Vaccine 26 (2008) 5595–5600

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Vaccine
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Efficacy and safety of cell associated vaccines against Marek’s disease virus
grown in a continuous cell line from chickens
Harm Geerligs a,∗ , Sandra Quanz a , Brenda Suurland a , Ine E.M. Spijkers a , Jeff Rodenberg b ,
Frans G. Davelaar a , Berend Jongsma a , Mahesh Kumar b
a
Fort Dodge Animal Health Holland, Bio R&D department, Van Houtenlaan 36, 1381 CP Weesp, The Netherlands
b
Fort Dodge Animal Health, Biological R&D, 800 fifth street N.W., Fort Dodge, IA 50501-0518, USA

a r t i c l e i n f o a b s t r a c t

Article history: The Marek’s disease virus (MDV) vaccine strains CVI 988 and herpes virus of turkeys (HVT) strain FC126,
Received 18 March 2008 usually are grown in primary chicken embryo fibroblasts (CEF). We found that the strains could be grown
Received in revised form 13 June 2008 also in the so-called JBJ-1 cell line to titres in the same range as when chicken embryo fibroblasts were
Accepted 29 July 2008
used. The JBJ-1 cell line is a fibroblast-like continuous chicken cell line, which can be grown in flat bottom
Available online 14 August 2008
tissue culture flasks, roller bottles and on micro carriers. We investigated the efficacy of experimental CVI
988 vaccines grown in JBJ-1 cells and the efficacy of combinations of CVI 988 grown in JBJ-1 cells with HVT
Keywords:
FC 126 also grown in JBJ-1 cells. The study was performed in accordance with European Pharmacopoeia
Marek’s disease
JBJ1 cell line
monograph 0589 for live MDV disease vaccines. Groups of 1-day-old SPF chicks were vaccinated subcu-
Efficacy taneously or intramuscularly, with 102.5 TCID50 per dose of CVI 988 alone or in combination with 500 PFU
per dose of HVT. As a control a group vaccinated with CVI 988 grown in CEF was included. One group was
not vaccinated. Five days after vaccination all chickens were challenged with the very virulent MDV strain
RB1B. After challenge the chickens were observed for a period of 70 days for signs of Marek’s disease (MD).
The protection induced by CVI 988 grown in JBJ-1 cells and the combination of CVI 988 and HVT-FC126
both grown in JBJ-1 cells, amply complied with the requirements of the European Pharmacopoeia which
prescribes that the protection index should be at least 80%. The safety of the vaccines grown in JBJ-1 cells
was tested in a field study in commercial layer chickens. No signs of MD were noticed during the study
and no other signs attributable to the vaccine. It is concluded that the JBJ-1 cell line is a suitable substrate
for the current vaccines against MD.
© 2008 Elsevier Ltd. All rights reserved.

1. Introduction
Marek’s disease virus (MDV) is the causative agent of a neo-
plastic lymphoproliferative disease of domestic chickens. The virus
causes mononuclear cell infiltration and the development of lym-
phomas, mainly in the peripheral nerves and visceral organs [1].
More intensive husbandry techniques, which were adopted for
poultry in the middle of the 20th century, resulted in the appear-
ance of more and more virulent forms of the virus. Economic losses
caused by MDV were great [1] and this stimulated the efforts to
isolate the virus and to develop effective vaccines. The most effec-
tive vaccine against MDV is based on an isolate by Rispens et al.
[2], i.e. strain CVI 988. This vaccine is used alone, or in combina-
tion with serotype 2 or serotype 3 (herpes virus of turkeys, HVT)
vaccine virus strains. These MDV vaccine strains usually are grown
∗ Corresponding author. Tel.: +31 294 478050; fax: +31 294 478115.
E-mail address: geerlih@fdah.com (H. Geerligs).
in primary chicken embryo fibroblast (CEF) cells. CEF are obtained
from embryos from fertilized chicken eggs which have been incu-
bated for approximately 11 days at 37 ◦ C in a humid atmosphere.
The embryos are washed and treated with trypsin. The cell sus-
pension, obtained after treatment of the embryos with trypsin, is
sieved, washed several times, suspended in tissue culture medium
and seeded in tissue culture bottles, in which they form confluent
monolayers after incubation at 37 ◦ C. For vaccine production the
monolayers are inoculated with the seed virus and incubated at
37 ◦ C. Subsequently, the infected cells are harvested by trypsiniza-
tion, washed and suspended in a cell culture medium with foetal
calf serum (FCS) and dimethyl sulfoxide (DMSO). The cells are filled
in glass ampoules and frozen and stored in liquid nitrogen. From
the above it is concluded that the production of MD vaccines in
CEF is a time and labor consuming process. Because a lot of aseptic
handlings are required, there are serious risks for contaminations.
Moreover, not all eggs are free from bacteria. In order to avoid risks
for contaminations the use of high concentrations of antibiotics is
inevitable. It would be preferable to have a continuous cell line for

0264-410X/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2008.07.080
5596 H. Geerligs et al. / Vaccine 26 (2008) 5595–5600
the production of the vaccine, but attempts to find continuous cell
lines suitable for growing these viruses have had limited success.
CHCC-OU2 cells are susceptible for MDV and a latent form of MDV
infection can be established [3]. However, the usefulness of this sys-
tem is limited by the fact that virus is reactivated after the infected
cell lines reach confluency. Quail cell lines CU447 and CU453 have
been tested and it appeared to be possible to detect multiplica-
tion of MDV strains in these cell lines [4], but no further data are
available on safety and efficacy of vaccine strains grown in these
cells.
We tested the so-called JBJ-1 cell line as substrate for strain
CVI 988 and HVT-FC126. The JBJ-1 cell line arose as a spontaneous
immortalization of chicken embryo fibroblasts made from embryos
of the ELL-0 genetic line of chickens [5]. A JBJ-1 cell bank was tested
according to the requirements of the European Pharmacopoeia. We
tested the cell line as substrate for MD strains CVI 988 and HVT-
FC126, and tested the efficacy and safety of the vaccine viruses
grown on this cell line in chickens.
2. Materials and methods
2.1. The JBJ-1 cell line
After arrival at the R&D laboratory of Fort Dodge labs, Fort Dodge,
IA, USA, a JBJ-1 cell bank frozen in liquid nitrogen was established.
Samples of the cell line were distributed to the R&D lab at Fort
Dodge, Weesp, The Netherlands, where also a cell bank was estab-
lished. Exemplary cells were designated UMNSAH-DF 1 cells and
were deposited with the American type culture collection (ATCC),
Rockville MD, USA, accession number CRL-12203.
We tested the growth of the JBJ-1 the cell line, which is an
adhesive cell line, in various types of tissue culture bottles, viz.
flat bottom tissue culture bottles, roller bottles, and tissue cul-
ture plates. Different cell seeding densities were tested. Various
media were tested for cell growth, for example Dulbecco’s min-
imal essential medium, supplemented with sodium bicarbonate,
10% FCS and a suitable concentration of gentamicine sulphate. We
also tested a custom made medium by Invitrogen Corporation,
New York, USA. This so-called QT35 medium had been developed
especially for the growth of QT35 cells. One liter of this medium
contains: 430 ml Ham’s F12, M199 43 ml (10× concentrated), 50 ml
LAH 2.5% in Hank’s solution, 10 ml Fructose 20%, 50% FCS, 20 ml
HEPES buffer, 9 ml sodium bicarbonate 7.5%, 0.25 ml gentamicin
sulphate 50 mg/ml and water to a total volume of 1 l. We incu-
bated the cells mainly at 37 ◦ C. After reaching confluency the cells
were treated with a trypsin solution in phosphate-buffered saline
with disodium triacetate, to have the cells detached from the sur-
face. The cell suspension was diluted further in tissue culture
medium with supplements and added to fresh tissue culture bot-
tles.
The cell banks were tested for cell culture characteris-
tics, absence of bacterial and fungal contamination, absence of
mycoplasma and absence of extraneous agents according to the
requirements of the European Pharmacopoeia, ed. 2003. The cell
banks also were subjected to karyotype analysis and tested for Mn2 +
and Mg2 + dependent reverse transcriptase activity.
2.2. Production of CVI 988 and HVT-FC126 vaccines in JBJ-1 cells
From a normal batch of Poulvac® Marek CVI 988, stored in liquid
nitrogen and containing 3 × 107 cells per ampoule, a sample was
taken and thawed. From a 490 cm2 roller bottle with a confluent
monolayer of JBJ-1 cells the cell culture medium was discarded.
Fifty % of the content of the thawed ampoule of Poulvac® Marek
CVI 988 was inoculated and incubated for 1 h at 37 ◦ C. Cell cul-
ture medium was added to the roller bottle and the bottle was
incubated further at 37 ◦ C. During incubation the monolayers were
examined regularly for cytopathogenic effect. If after a few days
no cytopathogenic effect was visible, the culture was harvested by
trypsinization and inoculated on fresh JBJ-1 cell monolayers. If it
was decided to store samples of a certain passage level, the har-
vested infected cell suspension was mixed with culture medium
with 10% FCS and 10% DMSO, 3 × 107 cells per ml, filled in 1 ml vials
and frozen and stored in liquid nitrogen. For HVT-FC126 the same
procedure was followed as for CVI 988. The starting material for
passaging was a batch of Poulvac® Marek HVT.
2.3. Titration
2.3.1. MD strain CVI 988
The MD strain CVI 988 was titrated using the same method as is
used for titration of commercial batches of Poulvac® Marek CVI, i.e.
a TCID50 titration method. Three ampoules were taken from storage
in liquid nitrogen and thawed. The content of each ampoule was
diluted in Poulvac® Marek diluent and 10-fold dilution series were
prepared in cell culture medium [6]. Of each dilution samples of
0.5 ml were inoculated on six confluent monolayers of CEF in 48-
well tissue culture plates. The plates were incubated for 10 days at
38.5 ◦ C ± 1 ◦ C/5% CO2 ± 0.5. At regular intervals the cultures were
examined for the presence of cytopathogenic effect typical for the
MD virus. The titer was calculated using the method of Reed and
Muench [7] and expressed in TCID50 per dose.
2.3.2. HVT-FC126
Strain HVT-FC126 was titrated according to the same method
as for the licensed product, i.e. a PFU (plaque forming units) titra-
tion method. Three ampoules were taken from storage in liquid
nitrogen and thawed. The content of each ampoule was diluted in
Poulvac® Marek diluent and 10-fold dilution series were prepared
in cell culture medium [6]. Of each dilution samples of 0.05 ml were
inoculated on four confluent monolayers of CEF. The plates were
incubated for 6 days at 38.5 ◦ C ± 1 ◦ C/5% CO2 ± 0.5. At regular inter-
vals the cultures were examined for the presence of cytopathogenic
effect. The titer was expressed in PFU.
2.4. Efficacy test of experimental MD vaccines produced in JBJ-1
cells in chickens
Prior to use the vaccines were thawed and diluted to the required
titer in Poulvac® Marek diluent at room temperature. For CVI
988/JBJ-1, a dilution containing 102.5 TCID50 per dose of 0.2 ml was
prepared. For CVI 988/JBJ-1 + HVT-FC126/JBJ-1, a dilution contain-
ing 102.5 TCID50 CVI 988/JBJ-1 and 500 PFU HVT-FC126 per dose
of 0.2 ml was made. For Poulvac® Marek CVI, a dilution containing
102.5 TCID50 per dose of 0.2 ml was prepared. Directly after dilution
the vaccines were cooled on melting ice and transported to the
test animal facilities and used for vaccination. It has been shown
that this procedure is optimal for the stability of the vaccine virus
[6].
Six groups, each consisting of 32–35 1-day-old-specific
pathogen free (SPF) chicks (Lohmann Tierzucht, Cuxhaven, Ger-
many) were ground housed in containment units under standard
housing conditions, such that mixing of different vaccine strains
was not possible and infection of the nonvaccinated control group
with the live vaccines was not possible. The chicks were fed ad
libitum, drinking water was freely available and further conditions
were standard. The chicks were vaccinated at 1 day of age, with the
above indicated vaccine preparations. CVI 988/JBJ-1 was injected
intramuscularly or subcutaneously, the combination of CVI 988/JBJ-
1 and HVT-FC126/JBJ-1 was injected intramuscularly only. Poulvac®
 H. Geerligs et al. / Vaccine 26 (2008) 5595–5600
Table 1
The JBJ-1 cell line as substrate for MDV strain CVI 988
Virus passage Period between cell seeding Inoculation of virus,
number and virus inoculation in days split ratio
1 5 NAa
2 3 1:10
3 4 1:20
4 3 1:20
5 3 1:20
a
NA, not applicable.
b
ND, not done.
Marek CVI also was injected intramuscularly only. One group con-
sisting of 35 chicks from the same flock was left nonvaccinated and
kept separately under the same conditions. After vaccination the
chickens were observed daily for clinical symptoms.
At 5 days after vaccination, all chickens were challenged with a
50-fold dilution of a spleen cell suspension from chickens, which
had been infected with the very virulent MD virus strain RB1B at
passage level 3 in CEF. After challenge the chickens were observed
for a period of at least 70 days for signs of MD, i.e. lameness. Dead
animals during the observation periods were investigated for gross
lesions of MD, i.e. enlargement of peripheral nerves or lymphomas
in kidneys, heart, lungs, liver, spleen, ovary or testicles. At the end
of the observation periods all surviving animals were killed and
investigated for gross lesions of MD. Animals with severe symptoms
of MD during the observation period were killed and investigated
for gross lesions of MD.
In the EP monograph for live vaccines against MD a method for
evaluation of the results is described and requirements for effi-
cacy are formulated. The EP prescribes that the minimal group size
should be 30 chickens. The EP prescribes that of the nonvaccinated
chickens at least 70% should show signs of MD, i.e. lameness or gross
lesions at autopsy. The vaccines comply with the requirements for
efficacy if the protection index (PI) is at least 80%. First, the per-
centage MD positive animals (% MD positives), after the 70 days
observation period in each group should be determined. Than, the
PI should be calculated as follows:
%MD positives in controls − %MD in vaccine group
PI (%) = × 100
%MD positives in controls
2.5. Safety of experimental MD vaccines produced in JBJ-1 cells in
a field study
Two thousand two hundred and twenty eight commercial 1-
day-old White Leghorn hens were divided over three groups. One
group of 716 chicks was vaccinated with MD strain CVI 988/JBJ-1,
103.5 TCID50 per dose of 0.5 ml. The vaccine virus had been pas-
saged nine times over JBJ-1 cells. Another group of 716 chicks was
vaccinated with HVT-FC126/JBJ-1, 6985 PFU per dose of 0.5 ml. The
vaccine virus had been passaged seven times over JBJ-1 cells. The
third group of 796 chicks was vaccinated with a commercial batch
of Poulvac® Marek CVI + HVT. The batch of vaccine was diluted
such that each dose of 0.5 ml contained 103.5 TCID50 of CVI 988
and 6985 PFU of HVT-FC126. The vaccines were injected intramus-
cularly in the thigh at 1 day of age, using a Pullet Injection Gun
(Veterinary Supplies, Mijdrecht, The Netherlands) with a pre-set
volume of 0.5 ml.
All chickens were vaccinated by coarse spray against Newcastle
disease and infectious bronchitis, at 1 day of age, with a commer-
cial batch of Poulvac® NDW and Poulvac® IB primer, respectively.
At 4, 10 and 18 weeks of age the chickens were booster vaccinated
against Newcastle disease with a commercial batch of Poulvac
NDW® using an Atomist spray. At 3 weeks of age the chickens were
5597
Virus incubation Cytopathogenic effect at TCID50 per cryovial
time in days moment of harvesting of 1 ml
3 None NDb
4 80% NDb
3 80% 106.50
3 80% 106.63
3 80% 106.85
vaccinated against Gumboro with a commercial batch of Poulvac®
Bursaplus via the drinking water and at 14 weeks against avian
encephalomyelitis virus with a commercial batch of Poulvac® AE
via the drinking water.
The chicks were group-housed on chopped straw in the animal
facilities of Fort Dodge, Muiden, The Netherlands, until 14 weeks
of age. All hens were debeaked at 7 weeks old. All chicks were
fed ad libitum and had free access to drinking water provided in
automatic drinkers. The chickens were observed daily for clinical
symptoms throughout the study. Special attention was given to
signs of MD. Postmortem examination was done on chicks that died
or were euthanized with signs of MD after 4 weeks of age. Diagnosis
for MD was made on clinical signs, lameness, and on postmortem
examination by the occurrence of lymphomas in various organs and
enlargement of peripheral nerves.
3. Results
3.1. Quality control testing and growth characteristics of the JBJ-1
cell line
The cell bank was free from bacteria, fungi, mycoplasma and
extraneous agents, and complied with the requirements of the
European Pharmacopoeia, ed. 2003. The JBJ-1 cell line was charac-
terized as female chicken. The cells did not show any Mn2+ and Mg2+
dependent RT activity. The cell line formed confluent monolayers in
various types of tissue culture bottles, viz. flat bottom tissue culture
bottles, roller bottles, and tissue culture plates. Dulbecco’s mini-
mal essential medium with the necessary supplements, appeared
to be a suitable medium for growing the cells. During cell culture
optimization studies we switched to QT35 medium. The cells grew
quicker and to higher densities with the QT35 medium in com-
parison to the Dulbecco’s medium mentioned above. The preferred
seeding density of the cells in tissue culture bottles is 4 × 104 to
5 × 104 cells per cm2 . Normally, the cells are incubated at 37 ◦ C, but
good growth characteristics were obtained also during incubation
at 39 ◦ C. The cells grew to confluency within 3–5 days. Cell densities
further increased during prolonged incubation till 7 days in total.
We used split ratios from 1–4.5 to 1–8.
3.2. JBJ-1 cells as substrate for MD strain CVI 988 and HVT-FC126
JBJ-1 cells were scaled up from storage in liquid nitrogen and
grown in QT35 medium. For virus growth always confluent mono-
layers in roller bottles were used. Summarized results of 2 virus
growth experiments are given in Tables 1 and 2. In Table 1 the
results can be found of a series of five passages of strain CVI 988.
For each passage the JBJ-1 cells had been seeded in 490 cm2 roller
bottles at a density of 5 × 104 cells per cm2 . For the first passage
we inoculated halve the contents of an ampoule of a commercial
batch of Poulvac® Marek CVI in one roller bottle. The cells had incu-
bated for 5 days after seeding. After 3 days no cytopathogenic effect
was detected. The culture was harvested and inoculated on new
5598 H. Geerligs et al. / Vaccine 26 (2008) 5595–5600

Table 2
The JBJ-1 cell line as substrate for HVT strain FC 126

Virus passage Period between cell seeding Inoculation of virus, Virus incubation Cytopathogenic effect at PFU per cryovial
no. and virus inoculation in days split ratio time in days moment of harvesting (%) of 1 ml

1 5 NAa 3 65 NDb
2 4 1:20 3 100 2573 × 103
3 3 1:7 2 90 7920 × 103
4 4 1:20 3 90 10,989 × 103
5 7 1:20 3 90 1750 × 103
a
NA, not applicable.
b
ND, not done.

monolayers at an age of 3 days (see Table 1) in 1–10 split ratio. This
time, a clear cytopathogenic effect was visible, which increased to
80% (80% of surface of monolayer affected) 4 days after inoculation.
We continued passaging till we had five passages. In all passages
a clear cytopathogenic effect was visible. Samples of the passages
were frozen and stored in liquid nitrogen and the TCID50 titers were
determined. The titers obtained were highly satisfactory and in the
same range as titers obtained with the same virus grown in CEF.
For commercial batches Poulvac® Marek CVI, titres range between
106.00 and 106.9 TCID50 per ampoule of 1 ml.
In Table 2, results are given of the growth experiments with
strain HVT-FC126. The results show that this virus strain caused a
clear cytopathogenic effect already in the first passage. In the sec-
ond passage a cytopathogenic effect of 100% was observed. In the
following passages a cytopathogenic effect of 90% was observed.
Samples of the harvests were frozen and stored in liquid nitrogen.
The samples were titrated, and the results in Table 2 show that the
virus titer increased over the passages. The titer of the fifth passage
is on the low side, which may have been caused by the fact that
the monolayer, on which the virus was inoculated, had had a rel-
atively long incubation period. Generally, the titers did not differ
from titers obtained for virus grown in CEF.
3.3. Efficacy of MD vaccines grown in JBJ-1 cells
Chicks were vaccinated at 1 day of age with CVI 988/JBJ-1, CVI
988/JBJ-1 + HVT-FC126/JBJ-1, via different vaccination routes and
using different dose sizes. In addition a group was vaccinated with
a commercial batch of Poulvac® Marek CVI. Another group was left
nonvaccinated. After vaccination none of the chickens died and also
none of the chickens showed signs of disease. Five days after vac-
cination all chickens were challenged with the virulent MD strain
RB1B. The results after challenge are summarized in Table 3.
In the nonvaccinated group 24 of the 35 chickens died because of
MD or had signs of MD. Three of the 35 chickens died without signs
of MD and were designated as non-specific deaths. The percentage
MD in the nonvaccinated controls was determined as 75%; this is
with the three non-specific deaths excluded. 75% complies with the
EP requirement of not less than 70%. In the vaccinated groups also
non-specific deaths occurred, which were excluded from the cal-
culation. In de group vaccinated with Poulvac® Marek CVI 2 of the
32 chickens showed signs of MD after challenge, which resulted
in a PI of 92%, and complies with the EP requirement of at least
80%. The PI in the groups vaccinated with vaccine viruses produced
on JBJ-1 cells, all complied with the EP requirements. A PI of 96%
was determined for CVI 988/JBJ-1 after intramuscular administra-
tion and a PI of 80% for CVI 988/JBJ-1 after subcutaneous injection.
The value of 80% is relatively low, but still in compliance with the
EP requirements. For the combination CVI 988/JBJ-1 + HVT-FC126
PI’s of 96 and 100% were determined for intramuscular and subcu-
taneous administration, respectively. It can be concluded that all
vaccines complied with the requirements for efficacy of the EP.
3.4. Safety of MD vaccines grown in JBJ-1 cells in a field study
The chickens in the three groups were vaccinated at 1 day of age
with the experimental vaccine CVI 988/JBJ-1, the experimental vac-
cine HVT-FC126/JBJ-1 and a commercial batch of Poulvac® Marek
CVI + HVT, respectively. After vaccination the birds were observed
for clinical signs and dead birds were autopsied and investigated
for enlargement of peripheral nerves and lymphomas in the vari-
ous organs. In Table 4 the number of dead animals and the cause of
these deaths are indicated. After 7 weeks all birds were debeaked
and, as can be seen from the data in Table 4, quite a number of chick-
ens died because of debeaking, especially in the groups vaccinated
with CVI 988/JBJ-1. As can be seen in Table 4 also, 74 other chick-
ens died in the group vaccinated with Poulvac® Marek CVI + HVT.
Most of these animals died because of Coccidiosis, i.e. 32, whereas
20 others died because they suffocated in a food container. It was
decided to medicate all birds with Baycox® in their drinking water
during 4 days in the 8th week of life. After exclusion of the ani-
mals which had died due to Coccidiosis and suffocation, 22 were
left, which is in line with the values that we found for the groups
vaccinated with CVI 988/JBJ-1 and HVT-FC126/JBJ-1, i.e. 22 and 24
deaths, respectively. In none of the groups there were deaths due to
MD. Also during daily observations of the live chickens no evidence

Table 3
Summarized results of vaccination challenge study

Vaccine Group size Vaccination route Non-specific deaths MDa positives % MD positive PI in %b
c
CVI/JBJ-1 34 IM 0 1 3 96
CVI/JBJ-1 35 SQd 2 5 15 80
CVI + HVT/JBJ-1 35 IM 3 1 3 96
CVI + HVT/JBJ-1 33 SQ 1 0 0 100
None 35 NAe 3 24 75 NA
CVI/CEF 32 IM 0 2 6 92
a
MD, Marek’s disease.
b
PI, protection index.
c
IM, intramuscular.
d
SQ, subcutaneous.
e
NA, not applicable.
 H. Geerligs et al. / Vaccine 26 (2008) 5595–5600
Table 4
Summarized results of a field safety test on experimental batches of Marek’s disease vaccine produced on JBJ-1 cells
Vaccine Number of
chicks used
Poulvac Marek CVI + HVT 716
HVT-FC126/JBJ-1 716
CVI 988/JBJ-1 796
a
If all deaths due to suffocation and Coccidiosis are excluded, the number is 22.
for the presence of an MD infection was noticed. As after more than
200 days still no signs of MD were noticed it was decided to dis-
continue the study. All chickens were euthanized 216 days after
vaccination.
4. Discussion
With respect to growth characteristics, the JBJ-1 cell line is not
different from other anchorage dependent cell lines. The same cell
culture vessels, trypsinization procedures and media can be used
as for other cell lines. While performing the experimental work
we found that a custom made medium, used for growing QT35
cells, gave somewhat better growth results than Dulbecco’s min-
imal essential medium. This custom made medium is very rich in
amino acids. QT35 cells, an avian cell line also, grew very well with
this medium. Split ratio’s for JBJ-1 cells ranged between 1–4 and
1–8, which is not very high, but similar with QT35 cells.
After inoculation of JBJ-1 monolayers with a commercial
batch of Poulvac® Marek CVI, which had been grown in CEF, no
cytopathogenic effect could be determined in the JBJ-1 cell cul-
tures. However, in the second and following passages a clear
cytopathogenic effect became visible. This phenomenon can be
observed also during culturing of strain CVI 988 in CEF. It is not
clear why a cytopathogenic effect does not become visible in the
first passage. The titres of the samples stored in liquid nitrogen,
were highly satisfactory. For Poulvac® Marek CVI a minimal release
titer of 103 TCID50 per dose has been registered. The preferred num-
ber of dosages per ampoule is at least 1000. The last passage has
a titer of 106.85 TCID50 per dose, which means that one ampoule
would contain at least 5000 doses. Samples of a commercial batch
of Poulvac® Marek HVT generated a clear cytopathogenic effect in
all five passages in JBJ-1 cells. Titres determined in frozen samples
from each passage were similar with titres obtained if CEF were
used as substrate. Especially the titer of the fourth passage was
high, i.e. 10,989 × 103 PFU per ampoule. The minimal release titer
for Poulvac® Marek HVT is 1500 PFU per dose, which means that
one ampoule would contain at least 7000 doses. From these results
it is clear that for production of these vaccines the potential of the
JBJ-1 cell line is great.
Especially vaccines based on strain CVI 988 are highly effective
in protection of chickens against MD. After administration at 1 day
of age chickens are protected against a challenge already within a
few days after vaccination. However, if for production of the vaccine
another substrate is used, properties of the virus may change. Kong
et al. [8] showed that subtype C avian metapneumoviruses (AMPV)
change after passages in Vero cells. There were several amino acid
differences in the fusion protein between viruses grown in Vero
cells and viruses grown in cells from the natural host of the virus,
i.e. the turkey. For influenza viruses the substrate has an effect on
the properties of the virus also. During growth in embryonated eggs
changes were generated around the receptor-binding site [9]. No
such changes have been reported for MDV when grown in other
substrates, although it has been found that during multiple pas-
sages in CEF there is an expansion of the 132-bp repeat sequences
flanking the long unique region of the virus genome [10]. This
5599
Deaths due to Deaths due to various reasons Deaths due to
debeaking except Marek’s disease Marek’s disease
6 74a 0
7 24 0
26 22 0
expansion has been associated with a decreased vaccine efficacy
of strain CVI 988. We tested the efficacy of MD vaccines produced
in JBJ-1 cells in chickens. We vaccinated the chicks at 1 day of age
and gave them a challenge already 5 days after vaccination. From
the results it can be concluded that the efficacy of the vaccines, the
current product Poulvac® Marek CVI as well as the experimental
vaccines produced in JBJ-1 cells, still was very good and exceeded
the EP requirement, i.e. a protection index (PI) of at least 80% after
a challenge 9 days after vaccination. It has to be remarked that the
JBJ-1 cell line originates from the chicken, just like CEF, and that a
minimal number of passages in JBJ-1 cells was sufficient to come
to good virus yields.
We tested the safety of the experimental vaccines in a field study.
The advantage of a field study for safety testing is that the vaccine
is tested in a realistic situation, that the effect of other vaccines
(chickens get a lot of different vaccines) is included and that a large
number of chickens is used. No signs of MD were noticed in any
of the groups, which indicates that the safety of the vaccines pro-
duced in JBJ-1 cells is highly satisfactory. It was decided to terminate
the study after 200 days. If MD is present in a flock of chick-
ens, this will become visible in the first 6 months of the chicken
life.
The JBJ-1 cell line is a highly promising candidate as new sub-
strate for vaccines against MD. The cell line does not have the
disadvantages of other alternative substrates for MD vaccines.
There are no special precautions needed in the use of this cell line,
with respect to media, incubators and cell culture vessels. The use
of this cell line opens the possibility to introduce new technolo-
gies in the production of conventional vaccines against MD, such as
bioreactors or other large-scale production techniques. Production
of MD vaccines on a larger scale with less risk for contaminations
will improve the economical situation of the poultry industry.
Acknowledgements
The authors thank Mrs. G. Groenveld-Hak of the Quality control
department of Fort Dodge Animal Health Holland for performing
the titrations of the experimental MDV vaccines, and Dr. Hans van
Dasler, head of the Department of animal investigations, for taking
care of the animals used in the studies in this article.
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