STUDY OF HUMORAL AND CELLULAR IMMUNE RESPONSE IN FMD VACCINATED SHEEP WITH DIFFERENT INACTIVATED FMD VACCINES

his study has been designed as a trial for comparing of ISA206 adjuvanted Foot and Mouth Disease (FMD) vaccine (commercial vaccine), commercial FMD vaccine containing vitamin (A), commercial FMD vaccine containing vitamin (ESelenium) and commercial FMD vaccine containing both vitamin (A) and vitamin (E-Selenium) together in eliciting immunity. The obtained results revealed that the duration of humoral immunity elicited by commercial vaccine only was 30 weeks, while, it was 32 weeks in commercial vaccine containing vitamin (A), 36 weeks in commercial vaccine containing vitamin (E-Selenium) and 38 weeks in commercial vaccine containing both vitamin (A) and vitamin (ESelenium) together in vaccinated sheep. Results of cellular immunity evaluation indicated that mean optical density (mean OD) correspond to non-specific mitogens using MTT kit reached its highest values by 21st day post vaccination in all vaccinated sheep and mean OD values correspond to FMDV as specific mitogen were higher than that of non-specific mitogen. Also, the highest mean OD values were obtained in vaccinated sheep with commercial vaccine containing both vitamin (A) and vitamin (E-Selenium) together. It could be concluded that usage of inactivated oil FMD vaccine using Montanide ISA 206 containing both vitamin (A) and vitamin (E-selenium) resulted in longer lasting immunity than that with Montanide ISA 206 only, besides the stimulating effect of both vitamin (A) and vitamin (E-selenium) in both humoral and cellular immunity. INTRODUCTION Foot and Mouth Disease (FMD) is a highly contagious disease and one of the most devastating diseases of cloven-hoofed animals, including domestic animals such as cattle, buffaloes, sheep, goats and pigs, as well as antelope, hison and other wild bovines and deer (OIE, 2012). This disease has a significant economic impact on livestock industry worldwide. The majority of the economic losses results either from mortality of young animals, loss of milk and meat and drastic fall in production performance or indirect losses due to the imposition of trade restriction (Verma, 2008). FMD virus is aphathovirus of Family Picornaviridae, There are seven recognized serotypes of FMD (O, A, C, Asia, SAT1, SAT2 and SAT3) which differ in T STUDY OF HUMORAL AND CELLULAR IMMUNE RESPONSE IN FMD VACCINATED SHEEP WITH DIFFERENT INACTIVATED FMD VACCINES 668 distribution across the world (Pereira, 1977) and comprise more than 65 subtypes (Sharma and Kakkar, 2005). In Egypt, the disease is enzootic and outbreaks have been reported since 1950. FMDV serotypes (SAT2, A) and (O) were last reported in the years 1950, 1972 and 2000, respectively (Aidaros, 2002). Type (O) was the most prevalent since 1960 and onwards (Farag et al., 2005). Since 1950, 1953 and 1956 serotype (A) was not recorded in Egypt (Zahran, 1960). Recently, serotype (A) of FMD virus was introduced to Egypt through live animals importation and severe clinical signs occurred among cattle and buffaloes (Abd El-Rahman et al., 2006). Also, FMDV serotype (SAT2) outbreaks in Egypt were officially reported by OIE (2012). In addition to this, endemic serotypes A and O continue to circulate in country (Lockart et al., 2012 and Heba Attia, 2017). Most of Foot and Mouth Disease vaccines are made of binary ethyleneimine (BEI) inactivated virus. Oil adjuvants are generally preferred due to introducing longer lasting immunity (Hunter, 1998). The commercial FMD vaccine is adjuvanted with Montanide ISA206 which is double water-in-oil-in-water (W/O/W) adjuvant eliciting protective humoral immune response and cellular immunity in vaccinated animals (Clo et al., 2008). Kutukculer et al. (2000) reported that both vitamin A and vitamin E-selenium can be used as adjuvant inducing high serum neutralization antibody titers and stimulate cellular immunity. Also, Rajeesh et al. (2008) found that using vitamin Eselenium as an adjuvant elicits high and prolonged immune response in vaccinated animals. This study is carried out as an attempt to compare between (ISA206), (ISA206 and vitamin A), (ISA206 and vitamin E-Selenium) and (ISA206 and both vitamin A and vitamin E-Selenium together) vaccine in eliciting both humoral and cellular immunity. MATERIAL AND METHODS 1. Animals: Twenty five sheep of 6 months (local breed) were clinically healthy and free from antibodies against FMD as proved by using SNT and ELISA. 2. FMD virus strains: Vaccinal strains serotypes O Pan Asia 2012, A/Iran 05, and SAT2/Egy/2012 of FMDV were used for production of trivalent FMD vaccines and neutralization test. 3. FMD vaccines: a. Commercial FMD vaccine: Inactivated FMD oil trivalent vaccine. ABEER H.M. HASSAN, et al. 669 b. Commercial FMD vaccine containing vitamin (A): Inactivated FMD oil trivalent vaccine containing vitamin (A) 200,000 IU/dose (Semba et al., 1998). c. Commercial FMD vaccine containing vitamin (E-selenium): Inactivated FMD oil trivalent vaccine containing vitamin (E-Selenium) 500mg/dose (Milad et al., 2001). d. Commercial FMD vaccine containing both vitamin (A) and vitamin (Eselenium: Inactivated FMD oil trivalent vaccine containing both vitamin (A) 200,000IU/dose and vitamin (Eselenium) 500 mg/dose. 4. Experimental Design: Four groups of sheep (5 animals/group) were vaccinated with the tested vaccines. Five animals were kept without vaccination as a control negative group. Serum samples were collected weekly post vaccination for one month and then every 2 weeks until the end of the experiment (38 weeks). Heparinized blood samples for MTT assay of cell mediated immunity were also collected. 5. Estimation of the immune response: 5.1. Humoral immunity: It was estimated using SNT according to OIE Manual (2012) and ELISA according to Hamblin et al. (1986). 5.2. Cellular immunity: It was estimated using MTT kit according to Verma (2010). 6. Statistical analyses Results were analyzed using ANOVA test (Sendecor, 1971). RESULTS From Tables (1 to 6), the results of humoral immune response revealed that mean protective serum antibody titre evaluated using SNT and ELISA were as follows: The first group reached the SNT protective level (1.5 log10) at 3rd week post vaccination (WPV) with mean antibody titers of (1.68 log10/ml for "O" serotype, 1.68 log10/ml for "A" serotype) and at the 4th WPV (1.59 log10/ml) in case of "SAT2" serotype and to ELISA protective level (1.65 log10) at the 3rd WPV (1.89 log10/ml for "O" serotype and 1.68 log10/ml for "SAT2" serotype) and 1.59 log10/ml for "A" serotype at the 2nd WPV. The highest level of mean antibody titers were obtained at 10th week post vaccination (WPV) (2.64 log10/ml for "O" serotype, 2.67 log10/ml for "A" serotype and 2.46 log10/ml for "SAT2" serotype) by SNT and by ELISA (2.73 log10/ml for "O" serotype, 2.67 log10/ml for "A" serotype and 2.46 log10/ml for "SAT2" serotype). STUDY OF HUMORAL AND CELLULAR IMMUNE RESPONSE IN FMD VACCINATED SHEEP WITH DIFFERENT INACTIVATED FMD VACCINES 670 The duration of protection lasted for 30, 36 and 28 WPV for "O", "A" and "SAT2" serotypes respectively in SNT and for 28, 32 and 26 WPV in ELISA respectively. In the second group, FMD SNT antibody protective levels were obtained at the 3rd, 2nd and 4th WPV in "O", "A" and "SAT2" serotypes respectively while in case of ELISA they achieved at the 3rd WPV in "O" and "SAT2" serotypes, but at the 2nd WPV in "A" serotype. The highest level was recorded at 10th WPV (2.67, 2.73 and 2.43 log10/ml for "O", "A" and "SAT2" serotypes respectively) by SNT and (2.76 and 2.73 log10/ml for "O" and "A" serotypes respectively) and at the 12th WPV (2.55 log10/ml for "SAT2" serotype) by ELISA with protection duration for 32, 36 and 30 WPV for "O", "A" and "SAT2" serotypes respectively in SNT and for 30, 36 and 28 WPV in ELISA respectively. Regarding to the third group, SNT antibody protective levels were recorded at the 3rd, 2nd and 3rd WPV in "O", "A" and "SAT2" serotypes respectively while in case of ELISA they achieved at the 2nd WPV in "O" and "A" serotypes, but at the 3rd WPV in "SAT2" serotype. The highest level of mean SNT antibody titers were recorded at the 10th WPV (2.85 log10/ml for "O" serotype, 2.94 log10/ml for "A" serotype and 2.55 log10/ml for "SAT2" serotype) by SNT and (2.94 log10/ml for "O" serotype and 2.55 for "SAT2" serotype) and at the 12th WPV (3.00 log10/ml for "A" serotype) by ELISA with protection duration lasted for 36, 38 and 32 WPV for "O", "A" and "SAT2" serotypes respectively in SNT and for 32, 36 and 30 WPV in ELISA respectively. In the fourth group, the SNT protective levels reached at 3rd WPV with mean antibody titers of 1.77 log10/ml for "O" serotype and 1.53 log10/ml for "SAT2" serotype) and at the 2nd WPV (1.71 log10/ml) in case of "A" serotype and to ELISA protective level at the 2nd, 1st and 3rd WPV (1.68, 1.68 and 1.74 log10/ml for "O", "A" and "SAT2" serotypes respectively). The highest level of mean SNT antibody titers were gained at the 10th to 12th WPV (2.94 log10/ml for "O" serotype, 2.94 log10/ml for "A" serotype and 2.61 log10/ml for "SAT2" serotype) by SNT and (2.94 log10/ml for "O" serotype, 2.97 log10/ml for "A" serotype and 2.64 log10/ml for "SAT2" serotype) by ELISA with protection duration lasted for 38, 38 and 32 WPV for "O", "A" and "SAT2" serotypes respectively in SNT and for 38, 38 and 26 WPV in ELISA respectively. DISCUSSION From Tables (1 to 6), the results revealed that SNT and ELISA titers of FMD antibodies induced by oil Montanide ISA-206 trivalent FMD vaccines in group one went in hand with Barteling and Vreeswij, 1991 and Sonia, 2007) who reported that oil emulsion FMD vaccine (double oil emulsion) induced best results in comparison with alhydragel vaccine. Also agree with Patil et al. (2002) and Abeer et al. (2009) who ABEER H.M. HASSAN, et al. 671 found that FMD vaccines adjuvanted with Montanide ISA-206 can promote long lasting immunity. The obtained results in the 2nd group in which animals were vaccinated with oil Montanide ISA-206 tri