Preparation of Foot and Mouth Disease trivalent vaccine type A , O , SAT 2 with determination of the Guinea pig protective dose 50 ( GPPD )

Aim: To determine the minimal effective dose of Foot and Mouth disease (FMD) serotypes (A, O, SAT2) according to antigenic content (146S) in order to produce a potent trivalent FMD vaccine. Materials and Methods: Monovalent ISA 206 vaccines were prepared with 3 final concentration of 146S (1.6, 2.2, 2.8 μg/dose). The vaccine potency was evaluated by the determination of guinea pig protective dose 50 (GPPD )for each 50 concentration of 146S for each type of FMD monovalent vaccine where a fourfold dilution of the vaccines was constructed and each dilution was inoculated as 0.5 ml S/C in each of 5 guinea pigs. Results: The obtained results revealed that by using 1.6 μg of 146S for type O Pan Asia-2, A Iran O5 and SAT/EGY/2012, the GPPD was 40.4, 19.75 and 31.6 respectively, while the use of 2.2 μg of 146S resulted in GPPD 78.6, 78.6 and 105.8 for the 50 50 three types respectively, and by using 2.8 μg of 146S resulted in GPPD 161.7, 105.8 and 161.7 for the three subtypes (A, O, 50 SAT2) respectively. So it is clear that the lowest 146S dilution inducing good protection (more than 72 GPPD ) was 2.2 μg for 50 each serotype of used FMD monovalent vaccines. Depending on this result, the trivalent vaccine was formulated as 2.2 μg of 146S payload from each virus type/dose with equal volume of montanide ISA 206 oil as adjuvant. For more confirmation the prepared trivalent vaccine potency was evaluated by Guinea pig protective dose 50 which was found to be 88 GPPD .Also 50 mean SNT antibody titer was detected in serum of the test Guinea pigs 1.56, 1.68 and 1.68 log /ml against FMDV serotype O 10 Pan Asia-2, A Iran O5 and SAT/EGY/2012 respectively in a higher level than the recommended protective titer (PT=1.2). Also for further confirmation the formulated trivalent vaccine which contain 2.2 μg/serotype/dose were evaluated in cattle to measure the antibody titer against the three serotypes and the antibody against the three serotypes were found to be higher than the recommended titer (1.5) which extended for 32 WPV and these results came in parallel manner to the GPPD and antibody 50 titer of the Guinea pigs of the prepared trivalent vaccine with 146S (2.2 μg/serotype/dose). Conclusion: It could be concluded that the minimum content of antigenic 146S of FMDV serotype O pan Asia, A Iran O5 and SAT2/EGY/2012 should not be less than 2.2 μg/dose/ from each serotype in the trivalent vaccine aiming to induce the permissible protection in vaccinated livestock.

Introduction [4].There are seven antigenically distinct serotypes of FMDV (A, O, C, South African Territories (SAT) types Foot and mouth disease (FMD) is an economically 1-3 and Asia-1) and each serotype has many subtype devastating disease of livestock.Although vaccines, variants.This antigenic variation creates a major available since the early 1900s, have been instrumental problem for the control of FMD, as infection or in eradicating FMD from parts of the world, the disease vaccination with one serotype of FMDV does not still affects millions of animals around the globe and protect against other serotypes and may fail to protect remains the main sanitary barrier to the commerce of fully against other subtypes within the same serotype animals and animal products [1].
[5].Although mortality is usually low, morbidity can Foot and mouth disease harvest contain three reach 100% causing severe losses in production.virus specific particles: (i) the infective 146S virus Therefore, the disease remains a major economic particle, comprising one molecule of ss RNA and 60 concern for livestock-health in many developing countries copies of each of four polypeptides VP1,VP2,VP3 and and a continued threat to disease free countries [2,3].
VP4; ( ii) the empty 75S particles, devoid of RNA and The causative agent of FMD is a small positive comprising 60 copies of each of VP1,VP3 and VP0 sense ssRNA virus (approx.8.3 kb) which belongs to (precursor of VP2 and VP4); (iii) the 12S particle the Aphthovirus genus of the family Picornaviridae consisting of VP1,VP2 and VP3 but devoid of VP4 [6].
In Egypt, the disease is enzootic and outbreaks have been reported since 1950.FMD serotypes 'SAT2 ', 2000, respectively [7-10].Virus type 'O' was which is evaluated as GPPD , determination of 50 incriminated in the last two epidemic outbreaks, which antibodies in G. Pigs and cattle, so as to be safe and occurred in 1987 and 1993.potent.In addition, determination of the optimal 146S Vaccination is the only approach to control FMD.content aids to avoid the challenge of vaccinated The immunogenicity of foot and mouth disease (FMD) animals during the vaccine evaluation test which may vaccines depends to a large extent on the production of result in infection of animals with the virulent viral whole virions (146S particles), in tissue culture and the strains.stability of these particles after virus inactivation

Materials and Methods
procedures and formulation into vaccines.[11].
Experimental guinea pigs: Healthy adult male albino The quantity of 146S particles in inactivated guinea pigs approximately of 400-500 gm body weight FMD virus vaccine samples produced in FMD Vaccine were used for preparation of guinea pig adapted FMD Production Center is generally estimated by the virus to be used for Guinea pig challenge and for sucrose gradient ultracentrifugation and optical determination of GPPD of the prepared vaccines.density analysis by using the computer applying 50 system [12].
Ethical approval: The experiment was as per the The use of 146S assay only or some serological protocol of Institutional Animal Ethics Committee, the tests which together increase the reliance on estimating authors had taken permission of animal owners of potency of specific vaccine batch.They can also be private farm.assembled with an interconnected method which is based on the 146S concentration of the final vaccine O5 and SAT2 / EGY/2012 intradermoplanter in the However, up to now, none of the suggested method has metatarsal pads.After 24-48 hours the developed been found valid [13].
lesions were collected aseptically.The lesions extract Guinea pigs were chosen as experimental models was again re-inoculated in other Guinea pigs 5 times to develop concepts and techniques to study the until the virus became quite adapted to Guinea pigs as protective dose 50 (PD ) of FMD vaccines because of 50 recommended earlier [22].the similarities of clinical symptoms in these animals to Virus purification: Aseptically, the harvested culture those of swine and cattle to saving cost [15,16].Guinea medium from FMD virus infected BHK cell cultures pigs are susceptible animals to FMD and can be 21 protected by aqueous FMD vaccines.The methods of were centrifuged in a cooling centrifuge at 7000 rpm demonstrating the potency of such vaccines using for 20 minutes to remove cell debris [23].Guinea pigs have been described having a good

Virus inactivation
correlation with the protection afforded to cattle [17].
th Each FMD virus (O, A and SAT2) of the 7 So the present work was designed to determine passage on BHK monolayer with an infectivity titer of the minimal ideal dose of each of three monovalent 8 10 TCID /dose was treated with 1.5% chloroform 50 FMD vaccines depending on its antigenic content then the inactivation occur by using combination of (146S) and detection of vaccine potency which is BEI 1mM and 0.04% FA (BEI-FA) according to the expressed as 50% Guinea pig protective doses method described previously [24,25].2% of each of (GPPD ) aiming to produce a potent trivalent vaccine sodium thiosulphate (20%) and sodium bisulphite 4-5 days of the challenge test for the development of (20%) were added after inactivation process to primary and secondary lesions.If the virus generalizes neutralize the excess of BEI and formalin.
in the guinea pig's body, the vesicles on the uninoculated feet and the tongue are observed for a

Concentration of the FMD virus serotypes
positive reaction to the FMDV infection in guinea pigs.The tissue culture viral fluids of the three The observations were recorded and the Protection serotypes (O pan Asia-2, A Iran O5 and SAT2/ EGY/ Dose50 (PD ) was calculated in all groups and the 50 2012) were centrifuged at 7000 revolution/ minute for control groups.30 minutes and then concentrated by PEG-6000 to Preparation of trivalent FMD vaccine reach to 1/10 of its original volume.
The trivalent FMD vaccine (containing serotypes and weekly for 4 weeks post vaccination.Serum Vaccine formulation was done according to the neutralization test was performed on the serum method described earlier [28, 29] as follows: The oil samples as described earlier [31] and the antibody titer phase consisted of Montanide ISA 206, mixed as equal was expressed as log .
10 parts of an aqueous and oil phase weight/weight, and Field application to the prepared trivalent FMD oil mixed thoroughly.

vaccine in cattle farm
Each of FMD monovalent vaccines was prepared In Fayoum governorate, cattle farm was vaccinated with three concentrations of 146S finally adjusted to be with the experimental trivalent FMD oil vaccine batch 1.6µg, 2.2 µg, and 2.8µg viral particles/dose/serotype.
to determine the efficacy and the duration of immune Trivalent FMD oil vaccine was formulated from response which induced with the prepared vaccine minimum concentration of 146S from each serotype according to its 146S concentration.Serum neutralization which gave a high GPPD .The 4 group contained 5 Guinea pigs were kept was planned as a preliminary work to establish such as non vaccinated control group.
vaccine through the determination of the minimal 146S After 3 weeks, all Guinea pigs in all groups were content of each used serotype inducing the highest challenged with its specific Guinea pig adapted FMD GPPD .virus; by inoculation intra-dermoplanter.The 50 challenged Guinea pigs were observed for 7 days till The concentration of antigen per dose indirectly generalization of infection.
determines the duration of immunity.Further, the The GPPD was calculated according to previous quantity of antibody induced and particularly, initial  [14,36], where it was mentioned that infectivity FMD vaccine virus where in 2.2 µg of the antigen was of FMD virus samples was determined by cell culture required to obtain 1 PD50 although, protection was methods.All of the virus samples were inoculated on observed even with lower antigen dose.On the other BHK cells; also, their titration was obtained by 21 side, these results disagree with another report [38] TCID methods.The antigenicity of the virus samples 50 who mentioned that many trials have been conducted were studied by the Complement Fixation Test (CFT).
to correlate the quantity of 146S virus particles per vaccine dose and the protection and immunity respectively.Such titers appear to be higher than the achieved.The minimum effective dose of purified recommended protective titer (1.2).These results FMDV A-119 required for eliciting virus-neutralizing indicate that the prepared trivalent FMD vaccine able immune response in guinea pigs was about 1.6 µg.This to induce acceptable immune level in vaccinated disagreement could be attributed to the different Guinea pigs where the obtained antibody titers against subtype of FMDV type A used in this experiment.
the three types were found to be higher than the Also as in Table-5, the mean neutralizing recommended titer (PT=1.2) coming in a parallel antibody titer of the Guinea pigs vaccinated with FMD manner to that of GPPD .These finding coming in 50 vaccine with 1.6, 2.2, 2.8 µg indicated that the agreement with those of earlier report [36] who found neutralizing antibody was protected using 146S 2.2 that after the vaccines were inoculated subcutaneously and 2.8µg/ml with all serotype while the SNT was 0.9, on the back foot in two groups of guinea pigs, just two 0.7 and 0.7 using 1.6 µg/ml of 146S for (O Pan Asia-2, of ten animals showed antiserum titration above the A Iran O5 and SAT/EGY/2012) respectively but it was protective titer (PT=1.2),and the other 8 animals were 1.6, 1.55, 1.7 using 2.2 µg/ml of 146S for (O Pan Asiabelow the PT. 2, A Iran O5 and SAT/EGY/2012) respectively also it Finally we used the Guinea pigs as model for was 1.84, 1.75, 1.86 using 2.8 µg/ml of 146S for (O Pan vaccine evaluation instead of cattle to lower the cost Asia-2, A Iran O5 and SAT/EGY/2012) respectively.
during the vaccine evaluation, so to confirm these These findings are in agreement with previous report results we made field application to the experimental [36] that showed that the protective titer of SNT in prepared vaccine to confirm that the amount of 146S Guinea pigs is 1.2.
used is sufficient to protect cattle from FMDV, as The above mentioned results indicate that the shown in Table-8, the serum neutralizing antibody titer lowest concentration of 146S which resulted in good against the three serotypes (A,O,SAT) in cattle for 36 protection; through determination of GPPD (more week post vaccination (WPV).The results were 50 than 72 as mentioned earlier [39]); was 2.2 µg for each confirmatory to the previous results as the SN antibody serotype (O Pan Asia-2, A Iran O5 and SAT/ EGY/ titer against serotype (O) started as 0.3 at 0WPV and 2012).
reached its maximum antibody level at 10WPV (3.15)The new formulated prepared vaccine as 2.2 µg of and extended as a protective SN antibody titer from 2 to 146S payload from each virus type with equal volume 32 WPV while the antibody titer against serotype (A) from the oil was diluted (undiluted, ¼, 1/16, 1/64, started as 0.3 at 0 WPV and reached its maximum titer 1/256) and each dilution was inoculated in each of 5 at 8,10 and 12 WPV, the protective (1.5) extended from Guinea pigs.The obtained GPPD was found to be 88 2 to 34WPV.The SN antibody titer against FMDV 50 as shown in Table-6.Such obtained GPPD50 appeared serotype (SAT) was zero at 0 WPV and reached its to be higher than the recommended one, the thing that maximum antibody titer (3.15) at 10 WPV, the confirms the high potency of the prepared vaccine.
protective antibody titer (1.5) extended from 2 to 32 Testing of Guinea pigs serum samples, to confirm WPV.From these results, the antibody titer against the the results of GPPD using SNT, showed antibody three serotypes were found to be higher than the 50 recommended titer (1.5) which extended for 32 WPV titers of 1.56, 1.68 and 1.68 log /ml against FMDV 50test was performed on the serum samples as described Potency test of prepared vaccines earlier[31] and the antibody titer was expressed as log .It was carried out through determination of the 10 Guinea pigs protection dose 50 (GPPD ) according to 50Results and Discussionearlier reports[17,30] where Guinea pigs were divided FMD is still represent a non neglectable problem into 10 groups as following: st in livestock in many countries resulted in huge * Each of the 1 three groups was vaccinated with economic loses especially developing countries.FMD (O pan Asia-2) with either concentration of Regarding Egypt several outbreaks attack the country 146S, using four fold dilution from such vaccine due to the infection with either type O, A, SAT2 [7].(undiluted, 1/4, 1/16, 1/64, 1/256).Each dilution Through the last years monovalent FMD vaccine type was inoculated as 0.5ml S/C in each of 5 Guinea O was used for several years where it was the only pigs.nd recorded type in Egypt[32], after that and according to * Each of the 2 three groups was vaccinated with the introduction of type A, bivalent vaccine was FMD (A Iran O5) in the same manner as FMD (O successfully prepared containing both type O and A [8, pan Asia-2).rd 28, 29, 30,33].More recently FMDV type SAT2 was * Each of the 3 three groups was vaccinated with recorded in Egypt [10, 34] and this required the FMD (SAT2/EGY/2012) in the same manner as preparation of a trivalent vaccine containing the three FMD (O pan Asia-2).thpresent serotypes (A, O, SAT2).So, the present study * 10 and these results came in parallel manner to the GPPD serotype O Pan Asia-2, A Iran O5 and SAT/EGY/2012 50

There are many publications Guinea pig adapted virus: Albino Guinea pigs
Tissue culture: Baby hamster kidney cell cultures batch [13].(BHK ) were obtained from the World Reference Lab.21 It is well known that vaccination is the basic step Pirbright Surrey, U.K. The cells were serially passaged and corner stone in controlling FMD as other viral and maintained with Minimum Essential Medium infectious diseases.The used vaccine in Egypt was the (MEM) modified with Hank's salt with 1-2 % bovine cell culture inactivated bivalent vaccine prepared from serum in the FMD Research Department, Veterinary the local strain O /3/1993 and type A/1/EGY/ 2006).

Table - 1
. FMD virus titer, CF value, total protein and 146S concentration

Table - 3
GPPD of FMD virus type A Iran O5 with different 146S content vaccine.

Table - 5
. GPPD of different 146S concentrations of FMD virus serotypes and mean serum neutralizing antibody after 28 day

Table - 7
. FMD serum neutralizing antibody titters in Guinea pigs vaccinated with trivalent FMD inactivated ISA 206 oil vaccine Weeks post vaccination; 1-5 vaccinated with trivalent FMD oil vaccine and challenged with FMDV type A after 4WPV; 6-10 vaccinated with trivalent FMD oil vaccine and challenged with FMDV type O after 4WPV; 11-15 vaccinated with trivalent FMD oil vaccine and challenged with FMDV type SAT2 after 4WPV; 16-18 control positive inoculated with FMDV type A; 19-21 control positive inoculated with FMDV type O 22-24 control positive inoculated with FMDV type SAT2; 25-27 control negative non vaccinated non infected Guinea Pigs * WPV:

Table - 8
. Mean FMD serum neutralizing antibody titters in cattle vaccinated with trivalent FMD inactivated ISA 206 oil vaccine Antibody titer expressed in log ; Protective SN antibody titer is 1.5 [14]