Small Ruminant Research 45 (2002) 185–192

Immune response of sheep to foot and mouth disease

vaccines containing different adjuvants
W. Deghaidy∗ , A. Daoud, A. El-Molla
Veterinary Serum and Vaccine Research Institute, Abbasia, P.O. Box 131, Cairo, Egypt

Abstract
This study evaluated the immunogenicity and immune duration of the locally produced inactivated foot and mouth disease
(FMD) vaccine, currently used in Egypt as the main control of the disease. Three batches of FMD vaccines inactivated with
binary ethyleneimine were prepared: two double oil emulsion (DOE) lots with either Arlacel A or Spane 80 as emulsifier
and a 30% alhydragel vaccine. Evaluation and testing of FMD vaccine containing different adjuvants for safety, sterility, and
potency were carried out. Guinea pig protective dose 50 (GPPD50 ) was determined for each batch separately. The evaluation
of three different types of vaccines in sheep revealed that the DOE–FMD vaccine using the new emulsifier Spane 80 showed
much higher antibody titers and longer duration of immunity than the other two vaccine preparations.
© 2002 Published by Elsevier Science B.V.
Keywords: Sheep; Foot and mouth disease; Vaccine; Adjuvants; Immune response; Egypt

1. Introduction tion must also be applied to limit the spread of infec-

In Egypt, FMD is endemic, generally occurs annu-
ally, and infects susceptible animals (cattle, buffaloes,
sheep, goats and pigs) causing severe economic losses
in milk and meat production (Moussa et al., 1984). The
disease is caused by one of the picornaviruses (Franki
et al., 1991) which comprises seven types: O, A, C,
SAT1, SAT2, SAT3, and Asia1 (Bachrach, 1968).
There are about 65 subtypes within the seven types
(Pereira, 1977). Generally, there are no cross-reactions
between the various types, but a partial immunity
exists between the different subtypes within the same
types (Anon., 1978). The only type recorded in Egypt
in the last 30 years has been type O1 .
The first essential measure in the control of FMD
is a strong importation policy to prevent the introduc-
tion of animals from infected areas. Prompt vaccina-
tion around outbreaks (Waston, 1978). The disease has
occurred in Egypt in all susceptible livestock causing
drastic losses in milk and meat production with deaths,
especially among young animals (Daoud et al., 1988).
Essentially, there are three important factors for
the production of FMD vaccines. First, the viral anti-
gens should be propagated for large-scale production.
Second, the virus must be treated in such a way that no
residual infectivity remains. Third, a non-toxic adju-
vant should be added to enhance the immune response
to a satisfactory level. Current research in Egypt is
directed towards developing and evaluating adjuvants
that result in a high and long lasting immunity.
2. Material and methods
2.1. Animals and samples

∗ Corresponding author. Fax: +20-2-685-8321. Thirty-two sheep local breed (male and female)
E-mail address: svri@idsc.gov.eg (W. Deghaidy). about 35–50 kg body weight clinically healthy and free

0921-4488/02/$ – see front matter © 2002 Published by Elsevier Science B.V.

PII: S 0 9 2 1 - 4 4 8 8 ( 0 2 ) 0 0 0 9 9 - 8
186 W. Deghaidy et al. / Small Ruminant Research 45 (2002) 185–192
from antibodies against FMDV type “O,” as shown
using the serum neutralization test (SNT) according
to Ferreira (1976), and an ELISA test according to
Hamblin et al. (1986).
Unweaned baby mice were used for the isolation
and titration of FMD virus by intraperitoneal route
(I/P) and in safety test of the prepared vaccines.
Guinea pigs were used for preparation of guinea
pig-adapted FMD virus for virus challenge in deter-
mination of PD50 of the prepared vaccines.
Serum samples were collected from vaccinated
sheep weekly for 4 weeks, then every 2 weeks till
the protective antibody titer declined. The sera were
stored at −20 ◦ C and inactivated at 56 ◦ C for 30 min
before being used in the SNT and ELISA tests.
2.2. Virus and vaccines
The viruses employed were FMD virus strain
O1 /3/93 EGYPT, and guinea pig adapted virus.
Infectivity titration of FMD virus, type “O1 ” was
determined in tissue cultures and in baby mice.
A locally prepared FMD vaccine containing FMD
strain 01/3/93 Egypt was inactivated using binary
ethyleneimine (BEI) to prepare the BEI-inactivated
FMD vaccine. The 30% alhydragel FMD vaccine was
prepared according to Roshdy (1992). Preparation
of double oil emulsion (DOE) FMD vaccine (with
Arlacel A as a classical emulsifier) and preparation of
double oil emulsion (DOE) FMD vaccine (with Spane
80 as a new emulsifier) were performed according to
the method described by Barteling et al. (1990).
2.3. Safety and potency testing of three vaccine
preparations
To ensure freedom from viable microorgan-
isms samples were cultured in thioglycolate broth,
Sabaroud’s and nutrient agar, and also dextrose-
containing media. If any viable microorganisms were
detected, the vaccine was considered unsafe and not
used in the field.
The innocuity test of the prepared vaccine was
checked by intradermolingual inoculation of 1 ml in
10 sites of the tongue of susceptible sheep. If no local
or general lesions appeared and there was no rise of
temperature over 1 week, the vaccine was considered
safe (Henderson, 1970).
The potency of FMD vaccine was tested in guinea
pigs according to Terpestra (1974). The prepared alhy-
dragel vaccine was diluted in four-fold dilutions, but
the double oil emulsion vaccines with two different
emulsifiers were calculated and the dose which gave
1/4, 1/16, 1/64 concentration of the original dose was
used. Each group of five guinea pigs was inoculated
with one dilution of the vaccine tested. Twenty-one
days post inoculation, the animals were each chal-
lenged by 10,000 MLD50 guinea pig-adapted virus.
All animals were checked daily for signs of gener-
alization for 3–5 days. Guinea pig protective dose
50 (GPPD50 ) was calculated according to Reed and
Muench (1938).
2.4. Experimental design
Twenty-seven susceptible sheep free from FMDV
antibodies were divided into three groups of nine
sheep. Each group was vaccinated with one of the
three different vaccine types. Each groups was subdi-
vided into three subgroups each with three sheep. One
subgroup in each group was vaccinated and chal-
lenged after 21 days with 104 MLD50 virulent FMDV
strain O1 /3/93 Egypt. The second subgroup in each
group was vaccinated and boostered after 4 weeks
post primary vaccination (WPPV), while the last sub-
group in each group was vaccinated and received a
booster dose of vaccine after 4 months.
2.5. Serological tests
The SNT was performed using microtechnique as
described by Ferreira (1976). Pre- and post vacci-
nation sheep sera were tested and the positive titer
is expressed as log10 tissue culture infective dose50
(TCID50 ).
The enzyme linked immunosorbant assay (ELISA)
was carried out according to McCullough and Butcher
(1985) then modified and adapted according to
Hamblin et al. (1986) for quantification of FMD type
specific antibodies.
3. Results
Results of the estimation of potency of the three
vaccines in guinea pigs, based on the GPPD50 , are
shown in Table 1.
 W. Deghaidy et al. / Small Ruminant Research 45 (2002) 185–192 187

Table 1
Estimation of the potency of three different FMD vaccines in guinea pigs.
Types of vaccine Dilution of vaccinea Calculated GPPD50 Number of
in log10 b GPPD50
Undiluted 1/4 1/16 1/64 Control

1 DOE with Arlacel A 0/5c 0/5 2/5 4/5 5/5 1.39 24.55
2 DOE with Spane 80 0/5 0/5 1/5 3/5 5/5 1.70 50.12
3 Alhydragel vaccine 0/5 2/5 3/5 3/5 5/5 1.09 12.30
a Dilution of FMD vaccines inoculated and challenged 21 days post vaccination.
b Guinea pigs protective dose expressed in log10 .
c Numbers of guinea pigs showed generalization over total number of challenged animals.

Results of the potency testing of the three vac-
cine preparations in FMDV susceptible sheep, as mea-
sured by SNT and ELISA titer responses, are shown
in Table 2. The response of the sheep to challenge
with FMD virus strain O1 /3/93 Egypt relative to the
development of oral lesions is shown in Table 2 and
the response relative to body temperature is shown in
Table 3. The control animals showed clinical signs of
FMD virus infection after challenge at different sites
of the tongue, including pyrexia (40–40.7 ◦ C), saliva-
tion, and the appearance of vesicles on the mucous
membrane of the mouth and tongue, especially at the
sites of inoculation. Primary lesions at the site of in-
noculation were noted in two vaccinated sheep as well
as both primary and secondary lesions in both control
sheep. The temperature of vaccinated sheep remained
within the normal range (39–39.5 ◦ C) indicating the
protective effect of vaccines, while the temperature of
the control sheep was elevated in the first 4 days post
challenge, and then returning to normal levels.
The antibody responses of vaccinated sheep receiv-
ing a second dose of vaccine after 1 month or after
4 months following primary vaccination are shown in
Tables 4 and 5, respectively.

Table 2
Immune response of sheep vaccinated with two DOE vaccines and alhydragel vaccine and the results of challenge after 21 days with FMD
virus strain O1 /3/93 Egypt.
Type of vaccine Sheep Pre-vaccination Week post vaccination Results of challenge
number titer FMD lesions in mouth
SNT antibody titer ELISA-antibody titer

1 2 3 1 2 3 1a 2b

DOE + Arlacel A 1 0.3 0.6 0.6 1.2 1.00 1.5 2.3 − −

2 0.6 0.6 0.6 1.2 0.9 1.7 2.5 − −
3 0.6 0.6 0.9 1.2 1.1 1.8 2.3 + −
Average 0.5 0.6 0.7 1.2 1.00 1.66 2.4
DOE + Spane 80 4 0.6 0.6 0.9 1.2 0.9 2.1 2.4 − −
5 0.3 0.6 0.9 1.2 0.9 1.5 2.4 + −
6 0.6 0.6 1.2 1.5 1.2 2.1 2.3 − −
Average 0.5 0.6 1.00 1.3 1.00 1.9 2.36
Alhydragel 7 0.6 0.9 1.2 1.5 1.5 2.3 2.5 − −
8 0.6 1.2 1.5 1.8 1.7 2.1 2.6 − −
9 0.3 1.2 1.5 1.8 1.9 2.7 2.4 − −
Average 0.5 1.1 1.3 1.7 1.7 2.36 2.5
Control 1 0.3 0.3 0.3 0.3 0.3 0.3 0.9 + +
Animals 2 0.3 0.3 0.3 0.3 0.3 0.3 0.6 + +
a 1ry: primary lesions.
b 2ry: secondary lesions.
188 W. Deghaidy et al. / Small Ruminant Research 45 (2002) 185–192

Table 3 were tested by GPPD50 , results giving 15 PD50 per

Temperature reactions of vaccinated and non vaccinated control dose or more are considered good results while results
sheep to challenge with 104 LD50 FMD virus strain O1 /3/93 Egypt
giving less than 5 PD50 per dose are considered low.
Time Control Vaccinated sheep The PD50 of DOE vaccine ISA206 either with Arla-
(days) sheep mean cel A or with Spane 80 emulsifier vaccine gave higher
DPC temperature 1st groupa 2nd groupb 3rd groupc
values than the gel vaccine and these results agree with
1st 40.7 39.0 39.0 39.3 Bornett et al. (1996) who stated that the ISA206 oil
2nd 40.6 39.1 39.3 39.0
3rd 40.5 39.0 39.2 39.5
formulation has the greatest PD50 value, because it is
4th 40.0 39.2 39.4 39.1 a stable mineral oil and has low viscosity.
5th 39.3 39.0 39.2 39.2 The results of potency testing of the vaccines in
DPC: days post challenge.
sheep vaccinated and challenged with FMD virus
a 1st group: sheep vaccinated with double oil emulsion vaccine strain O1 /3/93 Egypt (Table 2) showed that there were
with Arlacel A, then challenged. no clinical signs except for primary lesions at the site
b 2nd group: sheep vaccinated with double oil emulsion vaccine
of innoculation in two vaccinated sheep. The mean
with Spane 80, then challenged. SNT titers after 21 days post vaccination were 1.2,
c 3rd group: sheep vaccinated with alhydragel vaccine, then

challenged.
4. Discussion
Improper vaccination programs and lack of vaccines
together with the presence of carrier animals and poor
disease control measures all contribute to difficulty
in endemic FMD control in Egypt. Regular vaccina-
tion and quarantine measures are usually applied spe-
cially in endemic areas as an effective control measure
(Shahan, 1962).
Sheep are present on almost all cattle farms and
their role in maintaining and transmitting FMD have
been somewhat neglected. A potent vaccine was pre-
pared for use, especially to protect both local and im-
ported animals. Progress in FMD vaccine production
has been directed towards the selection of an adjuvant
that can stimulate a high and long-lasting immunity.
Also, in order to adopt a good vaccination program to
control FMD, it is essential to know the best time for
vaccination and revaccination that leads to an effective
immunity.
The results obtained with the GPPD50 (Table 1)
showed that DOE vaccine with Spane 80 emulsifier
resulted in the highest protection level compared to
the other two vaccines, as the former gave 1.7 GPPD50
Log10 , which is equal to 50.12 GPPD50 per dose while
the other two vaccines gave 1.39 GPPD50 log10 , (equal
to 24.55 GPPD50 per dose) and 1.09 GPPD50 log10
(equal to 12.30 GPPD50 per dose), respectively. The
results obtained agree with those of Black et al. (1985)
who showed that when oil emulsion FMD vaccines
1.3 and 1.7 log10 in double oil emulsion (DOE) with
Arlacel A, DOE with Spane 80 and alhydragel vac-
cine, respectively. While the titers detected by ELISA
were 2.4, 2.36 and 2.5 log10 in the same sera. This in-
dicates that the vaccinated sheep had protective levels
of antibodies against FMD. These results agreed with
Sharma and Datt (1972), who reported that the peak
antibody titer were reached from 14 to 21 days post
vaccination in bulls, sheep and goats vaccinated with
FMD vaccine (alhydragel). The results also agreed
with those obtained by Moussa et al. (1976) who
found that cattle vaccinated with FMD vaccine did
not respond to challenge and had neutralizing anti-
body titers ranging between 1.2 and 1.6 log10 at 21
days post vaccination.
The data also revealed that there is good correlation
between the potency values obtained by sheep inoc-
ulation and those obtained by SNT and ELISA test,
similar to the findings of Lorenz and Wittmann (1983)
and Hamblin et al. (1986).
The antibody response of sheep vaccinated with the
three vaccine preparations and receiveing a booster
dose one month post vaccination (Table 4) revealed
that serum neutralising antibody titers started at the
3rd, 4th and 3rd weeks post primary vaccination
in DOE with Arlacel A, DOE with Spane 80 and
Aldyragel with titers of 1.2, 1.3 and 1.3 log10 , re-
spectively. The mean of antibody titers maintained
protective levels till the 24, 28 and 14th weeks post
vaccination for the three vaccine preparations, re-
specitvely and then started to decline. The duration
of immunity for each vaccine after receiving the
two doses (primary and booster dose after 1 month)
W. Deghaidy et al. / Small Ruminant Research 45 (2002) 185–192 189
190 W. Deghaidy et al. / Small Ruminant Research 45 (2002) 185–192
 W. Deghaidy et al. / Small Ruminant Research 45 (2002) 185–192
were 28, 32 and 18 weeks post primary vaccination
in DOE with Arlacel A, DOE with Spane 80, and
alhydragel, respectively. The higher antibody levels
following vaccination with oil emulsion vaccine con-
taining Spane 80 was attributed to lower viscosity and
highly homogenicity of a new emulsifier and these
findings agreed with Barteling and Vreeswijk (1991)
who found that the peak titer with oil vaccines was
not reached before 60–80 days post vaccination.
While both DOE vaccines gave better results than
alhydragel vaccine, the DOE with Spane 80 gave
the longest duration compared to the other two vac-
cines (Table 4). Our findings were in agreement
with Barteling and Vreeswijk (1991) who found
that oil vaccines gave greater and longer duration
than aluminium hydroxide vaccines. According to
Roshdy (1996), the effectiveness of an emulsified
vaccine could be influenced by its physical charac-
ter, so that the viscosity limitation of the oil vac-
cine is due to the antigen dispersion which attracts
lymphocytes.
The humoral immune response of sheep vaccinated
with two DOE vaccines and alhydragel vaccine receiv-
ing a booster dose after 4 months post primary vaccina-
tion showed that the mean serum neutralizing antibody
titers started at the 4th, 3rd and 3rd weeks post primary
vaccination in DOE with Arlacel A, DOE with Spane
80 and alhydragel vaccine with the titers of 1.2, 1.5 and
1.4 log10 , respectively. These titers maintained protec-
tive levels in case of DOE with Arlacel A, DOE with
Spane 80, 1.4 and 1.9 log10 until 16 weeks post pri-
mary vaccination, but declined to non-protective lev-
els in the case of alhydragel vaccine at 14 weeks post
primary vaccination. After revaccination the antibody
level began to increase again and reached the highest
titers 2.2, 2.8 and 1.7 log10 at 6, 10 and 8th weeks post
secondary vaccination. The duration of immunity for
each vaccine after receiving the two doses (primary
and booster) were 38, 40 and 34th weeks post vacci-
nation in DOE withArlacel A, DOE with Spane 80,
and alhydragel, respectively. The results of serum an-
tibody titers by ELISA showed a higher increase than
that obtained by SNT, these results being in agreement
with those obtained by Hamblin et al. (1986). Our
results also agreed with those obtained by Rouchdy
(1996), El-Garf (1997) and Saad (1998) with regard
to the immune response, duration and booster time for
using the oil and alhydragel vaccines.
191
Acknowledgements
We would like to express our appreciation to Prof.
Dr. Ahmed M. Daoud, director of Veterinary Serum
and Vaccine Research Institute, Abassia, Cairo, for
his encouragement, valuable assistance and his gener-
ous providence of essential requirements and facilities
to carry out this work. I am very grateful to Prof.
Dr. Amal A.A. El Molla, professor of Infectious
Diseases, Faculty of Veterinary Medicine, Cairo Uni-
versity, for her fruitful help and advice in bringing
this work to completion.
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