The Scientific World Journal
Volume 2012, Article ID 147158, 8 pages
doi:10.1100/2012/147158

The cientificWorldJOURNAL

Research Article

Characterization of the Immune Response Induced by
a Commercially Available Inactivated Bluetongue Virus
Serotype 1 Vaccine in Sheep

Ana Cristina Pérez de Diego,1 Pedro José Sánchez-Cordón,2 Ana Isabel de las Heras,1

and José Manuel Sánchez-Vizcaı́no1

1 VISAVET Health Surveillance Centre and Animal Health Department, Veterinary Faculty, Complutense University of Madrid,
Avenida Puerta de Hierro s/n, 28040 Madrid, Spain

2 Department of Comparative Pathology, Veterinary Faculty, University of Córdoba, Animal Health Building, Rabanales Campus,
14014 Córdoba, Spain

Correspondence should be addressed to Ana Cristina Pérez de Diego, anacristina@sanidadanimal.info

Received 18 October 2011; Accepted 22 December 2011

Academic Editors: E. Carrillo and K. E. Kester

Copyright © 2012 Ana Cristina Pérez de Diego et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

The protective immune response generated by a commercial monovalent inactivated vaccine against bluetongue virus serotype 1
(BTV1) was studied. Five sheep were vaccinated, boost-vaccinated, and then challenged against BTV1 ALG/2006. RT-PCR did not
detect viremia at any time during the experiment. Except a temperature increase observed after the initial and boost vaccinations,
no clinical signs or lesions were observed. A specific and protective antibody response checked by ELISA was induced after vaccina-
tion and boost vaccination. This specific antibody response was associated with a significant increase in B lymphocytes confirmed
by flow cytometry, while significant increases were not observed in T lymphocyte subpopulations (CD4+, CD8+, and WC1+),
CD25+ regulatory cells, or CD14+ monocytes. After challenge with BTV1, the antibody response was much higher than during the
boost vaccination period, and it was associated with a significant increase in B lymphocytes, CD14+ monocytes, CD25+ regulatory
cells, and CD8+ cytotoxic T lymphocytes.

1. Introduction

Bluetongue virus (BTV), a member of the Orbivirus genus in
the Reoviridae family [1], shows considerable genetic and
antigenic variability, with at least 25 different serotypes char-
acterized to date [2, 3]. These serotypes do not confer cross-
protective immunity, which means that specific vaccines
must be developed for each serotype [4].

Vaccination has proven very effective in BTV control and
eradication strategies [5, 6]. A wide range of vaccines, based
on either inactivated or modified live virus, are available
against different BTV serotypes [7]. Inactivated vaccines are
considered safer than vaccines based on modified live virus
because they do not allow the possibility of viral replication.
Therefore, they are useful for avoiding virus circulation
among susceptible species [8, 9]. Indeed, inactivated vaccines

have already been used successfully in field trials, and they are
the vaccines most recommended by EU authorities [8, 10].

Several inactivated BTV vaccines have been shown to
confer protection mainly by inducing production of neutral-
izing antibodies [11–14]. These antibodies protect primarily
against homologous serotypes, and they appear ineffective
at cross-protecting against heterologous serotypes [15, 16].
Some studies reveal that cell-mediated immunity could play
an important role [17] when vaccinating with individual
antigens [18], or concretely just in some sheep [19]. Also,
inactivated vaccines have been shown to protect against BTV
in the absence of neutralizing antibodies [4, 20], but it is well
known that the main effective response against BT is capable
to generate neutralizing antibodies [11].

Evaluating the cell-mediated immunity in animals vac-
cinated against BTV could provide valuable information for



2 The Scientific World Journal

Table 1: Antibodies used to analyze PBMC populations by flow cytometry.

Primary and
secondary
antibodies

Specificity Ig isotype
Amount per

tube (μg)
Source Reference

Anti-sheep B
lymphocytes

B lymphocytes IgM 2 VMRD BAQ44A

Anti-sheep CD4 T helper lymphocytes IgG1 2 VMRD 17D1

Anti-sheep CD8 Cytotoxic T lymphocytes IgG1 2 VMRD CACT80C

Anti-sheep WC1 γδ subset of T lymphocytes IgG1 2 VMRD IL-A29

Anti-sheep CD25 IL-2 receptor α-chain IgG1 2 VMRD CACT116A

Anti-sheep CD14 Monocytes IgG1 2 VMRD CAM36A

FITC-conjugated
anti-mouse IgG1
(γ1)

Mouse IgG1 (γ1) — 0.4 Invitrogen P-21129

PE-conjugated
anti-mouse IgM

Mouse IgM — 0.4 Sigma-Aldrich F-9259

assessing and improving the efficacy of BTV vaccines. To that
end, the present study aimed to examine, in vivo, the cellular
and humoral immune response generated by a commercial
monovalent, inactivated vaccine against BTV serotype 1 in
Merino sheep.

2. Materials and Methods

All procedures were carried out in accordance with the Code
of Practice for Housing and Care of Animals Used in Scienti-
fic Procedures, approved by the European Economic Com-
munity in 1986 (86/609/EEC) and amended by European
Commission Directive 2003/65/EC. The procedures were
also approved by the Animal Experimental Committee of
Complutense University of Madrid.

2.1. Animals. Five female Merino sheep of 9-10 months old
and negative for antigens or antibodies against BTV were
housed in Biosafety Level 3 facilities (VISAVET, Complu-
tense University of Madrid).

2.2. Vaccine. A commercial inactivated vaccine against BTV
serotype 1 was used (Zulvac1, Fort Dodge Veterinaria SA).
The active component per dose (2 mL) was BTV-1/ALG2006/
01 E1 ≥ 106.4 TCID50. This vaccine contains adjuvants such
as Quillaia bark, with the primary adjuvant being hydrated
aluminum hydroxide.

2.3. Vaccination, Boost Vaccination, and BTV Challenge. Vac-
cination was carried out SC on day 0 of the experiment.
Booster vaccination was performed by the same route on day
20. In both cases, the vaccine dose was 2 mL as recommended
by manufacturer. On day 48, all animals were challenged
with 1 mL of BTV1 ALG/2006 at a virus title of 1.9 × 106

TCID50 in BHK cells. The challenge inoculum contained
1.9×106 TCID50 (kindly provided by CISA-INIA), and it was
administered intravenously into the jugular vein. On day 68,
animals were euthanized.

2.4. Temperature Monitoring, Clinical Survey, and Necropsy.
Rectal temperature was measured on day 0 prior to vaccina-
tion, as well as on various days until the end of the trial on
day 68. On each of these occasions, clinical signs were scored
using the system described by Perrin et al. [21].

2.5. Sample Collection for Serology and BTV RNA Extraction.
Serum samples were collected on day 0 prior to vaccination,
as well as on days 3, 14, 16, 20, 21, 23, 26, 35, 42, 48, 51, 53,
54, 57, 58, 61, 62, and 68. Samples were analyzed by a double-
recognition ELISA (Ingezim BTV DR 12.BTV.K0, Ingenasa)
according to the manufacturer’s instructions. Antibody res-
ponse was measured as optical density.

Samples of EDTA blood were collected on day 0 prior to
vaccination, as well as on days 3, 20, 21, 23, 42, 48, 51, 53, 54,
57, 58, 61, 62 and 68. RNA was extracted from the samples
using the NucleoSpin RNA II kit (Macherey-Nagel). The
presence of BTV RNA was assessed using real-time RT-PCR
(RT-qPCR) targeting BTV segment 5. Briefly, this RT-qPCR
was able to detect up to 100 RNA copies. The relationship
between the Ct and the copy number was linear between 17
and 33 cycles, which correspond to 1 × 108 and 1 × 103

copies, respectively. The RT-qPCR had an efficiency of 96%,
and it was associated with an R2 of 0.99. The RT-qPCR was
able to detect the mRNA in all of the 128 biological sam-
ples from sheep, goats, and cattle tested [22]. This could be
supposed as a high sensitivity close to 100%. In our study, the
negative controls were not template controls, while positive
controls where sample from experimentally infected animals,
and their Cts were between 20 and 25.

2.6. Flow Cytometry Analysis of Peripheral Blood Mononuclear
Cells. EDTA blood samples were collected on day 0 prior to
vaccination, as well as on days 3, 14, 16, 20, 21, 23, 26, 35, 42,
48, 51, 53, 54, 57, 58, 61, 62, and 68. Flow cytometry using an
FACS scan cytometer (Becton Dickinson) was used to detect
different populations of peripheral blood mononuclear cells
(PBMCs) (Table 1).



The Scientific World Journal 3

2
1.8
1.6
1.4
1.2

1
0.8
0.6
0.4
0.2

0
30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(D.F.V.)

O
pt

ic
al

 d
en

si
ty

 (
45

0 
n

m
) D0–D20. (mean: 0.67 ± 0.55) D21–D48. (mean: 0.88 ± 0.1) D51–D68. (1.04 ± 0.12)∗∗

(V) (BV) (C)

(a)

2
1.8
1.6
1.4
1.2

1
0.8
0.6
0.4
0.2

0
30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(D.F.V.)

O
pt

ic
al

 d
en

si
ty

 (
45

0 
n

m
)

(V) (BV) (C)

Sheep 1 Sheep 2
Sheep 3 Sheep 4
Sheep 5

(b)

Figure 1: (a) Antibody response in serum samples (mean optical density ± SD) measured by ELISA during the experiment. The threshold
below which a response was considered negative was defined as 15% of the positive control optical density. Thus, samples with an optical
density > 0.252 were considered positive. The mean value after boost vaccination was significantly different from that after challenge (∗∗P ≤
0.05; Mann-Whitney U test for nonparametric distributions). (b) Individual measurements of antibody response. Abbreviations: V, day of
first vaccination; DFV, day after first vaccination; BV, day of boost vaccination; C, day of challenge.

2.7. Statistical Analysis. Data were analyzed using GraphPad
InStat 3.0 and IBM SPSS Statistics 19. Percentages of
PBMC subpopulations (CD4+, CD8+, WC1+, CD25+, B lym-
phocytes, and CD14+) are reported as mean± standard devi-
ation (SD). Differences between the percentages of PBMC
populations at different times and the values prior to vaccin-
ation on day 0 were analyzed by repeated-measures ANOVA
with the Huynh-Feldt correction.

Optical density results from ELISA testing of serum sam-
ples are reported as mean ± SD. Differences among mean
optical density values after vaccination (days 0–20), after
boost vaccination (days 21–48), and after challenge (days 51–
68) were analyzed using the Mann-Whitney U test for non-
parametric distributions.

For all comparisons, differences for which P < 0.05 were
considered significant.

3. Results

3.1. Temperature, Clinical Signs, and Lesions. Hyperthermia
(rectal temperature higher than 40◦C) was detected in three
sheep on day 1 after vaccination and in three sheep on day 21
after boost vaccination. After challenge, however, no increase
in temperature was detected in any animal.

No BTV clinical signs were observed during the experi-
ment. Moreover, necropsy failed to detect gross lesions char-
acteristic of BTV infection. Histopathology confirmed the
absence of microscopic lesions characteristic of BTV.

3.2. Determination of Antibody Response by ELISA. A specific
antibody response against BTV was detected in all vaccinated
sheep from day 14 through the end of the experiment
(Figures 1(a) and 1(b)). Antibody levels peaked on day 14,

decreasing moderately thereafter. Antibody levels began to
rise again from day 26, and the levels remained relatively con-
stant until day 51. From that point until day 62, the levels
increased again, showing a slight decrease only in the final
stage of the study.

Mean values of antibody response were compared for the
periods after vaccination (days 0–20), after boost vaccination
(days 21–48), and after challenge (days 51–68). No signifi-
cant differences were observed between antibody levels after
vaccination (0.67± 0.55) and after boost vaccination (0.88±
0.1). After challenge, however, the antibody level increased
significantly to 1.04± 0.12 (Figure 1(a)).

3.3. BTV RNA Detection by RT-PCR. No viral genome was
detected in the animals at any time in the study.

3.4. Analysis of PBMC Populations

3.4.1. CD4+. CD4+ T lymphocytes represented 32.87% of
PBMC before vaccination. Between days 14 and 21, the pro-
portion of CD4+ cells significantly declined, with the mini-
mum value of 21.7% occurring on day 14 (Figures 2(a) and
2(b)). Subsequently, the proportion returned to its prevacci-
nation level. After challenge, the level decreased significantly
again between days 51 and 53, reaching 22.7% on day 53. The
level then returned to prevaccination values for the rest of the
trial.

3.4.2. CD8+. The mean percentage of CD8+ T lymphocytes
prior to vaccination (day 0) was 9.89% (Figures 2(c) and
2(d)). This percentage did not vary significantly after vaccin-
ation or boost vaccination. It increased from day 57, nine
days after challenge, and peaked between days 62 and 68.



4 The Scientific World Journal

0
30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

60

50

40

30

20

10P
B

M
C

 C
D

4+
(%

)

∗
∗
∗ ∗ ∗ ∗

(D.F.V.)

(a)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

Sheep 1 Sheep 2
Sheep 3 Sheep 4

Sheep 5

0

60

50

40

30

20

10P
B

M
C

 C
D

4+
(%

)

(D.F.V.)

(b)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

P
B

M
C

 C
D

8+
(%

) ∗
∗

18
16
14
12
10

8
6
4
2
0

(D.F.V.)

(c)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

P
B

M
C

 C
D

8+
(%

)

18
16
14
12
10

8
6
4
2
0

Sheep 1 Sheep 2
Sheep 3 Sheep 4

Sheep 5

(D.F.V.)

(d)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

0

60

50

40

30

20

10P
B

M
C

 W
C

1+
(%

)

(D.F.V.)

(e)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

0

60

50

40

30

20

10P
B

M
C

 W
C

1+
(%

)

Sheep 1 Sheep 2
Sheep 3 Sheep 4

Sheep 5

(D.F.V.)

(f)

Figure 2: Mean percentages (± SD) and individual percentages of PBMC populations labeled by antibodies against CD4+ (a, b), CD8+ (c,
d), and WC1+ (e, f) throughout the experiment. ∗ is statistically significant difference (P ≤ 0.05) from prevaccination value on day 0, based
on ANOVA with the Huynh-Feldt correction. Abbreviations: V, day of first vaccination; DFV, day after first vaccination; BV, day of boost
vaccination; C, day of challenge.

3.4.3. WC1+. The percentage of γδ T lymphocytes labeled by
the anti-WC1+ antibody did not vary significantly from the
prevaccination value of 14.28% (Figures 2(e) and 2(f)).

3.4.4. CD25+. The percentage of CD25+ cells increased sli-
ghtly, but not significantly, from a pre-vaccination value of

7.44% to 14.18% on day 14 (Figure 3(a)). This percentage
did not change significantly after boost vaccination. After
challenge, however, all five animals showed an increase in the
proportion of CD25+ cells. For example, by day 51, the level
in sheep 4 and sheep 5 had increased, respectively, to 24.2%
and 31.3% (Figure 3(b)). The level increased significantly



The Scientific World Journal 5

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

0

60

50

40

30

20

10P
B

M
C

 C
D

25
+

(%
)

∗

∗

(D.F.V.)

(a)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

0

60

50

40

30

20

10P
B

M
C

 C
D

25
+

(%
)

Sheep 1 Sheep 2
Sheep 3 Sheep 4

Sheep 5

(D.F.V.)

(b)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

0

60

50

40

30

20

10

P
B

M
C

 B
+

(%
)

∗

∗
∗ ∗ ∗ ∗ ∗ ∗

(D.F.V.)

(c)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

0

60

50

40

30

20

10

P
B

M
C

 B
+

(%
)

Sheep 1 Sheep 2
Sheep 3 Sheep 4

Sheep 5

(D.F.V.)

(d)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

18
16
14
12
10

8
6
4
2
0

P
B

M
C

 C
D

14
+

(%
)

∗

(D.F.V.)

(e)

30 14 16 20 21 23 26 35 42 48 51 53 54 57 58 61 62 68

(V) (BV) (C)

18
16
14
12
10

8
6
4
2
0

P
B

M
C

 C
D

14
+

(%
)

Sheep 1 Sheep 2
Sheep 3 Sheep 4

Sheep 5

(D.F.V.)

(f)

Figure 3: Mean percentages (± SD) and individual percentages of PBMC populations labeled by antibodies against CD25+ (a, b), B+ cells
(c, d), and CD14+ (e, f) throughout the experiment. ∗is statistically significant difference (P ≤ 0.05) from the mean pre-vaccination value
on day 0, based on ANOVA with the Huynh-Feldt correction. Abbreviations: V, day of first vaccination; DFV, day after first vaccination; BV,
day of boost vaccination; C, day of challenge.

again between days 54 and 57, peaking on day 54 (31.2%).
Subsequently, the level decreased progressively and returned
to pre-vaccination values at the end of the experiment.

3.4.5. B Lymphocytes. The percentage of B cells decreased
significantly after vaccination, with the lowest level of 15.4%
occurring on day 14 (Figures 3(c) and 3(d)). The level signifi-

cantly increased thereafter between days 16 and 21, and again
after boost vaccination between days 35 and 48. After chal-
lenge, the percentage varied slightly, showing a significant in-
crease on day 58.

3.4.6. CD14+. The percentage of monocytes labeled by the
anti-CD14+ antibody did not change significantly from its



6 The Scientific World Journal

pre-vaccination value of 3.85% after vaccination or boost
vaccination (Figures 3(e) and 3(f)). After challenge, however,
the percentage increased significantly, reaching 10.28% on
day 57. Subsequently, it decreased until it returned to pre-
vaccination values on days 62 and 68. After challenge, all ani-
mals showed an increase followed by a marked decrease. This
response occurred on different dates for different animals,
though in all cases the levels reached similar values.

4. Discussion

The inactivated vaccine against BTV serotype 1 (Zulvac1,
Fort Dodge Veterinaria SA) induced an effective immune res-
ponse in all vaccinated sheep. Challenge virus was not de-
tected in blood by RT-PCR even at 20 days after inoculation.
Except for a temperature increase observed in most animals
after the vaccination and boost vaccination, no clinical signs
or lesions characteristic of BTV infection were observed.

The temperature increase observed in most animals after
vaccination and boost vaccination may reflect activation/
stimulation of the immune system. Similar increases were
observed after administration of inactivated vaccines against
different serotypes of BTV [23, 24]. However, such a temper-
ature increase was not observed in sheep after vaccination
or revaccination with vaccines containing virus-like particles
[25]. These vaccines lack genetic material and so are nonre-
plicative; instead, they contain complexes of structural pro-
teins (VP2, VP3, VP5, and VP7) [26]. Therefore, even though
inactivated BTV vaccines do not contain live virus, they seem
to have a greater ability to stimulate the immune system from
a very early stage due to the presence of potent adjuvants.

After subcutaneous administration of inactivated vac-
cines against BTV, the presence of viral RNA in blood should
not be detected [11, 27]. Moreover, such vaccines prevent
viral replication after challenge with homologous virus [28,
29]. Our results showed the absence of virus in blood after
both vaccinations as well as after challenge with BTV sero-
type 1. These findings suggest that this vaccine may prevent
both virus dissemination and disease spread from vaccinated
animals. Therefore, in the presence of vector-borne BTV
serotype 1, the inactivated vaccine not only prevents virus re-
plication but also effectively induces a protective immune
response. The specific protection conferred by these vaccines
appears to relate to the key role played by structural pro-
tein VP2 in stimulating protective immunity mediated by T
and B cells [18, 30]. However, the immunological mecha-
nisms behind the protection observed in vaccinated animals,
including the possible role of the cellular immune response,
remains unclear.

At 14 days after vaccination, a specific antibody response
against BTV was observed in all vaccinated sheep, and it per-
sisted through the end of the experiment. This antibody res-
ponse was shown to be the main factor in protecting sheep
against BTV serotype 1. Our results are in agreement with
previous studies [8, 11], wherein the specific antibodies
detected by ELISA after administration of inactivated BTV
vaccines in sheep were considered to be neutralizing anti-

bodies with the ability to protect against the virus. Never-
theless, how antibodies against BTV neutralize virus in vivo
remains unclear, despite attempts to demonstrate antibody-
dependent cellular cytotoxicity [17, 31].

The potential role of the cellular immune response dur-
ing BTV infection and after vaccination is not fully under-
stood [4, 18, 20, 32]. In the present study, the specific anti-
body response detected in sheep after vaccination and boost
vaccination against BTV serotype 1 was not associated with
either a significant increase in T lymphocyte subpopulations
(CD4+, CD8+, and WC1+) or in CD25+ regulatory cells.
Moreover, significant changes in the percentage of CD14+

monocytes were not observed. These results are in agreement
with previous studies of inactivated vaccines against BTV
serotype 1 in sheep [33].

The lack of cell-mediated immunity after vaccination and
boost vaccination in our experiment could be attributed to
different components of the vaccine, in which inactivated
virus was mixed with an aluminum-based adjuvant. These
adjuvants delay the elimination of antigens after vaccine
administration, prolonging the antigenic stimulus. These
adjuvants also promote antibody response, even though
they have little stimulatory effect on cell-mediated responses
[34–36]. This inability to stimulate a strong cell-mediated
response, together with the diversity of responses by antigen-
presenting cells following exposure to live or killed viruses
in vaccines [37, 38], may explain the moderate participation
of cell-mediated response after vaccination and boost vacci-
nation, as well as the absence of significant changes in the
proportions of these immune cell populations.

On the other hand, B cells can recognize most antigens
without prior processing, and certain antigens can provoke
antibody formation in the absence of helper T cells, provid-
ing sufficient signal for B cell proliferation and differentiation
into antibody-producing plasma cells [39, 40]. This may ex-
plain why the significant increase in B lymphocytes observed
after vaccination and boost vaccination in the present study
was associated with an increase in specific antibodies against
BTV serotype 1.

After challenge, viral genome was not detected in vac-
cinated sheep, confirming specific protection induced by
antibody response. In fact, antibody response after challenge
was significantly higher than after boost vaccination, and this
increase was associated with a significant increase in CD14+

monocytes, CD25+ cells, B lymphocytes, and CD8+ T lym-
phocytes. Thus, challenge with BTV serotype 1 significantly
increased the levels of CD14+ monocytes and CD25+ regu-
latory cells. CD14 has been defined as a central molecule in
antigen recognition and cellular interactions, where it plays a
key role in monocyte-mediated T-cell activation [18, 41]. In
addition to dendritic cells [32], monocyte macrophages play
an important role in antigen presentation and virus spread
during BTV infection [42, 43]. CD25 has been defined as a
low-affinity receptor for IL-2 that is expressed mainly on T
cells activated by interaction with antigens [44, 45]. Thus,
the increase in CD14+ monocytes and CD25+ regulatory
cells observed after challenge with BTV serotype 1 may
costimulate B cells, allowing their activation, proliferation,
and differentiation into antibody-producing plasma cells.



The Scientific World Journal 7

Finally, after challenge with BTV serotype 1, an increase
in CD8+ T lymphocytes was observed, which became signif-
icant toward the end of the trial. Similar increases in CD8+

T lymphocytes and the cytokine IL-2 in association with an
increase in CD25 expression have been described after chal-
lenge of sheep vaccinated against BTV serotype 1 [33]. Such
increases have also been observed during experimental infec-
tions [31, 32, 46]. Thus, the activation and proliferation of
CD8+ T lymphocytes, which play a key role in the Th1 res-
ponse dominated by cytotoxic T cells, may also be associated
with the increase in CD14+ monocytes and CD25+ regula-
tory cells observed after challenge.

5. Conclusions

Collectively, these results demonstrate that a specific and
protective antibody response was induced after vaccination
and boost vaccination of sheep with an inactivated vaccine
against BTV serotype 1 (Zulvac1, Fort Dodge Veterinaria
SA). This response was associated with a significant increase
in B lymphocytes. However, significant increases were not
observed in T lymphocyte subpopulations (CD4+, CD8+,
and WC1+), CD25+ regulatory cells, or CD14+ monocytes.
After challenge with BTV serotype 1, antibody response sig-
nificantly increased over the level observed after boost vac-
cination, and this was associated with significant increases in
B lymphocytes, CD14+ monocytes, CD25+ regulatory cells,
and CD8+ cytotoxic T lymphocytes.

Conflict of Interests

None of the authors has any financial or personal relation-
ships that could inappropriately influence or bias the content
of the paper.

Acknowledgments

This study was funded by projects BTVAC FP6-2005-SSP-5A
and AGL2009-13174-C02-01. A. C. Pérez de Diego holds a
scholarship of the FPU programme (Ministry of Education
and Science, Spain). P. J. Sánchez-Cordón is supported by
a contract of the “Ramón y Cajal programme” (Ministry of
Science and Innovation, Spain). The authors thank Amalia
for assistance during flow cytometry analysis. They also
thank Rocı́o Sánchez, Belén Rivera, and Pablo del Carmen
for dedicating their time and effort to this study.

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