oot-and-Mouth Disease Virus (FMDV) is the pathological agent of the most important diseases that affect cloven-hoofed livestock. It is a small, non-enveloped single-stranded, positive-sense RNA virus related to the family Picornaviridae. FMD has seven serotypes: O, A, C, Asia 1, and Southern African Territories (SAT) 1, 2 and 3, they cause a highly contagious disease (Alexandersen et al., 2003). There are over 60 subtypes within these serotypes. For that, there are no universal vaccines, thus presenting challenges in the selection of vaccine strains (Brown, 2003 andArzt et al., 2011). Infection with FMDV leads to an acute disease that spreads very rapidly. It characterized by fever, lameness, and vesicular lesions on the feet, tongue, snout, and teats, also characterized by high morbidity but low mortality (Grubman and Baxt, 2004). Although vaccines extensively used to control FMD, there was no antiviral therapy to treat ongoing infections with FMD virus (Grubman, 2005).

The most effective FMD vaccines are consist of chemically inactivated FMDV. They can only offer complete protection after seven days of vaccination because of the time needed to trigger an immune response (Pacheco et al., 2015 andZhang et al., 2015).

As oil as an adjuvants is absorbed more slowly than its gel equivalent, also can cause local reaction in the site of vaccination. To prevent such effect, can use other adjuvant types than the oil, such as nanoparticles (Batista et al., 2010). Recently nanoparticles and micro carriers are used in vaccine delivery to enhance the cellular and humeral immunity through an increased presentation of vaccine epitopes to the antigenpresenting cell (Singh et al., 2010). The particles in the nanometer size range are of particular interest may be due to their unique cellular uptake and bio-distribution properties. They also play an important role when using as vaccine antigen carriers and adjuvants (Perni et al., 2014).

Silver nanoparticles (AgNPs) have attracted significant interest among the emerging Nano products because of their unique properties and increasing use for various applications in nanomedicine (Gurunathan et al., 2009). The adjuvanticity effect of AgNPs on rabies vaccine potency shown for the first time, and the results clearly showed the effect of AgNPs on increasing the humoral response to the rabies vaccine (Vahid et al., 2016). The immunological adjuvant effect of AgNPs investigated both in vitro and in vivo. The in vivo adjuvant effect of AgNPs evaluated with model antigen ovalbumin (OVA) and bovine serum albumin (BSA) in mice by intraperitoneal and subcutaneous immunization and the results showed the remarkable adjuvant effect of AgNPs. The result is beneficial for the future applications, especially in biomedicine (Xu et al., 2013).

This study was carried out to determine the adjuvant effects of Silver nitrate nanoparticles when used as an adjuvant to improve the polyvalent FMD vaccine on the immune response of calves.

Material and Methods

Cell line of Baby Hamster Kidney (BHK21) clone 13 was maintained in the Department of Foot and Mouth Disease Vaccine Research (DFMDVR), Veterinary Serum and Vaccine Research Institute (VSVRI), Abbasia, Cairo, according to the technique described by Macpherson and Stocher (1962), used for virus propagation and application of serum neutralization test. Using Eagle's medium with 8-10% sterile new-born calf serum obtained from Sigma, USA.

FMD viruses O / PanAsia2, A/Iran 05, SAT2/VII/Lib-12 (SAT2/ Lib) and SAT2/VII/Ghb-12(SAT2/Ghb) are locally isolated strains of cattle origin. The viruses were typed at VSVRI and confirmed by Pirbright, International Reference Laboratories, United Kingdom and propagated on BHK cells then concentrated using polyethylene glycol 6000 (PEG-6000) according to Killington et al.(1996) and Hiam and Eman (2010). The viral suspension was concentrated at 25,000 rpm for 5 hours at 4? in a high-speed centrifuge (Avanti J25, Beckman Coulter, and Fullerton, CA, USA). The virus in the bottom was removed and polled. It was further concentrated in an ultracentrifuge at 35,000 rpm /min for 3 hours at 4?. The viral pelted was polled and preserved at -80? to be used in vaccine preparation. Virus concentrations provide virus titers of 10 9 ; 10 9 ; 10 8.5 , and 10 9 TCID 50 /ml for O/PanAsia2, A/Iran 05, SAT2/VII/Lib-12 (SAT2/ Lib) and SAT2/VII/Ghb-12(SAT2/Ghb) respectively.

Complete inactivation 0f the concentrated virus stock using Binary Ethyleneimine (BEI) according to Bahnemann (1975) and Ismail et al.(2013). 1%M BEI in 0.2N NaOH was added to the virus suspension to give a final concentration of 0.001M of BEI. Mixed well the virus and BEI mixture, and the pH then adjusted to 8.0 by sodium bicarbonate. Incubation of the mixture at 37 o C for 12 hours.

Sodium thiosulphate added to give a final concentration of 2% to neutralize the BEI action. The inactivated viruses used in the preparation of vaccine formulation with AgNPs, ISA 206 oil, and AgNPs with Montanide ISA 206oil adjuvants for animal immunization.

Sample of Silver nanoparticles (AgNPs) was prepared as 0.001M/ 10mls and subjected to continuous stirring for 6 hours at room temperature, followed by sonication for three times repeated cycles each of 15 minutes according to Udapudi et al.(2012).

Transmission Electron Microscopy (TEM) For transmission electron microscopy of the samples of Silver nanoparticles, prepared by dispersing in ultrapure H 2 O at about 10% concentration and ultrasonicated at 1000L for 15 minutes. One drop of this liquid immediately transferred by a micropipette to a 3 mm diameter Formvar coated copper TEM grid, slowly evaporated to dryness. The samples on the TEM grid analyzed using a 100cx JEOL TEM at 80 kV at CURP, Giza, Egypt.

Baby Hamster Kidney cell line used to investigate the adjuvant inhibitory adverse effect on cell proliferation as an indicator of safety to use Silver nanoparticles (AgNPs) as a biocompatible adjuvant in the vaccine formulation.

The mineral oil-based adjuvant from water-in oilin-water (double emulsion) mixed with antigen w/w supplied by Seppic, Paris, France.

Formulation with oil phase carried out according to the method described by Barnett et al. (2003), and Wael et al. (2014) where the oil phase consisted of Montnide ISA 206 mixed with the inactivated viruses as equal parts of an aqueous and oil phase (w/ w) and mixed thoroughly.

The inactivated viruses adjuvanted with ISA 206 oil (w/ w), and AgNPs in a concentration of0.1 mg/dose.

Twelve local breed healthy calves and free from antibodies against FMD viruses as proved by using SNT and ELISA were used in this study where they were divided into four groups (3calves/group) as follow:

Group(A): vaccinated with the inactivated polyvalent FMD vaccine adjuvanted with AgNPs vaccine.

Group (C): vaccinated with the inactivated polyvalent FMD vaccine adjuvanted with oil adjuvant vaccine.

All vaccinated animals received 3ml/animal of the used vaccine formula inoculated subcutaneously.

Blood samples were collected from all calf's groups on an anticoagulant for evaluation of cell 2 Year 2020 mediated immunity using Lymphocyte blastogenesis assay on the 3 rd day post-vaccination, then every week up to 10 weeks.

Serum samples were collected for the serological tests (SNT and ELISA), weekly for one month then every 2 weeks up to 40 weeks post-vaccination and stored at -20 o C until used. The test was performed by the micro titer technique as described by Ferreira (1976), and the antibody titer expressed as serum neutralization log10.

13. Indirect Enzyme-linked immunosrobent assay (ELISA) It was carried out according to the method described by Voller et al. (1976) and OIE (2012). Serum samples were examined for FMD viral specific IgG antibodies using in-house developed ELISA assay.

Complete viral inactivation checked by inoculation of BHK cells incubated for two days and compared to the virus-infected cell (virus control) and normal cell (cell control). The inactivated virus showed monolayer of BHK cells and positive control showed viral cytopathic effect at 24-hour post-infection. Complete virus inactivation was obtained by 16hours for O / PanAsia2, A/Iran 05, SAT2/VII/Lib-12 (SAT2/ Lib), and SAT2/VII/Ghb-12(SAT2/Ghb) respectively.

Transmission Electron Microscopy (TEM) showed the particle size of the Silver nanoparticles (AgNPs) adjuvant of 5-10 nm as shown in the photo ( 1)

The effect of Silver nanoparticles (AgNPs) adjuvant on the in vitro cell proliferation investigated in BHK cell line monolayers after its exposure to gradient concentrations of Silver nanoparticles (AgNPs) for 48 hours. The percentage of viable cells among all of the preparations was above 50% indicating the safety of Silver nanoparticles (AgNPs) adjuvant.

The obtained results of cell mediated immune response using lymphocyte proliferation test for all animal groups expressed by Î?"OD (Delta Optical Density) were as follow: Group(A) showed Î?"OD (0.521) by using FMD viruses at 3 rd -day post-vaccination and reached its highest level (1.572) at 3 rd -week post-vaccination, then declined after nine weeks post-vaccination.

Group(B) showed Î?"OD (0.566) by using FMD viruses at 3 rdday post-vaccination and reached its highest level (1.660) at 3 rd -week post-vaccination then declined after ten weeks.

Group (C) showed Î?"OD (0.486) by using FMD viruses at 3 rd -day post-vaccination and reached its highest level (0.973) at 3 rd -week post-vaccination, then declined after seven weeks. These results are demonstrated in a table (1) and fig (1). 3)it is clear that protective FMD-ELISA antibody titers induced by AgNPs only started at the 1 st week post-vaccination with average values of 1.95, 1.93, 1.82 and 1.82 log 10 for type O, A, SAT2/ Lib and SAT2/Ghb respectively reaching their peak level at the 10 th week post-vaccination with average titers of 3.17, 3.17 Montaind ISA 206 oil vaccine induced FMD ELISA antibody titers started at the 2 nd week postvaccination with average values of 1.93, 1.97, 1.96 and 1.96 log 10 for type O, A, SAT2/ Lib and SAT2/Ghb respectively with peak levels at the10 th week postvaccination with average titers of 3.16, 3.06, 3.14 and 3.12 log10 respectively continued with protective level till the 36 th week then declined as shown in fig (5, 6 & 7).

Table (3): FMD ELISA antibody titer in vaccinated calves with the prepared polyvalent FMD vaccine formulae

Group (B) =oil and AgNPs vaccine.

Nanoparticle-containing vaccines have attracted tremendous interest in recent years, and a wide variety of nanoparticles have been developed and employed as delivery vehicles or immune potentiates, allowing an improvement of antigen stability but also the enhancement of antigen processing and immunogenicity (Smith et al., 2015).

The control of FMD in animals was considered to be important to effectively contain the disease in endemic areas so that vaccination of animals is effective in limiting the spread of FMD. So, this study aimed to improve the inactivated polyvalent FMD vaccine by adding Silver nitrate nanoparticles as an adjuvant.

Table (1) showed that the results of cellmediated immune response using lymphocyte proliferation test for all animal groups expressed by Î?"OD appeared to be supported by Knudsen et al., (1979) and Sharma et al., (1984) who reported that cell-mediated immune response was a constitute of the immune response against FMD virus, and agreement-in some points with Mercedes et al.(1996) The tabulated results in tables (2 & 3) that SNT and ELISA titers for AgNPs, oil and AgNPs with oil FMD vaccine formulae agreed with Vahid et al. (2016) who showed that adjuvant properties of AgNPs as a potent adjuvant induced higher antibody and the protective function is the production of neutralizing antibodies, either IgM or IgG, which are able to prevent the entry of the virus into cells. The results are supported also by Malyala and Singh (2010) and Rebecca et al. (2010), who found that AgNPs might help the vaccine work more effectively, increasing antibody production, also agreed with Gurunathan et al., (2009); Kaba et al. (2009); Zhao et al. (2014) and Daniel et al. (2019), who found that AgNPs improved B-cells function, mucosal and humoral immunity and protective activity also helped vaccine for induction of strong immunity when used as an adjuvant. The results also go in hand with the results obtained by Hamblin et al. (1986). They explained that the SNT measures those antibodies which neutralize the infectivity of FMD virion, while ELISA probably measures all classes of antibodies even those produced against incomplete and non-infectious virus.

Depending on the present obtained results we could conclud that the usage of Silver nitrate nanoparticles (AgNPs) with Montanid ISA 206 oil in inactivated FMD trivalent vaccine induces long-lasting immunity than that induced by oil adjuvant alone and improve both cellular and humoral immunity resulted in earlier and more long-lasting immunity the thing which can aid in companying to control FMD.