Journal of Biosciences and Medicines, 2013, 1, 22-25 JBM
http://dx.doi.org/10.4236/jbm.2013.13005 Published Online December 2013 (http://www.scirp.org/journal/jbm/)
The comparison of the efficacy of swine FMD vaccine
emulsified with oil adjuvant of ISA 201 VG or ISA 206 VG
Dong Li*, Chunxue Zhou, Daliang She, Pinghua Li, Pu Sun, Xingwen Bai, Yingli Chen, Baoxia Xie,
Zaixin Liu*
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of Ministry of Agriculture, OIE/Na-
tional Foot and Mouth Disease reference laboratory of China, Lanzhou Veterinary Research Institute, Chinese Academy of Agricul-
tural Sciences, Lanzhou, China
Received September 2013
ABSTRACT
The Seppic Company developed a new adjuvant
Montanide ISA 201 VG, the upgraded version of
Montanide ISA 206 VG, which keep the advantage
and added some chemical components on the basis of
ISA 206 to improve the cellular responses. The aim of
the study is to compare the efficacy of swine FMD
(foot-a nd-mouth) vaccine emulsified with oil adjuvant
of ISA 201 or ISA 206 respectively. The pigs were
vaccinated with FMD vaccine emulsified with inactive
FMD type O antigen and adjuvant ISA 201 or ISA
206 respectively, according to 2.0 ml (1/1 dose), 0.67
ml (1/3 dose), 0.22 ml (1/9 dose) to calculate their
PD50. The sera were collected from the vaccination of
the day 0, 3, 7, 14, 21, 28 and the ELISA FMD type O
antibody were detected. Furthermore, the PD50 were
calculated after the pigs were challenged with viru-
lent FMDV type O on 28 days post vaccination. The
ELISA antibody titers of 201vaccine were signifi-
cantly higher than that of 206 (except the third time).
The fifty percent of protection dose (PD50) of 201
vaccine (PD50 = 15.59) was higher than that of 206
vaccine (PD50 = 10.05). The above data showed that
the efficacy of the FMD vaccine emulsified with ISA
201 was better than which with ISA 206.
Keywords: Swine FMD Vaccine; Adjuvant; ISA 201;
ISA 206; Immune Efficacy
1. INTRODUCTION
Foot-and-mouth disease (FMD is an acute, febrile, and
contagious vesicular disease affecting cloven-hoofed ani-
mals. The causal agent, FMD virus (FMDV), is a mem-
ber of the genus Aphthovirus in the family Picornaviri-
dae and occurs as seven distinct serotypes throughout th e
world: A, O, C, Asia1 and South African Terri torie s (SAT)
1-3 [1-3]. Vaccination is the most important control and
eradication strategy for FMD, esp ecially the oil-adjuvant
vaccine in developing countries. Significant advances in
recent years have led to the introduction of ‘‘ready-to-
formulate” oil adjuvants. Montanide ISA 206, the miner-
al based oil, which readily forms water-in-oil-in-water
emulsion. Its efficacy in eliciting immune response was
studied in FMD vaccines against FMDV [4-7].
The advantage of oil adjuvant was attributed to depot
formation at the site of injection, a vehicle for transport
of the antigen throughout the lymphatic system and slow
antigen release with the stimulation of antibody produc-
ing cells. Moreover, being oil emulsion, Montanide ISA
206 had various advantages, like low viscosity, easy ad-
ministration, longer term protection, greater stability and
production of smaller nodules at the site of injec tion [5],
compared to other oil adjuvants, making as an ideal ad-
juvant candidate for FMD vaccines. It was also sug-
gested that Montanide ISA 206 could prevent the loss of
potency wa s due to the proteolysis o f VP1 or possibl y the
physical breakdown of the virus followed adsorption to
the aluminum hydroxide gel [8] and agree with the usage
of Montanide ISA 206 ready to formulate oil adjuvant
can be sued in all target species is ideal for emergency
vaccination [9]. The usage of oil adjuvant (Montanide
ISA 206) improve, enhance cell mediated immunity and
give higher level and long lasting immunity [10,11].
Other researchers [12,13] detected that DOE vaccine
containing Montanide ISA 206 is highly efficient, fluidy
with low viscosity which is easily dispersed from the
place of injection and gave high Ab titers and longest
duration of immunity than alhdyragel vaccine and Daoud
reported that the duration of immunity elicited by gel
FMD vaccine was short lived and antibody concentra-
tions rapidly fall after administration, while oils adju-
ventated FMD vaccines gave a longer duration of im-
munity and suggested that the oil adjuvenated vaccines
*Corresponding a uthors.

D. Li et al. / Journal of Biosciences and Medicines 1 (2013) 22-25
23
had potential as an alternative to the conventional alu-
minum hydroxide FMD vaccine [14]. Moreover Patil
reported that the oil adjuvant elicited a better immune
response at any time than did aluminum hydroxide gel
FMD quadrivalent vaccines in goats, and the response
developed quicker. Local tissue reactions such as granu-
lomas and cysts to oil-adjuvants have been not detected
[15].
Now the Seppic developed a new adjuvant Montanide
ISA 201, the upgraded version of Montanide ISA 206,
which keep the advantage and added some chemical
components on the basis of ISA 206 to improve the cel-
lular responses. The aim of the study is to compare the
efficacy and safety of swine FMD vaccine emulsified
with oil ad juvant of ISA 201 or ISA 2 0 6 respecti ve l y.
2. MATERIALS & METHODS
2.1. The Animals
Fifty 2-month-old pigs, sero-negative for FMDV type O
antibodies (titre 1:4 by LPB-ELISA) were randomly di-
vided into four groups. 45 pigs in group 1-3 and 15 pigs
in each group. The vaccine with ISA 206 was the first
group. The vaccine with ISA 201 was the second group.
The vaccine with PBS as the adjuvant was the third
group. Three small groups (Five pigs in it) were divided
in each group for three vaccine dose 2.0 ml (1/1 full
dose), 0.67 ml (1/3 full dose), 0. 22 ml (1/9 full dose) to
determine the PD50 (protective dose 50%). The left five
pigs were in the 4th group as the control. All the pigs
were bred in the P3 animal laboratory respectively.
2.2. Vaccine Preparation
The FMDV type O antigen was inactivated and safety
checked by conventional method, and emulsified with
ISA 201 or ISA 206 separately according to the prospec-
tus guide . The vacc ine was stored at 4˚C.
2.3. Vaccination, Sera Collection and Virus
Challenge
Three types of FMD vaccines were intramuscular ino-
culated at the ear-root-neck area of 45 pigs with 2 ml,
0.67 ml, 0.22 ml inactivated vaccine respectively, and
five pigs were bred without vaccination as negative con-
trol. Blood and sera samples were collected at 0, 3, 7, 14,
21, 28 dpv (days post vaccination) to assay the FMDV
type O antibody by L PB -ELISA. To demonstrate vaccin e
efficacy, all 50 pigs were challenged intramuscularly
with 1000 PID 50/2 ml of FMDV type O suckling mice
passaged strain at the ear-root-neck area after 28 dpv and
FMD symptoms were monitored for 10 days, and the
PD50 was calculated with Karber method.
3. THE RESULTS
Define abbreviations and acronyms the first time they are
used in the text, even after they have been defined in the
abstract. Abbreviations such as IEEE, SI, MKS, CGS, sc,
dc, and rms do not have to be defined. Do not use ab-
breviations in the title or heads unless they are unavoida-
ble.
3.1. The ELISA Antibody Titer of FMDV Type O
after Pigs Vaccinated
The ELISA antibody titer of 201-vaccine (1/1 dose) was
186.12 on 21st dpv, 201.82 on 28th dpv, much higher
than which of 206-titer (p < 0.05), because the titer of
206-vaccine (1/1 dose) was 69.76 on 21st dpv, 57.28 on
28th dpv. The ELISA antibody titer of 201-vaccine (1/3
dose) was 133.48 on 21st dpv, 120.62 on 28th dpv, much
higher than which of 206-titer r (p < 0.05), because the
titer of 206-vaccine (1/3 dose) was 69.3 on 21st dpv,
71.96 on 28th dpv. The ELISA antibody titer of 201-
vaccine (1/9 dose) was 65.54 on 21st dpv, 84.36 on 28th
dpv, much higher than which of 206-titer r (p < 0.05),
because the titer of 206-vaccine (1/9 dose) was 30.9 on
21st dpv, 29.56 on 28th dpv (Figure 1). These data
showed that the effect of 201-vaccine is better than 206-
vaccine.
3.2. The FMD V irule nt Virus Challenge to
Immunized Pigs on 28dpv
The challenge result was presented in Table 1. Fifty pig s
were all protected in 201-vaccine group (PD50-201 =
15.59). Thirteen pigs were protected in 206-vaccine
group (2 pigs had clinic symptom with 1/9 dose, PD50-
206 = 10.05). Four pigs were protected in PBS-vaccine
group (5 pigs had clinic symptom with 1/9 dose, 3 pigs
had clinic symptom with 1/3 dose, 3 pigs had clinic
symptom with 1/1 dose, PD50-PBS = 1.39). Generally,
the FMD vaccine is qualified when PD50 comes up to
6.0. The above data showed that the PD50 of 201 emul-
sified FMD vaccine was higher than which of 206 emul-
sified vaccine.
Figure 1. Comparisons of the ELISA antibody titers among
201-vaccine, 206-vaccine, PBS-vaccine.

D. Li et al. / Journal of Biosciences and Medicines 1 (2013) 22-25
24
Table 1. The FMD clinical incidence after challenge.
Groups Pig No & clinical incidence
206 (1/1) 3051 (−) 3056 (−) 3057 (−) 3076 (−) 3098 (−)
206 (1/3) 4441 (−) 3074 (−) 3047 (−) 3075 (−) 3077 (−)
206 (1/9) 3072 (−) 3060 (+) 3063 (−) 3065 (+) 3083 (−)
201 (1/1) 3058 (−) 3059 (−) 4477 (−) 3079 (−) 3086 (−)
201 (1/3) 3097 (−) 3073 (−) 3094 (−) 3082 (−) 3043 (−)
201 (1/9) 3064 (−) 3092 (−) 3081 (−) 3070 (−) 3069 (−)
PBS (1/1) 3055 (−) 3067 (−) 3093 (+) 3084 (+) 3089 (+)
PBS (1/3) 3054 (−) 3061 (+) 3095 (+) 3068 (−) 3088 (+)
PBS (1/9) 3044 (+) 3090 (+) 3087 (+) 3085 (+) 3096 (+)
Control 3099 (+) 1040 (+) 1439 (+) 3080 (+) 3071 (+)
Note: (+) means FMD clinic sighs appeared.
4. DISCUSS
The ISA206 adjuvant has applied in FMD vaccine pro-
duction and other vaccines for 20 years, because it has
lower side-reactions and viscosity, easier to emulsify and
injection, and higher stability. Recen tly, T he ISA 201 has
developed on the base of ISA 206, which inherited ad-
vantages of ISA 206, and added some chemical sub-
stances with the hope of enhanci ng animals’ cellular im-
mune reactions [16]. In this report, the comparisons be-
tween ISA 201 and ISA 206 were done as the FMD vac-
cine respectively.
First the ELISA antibodies against FMDV type O
were compared. The antibody titer induced by 201-vac-
cine were higher than which of 206-vaccine on 3dpv,
7dpv, 14dp v, 21 dpv , 2 8dpv. This means that the effect of
201-vaccine in inducing antibody is better than which of
206-vaccine.
The virus challenge trial to vaccinated animals is a
very important index for evaluating the potency of FMD
vaccine, which is indicated with PD50 (50 percent pro-
tection dose) [17,18]. Here, fifty pigs were all protected
immunized with 201 vaccine, but thirty pigs were pro-
tected with 206 vaccine, which means that the potency of
201 vacci ne is better than that of 206 va c c i ne.
5. CONCLUSION
From above, the efficacy of the FMD vaccine emulsified
with ISA 201 is su pe rior to ISA 206.
6. ACKNOWLEDGEMENTS
This work has been supported by “Seppic, France”.
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