J. gen. Virol. (1988), 69, 1067-1077. Printedin Great Britain
1067
Key words: Marek's disease/herpesviruses/immunosuppression
Effect of lmmunocompetence on the Establishment and Maintenance of
Latency with Marek's Disease Herpesvirus
By C. B U S C A G L I A , * B. W. C A L N E K AND K. A. S C H A T
Department of Avian and Aquatic Animal Medicine, New York State College of Veterinary
Medicine, Cornell University, Ithaca, New York 14853, U.S.A.
(Accepted 26 January 1987)
SUMMARY
Marek's disease virus (MDV) infections normally have an early cytolytic phase in
lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection.
Little is known about the mechanisms that govern latency with herpesvirus infections,
including Marek's disease (MD). To investigate the importance of immunocompetence
for either the establishment or the maintenance of latency in MD, immunosuppressive
treatments were applied prior to infection with MDV or after latency was established.
These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal
thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age
before full competence. The effect of all the treatments was determined by examining
tissues and spleen cells for evidence of virus replication before and after cultivation
in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age)
incompetence resulted in prolonged and more widespread early cytolytic infection.
Immunosuppression by Cs after latency had developed resulted in a reappearance of
cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus
and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was
found mostly in T cells, although many of the virus-positive cells did not have markers
for either T cells or B cells. Immunosuppression by BM after latency had developed also
resulted in the reappearance of cytolytic infection in the spleen but only at a very low
level. These results suggest that immunocompetence is required for the establishment
and maintenance of latency.
INTRODUCTION
Marek's disease (MD) is a disease of domestic chickens caused by a cell-associated
herpesvirus and characterized principally by T cell lymphoma and a demyelinating peripheral
neuropathy (Calnek, 1980). The outcome of Marek's disease virus (MDV) infection is
determined by virus and host cell interactions. During the first 3 to 6 days post-infection (p.i.),
an early cytolytic infection of the primary lymphoid organs (bursa of Fabricius, spleen and
thymus) occurs; the predominant targets are B cells (Shek et al., 1983). This is followed by a
persistent latent infection of mostly Ia-bearing T cells (Calnek et al., 1984). Lymphomas may
develop in some~hickens several weeks or months later depending on factors such as genetic
constitution, age at infection of the host and the strain of MDV.
Herpesviruses have developed a unique relationship with the host whereby, following
primary infection, they remain in a latent form and may be reactivated throughout the life of the
host. In the latent phase of MD there is no evidence of virus-associated antigens or virus
particles in vivo, yet the virus can be recovered in vitro and short-term culture in vitro causes the
appearance of virions or viral internal antigens (VIA). Although very little is known about what
factors govern the induction and maintenance of latency with any of the herpesviruses, it is
reasonable to consider the possibility that there can be both intrinsic and extrinsic influences.
With many systems, derepression of latent infection generally occurs following immunosuppressive treatments in vivo or after cultivation in vitro oflatently infected cells. Also, it has been noted
0000-7958 © 1988 SGM
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1068
C. BUSCAGLIA, B. W. CALNEK AND K. A. SCHAT
that latency develops at the time of early immune responses in MD (Calnek, 1986). Schat et al.
(1980) showed that induction of latency was not affected by embryonal bursectomy; in other
words, latency develops in the absence of humoral immunity. It might be presumed, then, that
cell-mediated immunity (CMI) plays a major role, or that other mechanisms are involved in
latency. Therefore, the purpose of this study was to determine whether immune responses are
required to induce and maintain latency.
METHODS
Experimental chickens and holding conditions. Chickens ranging in age from 1 day to 7 weeks were obtained from
specific pathogen-free departmental flocks of two genetic strains: MD-susceptible P-2a (B19B19) and MDresistant N-2a (B21B21) (Weinstock & Schat, 1987). All experimental chickens were kept in isolation units on the
floor.
Viral inocula. Cloned MDV isolates CU-2 (low oncogenicity) and JM-16 (moderate oncogenicity) have been
described (Smith & Calnek, 1973; Calnek et al., 1984). All inocula for these experiments were from stocks of
infected chicken kidney cell cultures stored at - 196 °C. The dose was 500 to 1000 p.f.u./bird; inoculation was by
intra-abdominal injection.
Preparation o f lymphocyte suspensions. Spleens were collected aseptically and processed as reported by Calnek et
al. (1984). Briefly, spleen cells were gently forced through an autoclavable 60 ~tm mesh (Tetco, Elmsford, N.Y.,
U.S.A.), washed once in phosphate-buffered saline (PBS) pH 7.3 and centrifuged over Ficoll-Paque (Pharmacia)
at 400 g for 20 min. Lymphocytes, collected from the interface, were washed in PBS and resuspended in LM-Hahn
medium (Calnek et al., 1981) at a concentration of 5 x 106/ml for direct culture, or in PBS modified by addition of
1% bovine serum albumin and 0.1% sodium azide (MPBS) for staining of surface markers.
Cultivation in vitro. Lymphocytes were suspended in LM-Hahn medium at a concentration of 5 x 106/ml and
incubated in a humidified, 5 % CO 2 atmosphere at 41 °C for 24 or 48 h as described previously (Calnek et aL, 1982,
1984).
Mitogen stimulation. Stimulation in vitro by concanavalin A (Con A; Sigma) was carried out using methods
similar to those described elsewhere (Schat et al., 1978). Briefly, splenic lymphocytes were counted and seeded at
5 x 105 cells/well in a microtitre plate system (Linbro, Titertek cat. no. 76-247-05). There were two sets of
triplicates for each sample. Culture medium (LM 2) consisted of a modified LM-Hahn medium with only 2%
chicken serum and no foetal bovine serum, plus 0.025 M-HEPES and 0.1% sodium bicarbonate. An optimal
amount of Con A (25 gg/well) was added to one triplicate set, while the second set was left as a control. After
incubation at 41 °C for 48 h, the cells were labelled for 16 h with 1 ~tCiof[3H]thymidine (sp. act. 2.0 Ci/mmol) per
well. The ceils were then harvested with a multiple semi-automated sample harvester (Titertek cell harvester, Flow
Laboratories) on filter discs (Titertek, Flow Laboratories), and processed for liquid scintillation spectrophotometry. Results were expressed as the difference between c.p.m, of the means of the stimulated and control
triplicates. Based on previous experience, a difference of 2000 c.p.m, was arbitrarily considered positive. A
stimulation index (SI) was calculated as: SI = (c.p.m. in treated cultures - c.p.m, in untreated culture)/c.p.m, in
untreated culture. An SI of 1.0 or greater was considered to indicate a response to Con A (Calnek et al., 1985). To
be considered a responder, a donor chicken had to meet both criteria; i.e. there had to be a difference of at least
2000 c.p.m, and an SI of 1.0 or greater.
Examination of spleen cells for viral antigens. Direct fluorescent antibody (FA) tests for MDV-VIA were done
before cultivation (0 h) and at 24 or 48 h after cultivation as described previously (Calnek et al., 1984).
Dual staining o f spleen cells for detection o f surface markers on VIA-positive cells. For detection of T cell or B cell
surface markers, 1 x 106 to 2 x 106 cells were washed with MPBS and then sequentially treated (15 rain at 4 °C
followed by washing with MPBS) with 20 gtl of the desired mouse monoclonal antibody, 20 gl of rabbit IgG antimouse Ig (heavy and light chain; Miles-Yeda, Rehovot, Israel) and 20 ~tl of goat IgG anti-rabbit Igs, conjugated
with rhodamine (Cappel Laboratories). The monoclonal antibodies used for T cell markers and surface IgM (B
cells) were those used previously (Calnek et al., 1984); i.e. MAC T and MAC IgM, respectively. After the final
wash, 10 ~tl drops of cell suspensions were air-dried on glass slides, fixed in acetone and stained for MDV-VIA as
above. The Leitz microscope used for examining the smears was equipped with filters suitable for exciting either
conjugate, and by switching from one to the other it was possible to determine whether or not a given VIA-positive
cell had a given surface marker. The fluorescein isothiocyanate-stainedVIA was seen as granular intracytoplasmic
and intranuclear greenish fluorescence and the rhodamine-stained surface markers were observed as strong
patchy or solid-ring reddish fluorescence.
Examination o f tissue sections for viral antigens. Tissue samples were collected from bursa of Fabricius, thymus,
spleen, kidney and liver. They were sectioned on a cryostat, fixed in acetone and stained for MDV-VIA as
described by Calnek & Hitchner (1969). For the present study, tissues with fluorescing ceils were given a score of
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Latency in Marek's disease
1069
0.5 to 4.0 to denote the intensity of reaction which varied from rare, isolated, positive cells (0.5) to nearly all cells
positive (4.0). Negative tissues received a score of 0.
Immunosuppressive treatments. Cyclosporin (Cs) was kindly provided by Sandoz Pharmaceuticals, E. Hanover,
N.J., U.S.A. Cs was dissolved in a warm mixture of absolute ethanol and the semi-synthetic neutral oil, Mygliol
812 (Dynamit Nobel, Kay-Fries, Inc., Rockleigh, N.J., U.S.A.). Dosage was 50 mg/kg body weight injected
intramuscularly every 3 days as previously described by Nowak et al. (1982) for depression of T cell responses in
chickens. The treatment began at 1 day of age for chicks when the induction of latency was examined, or after
latency had been already established when the effect on maintenance of latency was to be tested.
One part of betamethasone (BM; Sigma) was dissolved in 75 parts of warm absolute ethanol. It was
administered as a single intramuscular injection of 4 mg/kg body weight, or as five daily doses of 2.2 mg/kg body
weight (Powell & Davison, 1986). BM was used only in attempts to break latency.
Neonatal thymectomy (Tx) was performed on the first day after hatching (Herbert et al., 1973) in conjunction
with cyclophosphamide (Cy) treatment. Cy treatment was carried out by intra-abdominal injection of 4 mg of
Cytoxan (Mead Johnson & Co., Evansville, Ind., U.S.A.) per day per chick, given on days 1 to 4 after hatching
(Linna et al., 1972). The combined treatment (Tx/Cy) had been shown in other studies to impair cellular immunity,
whereas Tx alone did not (Calnek et al., 1978a). At necropsy, all Tx/Cy-treated chicks were examined for the
presence of thymic tissues, and only those without remnants were included in the trial.
Statistical analysis. Where appropiate, data were compared for statistical significance by using Student's t-test
or the two-sample rank sum test performed by the Minitab computer program (Ryan et al., 1985). Statements of
significance are based on a probability level of at least 9 5 ~ (Snedecor & Cochran, 1980).
Experimental design
Three experiments were conducted to study the relationship between immunocompetence and the induction of
latency (experiments 1, 2, 3). In addition one experiment was conducted to examine the effect of chemical
immunosuppression on the maintenance of latency (experiment 4).
Experiment 1. Three trials were done in which MDV-inoculated and uninoculated groups of 3-week-old
chickens were subdivided into three subgroups respectively treated with Cs, untreated, or mock-treated with
carrier (Mygliol and ethanol). In the first trial, P-2a chickens were inoculated with CU-2 MDV, and in the second
and third trials N-2a and P-2a birds were inoculated with JM-16 MDV. Samples in trials 1 and 2 were collected at
5, 8, 12 and 15 days p.i., and in trial 3 at 8 and 15 days p.i. FA tests for MDV-VIA were done on frozen tissue
sections of bursa of Fabricius, thymus, spleen, kidney and liver. Spleen cell suspensions were examined for VIApositive cells before (0 h) and after 48 h of cultivation.
Experiment 2. Intact and Tx/Cy P-2a chickens were inoculated with JM-16 MDV at 2 weeks of age and
sampled at 8 and 15 days p.i. Intact birds in an uninoculated control group were sampled at 8 days p.i. only. FA
tests for MDV-VIA were performed on spleen cells before (0 h) and at 48 h after cultivation as well as on frozen
sections of bursa of Fabricius, thymus, spleen, kidney and liver.
Experiment 3. One-day-old, 2-week-old and 7-week-old P-2a chickens were inoculated with JM-16 MDV.
Samples were collected at 0, 5, 6, 7, 8, 11 and 14 days p.i. Samples at 0 days p.i. were obtained before inoculation.
Five 1-day-old chicks were left uninoculated as controls and sampled at 5 days p.i. FA tests for MDV-VIA were
done on spleen cells at 0 h and 48 h after cultivation as well as on frozen tissue sections of bursa of Fabricius,
thymus and spleen. Spleens obtained from the group of birds inoculated at l-day-old and sampled during the first
week were examined without separation over Ficoll-Paque. Mitogenic stimulation tests with Con A were also
performed on spleen cells collected at 0 days p.i.
Experiment 4. Three-day-old P-2a chickens were divided into two groups, one of which was inoculated with
JM-16 MDV. At 11 daysp~i., each group was subdivided into four subgroups. One subgroup was left untreated;
the others were given Cs,BM (slngle dose) or BM (five daily doses). Samplings were done at 0, 8 and 14 days after
the treatments were started. Frozen tissue sections of bursa of Fabricius, thymus and spleen, and spleen cell
suspensions before and after 24 h of cultivation in vitro were examined for the presence of VIA-positive cells by
FA. Smears were made at 0 h from spleen cells that were not separated over Ficoll-Paque so that dead cells would
not be removed from the samples. Positive cells in the suspension cultures were examined by dual fluorescence to
identify surface markers. Mitogen stimulation tests were performed on spleen cell samplings done at 0 and 14 days
post-treatment (p.t.).
RESULTS
Effects o f immunocompetence on the induction o f latency
T h e results f r o m e x p e r i m e n t 1 are p r e s e n t e d i n T a b l e 1. D a t a f r o m u n t r e a t e d a n d m o c k treated chickens were pooled because no differences were found between them. Birds without
d e t e c t a b l e cytolytic i n f e c t i o n s w e r e l a t e n t l y i n f e c t e d , o n t h e b a s i s o f t h e a b s e n c e o f V I A b e f o r e
c u l t u r e (0 h) a n d its p r e s e n c e a f t e r 48 h o f c u l t i v a t i o n in vitro o f s p l e e n cells. A l t h o u g h t h e r e w a s
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P-2a
N-2a
P-2a
1
2
3
MDV
JM-16
JM-16
CU-2
8
15
8
15
+
5
8
12
15
+
-
5
8
12
15
5
8
12
15
+
-
5
8
12
15
-
Cs
2
0
3
0
0
4
4
0
0
4
0
0
0
3
1
0
5
2
5
0
0
0 h
3
4
4
4
4
4
4
4
4
4
3
4
4
4
6
6
6
6
7
No.
examined
3
3
2
3
4
4
4
0
4
2
1
0
3
4
4
6
5
5
5
7
48 11
0
3 (1"0)b
0
1 (0-0)
4 (2.8)
0
0
[ (0.5)
3 (1.0)
0
0
0
4 (2-0)
2 (0.5)
0
1 (0.0)
4 (2.0)
0
0
0
•
Spleen
1 (0.0)
3 (1.0) b
0
1 (0.0)
4 (3.0)
1 (0.3)
0
1 (0.0)
3 (1.8) b
0
0
0
3 (2.5)
2 (0.5)
0
6 (1-5)a
6 (1.5)
0
0
0
3 (1.5)
2 (2.0) b
0
3 (1.0)
4 (2.5)
1 (0.3)
0
2 (0-5)
4 (2.5)
0
0
0
3 (2.5)
2 (0.5)
0
2 (0.0)
6 (2.5)
0
0
0
0
0
0
0
3 (1.0)
0
0
0
0
0
0
[ (0-0)
3 (1-0)
1 (0.0)
0
0
0
0
0
0
F A test on frozen tissues1"
"~
Thymus
Bursa
Liver
A
1 (0.0)
0
0
0
0
0
0
0
0
0
0
[ (0.0)
0
2 (0-5)
0
0
0
0
0
0
Kidney
* Spleen cell suspensions (0 h and 48 h cultures) and frozen tissue sections from all organs collected from uninoculated controls were negative in F A tests for MDV-VIA.
"~ Figures in parentheses are median F A test scores, statistical significance (two-sample rank s u m test) between CS-treated and untreated controls: a, P < 0.005; b, P < 0-05.
Strain
Trial
Days
p.i.
Spleen
culture
~
No. birds VIA-positive
T a b l e 1. Cytolytic and latent infection in Cs-treated and normal chickens*
;~
Z
Z
rn
;~
r-
Latency in Marek' s disease
1071
Table 2. Cytolytic and latent infection in Tx/Cy-treated compared with intact P-2a chickens
infected with JM-16 MDI/*
No. birds VIA-positive
f
Spleen
culture
x
() h
48 1~
Tx/Cy
No.
examined
-
8
5
0
5
0
0
2 (0.0)
0
0
15
6
0
4
0
0
4 (0.8)
0
0
8
15
5
8
4
4
4
5
1 (0.0)
2 (0-0)
No:~
ND
0
4 (0-5)§
0
2 (0.0)
0
3 (0"0)
+
•
Spleen
FA test on frozen tissues~"
x
Thymus
Bursa
Liver
Days
p.i.
Kidney
* Spleen cell suspensions (0 h and 48 h cultures) and frozen tissue sections from all organs collected from
uninoculated controls were negative in FA tests for MDV-VIA.
i" Figures in parentheses are median FA test scores, no statistical significance (two-sample rank sum test) was
found between Tx/Cy-treated and untreated controls.
:~ND, Not done.
§ In epithelial ceils.
some variation, it could generally be said that treatment with Cs caused a prolonged period of
early cytolytic infection characterized by a large amount of V I A in lymphoid organs, a more
widespread infection in liver and kidney evidenced by the presence of V I A and an enhanced late
(15 days p.i.) cytolytic infection, especially in trial 1. In contrast, birds not treated with Cs
generally displayed the pattern of a lytic infection followed by a latent infection as described
previously (Calnek, 1986). Statistical significance was shown between some of the scores given
to the tissues with fluorescing cells belonging to Cs-treated and the untreated controls.
The effect of the Tx/Cy treatment (experiment 2) is shown in Table 2. Cytolytic infection in
spleen at both 8 days p.i. and 15 days p.i. and in non-lymphoid organs (liver and kidney) at 15
days p.i. was present in the Tx/Cy-treated birds. In contrast, intact untreated controls had only
latent infection of spleen cells and no V I A was observed in non-lymphoid organs. V I A was seen
in bursas of chickens from both groups, but because of the depletion of lymphocytes by Cy, in
the Tx/Cy group it was in epithelial cells. Again, most birds which were not cytolytically infected
had latent infections.
The effect of age was tested in experiment 3 (Table 3). Cytolytic infection was prolonged in
birds infected at 1 day of age compared to those infected later. It was the only group with V I A in
thymus at 8 days p.i. and some bursas were positive at all sampling times. In contrast, 2-weekold birds were free of detectable V I A in spleen and thymus after 8 days p.i. and only two birds
had bursal infection at that time. None had any evidence of cytolytic infection in any organs
tested at 11 or 14 days p.i. The 7-week-old group also was essentially in complete latency after 7
days p.i. except for one bird each at the 11 and 14 days p.i. samplings which had VIA-positive
cells in the bursa. The presence of VIA-positive ceils in those two birds was not statistically
significant when compared to the other groups. Latent infections were demonstrated in most
birds not cytolytically infected after 7 days p.i. The immunocompetence of the birds was
evaluated by the blastogenic response of spleen cells to Con A. Table 4 shows the values of the
mitogen stimulation tests of the three different age groups prior to inoculation. Only three out of
eight 1-day-old chickens were responders compared to all 16 birds of the other two groups. Also,
the 1-day-old chickens which did respond had lower responses than 2- and 7-week-old birds.
Effects of immunocompetence on maintenance of latency
Table 5 summarizes the data from experiment 4 in which four, five or 10 birds per group were
sampled at 0, 8 and 14 days after treatment with Cs, five daily doses of BM or a single dose of
BM.
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1072
C. BUSCAGLIA, B. W. CALNEK AND K. A. SCHAT
T a b l e 3. Cytolytic and latent infection in I-day-, 2-week- and 7-week-old P-2a chickens infected
with JM-16 MDV*
No. positive (median FA score)
A
g
Age
1 day
Days
p.i.
No.
examined
5
6
7
8
5
5
5
5
Spleen
culture
x
0h
48 la
5
4
5
5
2
3
4
3
FA test on frozen tissuest
r
A
Spleen
Thymus
Bursa
5 (1.0)
5 (1.0)
4 (1.0)
0
5 (2-0)d
5 (1.0) e
5 (1.5)g
5 (2.5) h,~
3
3
5
3
(0.5)
(1.0) a,~
(2-5)¢
(1.5)
11
5
0
4
0
0
2 (0-0)
14
5
0
5
0
0
4 (1.0)
2 weeks
5
6
7
8
11
14
4
4
4
4
4
5
4
4
1
0
0
0
4
4
4
4
4
3
4 (1.0)
4 (2-0)
2 (0.5)
0
0
0
2 (1.8)
4 (4.0) e,e
4 (1.5)
Oh
0
0
2
4
4
2
0
0
7 weeks
5
6
7
8
5
5
5
5
2
5
3
0
5
5
3
4
3 (0.5)
5 (1.0)
3 (1-5)
0
2 (0-0)d
5 (1.0) f
4 (1.0)g
0i
1 (0.0)
3 (1-0)~
5 (1.0) c
0
11
14
5
5
0
0
2
5
0
0
0
0
1 (0.0)
1(0-0)
(1.8)
(3.0) a
(1.7)
(0.5)
* Spleen cell suspensions (0 h and 48 h cultures) and frozen tissue sections from all organs collected from
uninoculated controls were negative in FA tests for MDV-VIA.
t Figures having the same superscript letter in a certain tissue were significantly different (P < 0.05) in the twosample rank sum test.
T a b l e 4. Effect of age of P-2a chickens on the response of spleen cells to Con A
Age
No. responders/
no. tested
C.p.m. of responders
(Mean + S.E.M.*)
1 day
2 weeks
7 weeks
3/8
8/8
8/8
24422 + 9189
56703 + 9532
46114 _ 8453
SI of responders
(Mean _+S.E.M.)I"
7 + 2a,b
22 + 6a
32 _+ l i b
* Values are the arithmetic mean + standard error of the mean (S.E.M.)derived from counts on triplicate samples
from each bird.
t Figures having the same superscript letter were significantly different (P < 0-05).
S p l e e n cells for t h e 0 h s m e a r s w e r e e x a m i n e d w i t h o u t s e p a r a t i o n o v e r F i c o l l - P a q u e b e c a u s e
p r e v i o u s e x p e r i m e n t s h a d s h o w n t h a t t h e cytolytically i n f e c t e d cells s h o w n to b e p r e s e n t in t h e
frozen spleen sections were apparently lost after centrifugation over Ficoll-Paque, probably
b e c a u s e t h e y w e r e d e a d . U n t r e a t e d c o n t r o l s h a d n o e v i d e n c e o f cytolytic i n f e c t i o n in spleen,
w h e r e a s spleens f r o m C s - t r e a t e d b i r d s w e r e all p o s i t i v e for V I A .
A p r o p o r t i o n o f t h e u n t r e a t e d b i r d s d i d h a v e V I A - p o s i t i v e cells i n t h e b u r s a a n d t h y m u s , b u t
t h i s w a s s i g n i f i c a n t l y e n h a n c e d i n t h e C s - t r e a t e d birds. B M - t r e a t e d g r o u p s h a d s o m e b i r d s w i t h
V I A - p o s i t i v e spleens, b u t t h y m i c a n d b u r s a l i n f e c t i o n s w e r e s i m i l a r to t h o s e i n t h e c o n t r o l
group.
T o d e t e r m i n e w h i c h cells b e c o m e cytolytically i n f e c t e d in vivo a f t e r C s - i n d u c e d i m m u n o s u p p r e s s i o n , m o n o c l o n a l a n t i b o d i e s t h a t specifically r e a c t w i t h B ceils or ~I"cells w e r e used. D u a l
f l u o r e s c e n c e tests to d e t e c t s u r f a c e m a r k e r s a n d V I A w e r e c o n d u c t e d w i t h M D V - i n f e c t e d s p l e e n
cells freshly collected f r o m C s - t r e a t e d c h i c k e n s o r a f t e r in vitro c u l t i v a t i o n o f s p l e e n cells f r o m
o t h e r g r o u p s t h a t h a d few o r n o V I A - p o s i t i v e cells a t 0 h. A f t e r i m m u n o s u p p r e s s i o n w i t h Cs,
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Latency in Marek's disease
T a b l e 5.
1073
Cytolytic infections after Cs or BM treatment of P-2a chickens latently infected with
JM-16 MD V*
No. positive birds
~k
Days
p.i.
No.
examined
None
0
8
14
5
10
5
Cs
8
14
10
5
BM (5 doses)
8
14
10
4
BM (single dose)
8
14
10
4
Treatment
VIA in cultured
spleen cells
c
A
~
0 h
24 h
0
0
0
5 (12)
9 (6)
4 (3)
10 (11)
5 (9)
FA test on frozen
tissues~
•
~,
Thymus
Bursa
0
2 (0-0) d
2 (0"5)~
0
6 (0.6)a
5 (1.0)g
ND§
ND
9 (0"5) d'e'f
5 (0'5) j'k'l
10 (4-0) ",b,~
1 (<1)
2 ( < 1)
10 (5)
3 (11)
3 (0'3)e
0
7 (0-5) b
4 (0.5)h
2 ( < 1)
1 ( < 1)
10 (7)
4 (2)
5 (0"3)f
0 k,l
9 (0.8)e
4 (0"9) i
5 (3"5) o'ia'i
* Spleen cell suspensions (0 h and 48 h cultures) and frozen tissue sections from all organs collected from
uninoculated controls were negative in FA tests for MDV VIA.
l" Mean number VIA-positive/10 s cells or mean FA score.
:~ Figures having the same superscript letter were significantly different: a,b,c, P < 0.0005; d, P < 0 . 0 0 5 ;
e,f,g,h,i,j,k, P < 0.05 in the two sample rank sum test.
§ ND, Not done.
T a b l e 6.
Mitogen responsiveness of spleen cells from uninfected and MD V-infected P-2a chickens
before and after immunosuppressive treatments
Days
p.i.
Day
p.t.
MDV
infection
Treatment
11
0
No
Yes
25
14
No
Yes
Responders*
C.p.m.of
responders
(mean ___S.E.M.)af
SI of
responders
(mean + S.E.M.)
-
4/5
3/5:~
24462 + 13632
32589 + 16420
9+ 3
11 + 4
Cs
BM ( 5 x )
BM (1 x )
Cs
BM (5 x )
BM (1 x )
3/5
1/5§
1/5§
1/5§
3/5
1/5
4/4
1/4
12249 ___5101
2328 + 0
2224+0
4573 + 0
5253 ___741
2247 + 0
30529 + 9600
108605 + 0
18 + 0.4
2+ 0
12+0
6 +0
9+ 2
3+ 0
22 + 6
33 + 0
* Number positive/total number.
t Values are the arithmetic mean +_ standard error of the mean derived from counts on triplicate samples from
each bird.
One of the birds had a mean difference of 15308, but an SI of 0.6; for this reason it was not considered a
responder. It had a high background, probably due to the presence of tumour cells.
§ Three birds from each of the three groups had an SI greater than 1.0, but a mean difference smaller than 2000
c.p.m, because all values were very low.
c y t o l y t i c i n f e c t i o n w a s f o u n d m o s t l y in T cells, a l t h o u g h m a n y o f t h e V I A - p o s i t i v e cells d i d n o t
s h o w m a r k e r s f o r e i t h e r T cells o r B cells.
Mitogen stimulation tests were conducted in experiment 5 on uninoculated and inoculated
chickens before and after administration
o f t h e d i f f e r e n t t r e a t m e n t s . P r i o r to t h e
immunosuppressive treatments no differences were found between infected and control groups
a n d b o t h s h o w e d a m a j o r i t y o f r e s p o n d e r b i r d s ( T a b l e 6). F o u r t e e n d a y s p . t . , t h e n u m b e r s o f
r e s p o n d e r s f o u n d a m o n g all t r e a t e d s u b g r o u p s in t h e u n i n f e c t e d g r o u p a n d all b u t t h e five d o s e
B M t r e a t m e n t g r o u p in t h e i n f e c t e d g r o u p w e r e l o w e r t h a n i n t h e r e s p e c t i v e n o n - t r e a t e d g r o u p s .
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1074
c. BUSCAGLIA, B. W. CALNEK AND K. A. SCHAT
Birds treated with Cs were the lowest responders in the treated, infected group. Only one bird of
the single dose BM-treated subgroup was a responder.
DISCUSSION
This is the first time that immunocompetence has been shown to influence the establishment
and maintenance of latency in MDV infection. The results presented here suggest that latent
MDV infection, like that with other herpesviruses, can be activated by immunosuppressive
treatments. The results also suggest that treatments known to cause immunosuppression affect
the establishment of latency in MD.
In MDV infection, two types of virus-cell interactions have been described: productive,
where there is active synthesis of virus DNA, virus proteins and virions, and non-productive, in
which latent infection and transformation occur (Calnek, 1980). We chose as our working
definition of latency in MD, the condition in which the presence of the viral genome could be
demonstrated by the absence of viral antigens detected by immunofluorescence (Calnek, 1986).
Thus, latently infected cells were those which expressed VIA only after cultivation in vitro
(Calnek et al., 1981, 1984). There are no experimental data to show whether or not cells latently
infected with MDV transcribe parts of the viral DNA. However, with other herpesvirus
infections, it appears that latency is relative and that some D N A transcription may take place
(Stevens, 1980; Kieff et aL, 1982) but with incomplete or complete blocks at the transcriptional
and translational levels. The mechanisms responsible are not understood (Openshaw, 1984),
although Stevens et al. (1987) suggested that R N A transcripts complementary to a herpesvirus ~t
gene m R N A could be involved in maintaining the latent infection of herpes simplex virus
(nsv).
Some direct evidence for immune responses being linked to herpesvirus latency comes from
work by Stevens & Cook (1974) who showed that anti-HSV IgG bound to cell receptors was
involved in modulating latency. However, work by others (Rotula et al., 1984) indicated that
antibodies are not necessary to induce HSV latency and suggested that the transition from acute
to latent HSV infection might be mediated mainly both by unknown virus factors and by host
factors such as cellular immunity and interferon. Also, Schat et aL (1980) showed that chickens
which had been bursectomized as embryos and hence lacking B cells, and hence Igs, can become
latently infected with MDV. Consequently, the immunosuppressive treatments chosen for these
studies were those which are supposed to impair CMI. Cs treatment was selected because
responsiveness to Con A of peripheral blood lymphocytes (PBL) from Cs-treated birds had been
reported to be strongly depressed after 7 and 22 days of Cs administration (Nowak et al., 1982).
Corticosteroids are known to reduce CMI responses and BM was chosen because of a previous
report on its use in the MD system (Powell & Davison, 1986) where it increased the incidence of
MD in infected birds.
The various treatments were either shown, or could be inferred from other studies, to be
effective. Uninfected or infected birds treated with either Cs or BM in experiment 4 (Table 6)
clearly had reduced responses in terms of both number of responder birds and stimulation
indices. An exception was the infected group given five daily doses of BM; this could have been
because acute stress has been found to inhibit responsiveness while prolonged or chronic stress
can be stimulatory (Monjan & Collector, 1977). In experiment 1, although CMI responsiveness
was not tested, chickens were treated with Cs for at least 14 days before the first sampling.
Mitogen stimulation tests were not performed in experiment 2, but Tx/Cy had been shown
previously to depress markedly Con A responsiveness and to delay or prevent skin graft
rejection (Calnek et al., 1978 a). Cy reportedly eliminates, or seriously impairs, B cell functions
but it also has a transient depressive effect on the thymus (Linna et al., 1972).
The use of 1-day-old chickens, which are immunologically immature, was another approach
to test the effect of the immune response. Con A stimulation tests (Table 4) showed only three of
eight 1-day-old chicks to be responsive compared with eight of eight for each of the older groups.
Furthermore, the mean stimulation index for the three 1-day-old responders was low compared
to that of the others.
Immunocompetence seemed to be crucial in the establishment of latency with MDV. There
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Latency in Marek's disease
1075
was at least a delay in latency induction with all treatments. This was shown in the age effect
experiment where MDV infection in 1-day-old chickens failed to enter latency in all lymphoid
organs whereas it did in more competent older birds. The 7-week-old group was essentially in
complete latency after 7 days p.i. except for one bird each at the 11 and 14 days p.i. samplings.
These two exceptions may have represented the beginning of the second cytolytic phase which
can accompany permanent immunosuppression from MDV infection. The role of immunocompetence in the induction of latency is also suggested from the results with the Cs and the Tx/Cy
treatments. Cs-treated birds had prolonged and more widespread cytolytic infection than
untreated controls in trials 1, 2 and 3 (Table 1). Tx/Cy treatment similarly prolonged cytolytic
infection in spleen and resulted in more widespread infection (liver and kidney) at 15 days p.i. It
would have been interesting to determine which cells were cytolytically infected in the spleens of
Tx/Cy-treated birds at 8 and 15 days p.i., although it might be presumed that they were T ceils.
In spite of immunosuppression, latency developed in many cases. Also, latency in intact birds
was not always complete in all organs. The bursa was often the last organ to show cytolytic
infection before latency was 'complete' and was also generally the only one positive at late
sampling times. Perhaps latency can be expressed to a variable degree in various organs with
MDV. Many birds have a persistent, low-level productive infection in the feather follicle
epithelium but apparently only latent infections in other tissues. Latent infection of splenic
lymphocytes and PBL persists, probably for the lifetime of the birds, but virus may be shed from
the skin during the same period (Witter et al., 1971 ; B. W. Calnek, unpublished data). Certain
changes in internal environment and certain apparent non-specific stimuli may trigger
induction of virus production more easily in some tissues than in others. It seems that at the host
level complete latency may not occur with MDV.
Because latency developed in spite of immunosuppression, albeit in a delayed fashion, and
because we do not have a method for determining whether latently infected ceils are present
intermixed with cytolytically infected cells, one cannot rule out the possibility that an alternative
explanation for our findings is that immunocompetence is required for terminating cytolytic
infection but is irrelevant for the induction of latency. However, this explanation seems unlikely
because once latency was induced, immunosuppressive treatment clearly caused reactivation of
cytolytic infection (Table 5). The effects of induced immunosuppression might be clouded by the
fact that oncogenic MDV infection is in itself immunosuppressive and can cause permanent
suppression at 2 to 3 weeks p.i. Re-emergence of cytolytic infection coincides with this. Even so,
in experiment 5 (Table 5) substantial differences between untreated and treated groups were
observed wherein only treated groups had cytolytic infection in the spleen. Cs was more
effective than BM, but BM did have an effect, judging by the appearance of splenic infection in
at least a proportion of the birds in most of the sampling periods. The less striking effect of BM
could be because corticosteroids elicit their maximum immunosuppressive activity when
administered just prior to the antigen rather than after immunological challenge (Webb &
Winkelstein, 1982). Unfortunately, we only tested BM after latency was established. Although
we favour the conclusion that Cs or BM treatments had their effect indirectly by causing
immunosuppression we cannot exclude the possibility that these drugs had a direct effect by
activating the MDV genome. Additional studies are required to resolve this point.
The dual fluorescence tests on cells which were activated in vivo after Cs treatment showed
that the majority were T cells although some B cells were also observed. These results are in
agreement with those reported for latently infected cells by Calnek et al. (1984) and it seems
probable that the cells with cytolytic infection after immunosuppression represent the same
populations that were latently infected. Also, there were VIA-positive cells that did not react with
either the T cell or the B cell reagent. This could have been due to weaker than normal expression
of cell surface markers on the infected cells. Weak expression of T cell markers on MDVtransformed lymphoblastoid cell lines has been reported (Ross et al., 1977; Matsuda et al., 1976;
Calnek et al., 1978b).
Our observations on the effect of immunosuppression on the induction or maintenance of
latency are in keeping with findings in other systems. There are data suggesting that the host
immune response which limits productive HSV infection may also play a role in the
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1076
C. B U S C A G L I A , B. W . C A L N E K A N D K. A. S C H A T
establishment of latency protecting against it. CMI appears to be important in the control of
initial HSV disease as well as other animal herpesvirus infections (Stanberry, 1986; Harris et al.,
1984; Miller, 1985). In the guinea-pig model, Cs administration had adverse effects on the
pathogenesis of primary cytomegalovirus infection (Bia et al., 1985), as it did on the
pathogenesis of MDV infection in chickens (experiment 1, Table 1).
These results support the conclusion that immune responses are indeed involved in the
maintenance and probably also in the establishment of latency although the mechanism(s) is not
yet clear. Perhaps soluble factors such as interleukin 2 or gamma interferon, reported to be
affected by the immunosuppressive treatments chosen in this study (Hess et al., 1986; Nelson,
1984; Cohen et al., 1984; Hall & Goldstein, 1982; Besedovsky et al., 1986), play a role. HSVstimulated PBL were shown to produce a factor(s) that inhibits lymphokine activity (Sheridan et
al., 1985). The absence of lymphokines may interfere in some way at the molecular level by
derepressing transcription and translation. Alternatively, immunosuppression might provoke
new factors which could directly contribute either to prolongation or reactivation of cytolytic
infection. A combination of intrinsic and extrinsic influences may be involved in governing
latency.
Although the cell-mediated responses seem to be important, the question is unresolved and
additional studies will need to focus on identifying the specific components of the immune
system that contribute to the control of latency.
We thank M r R a y m o n d Harris for excellent technical assistance and Mrs G w e n Troise for helping in the typing
of the manuscript. This research was supported in part by Public Health G r a n t R01 CA06709-24 from the
National Cancer Institute. It was presented in part at the section on Avian Medicine, 123rd A n n u a l Meeting of the
American Veterinary and Medical Association, Atlanta, Ga., U.S.A., 20 to 24 July 1986 and at the 10th Latin
American Poultry Congress, Buenos Aires, Argentina, 29 September to 2 October 1987.
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