Académique Documents
Professionnel Documents
Culture Documents
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
∗ 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
from antibodies against FMDV type “O,” as shown The potency of FMD vaccine was tested in guinea
using the serum neutralization test (SNT) according pigs according to Terpestra (1974). The prepared alhy-
to Ferreira (1976), and an ELISA test according to dragel vaccine was diluted in four-fold dilutions, but
Hamblin et al. (1986). the double oil emulsion vaccines with two different
Unweaned baby mice were used for the isolation emulsifiers were calculated and the dose which gave
and titration of FMD virus by intraperitoneal route 1/4, 1/16, 1/64 concentration of the original dose was
(I/P) and in safety test of the prepared vaccines. used. Each group of five guinea pigs was inoculated
Guinea pigs were used for preparation of guinea with one dilution of the vaccine tested. Twenty-one
pig-adapted FMD virus for virus challenge in deter- days post inoculation, the animals were each chal-
mination of PD50 of the prepared vaccines. lenged by 10,000 MLD50 guinea pig-adapted virus.
Serum samples were collected from vaccinated All animals were checked daily for signs of gener-
sheep weekly for 4 weeks, then every 2 weeks till alization for 3–5 days. Guinea pig protective dose
the protective antibody titer declined. The sera were 50 (GPPD50 ) was calculated according to Reed and
stored at −20 ◦ C and inactivated at 56 ◦ C for 30 min Muench (1938).
before being used in the SNT and ELISA tests.
2.4. Experimental design
2.2. Virus and vaccines
Twenty-seven susceptible sheep free from FMDV
The viruses employed were FMD virus strain antibodies were divided into three groups of nine
O1 /3/93 EGYPT, and guinea pig adapted virus. sheep. Each group was vaccinated with one of the
Infectivity titration of FMD virus, type “O1 ” was three different vaccine types. Each groups was subdi-
determined in tissue cultures and in baby mice. vided into three subgroups each with three sheep. One
A locally prepared FMD vaccine containing FMD subgroup in each group was vaccinated and chal-
strain 01/3/93 Egypt was inactivated using binary lenged after 21 days with 104 MLD50 virulent FMDV
ethyleneimine (BEI) to prepare the BEI-inactivated strain O1 /3/93 Egypt. The second subgroup in each
FMD vaccine. The 30% alhydragel FMD vaccine was group was vaccinated and boostered after 4 weeks
prepared according to Roshdy (1992). Preparation post primary vaccination (WPPV), while the last sub-
of double oil emulsion (DOE) FMD vaccine (with group in each group was vaccinated and received a
Arlacel A as a classical emulsifier) and preparation of booster dose of vaccine after 4 months.
double oil emulsion (DOE) FMD vaccine (with Spane
80 as a new emulsifier) were performed according to 2.5. Serological tests
the method described by Barteling et al. (1990).
The SNT was performed using microtechnique as
described by Ferreira (1976). Pre- and post vacci-
2.3. Safety and potency testing of three vaccine
nation sheep sera were tested and the positive titer
preparations
is expressed as log10 tissue culture infective dose50
To ensure freedom from viable microorgan- (TCID50 ).
isms samples were cultured in thioglycolate broth, The enzyme linked immunosorbant assay (ELISA)
Sabaroud’s and nutrient agar, and also dextrose- was carried out according to McCullough and Butcher
containing media. If any viable microorganisms were (1985) then modified and adapted according to
detected, the vaccine was considered unsafe and not Hamblin et al. (1986) for quantification of FMD type
used in the field. specific antibodies.
The innocuity test of the prepared vaccine was
checked by intradermolingual inoculation of 1 ml in 3. Results
10 sites of the tongue of susceptible sheep. If no local
or general lesions appeared and there was no rise of Results of the estimation of potency of the three
temperature over 1 week, the vaccine was considered vaccines in guinea pigs, based on the GPPD50 , are
safe (Henderson, 1970). 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- sites of inoculation. Primary lesions at the site of in-
cine preparations in FMDV susceptible sheep, as mea- noculation were noted in two vaccinated sheep as well
sured by SNT and ELISA titer responses, are shown as both primary and secondary lesions in both control
in Table 2. The response of the sheep to challenge sheep. The temperature of vaccinated sheep remained
with FMD virus strain O1 /3/93 Egypt relative to the within the normal range (39–39.5 ◦ C) indicating the
development of oral lesions is shown in Table 2 and protective effect of vaccines, while the temperature of
the response relative to body temperature is shown in the control sheep was elevated in the first 4 days post
Table 3. The control animals showed clinical signs of challenge, and then returning to normal levels.
FMD virus infection after challenge at different sites The antibody responses of vaccinated sheep receiv-
of the tongue, including pyrexia (40–40.7 ◦ C), saliva- ing a second dose of vaccine after 1 month or after
tion, and the appearance of vesicles on the mucous 4 months following primary vaccination are shown in
membrane of the mouth and tongue, especially at the 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
challenged.
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
4. Discussion were 2.4, 2.36 and 2.5 log10 in the same sera. This in-
dicates that the vaccinated sheep had protective levels
Improper vaccination programs and lack of vaccines of antibodies against FMD. These results agreed with
together with the presence of carrier animals and poor Sharma and Datt (1972), who reported that the peak
disease control measures all contribute to difficulty antibody titer were reached from 14 to 21 days post
in endemic FMD control in Egypt. Regular vaccina- vaccination in bulls, sheep and goats vaccinated with
tion and quarantine measures are usually applied spe- FMD vaccine (alhydragel). The results also agreed
cially in endemic areas as an effective control measure with those obtained by Moussa et al. (1976) who
(Shahan, 1962). found that cattle vaccinated with FMD vaccine did
Sheep are present on almost all cattle farms and not respond to challenge and had neutralizing anti-
their role in maintaining and transmitting FMD have body titers ranging between 1.2 and 1.6 log10 at 21
been somewhat neglected. A potent vaccine was pre- days post vaccination.
pared for use, especially to protect both local and im- The data also revealed that there is good correlation
ported animals. Progress in FMD vaccine production between the potency values obtained by sheep inoc-
has been directed towards the selection of an adjuvant ulation and those obtained by SNT and ELISA test,
that can stimulate a high and long-lasting immunity. similar to the findings of Lorenz and Wittmann (1983)
Also, in order to adopt a good vaccination program to and Hamblin et al. (1986).
control FMD, it is essential to know the best time for The antibody response of sheep vaccinated with the
vaccination and revaccination that leads to an effective three vaccine preparations and receiveing a booster
immunity. dose one month post vaccination (Table 4) revealed
The results obtained with the GPPD50 (Table 1) that serum neutralising antibody titers started at the
showed that DOE vaccine with Spane 80 emulsifier 3rd, 4th and 3rd weeks post primary vaccination
resulted in the highest protection level compared to in DOE with Arlacel A, DOE with Spane 80 and
the other two vaccines, as the former gave 1.7 GPPD50 Aldyragel with titers of 1.2, 1.3 and 1.3 log10 , re-
Log10 , which is equal to 50.12 GPPD50 per dose while spectively. The mean of antibody titers maintained
the other two vaccines gave 1.39 GPPD50 log10 , (equal protective levels till the 24, 28 and 14th weeks post
to 24.55 GPPD50 per dose) and 1.09 GPPD50 log10 vaccination for the three vaccine preparations, re-
(equal to 12.30 GPPD50 per dose), respectively. The specitvely and then started to decline. The duration
results obtained agree with those of Black et al. (1985) of immunity for each vaccine after receiving the
who showed that when oil emulsion FMD vaccines 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) 185–192 191
Henderson, W.M., 1970. A comparison of different routes of Roshdy, H.O., 1992. Studies on inactivated foot and mouth
inoculation of cattle for detection of the virus of foot and mouth disease virus vaccine. M.V.Sc. Thesis (Microbiology), Faculty
disease. J. Hyg. Camb. 50, 182–194. of Veterinary Medicine, Cairo University.
Lorenz, J., Wittmann, G., 1983. The correlation between the Roshdy, H.O., 1996. Studies on inactivated oil foot and mouth
potency value obtained by challenge by SNT. Zbl. Vet. Med. disease vaccine. Ph.D. Thesis. (Microbiology), Faculty of
(Reihe B) 19, 45–54. Veterinary Medicine, Cairo University.
McCullough, K.C., Butcher, R., 1985. Monoclonal antibodies Saad, M., 1998. Preparation of foot and mouth disease vaccine
against foot and mouth disease virus 146/S and 125/S particles. with new oil adjuvant. M.V. Sc. Thesis (Virology), Faculty of
Arch. Virol. 74, 1–9. Veterinary Medicine, Cairo University.
Moussa, A.A.M., Ibrahim, M.H., Hussein, K., Stouraitis, P., 1976. Shahan, M.S., 1962. The virus of foot and mouth disease.
A preliminary study on antibody response of cattle after Agricultural Research Series, US Department of Agriculture,
experimental infection with foot and mouth disease virus. In: Greenport, NY, pp. 444–451.
Proceedings of the 13th Arab Veterinary Medicine Congress, Sharma, R.N., Datt, N.S., 1972. Studies on the effect of inactivated
November 1976, Cairo, pp. 121–131. gel, saponified gel and saponified vaccines against types A, O,
Moussa, A.A.M., Daoud, A., Huissen, K., Hassan, N.A., Fahmy, C and Asia1 of foot and mouth disease virus. Indian J. Anim.
F., Azab, A., El-Shehawy, L., 1984. Prevalence of foot and Sci. 42 (19), 686–692.
mouth disease in Egypt. Agric. Res. Rev. 62 (5B), 55–63. Terpestra, C., 1974. Comparison of laboratory techniques for the
Pereira, H.G., 1977. Subtyping of FMD virus. In: Proceedings evaluation of foot and mouth disease vaccines. Research Group
of the International Symposium on Foot-and-Mouth Disease of European Commission, FMD, Lelystad, The Netherland,
(II), Lyon, France, 5–8 October 1976. S. Karger AG, Basel, pp. 22–24.
Switzerland, pp. 167–174. Waston, J., 1978. The control of foot and mouth disease in Great
Reed, L.J., Muench, H., 1938. A simple method for estimating Britain. Vet. Rec. 102, 187–190.
fifty percent end point. Am. J. Hyg. 27, 493–497.