Veterinary Quarterly

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Biological characteristics of Marek's disease

vaccine CVI‐988 clone C

G. F. de Boer , J. M. A. Pol & H.L. Oei

To cite this article: G. F. de Boer , J. M. A. Pol & H.L. Oei (1987) Biological characteristics
of Marek's disease vaccine CVI‐988 clone C, Veterinary Quarterly, 9:sup1, 16-28, DOI:
10.1080/01652176.1987.9694135

To link to this article: https://doi.org/10.1080/01652176.1987.9694135

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 Biological characteristics of Marek's disease
vaccine CVI-988 clone C
G. F. de Boer2, J. M. A. Po12 and H. L. Oei3

SUMMARY Biological characteristics of Marek's disease virus (MDV) CVI-988 clone C, of

importance for vaccine application, are described. CVI-988 clone C was shown to be non-
pathogenic for highly MD-susceptible chickens and slightly more effective than prototype
CVI-988 vaccine. During plaque purification and serial cell-culture passages, reductions were
observed in the release of A' antigen from infected cell cultures, in spreading properties and in
virus replication in vivo. Pre-licensing batches of CVI-988 clone C vaccine afforded excellent
protection against challenge infection with virulent MDV and highly virulent MDV strains.
Groups of chickens with bivalent (HVT/ SB-1) vaccine-induced maternal antibodies were
equally protected by a double dose of CVI-988 clone C vaccine. Field trials performed in the
Netherlands and in the United States confirmed the safety andprotective efficacy of monovalent
CVI-988 clone C vaccine.

INTRODUCTION
Marek's disease (MD) in commercial fowl has been successfully controlled by the
use of live virus vaccines based on each of the three avian herpesvirus serotypes (9,
15, 32).
Avian herpesvirus serotype 1. Churchill et al. (12) and Rispens et al. (22) developed
vaccines by attenuation of, respectively, a virulent isolate of Marek's disease virus
(MDV HPRS-16) and a MDV of low virulence (CVI-988). Initially these vaccines
were used only in their countries of origin, England and the Netherlands, but today
both serotype 1 vaccines, especially CVI-988, have found a wider application.
Avian herpesvirus serotype 2. Vaccination with the HN strain was performed locally
in the United States by inoculation of blood from viremic birds (33). Cho and
Kenzy (11) isolated this strain in cell culture. More widely used are vaccines based
on MDV strain SB-1, isolated by Schat and Calnek (23). This non-oncogenic virus
is frequently used in bivalent vaccines composed of serotypes 2 and 3 (26, 27, 28).
Avian herpesvirus serotype 3. Vaccines based on herpesvirus of turkeys, strain HVT
FC126 isolated by Witter et al. (29), are most widely employed throughout the
world. In addition, some local HVT isolates are used, e.g. in Australia.
Since the seventies, prototype CVI-988 vaccine of serotype 1 (22), has been eagerly
accepted by the poultry industry in the Netherlands (16); however, application
abroad was delayed because of its pathogenicity for highly MD-susceptible
chickens, such as Rhode Island Red (RIR) and Cornell line S chickens (3, 8, 20).
We have extended the attenuation of the CVI-988 strain by plaque-purification in
combination with serial cell culture passages in duck embryo fibroblast (DEF) cell
cultures, and adaptation to chick embryo fibroblasts (CEF). Three plaque-purified
CVI-988 preparations were obtained in the 49th DEF passage (clones A, B, and C)
and another three at higher DEF passage levels (clones D, E and F). In comparative
pathogenicity studies, various derivatives from these virus preparations were
shown to be non-pathogenic for RIR chickens, as opposed to prototype CVI-988

1
Data partly presented at the 36th Western Poultry Disease Conference, Davis, California.
2 Central Veterinary Institute, Virology Department, P.O. Box 365, 8200 AJ Lelystad, The Netherlands.
3
Central Veterinary Institute, Department of Control and Standardisation, P.O. Box 65, 8200 AB
Lelystad, The Netherlands.

16S THE VETERINARY QUARTERLY, VOL, 9, SUPPLEMENT 1, NOVEMBER 1987

vaccine (3, 8, 20). Further studies were concentrated on clone C derivatives, because
CVI-988 clone C DEF51 afforded satisfactory protection in a pilot study
(unpublished). From these derivatives the most immunogenic preparation, being
slightly more immunogenic than prototype CVI-988 vaccine, was selected in
protective efficacy studies (2, 3, 18). CVI-988 clone C vaccine has recently been
licensed for use in a number of countries, including the United States. Up-dated
information on the biological characteristics of CVI-988 clone C vaccine are
presented here.

MATERIALS AND METHODS

Experimental chickens and accommodation
In the laboratory trials specific-pathogen-free (SPF) white leghorn strain A (WLA) chickens
were used, except in the protective efficacy study employing broiler chickens with bivalent
vaccine (HVT/SB-1)-induced maternal antibodies (Table 6). The field trials in the
Netherlands were performed with laying-type chickens produced by three poultry breeding
companies; in the United States, commercial broiler chickens were employed. In the
laboratory, the various groups of experimental chickens were kept either in filtered-air,
negative-pressure (FANP) chicken houses equipped with slatted floors (2, 3), or on a dirt
floor in spatially separated, conventional chicken houses (18). Testing for safety of the
master seed virus, for the absence of extraneous agents, as well as the backpassage studies
were performed in modified Horsfall-Bauer isolators.
Virus strains used for vaccination
Vaccines of all three avian herpesvirus serotypes (9, 15, 32) were utilised. Serotype 1.
Prototype CVI-988 vaccine (DEF1-4, CEF/DEF5-14, CEF15-35) (22); MDV CVI-988 clone
C CEF 65 (DEF1-51, CEF/DEF 52-57, CEF58-59, DEF/CEF60-65 (2, 3)), being the master
seed virus, and derivatives in various CEF passages. Serotype 2. MDV SB-1 vaccine (23) was
used as a component of bivalent vaccine (serotypes 2 and 3). Serotype 3. HVT FC126 vaccine
(29) was used in cell-associated form. This vaccine batch was prepared from a lyophilised
HVT FC126 preparation received from Dr. J. E. Pearson, National Animal Disease Center,
Ames, Iowa.

MDV strains employed for challenge

MDV
Challenge infection was performed with virulent MDV (vMDV) and very virulent layer
(vvMDV) strains: vMDV-K (22); vvMDV-RB/IB isolated from an HVT-vaccinated
States (3)
flock (24); vvMDV-Tun, the first vvMDV isolate originating outside the United
and vvMDV-Md5 (27, 30).

Laboratory techniques and materials

The techniques utilised for virus propagation, virus titration, fluorescent antibody staining
(FAT), etc. have been described earlier in detail (4, 7, 22). Immunoprecipitation studies,
employing techniques described by Van Zaane et al. (32), were performed to investigate the
release of 'A' antigen (MDV-gp54/70) in supernatant fluids of cell cultures infected with the
preparations: CVI-988 CEF36, CVI-988 clone C CEF65, CVI-988 clone E DEF99 and
CVI-988 clone F DEF166. A rabbit antiserum against MDV-gp54/70 was kindly provided
by Dr. L. Velicer, East Lansing, Michigan. Monoclonal antibody (MCA) M26-8A reacting
with 'A' antigen (13) were received from Dr. K. Hirai, Bohseidai, Isehara, Japan. The MCAs
H19 and 2BN90 provoking, respectively, cytoplasmic and nuclear staining of a large number
of serotype 1 virus strains (15, 31), were kindly provided by Dr. R. L. Witter, East Lansing,
Michigan.
Protective efficacy studies, employing a modified procedure of 50% protective dose (PD50)
assays (1, 2, 3, 6, 18), were performed with three first-production batches of CVI-988 clone C
vaccine, harvested in chick embryo fibroblast (CEF), passage nos 68, 69 and 74. Two of the
vaccines tested were produced in the Netherlands (Nos 1 and 3 of Table 5) and one in the
United States (No 2 of Table 5).

THE VETERINARY QUARTERLY, VOL. 9, SUPPLEMENT I, NOVEMBER 1987 17S

 Tests for extraneous agents
The batch of master seed CVI-988 clone C CEF65 was tested for absence of various viruses
that are pathogenic for chickens and for Salmonella pullorum infection, following the
procedures of the European Pharmacopoeia, 1983. In these tests fertilised eggs, cell cultures,
I-day-old and 2-week-old chickens were used, according to the monographs V.2.1.3.1,
V.2.1.3.4, V.2.1.3.2 and V.2.1.3.5. Tests for exclusion of reticuloendotheliosis virus were
performed by the fluorescent antibody staining technique (17). A MCA DAS-ELISA was
employed for avian leukosis virus testing (5). Electron microscopic examination for
parvovirus was kindly performed by Dr. J. Kisary, Brussels, Belgium. Chick anaemia agent
fluorescent antibody testing (10) was kindly performed by Dr. W. Hermann, Weesp, the
Netherlands. Tests for Mycoplasmas and for bacterial and fungal contamination were
performed according to the European Pharmacopoeia, V.2.1.1. (1980) and V.2.1.3.6. (1983).
Peking ducks at the age of 4 weeks, fertilised eggs and embryo fibroblasts from Muscovy
ducks were utilised for exclusion of duck and goose viruses.
Safety tests
Safety testing for application of the vaccine was performed in two trials. Each trial was
comprised of three groups of twenty-two 1-day-old SPF chickens each. In the trials, sixteen
chickens of one group were intramuscularly (i.m.) and intraocularly (i.o.) inoculated with
resp. 104 and 10 PFU of the master seed. Sixteen chickens of the other two groups received
105 PFU i.m. and 104 PFU i.o. In one isolator, the latter treatment was repeated at 14 days of
age. All birds were necropsied at the end of the observation periods, respectivelyat 10 and 6
weeks. Heparinised blood samples were collected for virus isolation and for serological
examination for antibodies against various chicken pathogens.
Tests for stability of attenuation
The master seed CVI-988 clone C CEF65 underwent twelve in vivo passages. Twenty-two
1-day-old chickens were i.m. inoculated with 5x104 PFU (first chick passage). On the 7th day
p.i., a washed white blood cell (WBC) preparation was prepared by low-speed centrifugation
in Ficoll metrizoate (7) and utilised for virus isolation by co-cultivation with CEF and for
inoculation of the 2nd chick passage. The above procedure was repeated during twelve chick
passages. Each chicken received 10 to 14 million WBC (or 1 million WBC in the 1 1 th and
12th passage) from the previous chick passage. At the end of the observation period, ranging
from 50 to 105 days, blood samples were again collected for virus isolation and antibody
detection by fluorescent antibody staining (FAT). Necropsy was performed on all animals.
The observation period of the 12th chick passage was extended to 390 days, necropsies were
performed after 58, 120 and 390 days.
Contact transmission studies
The spreading properties of CVI-988 clone C were investigated in nine trials by adding SPF
chickens to vaccinated groups.
a. Twelve hatch mates were added to the first chick passage of the first backpassage trial
(interrupted because of failure of virus isolation during two blind CEF passages). Virus
recovery from the recipient chickens, by cocultivation.of WBC and CEF, and antibody
detection by FAT were attempted at 5 weeks of age.
b. Six SPF chickens were added to all six groups of sixteen chickens employed for safety
testing. Virus recovery and FAT were attempted after 6 and 10 weeks.
c. Two groups of SPF chickens were added to the 12th chick passage of the backpassage
study described at 58 days of age. FAT was performed 9 weeks later.
Protective efficacy studies
a. Protective efficacy studies in the laboratory were performed in maternal antibody-free
SPF chickens and in those with bivalent vaccine (HVT/SB-1)-induced maternal anti-
bodies from a commercial breeder of broiler chickens (Table 6). Fifty percent protective
dose (PD50) assays (1, 6), modified for employing conventional chicken houses (2, 3, 18),
were applied. The various groups of chickens were inoculated immediately after hatching
with graded doses of vaccine (0.5 ml), either intramuscularly (i.m.) in the leg (Table 5) or
0.2 ml subcutaneously (s.c.) in the dorsal part of the neck (Table 6). Challenge infection

18S
THE VETERINARY QUARTERLY, VOL. 9, SUPPLEMENT I, NOVEMBER 1987
 procedure (1). The
was by i.m. inoculation at the age ot 9 days, according to the standard
challenge dose was pre-determined so as to cause MD lesions in at least 70% of the
unvaccinated controls during the observation period of 10 weeks. However, challenge
infection of the broiler chicks with HVT/SB-1 maternal antibodies was by the
intraperitoneal (i.p.) route at 5 days of age (Table 6). Autopsy was performed on all
chickens which showed clinical signs of Marek's disease during the experimental period
and on all surviving birds at the end of the experiment. The data from each trial were
subjected to probit analysis with the aid of a computer program developed by the UCLA
Health Sciences Computing Facility (BMDO3S). With this program the 50% protective
dose (PD50) and the slope of the probit regression line were calculated from the results in
the MD-susceptible part of the experimental flock. The PD50 is defined as the number
of
plaque-forming units (PFU) of vaccine virus, determined in an in vitro assay, required to
protect 50% of the MD-susceptible part of a group of chickens from clinical symptoms
and macroscopical lesions of MD (6). If the data were not suited for the computer
program, the protective index of every vaccine dose group was calculated according to
the formula:
% MD in controls % MD in vaccinated birds x 100
% MD in controls
testing in the field was performed by the Poultry
b. In the Netherlands, official pre-licensing
Health Service, Doom. Eleven randomly selected farms with a total of 124.320 laying-
study.
type chickens, originating from three breeder companies, co-operated in this
Three batches of CVI-988 clone C vaccine (Nos 1, 2 and 3 of Table 5) were utilised. All
parent flocks had been vaccinated with prototype CVI-988 vaccine. Progeny received 0.5
ml cell-associated vaccine i.m. at 1 day of age. The production and health performances of
the flocks were carefully monitored and reported to the Poultry Health Service. Necropsy
was performed on all chickens that died during the observation periods, which ranged
from 29 to 36 weeks. At the occasion of the 4-weekly clinical inspection, five chickens
were selected for necropsy. Histological examination was performed in case of suspicion
for MD lesions.
performed in Arkansas and five states along the East
c. In the United States, field trials were
Coast as part of the pre-licensing testing. Three batches of CVI-988 clone C vaccine of
three companies were utilised to vaccinate thirty-one flocks of commercial broiler
chickens, comprising a total of 537.000 birds. About 50% of the chickens were progeny
from flocks vaccinated with bivalent vaccine (HVT/SB-1). The chickens were injected
s.c. in the neck, using automatic vaccination equipment, with a 0.2 ml dose containing
1500 to 3000 plaque-forming units (PFU) cell-associated CVI-988 clone C vaccine. The
flock owners supplied all performance data to the veterinarians of the vaccine-producing
companies, who made a compilation.

RESULTS
Subdivision within serotype 1
By the courtesy of the Regional Poultry Research Laboratory, East Lansing, we
have available two monoclonal antibodies which react with various avian
herpesviruses of serotype 1 by fluorescent antibody staining (15,31). MCA 2 BN90
was broadly reactive, but MCA H19 failed to stain CVI-988 clone C CEF 65.
Twelve CVI-988 preparations grown either in DEF or CEF, HPRS-16/att,
vvMDV-Md5 and vMDV-K were tested with these two MCAs. MCA 2BN90
reacted by fluorescent antibody staining with all serotype 1 virus preparations
tested, but MCA H19 failed to stain all CVI-988 derivatives. The two MCAs H19
and 2BN90, therefore, can be utilised for differentiation between HPRS-16/att and
CVI-988 (clone C) vaccines.

THE VETERINARY QUARTERLY, VOL. 9, SUPPLEMENT I, NOVEMBER 1987 19S

Release of 'A' antigen
Employing one-dimensional radio-immunoprecipitation (SDS-PAGE) studies, the
excretion of 'A' antigen, composed of glycoproteins ranging from 54,000 to 70,000
daltons (MDV-gp54/70) (12, 13), was demonstrated not only in prototype CVI-988
vaccine, but also in CVI-988 clone C CEF65 and CVI-988 clone D DEF99 (20).
Both the rabbit serum and MCA M26-8A reacted with a broad band corresponding
with gp5 described by Van Zaane et al. (32). The amount of antigen 'A' released into
medium of CVI-988 clone C CEF65-infected cells was significantly less than that
produced by prototype CVI-988 vaccine. The expression of 'A' antigen was detected
by FAT in eleven CV1-988 preparations tested, but not in CVI-988 clone F DEF164
(Table 1).
Table I. Differentiation within avian herpesvirus serotype I by fluorescent antibody staining.

Specificity Serotype 1 'A' antigen

1 2 3
Virus strain MCA H19 2BN90 M26-8A

4
CVI-988 DEF4 + +
CVI-988 DEF33 _ + +
CVI-988 DEF87 + +
CVI-988 clone E DEF97 _ + +
CVI-988 clone F DEF164 _ +
CVI-988 CEF10 _ + +
CVI-988 CEF20 - + +
CVI-988 CEF36 - + +
CVI-988 clone C CEF59 - + +
CVI-988 clone C CEF65 - + +
CVI-988 clone C CEF71 - + +
CVI-988 clone C CEF65/Chick 6/CEF10
5 - + +
vvMDVMd5 + + +
vMDVK + + +
MDV HPRS-16/att + + +

1
MCA 1119, cytoplasmic staining of various serotype 1 viruses (15)
2
MCA 2BN90, nuclear staining of various serotype 1 viruses (31)
3
MCA 26-8A reacting with 'A' antigen (gp54/70) (13)
4
'A' antigen'detected by fluorescent antibody staining (FAT)
5
Reisolated from 6th chick passage of the backpassage study

Tests for safety and extraneous agents

The master seed virus was extensively tested for safety, at administration to
1-day-old chickens, and for the presence of extraneous agents. The large virus doses
in the inoculations caused no adverse effects in the experimental chickens. All
laboratory tests yielded negative results.
Tests for stability of attenuation
Twelve serial in vivo passages were performed to examine the possibility of
pathogenic reversion. The first backpassage trial was interrupted because of failure
of virus recovery during two blind CEF passages in chickens inoculated at 1-day-
old with 104 PFU CVI-988 clone C CEF65 (although FAT antibodies were detected
in the 4th chick passage). However, in the backpassage study reported here, in
which the inoculum had been increased five-fold, virus recovery was successful in
all twelve chick passages, at 7 days p.i. and at necropsy (50-105 days p.i.). After 7
days, virus recovery was always successful from individual birds, usually after three
to four blind passages (3-4 days in cell culture). At the end of the observation

20S THE VETERINARY QUARTERLY. VOL. 9, SUPPLEMENT I , NOVEMBER 1987

periods, 80-95% scores were obtained; these sometimes required six blind CEF
passages before herpesvirus cytopathological effects could be observed. The gross
and histological lesions observed during necropsy in the various passages are
presented in Table 2.

Table 2. Backpassage studies performed with CVI-988 clone C CEF65.

Necropsy Histology

Virus On No. Nerve Inflamma A-type Other

Chick No.of
dose day of swelling tory lesions lesions
pass. chicks

No. inoculated chicks (Btype)

4 - -
1 22 5x10 PFU 56 10
6
56 10 - - -
2 20 10x10 WBC
3 25 14x10
6
WBC 105 15 - * -
6
98 1 - -
4 21 14x10 WBC 10
6
22 - - - -
5 32 9x10 WBC 91
6
22 - -
32 10x10 WBC 91
6
9x10
6
WBC 84 25 1 1
- -
7 35
6 3 3 -
8 31 10x10 WBC 84 20
6 _ _
9 34 12x10 WBC 77 9 1 1
6 - _
10 26 16x10 WBC
6 - -
11
1
59 10 WBC 50 15 -
2 50 15 1 1 -
11 55 0.25ml blood _
1 6 _ -
12 144 10 WBC 58 23 - **
120 48 3
390 67 -
2 58 27 - - -
12 144 0.25m1 blood
120 52
390 63 -

1
Inoculum consisted of one million WBC
2 Inoculum consisted of 0.25 ml heparinised blood
** Paralytic chicken
Three cockerels with nerve swelling at 120 days p.i.

During the in vivo passages, only one chicken of the 4th chick passage, out of the
total 680 inoculated, developed paralysis. Nerve swelling was observed in nine
chickens and inflammatory B-type lesions were observed in seven chickens in total.
The mild reversion of virulence after seven chick passages disappeared when the
inoculum was lowered by one tenth. Thereafter, only one bird of the llth chick
passage showed nerve swelling at 50 days p.i. In the 12th chick passage, no
macroscopical lesions were observed at 58 days p.i., but three cockerels with nerve
swelling were detected during the necropsies at 120 days p.i.
Specific antibodies directed to MDV were demonstrated by FAT in all experimental
chickens of the various chick passages when tested in serum dilution 1:10. By testing
the serum saniples in 1:100 dilution, an increase in the intensity of immuno-
fluorescence was observed during four chick passages.

Contact transmission studies

and
The spreading potential of CVI-988 clone C was investigated by virus recovery
FAT antibody testing of SPF chickens that were added to nine groups of vaccinated
birds. The results are presented in Table 4.
In the groups of recipient chickens exposed at the time of vaccination, virus
demonstrated
recovery was successful in three of seven trials, and antibodies were

9, SUPPLEMENT 1, NOVEMBER 1987 21S

THE VETERINARY QUARTERLY, VOL.
Table 3. Fluorescent antibody staining of serum samples from the backpassage study performed with
the master seed virus.

Chick Sera No. of Intensity of fluorescence

passage collected samples in dilution 1:100

No. on day tested 4+* 3+ 2+ 1+ -

3
1 56 10 6 4
4
2 56 8 6 2
3 105 10 4 4 2

4 98 9 1 1 4 3

5 91 10 3 3 4
6 91 10 5 4 1

7 84 10 5 3 1 1

8 84 10 3 4 1 2

9 77 8 5 3
1
11 50 10 3 7
2 4 3 3
11 50 10
1
12 58 10 3 6 1
2 4 4 2
12 58 10

1
Inoculum consisted of one million WBC
2
Inoculum consisted of 0.25 ml heparinised blood
3
No. of chicks with FAT antibody, scored 2+ in dilution 1:100
4
All sera were FAT positive in dilution 1:10
4+:bright fluorescence; 1+:faint fluorescence

Table 4. Nine contact transmission trials performed with CVI-988 clone C.

No. of chicks Contact Testing

vacci- ex- exposure after Virus
3 4
nated posed on day x wks recovery FAT

Backpassage
5
No. 1 20 12 1 5 0/12 4 0/12
1
No. 12 . 144 20 58 9 ND 3/10
2
No. 12 144 20 58 9 ND 7/10
Safety tests
No. 1,2,3 3x16 3x6 1 10 10/10 18/18
No. 4,5,6 3x16 3x6 1 6 0/18 5/18°

1 1
Chick passage 12 of Table 2
2 2
Chick passage 12 of Table 2
3
Virus recovery test by cocultivation of WBC and CEF
4
Fluorescent antibody staining
5
Infection was established in these chickens as demonstrated by FAT
antibody in the 4th chick passage
6
Respectively two and three FAT-positive chickens of two groups

in seven of nine trials. Contact exposure of hatch mates of the 12th chick passage, at
the age of 58 days, resulted in MD-specific antibodies in 50% of the birds examined.

Protective efficacy studies

a. The protective efficacy of three pre-licensing batches of CVI-988 clone C
vaccine were tested in PD50 assays, employing maternal antibody-free chickens.

22S THE VETERINARY QUARTERLY, VOL. 9, SUPPLEMENT I, NOVEMBER 1987

 The comparative trial included the master seed, the 71th CEF passage, and a
re-isolate from the 6th chick passage of the backpassage study. The trial was
performed according to the described procedures (2, 3, 6, 18), except that in this
trial all six similar vaccine dose groups were kept on litter in the same
conventional chicken house. All chickens were necropsied at time of death or at
10 weeks post-challenge. All surviving chickens without macroscopic MD
lesions were considered to be protected. The results are presented in Table 5.

Table 5. Experimental data with probit analysis' from comparative PD50 assays performed with three
pre-licensing batches of CVI-988 clone C vaccine and three CVI preparations (Nos. 4, 5 and 6).
Challenge at the age of 9 days with vMDV-K.

Vaccine No. 1 2 3 4 5 6
2 3 4
Passage level in CEF 68 74 69 65 71 65/Ch6/10

Highest dose (PFU) 452 245 454 337 560 601

Serial dilutions in steps 1:5 1:5 1:5 1:5 1:5 1:5

Number of chicks protected/total

Highest dose 24/25 22/25 25/25 25/25 20/25 24/24
21/25 17/23 21/25 19/24 18/25 24/25
13/25 6/25 10/25 11/25 14/24 17/25
Lowest dose 7/25 7/25 9/25 9/35 5/25 12/25
No vaccine 26/103

PD50
6
31 48 43 32 74 14

95% Confidence limits of FD50 16-61 15-155 22-85 15-69 26-213 4-47

Slope of probit regression line 1.52 1.69 2.56 2.12 0.95 1.57

Standard error of slope 0.36 0.45 0.89 0.77 0.24 0.42

1
Probit analysis employing UCLA Health Sciences Computer Program BMD03S
2
Master seed virus
3
CVI-988 clone C CEF71
4
CVI-988 clone C CEF65/Chick 6iCEF10 (backpassage study)
5 Number of chickens protected/total number of test chickens
6
The number of PFU required to protect 50% of the MD susceptible chickens

The three pre-licensing batches yielded PD50 estimates that were similar to the
PD50 of the master seed. The five chickens with MD lesions in the highest dose
group of vaccine No. 5 caused a flat probit regression line and a wide 95%
confidence interval of PD50.
The CVI-988 clone C preparation recovered from the 6th chick passage yielded
a PD50 value that was significantly below the other five PD50 estimates
(P<0.05).
b. In the United States, graded doses of CVI-988 clone C vaccine were utilised for
vaccination of 1-day-old chickens that were progeny from commercial broiler
parent stock, vaccinated with bivalent vaccine composed of HVT FC126 and
MDV SB-1. The birds were challenged with 500 PFU of vvMDV-RB/1B at the
relatively early age of 5 days, at variance with the standard procedure for
vaccine evaluation (1). All birds were necropsied at time of death or at 8 weeks
post-challenge. The results are presented in Table 6. A protective index of 85 was
obtained in the highest vaccine dose group. The 80 PFU vaccine dose did not
provide protection.

THE VETERINARY QUARTERLY, VOL. 9, SUPPLEMENT I, NOVEMBER 1987 23S

Table 6. Protective efficacy of CVI-988 clone C' in broiler chickens with HVT/SB-I maternal
antibodies. Challenge at 5 days with vvMDV-RB/1B. Observation period of 8 weeks.

Vaccine No. of chicks Percentage Protective

2
dose protected/total protection index
(PFU)

1937 36/38 95 85
647 17/20 85 58
80 12/20 60
none 32/50 64

1
CVI-988 clone C vaccine no 2 of Table 5
2
Data provided by Dr. S. Klopp, Millsboro, Delaware

c. Field testing of three pre-licensing batches of CVI-988 clone C vaccine was

performed in the Netherlands, utilising eleven flocks of commercial laying-type
chickens. Progeny from prototype CVI-988-vaccinated flocks were utilised.
Only minor changes in production and health performances of the flocks were
reported. The observed percentages of MD lesions were low. A summary of the
collected data is presented in Table 7.
Table 7. Field trials in the Netherlands employing three CVI-988 clone C vaccine batches' 2.

No. Vaccine Observation Percentage MD lesions

Flock of dose period total No. of
No. Breeder chickens in PFU (in weeks) mortality % chickens

1 A 13.750 920 36 3.2 <0.1 (0)3

2 A 21.000 920 36 3.4 0.3 (57)
3 B 15.500 920 34 6.6 0.2 (31)
4
4 B 14.605 920 36 15.1 0.5 (71)
5 B 14.450 1000 35 5.1 <0.1 (1)
6 B 16.500 1000 35 4.2 <0.1 (10)
7 B 8.650 1150 30 3.4 0.2 (20)
8 B 7.270 1150 30 3.6 <0.1 (7)
9 B 3.600 1150 30 5.2 <0.1 (2)
10 C 4.000 1000 29 8.5 <0.1 (2)
11 C 5.000 1000 29 8.5 <0.1 (3)

1
Vaccines Nos 1, 2 and 3 of Table 5
2
Data provided by Dr. C. Fris, Poultry Health Service, Doorn
3
No. of birds detected with gross or microscopical MD lesions
4
Including 10% mortality caused by coccidiosis and drug intoxication

d. In the United States, three batches of CVI-988 clone C vaccine were utilised for
.

pre-licensing testing. More than half a million broiler chickens were s.c.
vaccinated with doses, varying between 1500 and 3000 PFU. About half of the
test chickens were progeny from flocks that had received HVT/SB-1 bivalent
vaccine. The overall health and production performances of all thirty-one flocks
were similar to the previous flocks on the same farm. The number of carcasses
that were condemned due to MD lesions ('leukosis') by the USDA inspection

24S THE VETERINARY QUARTERLY. VOL 9, SUPPLEMENT I, NOVEMBER 1987

 veterinarians in the processing plant were usually below 0.025%, and frequently
zero. Condemnation rates of 0.6, 0.1% and 3.4% were recorded for three flocks;
these figures compared favourably with results from previous 'grow-outs' from
the same farm, of which 2.6 to 16.8% of the chickens were condemned at
slaughter.

DISCUSSION
Comparative studies of CVI-988 clone C CEF65 and prototype CVI-988 vaccine
yielded further information on alteration of characteristics during the process of
attenuation. The improved safety of CVI-988 clone C for very MD-susceptible
chickens was recently confirmed by Witter et al. (31). In addition, the very low MD
mortality observed in the Dutch field trials suggests that the improvement of safety
is expressed under conditions with low infection pressure.
The difference between 'A' antigen release of CVI-988 clone C- and prototype
CVI-988-infected cell cultures has been reported previously (20). Complete loss of
'A' antigen production as detected by FAT, however, was only observed in the
164th DEF passage, which was shown to be highly over-attenuated. The 97th DEF
passage, shown to be over-attenuated as well, still demonstrated release of 'A'
antigen. Absence of 'A' antigen production, therefore, cannot be considered as a
marker for MDV attenuation. Further, the slow virus recovery in the backpassage
studies yielded the impression of a decreased in vivo replication of vaccine virus,
confirming similar observations during attenuation by cell culture passage of other
serotype 1 viruses by Witter and Lee (28) and Schat et al. (25).
Spread of vaccine virus to contact-exposed chickens was absent in the first trial, but
occurred to a varying extent in later trials. The spreading potential of CVI-988
clone C compared with prototype CVI-988 vaccine (22) is apparently reduced;
however, there was not a complete loss of spreading properties as was observed
during attenuation of the Mdll strain (28, 31).
Differential fluorescent antibody staining (FAT) between CVI-988 and various
other serotypes 1 virus strains did not alter during the process of attenuation. The
FAT experiments with the monoclonal antibodies MCA H19 and MCA 2BN90
provided a technique for distinguishing between the available serotype 1 vaccines,
CVI-988 (clone C), and HPRS-16/att. Witter et al. (31) recently described distinct
restriction enzyme digestion patterns of CVI-988 clone C and of vvMDV strain
Md 1 1 and derivatives. Restriction enzyme analysis, therefore, creates another
possibility for differentiation between serotype 1 viruses.

During the backpassage study, a slight reversion of virulence was observed. One
chicken of the 680 inoculated, became paralytic and showed inflammatory B-type
lesions upon histological examination. In the 7th and 8th in vivo passages, four
birds demonstrated nerve swelling accompanied with B-type lesions. After
reducing the inoculum, however, the mild pathogenicity detected during the 7-week
observation period disappeared, and it seems that the acquired pathogenic
properties were minor. In particular, the low score observed during the prolonged
observation period in the 12th in vivo passage was reassuring. The higher levels of
FAT antibody detected in serum samples collected from the 4th chick passage and
higher were suggestive of an increase of virus replication during the in vivo passages.
Comparative protective efficacy studies were performed with maternal antibody-
free SPF chickens, employing a modified procedure of 50% protective dose (PD50)
assays (3) with standard challenge exposure, by injection at 9 days of age as
proposed by an international committee (1). This experimental design provides the

THE VETERINARY QUARTERLY, VOL. 9, SUPPLEMENT I, NOVEMBER 1987 25S

 most accurate evaluation of vaccine efficacy (1, 6). PD50 assays demonstrate much
less variability than observed between protective index studies, in particular if
maternal antibodies, which decrease the sensitivity of the test model, are present
and if challenge is performed on day 5 (28, 31). When experiments are tried to be
brought in tune with realistic field circumstances, early contact exposure should be
preferred.
In previous protection studies, monovalent CVI-988 clone C vaccine afforded
excellent protection against challenge infection with vvMDV strains RB/1B, Md5
and Tun (2, 3). The protective efficacy of monovalent CVI-988 clone C was at least
as good as that achieved by bivalent vaccine composed of HVT and SB-1 (3). PD50
assays performed with three pre-licensing batches of CVI-988 clone C vaccine are
presented here. These afforded solid protection against vMDV-K challenge (Table
5). In this comparative trial was included virus recovered from the 6th chick passage
of CVI-988 clone C CEF65 (because of the above-mentioned results of FAT
staining of serum samples). This re-isolate, in its 10th CEF passage, provided
significantly better protection than the other five CVI-988 clone C preparations,
with that providing evidence for an increase of immunogenicity during the in vivo
passages.
Recently, we have demonstrated that bivalent vaccine-induced maternal antibodies
inhibit HVT/SB-1 vaccination more strongly (to be published) than these did in
cases of vaccination with heterologous CVI-988 clone C (2). This detrimental effect
is due to the combination of maternal antibodies against serotypes 2 and 3
inhibiting replication of vaccine virus. Homologous maternal antibodies have a
lower inhibitive influence on monovalent vaccines of each of the three serotypes
(14). In this study the negative influence of the combination of maternal antibodies
against serotypes 2 and 3 is shown in Table 6. The lowest dose of 80 PFU CVI-988
clone C, normally yielding significant protection in SPF birds (2, 3), was completely
ineffective in the presence of bivalent vaccine-induced maternal antibodies. The
highest vaccine dose of about 2000 PFU CVI-988 clone C, however, was shown to be
highly effective under such circumstances, even against early vvMDV challenge.
Quantitatively, the inhibitive influence of bivalent vaccine-induced maternal
antibodies exceeds the increased protection by higher vaccine doses. In addition, it
should be realised that in the presence of serotype 2 antibodies, the supporting
immunising influence of field virus strains is also diminished, and probably
eliminated. These negative influences are avoided when monovalent vaccines are
employed for MD vaccination.
The field trials performed in the Netherlands in laying-type chickens, with relatively
long periods of observation, and in the United States in broilers, indicate that
CVI-988 clone C is safe and effective and does not adversely affect the health of the
chickens. It may be concluded that the scaling-up of CVI-988 clone C vaccine
application did confirm the laboratory data. CVI-988 clone C vaccines will be a
valuable tool in the control of Marek's disease. Those in the poultry industry,
however, must keep in mind what has recently been reconfirmed in experimental
studies (21): namely, that the optimal effect of these vaccines can only be obtained
by employing hygienic management procedures in order to avoid early virus
exposure.

ACKNOWLEDGEMENTS

The skillful technical assistence of Mrs. H. M. Boerrigter, L. Hartog, J. E. Groenendal, A.

Kant, G. L. Kok, J. Maissan, and P. M. J. van Rossum is greatfully acknowledged. We thank
. the colleagues C. Fris, A. Heuff, J. Naber, and J. van Walsum for collecting the data of field

26S THE VETERINARY QUARTERLY, VOL. 9, SUPPLEMENT I, NOVEMBER 1987

trials in the Netherlands, and Drs. S. Klopp, H. N. Lasher, T. R. Mick le, E. Odor, and J.
Story for providing the data of the United States field trials. Ir. P. R. Defize, ITI-TNO, Delft,
is acknowledged for the statistical evaluation of test results.

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