Appendix 34
Importance of cell mediated immunity for protection against Foot and Mouth Disease
Yooni Oh1*, Bryan Charleston1, David J. Paton1, Jong-Hyun Park2, Paul V. Barnett1, Yi-Seok Joo2,
Satya Parida1
1
Pirbright Laboratory, Institute for Animal Health, Ash Road, Surrey, GU24 0NF, UK.
2
FMD Research Laboratory, FADRD, NVRQS, Republic of Korea
Abstract:
Introduction: The humoral antibody titre is not always fully predictive of vaccine-induced protection
against FMD. Therefore, this study has looked for a correlation between cell mediated immune
responses and post-vaccination protection against FMDV infection. Another aim has been to
evaluate the use of the FMDV whole blood IFN-γ assay to differentiate between vaccination and
infection.
Materials and Methods: Samples were collected from two vaccine potency challenge experiments (A
May 97 and SAT 2) conducted at Pirbright, UK, according to the European Pharmacopoeia. Using a
modified version of the BOVIGAM assay for tuberculosis, whole blood samples from FMDV
vaccinated, non-vaccinated and vaccinated-and-challenged cattle were re-stimulated overnight
with inactivated FMDV antigen and the level of induced IFN-γ was measured. PBMC from these
animals were also used in ELIspot and cell proliferation assays. Humoral antibody levels were
measured by virus neutralization test.
Results: All 15 vaccinated cattle were clinically protected in the A May 97 study, whereas 4 out of
15 vaccinated cattle in the SAT 2 experiment developed generalized infection, even though similar
levels of neutralizing antibody were detected in cattle from both experiments on the day of
challenge. However, IFN-γ production in the re stimulated whole blood samples was much stronger
in the A May 97 study than in the SAT 2 study both before and after challenge. A positive
correlation was found between IFN-γ response and protection against the clinical disease.
Therefore not only humoral antibody but also cell mediated immune responses might help to
predict clinical protection from FMDV in vaccinated cattle and might contribute to a method for
evaluation of vaccine efficacy without challenge.
Introduction:
Foot-and-mouth disease (FMD) affects cloven hoofed domestic and wildlife species, reducing
productivity and severely constraining trade in animals and their products. Vaccination using
inactivated and adjuvanted virions is an important method of FMD control. Measuring the efficacy
of FMD vaccines relies on either direct potency tests by challenge of vaccinated cattle or indirect
assessment based on the serological response induced by vaccination. However, serological tests
are not an absolutely reliable indicator of protection. Though immune defence against FMD has
been related to the antibody mediated compartment and humoral antibody responses have been
considered to be the most important factor in conferring protection against FMD (McCullough &
Sobrino, 2004), there are still instances where animals with medium or high neutralizing antibody
titre are not protected upon challenge with virus (McCullough et al., 1992). Similarly, animals with
low or no detectable virus neutralizing antibody on occasion do not succumb to disease (Sobrino et
al., 2001). However, there is limited knowledge on the role of cell mediated responses which might
play a role against protection. As B lymphocytes are responsible for humoral antibody production,
but are dependant on the interaction with the Th lymphocytes (McCullough & Sobrino, 2004), both
humoral as well as cell mediated immunity are considered very important for protection. Recently
we developed a whole blood IFN-γ assay and reported the existence of a correlation between IFN-γ
and FMDV persistence (Parida et al., 2006). Therefore, this present study looked at the correlation
of both IFN-γ and neutralizing antibody with vaccine induced protection.
Materials and Methods:
Two vaccine potency experiments (A May 97 and SAT2) were carried out according to European
Pharmacopoeia. 15 animals in each experiment, 5 in each group, were vaccinated with different
doses of vaccine and challenged by homologous virus on 21 days post vaccination. Two more
animals were included as unvaccinated controls for each experiment. Beside the 8 day monitoring
230
period post challenge, all the experimental animals were also monitored a further 22 days post
challenge to identify the carrier status.
Clotted blood samples for serology, heparinized blood for cell mediated immune responses, such as
whole blood IFN-γ re-stimulation assay, ELIspot, lymphocyte proliferation assay, FACS and T cell
depletion assay, and probang samples for virus isolation and real time PCR were collected at
different time points during pre- and post-challenge period until the end of the studies.
Results:
Clinical signs: Upon challenge with homologous virus, all the unvaccinated control animals in both
the experiments were infected and showed clinical lesions in all four feet and tongue as expected.
All vaccinates in A May 97 potency test were clinically protected and vaccine passed with a PD50
value ≥ 32, whereas three vaccinates from the 1/4th dose group (VL 87, VL88 and VL89) and one
from the 1/16th dose group (VL91) of the SAT2 potency experiment showed clinical lesions though
the vaccine passed with a PD50 value of 10.
Virus isolation and real time RT-PCR : Live virus as well as genome copy was recovered from the
oro-pharyngeal samples of all the four non-vaccinated control animals of both the experiments. Out
of these four, two from SAT 2 infected animals became carriers whereas virus could not be
recovered after 28 days post challenge in A-May 97 infected animals. Though all vaccinates were
clinically protected in the A May 97 experiment, live virus was isolated from two animals in the full
dose vaccine group, three animals in the 1/4 dose group and from all five animals in the 1/16 dose
group (Table 1). In contrast, live virus was isolated from all 15 vaccinates in the SAT 2 experiment
(Table 2). Out of a total of 10 sub-clinically infected animals detected in the A May 97 experiment
by virus isolation and PCR, one animal, each from the full dose and 1/4 dose group, and three
animals from the 1/16 dose group became carriers (Table 1). Similarly, out of all 15 SAT2
vaccinates two from each of the full dose and 1/4th dose group and four from the 1/16th dose group
were detected as carriers (Table 2).
Detection of neutralising antibody: Mean virus neutralization antibody titre for each vaccinated
group from both experiments was very similar (data not shown) before and after challenge. A
vaccine dose dependent VN titre was observed pre and post challenge in the SAT 2 experiment. In
the A May 97 experiment though a dose dependant response was seen in the pre-challenge period,
and after infection the 1/16 dose group animals revealed much higher VN titres than the other two
higher dose groups. However, in both experiments a minimum 10 times rise of VN antibody
response was observed in all groups after infection. The virus neutralizing antibody responses on
the day of challenge (21 days post vaccination) are shown in Figure 1 for both experiments. On the
day of challenge, mean VN titre of the different vaccine dose groups of A May 97 experiment were
compared with the corresponding groups of the Sat 2 experiment and no significant difference was
observed (P>0.593, 0.589 and 0.840 for full, 1/4th, and 1/16 dose group respectively). However,
when the mean titre VN value of different vaccine dose groups were compared within the
experiment, a high statistical significance was observed.
Whole blood IFN-γ re-stimulation assay: For the whole blood IFN-γ re-stimulation assay,
heparinized blood samples from all the cattle of both experiments were collected at various time
points and induced with inactivated vaccine antigen and positive/negative controls were also
included. The IFN-γ level was measured by ELISA with known standards and the results are
presented in Figure 2. Within seven days of vaccination a considerable amount of IFN-γ production
was observed (Fig. 2A) in all three vaccination groups in the A May 97 experiment whereas IFN-γ
production was not obvious in the SAT 2 experiment (Fig. 2B). Only in the full dose vaccinated
animals of the SAT 2 experiment was an IFN-γ response apparent on the 2nd week of vaccination
whereas the other two groups only produced an IFN-γ response on or after the day of challenge
(Fig. 2B). Despite individual animal variation, analysis of the group mean data did not show a
correlation between vaccine dose and IFN-γ production post-vaccination in the A May 97
experiment (Fig. 2A). In contrast, and throughout the SAT 2 experiment, a higher amount of IFN-γ
response was observed in the full dose vaccine group than the other two groups. Further, this
vaccine dose dependent IFN-γ response was not found when compared with the 1/4th and 1/16th
dose groups of the SAT 2 experiment, and more IFN-γ response was observed in 1/16th dose group
than the 1/4th dose group (Fig. 2B). However, a significant difference (P<0.05) in the IFN-γ
response was not observed in-between all the 3 vaccination groups of the SAT 2 experiment at any
of the time points for sample analysis was observed in the A May 97 trial. Unlike the A May 97
experiment, a rise of IFN-γ response was observed upon challenge, particularly on 1/4th and 1/16th
dose groups.
231
Throughout the post challenge period in both experiments was the IFN-γ level significantly higher
in the vaccinated animals compared to the control group animals. A high IFN-γ response was
detected after re-stimulation with PWM as a positive control in all the animals whereas stimulation
with PPDA as a negative control and non-stimulated blood, as a baseline control, induced no IFN-γ
response throughout the experiments (data not shown).
B
400
350
350
300
300
250
250
VNT titre
VNT titre
A
400
200
150
200
150
100
100
50
50
0
0
VJ
62
VJ VJ VJ VJ
63 64 65 66
full dose
VJ
67
VJ
68
VJ
69
VJ VJ
70 71
1/4 dose
VJ
72
VJ
73
VJ VJ
74 75
VJ VJ
76 77
1/16 dos e
VJ
78
VL VL VL VL VL VL VL VL VL VL VL VL VL VL VL VL VL
80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96
control
full dose
1/4 dose
1/16 dose
control
Figure1. Virus neutralizing antibody titre in the serum of A May 97 (A) and SAT2 (B) cattle on the
day of challenge. Full dose ( ), 1/4 dose ( ), 1/16 dose (□) and control ( ).
■
70
IFN-γγ (ng/ml)
B
80
full dose
1/4 dose
1/16 dose
control
60
60
50
50
40
30
full dose
1/4 dose
1/16 dose
control
40
30
20
20
10
10
0
▤
80
70
IFN-γγ (ng/ml)
A
▧
0
v
0dp
v
7dp
pc
pc
pv
dpc
14d
19d
10d
pv/0
21d
days post vaccination/challenge
p
31d
c
v
0dp
c
pv
pc
pc
pc
0dp
14d
10d
13d
19d
pv/
21d
days post vaccination/challenge
pc
31d
Figure2. Comparisons of group mean value of IFN-γ response in the whole blood re-stimulation
assay for A May 97 (A) and SAT2 (B) cattle. Full dose ( ), 1/4 dose ( ), 1/16 dose (□)
and control ( ).
▤
■
▧
ELISpot assay: For the ELISpot assay, PBMC were separated from all the animals in both
experiments at 3 time points (19th, 27th and 31st day post challenge) for A May 97 and 5 time
points (14th day post vaccination, 8th, 13th, 20th and 31st day post challenge) for the SAT 2
experiment. PBMC were re-stimulated with specific vaccine antigen in vitro and IFN-γ producing
cell spots were assessed by ELISpot. IFN-γ production was found to be vaccine dose dependant
(Fig. 3A) for the A May 97 experiment though there was no significant difference (P>0.05) between
the different vaccine groups and the non-vaccinated group (ANOVA). In contrast, this dose
dependant IFN-γ production was not observed in the SAT 2 experiment (Fig. 3B). However, a
significant difference (P<0.05) of IFN-γ producing cell spots were observed between the vaccinated
and non vaccinated groups apart from the 14th day post vaccination (ANOVA).
Since ELISpot is a well established assay to detect IFN-γ production, it was compared with the
whole blood IFN-γ assay in both experiments, particularly between different vaccine doses and the
control animals. The mean value of ELISpot and whole blood IFN-γ ELISA for the 3 time points in
the A May 97 (Fig. 4A) and the 5 time points in the SAT 2 trial (Fig. 4B) were calculated for each
vaccine dose group. These results from ELISpot assay (bar) were compared to IFN-γ ELISA results
(line) and found to be comparable (Fig. 4).
232
A
B
350
full dose
1/4 dose
1/16 dose
control
300
full dose
1/4 dose
1/16 dose
control
300
250
250
Spot number
Spot number
350
200
150
200
150
100
100
50
50
0
0
19dpc
27dpc
31dpc
14dpv
days post challenge
10dpc
13dpc
19dpc
31dpc
days post vaccination/challenge
Figure3 Comparison of mean value IFN-γ responses in ELISpot assay in different vaccine dose
groups following vaccination and subsequent challenge in A May 97 (A) and SAT2 (B)
animals. Full dose ( ), 1/4 dose ( ), 1/16 dose (□) and control ( ).
350
300
70
40
150
30
100
20
50
0
full dose
1/4 dose
1/16 dose
Group
control
80
ELISPOT
ELISA
70
60
250
Spot number
50
200
350
300
60
250
Spot number
▤
B
80
ELISPOT
ELISA
IFN-γ (ng/ml)
A
▧
50
200
40
150
30
100
10
50
0
0
IFN-γ (ng/ml)
■
20
10
0
full dose
1/4 dose
1/16 dose
control
Group
Figure4. Comparison of IFN-γ response in ELISpot assay (bar) and whole blood re-stimulation
assay (line) in A May 97 (A) and SAT2 (B) animal.
Lymphocyte proliferation responses: Lymphocyte proliferation assay was carried out only in full
dose group and non-vaccinated group in both the potency experiments after re-stimulating PBMC
with vaccine antigen and the results were plotted in Figure 5. On the 7th day post vaccination it was
possible to differentiate the proliferative responses between vaccinates and non-vaccinates of A
May 97 animals whereas it was only possible after 2nd week of vaccination in the case of the SAT 2
animals. However, a significant difference (P<0.05) in proliferative response between the
vaccinated and non-vaccinated groups was only observed after the 14th day post vaccination (2Sample T-test) in the case of the A May 97 experiment and at the 21st day post vaccination in case
of the SAT2 experiment respectively. Interestingly, as in the whole blood IFN-γ assay, a significant
difference (P<0.05) in the proliferative response was observed between the full dose groups of
both experiments starting from the first week post vaccination (2-sample t-test) apart from the
19th and 28th day post challenge.
233
400x103
Full dose; A May 97
Control; A May 97
Full dose; SAT2
Control; SAT2
200x103
3
H TdR Incorporation
300x103
100x103
0
v
0dp
v
7dp
pv
dpc
14d
pv/0
21d
pc
10d
pc
19d
pc
28d
days post vaccination/challenge
Figure5. Comparison of lymphocyte proliferation response in A May 97 and SAT2 full dose and
control group animals after re-stimulation with FMD vaccine antigen and incorporation of
3
H thymidine. Full dose; A May 97 ( ), control; A May 97 ( ), Full dose; SAT2 ( ) and
control; SAT2( )
▩
■
▤
■
Correlation of clinical protection with IFN-γ and VN antibody responses on the day of challenge :
On the day of challenge, all the A May 97 vaccinated animals showed a considerable high IFN-γ
response from the whole blood assay whereas only three of the full dose and one of the 1/16th
dose group of SAT2 animals conferred high IFN-γ response though significantly lower than A May
97 animals. All the 4 clinically unprotected animals in SAT2 experiment did not produce any, or
very little IFN-γ on the day of challenge. However, the level of humoral antibody was not
significantly different between the same vaccine dose treatment groups in the A May 97 and SAT 2
experiments. On the day of challenge, out of four infected animals (VL87, 88, 89 and 91), only
VL88 and 91 had low VN antibody titres, whereas the other two had high levels of VN antibody
titres in comparison to other protected animals in the same experiment.
To establish a correlation between clinical protection, IFN-γ and VN antibody responses on the day
of challenge, all 30 vaccinated animals from both experiments were re-grouped into clinically
unprotected, clinically protected, carrier and non-carrier, and their mean group value of IFN-γ and
virus neutralization antibody titre were also compared (Fig. 6). Out of a total of 4 clinically
unprotected animals, three became carriers whereas one did not. Out of 26 clinically protected
animals, 10 became carriers whereas 16 were non-carriers. Therefore a total of 13 carriers and 17
non carriers were confirmed. A significant difference (P=0.001) in the level of both IFN-γ and VN
antibody was observed between clinically unprotected and clinically protected groups (Fig. 6A).
The IFN-γ response was found to be significantly different (P=0.017) when compared between the
total carrier (n=13) and non-carrier (n=17) animals whereas for the VN antibody titre was not
significant (P=0.209) between these two groups. However, no significant difference was observed
for the IFN-γ response and VN titre when compared between carrier and non carrier animals in the
clinically protected and clinically unprotected animals separately (data not shown).
234
B
A
40
40
120
120
IFN-γ
VNT
IFN-γ
VNT
100
100
30
30
VNT titre
60
IFN-γ (ng/ml)
IFN-γ (ng/ml)
20
20
60
VNT titre
80
80
40
40
10
10
20
20
0
0
0
clinically unprotected
0
carrier
clinically protected
non-carrier
Figure6. Comparison of IFN-γ (bar) and VNT (line) responses in-between 30 vaccinated animals of
A May 97 and SAT2 on the day of challenge between clinically unprotected and clinically
protected animals (A) and carrier animals and non-carrier animals (B).
Identification of IFN-γ producing T cell: Various T cell populations were depleted from PBMC using
each T cell surface marker to identify which cells are responsible for the IFN-γ production. IFN-γ
production was measured before and after depletion of each T cell population. IFN-γ production
was drastically decreased in the CD4 depleted population compared to whole PBMC (Fig. 7A).
Similarly γδ T cell (WC1-) depletion reduced a considerable amount of IFN-γ production (Fig. 7B).
However, depletion of CD8 and NK cells (difference between CD3 and CD2) resulted in very little
IFN-γ reduction than observed with CD4 and γδ T cells (Fig. 7C and 7D). The purity of depleted
cells was checked by FACS analysis which showed more than 95% purity.
B
140
120
120
100
100
IFNg (ng/ml)
IFNg (ng/ml)
A
140
80
60
40
20
80
60
40
20
0
0
PBMC
CD4-
CD4/CD8-
CD4/CD8/CD2-
CD4/CD8/CD2/
WC1-
PBMC
WC1-
C
WC1/CD8/CD4-
PBMC
CD2-
D
140
140
120
120
100
IFNg (ng/ml)
IFNg (ng/ml)
WC1/CD8-
80
60
40
20
100
80
60
40
20
0
PBMC
CD8-
CD8/CD4-
CD8/CD4/CD2-
CD8/CD4/CD2/
WC1-
0
PBMC
CD3-
Figure7. IFN-γ production after re-stimulation of total PBMC and depleted PBMC.
Discussion:
The whole blood re-stimulation assay was developed for the diagnosis of tuberculosis as an
alternative to the tuberculin skin test (Wood and Jones, 2001). This assay measures IFN-γ
production by ELISA after overnight exposure of heparinized whole blood to either M. bovis specific
antigen or M. avium as a comparison. The whole blood IFN-γ assay is used as a measure of Thelper-1 activation of CD4+ and CD8+ T cells and can be readily used on a large number of
samples (Black et al., 2002). Parida et al, 2006 showed this assay with some modifications could
be used to differentiate between A Iran 96 vaccinated and vaccinated homologous challenged
animals. Further they reported the existence of a correlation between IFN-γ production and the
establishment of carrier status in cattle. These initial results prompted us to firstly, develop an easy
235
assay to correlate the cell-mediated immune response and protection and secondly, to develop an
assay which could be used as a diagnostic assay for differentiation of subsequent FMDV infection in
vaccinated animals.
In this study, blood samples were taken from two vaccine potency tests and analysed by the whole
blood IFN-γ assay to measure the cell mediated immune response in FMD vaccinated and infected
animals. The IFN-γ response was higher in vaccinated animals in comparison to non-vaccinated
control animals throughout the period of study which confirmed earlier findings (Parida et al.,
2006). The quantity of IFN-γ production was found to be further elevated after challenge. This IFNγ production may be triggered by vaccination and then boosted by infection. Therefore, this whole
blood assay may have potential as a supporting assay to the established serological assays in
confirming infection in a vaccinated herd. However, in the present A May 97 potency study, an IFNγ response was observed at a high level by 7 days post vaccination and remained constant
throughout the experiment, even after challenge, which makes it difficult in its use to differentiate
between vaccinated and vaccinated subsequently challenged animals. Similarly, in the SAT 2
experiment it was difficult to differentiate the vaccinated animals before and after challenge as
there was not much difference in IFN-γ response on the day of challenge and post challenge.
However, in all the animal experiments the non-vaccinated infected animals produced less IFN-γ,
which might be due to an immunosuppressive effect by the FMDV (Parida et al., 2006).
To evaluate the newly developed whole blood IFN-γ assay, the ELISA result was compared to the
well established ELISpot assay. The IFN-γ responses from both assays are very comparable in both
experiments. However, the whole blood IFN-γ assay is easier, less time consuming and more
feasible for large scale surveillance.
The lymphocyte proliferation assay was conducted on the full dose vaccine group and nonvaccinated controls in both potency experiments after re-stimulating the PBMC with FMD specific
vaccine antigen. As in the whole blood IFN-γ assay, a significant difference (P<0.05) in the
proliferative response was observed between the full dose groups in both experiments as from the
first week post vaccination.
On the day of challenge, there was no considerable difference in the neutralizing antibody titre of
the different groups from both the experiments. However, four out of 15 cattle were clinically
unprotected in the SAT 2 potency test whereas all the animals in A Malaysia 97 experiment were
clinically protected. Apart from the neutralizing antibody titre, the cell mediated immune responses
detected by many of the assays (whole blood IFN-γ assay, ELISpot assay and Lympho proliferation
assay) were considerably higher for the A May 97 vaccinated animals compared to the SAT 2
animals. This may indirectly be the reason for all the animals in A May 97 being clinically protected.
Though it is well established that more antigen pay load is required in vaccine formulations of other
serotypes than the A serotype (personal communication with Tim Doel, Merial, Pirbright), the SAT2
animals were nevertheless not protected fully. Another reason for the poorer protection results
may be the instability of the SAT 2 antigen following immunization in the target host.
Though humoral antibody response is very important factor in conferring protection against FMD
infection (McCullough & Sobrino, 2004), still animals with high or medium neutralizing antibody
titre were not clinically protected (McCullough et al., 1992). Our study supports the fact that
though similar level of VN titre was observed for both the experiments, four of the SAT 2 animals
were infected which showed none or very little IFN-γ response. Further this has been supported by
the fact that the clinically protected group of animals had high IFN-γ and VN antibody titre than the
clinically unprotected animals. Thus not only humoral antibody but also cell-mediated immune
response plays a key role in FMD vaccine induced protection. By measuring the mean value of
IFN-γ production on the day of challenge in the A May 97 and SAT 2 animals, a significant
difference was observed between carrier and non-carrier animals as was seen in A Iran 96
experiment (Parida et al, 2006) though such a difference was not observed for the VN titres.
However, when a comparison had been made between carrier and non-carrier animals separately
within the clinically protected and clinically unprotected, a significant difference in IFN-γ response
was not observed though always a high amount of IFN-γ level was seen in non carrier animals than
the carrier animals.
Different doses of vaccine (full dose, 1/4th and 1/16th dose) were used in both animal experiments
conducted. A correlation between the vaccine dose and clinical protection was demonstrated in this
study which corroborates well with the findings of Cox et al. (2005). Animals from 1/16th dose
group and from 1/4th dose group of SAT2 experiment were not protected from the development of
lesions. However, all the animals from the full dose group of SAT 2 study and all the 15 vaccinates
236
from A May 97 experiment were fully protected. Out of a total of 13 carriers observed in both SAT
2 and A May 97 experiments, three were from the full dose group, three from 1/4th dose group and
the remaining 7 from the 1owest dose (1/16th) group. Further, during the pre-challenge and post
challenge period a dose dependant IFN-γ response was observed in the SAT 2 animals where the
full dose group produced more IFN-γ than the other two groups. Some times 1/16th dose group
animals produced more and/or same amount of IFN-γ as the 1/16th dose group. This may be due
to the difficulty of measurement of accurate volume of 1/16th dose of vaccine which used to be
carried out according to the European Pharmacopoeia. A correlation between IFN-γ production post
vaccination and protection has also been reported by Parida et al. (2006). The present study
mainly concentrates on Th1 response (IFN-γ). However it might be useful to check the other FMD
specific cytokine profile for both Th1 and Th2 responses as suggested by previous researchers
(Barnard et al., 2005 and Eble et al., 2006).
Conclusions:
•
A positive correlation of IFN-γ and VN antibody responses with vaccine induced protection
has been observed.
•
The IFN-γ re-stimulation assay may be useful for vaccine potency evaluation.
•
The use of IFN-γ re-stimulation assay as a DIVA test for FMD may not be possible when
high potency vaccines are used.
Recommendations:
•
T cell stimulation assays such as the whole blood IFN-γ assay along with VNT are potential
candidates for vaccine evaluation and could reduce the need for in vivo challenge in the
future.
Acknowledgements:
We would like to acknowledge NVRQS, Korea and Defra, UK for the funding through PhD1065 and
SE1122 research grants respectively.
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tuberculosis. Tuberculosis (Edinb), 81, 147-55.
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Table1. Virus isolation and real time RT-PCR results for A May 97 potency test
full dose
1/4th dose
1/16th dose
control
VJ62
VJ63
VJ64
VJ65
VJ66
VJ67
VJ68
VJ69
VJ70
VJ71
VJ72
VJ73
VJ74
VJ75
VJ76
VJ77
VJ78
C/S
+
+
10dpc
*
+
+
+
*
+
+
12dpc
+
+
+*
*
+
+
+
+
19dpc
+
*
*
+
+*
+*
+*
+
+
28dpc
+
+*
*
+*
+*
+
+
+
31dpc
+
*
+
+*
+
-
C/S refers to clinical sign generalized animals, + indicates virus isolation positive, * indicates PCR
positive and – indicates virus isolation and PCR negative.
Table2. Virus isolation result for SAT2 potency test
full dose
1/4th dose
1/16th dose
control
VL80
VL81
VL82
VL83
VL84
VL85
VL86
VL87
VL88
VL89
VL90
VL91
VL92
VL93
VL94
VL95
VL96
C/S
+
+
+
+
+
+
8dpc
+
+
+
+
+
+
+
+
+
+
-
13dpc
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
16dpc
+
+
NS
+
+
+
+
+
+
+
+
+
20dpc
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
23dpc
+
+
+
+
+
+
+
+
+
+
+
27dpc
+
+
+
+
+
+
+
+
+
+
30dpc
+
+
+
+
+
+
+
+
+
34dpc
+
+
+
+
+
+
+
C/S refers to clinical sign generalized animals, NS refers to no sample, + indicates virus isolation
positive and – indicates virus isolation negative.
239