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Graduate School
1986
Molecular Studies of T-Lymphocytes From Bovine
Leukemia Virus-Infected Cattle.
Diana Lynn Williams
Louisiana State University and Agricultural & Mechanical College
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D issertation
Information S erv ice
U niversity Microfilms International
A Bell & Howell Inform ation C o m p a n y
3 0 0 N. Z e e b R o a d , A nn Arbor, M ich ig an 48 1 0 6
8629207
W illiam s, D iana Lynn
MOLECULAR STUDIES OF T LYMPHOCYTES FROM BOVINE LEUKEMIA
VIRUS-INFECTED CATTLE
The Louisiana State U niversity a n d A g ricu ltu ra l and M echanical Col.
University •
Microfilms
International
300 N. Zeeb Road, Ann Arbor, Ml 4B106
Copyright 1987
by
Williams, Diana Lynn
All Rights Reserved
Ph.D.
1986
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University
Microfilms
In te rn a tio n a l
MOLECULAR STUDIES OF
T LYMPHOCYTES FROM BOVINE
LEUKEMIA VIRUS-INFECTED CATTLE
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
Veterinary Medical Sciences
with an option in
Veterinary Microbiology and Parasitology
by
Diana Lynn Williams
M.S.. Louisiana State University.
May 1986
1979
©1987
DIANA LYNN WILLIAMS
All Rights Reserved
ACKNOWLEDGEMENTS
I wish to express my sincere graditude and appreciation
to my major advisor, Dr. Grace Amborski,
guidance with my research efforts.
for her support and
She afforded me great
opportunities to interact and learn from research groups
affiliated with the Louisiana State University system and
other institutions around the country.
Thanks are also due
to the members of my graduate committee) Drs. Ronald
Montelaro)
Charles Issel, Ota Barta, and Ken Sehnorr for
their constructive advice and help editing this
dissertation.
A special thanks is given to Dr. Ota Barta
for his help and support with several aspects of this
research.
I am also indebted to Drs. James Casey and Dave Derse
of the Department of Genetics of the National Cancer
Institute for their continued help and for giving me the
opportunity to learn molecular hybridization technology.
Appreciation also goes to Dr. William Davis of the
Department of Veterinary Microbiology and Pathology,
Washington State University.
1 wish to thank Jennifer Lo for her continued help and
support throughout my graduate program.
The assistance
given to me by Joel Walker, Rick Ramsey, Susan Pourciau and
Li Huang is greatly appreciated.
ii
I am indebted to the Department of Veterinary
Microbiology and Parasitology of the School of Veterinary
Medicine* Louisiana State University for its financial
support in the form of a graduate assistantship and for the
privilege to undertake advanced studies.
Additional funds for this investigation uiere provided
by the Louisiana Agricultural Experiment Station, Louisiana
State University Agricultural Center*
2296.
Ill
projects 1467 and
To my husband Joe.
Without your constant love,
support, and understanding, the dream to be the best that I
could be would never have become a reality.
To my son, Anthony.
Thank you for bringing more
happiness and love into my world than 1 could ever have
imagined.
iv
TABLE OF CONTENTS
Chapter
Page
TITLE
........................................
......................
ii
...................................
iv
ACKNOWLEDGEMENTS
DEDICATION
TABLE OF CONTENTS
...........................
v
..............................
xi
.............................
xii
.....................................
xvi
INTRODUCTION ..................................
1
A.
Statement of Research Problem
......
2
B.
Objectives
...............................
3
LIST OF
TABLES
LIST OF
FIGURES
ABSTRACT
I.
II.
i
LITERATURE REVIEW
A.
B.
Enzootic Bovine Leukosis
................
6
1.
Aleukemic State
...
6
2.
Persistent Lymphocytotic State ......
6
3.
Enzootic Bovine Lymphosarcoma
7
A.
Geographic Distribution of BLV ......
8
5.
Diagnosis of B L V .....................
8
6.
Transmission of B L V
9
....
.....
BLV Infection in Other Animal
S p e c i e s ................ .... ... .........
C.
Sporatic Bovine Leukosis
v
................
10
ii
Chapter
II.
Page
D.
Other Bovine Lymphotrophie
Retroviruses ............................
E.
1.
Bovine Syncytial V i r u s
.....
12
2.
Bovine Visna-like Virus ...............
13
Lymphocytes in Enzootic Bovine
L e u k o s i s ........
1.
F.
12
13
Lymphocyte Isolation
P r o c e d u r e s ..........................
14
2.
B Lymphocyte M a r k e r s ..................
15
3.
T Lymphocyte Markers ..................
16
4.
Frequency of T and B Lymphocytes
in Enzootic Bovine Leukosis ........
17
5.
BLV Target Cells .......................
19
6.
Immune Responses to BLV
I n f e c t i o n ...........................
21
Molecular Studies of B L V ..................
23
1.
Bovine Leukemia Virus .................
23
2.
BLV P r o t e i n s ...........
24
3.
BLV Genome .. . .........................
27
4.
Morphogeneous
5.
Viral P e r s i s t e n c e ....................
6.
Molecular Aspects of
...................... 27
BLV-induced Leukomogenesis
7.
28
....... 3D
Relatedness to Human
Lymphotrophie Retroviruses
vi
.....
32
Chapter
Page
III. ENUMERATION OF T AND B LYMPHOCYTES IN
ALEUKEMIC AND PERSISTENT LYMPHOCYTOTIC
CATTLE USING MONOCLONAL ANTIBODIES THAT
DISTINGUISH THESE CELLS ....................
A.
Introduction
.................
E.
Materials and Methods
34
34
....................
1.
Animals
.........................
2.
Isolation of Peripheral Blood
36
36
Mononuclear cells ................... 37
3.
Complete Blood Counts
4.
Staining Cells for B and T
.......
Lymphocyte Enumeration
5.
.......
C.
Statistical Analysis
Results
................
...............
1.
Hemograms
............
2.
Frequency of T and B Lymphocytes .....
3.
Absolute Numbers of T and B
L y m p h o c y t e s .........................
D.
38
Detection of Fluorescence— Positive
Cells ................................
6.
36
Discussion
............
Vii
39
40
40
4D
40
43
44
Chapter
IV.
•
Page
PURIFICATION OF T LYMPHOCYTES FROM BOVINE
PERIPHERAL BLOOD MONONUCLEAR CELLS USING
IMMUNO—AFFINITY DEPLETION 'PANNING- ........ 59
A.
Introduction
B.
Materials and Methods
1.
............................
....................
.................. 6i
Coating Plastic Flasks with
Immunoglobulin
.....
3.
Affinity “Panning- Procedure
A.
Determination of Lymphocyte
C. Results
61
........
62
.............
63
....... ............................
64
Subsets and Monocytes
1.
T Lymphocyte Yield after
'Panning- ...........................
2.
61
Isolation of Peripheral Blood
Mononuclear Cells
2.
59
64
T Lymphocyte Enrichment and
B Lymphocyte Depletion
after "Panning-
D. Discussion
...........
viil
....................
64
65
Chapter
V.
Page
ASSESSMENT OF THE PRESENCE OF BLV PROVIRUS
IN THE T LYMPHOCYTES OF BLV-INFECTED*
ALEUKEMIC CATTLE
...........................
A.
Introduction
B.
Materials and Methods
....................
Animals
2.
Isolation of PBMC
3.
Purification of T Lymphocytes
4.
Determination of Lymphocyte
......
...............
.....................
75
DNA Purification
6.
Restriction Enzyme Digestion
...........
7.
Gel T r a n s f e r ..........
8.
BLV-DNA Probe
9.
Nick Translation of BLV-DNA
.........
77
79
Filter Hybridization
11.
Autoradiography
................
80
......
80
....................
80
Purity of T Lymphocyte
Preparations ........................
2.
76
........................ 77
ID.
1.
74
74
5.
Results
.....
73
.............
and Gel Electrophoresis
C.
73
73
Subsets and Monocytes
Probe
71
....................
1.
71
80
Agarose Gel Electrophoresis
of Restriction Enzyme Fragments .... 81
lx
Chapter
Page
3.
D.
VI.
DNA Hybridization Analysis
Discussion
..........
........................
81
82
RESPONSE OF LYMPHOCYTES FROM ALEUKEMIC AND
PERSISTENT LYMPHOCYTOTIC CATTLE TO
SELECTED PHYTOMITOGENS
A.
Introduction
B.
Materials and Methods
.....................
.........
1.
Animals
2.
Isolation of Peripheral Blood
...........
VIII.
IX.
X.
97
97
.................
3.
Complete Blood Counts
4.
Enumeration of T/B Lymphocyte
Ratios
VII.
94
.....................
Mononuclear Cells
94
97
.................. 98
............................
5.
Serum Preparation
6.
Preparation of Mitogens
7.
Lymphocyte Blastogenesis Assay
6.
Statistical Evaluations
98
.................... 99
..............100
...... 100
............102
C.
Results
D.
Discussion... ................................104
SUMMARY
....................
..102
.........................................11?
A P P E N D I X ........................................ 123
BIBLIOGRAPHY
VITAE
................................... 126
.........
142
X
LIST OF TABLES
Table
III-
Page
1.
Statistical Analysis of UBC and
Lymphocyte Counts from Bovine Leukemia
Virus-Infected
III*
Cattle ....................
2. Frequencies of T and B Lymphocytes
56
from
Peripheral Blood of Bovine Leukemia
Virus Infected
III. 3.
Cattle
..................
57
Statistical Analysis of T and B Lymphocytes
from Bovine Leukemia Virus-Infected
Cattle
V.
...............................
1. Frequencies of T and B Lymphocytes
5B
in
Cell Preparations for DNA Hybridization
Analysis
VI.
........
93
1. Statistical Analysis of Lymphocyte
Blastogenesis of Lymphocytes from
Bovine Leukemia Virus-Infected Cattle
Using Stimulation Index
VI.
...............
115
2. Statistical Analysis of Lymphocyte
Blastogenesis of Lymphocytes from
Bovine Leukemia Virus— Infected Cattle
Using Corrected Counts per Minute .......
VI.
3. T/B Lymphocyte Ratios of Bovine
Leukemia Virus-Infected Cattle .........
VIII.
116
117
1. Assessment of viral infections in Animals
Used for Molecular'Studies of Bovine T
Lymphocytes
...............
xi
124
LIST OF FIGURES
Figure
III.
Page
1.
Procedure for Isolation of Bovine
Peripheral Blood Mononuclear Cells
Using Density Gradient
Centrifugation
III. 2.
............ .............
49
WBC and Lymphocyte Counts from
Peripheral Blood of Bovine
Leukemia Virus Infected Cattle .........
III. 3.
50
Fluorescence Activating Cell Sorting
Contour Plots of Lymphocytes
from Bovine Leukemia Virus Negative*
Control Com # 5 6 ............ ..........
III. 4.
51
Fluorescence Activating Cell Sorting
Analysis Profile of Lymphocytes
from Bovine Leukemia Virus Infected*
Aleukemic Com #75 .......................
III. 5.
52
Fluorescence Activating Cell Sorting
Analysis Profile of Lymphocytes
from Bovine Leukemia Virus Infected*
Persistent Lymphocytotic
III. 6.
Com # BIO .....
53
Fluorescence Activating Cell Sorting
Contour Plots of Lymphocytes from
Bovine Leukemia Virus Infected*
Persistent Lymphocytotic
Xii
Com # 632 .....
54
Figure
III. 7.
Page
Absolute Numbers of Peripheral Blood
T and B Lymphocytes from Bovine
Leukemia Virus Infected* Aleukemic
and Persistent Lymphocytotic Cattle ....
IV. 1.
Procedure for T Lymphocyte Purification
Using "Panning*
IV. 2.
55
......................
67
T Lymphocyte Yield After "Panning
of Bovine Peripheral Blood Mononuclear
Cells Using Anti—Bovine Immunoglobulin
Coated Flasks
IV. 3.
....................
6E3
T Lymphocyte Enrichment by "Panning*
of Bovine Peripheral Blood Mononuclear
Cells Using Anti-Bovine Immunoglobulin
Coated Flasks
IV. A.
.............
69
Depletion of B Lymphocytes by "Panning"
of Bovine Peripheral Blood Mononuclear
Cells Using Anti-Bovine Immunoglobulin
Coated Flasks
V. 1.
........
70
Procedure for Detection of Bovine
Leukemia Virus Provirus in the Peripheral
Blood Mononuclear Cells and Purified
T Lymphocytes of Bovine Leukemia Virus
Infected Cattle
.................
Xlii
87
Figure
V. 2.
Page
Bovine Leukemia Virus Provirus
Restriction Enzyme Map
V. 3.
. .,.............
88
Purification of BLV DNA Probe Using
Electroelution of BLV-DNA Sacl Fragment
t
from an agarose gel .....................
V. 4.
89
Restriction Enzyme Fragments of Purified
T Lymphocyte and Peripheral Blood
Mononuclear Cell DNA from Boyine
Leukemia Virus Infected* Aleukemic
Cattle Generated by Digestion uiith
Sac 1 Restriction E n z y m e
V. 5.
........
90
DNA Hybridization Analysis of Purified
T Lymphocyte and Peripheral Blood
Mononuclear Cell DNA from Bovine
Leukemia Virus Infected* Aleukemic
Cattle
V. 6.
............. ....................
91
DNA Hybridization Analysis of Purified
T Lymphocyte and Peripheral Blood
Mononuclear Cell DNA from Bovine
Leukemia Virus Infected* Aleukemic
Cattle
VI. 1.
............ .....................
Procedure for Lymphocyte Blastogenesis ...
xiv
92
110
F igure
VI. 2.
Page
Schema of Plate Arrangement for Lymphocyte
Blastogenesis with 3 Mitogens and 4
Different Serum Supplements (Cells Added
to All Wells) ...........................
VI. 3.
Ill
Response of Lymphocytes from Cattle to
Optimal Concentrations of Selected
Phytomitogens Using Stimulation
Index
VI. 4.
.............
112
Response of Lymphocytes from Cattle to
Optimal Concentrations of Selected
Phytomitogens Using Corrected Counts
per M i n u t e ...............................
VI. 5.
113
Response of Lymphocytes from Bovine
Leukemia Virus Negative Cattle to Optimal
Concentrations of Selected Phytomitogens
in the Presence of Sera from Bovine
Leukemia Virus
Infected Cattle ........
XV
114
ABSTRACT
MOLECULAR STUDIES OF
T LYMPHOCYTES FROM BOVINE LEUKEMIA VIRUS
INFECTED CATTLE
by
Diana Lynn Williams
Highly specific monoclonal antibodies and
raicrofluorimetry were used to determine the absolute number
of B and T lymphocytes in the peripheral blood of bovine
leukemia virus (BLV) infected cattle.
The peripheral blood
lymphocyte populations from BLV-infected cattle
were
elevated when compared to BLV-negative animals.
The
increase in the lymphocyte population in the aleukemic (AL)
animals was
due to an increase in T
lymphocytes.
In
animals the
increase was due mainly to an increase of B
lymphocytes
but also to an increase of T lymphocytes.
thePL
The PL animals showed a significantly higher number of
surface immunoglobulin positive cells (slg'*') than did the
animals.
AL
The majority of these cells contained surface IgM
and had large quantities of this marker on their cell
surfaces.
One PL animal (632) had a very high lymphocyte
count and contained lymphocytes that appeared to stain with
markers for both bovine B and T lymphocytes.
xvi
Bovine T lymphocytes were purified from the peripheral
blood Df BLV~infected, AL cattle using icnmuno-affinity
depletion technique ("panning") with anti-bovine
immunoglobulin-coated flasks.
Large yields of highly
purified T lymphocyte preparations (95-10054 T lymphocytes
and 0-3?. contamination with B lymphocytes) were obtained by
the use of this technique.
The BLV provirus was found in the DNA of peripheral
blood lymphocytes and 3 of A purified T lymphocyte
preparations from AL animals using hybridization analysis
uith radiolabeled BLV-DNA as a probe.
Hybridization
analysis also confirmed that the B lymphocyte is the major
target of BLV infection.
The blastogernic response of lymphocytes from PL and AL
animals to selected phytomitogens appeared to be normal when
compared to the responses of BLV-negative,
animals.
clinically normal
The sera from both AL and PL animals contained a
heat-stable* camitogenic blastogenesis-augmenting factor.
xvii
CHAPTER is
INTRODUCTION
Enzootic bovine leukosis (EBL) is a neoplasm of
lymphatic tissue in the bovine species (B).
The etiologic
agent is bovine leukemia virus (BLV) (10B)* an exogenous*
leukomogenic virus of the family Retroviridae.
This virus
may induce two clinical manifestations of disease either a
benign proliferation of lymphocytes referred to as
persistent lymphocytosis (PL) or the development of
malignant lymphosarcoma (LS)
(46).
The majority of animals
infected with BLV do not show any clinical signs of disease
(88).
These animals are referred to as inapparent carriers
or as being aleukemic (AL).
Some of these AL animals (25 X)
will develop PL* and a small percentage (0.1 to 1 X) will
develop LS (108).
Studies have been conducted to investigate the
relationship between the PL and LS stages of EBL (46*51)*
but* very few studies have been done to investigate the
relationship between the AL and PL stages of EBL. This may
be very important since the switch from the AL to PL stage
may be one of the primary steps in the progression of the
disease to the tumor phase.
Over 75X of LS cattle have a
chronic PL phase which precedes tumor development
(108).
A target cell of BLV-infection in the cow has been
reported to be the B lymphocyte (121). The majority of
lymphocytes found in the peripheral blood of PL cattle have
surface markers typical of B lymphocytes (110*156)* and a
1
percentage of these cells have been shown to exhibit viral
antigens on their cell surfaces (72).
A recent report
suggested that some tumors consisted of cells that not only
bear surface antigens of T lymphocyte lineage but also
contain the BLV provirus <9D).
The role of bovine T
lymphocytes in the progression of EBL has not been
investigated.
Reports in the current literature have focused on the
antigenic relatedness of BLV and the human T lymphotropic
viruses HTLV I & II.
These viruses are associated with
human T cell leukemias (116*128).
The T lymphocyte appears
to be the target of both of these viral agents.
Therefore*
if T lymphocytes are found to be target cells of BLV and/or
important in the progression of EBL* not only will this
information aid in the better understanding of bovine
leukosis*
it may also help to establish an animal model to
study human viral induced leukemia.
I. A.
STATEMENT OF RESEARCH PROBLEM
Bovine T Lymphocytes from AL and PL cattle are the
subject of these studies.
Definitive studies on these cells
in EBL have not been conducted mainly because purified
preparations and specific markers for these cells have not
been available.
Monoclonal antibodies directed toward the
surface antigens of bovine cells of the T lymphocyte lineage
have recently been produced and characterized (90)* but
purification techniques have not been perfected to give good
yields of highly purified lymphocyte subpopulations.
In
order to determine whether T lymphocytes may serve as
targets for BLV it is nessecary to have large quantities of
purified T lymphocytes for DNA hybridization analysis.
Further studies on T lymphocytes may help to define
how the AL to PL shift is mediated.
Information on the
absolute number of T lymphocytes in the peripheral blood of
AL and PL cattle may indicate that there is a modulation of
this cell population in these animals.
occur as a result of BLV— infection.
This modulation may
Information on the,
functional ability of T lymphocytes from AL and PL cattle to
respond to selected phytomitogens may indicate a modulation
of the functional ability of these cells (T-helper and Tsuppressor).
The uncontrolled proliferation of
undifferientiated B lymphocytes in the PL animal may be the
result of a lack of or enhancement of a particular T cell
subset function.
Therefore* studies on bovine T lymphocytes
are needed to attempt to define the role if any of these
cells in the AL and PL phases of EBL.
I. B.
OBJECTIVES
The different chapters of this dissertation describe
specific experiments that were employed to accomplish the
following objectives!
4
1,
To determine the numbers of bovine T and B
lymphocytes from AL and PL cattle using
monoclonal antibodies that distinguish these
lymphocytes.
2-
To purify T lymphocytes from bovine peripheral
blood mononuclear cells (PBMC) of cattle using
immuno-affinity depletion "panning".
3.
To assess the presence of BLV provirus in
purified T lymphocytes and PBMC of AL cattle and
the PBMC of PL cattle using DNA hybridization
analysis.
4.
To evaluate and compare the responses of
lymphocytes in AL and PL cattle to selected
phytomitogens using the lymphocyte
blastogenesis assay.
The aim of these investigations is to acquire a better
understanding of the role that bovine T lymphocytes play in
the maintainance of the AL and
PL stages of EBL.
The
results from these investigations are presented as
individual chapters.
Some of these have been submitted for
publication to different Journals.
indicated belou:
They are entitled as
"Enumeration of T and B lymphocytes in aleukemic and
persistent lymphocytotic cattle using monoclonal
antibodies that distinguish these cells.
Submitted for publication to the "Journal of the
National Cancer Institute".
Co-authored by U. Davis
and G. Amborski.
■Purification of bovine T lymphocytes from peripheral
blood mononuclear cells using an immuno-affinity
depletion technique ("panning").
Accepted for
publication in "Veterinary Immunology and
Immunopathology". Co-authored by J. Lo and
G. Amborski.
"Assessment of the presence of bovine leukemia
provirus in the DNA of purified bovine T
lymphocytes of BLV-infected* aleukemic cattle."
Manuscript in preparation for publication to
"Cancer Research".
Co—authored by G. Amborski.
"Evaluation of the responses of lymphocytes of
aleukemic and persistent lymphocytotic cattle to
selected phytomitogens".
Manuscript in preparation
for submission to the "American Journal of
Veterinary Research".
and G. Amborski.
Co-authored by 0. Barta
CHAPTER IIs
A-
LITERATURE REVIEW:
ENZOOTIC BOVINE LEUKOSIS
Enzootic bovine leukosis (EBL) is a lymphoproliferative
disease of the bovine species (6>.
The etiologic agent is
bovine leukemia virus (BLV), an exogenous leukemogenic virus
of the family Retroviridae (108,75).
The presence of this
virus appears to be a prerequisite for tumor development,
but, it is not by itself sufficient to cause the disease
(75).
The virus may induce either inapparent, aleukemic
(AL) infections or two clinical manifestations of the
disease,
persistent lymphocytosis (PL) or malignant
lymphosarcoma (LS)
(46).
II. A. 1. Aleukemic State
Once infected with BLV, cattle show no signs of overt
viremia,
however, they elicit a strong and persistent
humoral response to BLV structural proteins (49).
Most
infected cattle <75%) have no clinical signs of disease.
These animals are inapparent carriers or AL cattle.
gain and milk production is unaltered (88).
Weight
Lymphocyte
counts remain in the normal range between 2.5 to 7.5 x
ID3 /mm3 of whole blood (2).
There has been very little
information reported about the AL state of BLV— infection.
II. A. 2.
Persistent Lymphocytotic State
Approximately 30% of BLV infected cattle develop PL
(108).
This stage of BLV— infection is a nonmalignant
6
hematological disorder characterized by an increase of
circulating peripheral blood lymphocytes (PBL)
(> 7.5 x
lO^/mm3 of whole blood) for over a period of 3 months (2).
Weight gain and milk production appear to be unaltered (88).
In most of the PL animals tested*
it appears that the
increase in the number of PBL is attributed to an increase
in B lymphocytes (110*156).
An increase in T lymphocytes of
some animals has also been reported (142).
The PL state is
generally a chronic condition that may or may not lead to
the tumor stage of EBL (108*51).
The PL phase precedes the
tumor phase (LS) in approximately 75’
/, of the cases (51).
12. A. 3. Enzootic Bovine Lymphosarcoma
The adult form of bovine lymphosarcoma or EBL is most
prevalent in dairy cattle herds and typically occurs in
animals older than 2 years of age.
It is a chronic disease*
evolving over extended periods (1—8 years)* with tumors
developing in only a small number of infected animals (0.1—
1%) (108).
The majority of cattle affected by EBL are those
from 5 to 8 years of age.
Tumors observed in EBL cases are
always of lymphoid origin and may invade mammary glands*
subcutaneous tissues* abdominal muscles*
diaphragm* heart*
respiratory tract and spinal cord (19).
Tumors are
inconsistent in location and tumGrization is usually
asymmetrical
(53*17).
As tumors grow* they mechanically
impair normal function of organs* and the ultimate stage is
complete destruction of the original organ structure.
Any
lymph node may be affected* but the pelvic lymph nodes are
most often involved.
Lymphosarcoma induced by BLV
eventually leads to either death of the affected animal or
condemnation of the carcass at slaughter.
U.
A. 4. Geographic Distribution of EBL
The first cases of EBL were reported in Germany in 1B7B
and in the United States in 1897 (9,8).
world wide distribution (19).
The disease now has
In some parts of the world
such as Switzerland and other parts of Europe, eradication
programs are in progress (19).
In these areas the number of
cases of EBL is steadily decreasing.
Some sections of the
United States are endemic areas for EBL.
One such area is
Louisiana where many herds contain 75-100% BLV positive
reactors (71).
II. A. 5. Diagnosis of BLV Infection
The viral etiology of EBL was shown in 1969 (106).
Serological identification of reactors became possible in
1972 (45).
Diagnostic techniques presently available for
the detection of BLV reactors are virus neutralization (61),
complement fixation (140,31), agar gel immunodiffusion
(AGID) (33,34), enzyme-linked immunoabsorbent assay (ELISA)
(80), radioimmunoassay (RIA)
(125,89), and the syncytia
induction and syncytia induction neutralization assays using
bovine fetal spleen cells as indicator cells (40,62).
AGID is the most commonly used diagnostic tool.
The
This assay
detects antibodies to the major envelope and core proteins
of BLV in the serum of infected cattle.
Various serologic
diagnostic tests have demonstrated that BLV antibodies are
detectable in > 90’
A of LS and PL animals (19).
II. A. 6. Transmission of BLV
Studies conducted in mant) laboratories indicate that
contact transmission is the principal method by which BLV is
spread (122*AS).
vivo (42,3),
Since BLV particles are seldom produced in
it is likely that in most cases BLV infection
in cattle is the result of the transfer of infected
lymphocytes rather than viral particles. The transfer of as
few as 1 x ID4* blood lymphocytes from infected to non—
infected animals results in virus transmission (20).
Penetration of the skin appears tc be the mode of entry of
the virus,
therefore*
infection can be easily spread through
multiple use of needles* ear tagging devices* dehorning and
castration equipment (154*160).
Transmission studies using
potential insect and tick vectors have been conducted
(21*114*130).
These investigations indicate that BLV may be
spread by stable flies (Stomoxys calcitrans)* horn flies
(Haematobia irritans)* horse flies (Tabanidae) (21*114)* and
some species of ticks (Boophilus microplus) (130).
Another possible means of BLV spread is in utero
transmission. The incidence of transmission of BLV from
infected dam to fetus is very low (46* 123).
Usually less
than 155i of BLV-infected cows give birth to infected calves
(123).
Colostrum and milk are potential sources of
infection (50*119).
Both contain BLV-infected lymphocytes*
but the amount is minimal when compared to tnat of the blood
(109).
Usually antibody present in the colostrum of
10
infected cows is adequate for protecting calves until about
a months of age <50, 49).
Pasteurization of infected milk
totally destroys BLV (5).
Vertical transmission xn the gametes has never been
observed (109).
In Sweden* transmission of BLV has occurred
through the use of a BLV— contaminated babesiosis vaccine
<19).
All the above observations confirm that natural
spread of BLV occurs mainly through the horizontal route.
II. B.
BLV Infection in Other Animal Species
Natural cases of viral-induced lymphomas in sheep are
rare.
The virus isolated from these cases appear
indistinguishable from BLV (19).
Sheep have been
experimentally infected with either cell— free virus or BLVinfected lymphocytes from infected cattle.
not occur in sheep.
Typical PL does
Lymphosarcoma usually occurs quite
rapidly after infection (1-3 years) and at a much higher
frequency than in cattle (19).
Anatomopathological lesions
are comparable for tumors of sheep and cattle.
BLV
sequences are present in tumor tissue but absent from normal
tissue.
Recent evidence seems to indicate that, unlike BLV-
induced tumor cells in cattle, tumor cells in sheep are of T
lymphocyte origin (67).
BLV-infected sheep have antibodies
to structural proteins of the virus.
been demonstrated in sheep (19).
No viremia has ever
BLV-induced lymphosarcoma
of sheep does not seem to be naturally transmitted from
11
animal to animal.
BLV-induced lymphosarcoma has been demonstrated in only
one other animals species.
After numerous attempts to
induce LS in goats with BLV experimentally*
reported (115).
Lymphoid tumors appeared in many organs 6
years postinfection.
chimpanzees*
one case was
Many other animal species such as
pigs* domestic rabbits* cats, dogs, deer, rats*
and guinea pigs have been experimentally infected with BLV*
but in none of these species were viremia or pathological
disorder observed (97*153).
The only species which 'remained
antibody positive was the chimpanzee (153).
BLV infection
in humans has never been documented (6*153*97).
II. C. SPORADIC BOVINE LEUKOSIS
EBL induced by BLV should be distinguished from
sporadic bovine leukosis (SBL) which includes the Juvenile*
skin* and thymic forms of the disease (1).
associated with BLV infection.
Only EBL is
The SBL forms occur at
random* having no proven association with an infectious
etiology.
The possibility exists that other bovine
retroviruses may be associated with this condition* although
this has not been thoroughly investigated.
A transient
lymphocytosis may be detected in some SBL cases.
The target
cell of SBL appears to be the lymphocyte stem cell because
tumors consist of cells of both T and B lymphocytes lineage
(19).
The calf or Juvenile form generally occurs in calves
12
less than 6 months of age* however*
animals.
it can occur in older
Tumor cells can be found in lymph nodes*
spleen and bone marrow.
liver*
In contrast the thymic form,
typically restricted to the region of the thymus* the calf
form typically occurs in young animals (6 to 3D months of
age) and appears mainly in beef herds rather than in dairy
herds (43).
The skin form is extremely rare* and the only
form of lymphosarcoma that is not fatal.
Lesions of this
form may regress but a generalized lymphadenopathy may occur
(19).
II. D. OTHER BOVINE LYMPHOTROPHIC RETROVIRUSES
Studies on EBL have resulted in the isolation of two
other bovine retroviruses*
bovine syncytial virus (BSV) and
bovine visna-like virus (BVV) which also produce syncytia in
cell culture (54*96).
However,
Their relationship to EBL is unclear.
neither agent is considered to be a significant
factor in the cause of EBL (149*150).
II. D. 1. Bovine Syncytial Virus
BSV is a bovine lymphotrophic retrovirus that was
originally isolated from buffy coat cells* spleen*
lymph
nodes and milk of lymphosarcomatous and normal cattle (150).
The target cell(s) of this virus is not known nor is the
effect of infection on susceptible animals. This viral agent
induces syncytia formation in monolayer cultures of bovine
fetal spleen cells and has been morphologically
13
characterized as a foamy virus (96*150).
BSV was
originally suspected of being the etiologic agent of EBL
(96).
Subsequent studies demonstrated that only 22% of LS
animals had detectable levels of serum antibodies to BSV*
whereas* over 90% of LS animals had detectable antibodies to
BLV (148,19).
Observations of experimentally infected
calves and immunodiffusion tests of sera from normal and
lymphosarcomatous cattle as well as cattle from multiple
incidence lymphosarcoma herds have not provided evidence for
a direct etiologic involvement of BSV in either the PL or LS
stages of EBL (148).
II. D. 2
Bovine Visna—Like Virus
A syncytia-inducing virus (BVV) isolated from a cow
with PL was found to be similar in structure to maedi,
visna, and progressive pneumonia viruses of sheep (149).
Calves inoculated with virus isolated from this PL cow
developed a mild nonpersistent lymphocytosis and
subcutaneous lymphatic nodules.
The relationship of BVV to
the induction of PL is unclear, but it does not appear that
this viral agent is a significant factor in the development
of PL or LS (149).
II. E. LYMPHOCYTES IN ENZOOTIC BOVINE LEUKOSIS
Lymphocytes in man and animals are composed of two
subpopulations: thymus—derived (T) lymphocytes, which are
involved in cell—mediated immunity and bursal—equivalent
(B)
14
lymphocytes*
primarily involved in humoral immunity.
An
understanding of the recognition and effector functions of
these lymphocytes in disease processes is clearly dependent
on the ability to enumerate and separate these cells for
functional analysis.
Various techniques
have been used for
the isolation and enumeration of T and B lymphocytes in the
bovine species and have been applied to BLV— infected
animals.
11. E. 1 Lymphocyte Isolation Procedures
Bovine peripheral blood mononuclear cells (PBMC) have
been isolated routinely using a density gradient of ficoll
(146).
This technique gives highly purified populations of
mononuclear cells with an average of 2% polymorphonuclear
leukocytes.
Numerous other techniques have been utilized to
isolate lymphocyte subpopulations.
Nylon wool columns have
been used to fractionate B and T lymphocytes (121) and yield
highly purified T (nonadherent) and B (adherent) lymphocyte
populations (95%)*
but recoveries are very low (2 to 20%).
Antibody— complement immunolysis has been used to selectively
deplete B lymphocytes from PBMC and hence enrichs the T
lymphocyte population (146).
This technique also gives low
yields of purified T lymphocytes.
Bovine serum albumin
gradients have been used to isolate subpopulations of bovine
lymphocytes on the basis of their bouyant density.
The
purity and yield of these preparations are also low (73).
The techniques currently used to isolate bovine
lymphocyte subpopulations do not give large quantities of
15
highly purified T or B lymphocytes.
Recently, an immuno-
affinity depletion technique using immunoglobulin-coated
surfaces has been used to isolate and purify murine
peripheral blood T and B lymphocytes (92, 95). This
technique has not been applied to purification of large
quantities of bovine lymphocyte subpopulations.
II. E. 2. B Lymphocyte Markers
The B lymphocytes have 3 generally recognized cell
surface receptors:
(37.36),
(2)
(1) surface membrane immunoglobulin (slg)
receptor for the Fc portion of immunoglobulin
(38.36) and (3) the receptor for complement (29).
These
markers have been used to distinguish bovine B lymphocytes.
The slg— bearing lymphocytes (slg'*') have been detected
by an immunofluorescence antibody technique which uses
fluorescein—conjugated F(ab')2 fragments of rabbit antibovine IgG (heavy and light chain specific)
recently, monoclonal anti— IgM (127,110,90).
(Rablg—FITC) and
Rablg-FITC is
specific for cells which contain slg (IgA, IgM, IgG) on
their surface since this marker is directed against the
neavy chains of all the immunoglobulin classes.
The anti—
IgM is specific for cells which contain surface IgM because
this antibody is directed towards the heavy chain of IgM (u
chain).
Isotype specific lymphocyte surface immunoglobulins
have not been as well studied in the cow as in certain other
species.
The isotypes on slg* bovine peripheral blood
lymphocytes appear to be mainly IgM and IgA (85, 55).
It
was found that anti—bovine IgM is a highly specific marker
16
for bovine peripheral blood B ly m p h o c y t e s <55* 90).
Detection of the Fc receptor on the surface of bovine B
lymphocytes has been accomplished using an
immunofluorescence technique* erythrocyte—antibody
complement rosetting (EAC rosette) technique and slg
staining (B5,142*110*156).
The complement receptor has also
been used to detect B lymphocytes (10).
The use of these
techniques has led to a wide range of values for bovine B
lymphocytes in the PBMC of normal cattle. Many of these
techniques are very cumbersome to perform.
II. E. 3. Bovine T Lymphocyte Markers
Markers which have been used for enumeration of bovine
T lymphocytes include:
cells (sRBC)
(127*142)*
(PNAR) (147)*
(1) receptors for sheep red blood
(2) receptors for peanut agglutinin
(3) alpha-naphthy1 acetate esterase (ANAE)
positive granules (71)* and (4) T lymphocyte surface
antigens (B4).
The frequency of lymphocytes which contain receptors
for sRBC has been measured by the use of a sheep erythrocyte
rosetting technique (E-rosette)
(73*127*142).
Peanut
agglutinin receptor—positive cells (PNAR*) have been
detected by a direct fluorescence technique using
fluorescein-conjugated peanut agglutinin.
This technique
has been reported to be a very specific means of enumerating
□ovine T lymphocytes (147* 55).
Recently* with the use of
monoclonal antibodies (MAb) directed towards T lymphocyte
surface markers*
it was found that PNAR can be found not
17
anly on T lymphocytes but also on other leukocytes (90).
ANAE staining has been used as a cytochemical marker to
enumerate bovine T lymphocytes (71), and the frequency of
bovine T lymphocytes has been measured by direct
immunofluorescence using anti— bovine T lymphocyte serum
(ATS)
(04).
Many of the above markers and detection techniques have
been used to enumerate T and B lymphocytes in normal and
BLV-infected animals.
The use of the above markers for
enumeration of bovine T lymphocytes has led to great
variation in the numbers of these cells in the PBMC of
normal as well as BLV-infected animals.
Recently, MAb
specific for the sRBC receptor on the surface of bovine T
lymphocytes have been prepared, characterized and used to
enumerate the frequency of T lymphocytes in the PBMC of
normal cattle (90).
II. E. 4. Frequency of T and B Lymphocytes in Enzootic
Bovine Leukosis
Some of the above techniques for B and T lymphocyte
enumeration have been used to study the frequency of these
cells in PBMC from cattle with AL, PL and LS.
Very little
information is available on the lymphocytes of AL animals.
One study of 8 cattle in the AL stage showed that lymphocyte
counts averaged between 3.4 x lO3 to 7.5 x 103 /mm3of whole
blood and between 12 to 50% of these cells were slg'*' (72).
An increase in slg^ lymphocytes in the PL phase of the
disease has been observed (110, 85, 72).
The values
averaged 2B% for BLV-negative <normal) and 63% far PL cattle
(110).
One report indicated that PL cows had 3.85 times
more slg*lymphocytes than did normal cows (85).
When EAC
rosetting was used to enumerate the frequency of £
lymphocytes in the PBMC of PL animals* values of 10% to 20%
for normal cattle* and 55% to 81% for PL animals were
observed (72).
Lymphocyte counts in the PL group ranged
from 11.0 x 103 to 22 x ID3 /mm73 of whole blood* and there
was a significant strong correlation between the percentages
of cells bearing the B lymphocyte markers and the absolute
lymphocyte counts (72).
Tumor development correlates with a drastic
modification of lymphocytosis (rapid increase or decrease of
the total lymphocyte count) and the presence in the
peripheral blood of lymphoblasts and lymphocytes with
altered morphological and karyotypic characteriseics (104).
Many animals with LS have lower percentages of circulating
slg + cells and lymphoid cells from tumorous lymph nodes of
these animals have a higher percentage of slg bearing cells
than normal lymph nodes (142).
Studies using E-rosette formation as a marker for
bovine T lymphocytes indicated that between 12.5 to 26% of
PBMC of normal cattle* 3.5 to 26.5% of PBL from BLV-infected
cattle (AL or PL status unknown) and 0.5 to 50% of PBL from
LS animals formed E-rosettes (142).
This study also
reported that in tho lymph nodes of BLV-negative animals*
8.5 to 30.5% of lymphoid cells formed E—rosettes.
E—rosette
19
positive cells from lymph nodes of BLV-infected animals
ranged from 7 to 43.5%.
Lymph node cells from LS animals
had 10% or fewer positive cells (142).
Investigations using
(ANAE) staining to identify T lymphocytes in cattle
concluded that an average of 47.7% ANAE positive cells were
present in the PBMC of normal cattle* and 20.5%
present in the PBMC of PL cattle
concluded that in the PBMC of PL
were
(71). Therefore, this study
there was a decrease in the
number of T lymphocytes.
Studies were conducted using an indirect
immunofluorescent antibody technique with rabbit anti— bovine
thymocyte serum (ATS) to detect T lymphocytes in the PBMC of
BLV-negative,
(84).
PL and LS cattle and tumor cells of LS cattle
The frequency of T lymphocytes in the PBMC of BLV-
negative animals ranged from 55 to 80%,
to 45%, and LS animals from 5 to
PL animals from 4
38%. The percentage of T
cells found in tumors ranged from 2 to
30%.
As described above, the use of such a variety of
techniques for T and B lymphocyte enumeration has led to a
wide range of values for these cells in both normal and BLVinfected cattle.
The MAbs recognizing bovine T lymphocytes
have not yet been used to assess the absolute numbers of T
lymphocytes in BLV-infected cattle.
II. E. 5.
BLV Target Cells
Since tumors present in EBL are neoplasms of the
lymphoid system, cells of the lymphocyte lineage were the
first candidates to be suspected as BLV-preferred targets.
20
This bias shown to be correct by demonstration of BLV
production in vitro from cultures of lymphocytes from BLVinfected cattle (10B* 120).
The target cell of BLV
infection has been reported to be the B lymphocyte as
evidenced by the presence of slg on the majority of tumor
cells and BLV-infected lymphocytes in the peripheral blood
of PL and LS cattle (72* 120).
However* a recent report
which investigated the phenotype of lymphoblastoid cell
lines derived from two cases of LS indicates that BLV may
also infect cells of the T lymphocyte lineage (91).
It appears that BLV is present in a subpopulation of B
lymphocytes of PL cattle (73).
A nylon wool separation
procedure was used to fractionate the B and non—B
populations of peripheral blood of BLV-infected cattle.
It
was discovered that the capacity to produce C-type particles
resided with the B lymphocytes (119).
This was demonstrated
by the use of transmission electron microscopy and the
induction of syncytia in bovine fetal spleen cells
(120*121*73).
From these studies*
it was concluded that 397.
of the B lymphocytes produced BLV particles.
This implies
that BLV may be associated with a certain subpopulation of B
lymphocytes.
Isolation of a subpopulation of BLV-infected
lymphocytes from AL and PL animals was accomplished by the
use of density gradients of bovine serum albumin (B5A) (73).
The B lymphocytes were reportedly separated into two
distinct subpopulations* one which produced BLV and the
21
second which did not produce BLV* but underwent spontaneous
blastogenesis when cultured in vitro <73).
The BLV-
nonproducer accounts for most of the cell—count increase in
PL as assayed by the uptake of tritiated thymidine which
measures DNA synthesis.
It is not known if the lymphocytes
which incorporate '~*H thymidine* but do not produce BLV*
contain the BLV provirus in a repressed state.
No further
characterization of either of these two cell populations has
been reported.
II. E. 6.
Immune Responses to BLV Infection
Humoral response to BLV antiyens has been observed
shortly after infection (49). The presence of antibodies
directed against the major glycoprotein of the viral
envelope (gp51) and internal viral core protein <p 24) is
detectable shortly after experimental BLV infection (49).
The anti—gp51 antibodies are consistently formed at a higher
titer than anti— p 24 antibodies and display virus
neutralizing activity and a strong cytolytic effect on BLV—
producing cells in the presence of rabbit complement (47).
Recently* a study was conducted to assess the humoral
and cellular responses in calves experimentally infected
with BLV (107).
The results showed that there was an
increase in the number of B lymphocytes* a decrease in the
number of T lymphocytes and reduced levels of IgM in the
sera of BLV infected calves.
These findings seemed to
indicate that the immune system of these calves was affected
by the viral infection and a state of immune—suppression was
22
induced (107).
Responses of lymphocytes from normal) AL* PL and LS
cattle to phytomitogens have been evaluated using the
lymphocyte blastogenesis assay (111*
112* 157* 132).
This
assay has been used to determine the functional ability of
both T and B lymphocytes to respond to mitogenic stimuli.
The T lymphocytes of normal cattle were found to be
stimulated with both phytohemagglutinin (PHA)
Concanavalin A (ConA)
(111)
132) and
(157). The T and B lymphocyte
populations responded to pokeweed mitogen (PWM) (111)132).
The mitogenic activity of PHA and PWM was studied on the
PBMC from cows with PL, and it was found that when using
stimulation indices)
the degree of stimulation was markedly
less for PBMC of PL cattle than for normal cows. Lymphocytes
from cows with PL cultured without phytomitogens
incorporated 5 times more tridiated thymidine than did
lymphocytes from normal cows (112).
It has been reported
that this spontaneous blastogenesis is BLV—antigen driven
because it can be inhibited by serum containing BLV
antibodies but not by normal serum (143).
The response of cultured PBMC from cattle affected with
EBL to the mitogenic activity of ConA has been determined
(157).
It was found that the stimulatory effect of ConA was
significantly decreased in lymphocytes from LS cattle in}
comparison to lymphocytes from normal animals.
The sera from cows with LS have been shown to inhibit
the blastogenesis of normal lymphocytes (6B). This
23
suppressive activity was not observed in sera from animals
that were infected with BLV but did not have LS.
These
results indicated that sera from l.S animals contained
suppressive factor(s).
No further characterization of this
serum factor(s) was reported.
The above studies indicated that the functional ability
of T lymphocytes from AL animals to proliferate in response
to mitogens appeared to be intact* but the functional
ability of T lymphocytes in the PL and LS stages of EBL
appeared to be suppressed.
It is known that the percentage
of T lymphocytes in the peripheral blood is significantly
decreased in the PL animal.
Therefore*
the reported
reduction in the response of the T cells in these animals
may be due at least in part to the low number of these cells
present in the lymphocyte blastogenesis assays.
Suppression of natural cytotoxic activity of
lymphocytes from PL and LS animals has been observed (164).
It was demonstrated that the cytotoxic activity of PBMC was
closely related to the stage of the disease.
The activity
of PBMC from cattle with PL and LS was decreased markedly
when compared to that of normal or AL animals (164).
II. F. MOLECULAR STUDIES OF BLV
IX. F. 1. Bovine Leukemia Virus
The putative etiological agent of EBL is BLV.
This
virus has been identified as an RNA virus of the family
24
Retroviridae and is characterized as a C-type oncornavirus
(158,75). Since the original discovery of BLV in the
supernatant fluids of short—tern* cultures of PL lymphocytes*
long-term cultures producing BLV have been obtained.
These
include cell lines derived from explanted tumor tissues or
various epithelioid and fibroblastoid cell lines infected
with BLV after co-cultivation with BLV-infected bovine
lymphoid cells (151*98*44).
Most of the information on the
structure and biosynthesis of BLV components has been
obtained from the fetal lamb kidney-BLV-producing cell line
(FLK-BLV)
(152).
The BLV virus has a sedimentation coefficient of 600S to
70QS with a buoyant density of 1.15 to 1.18 g/ml in sucrose
and 1.12 g/ml in metrizamide solutions (39*59*74*75).
The
diameter of mature BLV particles ranges between 68 and 125
nm (22*23*15B).
Particles contain a central electron— dense
nucleoid with a diameter of 40 to 90 nm* and it appears that
the nucleocapsid is icosahedral
a 70S RNA.
(155).
The virion contains
Denaturation of this virion RNA yields poly <A>-
containing subunits that sediment at 38S and are found to be
of molecular weight 2.95 x 10** daltons (ca.8.8 kb) in sodium
dodecyl sulfate polyacrylamide gels (57*25).
II. F. 2.
BLV Proteins.
The internal structural protein* p2* was the first
recognized BLV protein.
It has been purified and
extensively characterized (58*106*32).
This protein has
been partially sequenced and appears to share the
25
aminoterminal praline and carboxyterminal leucine of the p30
of all mammalian retroviruses.
However*
it has been found
that BLV p24 is antigenically distinct from the major core
protein of most other retroviruses.
Nucleic acid and amino
acid sequence homology has been found between BLV p24 and
the major p£4 core protein of HTLV I (118*128).
The major phosphoprotein of BLV* pl5* is a slightly
basic protein that exists in two populations of molecules
(70).
One is linked to the lipid bilayer of the viral
membrane* and the other is linked to viral RNA.
basic protein of BLV is pi2.
Another
This protein has been shown to
be in close proximity to viral RNA in BLV particles (144).
Zt has been reported that there is amino acid homology
between BLV pl2 and human T— cell Leukemia virus (HTLV I) pl2
(118*128).
The other basic protein plO is able to bind to
single-stranded DNA (93) Nature BLV particles also contain a RNA-dependent DNApolymerase (reverse transcriptase) of an approximate
molecular weight of 70*000 dal tons (41).
This enzyme is
strictly dependent on RNA and all four deoxyribonucleoside
triphosphates and*
irrespective of template-primer used*
displays a strong preference for Hg’*"*" over Mn*'*' (59*58*75).
The major envelope glycoproteins of BLV are gp51
gp60) and gp3D.
(or
These proteins are found associated as a
1*1 complex linked by disulfide bonds in viral particles
isolated under nonreducing conditions (18).
It appears that
these proteins are both derived from a precursor protein
gPr72"‘r"’
' and appear as a tetramer with two g p ^ molecules
buried in the membrane structure of the virus* while the two
gp51 molecules protrude outside (56*99).
The overall
structure resembles that of an inununoglobulin molecule.
The
gt*51 appears to function in the attachment of the virus to
the target cell.
Monoclonal antibodies to BLV gp51 have
been obtained in the mouse using either nondisrupted BLV
particles or purified gp51 as immunogens (16,15).
Antigenic
determinants on gp51 were shown to be involved in
determining both virus infectivity of and syncytia formation
by BLV—producing cells as well as functioning as a target
for antibody-mediated complement-dependent cytotoxicity
(15).
It appears that the carbohydrate residues on gp51 are
very important virus neutralization epitopes (15).
From recent reports*
it appears that the BLV genome
like that of the HTLV's (HTLV I,HTLV II, and HTLV III) may
produce another as yet uncharacterized protein referred to
as a trans—acting protein (tat) (138*137,131).
It has been
proposed that this protein is coded for by the long open
reading frame (LOR) region of approximately 16DD nucleotides
3' to the envelope gene.
In HTLV I, it has been proposed
that the HTLV tat protein mediates transcriptional trans
activation of the HTLV long terminal repeat (LTR) (138*137).
The transcriptional initiation signals for these
retroviruses lie within the LTR sequences which flank the
integrated provirus (137).
A 2— kilobase message capable of
encoding such a protein has been detected in BLV-infected
27
celIs* but the protein has not been found as an in vitro
translation product of the viral genome (131).
II. F. 3. BLV Genome.
The genome contains the coding capacity for at least
four internal structural proteins (p24* pl5, pl2* plO>,
reverse transcriptase (p70)* and two envelope glycoproteins
<gp51 and gp30> (99*100).
It appears that the BLV gene
order (5' —gag-pol—env—3') is similar to that of other
replication-competent lymphoid leukemia viruses (56).
It is
not clear whether other proteins are encoded by the viral
genome.
Recent studies seem to indicate that the BLV genome
contains ah additional coding region or LOR 3' to the
envelope gene (131).
This gene may code for a tat protein
which may play a vital role in the ability of the virus to
induce transformation (131).
Nucleic acid hybridization experiments run with single­
stranded BLV tP-cDNA have shown no homology between BLV and
any other retrovirus with the exception of HTLV I and II
(24*74,75*11B»12B).
Significant homology has been found
with HTLV I (118*128).
Up to 45 7. nucleic acid homology
exists between DNA coding for p24 internal core proteins of
BLV and HTLV I (113).
II. F. 4.
Morphogenesis.
Detailed investigations in various laboratories have
led to the conclusion that BLV has distinct morphogenesis
events from most other retroviruses (22*23).
Some
investigators have shown peculiarities in budding of BLV
viruses when comparing the budding processes of murine and
feline oncornaviruses to that of BLV <2B).
In short-term
lymphocyte cultures* packages of virus particles accumulate
in cytoplasmic bags surrounded by plasma membrane.
BLV
particles are rarely observed to be released from the plasma
membrane into the pericellular spaces or into cytoplasmic
vacuoles (151).
It was suggested that virus particles do not
mature at the cell surface but leave the cell by unusual
routes (22*23). This mechanism of virion maturation may be
responsible for the persistent infections observed with BLV.
Since viral antigens may not be present in large quantities
on the infected cell surfaces* BLV-infected cells may be
protected from lysis mediated by antibody— dependent
cytolysis mechanisms.
II. F. 5.
Viral Persistence
The presence of antibodies directed against the major
glycoprotein of the viral envelope (gp51) and internal viral
core protein (p24) is detectable shortly after experimental
BLV infection (49).
The gp51 antibodies are consistently
formed at a higher titer than p24 antibodies and display
virus neutralizing activity and a strong cytolytic effect on
BLV—producing cells in the presence of rabbit complement
(47).
Antigens do not appear to be expressed at detectable
levels on infected cell surfaces in vivo.
Consequently*
production of virus in the blood plasma appears to be
prevented.
It is not known where virus replication and or
29
viral antigen production actually takes place in infected
animals.
The BLV provirus can be demonstrated in the genome of
peripheral blood mononuclear cells (PBMC) and tumor cells
but not nonlymphoid tissues by Southern blot hybridization
analysis of the DNA of these cells (22,73).
There appears
to be host modulation of BLV antigen expression and
efficient control of virus spread.
The BLV proviral genome
can be activated by mitogens (108) or short— term cultivation
(48-72 hours)
(120).
Viral antigens can then be
demonstrated on the surface of infected cells by fluorescent
microscopy techniques (140), and mature virions can be
detected in the .pericellular spaces by transmission electron
microscopy (120).
The control of BLV antigen expression appears to be at
the level of transcription (77).
BLV mRNA transcripts
cannot be detected in noncultivated circulating lymphocytes
or tumor cells from PL or LS animals using solution
hybridization techniques (77,76).
Viral mRNA transcripts
can be detected in these cells using highly sensitive in
situ hybridization techniques.
The amount of viral mRNA
increases with short—term cultivation of these cells (77).
Therefore, there appears to be control of BLV antigen
expression.
This lack of expression of gp51 on the BLV-
infected cell surface could clearly prevent cell lysis by
antibody mediated cytolytic mechanisms and allow for the
expansion of BLV-carrying cells.
The mechanism by which the BLV provirus is
transcriptionally repressed in the lymphocytes of PL and AL
animals is not understood.
A plasma protein has been
reported to be responsible for this repression (63).
This
protein has an approximate molecular weight of 150*000
daltons.
It does not appear to be immunoglobulin or
interferon* but a unique protein contained in the plasma
fraction of BLV— infected bovine blood.
When cel Is are
separated from whole blood using density gradient
centrifugation and washed* this protein is diluted or
completely removed from the cell preparation as assessed by
the increase of BLV p24 production using competitive
radioimmunoassay on cell culture supernatant fluids.
Transcription of the BLV genome can then proceed.
II. E. 6.
Molecular Aspects of BLV— induced
Leu kemogenes is
The exogenous nature of the BLV genome to the DNA of
bovine cells as well as to the DNA of several other
vertebrate species has been well documented (75*30).
It has
also been established that the BLV genetic information
includes no one genes (80).
Liquid hybridization and
Southern blot analysis using a molecularly cloned BLV
proviral—specific probe show that most of the cells of all
BLV— induced tumors contain proviral genetic information
(78).
In individual tumors the site of proviral integration
appears to be mono- or oligoclonal
(78).
However* no
preferential integration site for BLV provirus* such as* in
31
close proximity to a cellular one gene sequence has ever
been demonstrated either in tumor cells or PBL of AL or PL
cattle (60*79).
Proviral insertion sites in the
lymphocytes of AL and PL cattle have been observed to be
polyclonal.
The BLV-infected lymphocytes of animals in PL
do not display abnormalities in their chromosomal ploidy as
do tumor lymphocytes (66).
There appears to be a multi-step process for tumor
development. The presence of the BLV provirus appears to be
necessary but* by itself*
insufficient to cause tumors (75).
The role of BLV proviral information in the development
and/or maintenance of the tumor state has been evaluated
(78*76). Cells of BLV— induced tumors contain at least one or
a portion of one provirus.
When deletions occur, they seem
to affect only the 5' half* as the 3' proviral sequences
seem to be conserved.
The conservation of the 3' proviral
sequences may be of functional importance at a particular
stage of disease development.
The tat gene appears to be
located in this portion of the genome (131).
Experiments
indicate that maintenance of the tumor state does not
require a continuous expression of BLV proviral DNA (79).
This may suggest that proviral transcription is important in
a specific stage of EBL. Possibly* the PL stage of EBL is a
manifestation of proviral gene expression.
Gene product(s)
such as the tat protein may induce rapid proliteration of
lymphocytes.
Tumor development may then be a consequence of
chromosomal rearrangements which occur when cells
32
proliferate uncontrollably.
This is one mechanism
hypothesized in the tumorgenesis of Burkett's lymphoma (35).
However* restriction enzyme analysis of a panel of ten tumor
DNAs has failed to demonstrate rearrangements of the
cellular homologues of several retroviral one genes
including c-myc, c-myb, c-erbA, c-erbB, c-abl. c-src, crasH » c-fes* and c-sis (BO).
Furthermore, the DNA of most
BLV induced tumors do not induce foci of transformed cells
when transfected into. NIH 3T3 cells.
II. F. 7. Relatedness to Human Lymphotrophic Retroviruses
The importance of studying the parameters involved in
the development of EBL has been emphasized by the close
genetic and antigenic relationship of BLV to some of the
members of the human T lymphotrophic virus family (HTLV)
(128,74,27). This virus family consists of three
retroviruses, HTLV I, II and III (162).
BLV is the closest
known relative of HTLV I and II (94,128,27,133,).
HTLV-I is
the etiologic agent of adult T-cell leukemia (ATL) and HTLVII has been isolated from one patient with hairy T-cell
leukemia (129,162).
Both viruses appear to induce chronic
leukemias and malignant lymphomas in man (165).
These
viruses have a tropism for the helper T lymphocytes
possessing the cell—surface antigen 0KT4 and have the
capacity to transform these cells and like BLV, induce
syncytia formation in vitro (162,68).
HTLV I, II and BLV
appear to have a similar mechanism of transcriptional
activation and may have similar mechanisms of neoplastic
33
transformation (137,138,135,131).
A better understanding of the parameters involved in
the development of BLV— induced lymphosarcoma may aid in the
understanding of the mechanismts) of leukemogenesis and
lymphomagenesis with the HTLV viruses.
CHAPTER III:
ENUMERATION OF T AND B LYMPHOCYTES IN
ALEUKEMIC AND PERSISTENT LYMPHOCYTOTIC CATTLE USING
MONOCLONAL ANTIBODIES THAT DISTINGUISH THESE CELLS.
HI.
A. INTRODUCTION
Limited information has been obtained on the immune
response of cattle to bovine leukemia virus (BLV) infection
and the sequence of cellular events that leads to
lymphocytosis and the development of lymphosarcoma.
In
animals which develop a persistent lymphocytosis (PL)* there
appears to be a loss of control of B lymphocyte
proliferation (72*84).
is unclear.
The reason for this lack of control
It may be mediated* at least in parti by a
decrease or increase in T lymphocyte numbers and* possibly*
alteration of the function of these cells.
Since T
lymphocytes help control B lymphocyte proliteration and
differentiation* any alteration in the T lymphocyte
population may have profound effects on the B lymphocyte
population (142).
In a recent report* evidence was
presented which demonstrated that calves experimentally
infected with BLV had an increase in the B lymphocyte
population as determined by the use of iromunofluorescence
assay using fluorescein-conjugated rabbit anti-bovine IgM
(107).
There was a corresponding decrease
in the absolute number of T lymphocytes using E—rosette
formation for enumeration of these cells.
34
A decrease in
35
the concentration of IgM in the serum was also observed
using quantitative radial immunodiffusion.
The data
reported suggest that BLV-infection may induce a state of
immunosuppression.
A comparison of the absolute numbers of bovine T
lymphocytes in the peripheral blood of adult asymptomatic or
aleukemic (AL), PL and BLV-negative cattle may indicate that
there is an expansion or depletion of these cells m
AL or PL animals.
either
This alteration in T lymphocyte numbers
may indicate that the T lymphocyte population is affected by
BLV infection.
Identification of peripheral blood lymphocytes of
cattle has been performed mainly on animals which exhibit PL
or LS <110*72,51,156*46).
Very few studies have been
reported on the AL animal (72).
Techniques which have been
used to identify bovine B lymphocytes in BLV-infected cattle
include the EAC resetting technique (142) and Fc receptor
(85) and surface immunoglobulin (slg) immunofluoresence
assays (110,B5,).
Techniques which have been used for
bovine T lymphocyte enumeration include E-rc-sette formation,
(142) an assay for terminal deoxynucleotidy1 transferase
activity (71), anti— thymocyte serum (ATS) and peanut
agglutinin (PNA) cell surface fluorescence assays <B4,147>.
The
use of the above techniques for T lymphocyte
enumeration has led to conflicting reports as to the numbers
of these cells in normal and BLV-infected cattle.
Recently, specific monoclonal antibodies (MAbs) that
36
distinguish bovine T and B lymphocytes have been developed
and characterized (90).
An anti-pan T cell antibody (B26a)
that reacts with the sheep red cell receptor on the surface
of T lymphocytes and an anti— IgM antibody'(PIgASA) that
reacts with cell surface IgM (slgM) have been characterized
as markers for bovine T and B lymphocytes) respectively
(90).
The specificities of these two MAbs mere determined
using microcytotoxicity)
dual fluorescence microscopy) and
flow microfluorimetry (FMF).
The present investigation was undertaken to determine the
frequencies and absolute number of bovine T and B
lymphocytes in the peripheral blood of AL and PL animals by
FliF using MAbs that distinguish these cells.
MAbs and
fluorescein labelled PNA (PNA—FITC) were used for T
lymphocyte enumeration anti— IgM and fluorescein labelled
rabbit anti-bovine immunoglobulin (Rblg—FITC) for B
lymphocyte enumeration.
XII. B. MATERIALS AND METHODS
III. B. 1. Animals
A total of 12 cows at least 3 years of age were used in
this study.
Animals were acquired from sale barns in
Southern Louisiana and were maintained at the Veterinary
Medical Farm of the Louisisana State University Agricultural
Center) Louisiana State University School of Veterinary
Medicine) Baton Rouge) Louisiana.
The BLV infection status
was monitored by detection of serum antibodies to BLV
structural antigens using the agar gel immunodiffusion assay
<49).
Of the 12 cattle tested* 4 were negative for BLV
antibodies* 4 were clinically normal but had BLV antibodies
<AL), and 4 were diagnosed as having PL using the criteria
of the International Conference on Bovine Leukosis (> 7.? x
ID3* lymphocytes/mm3 of whole blood for over a period of 3
months)
<2).
Animals were also vaccinated for various
bovine viral diseases and screened for infection by other
lymphotrophic retroviruses*
bovine syncytial virus (BSV) and
bovine visna-like virus <EVV).
VIZI and Table VIII.
(For details* see Chapter
1.)
III. B. 2. Isolation of Peripheral Blood Mononuclear
Cells
Peripheral blood was collected by Jugular venipuncture
into 20 ml vacutainer tubes containing 10 units/ml of
Pan he pr in’* (Abbott Laboratories).
The heparinized blood was
then diluted in an equal volume of phosphate buffered saline
(PBS)* pH 7.4.
Twenty ml aliquots were layered onto 20 ml
of sodium diatrizoate/ficoll (Histopaque” 1077* Sigma
Chemical Co.USA) in 50 cc conical centrifuge tubes (Costar*
Cambridge MA).
The gradients were centrifuged for 30 min at
1DO0 x g at room temperature* and the bands at the interface
between the plasma layer and HistopaqueR were collected and
uashed twice in PBS at 500 x g for 10 min at room
temperature.
Viability counts were performed using the
trypan blue exclusion method.
The cell concentration was
3B
adjusted to 10 x 10^ cells/ml with PBS.
The peripheral
blood mononuclear cell (PBMC) preparation usually contained
greater than 95X viable cells and less than 2X contamination
with granulocytes.
(Refer to Figure 111. 1. for flow
diagram.)
III. B. 3. Complete Blood Counts
Animals were bled by venipuncture every 2-3 weeks for a
period of 3 months to give a total of A counts per animal.
Blood was collected in tubes containing
ethylenediaminetetraacetic acid as an anticoagulant.
Total
leukocyte counts were made on an automatic cell counter.
Blood smears were prepared and stained by the Wright method*
and differential counts were made.
III. B. 4. Staining CelIs for B and T Lymphocyte
Enumeration
PBMC were isolated from each animal twice at 30 day
intervals.
To identify B lymphocytes*
3 x 10* PBMC were
incubated with either 100 ul (0.74 mg/ml) fluoresceinconjugated F(ab')=. fragments of rabbit anti— bovine IgG* H &
L chains specific (Rblg—FITC)
(Cappel Labs* Cochranvi1le*
PA> or 10 ul (1 mg/ml) of hab PIg45a (14. Davis* Washington
State University*
Pullman* U A . ).
To identify T lymphocytes*
3 x lO* PBMC were incubated with either 100 ul (1 mg/ml)
PNA-FITC (E.Y. Laboratories, U.S.A.) or 10 ul (1 mg/ml) pan
T cell Mab
B26a (W. Davis* Washington State University,
Pullman* W A . ).
Cells were incubated for 3D min at 4 C in
the dark* then washed twice in 1 ml aliquots of PBS with
39
0.02% Nahl3 to prevent antibody‘capping.
The washed
preparations of antibody coated cells (PIg45a and E26a) were
then reacted with 100 ul cf fluorescein-conjugated goat
anti-murine IgGf Igrt, IgG mix (GaM-FITC)
Cochranville, PA>,
described above.
(Cappel Labs.,
incubated for 30 min at 4 C and washed as
All cells were fixed with 1 ml 0.5%
formalin in PBS NaN3 .
An aliquot of cells were reacted with
GaM-FITC as a control for nonspecific binding.
III. B. 5. Detection of F 1uorescence—positive CelIs
Fluorochrome labelled cells were enumerated by FMF
(90).
The instrument used for screening and analysis was
the mercury arc lamp based flow cytometer (Becton Dickinson
Hg— arc F A C S ™ Research Analyzer, Becton Dickinson
Immunocytometry Systems Inc.* Mountain View, CA).
The
instrument measures individual cells simultaneously for
fluorescence (autofluorescence and fluorescence imparted by
fluorochrome— coupled antibodies or other specific
fluorochrome reagents bound to a cell membrane), size
(measured by electronic volume) and granularity (measured by
wide angle light scatter).
Data on 5000 cells were collected in list mode on each
sample for analysis.
Two parameter data, scatter versus
fluorescence were recorded in dot plot, contour plot (with
concentric lines outlining areas of highest cell density)
and isometric display using the data analysis programs
provided for the Beckton Dickinson Consort 30.
The percent
of labelled cells was determined with the computer program
40
by setting a marker at the appropriate channel on a contour
profile of control cells (exposed to GaM-FITC alone) such
that 95% of the signal imparted by cells fell between
channels 10 and 80.
Erythrocytes and platelets were
excluded from analysis by setting the trigger threshold and
gates to exclude low scatter and low volume signal* thereby
excluding cells smaller than lymphocytes.
III. B. 6.
Statisiteal Analysis
The statistical significance of the total UBC*
lymphocyte* and absolute B and T lymphocyte counts was
assessed with an unpaired Student's t test (136).
III. C.
RESULTS
III. C. 1.Hemogram
The total UBC and lymphocyte counts used for
statistical evaluation were obtained from 4 individual
bleeding times (2-3 weeks apart) for each animal.
Four
animals were evaluated from each group of AL* PL and BLV—
negative control animals.
The mean UBC from BLV—negative
cattle was 6.7 x 103/mm3 of whole blood* from AL animals*
9.7 x 103/mm3 * and from PL animals* 26.5 x lCP/mnr* (Fig.
III.2).
The mean lymphocyte counts from BLV—negative cattle
was 3.3 x lCP/mm3 * from AL animals* 5.0 xlO^/mm3 * and from
PL animals was 23.5 x lO^/mwr* (Fig. III. 2).
These data
show that there was a significant increase (p < 0.005) in
the both the UBC and lymphocyte populations in the
peripheral blood of AL and PL cattle tested (Table III. 1).
41
III. C. 2. Frequencies of T and B Lymphocytes
A FMF analysis of one representative animal from each
BLV group (BLV-negative* AL and PL animals) was selected to
demonstrate the staining characteristics of that group.
Animal R 56 represents the BLV—negative group of animals
(Fig.
III.3).
Animal Y 75 represents the AL group (Fig.
III.4) and animal B 10 represents the PL group (Fig. III.5).
When PNA was reacted with PBMC from BLV-negative, AL and
animals,
three patterns of labelling were observed.
PL
In
uninfected animals* T lymphocytes invariably exhibited high
expressions of the PNA receptors (Fig.
III. 3a).
B
lymphocytes and null cells exhibited low expression or
inapparent expression (i.e.*
detected by FMF).
levels of expression not
Cells from animals in the AL phase
contained a high percentage of brightly stained PNA positive
cells and a small population of the dimly stained PNA
positive cells (Fig. III. 4a).
In contrast*
in cell
preparations from PL animals* a large population of dimly
stained PNA positive cells were present and there was only a
small population of brightly stained cells (Fig. III. 5a>.
When PBMC from normal* AL and PL cattle were reacted
with B26A (pan T cell antibody)* the positive cells
corresponded to the brightly staining PNA cells (Figs.
3b*4b*5b).
III.
The percentage of B26a positive cells ranged
from 10—20% fewer than PNA positive cells (Table III. 2).
There was a decrease in the percentage of labelled cells (
PNA or B26a) in the PBMC of PL animals.
Cells which reacted with Rablg—FITC (sly*) and PIg45A
(slgM-*") corresponded with the percent of dimly staining PNA
positive cells in all animals tested (Figs.
III.3c,d,4c,d,5c,d).
The percentage of slgM* cells
corresponded with the slg"~ cell population (Table III.2).
In the PL animals,
the staining intensity was much greater
for the slgli* bearing cells than for the slg* cells (Fig.
III. 4c,d>.
This indicated that the majority of B
lymphocytes in the PBMC of these animals were IgM— bearing
cells and that these cells contain large quantities of IgM
on their surfaces.
The percentage of slgM* cells equaled
the percentage of slg* cells in most cases (Table III. 2).
These results indicated that even though the anti— IgM
antibody was an intact Ig molecule,
it was not binding to
cells other than B lymphocytes via the receptor for the Fc
portion of the Ig molecule.
There was an increase in the
percentage of both slg“" and slgM* cells in the PBMC of PL
animals when compared to either AL or BLV—negative control
animals.
The pattern of labelling differed only in one animal,
632 (Fig.
III. 6).
This was a PL animal with a high level
of lymphocytes (72 x 103 /mm3 of whole blood) of which B75i
were positive for the PIg45A marker (Table III.2). A number
of slg~ cells appeared to react with both B26A and PIg45A
suggesting the cells may co-express slg and the B26A
antigen. The staining characteristics of this animal's PBMC
showed that the majority of its cells stained very intensely
43
with the PIg45a marker,
indicating that these cells had
large quantities of IgM on their surfaces (Fig. Ill, 6d)
III. C. 3.
Absolute Numbers of T and B Lymphocytes
The absolute numbers of T and B lymphocytes (B26a"‘ and
slgM*) in the PBMC of animals tested were determined by
multiplying the percentage of these cells by the total
lymphocyte counts.
Four animals in each group of AL and
BLV-negative control animals and 3 PL animals were used.
Lymphocytes harvested from 2 bleedings (30 days apart) were
used in this study.
Data on 500P cells were collected for
each sample tested.
The statistical information was
obtained by using raw data for each group (Table III. 3).
The absolute numbers of B and T lymphocytes in the
peripheral blood of the BLV-negative* AL and PL animals
tested are summarized in Figure III. 7.
The number of T
lymphocytes in the PBMC of AL and PL animals was
significantly higher (significance at the level of (p <
0.DOS) than those of BLV— negative animals (Table III. 3).
The number of B lymphocytes in the FBMC of PL animals was
significantly higher than that of BLV—negative control
animals (p < 0.005).
There was no significant difference in
the number of B lymphocytes in the PBMC of AL animals when
compared to BLV—negative control animals.
Absolute numbers of B and T lymphocytes from the PBMC
of animal 632 were not averaged into the counts for PL
animals because under the labelling conditions used in this
study we were unable to distinguish B from T lymphocytes
44
because a cell population appeared to stain with both T and
B lymphocyte markers.
III. D. DISCUSSION
Monoclonal antibodies were used to determine the
frequencies of T and B lymphocytes in the peripheral blood
of AL and PL cattle using monoclonal antibodies that
distinguish these cells.
An overall increase in the
lymphocyte population in AL and PL animals tested when
compared to BLV—negative control animals.
The increase in
the lymphocyte numbers of AL animals was attributed to an
increase in the T but not the B lymphocyte population.
In
the PL animals* an increase in both the T and B lymphocyte
populations was observed.
These experiments indicate that
not only the B lymphocyte population but also the T
lymphocyte population may be affected by BLV infection.
In order to assess the increase of B and T lymphocytes
in the peripheral blood of AL and PL cattle*
it was
important to look at absolute numbers of these cells based
on their frequencies in the PBMC and relative to the total
lymphocyte counts.
Relying on the percentage of cells alone
leads to an inaccurate assessment of the number of cells
present in the circulation of individual animals because of
the increase in the total lymphocyte numbers in AL and PL
animals.
Most of the information reported on T and B
lymphocytes in BLV-infected animals is reported as
percentages not as absolute numbers (72*B5*142).
This is
45
probably the main reason that the increase of T lymphocytes
in these animals has gone unnoticed.
The use of MAbs to assay bovine T and B lymphocytes has
many advantages over other methods of evaluation (i.e. SRBC
rosetting, enumeration of the Fc receptor positive cells*
ANAE staining).
These methods are tedious* and the results
have been difficult to reproduce in different laboratories.
In cattle* these techniques have yielded wide ranges of
values for peripheral B and T lymphocytes (72*142*84).
Moreover*
it has been demonstrated that up to 10% of E—
rosetting bovine PBMC may be slg~ (147).
The existence of a
population of T lymphocytes having receptors for the Fc
fragment of IgG has also been demonstrated (147).
These
findings could account for some for the variability
previously observed in the characterization of the
frequencies of T and B lymphocytes in BLV-infected animals.
In the experiments reported here* an anti— IgM antibody of
the IgM subclass and F(ab')2 fragments of IgG mere used to
enumerate bovine B lymphocytes and to avoid possible
nonspecific staining of other PBMC.
PNA-FITC mas found to stain T lymphocytes brightly and
B lymphocytes and null cells dimly* forming two distinct
populations with cells from most animals tested.
Cells
labelled with B26A and Rablg and Pig45A corresponded with
the brightly and dimly PNA-FITC labelled cells respectively.
Preliminary studies using dual fluorescence analysis with
granulocyte free HistopaqueR purified PBMC* fluorescein
labelled PNA versus phycoerythrin—avidin biotinated GaMIg
revealed B26A reacts with cells expressing a high
concentration of the PNA receptor and that RbXg—FITC and
PIg45A react with cells expressing a low concentration of
PNA (90).
Therefore* the data presented in this report is
in agreement with these findings.
The staining
characteristics of PIg45a closely parallel those of Rablg
indicating that this marker is highly specific for bovine B
lymphocytes. These results give further evidence that the
MAb markers are specific for the lymphocyte subsets.
The staining characteristics of lymphocytes from animal
632 seem to indicate that there is a subpopulation of
lymphocytes in some BLV-positive cattle with very high
lymphocyte, counts that carries both B and T lymphocyte
markers on their cell surfaces.
This is an observation
consistent with SRBC resetting studies reported previously
(147).
Additional studies by dual fluorescence analysis are
needed to analyse carefully the antigen expression on the
cells in this animal.
There is also an increase in the
number of cells that bind the PIg45A MAbs alone.
antibody is an anti-bovine IgM antibody*
Since this
it suggests that PL
is attributable* at least in part* to an ‘increase in the
number of slgM-bearing lymphocytes. These cells are most
likely immature (virgin) or memory B lymphocytes (142).
Perhaps this is the BLV-infected population.
This
hypothesis may be proven by isolating this population of
cells and performing DNA hybridization on its purified DNA
47
with a specific BLV-DNA probe.
Some of the BLV—negative and BLV-infected animals used
in this study
had concurrent infections with other bovine
lymphotrophic viruses (Table VIII.
1).
There did not appear
to be any correlation between the infection with other
retroviruses* BSV and B W
and the BLV status (AL or PL) of
the animals used in the above study.
that both BSV and BVV
(148* 149*11*96).
Ithas been reported
have been found in
PL and LS animals
It was once considered that these viruses
were responsible for EBL (148*149*11).
However*
it was
found that the incidence of infection by these viral agents
in PL or LS animals was low (<2570* whereas* greater than
9DTi of animals with EBL have antibodies to BLV antigens.
No
direct connection between either of these virus infections
and the AL or PL state has ever been established (96).
All AL and PL animals were positive for BLV antibodies
throughout the course
of this study. In
increase in the total
WBC* lymphocyte
cattle with PL* an
count and B lymphocyte
population in the peripheral blood has been documented
<110*85*72).
An elevation in the T lymphocyte population in
PL animals has not been previously reported.
This study is
also the first documentation of an elevation of the T
lymphocyte population of AL cattle.
Therefore* the data
obtained from the experiments reported herein suggest that
there is an alteration in the T lymphocyte numbers of BLVinfected animals.
Identification of the particular T
lymphocyte subset that is affected by BLV-infection must
AB
wait for the development of additional monoclonal antibodies
to bovine T lymphocyte markers.
The reason for increase in the numbers of T lymphocytes
in the peripheral blood of AL and PL animals is unclear.
The T lymphocyte may be a target cell for BLV infection and
as a result of infection there could be a T-cell
lymphocytosis.
To address this hypothesis* DNA from
purified T lymphocytes of BLV-infected animals must be
acquired and DNA hybridization analysis must be performed
using a highly specific BLV—DNA probe.
The T lymphocyte
population may be responding to the presence of a serum
protein(s) which may augment the mitogenesis of these or a
subpopulation of T lymphocytes.
It has been shown that the
BLV proviral genome codes for a trans activational
transcriptional protein (tat? which mediates transcriptional
activation of BLV proviral gene sequences and perhaps host
cellular genes (131).
Perhaps* this gene product is
responsible for augmenting the T and B lymphocytosis
observed in BLV-infected animals.
49
©
©
©
HISTOPAQUE®
SEPARATION
©
\ 7
©
DILUTED PERIPHERAL BLOOD
1 :2 WITH P B S
LAYERED 2 0 m l ALIQUOTS ON
2 0 m l HISTOPAQUE ( d e n s i t y ,
1 .0 7 7 g /m l)
CENTRIFUGED 1 0 0 0 x g , 3 0 min,
25C
REMOVED BANDED CELLS AT
INTERFACE
WASHED X 2 WITH P B S ,
SOOXg 1 0 min, 2 5 C
\
PERIPHERAL BLOOD MONONUCLEAR CELLS
(PBMC)
Figure I I I . l .
Procedure f o r Is o la tio n o f Bovine P e rip h e ra l
Blood Mononuclear C e lls Using D en sity G radient C e n trifu g a tio n .
50
60
?0
60
|
u
22
H A leu k em ic
of
C e 11s
o
o 20
18
Number
m
£
E
\
tn
NBC
I PL ye mr spi hs ot ecny tot t1c
m
0
j:
2C
BLV Neg
|J L y m phocytes
16
14
I
12
1
03
10
I
6
6
/
/
/
4
2
/
/
/
0
2
^54 ^ 6 Y73 Y12 *32 Y?4 Y?S ®10 Y11 Y13 Y632
Animal
Numbers
Figure III.2. WBC and Lymphocyte Counts fran Peripheral Blood
of Bovine Leukemia Virus Infected Cattle
51
7/2G/8S
FACS Analyzer I
Sample 10 : SG/PtIA
Sample t a g : 002
7/2G/85
FACS Analyzer I
1*
1
0
ma
m
«w
t>
Sample ID : SG/B26
Sample tag ; 004
ma
(l
j
u
JL
a
a
a
rw
7/2G/8S
FACS Analyzer 1
Sample ID : PIg45A/56
Sample tag : 007
7/2670S
FACS Analyzer I
Sample ID : 56/F<ab
Sample tag : 003
n
a
a
ma
u
u
a
fl
is
oa
**io°
oa
•D>
Figure III.3. Fluorescence Activating Cell Sorting Contour Plots
of Lymphocytes from Bovine Leukemia Virus Negative,
Control Cow #56
52
'4
!“ «
• *
."■vjr-v */y
PNR-Y 75
V*
T
• ••;•/** /.
?.s?*IB-Y75
B26R-Y75
BCRTTER,
TLti
PNA-Y 75
B26R -Y 75
PIB45fl-Y?5
d
c
t)
y
* v!ofy=*
*
■
i;
.* .A'-
*tu*
*
<i^V
B‘3«
•I9-Y7S
P I g « 3fl-Y 73
6
Figure III.4.. Fluorescence Activating Cell Sorting Analysis Profile
of Lymphocytes from Bovine Leukemia Virus Infected,
Aleukemic Cow #75
SCATTER.
53
SCATTER
PN A -B 1B
B26R -B I 0
P I g 45ft-810
u
IA
Figure III.5. Fluorescence Activating Cell Sorting Analysis Profile
of Lymphocytes from Bovine Leukemia Virus Infected,
Persistent Lymphocytotic Cow #B10.
54
7/26/8S
FACS Analyzer I
Sample ID : PNA/632
Sample tag : 010
7/26/B5
FACS AnaJyier I
Sample ID : B2SA/632
Sample tag : 012
ma
Ma
u
£
o
s
a
a
aa
n.i IB
7/26/85
FACS Analyzer I
Sample ID : PIg45A/632
Sample tag : 015
7/26/85
FACS Analyzer I
Sample ID : F(ab‘12/632
Sample tag : 011
W
HUT
TTr
ie'
IB
IQ'
ID
:-"h M* f«
i^n*
.
‘
z
j
* 1»*
| -1 11muj i
a°
IB
IB*
IXI
IB
F ig u re I I I . 6 . Fluorescence A c tiv a tin g C e ll S o rtin g Contour
P lo ts o f Lymphocytes from Bovine Leukemia Virus
In fe c te d , P e rs is te n t Lymphocytotic Cow #632
55
12
^ BLV Nag
^ Aleukemic
Persistent
Lymphocytot1c
C e lls
10
*TJ
O
O
m
I
P B Lymphocytes
}
T Lymphocytes
e
0
of
X
2
6
Number
CO
E
E
\
CO
1
G>
Id
0
R
R
R
a
ti l
Y
52 54 56 73
Y
Y
Y
Y
12 32 74 75
Animal
B
Y
10 11
Y
Numbers
Figure III.7. Absolute Numbers of Peripheral Blood T and B
Lymphocytes fran Bovine Leukenia Virus Infected
Aleukemic and Persistent Lymphocytotic Cattle
13
Table III. 1. Statisical Analysis of VJBC and
Lymphocyte Counts from Bovine Leukemia
Virus Infected Cattle
BLV* Status
T Value
Degrees of
Freedom
P Value
WBC
BLV (-) Vs BLV (AL)
4.942
29
'0.005
BLV (-) VS BLV (PL)
3.322
30
0.005
BLV (-) Vs BLV (AL)
5.439
28
0.005
BLV (-) VS BLV (PL)
3.268
29
0.005
Lymphoc\rtes
BLV (-) = Cattle with no detectable serum antibodies
to BLV antigens using- iitmunodiffusion test.
BLV (AL) - Cattle with detectable serum antibodies to
to BLV antigens but have no clinical signs of disease.
BLV (PL) = Cattle with detectable serum antibodies to
BLV antigens and have persistent lymphocytosis.
Table III. 2. Frequencies of T and B Lymphocytes from
the Peripheral Blood of Bovine Leukemia
Virus Infected Cattle
*
BLV
Status
No. of
Animals
Percent o£^Positive Lymphocytes
% T
% B **
PNA
B26a
SIg
SIgM
BLV (AL)
4
82 + 6+ 66 ± 9
16 + 11 21 + 13
BLV (PL)
3
45+3
53 + 16 58 + 11
BLV (-)
4
79 + 11 56 + 12 26 +, 6
26 + 7
1
44 + 11 57 + 6
77 ± 11
632++
34+6
87 + 7
*
BLV Status indicates: BLV (-) = Cattle negative for BLV
antibodies. BLV (AL) = Cattle positive for BLV antibodies
but no clinical signs. BLV (PL) - Cattle positive for
BLV antibodies and have persistent lymphocytosis.
**
Frequency of T lymphocytes detected by peanut agglutinin
(PNA) and monoclonal antibodies to pan T cells using
microfluorimetry. Frequency of Blymphocytes detected by rabbit anti-bovine
IgG (h & L chains specific) (F(ab’)2 fragments and
monoclonal antibodies to surface bound IgM using
microfluorimetry.
+
Mean ar standard deviation
++
PL animal with high lymphocyte count.
58
Table III. 3. Statistical Analysis of T and B Lymphocytes
frctn Bovine Leukemia Virus Infected Cattle
BLV*
Status
T Value
Degrees of
Freedom
P Value
T Lymphocytes
BLV (-) vs BLV (AL)
5.599
12
0.005
BLV (-) vs BLV (PL)
5.382
14
0.005
BLV (-) vs BLV (AL)
0.132
14
BLV (-) vs BLV (PL)
6.250
12
B Lymphocytes
**
N.S.
0.005
*
BLV (-) = Cattle with no detectable serum antibodies
• to BLV antigens using immunodiffusion test.
BLV (AL) = Cattle with detectable serum antibodies to
BLV antigens but have no clinical signs of disease.
BLV (PL) = Cattle with detectable serum antibodies to
BLV antigens and have persistent lymphocytosis.
**
Not significant at the level P = 0.1.
CHAPTER
IV:
PURIFICATION OF T LYMPHOCYTES FROM BOVINE
PERIPHERAL BLOOD MONONUCLEAR CELLS USING IMHUNO
AFFINITY DEPLETION (“PANNING")
IV. A.
INTRODUCTION
It has been documented that B lymphocytes are target
cells for bovine leukemia virus (BLV), the etiologic agent
of enzootic bovine leukosis (EBL> (8,108,75).
The role that
T lymphocytes play in the disease process is unknown.
It
was demonstrated that there is an increase in the number of
circulating T lymphocytes in BLV-infected aleukemic (AL> and
"s.
persistent'lymphocytotic (PL) animals (Chapter III).
Purified T lymphocyte populations are necessary to determine
the significance of this increase in the progression of EBL.
There are numerous techniques for purification of T
lymphocytes from human and mouse peripheral blood
(£4,86,134).
However, no techniques are currently available
for the efficient separation of large quantities of bovine
lymphocyte subsets.
Purification of peripheral blood
mononuclear cells (PBMC) is done routinely by centrifugation
on ficoll of density 1‘.077 g/ml
(146).
Attempts to
accomplish further separation of bovine T lymphocyte subsets
using various techniques have failed to give reproducible
separation results.
Purification of bovine T lymphocytes by
nylon wool adherence has given high purity of these cells,
59
6D
however the yield of purified cells uias less than 37. of the
original cells applied (121).
Antibody-complement
immunolysis has been used to selectively deplete B
lymphocytes from PBMC of cattle and enrich the T lymphocyte
population but recoveries are also law (146).
The efficient separation of murine T lymphocytes has
been reported using plastic surfaces coated with anti-murine
immunoglobulin antibodies.
This technique is referred to as
immuno-affinity depletion ('panning")
(92).
This technique
has' £-iven good yields of highly purified murine T
lymphocytes.
Polystyrene surfaces have also been employed
as an insoluble matrix for the affinity reagents used to
separate lymphocyte subpopulations from other species
(95jl63).
A procedure using “panning" has not been reported
for the efficient separation of bovine T lymphocytes.
Surface markers have been used to identify bovine
lymphocytes (Refer to Chapter III).
Those cells staining
with anti-bovine immunoglobulin conjugated with fluorescein
isothiocyanate (slg-bearing cells) are suggested to be B
lymphocytes (B5*55)» and those staining with peanut
agglutinin conjugated with fluorescein isothiocyanate (PNAFITC), T lymphocytes (124*55).
The purpose of the present investigation was to explore
the application of "panning" to enrich T lymphocytes from
the PBMC of BLV— infected cattle so that large numbers of
these cells could be obtained for further testing.
The
"panning" technique is based on the fact that bovine B
61
lymphocytes have immunogiobulin
<Ig> on their cell surfaces
and that bovine T lymphocytes do not.
Therefore*
plastic
surfaces coated uiith anti-bovine Ig will specifically bind
and therefore deplete B lymphocytes from PBMC.
The T
lymphocytes can then be recovered from the nonadherent cell
fraction.
IV. 3.
MATERIALS AND METHODS
IV. B. 1.
Isolation of Peripheral Blood Mononuclear
Cells
Forty ml of peripheral blood were obtained by
venipuncture of the Jugular vein from adult AL cows (Y 12* Y
32, Y 74) and BLV-negative cows (R 52, R 56)
Chapter VIII).
(Refer to
The blood was diluted in an equal volume of
phosphate buffered saline (FBS), pH 7.4.
Twenty ml of
diluted blood were layered on 20 ml of Histopaque" (density
1.077 g/ml)
(Sigma* U.S.A.),
in 50 ml polypropylene*
conical
centrifuge tubes (Costar* U.S.A.) and centrifuged at 1000 x
g for 30 min at 25 C.
Cells at the interface between the
plasma and Histopaque" phases were removed with sterile
Pasteur pipettes* washed twice with PBS <500 x g for 10 min
at 25 C), pooled and resuspended in 2 ml PBS.
Cell
viability was determined by the trypan blue exclusion test*
and the cell suspension was diluted to a concentration of
2.0 x IQ7 cells in PBS.
diagram.)
(Refer to Figure III.l. for a flow
62
IV.
B. 2. Coating Plastic Flasks with Immunogiobulin
To coat polystyrene tissue culture flasks with antibovine Ig a previously described procedure (92) was used
with the following modifications.
Two ml (2.5 mg/ml) of
F ( a b ')s fragments of rabbit anti— bovine IgG, heavy and light
chains specific (anti— bovine Ig) (Cappeli U.S.A.) were added
to each 25 cm2 polystyrene tissue culture flask (Costar,
U.S.A.).
The concentration of anti-bovine Ig was critical?
if less than 5 mg per flask were used* the yield and purity
of the T lymphocyte preparation were low.
The flasks were
incubated on a level table for 19-24 hours at 4 C to
optimize Ig binding.
Immediately before use* the unbound
anti-bQvine Ig was transferred to a new, sterile flask which
was then stored at 4 C for future use.
The Ig-coated flask
was then washed four times, each time with 5 ml PB5.
IV. B. 3.
Affinity **Panning" Procedure
The PBMC were gently agitated to assure a single cell
suspension.
coated flask.
Two ml (4.0 x 107 cells) were added to an IgThe flask was placed on a level table and
gently swirled to assure even distribution of the cells over
the entire dish.
After incubation for 15 min at 25 C, the
nonadherent cells were carefully resuspended by rocking the
flask, then removed with a pipette and washed twice with PBS
(500 x g for 10 min at 25 C).
Maximum binding of cells to
precoated surfaces was determined to occur within fifteen
minutes of incubation at 25 C.
Longer incubation times did
not increase the cell binding capacity.
Shorter incubation
63
periods resulted in lower T lymphocyte yield and higher slg—
bearing cell contamination.
Cell viability was determined
by trypan blue exclusion test, and the cells were
resuspended to a concentration of 10 x ID*" viable cells/ml
in PBS to prepare for quantitation of lymphocyte subsets and
monocytes.
(Refer to Figure IV. 1. for a flow diagram.)
IV. B. 4. Determination of Lymphocyte Subsets and
Monocytes
For detection of contamination with slg bearing cells,
nonadherent cells were incubated with fluorescein-conjugated
F(ad')a
fragments of rabbit anti-bovine IgG, heavy and
light chains specific <Rablg-FITC>, (Cappel, U.S.A).
To
detect T lymphocytes, nonadherent cells were incubated with
PNA-FITC (E.Y. Laboratories,
U.S.A.).
Approximately, 0.3 ml
(3.0 x 10^ ) cells were mixed with either 0.1 ml <0.74
mg/ml) of Rablg-FITC or 0.1 ml
(2.5 mg/ml) PNA-FITC.
The
cells were incubated for 30 minutes at 4 C then washed twice
(500 x g for 10 min at 25 C) with 1 ml of PBS containing
0.02% NaN3 . This concentration of NaN? was used to insure
that capping and shedding of Igs did not occur on the slgbearing cells.
Rablg-FITC and PNA-FITC positive cells were enumerated
by counting 200 cells at 1000 x magnification with the aid
of a fluorescent microscope equipped for phase contrast and
incident UV light i 11 uniin at ion (Leitz, Germany).
The
detection of monocytes in the nonadherent cell population
was accomplished by using a nonspecific esterase staining
64
procedure (83).
IV. C.
RESULTS
IV. C. 1.
T Lymphocyte Vield after "Panning*
The results Df T lymphocyte recovery or yield after
■panning* using the PBMC of coins are summarized in Figure
IV. 2.
An average of 31 x 10** PNA-positive (T lymphocytes)
were added to an anti— Ig coated flask.
15 minutes,
After incubation for
the T lymphocyte recovery in the nonadherent
fraction averaged 15 x 10** cells.
This was an average yield
of 48%.
IV.
C. 2.
T Lymphocyte Enrichment and B Lymphocyte
Depletion
The results of T lymphocyte enrichment from PBMC using
■panning* are summarized in Figure IV. 3.
After separation
on a density gradient of HistopaqueR , the PBMC fraction
consisted of an average of 77% T lymphocytes.
After
“panning", the nonadherent cell fraction was composed of an
average of 95% T lymphccyte population.
An average of 85% of the slg-bearing cells were
depleted from the PBMC using "panning"
(Fig.
IV. 4).
B
lymphocytes accounted for an average 3% of the contaminating
cells in the nonadherent fractions.
The rest of the cells
were not identified by the methods used and were assumed to
be null cells.
Non—specific esterase— positive cells
(monocytes) were not detected in the nonadherent cell
65
fractions.
IV. D. DISCUSSION
An affinity depletion technique for the efficient
enrichment of bovine T lymphocytes from peripheral blood has
been described.
Antibodies to bovine Igs attached to
polystyrene tissue culture flasks mere used to separate slgbearing cells from T lymphocytes.
The T lymphocyte yield
and purity using "panning" on the PBMC of cattle is higher
than any method previously reported. For example*
the use of
nylon wool adherence to deplete B lymphocytes from PBMC
resulted in high T lymphocyte purity (96%) but low cell
recovery (2.5%) in the nonadherent fraction (121).
However*
the use of "panning" resulted in a 95% T lymphocyte purity
and 48% recovery.
This technique gave reproducible results with BLVinfected AL animals (Y 12* Y 32 and Y 74) and BLV-negative
control animals (R 52 and R 54) which had normal white cell
counts in the range of 6.8-9.B x lCF/mm3 in whole blood and
52—64% lymphocytes (121).
“Panning" did not give good
yields (< 10%) of purified T lymphocytes from PBMC of BLVinfected cattle which exhibited a persistent B cell
lymphocytosis (PL).
These animals typically had elevated
WBC (> 7.5 x lCP/mm3 in whole blood).
They also had a high
percentage of lymphocytes (> 50%)* of which the majority
60%) were slg-bearing cells.
(>
When PBMC from these animals
were added to an anti-Ig coated flasks, the number of slgbearing cell binding sites on the flask apparently became
saturated* and the nonadherent cell fraction mas highly
contaminated with slg-bearing cells.
Therefore,
in order to
obtain an efficient T lymphocyte recovery with high purity,
modification of the above procedure was attempted.
A second
■panning" step to remove the slg— bearing cells not bound by
the first "panning" was employed.
This technique did not
give good yields of highly purified T lymphocytes.
0 trier
separation techniques such as B lymphocyte depletion by
antibody, complement-mediated cytolysis (14&), and
separation on PercollR density gradients as well as affinity
column chromatography using Affi-Gel anti—FITC columns <
BioRad, U.S.A.) were used to attempt to purify T lymphocytes
from the PBMC of PL cattle.
No procedure gave good yields
of purified T lymphocytes from these animals.
The affinity "panning" for efficient enrichment of
bovine T lymphocytes from PBMC of normal and AL cattle was a
simple, reproducible method that gave a highly purified,
viable T cell population in as little as two hours.
The
procedure used a minimal amount of reagents, and the unbound
anti— Ig could be transferred and used up to three times
without loss of T lymphocyte purity or yield.
The technique
yielded 20-40 >: ID*5* T lymphocytes from 40 ml of whole blood.
67
ADD 2 ML (2.5 MG/ML)
F(AB’)2 FRAGMENTS OF
ANTI-BOVINE IgG
H&L CHAINS
SPECIFIC
INCUBATE 18 H AT 4°C
........
s
REMOVE UNBOUND IgG
WASH X 4 - 5 ML PBS
ADD 2 ML
(4 .0 X 107) PBMC
INCUBATE X 15 MIN AT 2 5 °C
TO BIND IgG BEARING CELLS (B CELLS)
NONADHERENT
CELLS
(T CELLS)
GENTLY
AGITATE
WASH X 2 WITH PBS
( 5 0 0 X G FOR 1 0 MIN )
Figure IV . 1. Procedure f o r T Lymphocyte P u r if ic a tio n
using "Panning".
68
40
A verage
C l 31
B efore
30
■
15
A fter
20
T-C ells
X 10“
CO
10
R52
R56
Y12
Y32
Y74
A nim al N u m b er
Figure IV.2. T Lymphocyte Yield after "Panning" of Bovine
Peripheral Blood Honnuclear Cells using AntiBovine Immunoglobulin Coated Flasks
69
T -C ells
A verage
Percent
A fter
R52
R56
yi2
Y32.
Y74
A nim al N u m b er
Figure IV.3. T Lymphocyte Enrichment by "Panning"of Bovine Peripheral
Blood Mononuclear Cells using Anti-Bovine Immunoglobulin
Coated Flasks.
70
25
A verage
□
20
20
Percent
B -C ells
B efore
15
A fter
10
R52
R56
Y 12
Y32
Y74
A nim al N u m b er
Figure IV.4. Depletion of B Lymphocytes by "Panning" of Bovine
Peripheral Blood Mononuclear Cells using Anti-Bovine
Immunoglobulin Coated Flasks.
CHAPTER V:
ASSESSMENT OF THE PRESENCE OF BLV PROVIRUS IN
THE T LYMPHOCYTES OF BLV-INFECTED* ALEUKEMIC CATTLE
V. A.
INTRODUCTION
The bovine leukemia virus (BLV)* an exogenous (14)
leukemogenic virus of the family Retroviridae*
has been
implicated as the etiologic agent of enzootic bovine
leukosis (EBL)
(108).
This virus may infect cattle and not
produce any clinical signs of disease.
referred to as aleukemic (AL) cattle.
These animals are
The virus may also
induce two clinical manifestations of diseasepersistent
lymphocytosis (PL) or malignant lymphosarcoma (LS)
Since* EBL is a neoplasm of the lymphoid system*
(46).
cells of
the lymphocyte lineage were the first candidates to be
suspected as preferred BLV targets.
This was shown to be
correct by demonstration of BLV production in vitro from
cultures of peripheral blood mononuclear cells (PBMC) from
BLV-infected cattle (108).
Also* molecular hybridization
analysis using a single-stranded BLV cDNA probe for
detection of BLV proviral sequences in the white blood cells
of PL cattle confirmed the presence of BLV in the
lymphocytes of these animals (76).
By using the nylon wool separation procedure to
fractionate the B and non—B populations of bovine peripheral
blood and subsequent in vitro cultivation of these cells*
was shown that the capacity to produce C-type particles
resided with the B lymphocytes (124).
71
it
72
It
was also found that the majority
of lymphocytes present
in
the peripheral blood of PL and LS cattle and in
had markers typical of B lymphocytes (110,85,72).
tumors
This
information led to the conclusion that the B lymphocyte is
the target cell of BLV infection.
A recent study indicated
that lymphoblastoid-l ike cells derived from two EBL tumors
had cell surface markers typical of T lymphocytes and that
the cells from these cultures contained BLV provirus (91).
It
has been demonstrated that there
is an increase in the
absolute number of T lymphocytes in
AL and PL animals (Chapter 111).
the peripheral blood of
These studies suggest that
T lymphocytes of BLV-infected animals may either be infected
by BLV or are responding to the presence of the virus or
viral proteins.
Definitive studies on highly purified T lymphocyte
populations from the peripheral blood of animals with AL or
PL have not been conducted.
The main reason that this
research has not been done is that purification techniques
which yield large quantities of highly purified T lymphocyte
preparations have not been available.
A technique using
immuno-affinity depletion "panning* to deplete B lymphocytes
from PBMC of cattle has recently been reported (Chapter IV,
ld»l).
This technique gives good yields of highly purified
bovine T lymphocytes from AL but not PL animals.
The purpose of this investigation was to assess the
presence of the BLV provirus in the DNA of immuno—affinity
purified bovine T lymphocytes from AL cattle using DNA
hybridization with a highly specific* nick-translated 3ap—
BLV-DNA cloned probe.
V- B.
MATERIALS AND METHODS
V. B. 1. Animals
A total of 7 animals were used in this study (R 52*
73* Y 12* Y 32* Y 74, Y 75 and B 10).
(For more details
refer to Chapter VIII and Table VIII.
1.)
Y
The cattle were
examined clinically and hematologically for signs of bovine
leukosis using the guidelines of the International
Conference of Bovine Leukosis (2).
BLV infection was
monitored by detection of serum antibodies to Bi_V structural
antigens using the agar gel immunodiffusion assay (33).
Of
the 7 animals* 2 were BLV-negative* 4 were AL and 1 one was
a PL animal.
V. B. 2. Isolation of PBMC
Large quantities of cells were required <1 x 10B
cells) for DNA purification.
Therefore* 5DD ml of
peripheral blood were collected by venipuncture into vacuum
bottles containing Panheparinw * 10 units/ml whole blood
(Abbott Laboratories, U.S.A.).
The heparinized blood was
then diluted in an equal volume of phosphate buffered saline
(PBS)* p H 7.4.
Twenty ml aliquots were layered onto equal
volumes of sodium diatrizoate/ficol1 * 1.077 g/ml
(Histopaque** 1077* Sigma Chemical Co. * U.S.A. ) in 50 cc
conical centrifuge tubes (Costar* U.S.A.).
The gradients
74
uere centrifuged for 30 min at 1000 x g at room temperature.
The bands at the interface between the plasma layer and
HistopaqueR were collected and washed twice in PBS at 500 x
g for 10 min at room temperature.
Viability counts were
performed using the trypan blue exclusion method.
The
lymphocyte preparation usually contained greater than 7b5i
viable cells.
V. B. 3.
(Refer to Figure III.
1 for flow diagram).
Purification of T Lymphocytes
The T lymphocytes were purified from PBMC of BLV—
negative and AL cattle by immuno—affinity depletion
("panning*)i a method previously described (Refer to Chapter
IV.).
Briefly* 4 x IG'7’ PBMC were added to each of six 25
cm2 * polystyrene tissue culture flasks (Costar* U.S.A.).
These flasks had been previously coated with 5 mg of F(ab')2
fragments of rabbit anti— bovine IgG* H and L chains specific
(anti—bov IgG)
(Cappel* U.S.A.)* and incubated for at least
IB hours at 4 C.
The unbound anti-bov IgG was then removed*
and the coated flasks were washed x 4 with 5 ml of PBS.
The
cells were incubated in the immunoglobulin-coated flasks at
25 C for 15 min.
After incubation* the nonadherent cells
were carefully resuspended by rocking the flask* then
removed with a pipette and washed twice with PBS.
Figure IV. 2. for flow diagram).
(Refer to
The nonadherent cell
preparation usually contained 40-502 of the original number
of cells applied.
V. B. 4. Determination of Lymphocyte Subsets and
Monocytes
For detection of B lymphocytes in PBMC and purified T
lymphocyte preparations, 3 x 10** cells were mixed with 100
ul CO.74 mg/ml) of fluorescein— conjugated F(ab')2 fragments
of rabbit anti— bovine IgG* H and L chains specific (RablgFITC) (Cappel* U.S.A.).
This marker has been shown to be
highly specific for bovine B lymphocytes (refer to Chapter
III).
For detection of T lymphocytes!
nonadherent cells
were mixed with 100 ul (2.5 mg/ml) of fluorescein-conjugated
peanut agglutinin—FITC (PNA-FITC)
(Cappelf U.S.A.).
This
marker has been shown to label bovine T lymphocytes (Refer
to Chapter III).
All cell preparations were incubated for
30 minutes at 4 C* then washed twice with 1 ml of PBS
containing 0.02% NaN3 to prevent antibody capping.
Fluorescence-positive cells were enumerated by counting
200 cells at 1000 x magnification with the aid of a
fluorescent microscope equipped for phase contrast and
incident UV light illumination (Leitz* Germany).
The
detection of monocytes in the T lymphocyte preparations was
accomplished by using the nonspecific esterase staining
procedure <63).
V. B. 5. DNA Purification
DNA purification of PBMC and T lymphocyte preparations
was performed as previously described with the following
modifications (101).
Briefly*
1 x 10s cells were lysed in
DNA lysing medium (10 mM Tris buffer pH 7.4*
lOmM
ethylenediaminetetraacetic acid (EDTAf 0.1 M NaCl*
1.0%
Sarcosyl and 10D ug/ml Protease K* Sigma Chemical Co
U.S.A.).
The lysate was digested for 45 min. at 37 C then
either promptly extracted or frozen at —70 C.
The lysates
were extracted 2 x with equal volumes of chloroform:isoamyl
alcohol mixture (24:1) (centrifuged 2*500 x g for 20 min at
25 C).
The chloroform was removed by two cycles of ether
extraction using an equal volume of H20 saturated ether.
Residual chloroform and ether was removed by heating the
preparation to 6B C for 10 min. The salt concentration of
the DNA preparation was adjusted to 0.5 M NaCl.
The DNA was
ethanol precipitated with two volumes of ethanol for at
least 2 hrs at -20 C.
The precipitate was pelleted at
15*000 x g for 15 min at 4 C and resuspended in 1-2, ml Tris
EDTA (TLE) buffer (lOmM Tris, pH 7.4 and 0.1 M EDTA).
The
concentration of DNA was assessed by spectrophotometric
means using 0D2M3 for DNA concentration quantitation and
0D2 M ,/0D2OO ratio as an estimate of purity (135).
One
optical density at 260nm was equal to 50ug of DNA.
A ratio
of from 1.8 to 2.0 (0D2<>o/0D2tsc,) was considered of good
purity.
The DNA was stored at —20 C until used.
(Refer to
Figure V. 1. for flow diagram.)
V.
B. 6. Restriction Endonuclease Digestion and Gel
Electrophoresis
Sac 1 restriction endonuclease (R.E.) (Cooper
Biomedical* U.S.A.) digestions were carried out on the
bovine PBMC and T lymphocyte DNA's using the following
method.
Approximately 5 ug of DNA was mixed with 5 ul of
ID X Sac 1 buffer <60 mM NaCl, 6D mM Tris, pH 7.4, 60 mM
77
MgCl, 60 mM 2-mercaptoethanol, and 100 ug/rol bovine serum
albumin), 5 ul of Sac 1 R.E.
a 50 ul reaction mixture.
hr at 37 C.
(10
units/ul), and H^O to make
This mixture was incubated for 2
After digestion was complete, 5 ul of DNA
loading dye (0.25% bromophenol blue, 0.25% xylene cyanol,
and 15% ficoll type 400) was added tD the digests.
The
reaction mixtures were loaded into individual wells of
horizontal, 0.7% agarose (SeaChem agarose FMC U.S.A.) slab
gels.
The Hind III digest of lambda DNA and the Haelll
fragments of 0X174 RF DNA (New England BioLabs, MA) were
used as molecular-weight markers.
Sac 1 digest of DNA from
the fetal lamb kidney cell line which is persistently
infected with BLV (FL.K—BLV)
(153) as well as DNA from PBMC
of PL animal B ID were used as BLV-DNA positive controls.
Sac 1 digests from PBMC and purified T lymphocyte
preparations from BLV-negatiive cows were used as negative
controls.
The DNA was electrophoresed in Tris acetate
buffer (TAE) (0.04 M Tris acetate and 0.002 M EDTA). at 30 V
for 16 hr at 25 C. The gel was then stained for 30 min using
ethidium bromide (EtBr)
(5 ug/ml) and photographed using
long wave UV light illumination and Polaroid Type 57 film.
V. B. 7. Gel Transfer
DNA fragments were transferred from agarose gels to
nitrocellulose filters using the Southern transfer technique
(137).
Nitrocellulose filters were then dried for 2 hr at
60 C in a vacuum oven.
V. B. 8. BLV-DNA Probe
The BLV DNA probe was provided by Dr. James Casey
(Frederick Cancer Res. Inst. Fredrick* MD . ) as a cloned
provirus probe consisting of an EcoRl fragment of FLK-BLV
DNA which contained almost the entire BLV provirus excluding
the 3' long terminal repeat and included 20D base pairs of
cellular flanking sequence DNA (Fig. V. 2).
This fragment
was ligated into a PUC-S vector plasmid and cloned in E.
coli HB101.
This probe hybridized to the 7.5 kilobase (Kb)
fragment of the BLV-provirus DNA in BLV-infected lymphocytes
but hybridization also occurred with a variety of other
fragments in these DNAs and in BLV—negative control animals.
Therefore* the cloned probe was further purified as
described below.
The BLV-DNA probe (ID ug) was digested with Sac 1 to
purify almost the entire BLV proviral genome as a 7.5 Kb
fragment ( Fig. V. 2).
'
The R.E. fragments were then
separated by agarose gel electrophoresis using a 0.7'i
horizontal agarose gel at 30 V for 16 hr in TAE buffer. The
gel was then stained with EtBr to visualize the R. E.
fragments.
A 2 mm wide trough was cut in front of the
leading edge of the 7.5 Kb fragment* and the trough was
filled with electrophoresis buffer.
The band was then
observed by UV light as it was electroeluted from the gel
into the trough at 200 V.
Every 30 to 40 seconds the fluid
from the trough was recovered* and the trough was refilled
with fresh buffer.
Electrophoresis was then continued until
all the DNA in the band was removed from the gel and was
79
recovered in the fluid taken from the trough (Fig. V. 3).
The probe was further purified of contaminating gel
components by ion—exchange chromatography using a nucleic
acids chromatography column (NACS P R E P A C ™ mini-column)
(Bethesda Research Laboratories* MA).
The DNA mas
precipitated in two volumes of cold ethanol at -20 C for 2
hr and then pelleted at 15*000 x g* 15 min* A C.
The pellet
was resuspended in 20 ul TLE.
V. B. 9. Nick Translation of BLV-DNA Probe
The BLV-DNA probe was radiolabelled with a-32P labelled
deoxycytosine triphosphate (dCTP Ca-^^Pl) by nicktranslation using a Nick Translation Kit” (Cooper
Biomedical* PA).
Briefly* 2 ug of BLV-DNA* 5 ul of nick
translation buffer* 1 ul of each of dATP* dTTP and dGTP* 10
ul of dCTP Ca—33rP3 (10 mCi/ml)
(New England Nuclear* MA), 2
ul of DNAase 1/ DNA polymerase I mixture and H^O were added
to make a 50 ul reaction mixture.
The mixture was incubated
at 18 C for 1 hour* and the reaction stopped with 20 mM
EDTA.
The unincorporated isotope was removed from the nick-
translated DNA using gel filtration chromatography using a
MINI—SPIN” column (Cooper Biomedical, PA).
This column
consisted of a pre— hydrated* prepacked column of Sephadex*
G-50.
The nick-translated probe was then dissociated into
single-stranded DNA by the addition of 1/10**' volume of 2 M
NaOH.
The specific activity of the radiolabelled probe was
determined by scintillation counting of the probe using Hz0
as a scintillation medium (Packard Tricarb Scintillation
ea
Counters USA).
V. B. 9. Filter Hybridization
Nitrocellulose filters were prehybridized in
hybridization mixture (103) for 48 hr at 37 C.
Hybridization was carried out by adding 1.5 x ID7 cpm of the
single-strandedt nick translated 33P-BLV DNA cloned probe in
B ml of hybridization buffer.
The filters were wrapped
in SaranR wrap and once in aluminum foil.
incubated for 48 hr at 37 C*
x 2
The filters were
washed for 1 hr in a .high salt
solution <2x SSC and 0.5% sodium dodecyl sulfate (SDS) at 37
C and for 2 hr in a low salt solution (0-lx SSC and 0.5%
SDS) (104) at 45 C.
V. B
10.
Filters were dried at 60 C for 1 hr.
Autoradiography
Filters were exposed at -70 C to Kodak X RP-5 X—Omat
film in the presence of 2 Cronexj Lightning Plus'S
intensifying screens (Dupont( U.S.A.) for 2-5 days.
The
exposed film was then developed using a Kodak X-Omat
Processor (Kodak T U S A ).
V. C. RESULTS
V. C. 1. Purity of T Lymphocyte Preparations
The frequency of T and B lymphocytes in the PBMC and
purified T lymphocyte preparations used for DNA
hybridization analysis can be found in Table V. 1.
The
frequency of T lymphocytes in the PBMC of AL and BLVnegative control animals ranged from 69 to 89%.
The
81
frequency of B lymphocytes in the PBMC of these animals
ranged from 15 to 23%.
In the purified T lymphocyte
preparations* frequencies of T lymphocytes ranged from 91 to
97%* and frequencies of B lymphocytes ranged from 0 to 4%.
Based on 1 x 10s cells (quantity of cells iysed for DNA
purification) the absolute number of contaminating B
lymphocytes were estimated for each DNA preparation.
Animals Y 12 and Y 74 were assumed to have no B lymphocytes
in their purified T lymphocyte preparations* whereas*
in the
PBMC of these animals there were 5 x 10* and 13 x 10* B
lymphocytes* respectively.
In the purified T lymphocyte
preparation of animal Y 75* there were 3 x 10* B lymphocytes
f
versus 23 x 10* B lymphocytes found in the PBMC of this
animal.
In the T lymphocyte preparation of animal Y 32*
there were 4 x 10* Blymphocytes versus
27 x 10* B
lymphocytes found in the PBMC of this animal.
V. C. 2. Agarose Gel Electrophoresis
Results of the agarose gel electrophoresis of R. E.
fragments show that test DNA was present in relatively equal
concentrations in each of the lanes of the gel.
Also* the
enzyme Sac 1 effectively digested the DNA's as evidenced by
detection of the highly repetitive DNA sequences (Fig. V. 3)
V. C. 3. DNA Hybridization Analysis
As shown in Figures V. 5 & 6* ^“ P-labeled BLV DNA
hybridized on the 7.5 Kb Sac 1 fragment from the PBMC of all
BLV-positive AL and PL cattle and the FLK-BLV positive
control DNA.
No hybridization was observed to the DNA
fragments from BLV—negative cattle.
DNA from the purified T
lymphocyte preparations of 3 AL animals (Y 74, Y 75, and Y
32) were positive for hybridization on the 7.5 Kb Sac 1
fragment with the BLV-DNA probe.
The quantity of DNA which
hybridized with the probe appeared to be much lower in the
purified T lymphocyte DNA preparations than in the
respective PBMC DNA preparations because the respective
bands on the autoradiographs were much less dense than those
of the PBMC DNA preparations.
Cow Y 74 contained no
detectable B lymphocytes in the purified T lymphocyte
preparation but its DNA gave a positive hybridization signal
with the BLV-DNA probe.
Cow Y 12 which also contained no
detectable B lymphocytes in the purified T lymphocyte
preparation gave negative hybridization results.
V. D. DISCUSSION
The purpose of this study was to determine if bovine T
lymphocytes were targets for BLV infection.
Results from
the experiments reported herein suggest that bovine T
lymphocytes of some AL cattle contain the BLV provirus.
The
BLV provirus was detected in the DNA of PBMC from all AL
animals and also in the DNA of 3 of 4 purified T lymphocyte
preparations from AL animals.
Two of the T lymphocyte
preparations which gave positive hybridization results
contained only small amounts of contaminating B lymphocytes
(3 and 4%).
It has been well documented that the B
lymphocyte is a target for BLV infection (45,108,121).
Therefore,
it is possible that the positive hybridization
results obtained from the DNA of these 2 cel) preparations
were due to the DNA from contaminating BLV-infected B
lymphocytes.
Less than 25% of B lymphocytes from BLV-
infected animal5 have been shown to produce viral particles
or display viral antigens on their cell surfaces after short
term incubation (121,73).
Therefore,
less than 10 x 10s
BLV-infected B lymphocytes were possibly present in the
purified T lymphocyte preparations.
It is possible that
this amount of BLV-infected lymphocyte DNA can give a
hybridization signal because of the sensitivity of the solid
Phase hybridization procedure.
The sensitivity of the solid
phase DNA hybridization assay is such that it can detect 1
proviral copy in 10. (personal communication Dr. James
Casey, National Cancer Institute, Fredrick, tiD).
When
comparisons of the percentage of B lymphocyte DNA in the
purified T lymphocyte preparations and the percentage of B
lymphocyte DNA in the PBMC of the same animals were made
with their respective hybridization signals on the
autoradiographs,
it appeared that the signals generated by
the purified T lymphocyte DNA preparations were greater than
what would be expected if only B lymphocytes contained the
BLV provirus.
One purified T lymphocyte preparation (animal Y 74)
contained no detectable B lymphocytes.
The DNA from this
cell preparation gave positive hybridization results.
It is
possible that a population of slg- B lymphocytes exists in
cattle* but this has not been reported.
The marker used in
this study to enumerate bovine B lymphocytes has been shown
to be highly specific for bovine peripheral blood B
lymphocytes (55*90*85).
The lymphocyte surface marker
profile of this animal appeared normal using flow
microfluorimetry (FMF) (Chapter III).
Monoclonal antibodies
(MAbs) against surface bound IgM (PIg45a> and Rablg-FITC
markers for B lymphocyte enumeration and MAbs against T
lymphocyte surface antigens and PNA receptors for T
lymphocyte enumeration were used in the FMF assays (Chapter
III).
One additional AL animal (Y 12) had no detectable B
lymphocytes in the purified T lymphocyte preparation.
However*
the DNA from this preparation' gave no hybridization
with the BLV—DNA probe.
The DNA of the PBMC from this
animal gave a weaker hybridization signal than the DNA from
other AL animals and this indicated that fewer cells
contained the BLV provirus.
The BLV-infected T lymphocytes
may have been depleted in the “panning* procedure since the
average yield of T lymphocytes from “panning" is 48 % of the
original number of T lymphocytes applied to the “panning"
flask (Refer to Figure VI. 2).
Another possibility is that
not all AL animals have detectable levels of BLV-infected T
lymphocytes.
This study documents the first attempt to assess the
presence of the BLV provirus in highly purified peripheral
blood T lymphocyte populations from BLV-infected AL cattle.
Hybridization studies reported to date using highly specific
BLV-DNA probes and buffy coat preparations or PBMC*
demonstrated that the BLV provirus was present in the
lymphocyte population (75*76).
It was also shown that the
ability to produce C-type viral particles resided with the B
lymphocyte subset (121).
However* definitive studies on
highly purified peripheral blood lymphocyte subsets had not
been reported.
A recent report showed that lymphoblastoid-
like cells derived from two EBL tumors not only had markers
of the T lymphocyte lineage but also contained the BLV
provirus (91).
Therefore,
it appeared that bovine T
lymphocyte population of BLV-infected animals may be
infected with the BLV provirus.
The results of this study suggest that the B lymphocyte
is the major target cell of BLV infection in the AL animal.
After B lymphocytes were depleted from PBMC using "panning"
with anti-bovine Ig* the amount of DNA in the "panned” cell
preparation which hybridized with the 32P BLV—DNA probe was
greatly reduced.
This indicates that a large number of BLV-
infected cells (B lymphocytes) were removed by the "panning”
procedure.
This study also suggests that the BLV provirus
can be detected in the DNA of T lymphocytes of some AL
animals.
It appears that only a small number of these cells
are infected by the virus* as evidenced by the weak
hybridization signal from the purified T lymphocyte DNA
preparations.
Therefore, BLV may infect only a
subpopulation of T lymphocytes in the peripheral blood of AL
animals.
Infection of this cell population may alter
function of these cells and thereby facilitate the
transition from the AL to PL stage of EBL.
Possibly*
infection of the T lymphocytes is a prerequisite for the
progression to the PL state.
In order to address this
hypothesis* purified T lymphocytes from PL animals must be
screened for BLV provirus.
The purification techniques
which were used in this study for T lymphocyte purification
were not applicable to PBMC of PL animals (Refer to Chapter
IV).
This was probablly due to the very low frequencies of
T lymphocytes present in the PBMC preparations.
The question of whether BLV infects cells of the T
lymphocyte lineage is of importance for the use of EBL as a
model for the viral-incuced human adult T cell leukemia
(ATL) associated with human T cell leukemia virus (HTLV I)
(162).
Many aspects of the disease induced by HTLV I
parallel those of EBL (162*121).
chronic.
The disease is typically
Lymphocytosis occurs in some cases as do
immunodefficiencies.
Lymphoma occurs in a small percentage
of infected individuals.
A major difference between ATL and
EBL is that the target cell of HTLV I appears to be the T
lymphocyte and the major target cell of BLV appears to be
the B lymphocyte (162* 121).
The data presented here
indicate that bovine T lymphocytes may also act as targets
for infection by BLV and therefore* this information may
contribute to the BLV model of HTLV— induced leukemogenesis.
87
PURIFIED CELLS
(T CELLS + PBMC)
© LYSED CELLS WITH
DNA LYSING MEDIUM
^
®.DIGESTED
DNA PURIFICATION:
CHLOROFORM EXTRACTED X 2
ETHER EXTRACTED X 1
ETHANOL PRECIPITATED
~)Z Z Z Z-Z' ~)ZZ Z Z
DNA
WITH R.E. SAC 1
U'UU u u u u
©SEPARATED R.E, FRAGMENTS
BY AGAROSE GEL
ELECTROPHORESIS
BLOTTED DNA ONTO
NITROCELLULOSE
PAPER
©HYBRIDIZED WITH 3 2 P
LABELED BLV-DNA PROBE
(S u pplied by Dr. Jim C a se y )
AUTORADIOGRAPHY
Figure V . l . Procedure f o r D etectio n o f Bovine Leukemia Virus
P ro viru s in the P e rip h e ra l Blood Mononuclear C e lls
and P u r ifie d T Lymphocytes o f Bovine Leukemia Virus
In fe c te d C a ttle
88
Kb
3
2
I
*
Sac1
T
4
—
i-
Bam
7
-i.
Bam
Y
i
Pst1
5
_i_
T
I
Bgl1
8
Bam S a c 1
S a c 1 |R 1
v
T TT r
—
A
A
Bgl1
Bgl1
-
Figure V .2 . Bovine Leukemia Virus P rovirus R e s tr ic tio n Enzyme Hap
89
B L V DNA
PROBE
M.W.
MARKERS
7.5 Kb
BEFORE
ELECTROELHTION
7.5 Kb
AFTER
ELECTROELOTION
Figure V.3. Purification of BLV DNA Probe Using Electroelution
of BLV DNA Sac 1 Fragment from an Agarose Gel.
90
Figure V.4.
Gel Electrophoresis of Sac 1 Restriction Enzyme
Fragments of Lymphocyte DNA.
BLV
(PL)
(AL)
(AL)
B 10
PBMC
Y 74
T Lymph
Y 74
PBMC
(AL)
(-)
(AL)
status]
FLK
BLV
Y 75
R 52
T Lymph PBMC
Y 75
PBMC
—
A^
«
4.-5? * f
*§sv'r- ,
Figure V.5.
DNA Hybridization Analysis of Peripheral Blood
Mononuclear Cells and Purified T Lymphocytes from
Bovine Leukemia Virus-Infected Cattle Using a -*2p
BLV-DNA Probe.
7.5 Kb
BLV
STATUS
J
FLK
BLV
(PL)
(— )
(AL)
B 10
PBMC
R 52
PBMC
Y 32 Y 32
Y 73 Y 73
Y 12
P BMC T Lymph PBMC T Lymph T Lymph
(AL)
(— )
(— )
(AL)
(AL)
Y 12
'PBMC
7.5 Kb
^ 4
Figure V. 6. DNA Hybridization Analysis of Peripheral Blood
Mononuclear Cells and Purified T Lymphocytes from
Bovine Leukemia Virus-Infected Cattle Using a 33p
BLV-DNA P r o b e .
v£>
ro
93
Table V. 1 — Frequencies of T and B Lymphocytes
for DNA Hybridization Analysis
Animal
No.
Peripheral Blood
Mononuclear Cells
% T a
B L V (AL)c
Y 74
% B b
Purified
T Lymphocytes
% T
% B
79
13
97
0
Y 75
76
23
91
3
Y 32
69
27
95
4
Y 12
79
5
95
0
R 52
69
20
ND
ND
Y 73
73
22
94
2
BLV
(— )d
aCells positive for fluorescence using PNA-FITC.
^Cells positive for fluorescence using Rablg-FITC.
cBovine leukemia virus positive, aleukemic state.
No detectable antibodies to bovine leukemia virus.
CHAPTER VI: EVALUATION OF THE RESPONSES OF LYMPHOCYTES OF
ALEUKEMIC AND PERSISTENT LYMPHOCYTOTIC CATTLE TO
SELECTED PHYTOMITOGENS
VI- A. INTRODUCTION
Approximately 30% of cattle infected with bovine
leukemia virus (BLV) develop a benign condition referred to
as persistent lymphocytosis (PL)
(10B). The PL state is
characterized as a persistent elevation of primarily
lymphocytes in the peripheral blood (108).
B
Animals with PL
develop clinical lymphosarcoma (LS) more frequently than do
BLV-infected) aleukemic (AL) cattle which have no clinical
signs (51).
Therefore) the transition from the AL to PL
state may be a primary step in the progression of the
disease to the tumor state.
It is unclear what factor(s) mediate the switch from
the AL to the PL stage or allow for the uncontrolled B-cell
lymphocytosis.
A possible factor may be a modulation of a
specific T-helper or suppressor cell population that elicit
soluble factors which help to regulate B lymphocyte
proliferation (81).
However)
little is known about the
immunological and functional properties of lymphocytes of AL
or PL cattle.
It has been demonstrated in other leukomogenic
retroviral infections (human adult T cell leukemia (ATL))
feline leukemia) murine leukemia) avian leukemia)
94
that immunosuppression takes place as a result of retroviral
modulation of immune cells (13* 159* 26).
Immunosuppression
may occur as a result of the interaction of BLV with its
target cells.
It has been documented that the B lymphocyte
is a target cell for BLV infection (45*73*76).
It has also
been demonstrated that the T lymphocytes from the peripheral
blood of some AL animals and tumors of some LS animals
contain the BLV provirus (Chapter V f9i).
An increase in
the absolute T lymphocyte numbers in AL animals and an
increase in the absolute number T and B lymphocytes in PL
animals has also been observed (Chapter 111*142).
It is
unclear whether these factors affect T lymphocyte function
in BLV-infected animals.
The mitogenic stimulation of lymphocytes as measured by
uptake of 3H thymidine is used to evaluate inherited and
acquired immunodeficiencies and the effect of the
iiwnunoenhancing or immunosuppressive action of serum factors
(4* 112*157).
This technique can therefore be used as an
indicator of lymphocyte function.
Mitogens such as
phytohemagglutinin (PHA>* Concanavalin A (Con A) and
pokeweed (PWW) have been used tD assess the mitogenic
response of bovine lymphocytes (111*112*132*157).
The PHA
and Con A responsiveness is a property of bovine thymusderived lymphocytes <T lymphocytes)
(111*132) and the PWM
responsiveness is a property of both T and B lymphocytes
because a B lymphocyte mitogen requires the presence of T
lymphocyte soluble factors to obtain a full blastogenic
96
response (111,132).
In studies with BLV-infected animals* the lymphocyte
response to PWM and PHA* as indicated by the use of
stimulation index (S.I.), was markedly less for those cells
obtained from animals with PL than for those obtained from
hematologically normal animals (112).
The lymphocytes from
cows with PL that were cultured without phytomitogens
incorporated approximately 5 times more tritiated thymidine
than did lymphocytes from normal animals (112).
It was
also shown that the lymphocyte stimulation of AL animals
appeared to be normal when blastogenesis assays were
conducted using PHA as the mitogen (68).
Another study
suggested that the stimulatory effect of Con A on
lymphocytes of PL- cattle was significantly depressed when
compared to response using normal lymphocytes (157).
These
results suggested that the T lymphocyte population of PL
animals may be functionally affected in the ability to
respond to mitogens.
However*
the depressed stimulation may
have been the result of low numbers of T lymphocytes present
in the lymphocyte preparations of PL cattle used in the in
vitro blastogenesis assays or because stimulation index was
used to assess the responsiveness to mitogens.
These
questions have not been fully considered.
Inhibition of lymphocyte blastogenesis of normal
lymphocytes by sera from cows with LS has been shown but
suppression has not been demonstrated with the sera from PL
or AL cattle (164).
If present*
these serum factors may
97
affect immunoregulation.
The purpose of the present investigation was to compare
the response of lymphocytes from AL and PL cattle to
phytomitogens PHA, ConA* and PWM using the in vitro
lymphocyte blastogenesis assay.
The responsiveness of the
cells to mitogens were compared to the T/B lymphocyte ratios
from these animals.
The effect of sera from AL and PL
cattle on lymphocyte blastogenesis of BLV—negative control
cattle was also evaluated to determine if suppressive or
augmenting factors were present.
VI. B.
MATERIALS AND METHODS
VI. B. 1. Animals
A total of 9 cows of at least 3 years of age mere used
in this study.
The animals were examined clinically and
hematologically for symptoms of bovine leukosis* and the BLV
infection status was monitored by detection of serum
antibodies to BLV structural antigens using the double
immunodiffusion assay (33).
Of the 9 animals* 3 were BLV-
negative on repeated tests* 3 were AL and 3 were PL as
determined by the critera of the International Committee of
Bovine Leukosis (2).
VIII* Table VIII.
(For more details refer to Chapter
1.)
VI. B. 2. Isolation of Peripheral Blood Mononuclear
Cel Is
Peripheral blood was collected by Jugular venipuncture
into 20 ml vacutainer tubes containing 20 units/ml of
preservative-free heparin (Sigma Chemical Co.* U.S.A.).
The
heparinized blood was then diluted in an equal volume of
calcium and magnesium free (CMF) Hanks buffer* pH 7.4.
Twenty milliliter aliquots were layered onto equal volumes
of sodium diatrizoate/ficoll
(Histopaque" 1D77* Sigma
Chemical Co.* U.S.A.) in 50 cc conical centrifuge tubes
(CDstar* U.S.A.).
The gradients were centrifuged for 30 min
at 1000 x g at room temperature* and the cells banded at the
Histopaque"— plasma interface were collected and washed twice
in CMF Hanks buffer at 50D x g for ID min at room
temperature.
Viability counts were done using 900 ul of 10%
trypan blue mixed with 100 ul of cells* and the peripheral
blood mononuclear cell
(PBMC) concentration was adjusted to
2 x 10* viable cells/ml in RPMI 1640 medium (GIBC0*
Burlington* Ontario) supplemented with NaHCo3 and
antibiotics (100 units/ml of penicillin and 100 ug/ml of
streptomycin* 5 ug/ml gentamycin).
VI. B. 3. Complete Blood Counts
Blood was collected in tubes containing
ethylenediaminetetraacetic acid as an anticoagulant.
Total
leukocyte counts were determined on an automatic cell
counter and differential counts were performed.
VI. B. 4. Enumeration of T/B Lymphocyte Ratios
Aliquots of PBMC which were isolated for the lymphocyte
blastogenesis assays were stained for B and T lymphocyte
enumeration using the following procedure.
To identify B
lymphocytes* 3 x 10* PBMC were mixed with 10D ul (G.74mg/ml)
99
of fluorescein— conjugated Ftab'J^. fragments of rabbit antibovine IgG, H & L chains specific <Cappel Labs,
Cochranvi1le, PA).
To identify T lymphocytes, 3 x 10fc PBMC
were mixed with 10 ul B26a (1 mg/ml, bovine pan T cell
monoclonal antibody which recognizes the sheep RBC receptor
on T lymphocytes)
(courtesy of Dr. William Davis, Washington
State University, Pullman* WA>.
The PBMC were incubated at
A C for 30 min in the dark, then washed twice in 1 ml
aliquots of phosphate buffered saline (PBS) pH 7.4 with
0.05% NaN3 (PBS NaN3 ) to prevent antibody capping.
PBMC
pretreated with B26a were stained with 100 ul (1 mg/ml) of
fluorescein-conjugated goat anti-murine IgG, IgA, IgM
mixture (goat anti-mouse-FITC) (Cappel Labs., Cochranvi1le,
PA),
incubated and washed as described above.
then fixed in 0.551 formalin in PBS-NalM3 .
The PBMC were
An aliquot of PBMC
was also stained with goat anti—mouse FITC as a control.
The method used for the detection of fluorescencepositive celIs was fluorescence microscopy.
Positive cells
were enumerated by counting 200 cells at 1000 x
magnification with the aid of a fluorescence microscope
equipped for phase contrast and incident UV light
illumination (Leitz, Germany).
The T/B ratio was then
determined by dividing the average of the percent T
lymphocytes by the average of the percent B lymphocytes in
the peripheral blood of each animal.
VI. B. 5. Serum Preparation
Twenty ml of blood was collected from each animal into
lOD
vacutainer tubes at sane tine blood was removed for
blastogenesis assays.
The blood was allowed to clot at room
temperature for one hour, and centrifuged at 1000 x g for 15
min.
The harvested serum was filter sterilized by
ultrafiltration with a 0.45 um disposable syringe filter
(AcrodiscR, Gelman, U.S.A.).
One half of the serum was heat
inactivated at 56 C for 30 min* and the remaining serum was
kept at 25 C until used.
The sera (both heat inactivated
and fresh) were then diluted in RPMI 1640 medium to a final
concentration of 40% which was mixed in the lymphocyte
blastogenesis test with an egual volume of cell suspension
to yield 20% final concentration.
Heat inactivated fetal
calf serum (FCS) and heat inactivated sera from BLV-negative
cows were diluted to 40% with RPMI 1640 medium and used as
serum controls.
VI. B. 6. Preparation of Mitogens
Suboptimal and optimal concentrations of Con A* PHA,
and PWM as determined by pre-testing on the PBMC of BLVnegative, clinically normal animals were prepared in RPMI
1640 medium.
VI. B. 7. Lymphocyte Blastogenesis Assay
The lymphocyte blastogenesis assay was performed as
previously described (143).
Briefly, the test was conducted
in flat-bottom, 96-well,
tissue culture plates with 2 x 10=
viable PBMC cells/well.
Medium in the wells contained a
final concentration'of 20% (v/v) of serum.
Each animal's
lymphocytes were tested with the following sera added: fresh
ioi
autologous sera, heat-inactivated autologous sera* heatinactivated control BLV-negative sera* and FCS (Fig. VI.2.).
The total well content was 0.2 ml* to which 1 of the
following had been added (final concentrations): Con A* 5
and 30 ug/ml; PHA* 2 and 5 ug/ml; and PWM* 0.05 and 0.2
ug/ml. The lower concentrations represented the subDptimal*
and the higher concentrations* the optimal doses of mitogens
to stimulate lymphocytes from a majority of clinically
normal cows.
For each animal's cells* there were 6 control
wells used with PBMC without addition of the mitogens* and 3
wells for each mitogen concentration and the given serum
addition.
(Refer to Figure VI.1 for flow diagram.)
To test for the presence of blastogenesis augmenting or
suppressive factors in the serum of BLV-infected animals*
serum was obtained from 3 AL and 3 PL animals and 2 BLVnegative control animals at 2 intervals 30 days apart and
processed as above then applied to lymphocytes of BLVnegative animals.
Heat— inactivation was used to determine
if possible augmenting or suppressive factors present in the
serum were heat stable.
The PBMC were incubated at 37 C in 5Z CCL. for 3 days*
labeled with [3HDthymidine (1 uCi/well)* and incubated for
an additional A hours.
The PBMC were harvested onto Skatron
filter mats* using a multiple-well cell harvester (Skatron*
Norway).
After treating each sample with Concifluor”
toluene scintillation cocktail (Fisher Scientific* U.S.A.)*
the radioactivity or counts per minute (cpm) was determined
102
using a scintillation counter (Packard TriCarb, Union
Carbide* U.S.A.).
VI. B. 7.Statistical Evaluations
Statistical evaluations were performed on log
transformed data using standard programs of the Prophet
Computer System (U.S. Dept, of Health and Human Resources*
Bethesda* HD.).
To test the hypothesis of homogeneity of
variance the Bartlett's test was used (14).
A two-tailed T—
test using unpooled variances was performed on all data
(136* 14).
The nearest P value was calculated.
Data were
considered significant at a level of P < 0.05.
VI. C. RESULTS
The results of blastogenesis assays were expressed as
the log of the stimulation index (S.I. = cpm of mitogenstimulated PBMC divided by cpm of unstimulated PBMC grown in
the presence of the same serum)
(Fig. VI. 3) and as the log
of delta counts per minute (cpm of mitogen—stimulated PBMC
minus cpm of unstimulated PBMC grown in the presence of the
same serum ) (Fig. VI 4.).
The responses of lymphocytes
from the animals used in blastogenesis assays were
determined using FCS as the serum source because of the
variability encountered when autologus and homologus sera
were used.
There were no significant differences noted between the
responsiveness of PBMC from AL and BLV-negative animals when
SI was used to measure the response to Con A* PHA* and PWM
103
(Table VI. 1).
The PBMC of PL animals appeared to have
a
significant decrease in the blastogenic response to Con A (p
< 0.01), PHA (p < 0.01) and PWM (p < D.G2) when SI was used
as a measure of the mitogenic response.
It was also
observed that the spontaneous blastogenesis (PBMC cultured
with no mitogen) was significantly higher for the BLVinfected anixals when compared to that of BLV-negative
animals (Table VI. 2).
When delta cpms were used as a measure of the mitogenic
responsiveness of lymphocytes from AL and PL animals*
it was
found that there were no significant differences in the
responses to any of the mitogens when compared to the
mitogenic response of lymphocytes from BLV—negative animals
(Table VI. 2).
T/B lymphocyte ratios from BLV-negative control* AL and
PL animals are shown in Table VI. 3.
The T/B ratios from PL
animals were lower than those from either AL
control animals.
or BLV-negative
There were fewer T and more B
lymphocytes
in the PBMC preparations used in the blastogenesis assay for
these animals (B 10, Y 11* Y 13). There was
the T/B ratio of
an increase in
AL animals when compared to PL or BLV-
negative animals..
There were fewer B and more T lymphocytes
in the PBMC preparations used in the assay.
When lymphocyte
responses to phytomitogens were compared to T/B ratios using
linear regression analysis,
it was found that there was no
correlation between
The presence of possible suppressive or augmentative
IDA
factors for blastogenesis in the sera of BLV-infected
animals was assessed by the addition of these sera to PBMC
from BLV—negative control animals in the presence of
mitogens.
The data was expressed as corrected counts per
minute (cpm of mitogen—stimulated BLV-negative PBMC in the
presence of BLV-infected serum minus cpm of mitogenstimulated BLV-negative PBMC in the presence of BLV-negative
serum) (Figure VI. 5).
The sera from AL and PL animals
augmented the mitogenic response of lymphocytes to optimal
concentrations of Con Ai PHA and PWM in comparison to the
reponses observed BLV-negative control sera.
No
augmentation was observed in cultures without mitogen. The
blastogenic response of the lymphocytes to all mitogens was
greater in sera from PL animals than sera from AL.
The
blastogenesis augmenting factor was active on both T and B
lymphocytes and was heat stable at 56 C for 30 min.
VI. D. DISCUSSION
The stimulatory effect of optimal concentrations of PHA*
Con A and PWM on the PBMC from BLV-negative control animals
could be demonstrated clearly in the experiments reported
herein.
The values obtained in lymphocyte blastogenesis
studies with optimal concentrations of mitogens were used in
this study to assess the blastogenic responsiveness of
lymphocytes.
The use of suboptimal concentrations of
i
mitogens gave no different information on the response of
105
lymphocytes to various mitogens. These data are not
presented.
When SI was used to measure the mitogenie
responsiveness of lymphocytes from BLV— infected animals*
there appeared to be a false assessment of the response due
to the high spontaneous blastogenesis observed in the
unstimulated PBMC cultures.
SI was calculated by dividing
the cpm obtained from mitogen—stimulated PBMC by the cpm of
unstimulated PBMC.
If spontaneous blastogenesis was high
and the response to mitogens was within the normal range
then the SI was low.
Therefore* the PL animals which had
high spontaneous blastogenesis had low SI.
The observations that lymphocytes from PL animals
undergo high levels of spontaneous blastogenesis and have
low SI are in agreement with previous reports (112*143).
This information led to the original conclusion that
lymphocytes from PL animals have depressed responses to
mitogenie stimulation. This does not appear to be a correct
conclusion as discussed below.
The observation that the
lymphocytes of AL animals undergo high levels of spontaneous
blastogenesis has not been previously reported.
When delta counts per minute were used to measure the
mitogenie responsiveness of lymphocytes from AL and PL
animals* no significant difference was noted when compared
to the response observed for the lymphocytes of BLV-negative
animals.
This suggested that the lymphocytes of BLV/
infected AL and PL animals are capable of normal mitogenie
106
response to both T and £ lymphocyte mitogens.
It has been observed that there is an increase in the
absolute number of T lymphocytes in the peripheral blood of
AL animals (Chapter 111).
It was shown in this study« that
T/B ratios were higher in AL animals than either PL or BLVnegative animals.
More lymphocytes with T lymphocyte
markers were present in the PBMC preparations of AL animals
used for the blastogenesis assays.
This increase in T
lymphocyte numbers did not appear to affect the overall
response to optimal concentrations of the mitogens used in
this study.
It has been shown that the response of
lymphocytes to Con A is a property of the T lymphocyte
population but is dependent on accessory cells (monocytes)
(B2*86).
Therefore* even though T lymphocyte numbers were
more numerous in the cultures from AL animals used for the
mitogenie studies*
the limiting factor may have been the
concentration of monocytes in the culture.
It was also observed that as the T/B ratios decreased
(PBMC of PL animals)* there was no significant increase or
decrease in the ability of the cultured cells to respond to
mitogens.
Even though there were more B lymphocytes in the
cultures of PL animals used in the blastogenesis assay* the
response to PVNi was not elevated.
Responsiveness to PWM is
predominantly a property of B lymphocytes (111*112).
The
ability of B lymphocytes to respond to PWM is T lymphocyte
(T— helper) dependent (112*111).
Even though there was an
/
increase in the number of lymphocytes with B lymphocyte
107
markers in the cultures used for mitogenie studies* no
increase in the response was observed because the
concentration of T— helper cells might have been the limiting
factorThe increase of peripheral blood T lymphocytes in AL
animals and Blymphocytes in PL animals mag be the result of
antigen-induced expansion of T and B lgmphocgte clones*
respectivelg.
These cells mag also be responding to
infection bg BLV (Chapter V* 91)-
The high spontaneous
blastogenesis as measured bg the synthesis of DNA indicates
that cells in the cultures from AL and PL animals were
undergoing high levels of mitosis.
These stimulated cell
populations might have been refractory to further mitogenie
stimulation therefore* no increase in the response to
mitogens was observed.
It was concluded that the increase in
the relative numbers of T and B lymphocytes in the cultures
used for blastogenesis assays did not affect the response to
selected phytomitogens.
Therefore* the lymphocytes of AL
and PL animals were considered as responsive to mitogenie
stimulation as lymphocytes from BLV-negative* clinically
normal animals
Sera from AL and PL animals contained a heat—stable
blastogen'esis-augmenting factor(s).
This factor enhanced
the mitogenie response of lymphocytes from BLV-negative
animals for Con A* PHA and PWM.
No augmentation was
observed with BLV-negative control sera nor was
/
blastogenesis augmentation observed in the absence of
10B
mitogenie stimulation.
Therefore*
it was assumed that this
factor was a comitogenic serum factor or one that works in
the presence of mitogens or exhibits its activity on mitogen
and /or antigen—activated cells.
Sera from PL animals gave
a higher degree of stimulation than did sera from AL
animals.
Since the augmenting factor enhanced the blastogenic
activity of all three phytomitogens*
it was assumed that the
effect was on both B and T lymphocytes.
heat stable.
This factor was
No further characterization of the serum
factor was attempted in this study.
Uhen HTLV— III— infected T lymphocytes are stimulated to
divide by antigen activation* the viral genome is also
activated (105).
There is then production of the trans-
activation of transcription <tat) viral gene product.
This
protein enhances the expression of not only the proviral
genes but also the genes for interleukin—2 and its receptor
(105).
divide.
The infected cells are then further activated to
This tat enhancement of antigen— driven lymphocyte
blastogenesis also appears to occur in the other HTLVs (I
and II)
(105).
BLV is closely related to HTLV I and II and
>
has been found to contain a tat gene (128*131).
The co­
rnitog enic factor found iii the sera of BLV— infected animals
may be the tat protein.
The enhancement of mitosis was only
apparent when PBMC of BLV-infected animals are cultured in
the presence of mitogens and sera from BLV— infected animals.
The mitogenie activation of lymphocytes appeared to be
10?
necessary before this protein bias able to enhance the
blastogenic response.
An increase in concentration of this
factor in the sera of PL animals may be responsible for
maintenance of the PL state.
The T and B lymphocytes of BLV-infected animals used in
this study
appeared to be responsive to the selected
phytomitogens.
Responsiveness to mitogens is used as an
indicator of functional competence of lymphocytes
(4,111*132).
This study indicates that at least a portion
of lymphocytes from AL and PL animals respond to mitogenie
stimulation at least to a level comparable to that of
lymphocytes from BLV-negative,
clinically normal animals.
The blastogenenesis augmenting factor present in the sera of
PL animals may be important in maintaining the PL stage of
EBL.
The observation that BLV-infected animals have a
blastogenesis augmenting factor present in their sera is
important for the establishment of BLV-induced enzootic
bovine leukosis as a model for HTLV I induced ATL.
lymphocytosis occurs in some cases of ATL (162).
A T— cell
The tat
protein appears to be responsible for the rapid
proliferation of antigen-activated lymphocytes in this
disease (105).
If the tat protein of BLV is found to be
responsible for the B-cell lymphocytosis observed in PL
animals, BLV-induced persistent lymphocytosis in cattle may
be used as an animal model for HTLV— induced ATL.
110
BLV(+)-Aleukemlc
BLV(+)-Persistent Lym phocytotlc
BLV(-j-Control
1) WBC
2) Lym phocyte
C ounts
Serum
H Istopaque
S eparation
F resh
Heat
Inactivated
56C
Dilute in
RPMI)640
PBMC
ADD
2 X 1 0 s ceIls/w efl
In RPMI]g4Q
3X10® cells to:
1) anti-bovine pan T cell
m onoclonal antibody
2} anti-bovine tgM
Phytom itogens
DPNA
2JCONA
3JPWM
Incubated 37C X 3 days
* T ♦ B cells
ADD3 H-thymldlne
|3 7 C X 4 hours
H arvest Cells
on Cell H arv ester (Skatron)
cpm
S cintillation Counting
Figure V I. I . Procedure f o r Lymphocyte Blastogenesis
/
SI
111
x © tim o o cD >
1 2 3 4 5 6 7 8 9 [101112
Mitogen:
Without
mitogen
Con A
PHA
PWM
C oncentration:
s u b o p ti m a l
ODtima!
su b o p tim a l
optim al
su b o p tim a l
optim al
Auto I' Dgous
H om o ­ Bovine
f r e s h | inactiv. lo g o u s f e t a l
inactiv. inactiv.
Figure V I. 2.
Schema o f P la te Arrangement f o r Lymphocyte
Transform ation w ith 3 Mitogens and 4 D if fe r e n t
Serum supplements (C e lls added to a l l w e l l s ) .
/
112
£J Con R 30ug/n1
^
PHR Sug/ml
3.0
2.0
8888
L O G ( 10)
STIMULATION
INDEX
(S.I
| PWM 0.2ug/ml
1.0
*%2 R 56 Y 73
Y 32 Y 74 Y 75 B 10 Y 11 Y 13
I__________ I I__________ I I---------- 1
BLVCNeg)
BLV(RL)
Animal
BLV(PL)
N u m/b e r s
Fiqure VI. 3. Response of Lyrrphocytes fran Cattle to Optimal
Concentrations of Selected Phytcmitogens Using
Sirmilation Index
113
D
7.0
C on R 3 0 u g /m l
| PWM 0.2ugSml
6.0
-
5.0
L O G ( 10)
DELTR
COUNTS
PER
MINUTE
^ PHR 5 u g / m l
4.0
3.0
36
Y
73
Y
32
Y
74
Y
I I
BLVCNeg)
75
B
10
Y
U
Y
13
I I---------- 1
BLVCRL)
Rnlmal
BLVCPL)
Numbers
Figure VI. 4. Response of Lyirpbocytes frcm Cattle to Optimal
Concentrations of Selected Phytanitogens Using
Delta Counts Per Minute
114
6
|~| Con fl 30ug/m1
0
PHR 5ug/m1
|
PWH 0.2ug/ml
-
COUNTS
PER
MINUTE
7 r
5 -
LOGU0)
I
4 -
Y
32
Y
74
Y
73
I____________________ I
BLV(RL)
Rnlmal
B
° ie
Y
it
Y
13
_____________________ i
BLV(PL)
N um bers
Figure VI. 5. Response of Lymphocytes fran Bovine Leukania Virus
Negative Cattle to Selected Phytanitogens in the
Presence of Sera frcm BLV-Infected Cattle
115
Table VI. 1. Statistical Analysis of Lymphocyte Blastogenesis
of Lymphocytes from Bovine Leukemia Virus
Infected Cattle Using Stimulation Index*
**
BLV
Status
BLV (-) Vs
BLV (AL)
BLV (-) vs
BLV (PL)
Mitogen
T Value
Con A
1.391
16
N.S.-^
PfiA
1.495
16
N.S.
EWM
0.631
16
N.S.
Con A
2.654
16
0.01
FHA
2.257
16
0.01
PWM
3.985
16
0.02
Degrees of
Freedom
P Value
*
Stimulation Index = cpns of mitogen treated cultures divided
by cpns of cultures with no mitogen.
**
BLV Status indicates: BLV (-) = Cattle negative for BLV
antibodies. BLV(AL) = Cattle positive for BLV antibodies but
have no clinical signs. BLV (PL) = Cattle positive for BLV
antibodies and have persistent lymphocytosis.
+
Optimal Concentrations.
Not significant at the level of p = 0.05.
116
Table VI. 2. Statistical Analysis of Lymphocyte Blastogenesis
of Lymphocytes from Bovine Leukania Virus
A
Infected Cattle Using Corrected Counts per Minute
**
BLV
Status
BLV (-) vs
BLV (AL)
+
Mitogen
■
T Value
Degrees of
Freedom
p Value
None
2.015
16
0.05
Con A
1.629
16
N.S.
PHA
1.894
16
N.S.
PWM
1.858
16
N.S.
None
2.677
Con A
0.3512
16
N.S.
PHA
0.3522
16
N.S.
PVM
0.1246
16
N.S.
I. I.
BLV (-) vs
BLV (PL)
0.02
Corrected counts per minute = cpns of mitogen treated cultures
minus cpns of cultures with no mitogen treatment.
**
BLV Status indicates: BLV (-) = Cattle negative for BLV
antibodies. BLV(AL) = Cattle positive for BLV antibodies
but have no clinical signs, BLV (PL) = Cattle positive
for BLV antibodies and have persistent lymphocytosis.
+
Optimal concentrations.
++
Not significant at the level of p = 0.05.
117
Table VI. 3. T / B Lymphocyte Ratios of Bovine
Leukemia Virus-Infected Cattle
Animal
Numbers
R 52
R 54
Y 73
.BLV
Status
mm
-
T /B a
Ratio
1.9
2.0
2.0
Y 32
Y 74
Y 75
AL
AL
AL
3.2
5.1
3.3
B 10
Y 11
Y 13
PL
PL
PL
0.5
0.8
0.7
T/B ratio = %T lymphocytes in peripheral blood
divided by the % B lymphocytes in the peripheral
blood.
CHAPTER VIis SUMMARY
The aim of the investigations reported herein was to
obtain a better understanding of the numbers and function of
bovine T lymphocytes in bovine leukemia virus (BLV) infected
cattle in the aleukemic (AL) and persistent lymphocytotic
(PL) stages of enzootic bovine leukosis.
Highly specific monoclonal antibodies that distinguish
bovine T and B lymphocytes and flow microfluorimetry (FACS
analysis) were used to determine the absolute number of B
and T lymphocytes in the peripheral blood of ELV-infected
cattle.
The peripheral blood lymphocyte populations from A
AL and 4 PL cattle appeared to be increased when compared to
that of 4 BLV negative control animals.
The increase in the
lymphocyte population in the AL animals was due to an
increase in T lymphocytes but not B lymphocytes* whereas*
in
the PL animals the increase appeared to be due to an
increase in both T and B lymphocyte populations.
This
information indicates that there is an alteration in the
lymphocyte populations of BLV-infected cattle.
The PL animals showed a significantly higher number of
surface immunoglobulin positive cells (slg"’') than did the AL
animals.
The majority of these cells had IgM on their cell
surfaces* which is typical for virgin (res'ting)*
intermediate or memory B lymphocytes. Therefore*
it appears
that these cells have been induced to proliferate but not to
differentiate into antibody secreting (plasma)
118
cells because plasma cells typically do not have surface
bound IgM as do intermediate mature (activated) B
lymphocytes and are rarely found in the peripheral blood.
This observation indicates that the increase in the numbers
of lymphocytes in the peripheral blood of PL animals is not
totally an antigen driven event.
It is possible that the
uncontrolled lymphocytosis is at least in part due to the
activity of a trans activational transcriptional protein
(tat protein)
(a product of the BLV genome).
It was shown
in this study that there is a comitogenic blastogenesis
augmenting factor in the sera of all AL and PL animals
tested.
This factor may be responsible for the further
proliteration of antigen—activated lymphocytes.
It has been
observed that antigen-activated HTLV III virus infected
cells are further induced to proliferate as a result of the
tat protein.
PL animal 632 had an elevated lymphocyte count (72 x
lCP/mm3 of whole blood) and contained a subpopulation of
lymphocytes that appeared to stain for markers of both
bovine B and T lymphocytes.
mere immature.
This suggested that these cells
It has been observed that animals with LS
contain immature lymphocytes circulating in the peripheral
blood.
Perhaps this is the BLV— infected cell population.
We were unable to guantitate the number of T lymphocytes in
this animal using cell surface markers. The lymphocytes of
this animal stained much more intensely with the anti-bovine
Igli monoclonal antibody than lymphocytes from the other PL
120
animals tested.
This suggests that there is modulation of
the expression of this immunoglobulin marker in animal 632.
Perhaps the gene that codes for cell surface bound IgM is
enhanced in its expression in PL animals as a response of
the tat protein effect on lymphocytes in the peripheral
blood.
Bovine T lymphocytes were purified from the peripheral
blood of BLV-infected* AL cattle using immuno-affinity
depletion <"panning") with anti-bovine immunoglobulin-coated
flasks.
Large yields of highly purified T lymphocyte
preparations (95-100X T lymphocytes and 0-35C contamination
with B lymphocytes) were obtained from BLV-negative and AL*
but not PL animals by the use of this technique.
This
purification technique was easy to perform* required minimal
amounts of reagents and gave higher yields of purified T
lymphocytes than any
technique.
other currently available purification
It was then possible to assess the presence of
BLV provirus in highly purified T lymphocyte populations of
AL animals.
A 7.5 kilobase fragment of the original BLV— provirus
probe (EcoRl fragment of the FLK-BLV DNA ligated into PUC-S
vector plasmid) was purified by Sac 1 restriction enzyme
digestion* agarDse gel electrophoresis and subsequent
fragment electroelution.
The presence of BLV provirus was
then demonstrated in the DNA of the peripheral blood
mononuclear cells of
all AL animals tested and 3 of the 4
purified T lymphocyte preparations using DNA hybridization
121
techniques with the highly specific radiolabelled (3=P) BLVDNA probe.
This study documents the first attempt to assess
the presence of the BLV provirus in highly purified
peripheral blood T lymphocytes from BLV— infected* AL cattle.
These experiments shouted that bovine T lymphocytes are
possible targets of BLV infection.
The significance of this
finding is that infection of T lymphocytes or a specific
subpopulation of T lymphocytes may lead to modulation of
this cell population and may facilitate the progression of
the disease to the PL and ultimately the tumor state.
This study also confirmed that the B lymphocyte is the
major target of BLV infection.
As these cells were depleted
from the PBMC by "panning", the hybridization signal that
was obtained from purified DNA of the "panned" cells was
much less dense than the signal obtained from the PBMC
preparation.
The lymphocyte blastogenesis assay was used as an
indicator of the functional ability of lymphocytes of AL and
PL animals to respond to mitogenie stimuli.
It appeared,
that the responsiveness of lymphocytes from AL and PL
animals to Con A, PHA, and PWM mitogens was comparable to
that of lymphocytes from BLV-negative animals when delta
cpms were used as measurement of mitogenic— induced
blastogenesis.
This indicated that the infection of some
cells of the lymphocyte population does not appear to alter
the overall response of the lymphocytes to mitogenie
stimuli.
122
High levels of spontaneous blastogenesis in the absence
of mitogenie stimulation was observed for lymphocyte
preparations of both AL and PL animals.
proliferation of lymphocytes is unclear.
The reason for this
Sera from AL and
PL animals was found to contain a blastogenesis—augmenting
factor(s) when added to lymphocytes from BLV-negative
control animals in the presence of Con A* PHA and PWM.
Therefore*
it appeared that the effect of augmentation was
on B and T lymphocytes.
tat protein.
Perhaps this serum factor is the
The PL animals appeared to have more of this
factor or a more active form of this factor in their sera
than the AL animals.
The PL animals also had higher levels
of spontaneous blastogenesis and higher rates of lymphocyte
proliferation in vivo as Judged by peripheral blood
lymphocytes counts.
The presence of the blastogenesis-
augmenting factor(s) in tne serum of BLV— infected animals
may facilitate the transition from AL to PL phase of EBL and
/or is at least partly responsible for maintenance of the PL
phase of EBL.
CHAPTER VIIis APPENDIX
The animals used in this project mere cows of various
breeds between the ages of 3 and 5 years old (Table VIII.
i>.
They were purchased from sale barns in southern
Louisiana and maintained at the Veterinary Medicine Farm*
Lousiana State University Agricultural Center* Louisiana
State University* School of Veterinary Medicine* Baton
Rouge* Louisiana for at least 3 months before being used in
the project.
lactating.
The animals were neither pregnant* nor
They were judged healthy on physical exam by the
staff veterinarian.
All BLV positive animals were naturally
infected* therefore*
it was not known when infection
originally occurred.
It was not known when persistent
lymphocytosis developed.
Peripheral blood was collected from all test animals at
monthly intervals prior to and during the project.
Serum
was harvested* and the agar gel immunodiffusion test (AG2D)
was performed for the detection of antibodies to BLV
antigens. Total WBC and differiential counts were performed
at monthly intervals to establish the BLV status (AL or PL).
Animals with detectable serum antibodies to BLV antigens but
no clinical signs of disease were considered to be aleukemic
animals (AL).
Animals with detectable serum antibodies to
BLV antigens and a persistent elevation of peripheral blood
lymphocytes (> 7.5 x lCPcelIs/mm3 whole blood over a three
month period) were considered persistent lymphocytotic
123
124artimale <PL) (2).
Animals with no detectable antibodies to
BLV antigens on repeated testing were BLV-negative animals.
These BLV negative animals were used as control animals in
all studies.
All animals were also palpated for the
presence of tumors.
No tumors were observed in any of the
test animals.
Animals were screened for Brucellosis and Anaplasmosis
and found negative.
diarrhea virus*
They were also vaccinated for bovine
infectious rhinotracheitis virus and
parainfluenza virus type III by the Louisiana State
University School of Veterinary Medicine clinical staff.
The presence of antibodies to bovine lymphotrophic
retroviruses* bovine syncytial virus (BSV) and bovine visnalike virus (BVV) was also evaluated.
Antibodies to these
viruses were detected by the use of indirect
immunofluoresence test assays utilizing bovine fetal spleen
cells infected with either BSV or BVV (Table VIII.
1).
125
Table VIII. 1. Assessment of Viral Infection in Animals
Used for Molecular Studies of Bovine
T Lymphocytes
Animal
Number
Breed
Antibodies
BLV+
BSV**
+
R
R
R
Y
52
54
56
73
Holstein
Holstein
Holstein ,
Jersey
Y
Y
Y
Y
12
32
74
75
Holstein
Holstein
Jersey
Jersey
+
+
+
+
(AL)
(AL)
(AL)
(AL)
B
Y
Y
Y
10
11
13
632
Hereford
Holstein
Holstein
Holstein
+
+
+
+
(PL)
(PL)
(PL)
(PL)
bvvh
+
+
-
-
-
*
-
—
—
—
_
+
-
—
+
+
—
-
+
—
—
+
+
-f
BLV antibodies determined by the use of agar gel
iimtunodiffusion. BLV status determined by clinical
signs.
BLV (-) - Animals with no detectable BLV antibodies
BLV (+)AL - Animals with detectable antibodies to
BLV antigens but no clinical signs of disease
BLV (-f)PL - Animals with detectable BLV antibodies
and exhibit persitent lymphocytosis.
++BSV - Bovine syncytial, virus
I||
BVV - Bovine visna-like virus
126
CHAPTER IX: BIBLIOGRAPHY
1.
Anderson, L . , Jarrett, W.F. and g.W. Crighton 1968.
A classification of lymphoid neoplasms of domestic
animals. Natl. Cancer Inst. Monogr.* 32:343-353.
2.
Anonymous, (i960). International Committee on Bovine
Leukosis. Criteria for the determination of the
normal and leukotic state in cattle. J. Natl.
Cancer Inst. 41:243-263.
3.
Baliga, V. and J.F. Ferrer. 1977. Expression of the
bovine leukemia virus and its internal antigen in
blood lymphocytes. Proc. Soc. Exp. Biol. Med.
156:38B.
4.
Barta, O. , Oyekan,P. and L., Shaffer. 19B4.
Lymphocyte transformation (Activation) test.
In:Laboratory Techniques of Veterinary
Clinical Immunology edited by 0. Barta.
Charles C. Thomas. Springfield, Illinois. pp.B6-100.
5.
Baumgartener, L. , Olson, C. and M. Onuma. 1976.
Effect of pasteurization and heat treatment
on bovine leukemia virus. J. Am. Vet. Med. Assoc.
169:1189-1191.
6.
Baumgartener, L.E., and C. Olson. 19B2. Host range of
bovine leukosis virus: preliminary report. Curr.
Top. Vet. Med. Anim. Sci. 15:338-347.
7.
Bech-Neilsen, S., Piper, C.E., and J.F. Ferrer. 1978.
Natural mode of transmission of the bovine leukemia
virus; role of blood—sucking insects. Am. J. Vet.
Res. 39:1089.
8.
Bendixen, H.J. 1965.
Bovine enzootic leukosis.
Adv. Vet. Sci. Comp. Med. 10:129.
9.
Bendixen, H.J.
1:422.
1965. Natl. Cancer Inst. Monogr.
ID.
Bianco, C., Patrick, R . , and V. Nussenzweig. 1975.
A population of lymphocytes bearing a memgrane
receptor for antigen-antibody-complexes, I.
Separation and characterization. J. Exp. Med.
132;702-707.
11.
Boothe, A. D. and M.J. Van der Maaten. 1974.
U1trastructural studies of a visna-like
syncytial— producing virus from cattle with
lymphocytosis. J. Virol. 13:197-204.
127
12.
Boothe* A.D. * Van der Maaten, M.J.* and W.A.
Malmquist. 1970.
Morphological variation of a
syncytial virus from lymphosarcomatous and
apparently normal cattle. Archiv fur die gesamte
Virusforschung. 31:373-384.
13.
Broder* S.* Bunn* P. A. * Jaffe* E.S. * et al. 1984.
T—cell lymphoproliferative syndrome associated with
human T-cell leukemia/lymphoma virus. Ann. Intern.
Med.
100s543.
14.
Brown* B.U. and N. Kolander. 1977. Statistics:
£ Biomedical Introduction. John Mi ley & Sons*
New York* pp. 328-331.
15.
Bruck* C.* Mathot, S.* Portelelle, D . » Berte* C.*
et al. 1982.
Monoclonal antibodies define eight
independent antigenic regions on the bovine leukemia
virus (BLV) envelope glycoprotein gp 51. Virology
122:342-352.
It.
Bruck* C . * Rensonnet* N. * Portetelle* D . * Cleuter*
Y., et al. 1984. Biologically active epitopes of
bovine leukemia virus glycoprotein g p 5 1 :their
dependence on protein glycosylation and genetic
variability.
Virology* in press.
17.
Burny* A.* Bex* F . * Chantrenne* H . * Cleuter, Y.,
et al. 1978.
Bovine leukemia virus involvement
in enzootic bovine leukosis. Adv. Cancer Res.
2B:251—311.
18.
Burny* A.* Bex* F. * Bruck* C.* Cleuter* Y . * et a l .
1978.
Biochemical studies on enzootic and sporadic
types of bovine leukosis. InsAntiviral mechanisms
in the control of neoplasia. Chandra*.P. editor.
Plenum* New York* p p . 83-99.
19.
Burny* A.* Bruck* C.* Chantrenne* H. * Cleuter* Y . *
et al. 198D.
Bovine leukemia virus: molecular
biology and epidemiology. In: Viral Oncology,
editor George Klein. Raven Press* New York. 234.
20.
Buxton* B.A. and R.D. Schultz. 1984. Factors
affecting the infectivity of lymphocytes from
cattle with bovine leukosis virus. Can J. Comp.
Med. 48:365-369.
21.
Buxton B.A.* Hinkle* N.C.» and R.D. Schultz. 1985.
Role of insects in the transmission of bovine
leukosis virus: Potential for transmission by stable
flies* horn flies* and tabanids. Am J Vet. Res.
46:123-126.
128
22.
Calafat* J. , Hageman* P.C.* and A.A. Ressang. 1974.
Structure of C— type virus particles in lymphocyte
cultures of bovine origin. J. Natl. Cancer Inst.
52:1251-1257.
23.
Calafat* J. and A. A. Ressang. 1977. Ultrastructure
of bovine leukemia virus. Comparison with other RNA
tumor viruses. In: Bovine Leucosis: Various
Methods of Molecular Virology* edited by A. Burny*
pp13-30. Commission of European Communities*
Luxembourg.
24.
Callahan* R.* Lieber* M.M.* Todaro* G.J.* et al.
1976. Bovine leukemia virus genes in the DNA of
leukemic cattle. Science 192:1005.
25.
Casey* J,W. and M.O. Nicholson. 19B2.
Susceptibility
to productive infection by the bovine leukemia virus
is governed at the proviral level. Fed. Pro 41:1275
26.
Cockerell* G.L. and E.A. Hoover. 1977. Inhibition
of normal lymphocyte mitogenie reactivity by serum
from feline leukemia virus— infected cats.
Cancer Res. 3 7 :3985-39B9.
27.
Copeland* T.D.* Morgan* M.A.* and S.Oroszlan.
19B3.
Complete amino acid sequence of the nucleic acid
binding protein of bovine leukemia virus FEBS Lett.
156:37-40.
28.
Dekegel* D.* Mammerickx* M . * Burny* A.* Portetelle*
D. * et al. 1977. Morphogenesis of bovine leukemia
virus <BLV>. In: Bovine Leucosis: Various Methods
of Molecular Virology.edited by A. Burny*
PP. 31-42.
29.
Dermont* E . * Clarke* J.K. and J. Samuels. 1971.
The morphogenesis and classifcation of bovine
syncytial virus.
Gen. Virol. 12:105-119.
30.
Deschamps* J . * Kettmann* R.* and Burny* A. 1981.
Experiments with cloned complete tumor— derived
bovine leukemia virus information prove that the
virus is totally exogenous to its target animal
species.
J. Virology* 40:605—609.
31.
Deshayse» L . * Levy* D.*Parodi* A.L.*and J. Levy.
1977.
Proteins of bovine leukemia virus. I.
Characterization and reactions with natural
antibodies. J. Virol. 21:1056.
129
32.
Devare, S.G.« Stephenson* J.R.* Santa* P.S.* et al.
1976. Bovine lymphosarcoma: Development of a
radioimmunologic technique for detection of the
etiologic agent. Sci. 194:1428-1430.
33.
Devare* S.* Chandler* S.* Samagh* B.S.* and J.R.
Stephenson. 1977. Evaluation of radioimmunoprecipitation for the detection of bovine leukemia
virus infection in domestic cattle. J. Immunol.*
119:277-282.
34.
Devare* S.* Oroszlan, S.* and J.R. Stephenson. 1978.
Application of radioimmunologic techniques to
epidemiologic studies of lymphosarcoma in domestic
cattle. Ann. Rech. Vet. 9:6B9-697.
35.
Diamond* A.* Cooper* G.M., Ritz* J. and M. Lane.
1983. Identification and molecular cloning of the
human BLym transforming gene activated in Burkett's
lymphoma. Nature 305:112-116.
36.
Dickler, H.B. and H.G. Kunkel. 1972. Interaction
of aggregated y—globulin with B-lymphocytes* J. Exp.
Med. 136:191-196.
37.
Dickler, H.B., Siegal* F.P., Bentwich* Z.H.* et al.
1973. Lymphocyte binding of aggregated IgG and
surface Ig staining in chronic lymphocytotic
leukemia. Clin Exp. Immunol. 14:97— 106.
38.
Dickler, H.B. 1974. Studeis of the human lymphocyte
receptor for heat aggregated or antigen— complexed
immunoglobulin. J. Exp* Med. 140:508-522.
39.
Dietzschold, B . * Kaaden, 0.R . * Uberschar*S. Wei land,
F.» and O.C. Straub. 1974. Suggestive evidence for
an oncornavirus-specific DNA polymerase from C-type
particles of boivne leukosis. Z. Naturforsch.
29:72-75.
40.
Diglio* C.A.* and J.F. Ferrer. 1976. Induction of
-syncytia by the bovine C— type leukemia virus. Cancer
Res. 36:1056-1067.
41.
Drescher, B . * Rossler, H . * Venker, P., and W.
Wittman. 1979.
Purification and characterization
of bovine leukemia virus DNA polymerase. Arch.
Geschtuulstforsch.
49:569—579
42.
Driscoll, D. M. and C. Olson. 1977.
Bovine leukemia
virus associated antigens in lymphocyte cultures.
Am J. Vet. Res. 38:1897-1898.
130
43.
Dungworth, D.L. Theilen, G.H. , and J. Lengyel. 1964.
Bovine lymphosarcoma in California.
II. The thymic
form.
Path. Vet., 1:323-350.
44.
Ferrer, J.F. Stock, N.D., and P.S. Lin. 1971.
Detection of replicating C— type viruses in
continuous cell cultures established from coins with
leukemia: effect of the culture medium. J. Natl.
Cancer Inst. 47:613-621.
45.
Ferrer, J.F., Avila, L . , and N.D.Stock. 1972.
Serological detection of type C viruses found in
bovine cultures. Cancer Res. 32:1864— 1870.
46.
Ferrer, J.F., Abt, D.A., Bhatt,D.H.* and R.R.
Marshak. 1974.
Studies on the relationship between
infection with bovine C— type virus, leukemia, and
persistent lymphocytosis in cattle.
Cancer Res.
3 1 :093.
47.
Ferrer, J.F., and C.A Diglio. 1976. Development of an
in vitro infectivity assay for the C-type bovine
leukemia virus. Cancer Res. 36:1068.
48.
Ferrer, J.F., Piper, C.E., Abt, D.A., Marshak, R.R.,
and D.M. Bhatt. 1976. Natural mode of transmission
of the bovine C-type virus (BLV). Bibl. Haematol.
43:235.
49.
Ferrer, J.F., C.E. Piper, D.A. Abt, and R.R Marshak.
1977. Diagnosis of bovine leukemia virus infection;
evaluation of serological and hematological tests
by means of a direct infectivity assay.
Am. J. Vet. Res. 38:1977.
50.
Ferrer, J.F. and C.E. Piper. 1978. Role of milk in
the transmission of BLV. Ann. Rech. Vet. 9:803-807.
51.
Ferrer, J.F., Marshak, R.R., Abt, D.A., and S.J.
Kenyon. 1979.
Relationship between lymphosarcoma
and persistent lymphocytosis in cattle: A Review.
J.A.V.M.A. 175:705-708.
52.
Froland, S.5. and J.B. Natvig. 1973.
Identification
of three different human lymphocyte populations by
surface markers.
Transplant Rev. 16:114-162.
53.
Fujimoto, Y. Miller, J, and C. Olson. 1969. the fine
structure of lymphosarcoma in cattle.
Pathol Vet
6:15-29.
131
54.
Georgiades* J.A. * Billiau* A. and B. Vandershueren.
1970.
Infection of human cell cultures with bovine
visna virus. J. Gen Virol. 3B:375-3B1.
55.
Gershwin* L.J.* Lance* P. and A.S. Rokito. 1983.
Cottiparison of analysis of bovine surface
immunoglobulin bearing and peanut agglutinin
binding lymphocytes by flow cytometry and
fluorescence microscopy. Vet. Immunol. Immunopath.
5:185-196.
56.
Ghysdael* J . * Kettmann R.* A. Burny. 1978.
Translation of bovine leukemia virus genome
information in heterologous protein synthesizing
systems programmed with virion RNA and in
cel 1-lines persistently infected by BLV.
Ann. Rech. Vet. 9:627-634.
57.
Ghysdael* J . * Kettmann* R . * and A. Burny. 1979.
Translation of BLV virion RNAs in heterologous
protein synthesizing systems. J. Virol.
29:1087-1098.
58.
Gilden* R.V.* Long* C.U.* Hanson* M . * et al. 1975.
Characteristics of the major internal protein and
DNA—dependent DNA polymerase of bovine leukemia
virus.
J. Gen. Virol. 29:305-314.
59.
Graves* D. C.* Diglio* C. A . * and Ferrer* J.F.* 1977.
A reverse transcriptase assay for detection of t.he
bovine leukemia virus.
Am. J. Vet. Res.
38:1739-1744.
60.
Graves* D.C. and L. V. Jones. 1981. Early
syncytium formation by bovine leukemia virus.
J. Virol. 38:1055-1063.
61.
Greig* A.S.* Chandler* S . * Samagh* B . * and A. M.
Bouillant*. 1978. A simple* rapid
syncytial-inhibition test for antibodies to bovine
leukemia virus. Can. J.
Comp. Med. 42*446-451.
62.
Guillemain* B . * Mamoun* R.* Astier* T . * Duplan* J.F.
et al. 1978. Early polykaryocytosis inhibition
test: Evaluation of its performance in a
sercepidemiologicaL survey of bovine leukemia
virus induced antibodies in cattle. Ann. Rech.
Vet. 9:709-720.
132
63-
Gupta, P.* Kashmiri, S.V. , and J.F. Ferrer. 1984.
Transcriptional control of the bovine leukemia virus
genome: role and characterization of a
non-immunoglobulin plasma protein from bovine
leukemia virus-infected cattle. J. Virology
50:267-270.
64.
Gutierrez, C., Bernabe, R.R*, and J. Vega. 1979.
Purification of human T and B cells by a
discontinuous density gradient of Percoll.
J. Immunol. Methods.
29:57-63.
65.
Halseltine, W. A., Sodroski, J . , Patarca, R. , et al.
.1984. Structure of 3' terminal region of type XI
human T lymphotrophic virus: Evidence for new
coding region.
Sc i .225:419-421.
66.
Hare, W.C., Yang, T.J., and R.A. McFeely. 1967. a
survey of chromosome findings in 47 cases of bovine
lymphosarcoma <leukemia). J. Natl. Cancer Inst.
38:383-387.
67.
Horvath, Z. 1982. Experimental transmission of bovine
leukosis to sheep (Clinicopathological
considerations). Curr. Top. Vet. Med. Anim.
Sci.15:269-281.
68.
Jacobs, R.M., Valli, V.E. and B.N. Wilkie. 1979.
Inhibition of lymphocyte blastogenesis by sera from
cows with lymphoma. Am. J. Vet. Res. 41:372-376.
69.
Janossy, G., and M.F. Greaves. 1971. Lymphocyte
activation.I. Response of T and B lymphocytes to
phytomitogens. Clin. Exptl Immunol. 9:483-498.
70.
Kaaden, O.R., Frenzel, B . , Dietzschold, B . , et al.
1977. Isolation of a pl5 polypeptide form bovine
leukemia
virus and detection of specific antibodies in
leukemic
cattle. Virology, 77:501-509.
71.
Kajikawa, 0., Koyama, H. , Yoshikawa, T. , et al. 1983.
Use of alpha-naphthyl acetate esterase staining to
identify T lymphocytes in cattle. Am. J. Vet. Res.
44:1549-1552.
72.
Kenyon, S.J., and C.E. Piper. 1977. Cellular basis of
persistent lymphocytosis in cattle infected with
bovine leukemia virus. Infect. Immun 16:891.
133
73.
Kenyon* 5.J.T and C.E. Piper. 1977.
Properties of
density gradient-fractionated peripheral blood
leukocytes from cattle with bovine leukemia virus.
Infection and Immunity 16*898-903.
74.
Kettmann* R. * Mammerickx, M. * Dekegel, D. * et al.
1975.
Biochemical approach to bovine leukemia.
Acta Haematol.
54:201—209.
75.
Kettmann* R.* Portelle, D . * Mammerickx, M . * Cleuter,
Y.* et a l . 1976.
Bovine leukemia virus* An
exogenous RNA oncogenic virus. Proc. Natl. Acad.
Sci., 73:1014-1018.
76.
Kettmann, R., Marbaix, 6., Cleuter, Y . , et. a l . 1980.
Genomic intergration of bovine leukemia provirus and
lack of viral RNA expression in the target cells of
cattle with different responses to BLV infection.
Leukemia Res.
77.
78.
Kettmann, R. Marbaix, G . , and A. Burny, et. al. 1980.
In: Viruses in naturally occurring cancer. CDld
Spring Harbor Conference on Cell Proliferation
?: 927-941
Kettmann, R., Cleuter, Y . , Mammerickx, M . , et al.
1980. Genomic integration of bovine leukemia
provirus: comparison of persistent lymphocytosis
with lymph node tumor form of enzootic bovine
leukosis. Proc. Natl.
Acad. Sci. USA 77:2577^25B1.
79.
Kettmann, R . , Deschamps* J . , Cleuter, Y. , Couez, D . ,
et a l . 1982. Leukemogenesis by bovine leukemia
virus: proviral DNA integration and lack of RNA
expression of viral long terminal repeat and 3'
proximate cellular sequences. Proc. Natl. Acad.
Sci. USA. 79:2465-2469.
80.
Kettmann R., Westin, E.H., Marbaix, G. , Deschamps,
et a l . 1983. Lack of expression of cellular
homologues of retroviral one genes in bovine
tumors. In: Modern Trends in Human Leukemia,
edited by Neth* R., et al. V. Springer, Berlin
Heidelberg New York, pp 21B-221.
81.
Kishimoto, T. 19B5. Factors affecting B-cell growth
and differentiation. Ann. Rev. Immunol. 3:133— 157.
82.
Knop, J. 1980.
Influences of various macrophage
populations on Con A induced T-cell proliferation.
Immunol. 41: 379—385.
134
83.
Koskif I.R. * Poplack* D.G.* and R.M. Blaese. 1776.
A nonspecific esterase stain for the identification
of monocytes and macrophages.
In: jU) Vitro
Methods of Cel1 Mediated and Tumor Immunity, eds.
B. Bloom and J. David. Academic Press Inc.*
New York pp. 359—362.
84.
Koyama* H . * Nakanishi* H. * Kajikawa* O. * et al. 1983.
T and B lymphocytes in persistent lymphocytic and
leukemic cattle. Jpn. J. Vet. Sci. 45:471-475.
85.
Kumar* S.P.* Paul* P.S.* Pomeroy* K.A. Johnson* D.U.
et al. 1978. Frequency of lymphocytes bearing Fc
receptors and surface membrane immunoglobulins in
normal* persistent lymphocytotic and leukemic cows.
Am. J. Vet. Res. 39:45-49.
86.
Kumar* S.P.* Muscoplat* C.C.* Thoen* C.O.* and D.U.
Johnson. 1979. Potentiation of lymphocyte mitogen
responses to Con A by antigen—activated peripheral
blood monocytes in bovine.
B7.
Kurnick* J.T.* Ostberg* L. and M. Stegagno. 1979. A
rapid method for the separation of functional
lymphoid cell populations of hyman and animal origin
on PVP—silica (Percoll) density gradients. Scand. J.
Immunol. 10:563-573.
88.
Langston* A . * Ferdinand* G.A.* Ouppanner* R . * et.al.
1978. Comparison of production variables of bovine
leukemia virus antibody-negative and antibody
positive cows in two California dairy herds.
Am. J. Vet. Res. 39:1093-1098.
89.
Levy* D.* Deshayes* L . * Parodi* A.* Levy* J.P. et al.
1977. Bovine leukemia virus specific antibodies
among French cattle. II. Radioimmunoassay with the
major structural protein (BLV p24).
Int. J. Cancer.
20:543-550.
90.
Lewin* H. A.* Davis* W. and D. Bernoco. 1985.
Monoclonal antibodies that distinguish bovine t
and B lymphocytes. Vet. Immuno.* and Immunopath.
9:87-102.
91.
Lewin* H. A.* Casey* J.W.* and D. Bernoco. 1985.
Lymphoblastoid cell lines derived from two cases
of enzootic bovine lymphosarcoma express a T— cell
phenotype. Abstract* Proceedings of Federation of
Biological Research * 1985.
135
92.
Lewis* 6.K.* and J.W. Goodman* 197B. Purification of
functional* determinant specific idiotype-beering
murine T cells. J. Exp. Med. 14B:915—924.
93.
Long* C.U.*
Henderson* L.E.* and S. Oroszlan. 19BD.
Isolation and characterization of low molecular
weight DNA binding proteins from retroviruses.
Virology 104:491—496.
94.
Oroszlan* S.* Sarngadharan* M.G.* Copeland* T.D.,
et al- 19B2.
Primary structure analysis of the
major internal protein p24 of human type C T-cell
leukemia virus. Proc. Natl. Acad. Sci. USA
79:1291-1294.
95.
Mage* M.G.*
McHugh* L.L. * and T.L. Rothstein. 1977.,
Mouse lymphocytes with and without surface
immunoglobulin: Preparative scale separation on
polystyrene tissue culture dishes coated with
specifically purified anti-immunoglobulin.
J. Immunol. Methods. 15:47-56.
96.
Malmquist* 14.A.* Van der Maaten* M.J. and A. D.
Boothe. 1969.Isolation* Immunodiffusion*
Immunofluorescence* and electron microscopy of a
syncytial virus of lymphosarcomatous and apparently
normal cattle. Cancer Res. 29:18B-2DD.
97.
Mammerickx*
M.* Portetelle* D. * and A. Burny. 19B2.
Experimental cross-transmissions of bovine leukemia
virus (BLV) between several animal species.
Zentralbl Veterinaermed (B) 28:69-81.
9B.
Mamoun R.Z., Astier* T.* and B. Guillemain. 1991.
Establishment and propagation of a bovine leukemia
virus-producing cell-line derived from the
leukocytes of a leukemic cow. J. Gen. Virol.
54:357-365.
99.
Mamoun* R.Z.* Astier*T.<, Guillemain* B . * and J.F.
Duplan.
1983.
Bovine lymphosarcoma: expression
of BLV-related proteins in cultured cells. J. Gen.
Virology. 64:1B95-19D5.
100.
Mamoun* R.Z.* Astier* B . * Guillemain* B . * J.F.
Duplan.
1983.
Bovine lymphosarcoma: processing of
bovine leukemia virus— coded proteins. 64:2791-2795.
101.
Maniatus*T.* Fritsch* E.F.* and J. Sambrook. 19B3.
Purifications of nucleic acid.
In:Molecular
Cloning Cold Spring Harbor Press, pp.458-462.
136
1D2.
Maniatus,T., Fritsch, E.F.,and J. Sambrook. 1983.
Spectrophometric determination of DNA or RNA. In:
Molecular Cloning. Cold Spring Harbor Press.
P.46B.
103.
Maniatus, T . , Fritsch, E.F. and J. Sambrook.
1983.
Preparation of stock solutions. In: Molecular
Cloning. Cold Spring Harbor Press. P P .4 4 7 - 4 4 B .
104.
Marshak, R.R., Coriell, L.L., Laurence, W.C. et al.
1962. Studies on bovine lymphosarcoma. I. Clinical
aspects. II. Pathological alterations and herd
studies. Cancer Res. 22:202-217.
105.
Marx, J. 1986. The slou, insidious natures of the
HTLV's. Sci. 231:450-451.
106.
McDonald, H.C., and J.F. Ferrer. 1976. Detection,
quantitation and characterization of the major
internal virion antigen of the bovine leukemia virus
by radioimmunoassay. J. Natl. Cancer Inst.,
5 7 s875-882.
107.
Merion , R. Brenner, J., Gluckman, A., Avraham, R.,
et al 1985. Humoral and cellular responses in
calves experimentally infected bovine leukemia
virus (BLV). Vet. Immuno and Immunopathol.
9:105-114.
108.
Miller, J.M., Miller, L.S., Olson,S., and D.G.
Gillette. 1969. Virus-like particles in
phytohemagglutinin- stimulated lymphocyte cultures
with reference to bovine lymphosarcoma. J. Natl.
Cancer Inst. 43:1297-1305.
109.
Miller, J.M. and M.J. van der Maaten. 1978.
Infectivity tests of secretions and excretion from
cattle infected uith bovine leukemia virus. J. Natl,
Cancer Inst. 62:425-428.
110
.
111.
Muscoplat, C.C., Johnson, D.W.» K.A. Pomeroy, et. a l .
1974. Lymphocyte surface immunoglobulin: Frequency
.in normal and lymphocytotic cattle. Am. J. Vet. Res.
35:593-595.
Muscoplat, C.C., Wu Chen, A., Johnson, D.W., and I.
Alhji. 1974. In vitro stimulation of bovine
peripheral blood lymphocytes: standardization and
kinetics of the responses. Am. J. Vet. Res.
35:1557-1561.
137
Muscoplat* C.C.* Alhaji* 1.* Johnson* D.U.* et al.
1974.
Character1stics of lymphocyte responses to
phytomitogens: comparison of responses of
lymphocytes from normal and lymphocytotic cows. Am.
J. Vet. Res.
35:1053-1057.
113.
Notzel* U . * Drescher* E . * and S. Rosenthal.
1962.
Detection of bovine leukaemia virus RNA sequences in
non-cultivated peripheral lymphocytes by in situ
hybridization with 3H-labelled viral cDNA. Acta
Virol 26:33-40.
114.
Ohshima* K* Okada* K.* Numakunai* S. et a l . 1981.
Evidence of horizontal transmission of bovine
leukemia virus due to blood sucking tabanid flies.
Jpn. J. Vet,
Sci. 43:79-81.
115.
Olson, C., Kettmann, R., Burny, A., and R. Kaja.
1981.
Goat lymphosarcoma from bovine leukemia
virus. J. Natl. Cancer Inst. 67:671—675.
116.
Onuma, M . , and C. Olson. 1977. tumor-associated
antigen in bovine and ovine lymphosarcoma. Cancer
Res. 37:3249-3256.
117.
Onuma, M . , Takashima, I., and C. Olson. 1978.
Tumor-associated antigen and cell surface marker
in cells of bovine lymphosarcoma. Ann. Rech. Vet.
9:825—83D.
118.
Oroszlan, S., Sarngadharan M.G., Copeland, T.D.,
et al.
1982.
Primary structure analysis of the
major internal protein p24 of human type-C T-cel1
leukemia virus.
Proc. Natl. Acad. Sci. USA
79:1291-1294.
119.
Parfanovitch, M.I., Zhdanov, V.M., Kolomiets, A.G.
et al. 1978. Leukogenic properties of purified BLV
and possible routes of transmission with leukocytes
of milk. Ann. Rech. Vet. 9:781-796.
120
.
Paul, P.S., Pomeroy, K.A., Johnson, D.W., et al.1977.
Evidence for the replication of bovine leukemia
virus in the B-lymphocytes. Am J. Vet. Res. 38:B73.
121.
Paul, P.S., Pomeroy, K.A., Castro, A.E, et. al. 1977.
Detection of bovine leukemia virus in B-lymphocytes
by the syncytia induction assay. J. Natl Cancer
Inst.
59:1269-1271
122.
Piper, C.E., Abt, D.A., Ferrer, J.F., and R.R.
Marshak. 1975. Seroepidemiological evidence of the
horizontal transmission of the bovine C-type virus.
Cancer Res.
35:2714.
138
123.
Piper, C.E. Ferrer, J.F. , Abt, D.D., and R.R.
Marshak.
1979.
Postnatal and prenatal transmission
of the bovine leukemia virus under natural
conditions. J. Natl. Cancer Inst. 62:165-16B.
124.
Pomeroy, K.A., Paul, P.S., Weber, A.F., Sorensen,
D.K., and D.W. Johnson. 1977.
Evidence that
B-lymphocyt.es carry the nuclear pocket abnormality
associated with bovine leukemia virus infection. J.
Natl. Cancer Inst. 59:281-283.
125.
Portetelle, D . , Mammerickx, M . , Bex, F . , et a l . 1 9 7 7 .
Purification of BLV 9 P 7 0 and BLV p24. Detection by
radioimmunoassay of antibodies directed against
these antigens. In : Bovine Leucosis: Various
Methods of Molecular Virology, edited by A. Burny,
PP. 1 3 1 —1 5 2 .
Commission of European Communities,
Luxembourg.
126.
Ressang, A.A., Gielkens, A.L., Quak, S., et al.l?78.
Studies on bovine leukosis. VI. Enzyme linked
immunosorbent assay for the detection of antibodies
to bovine leukosis virus. Ann. Rech. Vet. 9:663-666.
127.
Reeves, J.H. and H.U. Renshau. 1978.
Surface
membrane markers on bovine peripheral blood
lymphocytes. Am J. Vet. Res. 39:917—923.
128.
Rice, R.R., Stephens, R.M., Burny, A., and R.V.
Gilden. 1985. The gag and pol genes of bovine
leukemia virus: nucleotide sequence and analysis.
Virology 142:357-377.
129.
Robert-Guroff, M . * Nakao, Yl, Notake, K . , et a l .
1982.
Natural anitbodies to human retrovirus
HTLV in a cluster of Japanese patients with
adult T cell leukemia.
Sci. 215:975—97B.
130.
Romero, C.H., Abaracon, D . , Rowe, C.A., and A. G.
Silva.
1984. Bovine leukosis virus infectivity in
Boophilus microplus ticks. Vet.Record.115:440—441.
131.
Rosen, C.A., Sodroski, J.G., Kettmann, R. , Burny, A.,
and W. Haseltine. 1985. Trans activation of the
bovine leukemia virus long terminal repeat in
BLV-infected cells. Sci. 227. 320-322.
132.
Rouse, B.T. and L.A. Babiuk. 1974. Host responses to
infectious bovine rhinotracheitis virus III.
Isolation and immunologic activities of bovine T
lymphocytes.
J. Immunol. 113:1391-1398
139
133.
Sagata* N . * Yasmaga* T.* Tsuzuku* I.* et.al. 19B5.
Complete nucleotide sequence of the genome of BLV:
Its evolutionary relationship to other retroviruses.
Proc. Natl. Acad. Sci. U>S>A> 82:677-681.
134.
Salisburg* J.G. * Graham* J.M.* and C.A. Pasternak.
A rapid method for the separation of large and small
thymocytes from rats and mice.
1979. J. Biochem.
Biophys. Methods. 1:341-347.
135.
Slanon* D.J.* Press* M.F.* Souza* L.M.* et al.
1985.
Studies of the putative transforming protein of the
Type I human T-cell leukemia virus. Sci.
228:1427-1430.
136
.
Snedecor G. and W.G. Cochran. 1967.
Statistical
Methods. Sixth Ed.
Iowa State Univ. Univ. Press.
Anesi Iowa.
137.
Sodroskit J. G.. Rosen* C.A.* and W.A. Haseltine.
1984.
Trans-Acting transcriptional activation of
the long terminal repeat of human T lymphotropic
viruses in infected cells.
Sci. 225:381-385.
138.
Sodroski* J.G.* Rosen C.* Goh* C.W.* and U. Haseltine
1985.
A transcriptional activator protein encoded
by the x— lor region of the human T-cell leukemia
virus. Sci. 228:1430-1434.
139.
Southern* E. 1975. Detection of specific sequences
among DNA fragments separated by gel
electrophoresis.
J. Mol. Biol. 98:503.
140.
Stock* N.D.* and J.F. Ferrer. 1972. Replicating
C-type virus in PHA treated buffy coat cultures of
bovine origin. J. Natl. Cancer Inst. 48:985—996.
141.
Tabel* H . * Chander* S.* Van der Maaten* h.J.* and
J.M. Miller. 1976.
Bovine leukosis virus.
Epidemiological study of bovine C-type virus by the
use of the complement fixation test. Can J. Comp.
Med. 40:350-354.
142.
Takashima* I.* C.* Olson* Driscoll* D.M . * and L.E.
Baumgartnener. 1977. B lymphocytes and T lymphocytes
in three types of bovine lymphosarcoma. J. Natl.
Cancer Inst. 59:1205-1209.
143.
Thorn* R.M.* Gupta* P.* Kenyon* S.J.and J.F. Ferrer.
1981.
Evidence that the spontaneous blastogenseis
of lymphocytes from bovine leukemia virus-infected
cattle is viral antigen specific. Infection and
Immunity 34:84-89.
140
144.
Uckert* W . * and V. Wunderlich. 1979. Proteins of
bovine leukemia virus! pis is the major
phosphoprotein. Acta Biol Med Ger 30=K42.
145.
Uckert* W.i and Wunderlichf V. * Ghysdael* J . *
Portetelle*
D.* and A. Burny. 1964. Bovine leukemia
virus (BLV) structural model based on chemical
cross-linking studies. Virology* in press.
146.
Usinger* W.R.* and G.A. Splitter. I960. A simple
method for specific depletion of B lymphocytes from
bovine peripheral blood mononuclear cells.
Immunol.
Commun. 9 s693-703.
147.
Usinger* U.R.* and G.A. Splitter. 1981.
Two
molecularly independent surface receptors identify
bovine T lymphocytes. J. Immunol. Methods.
45:209-219.
146.
Van der Maaten* Boothe* A.D. and W.A. Malmquist.1969.
Further Studies of bovine syncytial virus.
Comparative Leukemia Res. 36:446-452.
149.
Van der Maaten* Boothe* A. D . * and C. L. Seger.
1972. Isolation of a virus from cattle with
persistent lymphocytosis. J. Natl. Can. Inst.
49: 1649-1657
150.
Van der Maaten* M. J. Hubbert* W. T . * Boothe* A. D. *
et al. 1973. Isolations of bovine syncytial virus
from maternal and fetal blood. Am. J. Vet. Res.
34:341-343.
151.
Van der Maaten* M.J.* J. M. Miller and A.D. Boothe.
1974. Replication type-C virus particles in
monolayer cell cultures of tissue from cattle with
lymphosarcoma. J. Natl. Cancer Inst. 52:491-497.
152.
Van der Maaten* M.J. and J.M Miller. 1976.
Replication of bovine leukemia virus in monolayer
cell cultures.
Bibl.
Haematol. 43:341—343.
153.
Van der Maaten* M.J.* and J.M. Miller. 1976.
Serological evidence of transmission of bovine
leukemia virus to chimpanzees. Vet. Microbiol.
1:351-357.
154.
Van der Maaten*
M.J.* and J.M. Miller. 1978.
Susceptibility of cattle to bovine leukemia virus
infection by various routes of exposure. In:
Advances in Comparative Leukemia Research, edited
by P. Beutvelzen. Elsevier* North Holland*
Amsterdam, pp. 29—32.
141
155.
Wei land* F . , Uberschar, S., Straub, O . , et al. 1?74.
C— type particles in cultured lymphocytes from
highly leukemic cattle. Intervirology, 4:14D-149.
156.
Weiland, F . , and O.C. Straub. 1975. Frequency of
surface immunoglobulin bearing blood lymphocytes in
cattle affected with bovine leukosis. Res. Vet. Sci.
2 0 i340-41.
157.
Weiland, F. and O.C. Straub. 1976. Differences in
the in vitro response of lymphocytes from leukotic
and normal cattle to Concanavalin A.
Res. Vet. Sci.
20:340-341.
158.
Weiland, F . , and S. Ubershar. 1976. U1trastructural
comparison of bovine leukemia virus (BLV) with
C-type particles of other species. Arch. Virol.,
54:187-190.
159.
Weiss, A., Hollander H. and J. Stobo. 1985. Acquired
Immunodeficiency syndrome: Epidmiology, virology,
and immunology. Ann. Rev. Med. 36:545-562.
160.
Wilesmith, J.W., Straub, O.C. and R.J. Lorenz. 1979.
Some observations on the epidemiology of bovine
leukosis virus in a large dairy herd. Res. Vet. Sci.
161.
Williams, D.L., Lo, J . , and G. F.
Enrichment of T lymphocytes from
blood mononuclear cells using an
depletion technique ("panning").
Immunopathol. (in press).
162.
Wong—Stal1, F. and R. C. Gallo. 1985.
Human
T-lymphotrophic retroviruses. Nature 317:393-403.
163.
Wysocki, L.H., and V.L. sato. 1978.
“Panning* for
lymphocytes: A method for cell separation. Proc.
Natl. Acad. Sci. (USA). 75:2844-284B.
164.
Yamamoto, S., Onuma, 11., Kosama, H. , Mikamai, T . ,
and H. Izawa. 1984. Suppression of natural cytotoxic
activity of lymphocytes from cattle and sheep during
the progress of bovine leukosis. Vet. Micro.
9:105— 111.
165.
Yamantota, N . , Okada, M . , Koyanagi, Y. , et al. 1982.
Transformation of human leukocytes by cocultivation
with an adult T cell leukemia virus producer cell
line. Sci. 217:737-739.
•f'
Amborski. 1985.
bovine peripheral
immuno—affinity
Vet. Immunol.
CHAPTER X:
NAME:
CURRICULUM VITAE
Diana Lynn Brubach Williams
DATE AND PLACE OF BIRTH:
July 7, 1951
Pittsburgh, Pennsylvania
CITIZENSHIP:
United States of America
HOME ADDRESS:
13B06 Broad Ave.
PHONE (504) 293-3815
Baton Rouge, Louisiana
70810
MARITAL STATUS:
Married
CHILDREN:
Anthony,
19B0
UNIVERSITY EDUCATION:
Institution
Louisiana State
Dates
Speciality
Degree
1974-1977
Microbiology
B.S.
1977-1979
Veterinary and
M.S.
University
Baton Rouge, La.
Louisiana State
University
Medical Entomology
Baton Rouge, La.
School of Veterinary
1982-1985
Medicine, Louisiana
Veterinary
Microbiology
State University,
Baton Rouge, La.
142
Ph.D.
D OC TO R AL EXAMINATION A ND DISSERTATION REPORT
Candidate:
Major Field:
D iana L. W illiam s
V e t. M edical S c ie n c e s
Title of Dissertation:
M o lec u la r S tu d ie s on T Lymphocytes from B ovine Leukemia V iru s
I n f e c te d C a t tle
Approved:
Major Professor and Chairman
Dean of the Graduate
uaye School
Sctux
EXAMINING COMMITTEE:
AT
OVq-
Date of Examination:
fj»..