Classical swine fever: comparison of oronasal immunisation with CP7E2alf marker and C-strain vaccines in domestic pigs.

Abstract

Effective oronasal vaccination against classical swine fever (CSF) is essential to achieve protection in wild boar. However the currently available live CSF vaccines, e.g. C-strain, do not allow serological differentiation between infected and vaccinated animals (DIVA). A modified live marker vaccine candidate (CP7E2alf) has been recently developed (Reimann et al., 2004). This communication reports the comparison of CP7E2alf and C-strain virus vaccines during 98 days following oronasal immunisation in domestic pigs. C-strain vaccine virus was consistently detected in tonsils of all (n=30) animals from 3 to 77 days post vaccination (dpv) and in blood (n=36) between 3 and 13dpv by CSFV-specific rRT-PCR. CP7E2alf virus RNA was detected in 6 animals slaughtered between 4 and 63dpv by a BVDV-specific rRT-PCR. The chimeric virus was not detected in blood samples. As detected by CSFV E2-specific antibody ELISA and virus neutralisation tests, seroconversion first occurred at 11dpv in the C-strain vaccinated group and between 11 and 15dpv in the CP7E2alf vaccinated group. The serological response was still present at 98dpv. The CP7E2alf serological response remained negative using the CSFV E(rns) ELISA whereas seroconversion occurred in the C-strain vaccinated group. In conclusion, the primary replication site of CP7E2alf vaccine virus was found to be the tonsils as in the C-strain and virulent field strains. Persistence of CP7E2alf in the tonsils was also demonstrated up to 63dpv. Both vaccines showed immunogenicity after oronasal administration in domestic pigs. In contrast to the C-strain, CP7E2alf vaccine allowed the use of DIVA approaches in serological tests. This study confirms CP7E2alf as a promising marker vaccine candidate for oronasal vaccination programmes to control CSF in domestic pigs and wild boar.

Full text

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Accepted Manuscript
Title: Classical swine fever: comparison of oronasal
immunisation with CP7E2alf marker and C-strain vaccines in
domestic pigs
Authors: Maryl` ene Tignon, G´ abor Kulcs´ ar, Andy Haegeman,
Timea Barna, Katalin F´ abi´ an, R´ eka L´ evai, Yves Van der
Stede, Attila Farsang, Robert Vrancken, Katinka Bel´ ak, Frank
Koenen
PII:
DOI:
Reference:
S0378-1135(09)00461-1
doi:10.1016/j.vetmic.2009.09.044
VETMIC 4601
To appear in:
VETMIC
Please cite this article as: Tignon, M., Kulcs´ ar, G., Haegeman, A., Barna, T., F´ abi´ an,
K., L´ evai, R., Van der Stede, Y., Farsang, A., Vrancken, R., Bel´ ak, K., Koenen,
F., Classical swine fever: comparison of oronasal immunisation with CP7E2alf
marker and C-strain vaccines in domestic pigs, Veterinary Microbiology (2008),
doi:10.1016/j.vetmic.2009.09.044
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DOI : 10.1016/j.vetmic.2009.09.044

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(4) National Veterinary Institute (SVA), Departments of Pathology
1
Title:
1
Classical swine fever: comparison of oronasal
2
immunisation with CP7E2alf marker and C-strain vaccines
3
in domestic pigs
4
Authors’ full names:
5
Marylène Tignon (1), Gábor Kulcsár (2), Andy Haegeman (1), Timea
6
Barna (2), Katalin Fábián (2), Réka Lévai (2), Yves Van der Stede (3),
7
Attila Farsang (2), Robert Vrancken (1) Katinka Belák (4) and Frank
8
Koenen (1)
9
Institutions:
10
(1) Veterinary and Agrochemical Research Centre (VAR),
11
Department of Virology, Groeselenberg 99, B-1180 Brussels, Belgium;
12
(2) Central Agricultural Office, Directorate of Veterinary Medicinal
13
Products (DVMP), Szállás utca 8, H-1107 Budapest, Hungary;
14
(3) Veterinary and Agrochemical Research Centre (VAR),
15
Coordination Centre for Veterinary Diagnostics, Groeselenberg 99,
16
B-1180 Brussels, Belgium;
17
18
and Wildlife diseases, Ulls väg 2B, SE-751 89 Uppsala, Sweden.
19
20
Corresponding author:
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2
Marylène Tignon, Veterinary and Agrochemical Research Centre
1
(VAR), Department of Virology, Groeselenberg 99, B-1180 Brussels,
2
Belgium;
3
Phone: +32 2 379 0519; Fax: +32 2 379 0666; e-mail address:
4
Marylene.Tignon@var.fgov.be
5
6
Short running title:
7
Classical swine fever: CP7E2alf and C-strain vaccines
8
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dpv. CP7E2alf serological response remained negative using the
3
Abstract
1
Effective oronasal vaccination against classical swine fever (CSF) is
2
essential to achieve protection in wild boar. However the currently
3
available live CSF vaccines, e.g. C-strain, do not allow serological
4
differentiation between infected and vaccinated animals (DIVA). A
5
modified live marker vaccine candidate (CP7E2alf) has been
6
recently developed (Reimann et al., 2004). Here we report on the
7
comparison of CP7E2alf and C-strain virus vaccines during 98 days
8
following oronasal immunisation in domestic pigs.
9
C-strain vaccine virus was consistently detected in tonsils of all
10
(n=30) animals from 3 to 77 days post vaccination (dpv) and in
11
blood (n= 36) between 3 and 13 dpv by CSFV-specific rRT-PCR.
12
CP7E2alf virus RNA was detected in 6 animals slaughtered between
13
4 and 63 dpv by a BVDV-specific rRT-PCR. The chimeric virus was not
14
detected in blood samples.
15
As detected by CSFV E2-specific antibody ELISA and virus
16
neutralisation, seroconversion first occurred at 11 dpv in the C-strain
17
vaccinated group and between 11 and 15 dpv in the CP7E2alf
18
vaccinated group. Serological response was still observed at 98
19
20
CSFV Erns ELISA whereas seroconversion occurred in C-strain
21
vaccinated group.
22
In conclusion, the primary replication site of CP7E2alf vaccine virus
23
was found to be the tonsils similarly to C-strain and virulent field
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4
strains. Persistence of CP7E2alf in the tonsils was also demonstrated
1
up to 63 dpv. Both vaccines showed immunogenicity after oronasal
2
administration in domestic pigs. In contrast to the C-strain, CP7E2alf
3
vaccine allowed the use of DIVA approaches in serological tests.
4
This study confirms CP7E2alf as a promising marker vaccine
5
candidate for oronasal vaccination programmes to control CSF in
6
domestic pigs and wild boar.
7
Keywords
8
Classical swine fever virus (CSFV), DIVA marker vaccine, CP7E2alf,
9
C-strain, oronasal vaccination, diagnostic tests
10
11
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disease control in wild boar (von Rüden et al., 2008). Therefore, strict
5
Introduction
1
Classical swine fever (CSF) is a highly contagious and often fatal
2
disease of domestic pigs and wild boar. Outbreaks of CSF usually
3
cause important economic losses, and impair internal and
4
international trade of pigs and pig products (Vandeputte and
5
Chappuis, 1999; Edwards et al., 2000). The aetiological agent of CSF
6
is classical swine fever virus (CSFV), a member of the Pestivirus genus
7
of the Flaviviridae family (van Regenmortel et al., 2000).
8
The effectiveness of modified live vaccines, such as the Chinese
9
strain (C-strain), has been frequently reported in domestic pigs and
10
in wild boar (Terpstra and Robijns, 1977; Chenut et al., 1999; Kaden
11
et al., 2000; Kaden and Lange, 2001; Dewulf et al., 2004b; Dong and
12
Chen, 2007). Such vaccines induce protection shortly after
13
administration. Prophylactic mass vaccination with modified live
14
vaccines, combined with culling of infected pigs, successfully
15
resulted in the eradication of the disease in most countries of the
16
European Union (EU) (Vandeputte and Chappuis, 1999; Greiser-
17
Wilke and Moennig, 2004). Currently, modified live vaccines are still
18
in use where the disease is endemic in domestic pigs and for
19
20
restrictions on the international trade in pig products are
21
implemented from countries using vaccination (Greiser-Wilke and
22
Moennig, 2004). The lack of serological discrimination between
23
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6
naturally infected animals and animals vaccinated with a modified
1
live vaccine hampers disease control relying on serology (van
2
Oirschot, 2003; Pasick, 2004). To overcome this problem, marker
3
vaccines which allow differentiation of infected from vaccinated
4
animals (DIVA) have been developed.
5
The first subunit vaccine was developed in 1993 based on the
6
expression of the E2 envelope protein of the Brescia strain within the
7
baculovirus system (Hulst et al., 1993). Subunit vaccines induce a
8
DIVA serological response and reduce morbidity and mortality
9
following subsequent challenge with a virulent CSFV field strain
10
(Moormann et al., 2000). However, horizontal or vertical virus
11
transmission is not completely prevented when challenge occurs in
12
the first week post vaccination (Dewulf et al., 2001, 2004b).
13
Moreover, the subunit vaccines are not applicable for CSF control in
14
wild boar by oral immunization.
15
A new generation of live marker vaccines was developed based on
16
the construction of chimeric pestiviruses (for review see Beer et al.,
17
2007). The E2 or Erns-coding region of CSFV or cytopathogenic
18
bovine viral diarrhoea virus (BVDV) strain CP7 has been replaced by
19
that of a heterologous strain (van Gennip et al., 2000; Reimann et
20
al., 2004; Rasmussen et al., 2007; Wehrle et al., 2007). One of these
21
constructs, CP7E2alf, is based on the infectious full-length clone of
22
BVDV strain CP7 in which the BVDV E2 gene is replaced by that of
23
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properties of the vaccines were compared.
7
CSFV strain Alfort/187 (Reimann et al., 2004). CP7E2alf proved to be
1
completely safe in pigs following intramuscular inoculation. Neither
2
viraemia, nor virus transmission to contact animals were detected.
3
Furthermore, immunogenicity and protective effectiveness have
4
been demonstrated after intramuscular administration in pigs
5
(Reimann et al., 2004; Koenig et al., 2007a) and oral vaccination in
6
wild boar (Koenig et al., 2007b; Kaden et al., 2008). Additionally, the
7
DIVA properties of this live vaccine have been described (Reimann
8
et al., 2004). Nevertheless additional data regarding
9
immunogenicity after oronasal administration, dose effect and
10
persistence over a longer period are required to improve vaccine
11
characterisation and application efficiency.
12
The aim of the present study was to evaluate the live marker
13
vaccine CP7E2alf following oronasal administration to domestic pigs
14
in comparison to the C-strain. The presence and persistence of
15
vaccine virus was investigated in tonsils and blood samples by
16
comparison with the C-strain. The serological response observed
17
after oronasal vaccination was studied up to 98 days post
18
vaccination (dpv) in the two vaccine groups and the DIVA
19
20
21
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8
Materials and methods
1
Vaccine
2
The conventional C-strain vaccine (‘Riems’) was kindly provided by
3
Dr. V. Kaden (Friedrich-Loeffler-Institut, Germany). The CP7E2alf
4
marker vaccine (Reimann et al. (2004) was produced using Good
5
Manufacturing Practice by Fort Dodge Veterinaria (Spain).
6
Vaccines were diluted in saline solution to 104.5 50% tissue culture
7
infective dose per ml (TCID50/ml) and virus titre was checked by
8
back-titration (104.48 and 104.59 TCID50/ml for CP7E2alf and C-strain
9
viruses respectively).
10
Animals
11
For the experiment, pigs were recruited twice from the same
12
conventional breeding farm with an interval of six months. Each
13
group was constituted by 46 Kahyb breed pigs (Hungarian
14
landrace-typed hybrid), two months old and weighing
15
approximately 12 kg. These animals were free of pestivirus
16
antibodies, as tested by standard diagnostic procedures.
17
Experimental design
18
The study design, the administration dose and routes were similar to
19
a previous experimental infection with a field isolate (‘WIL-11722’)
20
(Tignon et al., 2008). Vaccination with CP7E2alf and C-strain were
21
conducted separately with an interval of six months.
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All the living animals were clinically monitored using a scoring
9
Briefly, weaner pigs were housed in a biosecure unit in agreement
1
with EU directive 91/630/EEC and its amendments 2001/88/EC and
2
2001/93/EC. Following a seven-day acclimatization period, the pigs
3
were clinically inspected and blood sampled. On the first day (day
4
0), CP7E2alf live marker vaccine was administrated via the
5
intranasal (1 ml) and the oral (1 ml) routes by means of a syringe to
6
all animals of the first group (n=46), except two pigs kept as
7
negative controls. The two non-vaccinated controls were
8
slaughtered on the day of vaccination by an intravenous
9
pentobarbital injection, followed by exsanguination. Subsequently,
10
two randomly selected vaccinated animals were slaughtered on a
11
daily basis between 1 and 8 dpv, at 13 and 18 dpv, and then
12
weekly from 21 to 98 dpv.
13
Six month later, the second group of animals (n=46) was vaccinated
14
with the C-strain modified live vaccine, except two pigs kept as
15
negative controls. Vaccination and slaughtering protocols were
16
conducted in the same way as previously described.
17
Clinical examination and sample collection
18
19
system (Mittelholzer et al., 2000) based on the observation of ten
20
parameters (breathing, liveliness, body tension, body shape,
21
walking, skin, eyes, appetite, defecation and leftovers) and the
22
rectal temperatures were recorded on a daily basis, before sample
23
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10
collection, during the first 8 days and subsequently bi-weekly until 98
1
dpv. Animals were considered clinically ill when their daily
2
cumulative clinical score exceeded the value of 6. Pigs were
3
considered febrile when rectal temperatures exceeded 40.5°C.
4
Blood samples with addition of EDTA as anticoagulant and serum
5
samples were collected from each animal in the group during the
6
experiment. Samples were collected on slaughter days and
7
additionally at 11 and 15 dpv. At necropsy, the tonsils of the two
8
slaughtered animals were aseptically collected for virological
9
examination. The samples were stored at -80°C until analysis.
10
Haematology
11
The total number and differentiation of peripheral blood leucocytes
12
(PBLs) were determined in EDTA-treated blood samples by using a
13
Sysmex E-4000 automatic haemocytometer (Toa Medical
14
Electronics, Kobe, Japan). Counts below 10,000 leukocytes/µl were
15
considered as leucopaenia. From 0 until 70 dpv, data were
16
collected from 10 animals and later from the remaining animals.
17
Virological analysis
18
Virus isolation (VI) from the tonsil samples and from EDTA-treated
19
blood was performed on semi-confluent monolayer of porcine
20
kidney cells (PK15, ATCC-CCL33) (Dewulf et al., 2004a). After 48
21
hours, the cells were fixed with isopropanol and stained with
22
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HerdChek CSFV ELISA (IDEXX Europe B.V., Schiphol-Rijk, The
11
polyclonal fluorescein-conjugated anti-CSFV immunoglobulins.
1
Cultures were passaged one to three times.
2
After RNA extraction with the Viral RNA kit (Qiagen Benelux B.V.,
3
Venlo, The Netherlands), the presence of viral RNA molecules was
4
investigated in the tonsils and blood samples by real-time RT-PCR
5
(rRT-PCR). The rRT-PCR described by Hoffmann et al (2005) and the
6
commercial TaqVet CSF rRT-PCR kit (Laboratoire Service
7
International, Lissieu, France) were used for CSFV-specific genome
8
detection. In addition, one BVDV-specific (Letellier and Kerkhofs,
9
2003) and one Pestivirus-specific (pan-pesti) (Hoffmann et al., 2006)
10
rRT-PCR assays were used for CP7E2alf virus RNA detection. The rRT-
11
PCR assays were performed using an ABI 7500 FAST machine
12
(Applied Biosystems, Lennik, Belgium) and analyzed using the
13
Sequence Detection Software 1.4. For CSFV-, BVDV- and pan-pesti-
14
specific rRT-PCRs, threshold cycle (Ct) values lower than 42 were
15
considered positive.
16
Serological analysis
17
The CSFV E2 and Erns antibody responses were investigated with the
18
19
Netherlands) and PrioCHECK CSFV Erns ELISA (kindly provided by
20
Prionics A.G., Zurich, Switzerland) respectively. Values obtained in
21
the ELISAs were expressed as blocking percentages. Blocking
22
percentages between 30 and 40% in the E2 ELISA were considered
23
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12
as doubtful while those above 40% were considered positive.
1
Blocking percentages in the Erns ELISA were considered as positive
2
when above 50%. The neutralising response was determined in the
3
virus neutralisation test (VNT) according to the OIE manual
4
(Anonymous, 2004) using two viral strains: Alfort/187 and CP7E2alf.
5
For VNT, 50% neutralising doses (ND50) were expressed as Log10-
6
transformed values of neutralising antibody titres, with values
7
greater than one regarded as positive.
8
Statistical analysis
9
The data were presented as medians with either the corresponding
10
range of values or the 25th and 75th percentiles. The temperature,
11
haematological and serological data (E2 and Erns ELISA blocking
12
percentage, virus neutralising titres) collected after vaccination with
13
CP7E2alf and C-strain were subject to statistical test for difference
14
between groups. The two vaccines groups were considered as two
15
independent populations. The data were compared per day after
16
linear ranking with the Wilcoxon-Mann-Whitney test for
17
nonparametric samples (XLstat, Addinsoft). The null hypothesis was
18
considered rejected at P0.05.
19
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The initial median PBL population before infection was 20.5 x 103
13
Results
1
Clinical examination
2
Clinical disease was not recorded in the two groups vaccinated
3
with the CP7E2alf and C-strain using the referred scoring system. The
4
maximal daily cumulative clinical score per animal observed was 2
5
and 5 in CP7E2alf and C-strain groups respectively. Median rectal
6
temperatures remained below 40.5°C during the 98 dpv (Figure 1).
7
Between 2 and 21 dpv, 16 febrile animals with rectal temperatures
8
below 42°C were recorded in the CP7E2alf group. Fever generally
9
disappeared after one to five days. During this time, 12 transiently
10
febrile animals, with fever during one to two days, were observed in
11
the C-strain group. In the two vaccine groups, fever could not be
12
linked with an increased clinical scoring. Temperatures of both
13
groups were statistically distinct using the Wilcoxon-Mann-Whitney
14
test at 3, 7 to 10, 12 to 28, 49 to 67, 74 to 77 and 84 to 91 dpv with
15
P≤0.001 from 8 to 10, 25, 49 and 56 dpv, with P≤0.01 at 7, 18 and 67
16
to 84 dpv and P≤0.05 at 3, 21, 28, 60 to 63 and 91 dpv.
17
Haematology
18
19
cells/µl (with range from 15.7 to 25.5 x 103) and 20.7 x 103 cells/µl
20
(with range from 17.8 to 28.3 x 103) for the C-strain and CP7E2alf
21
groups, respectively (Figure 2). Leucopaenia was not observed in
22
either vaccinated group. PBL populations of both groups were
23
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Vaccine virus detection in sequentially collected blood samples
14
significantly distinct at 4 (P≤0.01), 5 (P≤0.05) and 7 dpv (P≤0.01), as
1
well as at 28 (P≤0.001) and 63 dpv (P≤0.01) by the Wilcoxon-Mann-
2
Whitney test.
3
Vaccine virus detection in sequential tonsil samples
4
The CP7E2alf virus was only isolated on cell culture from the tonsils of
5
two animals at 4 and at 5 dpv, respectively (Table 1). The marker
6
vaccine virus genome was detected in the tonsils of four more
7
animals at 7, 18, 21 and 63 dpv by BVDV-specific rRT-PCR. However,
8
CP7E2alf vaccine genome was not detected in the tonsils by the
9
pan-pesti-specific rRT-PCR, nor could the vaccine virus be detected
10
in the tonsils by the two CSFV-specific rRT-PCRs.
11
The C-strain vaccine virus was isolated from the tonsils collected
12
between 3 and 13 dpv except for one animal at 3 dpv and another
13
at 8 dpv. CSFV-specific rRT-PCRs detected the vaccine virus
14
genome in the tonsils from 3 until 77 dpv with only one of the two
15
collected samples being positive at 3, 63, 70 and 77 dpv. At the end
16
of the experiment (98 dpv) the C-strain vaccine virus was still present
17
in one of the two remaining animals.
18
19
The VI and rRT-PCR assays revealed no viraemia in blood samples
20
from the CP7E2alf vaccinated group (Table 2). In contrast, viraemia
21
started between 3 and 8 dpv in the C-strain group, as detected by
22
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