
distinguished from one another by the AGP test.

19. The MDV and HVT could be distinguished from
one another, as could antibody to each virus, by the intra-
cellular location of antigen by the IFA test.

20. Cultures of fibroblasts from chicken, duck,
bobwhite and Japanese quail, turkey, pheasant, pigeon and
goose embryos, cultures of chicken and duck kidney cells
and the chorioallantoic membranes of embryonating chicken
eggs were all susceptible to low and high cell culture
passaged MDV and the HVT.

21. HeLa cells were not susceptible to MDVs or

HVT.

131

22. CEF were much less susceptible to LMD, equally
susceptible to HMD, and more susceptible to the HVT than
were DEF.

23. The three viruses could best be differentiated
by the morphological appearance of cytopathic areas in CK
cultures.

24. A cytoplasmic virus-induced antigen could be
detected by the IFA test in cells of all types infected with
the HVT using homologous antiserum but not by using MDV

antiserum. Nuclear antigens were detected by both sera.

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132

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