Avian Pathology

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Isolation of Marek's disease virus: revisited

K. A. Schat

To cite this article: K. A. Schat (2005) Isolation of Marek's disease virus: revisited, Avian
Pathology, 34:2, 91-95, DOI: 10.1080/03079450500059289

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Avian Pathology (April 2005) 34(2), 91
/95

Isolation of Marek’s disease virus: revisited

K. A. Schat*

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853, USA

Splenocytes from chickens infected with low-passage stocks of Marek’s disease virus (MDV) RB-1B, a very
virulent (vv) strain and vv RK-1 were used to compare the efficacy of chick kidney cells (CKC), chicken
/

embryo fibroblasts (CEF) and chicken embryo kidney cells (CEKC) for virus isolation. CKC were superior
to CEF and CEKC. MDV foci were present at 4 days post infection in CKC but not until 6 days post
infection in CEF or CEKC. Virus yield was higher in CKC than in CEF or CEKC at 6 days post infection.
Passage of RB-1B in CKC yielded a significantly higher virus increase than with CEF or CEKC. The same
was true for RK-1 comparing CKC with CEKC. Interestingly, RK-1-infected CEF were negative or had very
low number of foci in passage 1, but virus yield increased 500-fold to 600-fold on passage in CKC, CEF, and
CEKC. Recommendations on procedures for successful virus isolation are provided.

Introduction

Marek’s disease virus (MDV), the causative agent of
Marek’s disease (MD), was first isolated in chick kidney
cell (CKC) cultures by Churchill & Biggs (1967) and
shortly afterwards in duck embryo fibroblasts (DEF)
(Nazerian et al. , 1968; Solomon et al. , 1968). In both
culture systems, characteristic cytopathic effects (CPE)
developed and incomplete herpesvirus particles were
detected in the nuclei of infected cells. Calnek & Madin
(1969) indicated that inoculation of CKC at 24 to 48 h
with monolayers 70% to 80% confluent yielded higher
virus isolation rates using JMCT (a MD-derived tumour
transplant) cells than with monolayers that were 30% to
40% complete. The use of chicken embryonal fibroblasts
(CEF) yielded conflicting results for virus isolation.
Kottaridis et al. (1968) reported the development of
CPE after the third passage in CEF, and inoculation of
these cells into chickens induced MD. However, Witter et
al. (1968) reported that CEF were refractory to MDV
infection. Chicken embryo liver cell, chicken embryo
lung cell, and chicken embryo kidney cell (CEKC)
cultures did not show CPE after inoculation with blood
from JM-infected chickens, but chickens inoculated with
these cells developed MD. Calnek (1967) also concluded
that different types of chicken embryonal cells were not
able to replicate MDV. Schat (1985) mentioned unpub-
lished data indicating that replication of MDV in CEKC
was abortive and that virus would be lost after several
passages in CEKC. Unfortunately, the negative virus
isolation results using different chicken embryonal cells
were mostly referred to as unpublished data or were
obtained prior to the isolation of MDV and therefore
lacked positive controls.
These results clearly suggest that CKC or DEF are the
optimal cell cultures to isolate MDV (Sharma, 1998).
The genetic susceptibility of the SPF chickens used for
the preparation of CKC does not influence virus
replication rates (Spencer, 1969). However, CEKC and
CEF are frequently used (De Laney et al. , 1995, 1998;
Lee et al. , 1999), often resulting in low or very low virus
isolation rates. In the summing-up of the 6th Interna-
tional Marek’s Disease Meeting in 2000, Calnek stated
‘‘I found it disheartening that some of the techniques
used for virus isolation were so woefully inefficient. For
instance, the use of CEK rather than CK cultures could
certainly account for the failure to detect viraemia in
many of the birds’’ (Calnek & Morgan, 2001, p. 332).
Similarly, during the Molecular Workshop on Marek’s
Disease in Cyprus in 2002 it was mentioned that virus
isolation often failed when samples were obtained from
MD-positive chickens that were suspected to be infected
with ‘‘hypervirulent’’ MDV strains in Europe. The
question was raised of whether recent isolates had
changed and that virus isolation of these strains had
become more difficult. This paper presents comparative
studies on isolation of MDV in CKC, CEF and CEKC
from chickens infected with very virulent (vv) RB-1B,
vv/ RK-1 MDV strains, and viruses of undetermined
virulence from recent field cases. DEF were not included
in this study because there are very few specific pathogen
free (SPF) duck flocks available.
Materials and Methods
Cell cultures. Primary CKC, CEF, and CEKC were prepared from 14-
day-old chicks, 10-day-old embryos, and 19-day-old to 20-day-old
embryos, respectively, following standard procedures (Schat & Pur-
chase, 1998). Cells were seeded in 35 mm dishes and incubated at 38.5 to
398C in 5% CO2 to establish nearly confluent monolayers at 24 h.
Growth medium consisted of M199 supplemented with 10% tryptose-
phosphate broth (TPB), 0.63% of a 10% NaHCO3 solution, 1% of
Antibiotic-Antimycotic Mixture 100X (Gibco, Grand Island, New
York, USA), and 4% foetal bovine serum (FBS). For CEF and
CEKC, this medium was replaced after 24 h by the same medium but
supplemented with only 0.2% FBS. The maintenance medium for CKC
consisted of an equal mixture of Ham’s F10 and 199 supplemented with

*To whom correspondence should be addressed. Tel: /1 607 253 4032. Fax: /1 607 253 3384. E-mail: kas24@cornell.edu
Received 8 September 2004. Provisionally accepted 12 November 2004. Accepted 16 December 2004
ISSN 0307-9457 (print)/ISSN 1465-3338 (online) # 2005 Houghton Trust Ltd
DOI: 10.1080/03079450500059289
92 K. A. Schat
7.5% TPB, 0.63% of a 10% NaHCO3 solution, 1% of Antibiotic-
Antimycotic Mixture 100X, and 0.2% FBS. Embryonated eggs and
chicks were obtained from SPF N2a (Weinstock & Schat, 1987) flocks
maintained by the Department of Microbiology and Immunology at
Cornell University.
Virus strains. The vv MDV strain RB-1B (Schat et al. , 1982) passage (p)
5 was passed twice (experiment 1) or three times (experiment 2) in SPF
chickens. The vv/ RK-1 strain (Calnek et al. , 1998) p9 was passed
once (experiment 1) or twice (experiment 3) in SPF chickens. Spleen
cells from individual chickens were harvested 7 to 14 days post infection
(d.p.i.), red blood cells were removed by centrifugation over Ficoll-
Paque (Amersham Biosciences, Sweden), and lymphocytes were stored
in liquid nitrogen using medium containing dimethyl sulphoxide (Schat
& Purchase, 1998). Dr Patricia Wakenell (College of Veterinary
Medicine, University of California at Davis, California, USA) kindly
provided CKC infected with p2 of an unidentified MDV isolate
obtained from tumour material from a Silkie hen. Tumour material
(NC) from a recent outbreak of MD was obtained from Dr Andrea
Miles (College of Veterinary Medicine, North Carolina State Univer-
sity, Raleigh, North Carolina, USA). The latter two samples were added
to determine the efficacy to isolate MDV from field material.
Monoclonal antibodies and indirect immunofluorescence assays. Mono-
clonal antibodies (mAb) specific for serotype 1, serotype 2, and serotype
3 (Lee et al. , 1983) were kindly provided by Dr Lucy Lee (ADOL, East
Lansing, Michigan, USA). CEF grown on glass coverslips infected with
p5 of the Silkie chicken were harvested and fixed in acetone, when CPE
was visible. CEF containing p3 of the NC isolate were placed in 18 wells
of a glass slide, air-dried, and acetone fixed. The fixed cells were
incubated for 30 min with the appropriate dilutions of the three mAbs,
washed, incubated for 30 min with fluorescin isothiocyanate-labelled
goat anti-mouse IgG H and L chain, washed, and examined for
fluorescent cells.
Experimental design. Primary cell cultures were inoculated at 24 h after
seeding when the monolayers were between 90% and 100% confluent.
Media were changed at 3 and 5 d.p.i. and foci were enumerated at 4 and
6 d.p.i. All cultures were trypsinized at 6 d.p.i. and the cells were
inoculated onto new primary cultures as outlined in the following for
each individual experiment.
Experiment 1. Four 35 mm Petri dishes with CKC, CEKC, and CEF
were inoculated with 0.5 /106 or 0.25 /106 splenocytes from a RB-1B-
infected chicken or 0.3 /106 splenocytes from a RK-1 infected chicken.
At 6 d.p.i., each CKC and CEKC culture was divided over two CKC
and two CEKC cultures and the foci were enumerated at 5 d.p.i.
Selected cultures of p2 were trypsinized, cells were divided over two new
CKC and CEKC, and foci were counted at 5 d.p.i. CEF infected with
splenocytes were not used for additional passage. Foci were enumerated
at 5 d.p.i.
Experiment 2. Four CKC, CEKC, and CEF cultures/bird were
inoculated with 0.5 /106 splenocytes from three individual RB-1B-
infected chickens and with the NC tumour material. At 6 days, cells
were trypsinized and each CKC and CEKC culture from 2 RB-1B-
infected chickens and the NC isolate were divided over two CKC and
two CEKC. CEF cultures from two RB-1B-infected birds and the NC
isolate were also trypsinized, and the cells from each culture were
divided over two CKC and two CEF cultures. Foci in all p2 cultures
were enumerated at 5 d.p.i.
Experiment 3. Splenocytes (0.5 /106) from three RK-1-infected chick-
ens and p2 of the Silkie hen isolate were inoculated onto four CKC,
CEF, and CEKC cultures/bird. At 6 d.p.i., each dish of each cell type
was trypsinized and 50% of the cell yield of each dish was used to
inoculate two dishes with the same cell type. The remaining cells of each
dish were used to infect two dishes of one of the other two cell types.
Foci were counted at 5 d.p.i.
Statistical analysis. The numbers of foci for each bird were expressed as
the mean number of foci/0.5 /106 splenocytes in p1. The number of foci
for each cell type in p2 and p3 were expressed as the estimated total
number of foci if four dishes with that cell type had been inoculated by
multiplying the total number of foci in two dishes with a factor 2. The
numbers of estimated foci for each cell type were compared for
statistical differences using the two-tailed Student’s t test.
Results
Primary isolation of RB-1B and RK-1 strains. The results
for the isolation of RB-1B and RK-1 are summarized in
Figure 1 (experiments 1 to 3), respectively. At 4 d.p.i.,
CPE was detected in CKC but, with one exception, not
in CEF and CEKC. At 6 d.p.i., virus was isolated in
CKC from all birds, but only 50% of the birds were
positive in CEF or CEKC. The number of focus-forming
units (FFU)/0.5 /106 splenocytes was always lower in
CEF and CEKC than in CKC. For each bird four dishes
of each cell type were inoculated and there was little
variation in the number of foci/dish, as illustrated in
Figure 2 for experiments 2 and 3 in CKC.
Primary isolation of virus from field samples. Two field
samples were tested and in both cases the number of foci
was higher in CEF than in CKC or CEKC (Figure 3).
However, the foci included HVT, which was confirmed
by immunofluorescence assays. The isolation of HVT
was not unexpected because these birds had been
vaccinated with HVT.
Passage of RB-1B and RK-1. In order to prepare stocks
of virus representing the original isolate it will be
important to increase titres of new isolates with the
smallest number of passages. To determine whether there
are differences between cell types in increasing titres, the
first passage of RB-1B-infected and RK-1-infected
CKC, CEF and CEKC were trypsinized and the cells
were used to inoculate two 35 mm dishes with CKC,
CEF, and/or CEKC. In experiment 1, CKC and CEKC-
isolated RB-1B and RK-1 were passed to these two cell
types. Passage of CKC-isolated virus to CKC and CEKC
increased the titre, while passage of CEKC-isolated virus
resulted in low virus yields. Similar results were obtained
when RB-1B p2 in CEKC was used to generate p3 in
CKC and CEKC (Table 1). The p2 CEKC were obtained
from CEKC infected with p1 RB-1B in CKC, because
inoculation of CEKC with p1 RB-1B in CEKC did not
yield enough foci to warrant making a passage. In
experiments 2 and 3, there was a significant increase in
the number of foci when virus-infected CKC were
inoculated onto CKC, but when these cells were inocu-
lated onto CEF or CEKC the yield remained similar to
the input value or slightly decreased (Figure 4). RB-1B
isolated in CEKC could be passed to CKC cultures with
a significant increase in virus yield (Figure 5a), confirm-
ing the data of experiment 1. However, passage of RK-1
CEKC did not increase the virus yield significantly in
any of the three cell types (Figure 5b). The passage of
CEF-isolated RB-1B to CEF and CKC resulted in a
marked increase in FFU yield in CKC and a lower yield
in CEF, especially in the bird that was negative for virus
isolation (Figure 6a). CEF was not a good cell type for
primary isolation of RK-1 with 0 or very few foci for
individual birds in contrast to isolation on CKC (Figure
1b). However, passage of the RK-1-inoculated CEF to

50 (a)
No. FFU / 0.5X106
40
Splenocytes
30
20
10
Bird 1 2 34 1 2 34
CKC CEF
Figure 1. Number of FFU of MDV isolated in CKC, CEKC, and CEF cultures inoculated with 0.5 /106 splenocytes/culture infected
with RB-1B (1a) or RK-1 (1b). The FFU represent the average for four cultures/bird. Foci were enumerated at 4 d.p.i. (open bars) and 6
d.p.i. (closed bars). The absence of a bar indicates that foci were not present.
CEF, CKC, and CEKC yielded on average between 500
and 700 FFU, with the highest number in CKC, but the
differences between CKC, CEF, and CEKC were not
statistically significant (Figure 6). This yield is signifi-
cantly higher than when RK-1 was isolated in CKC and
passed to CKC (compare Figures 4 and 6).
Discussion
The results confirm that CKC are superior for isolation
of serotype 1 MDV compared with CEF and CEKC. The
latter are not acceptable for virus isolation as was
suggested previously by Schat (1985), and although
virus can be isolated in CEKC, passage of CEKC-
isolated virus onto CEKC resulted in minimal increases
to actual decreases in virus yield. CEF are also not
recommended for virus isolation because titres are lower
than in CKC and foci could not be detected at 4 d.p.i. in
contrast to CKC. This is especially important if vaccine
viruses are present in the inoculum, because HVT but
also CVI988 and serotype 2 strains may replicate faster
than, and outgrow, wild-type viruses if foci can only be
identified at 6 d.p.i. Moreover, HVT, serotype 2 viruses,
and CVI988 vaccines can be readily isolated on CEF.
Identification of foci in CKC at 4 d.p.i. will facilitate the
selection of individual foci of wild-type virus using
morphological characteristics such as small clusters of
round cells (Schat, 1985; Witter & Schat, 2003). These
foci can be harvested from CKC using Eppendorf
pipettes while the cultures are observed through a tissue
60
50
No of FFU / dish
40
30
20
10
Bird 1 2 3 1 2 3
RK-1 RB-1B
Figure 2. Number of FFU isolated in individual CKC cultures
inoculated with 0.5 /106 RB-1B-infected or RK-1-infected
splenocytes/culture using the results from experiments 2 and 3
(birds 2 to 4 from Figure 1a and Figure 1b, respectively). Each
symbol represents one culture/bird. Foci were enumerated at 6
d.p.i.
Isolation of Marek’s disease 93
50
(b)
40
30
20
10
1 2 34 Bird 1 2 34 1 2 34 1 2 3 4
CEKC CKC CEF CEKC
culture microscope. The harvested cells can be collected
in tubes containing a small amount of trypsin-versene
(Schat & Purchase, 1998) to disperse the cells and
inoculated onto new CKC cultures. This approach will
enrich for the virus present in the harvested cells, but this
approach does not constitute biological cloning (see
later).
The finding that RK-1 was present in a large number
of CEF after inoculation without causing CPE was
unexpected and the reasons for this observation are not
clear. Moreover, inoculation of CKC, CEF, and CEKC
with the first passage in CEF yielded equally high titres.
It is not clear whether this is specific to RK-1 or that
other vv/ strains will show the same pattern.
Recommendations for virus isolation. The following
procedure for virus isolation is recommended. Prepare
CKC from 14-day-old chickens or DEF from 12-day-old
duck embryos as described by Schat & Purchase (1998).
The use of SPF chickens or SPF duck eggs is essential to
reduce the risk of introducing extraneous viruses.
Because there is no egg transmission of MDV, placement
of 1-day-old SPF chickens in isolators is sufficient to
provide a source of kidneys. However, if isolators are not
available, it may be possible to maintain a source of SPF
chickens free of MDV and other agents using a clean
room and proper management rules, but it will be
essential to check the CKC cultures for possible con-
tamination. Spleen cells, tumour cells, or peripheral
blood cells are all good sources for virus isolation, but
5
No. FFU / 0.5X106
4
Splenocy tes
3
2
1
Sample 1 2 1 2 1 2
CKC CEF CEKC
Figure 3. Number of FFU isolated in CKC, CEKC, and CEF
cultures inoculated with 0.5 /106 cells/culture of tumour material
(p2 in CKC) from a Silkie hen (sample 1) and NC tumour cells
(sample 2). The FFU represent the average for four cultures/
sample. Foci were enumerated at 4 d.p.i. (open bars) and 6 d.p.i.
(closed bars). The absence of a bar indicates the absence of foci.
94 K. A. Schat

Table 1. Isolation and passage of MDV strains RB-1B and RK-1 in CKC and CEKC cultures

Inoculum Number of FFUa detected

Passage Virus Cell type Numberof FFU CKC CEKC

1 RB-1B CKC 5 33 11
CEKC 1 0.5 0.5
RK-1 CKC 1.5 6.5 1.5
CEKC 0 1 0.5
2 RB-1Bb CKC 9.3 93 1.3
RB-1Bc CEKC 2.8 42.2 0

a
Number of FFU if all cells of p1 would have been used to inoculate these cells.
b
Average number of FFU using three 35 mm dishes of p2 to generate p3.
c
Average number of FFU using eight 35 mm dishes of p2 to generate p3.

9x (a) (b) (a) (b)

CKC *** CKC* 300 CKC* 800
Relative No of FFU

Average N o of FFU
2x CKC
CEF 600
6x 60
CEF
CKC* CEF
35 CEKC
400
1x CEF
3x 10
CEKC
5 200
1x CKC
CEKC
CEF
P1 P2 P1 P2
P1 P2 P1 P2
Figure 4. Relative increase of the number of MDV FFU in p2.
4a: p1 RB-1B-infected CKC were trypsinized and the cells were Figure 6. Increase in the number of MDV FFU in p2 using
passed to CKC or CEKC (experiment 2). 4b: p1 RK-1-infected MDV-infected CEF. 6a: p1 RB-1B-infected CEF were trypsi-
CKC were trypsinized and the cells were passed to CKC, CEF, or nized and the cells were passed to CEF or CKC (experiment 2).
CEKC (experiment 3). The values are based on the average 6b: p1 RK-1-infected CEF were trypsinized and the cells were
counts of FFU for three individual bird samples and are expressed passed to CKC, CEF, or CEKC (experiment 3). The values
as the relative number of FFU in p2 compared with the number of represent the average number of FFU based on four dishes for two
FFU in p1. The average number of FFU for the three birds in p1 individual bird samples (#1 /open symbols and #2 /closed
was set at 1. The open and closed symbols in (b) are representing symbols (a) or the average value for three individual birds (b).
the same birds in p1, but cells were passed to CKC and CEKC The open and closed symbols in (b) are representing the same
(open symbols) or CKC and CEF (closed symbols). Note that birds in p1 but cells were passed to CEF and CEKC (open
the scales for (a) and (b) are different. Significant differences: symbols) or CEF and CKC (closed symbols). Significant
*PB/0.05 and ***PB/0.001 using Student’s t test; vertical bars differences: *P B/0.05 using Student’s t test; vertical bars indicate
indicate the standard deviation. the standard deviation.

removal of red blood cells is strongly recommended to

facilitate the detection of foci. The number of lympho-
220 (a) (b)
CKC** cytes to be inoculated depends on the size of the culture
Average N o of FFU

180
20
2 4
2 CEF
P1
Figure 5. Increase in the number of MDV FFU in p2 using
MDV-infected CEKC. 5a: p1 RB-1B-infected CEKC were
trypsinized and the cells were passed to CEKC or CKC
(experiment 2). 5b: p1 RK-1-infected CEKC were trypsinized
and the cells were passed to CEKC, CEF, or CKC (experiment
3). The values represent the average number of FFU based on
four dishes/bird for two individual bird samples (#1/open
symbols and #2 /closed symbols (a) or the average value for
three individual birds (b). The open and closed symbols in (b)
are representing the same birds in p1, but cells were passed to
CEKC and CEF (open symbols) or CEKC and CKC (closed
symbols). Significant differences: **PB/0.01 using Student’s t
test; vertical bars indicate the standard deviation.
8 dish. Between 0.5 /106 and 106 cells is recommended for
CEKC
6
dishes with a diameter of 35 to 60 mm. Higher numbers
CEKC CKC of lymphocytes may inhibit the formation of foci and
CEKC decrease the virus yield per 106 lymphocytes (Calnek et
al. , 1982). Media changes 2 and 4 d.p.i. will be sufficient
CKC
2 and allow the rescue of virus from latently infected
CEKC
lymphocytes. If whole blood is used for virus isolation,
P2 P1 P2
media changes and extensive washing with phosphate-
buffered saline is recommended at 1 d.p.i. Inoculation of
three to four dishes with a single sample from a chicken
will be enough to isolate virus if MDV is present, even if
present at low levels. Selection and passage of individual
foci at 4 d.p.i. will strongly reduce the chances of
contamination with vaccine viruses or wild-type serotype
2 viruses. Once virus is isolated it is recommended to
expand the virus by inoculating SPF chickens with p2 or
p3 and to harvest splenocytes from these birds between 7
and 14 d.p.i. These cells can be used to reisolate virus
and obtain standardized stocks of low passage for
experimental purposes. This approach will yield an
‘‘unlimited’’ source of very low-passage material. It is

also recommended to produce biological clones by
inoculation of CKC with cell-free virus followed by
selection of a single focus in cultures with a small
number of foci (Calnek, 1973). Cell-free virus can be
obtained from feather follicle epithelium (Calnek et al. ,
1970a) harvested between 14 and 21 d.p.i. from chickens
inoculated with p2 or p3 using SPGA as a diluent
(Calnek et al. , 1970b). The addition of 5 to 10 mM
ethylenediamine tetraacetic acid to the medium will
increase the virus yield (Adldinger & Calnek, 1971).
Cloned virus can be amplified in birds once more and
cell-culture-propagated virus with adequate titres can be
obtained in three to four passages in CKC starting with
cloned virus in splenocytes.
Acknowledgements
The author thanks Ms P. H. O’Connell for technical
support. This work was supported in part by the USDA
Regional Research NE-1016.
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Avian Pathology (April 2005) 34(2), 1
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Non-English Abstracts
Isolation of Marek’s disease virus: revisited
K. A. Schat*

Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853, USA

Les splénocytes de poulet infectés par du virus de la maladie de Marek (MDV) correspondant aux premiers
passages d’une souche très virulente (vv) RB-1B et d’une souche hypervirulente vvRK-1, ont été utilisés /

pour comparer l’efficacité des cellules de rein de poussin (CKC), des fibroblastes d’embryon de poulet (CEF)
et des cellules embryonnaires de reins de poulet (CECK). Les CKC ont été supérieurs aux CEF et aux
CECK. Les foyers de MDV ont été mis en évidence le 4è jour après l’infection (dpi) pour les CKC mais pas
avant le 6è dpi pour les CEF et CECK. La récolte de virus a été supérieure avec les CKC qu’avec les CEF ou
CECK au 6è dpi. Le passage de RB-1B sur CKC a donné des quantités significativement supérieure en virus
par rapport au passage sur CEF ou CECK. Les mêmes résultats ont été obtenus avec RK-1 en comparant
CKC et CECK. D’une manière intéressante, les CEF infectés par RK-1 ont été négatifs ou présentaient des
foyers en nombre très faible au premier passage, mais la quantité de virus récoltée a augmenté de 500 à 600
fois en passant sur CKC, CEF et CECK. Les recommandations sur les procédures de l’isolement de virus
avec succès sont fournies.

Für den Vergleich der Eignung von Hühnernierenzellen (CKC), Hühnerembryofibroblasten (CEF) und
Hühnerembryonierenzellen (CEKC) zur Virusisolierung wurden Milzzellen von Hühnern, die mit einer
niedrigen Passage des hochvirulenten (vv) Stammes RB-1B des Virus der Marekschen Krankheit (MDV)
infiziert worden waren, und der vv RK-1-Stamm verwendet. CKC waren den CEF und den CEKC
/

überlegen. In CKC traten MDV-Plaques am 4. Tag nach der Infektion (dpi) auf, in CEF und CEKC jedoch
nicht vor dem 6 dpi. Die Passagierung von RB-1B führte in CKC zu einem signifikant höheren
Virustiteranstieg als in CEF oder CEKC. Das gleiche galt für RK-1 beim Vergleich von CKC und CEKC.
Interessanterweise waren RK-1- infizierte CEF in der 1. Passage negativ oder wiesen nur eine sehr geringe Zahl
von Plaques auf, aber der Virusgehalt stieg nach Passagierung in CKC, CEF und CEKC um das 500- bis 600-
fache an. Die Anzüchtungsanforderungen für eine erfolgreiche Virusisolierung werden beschrieben.

Se utilizaron esplenocitos de pollos infectados con estoc de pocos pases de virus de Marek (MDV) RB-1B,
una cepa muy virulenta (vv) y vv RK-1 para comparar la eficacia de células de riñón de pollo (CKC),
/

fibroblastos de embrión de pollo (CEF) y células de riñón de embrión de pollo (CEKC) para el aislamiento
del virus. Las CKC fueron superiores a las CEF y CEKC. Se observaron focos de MDV a los 4 dı́as post
infección (dpi) en CKC, pero no hasta 6 dpi en CEF o CEKC. Los rendimientos vı́ricos fueron superiores en
CKC respecto a CEF o CEKC a los 6 dpi. Pases de RB-1B en CKC produjeron un incremento del virus
significativamente más elevado que con CEF o CEKC. Interesantemente, CEF infectados con RK-1 fueron
negativos o presentaron un bajo número de focos en el pase 1, pero el virus incrementó 500 o 600 veces en
pases en CKC, CEF y CEKC. Se proponen algunas recomendaciones sobre los procedimientos para poder
aislar satisfactoriamente este virus.

*To whom correspondence should be addressed. Tel: /1 607 253 4032. Fax: /1 607 253 3384. E-mail: kas24@cornell.edu
Received 8 September 2004. Provisionally accepted 12 November 2004. Accepted 16 December 2004
ISSN 0307-9457 (print)/ISSN 1465-3338 (online) # 2005 Houghton Trust Ltd
DOI: 10.1080/03079450500059289