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Journal of Veterinary Advances

Optimization of the Inactivation Process of FMD Virus Serotype SAT-2 by Binary Ethyleneimine (BEI) Ismail A. H., El-Mahdy S.A., Mossad W. G., Abd El-Krim A. S., Abou El-Yazid M and Ali S. M.

J Vet Adv 2013, 3(3): 117-124 DOI: 10.5455/jva.130322070526

Online version is available on: www.grjournals.com

ISSN: 2251-7685

ISMAIL ET AL.

Original Article

Optimization of the Inactivation Process of FMD Virus Serotype SAT-2 by Binary Ethyleneimine (BEI) 

Ismail A. H., El-Mahdy S. A., Mossad W. G., Abd El-Krim A. S., Abou El-Yazid M. and Ali S. M. Department of Foot and Mouth Disease, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo, Egypt.

Abstract Complete inactivation of FMDV new isolate type SAT2 Egypt 2012 at 37ºC using BEI with morality 0.1, 0.4, 0.8, 1.2 and 1.6 mM was obtained at 16,14, 10, 8 and 8 hours respectively, with inactivation rate ranged from 0.53 to 1.15 log10/hour. At 25º C the complete virus inactivation was occurred at 52, 44, 40, 36 and 36 hours post inactivation using a concentration of 0.1, 0.4, 0.8, 1.2 and 1.6 mM respectively with inactivation rate ranged between 0.16-0.24 log10/hour. Also the result of complement fixation test of FMDV type SAT2 using different BEI morality at 37ºC, 25ºC showed that Antigen complement fixation titer were changed before and after inactivation from 1/32 to 1/16 in 0.1, 0.4 and 0.8 Mm while from 1/32 to 1/8 in 1.2 and 1.6 mM. There was no significant difference between the use of BEI in concentration of 0.1 and 0.4 mM, and between 1.2mM and 1.6 mM, while the inactivation time decreased by approximately 8 h in the concentration of 1.2 rather than 0.1 mM, this may be very useful to finish the process of inactivation during working day or just over night. Evaluation of the immune response in Guinea pig of the vaccine prepared from different inactivated virus using different molarities of BEI and different incubating temperature was carried out and showed that the best antibody titer appeared after 28 days post vaccination using inactivated virus with molarity 0.1M BEI at incubation temperature, 37ºC it reached 1.8 and 2 log10 using Serum Neutralization Test (SNT) and Indirect ELISA respectively. On the other side there was a significant reduction in the post vaccinal antibody titer appeared using inactivated virus with 0.4, 0.8, 1.2 and1.6 mM BEI while at 25ºC it was 1.8 and 2.1 log10 in 0.1mM using SNT & ELISA respectively. It was clear that the optimal condition used for inactivation of FMDV SAT2 is by using BEI with a molarity of 0.1mM and incubational temperature 37ºC for 16 hours as this provide a good and safe antigenic content and reduce the inactivation time.

Keywords: FMDv, inactivation, vaccine, BHK



Corresponding author: Department of Foot and Mouth Disease, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo, Egypt. Received on: 15 Mar 2013 Revised on: 23 Mar 2013 Accepted on: 25 Mar 2013 Online Published on: 31 Mar 2013

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OPTIMIZATION OF THE INACTIVATION PROCESS OF FMD …

Introduction Foot-and-mouth disease (FMD) is a highly contagious and devastating disease affecting livestock that causes significant financial losses. Therefore, safer and more effective vaccines are required against Foot-and-mouth disease virus (FMDV) (Liu et al., 2011). Foot-and-mouth disease (FMD) is perhaps the most contagious animal disease known and therefore one of the most important transboundary animal diseases. Economic losses caused by FMD include reduction in production parameters such as decreases in milk production, weight gain, reproductive inefficiencies and death in young ruminants FAO (2012) Foot-and-mouth (FMD) vaccines used around the world are inactivated vaccines for prophylactic or emergency use, generally manufactured by the same basic methodology outlined in the OIE manual (Declarq et al., 2008). Virus inactivation and safety tests are the most critical steps in the production of inactivated vaccines. For FMD vaccines in particular, guaranteed safety is essential because any occurrence of the disease will have great economic consequences e.g. by a blockade of all export trade of animals and animal products (Barteling and Cassim, 2004) The first widely used FMD Vaccine was in the form of virus inactivated with formalin, which is still was resided in use in several vaccine producing companies. A residual remained or the preparation lost immunizing potency by using formalin inactivation (Waldman et al., 1937). It has been recognized early that formalinsed vaccines, although innocuous, might contain effective virus Moosbrugger (1948). Also formalin does not produce a first order kinetic reaction when inactivating the virus (Wesslon and Dinter, 1957). Also the using of formalin alone as inactivator may alter the structure of the virion Brown (1968). For all these reasons, on the less confidence in the formaldehyde treatment forced many production laboratories changed to aziridine group inactivators like Acetylethyleneiemine (AEI) as inactivaator (Brown and Newmann, 1963a) and (Brown et al., 1963b). More recently Bahnemann (1975) used 2118

bromoethyleneamine (BEA) in alkaline solution to reduce the toxicity of ethyeneimine (EI), which is called binary ethyleneimine (BEI), it was used in FMD vaccine preparation, (Bahnemann et al., 1974). Inactivation with aziridine became the method of choice. Binary ethylenimine (BEI) is used in particular, because this method-developed by (Bahnemann 1975 &1990) circumvents the direct handling of the very toxic other aziridine. The aim of the present study is directed to determine the optimal inactivation time of FMD virus Type (SAT-2) recently isolated in 2012 outbreak in Egypt using BEI as inactivator. Materials and Methods FMD Virus (SAT-2) The virus strain used in this study was SAT-2/ Egypt /2012 isolated from EL-Garbia Governorate. The virus was isolated, identified and typed by the Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo, Egypt and was confirmed by “The Animal Research Institute, Pirbright, UK”. It was kept at -70 °C until used (seed virus). The virus was propagated in roller bottles as described by Panina (1976). Tissue cultures Primary Bovine Kidney Cell Cultures (BK) It was prepared according to the method described by Patty (1965) it was used for first passage during virus isolation. Baby Hamster Kidney cell line (BHK21) It was obtained from the World Reference Lab. Pirbright Surrey, U.K. The cells were serially passage maintained in the FMD Department at the Veterinary Serum and Vaccine Research Institute (VSVRI), Cairo, As an approximation, the main culture is seeded to give an initial density of 0.5-2.0 * 106 cells/ ml, which is allowed to multiply to 2-5 * 106 cells/ml before being infected with the virus according to OIE Terrestrial Manual (2009). Chemicals 2-Bromoethylamine hydrobromide (BEA) 95% (molecular weight = 204.9) J. Vet. Adv., 2013, 3(3):117-124

ISMAIL ET AL.

It was obtained from Aldrich Chemical Company Limited Gillinham. Dorest, U.K.

weight were used for testing of FMD bivalent oil vaccines.

Sodium Hydroxide (Analar) Na OH (Molecular weight =40) It was obtained from PRATAP chemical industries PVT. LTD (INDIA) and used in a concentration of 0.2 Normal as dissolving agent for (BEA) in inactivation process according to Bahnemann (1975).

Preparation of inactivated FMD vaccines The vaccine formulation was carried out according to the method described by Gamil (2010) where the oil phase consisted of Montanide ISA 206 mixed with the inactivated viruses as equal parts of an aqueous and oil phase (weight/ weight) and mixed thoroughly. The vaccine was prepared on the base that each dose (2 ml) of vaccine contains not less 108 TCID50/ dose of each virus type.

Sodium Thiosulphate (Na2S2O3. 5H2O) The chemical was obtained from Merk Company, Germany. It was prepared as 20% solution in double distilled water, sterilized by autoclaving. It was used in final concentration of 2% to neutralize the excess of BEA after the inactivation process; its molecular weight was 248.18 as described by (Girared et al., 1977). Titeration of FMDV type (SAT-2) It was carried out in BHK21 tissue culture and the infectivity titer was calculated according to Karber (1931). Complement Fixation Test (CFT) It was carried out according to (Traub and Manso 1944) and Health protection Agency (2009) to estimate the antigenecity of the virus. Inactivation of FMD virus (Type SAT-2) by using BEI BEI can be easily prepared by adding the BEA salt in prewarmed 0.2 N NaoH. This BEI solution can be easily kept at room temperature, different final morality of BEI (0.1, 0.4, 0.8, 1.2& 1.6mM) were used in inactivation process and kept at 37o C and 25ºC for 24 hours at ph 8.0 according to Bahnemann (1990), sodium thiosulphate 20% was added to treated virus samples during and after inactivation in final concentration of 2%. Samples were taken prior, during and after inactivation for virus infectivity and testing its antigenicity using CFT. Guinea pigs Sixty Albino male apparently healthy adult Guinea pigs of approximately 500 grams body 119

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Testing of FMD vaccine SAT2 produced from inactivated virus with different molarity of BEI and different temperature in guinea pigs Sixty adult male guinea pigs approximately 500 gm weight from VSVRI, Foot and mouth disease department were randomly divided in to 12 groups (5 guinea pigs). Five groups were inoculated S/C by inactivated vaccine of FMD SAT-2 strain using different molarities as follow 0.1 mM, 0.4 mM, 0.8 mM, 1.2 mM and 1.6mM of BEI at 37º C and one group was kept as control without vaccination. On the other hand, another five groups of guinea pigs were inoculated S/C by inactivated vaccine of FMD SAT2 strain using different molarities as follow 0.1 mM, 0.4 mM, 0.8 mM, 1.2 mM and 1.6 mM BEI while the inactivation process was done at 25º C, and also one group (of five guinea pigs) was kept as control group without vaccination. All groups of guinea pigs were kept for 28 days and then blood samples were collected for antibody detection against FMD virus SAT-2 strain using serum neutralization test (SNT) and ELISA. Serum Neutralization Test (SNT) It was performed using the micro titer technique as described by Ferreira (1976) to estimate antibodies against FMD in sera of guinea pigs. Indirect enzyme linked immune sorbent assay ELISA ELISA and its reagents were prepared according to (Hamblin et al., 1986 a & b) used to follow up the immune response in animals.


Results and Discussion The complete inactivation of Foot-and –Mouth Disease (FMD) virus is a critical requirement in the production of FMD vaccine to ensure the safety of the product. Binary ethyleneimine (BEI) is an aziridine compound, produced from bromoethylamine hydrobromide (BEA) commonly used for the inactivation of FMD virus during vaccine manufacturing. The validation of BEI, when used as inactivator, is essential to ensure the quality of the inactivating agent and the validity of the process (Aarthi et al., 2004). FMDV inactivation with BEI is usually carried out at 35- 37ºC with a concentration of 1.0 – 1.5 mM applied either once for 24 hours or repeated for an additional 24 hours period (i.e. total 48 h) (Rweyemamu, et.al 1989). The tabulated results in Table (1), Figure (1) and table (3), Figure (3) showed that the complete inactivation of FMDV type SAT2 with no residual virus using BEI of moralities 0.1, 0.4, 0.8, 1.2 and 1.6 mM as detected at 16hr, 14hr, 10hr, 8hr, 8hr respectively at 37ºC, with inactivation rate ranged from 0.53 -1.15 log10TCID50 this results agreed with (Aarthi et al., 2004) and Bahnemann (1990). Table (2), Figure (2) represented that the inactivation kinetics of FMDV type SAT-2 with BEI at 25º C showing that the complete inactivation with no residual virus was occurred at 52, 44, 40, 36 and 36hr post inactivation using BEI of morality of 0.1, 0.4, 0.8, 1.2 and 1.6 respectively, the results in table (3),Figure (3) showed that the inactivation rate ranged between 0.16-0.24 log10TCID50 explained with (Uddowla et al., 2012) also Bahnemann (1990) who said that When FMDV inactivation with BEI at 26º C you must duplicate the BEI concentration to obtain inactivation time the same with inactivation at 37º C . Table (4) represented that the results obtained by complement fixation test of FMDV type SAT2 using different BEI morality at 37ºC, showed that antigen titers were changed from 1/32 to 1/16 with 0.4, 0.8 moralities, while it changed from 1/32 to 1/8 in 1.2 and 1.6 mM while no changes in case of 0.1mM at 37ºC, these results came in agreement with (Ali et al., 2009), while the CFT changed from 1/32 to 1/16 with 0.1mM and from 1/32 to 1/8 with 120

0.4, 0.8 moralities also from 1/32 to 1/4 with 1.2, 1.6 moralities at 25ºC. The results showed that there is no significant difference between the use of BEI in morality of 0.4 and 0.8 mM, and between 1.2mM and 1.6mM, while the inactivation time decreased by approximately 8 h in the morality of 1.2 rather than 0.1 mM and the result supported by Aarthi (2004). Vaccination different groups of Guinea Pigs with such preparations showed that the highest antibody titer appeared after 28 days post vaccination was 1.8 and 2.1 log10 using SNT and ELISA respectively using inactivated virus with 0.1M BEI at 37ºC, while the antibody titer appeared after 28 days post vaccination was 1.8 & 2.1 log10 using SNT & ELISA respectively using inactivated virus with 0.1mM BEI at 25ºC as reported by Eman (2012). The antibody titer appeared after 28 days post vaccination was of bad results using inactivated virus with 0.4, 0.8, 1.2 & 1.6 mM BEI either incubated at 37ºC or at 25ºC. This means that the best moralities of BEI used for inactivation to FMDV is 0.1mM at37ºC because the inactivation process take more time (52hrs) at 25ºC with the same concentration (0.1mM) while take 12 hrs at 37ºC. From all above results we can conclude that the best morality of BEI used for inactivation of FMDV is 0.1mM with final concentration 3% and incubate at 37ºC for 16 hours for production of FMD SAT2 vaccine with the best quality. Conclusion This study can be used as a guide line for routine procedures for using BEA as an inactivator, and it was clear that the optimal condition used for inactivation of FMDV SAT2 with a molarity of 0.1 mM and at 37ºC incubational temperatures for 16 hours provides a good and safe method of inactivation and vaccine production. Acknowledgment The authors are grateful to director of veterinary serum and vaccine research institute (VSVRI) for providing all facilities to perform with research specially Prof. Dr. Mohamed Hassan Khoudier, Prof. Dr. Khayrat Abdel Mageed Elia J. Vet. Adv., 2013, 3(3):117-124

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Table 1: Inactivation of FMD virus type SAT2 with BEI at 37ºC. Virus titer (log10 TCID50/ml) with different BEI molarities Hours of inactivation 0.1mM 0.4mM 0.8mM 1.2mM 1.6mM 0 8.5 8.5 8.5 8.5 8.5 2 7.3 7.3 7 6.6 6.2 4 6.2 6.2 5.4 4.1 3.9 6 5 5 3.2 1.9 1.9 8 4.6 3.8 1.2 0 0 10 3.3 2.5 0 0 0 12 2.2 1.2 0 0 0 14 1 0 0 0 0 16 0 0 0 0 0 Titer expressed in log10

Fig. 1: Inactivation of FMD virus type SAT2 with different molarities of BEI at 37ºC. Table 2: Inactivation of FMD virus type SAT2 with BEI at 25ºC. Virus titer (log10 TCID50/ml) with different BEI molarities Hours of inactivation 0.1mM 0.4mM 0.8mM 1.2mM 1.6mM 0 8.5 8.5 8.5 8.5 8.5 4 8 7.7 7.4 7.4 7.3 8 7.4 6.4 6.4 6.2 6.2 12 6.6 5.5 5.5 5.3 5.3 16 6 4.8 4.5 4.3 4.2 20 5.3 4.1 3.4 3.1 2.9 24 4.6 3.5 2.6 2.5 2.1 28 4.1 2.9 1.7 1.2 1.6 32 3.6 2.1 0.9 0.6 0.9 36 3 1.3 0.3 0 0 40 2.4 0.6 0 0 0 44 1.7 0 0 0 0 48 1.2 0 0 0 0 52 0 0 0 0 0 Titer expressed in log10 121

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Fig. 2: Inactivation of FMD virus type SAT2 with different molarities of BEI at 25ºC.

Table 3: Inactivation rate (log10/hr) with BEI (0.1 and 1.6 mM). Virus inactivation rate with different BEI molarities Temperatures 0.1mM 0.4mM 0.8mM 1.2mM 1.6mM 37ºC 0.53 0.57 0.8 1.15 1.15 25ºC 0.16 0.18 0.21 0.24 0.24

Fig. 3: Inactivation rate (log10/hr) with BEI (0.1 and 1.6 mM). 122

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Table 4: Complement fixing content of antigen of FMDV type SAT2 before and after inactivation with BEI. CF content before & After inactivation at BEI 37ºC 25ºC Molarity Before After Before After 0.1 mM 1/32 1/32 1/32 1/16 0.4 mM 1/32 1/16 1/32 1/8 0.8 mM 1/32 1/16 1/32 1/8 1.2 mM 1/32 1/8 1/32 1/4 1.6 mM 1/32 1/8 1/32 1/4 Table 5: Mean antibody titers in Giunea pigs vaccinated with vaccine preparation with different molarity of BEI at 37ºC & 25 ºC at 28 day post vaccination. FMD SAT-2 antibody titers obtained by prepared vaccine at BEI 37ºC 25ºC Molarity SNT ELISA SNT ELISA 0.1 mM 1.8 2 1.8 2.1 0.4 mM 1.65 1.9 1.65 1.98 0.8 mM 1.5 1.75 1.5 1.8 1.2 mM 1.2 1.45 1.2 1.5 1.6 mM 0.9 1.18 0.9 1.2

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