1-Animals enrolled in virology studies: The present study performed on 100 apparently sick cattle and buffaloes from different ages (from 3 months up to 1.5 years old) in different localities in delta governorates.

Samples for virological investigation; a total of 300 samples (sera, tissues) were collected from cattle and buffaloes, 60 buffaloes and 40 cattle ages 3 months up to 1.5 years, animals included in this study were not vaccinated against FMD.

2- Animals enrolled in clinical pathology studies: Fifty cattle aged between 1 and 1.5 years were used in this study and divided into two groups. First group (gp-1) forty (40); diagnosed cattle with FMD. The second group 10 cattle were tested free from FMD were used as control.

Samples for haematology; peripheral blood samples collected from the jugular vein into EDTA treated tubes used to establish total red blood cells (RBCs), haemoglobin concentration (Hb), packed cell volume (PCV), erythrocyte indices ^{mean corpuscular volume (MCV) mean corpuscular haemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)}, total white blood cell and differential leucocytic count manually (Feldman et al., 1986).^[12]

The obtained data were analyzed by using t- student test according to SPSS 14 (2006).^[13]

3- Histopathology: Tissues specimens collected (5 mm diameter, kept in 10 % formalin solution) from the Dead animals that suffering FMD were undergoing pathological examination. Only the positive FMD tissue specimens were undergoing pathological tests. However, about 50 tissues samples were collected from dead cattle and buffaloes aged 6 months to 1year old. These samples are liver, tongue, heart, kidneys, skin, spleen, and intestine. The samples were processed to prepare 5 micron thickness of histopathological slides, and then stained by H&E (Hematoxlyin and eosin), special stains (Hematoxylin and fuchsin). The slides were examined by light microscope. The procedures were referenced according to (Clayden, 1971), (Lillie &Fullmer, 1976).

3- BHK Cell lines: Darby hamster kidney cells (BHK-21) were prepared and provided by VACSERA Egypt. The BHK cells were used for adaptation of FMDV, virus titration as well as serum neutralization test (SNT). ^{Figure 14}

6- Tissue specimens for FMDV isolation in tissue culture:Skin biopsies, comprising epidermis and dermis of the ulcerated mouth lesions, were collected from local cattle and buffaloes. Also, samples and tissues specimens from dead animals were collected aseptically in 15 ml sterile tubes and stored at -20°C until used. The procedures were performed according to Payment &Trudel, 1993.

5- Infectious FMDV: The viruses used in this study are the locally isolated FMDV strains (O/ ME-SA/ Sharqia-72), (SAT2/V11/ Ghb-12), (A/ Africa/ G-1V) that identified by reference laboratory of FAO (WRLFMD) in the Institute for Animal Health, Pirbright, England.

6- Titration of FMDV and determination of infectious dose (ID50): Titration of the virus was according to Reed and Muench’s method. (Reed and Muench, 1938)

7- Virus Neutralization test:This test was used to determine the neutralizing antibodies of FMDV in sera samples of infected animals that able to neutralize FMDV and prevent infection of culture cells. (Payment &Trudel, 1993)

9- Manual ELISA method for Detection of anti-FMDV antibodies in blood of cattle and buffaloes:

this test was performed according to (Payment &Trudel, 1993)

The anti-FMDV antibodies present in serum dilution react with FMDV coated on micro-titer-plates. Unbound reactants are then washed out subsequently, peroxidase conjugated antibodies to (cattle and buffaloes) are added, followed by incubation period and washing, then substrate is added and the peroxidase conjugate bound to the anti- FMDV present in the serum samples reacts with the substrate producing a color. The enzymatic reaction and the resulting color is measured by ELISA reader at 492 nm.

8- PrioCHECK FMDV NS Antibody ELISA Kit (Thermo Fisher Scientific, Inc):It is an ELISA that detects antibodies against the highly conserved non-structural (NS) protein of the FMD virus. The test can therefore be used for all species.

5- FMDV antigen detection and serotyping: Enzyme linked immunosorbent assay (indirect sandwish ELISA): (Instituto Zooproflattico Sperimental della Lombardia dell ‘Emilia Romagna (IZSILER), Brescia, Italy; Ferris et al., 2011.)

The component of indirect sandwich ELISA kit design to detect FMDV serotypes O, A, C, and Asia 1, and SAT-2. {BDSL, IAH, Pirbright, UK; Ferris and Dawson, 1988}.The product is produced by the reference laboratory of FAO, IZSLER: Brescia, Italy.

The present study was performed on 100 clinically diseased cattle and buffaloes of different ages (from three months to 1.5 years old) from different localities in delta governorates years 2015 and 2016. The animals suffered fever (40^oC - 41^oC), lameness, lacrimation, excessive salivation, bloody mouth discharges, oral erosions, different lesions (vesicles, ulcerations, salivation), foot lesions (ulcerations on the inter-digital space with lameness), teat lesions (vesicles and ulcerations on the teat with difficulty on milking due to pain), and sudden death mostly in young animals. Mortalities were recorded high among cattle and buffaloes less than 3 months old. Figures (1-5).

Macroscopic lesions of FMD in young animals are in the myocardium, liver, kidneys, intestine, tongue, mouth cavity, nostrils, blood vessels and skin. Lesion of heart are forming whitish streaks on myocardium separated by dark and congested areas in longitudinal shapes giving the pathgnomonic lesion of FMD in the young infected animal. Skin showed vesicles and erosions usually seen in the areas of soft tissues: mouth, muzzle, nostrils, foot, udder, vagina and anal area, base of horns, conjunctiva.

A field strains (serotypes O, A& SAT 2) of FMDV were isolated during the outbreaks of the disease in Egypt by Animal Health research Institute, Egypt. These virus strains were identified by the FAO World Reference Laboratory for Foot-and-Mouth Disease (WRLFMD) of The Pirbright Institute, England. They were used for cell culture adaptation and propagation, serum neutralization test and as reference guide of other tests.

The data in table (1) illustrates the procedure used in the Reed-Muench method for calculating the accumulated values of infected and uninfected cell culture tubes. The dilution which would be expected to yield 50% positive (CPE) tubes is seen to lie between 10-3 and 10 -4 and will, in fact, be located at the proportionate distance from 10-3. The necessary proportionate distance (PD) of the 50 % infectively end point is obtained. The dilution of reference virus selected for the neutralization test is usually 100 times stronger than the 50% end point.

Samples used for isolation were collected from animals which showed severe clinical symptoms and FMDV positive infections with FMDV by using various serological examination. FMDV isolation trials from tissue samples were using BHK-21 cell lines. All samples were collected during active infection and from feverish animals (i.e. during circulation of viruses in blood; vireamia). Tissue culture infected cells showed cytopathic effects (CPE) in the form of rounding of cells, abnormalities on cytoplasm and cytolysis.

The cultures showed CPE were subjected to test by antigen detection ELISA (IZLER), the results were shown in table (2).

This test performed to detect anti-FMDV antibodies in sera samples. The results were shown in table (2). However, it was observed that sera gives positive reaction in this test is sometimes but not always gives positive reaction with PrioCHECK ELISA.

Results were shown in table (2). It was observed that the Sera samples which exhibits positive reactions by conventional ELISA were giving positive reactions by SNT test.

It is an ELISA that detects antibodies against the highly conserved non-structural (NS) protein of the FMD virus. The test can therefore be used for all species. The positive reactions mean the presence of infectious agent of FMDV. The indicate anaemia. Al-Rukibat et al., (2015) recorded significant increase in PCV in FMD infected sheep. Also, Elitok et al., (2005) found that PCV was higher in FMD infected animals than in healthy free ones. However, from our histopathology of kidneys of infected animals it was observed that it suffers degenerations and focal necrosis in both cortex and medulla, and as erythropoietin is a vital substance produced by the kidneys and is responsible for synthesis of RBCs (erythropoiesis), one could explain main reason for anemia in FMD infected animals (Jubb et al., 1991).

Leucogram of FMD affected animals showed leukocytosis with lymphocytosis. Our work is in accordance with Mohapatra et al., (2005) who stated the same results in all ages of infected animals with FMD. Elitok et al., (2005) 11 found that leucocyte count in FMD infected sheep was higher than normal level (over 10.000 cells/ul), mean neutrophil count (22.2%) less than control (34.7%), lymphocytosis was (75.4). Al-Rukibat et al., (2015) mentioned that there was a significant increase in eosinophil percentage in FMD positive sheep. The increase in total leucocytes count was mainly due to increase in lymphocytes; and this findings are in agreement with Ghanem & Abd El-Hamid (2016).