3.1. Clinical signs
Clinical signs in cattle, buffaloes, and calves were summarized in (Table.3). Animals expressed variable clinical signs (fever, lameness, and oral lesions) and were negative by viral isolation, and RT-PCR was culled from this study.
In cattle, clinical signs were slightly different depending on age; in adult cattle (above two years), fever ranged from (39°C – 40°C) and continued for 3–4 days with anorexia; however, in calves, less than six months age, fever reached up to 41°C in some cases, and some animals were suddenly found dead without previous clinical signs. Sever salivation with mouth lesions varied from an elevated area of hydropic degeneration and vesicular formations on the tongue's upper surface, lips, and red area of submucosal hemorrhage on the oral commissar of calves aged 1 to 6 months. Ulceration was developed later in the upper lips, tips of the tongue, dental pad, and upper third of the tongue, leading to severe salivation. The foot lesion represented by hyperemia and vesicular lesion between the digits ended by severe ulceration in interdigital space and takeoff of the claw in some seriously affected cases. These foot lesions were obvious in adult animals above two years and calves aged more than six months, while these lesions were absent in calves aged less than six months. Obvious lameness was exhibited by animals that suffered severe ulceration in digits. Mortality rates were high, especially in young animals of 1 month to 2 years, and moderate in animals above two years. All FMD positive calves less than six months of age had died.
On the other side, signs in buffaloes were mild to moderate in the form of viremia ranged from 39°C – 40°C. Mouth lesions were mild in the form of blanched areas in 1 month to 2 years age group. Moderate lesions in the form of the small shallow vesicle with scanty fluid were also detected. The foot lesion is absent in calves aged from 1 to 6 months while calves (6 months to 2years) and adults (above two years) showed only mild vesicles and erosion mainly at the bulb of the heal, so lameness was not clear. Mortalities were 100% in calves less than six months in both cattle and buffaloes (Table.1). Some animals displayed a cardiac arrhythmia, followed by dyspnea and grunting, just before death.
3.2. Gross pathology
Lesions scoring were summarized in (Table.3). The adult cattle and buffalo showed several gross lesions in the mouth and interdigital space as blisters, erosions, and ulcerations. Vesicles, irregular-shaped erosions/ulcers of variable size, were usually located on the torus lingua and the anterior third of tongues and more frequently on the gingiva, lip, and dental pad. Hyperemia, ulceration, detachment of the heel's bulbs and the soles were also detected. Similar ulcers were observed in the abomasum, ruminal pillars (Fig. 1C). Myocardial hemorrhage (petechial and ecchymotic) with various degrees of myocardial necrosis were also recognized grossly.
Young calves exhibited gross oral, digestive, and foot lesions mentioned above but increased severity. Pronounced myocardial hemorrhage with yellowish-grayish streaks in the myocardium (tiger heart appearance) were constantly detected in all dead calves (Fig. 1 A, B).
3.3. Histopathological findings
Histopathological examination and lesion scoring of young and adult affected animals were recorded in (Table.3). Briefly, in cattle, histopathological changes in the stratified squamous epithelium began with hydropic degeneration with increased cytoplasmic eosinophilia; followed by necrosis and subsequent mononuclear cell and granulocyte infiltration in the cells of the stratum spinosum; the lesions further develop into vesicles after separation of the epithelium from the underlying tissue and filling of the cavity with vesicular fluid. The formerly mentioned lesions of cornified epithelial tissues in buffalo were mild in adult buffaloes or completely absent in the affected young calves.
The heart of the affected adult buffaloes showed mild to moderate non-suppurative myocarditis (Fig.1D), while the young calves were is severely affected, especially in younger calves (3-6 months old age). The young calves died from acute disease; the heart muscles showed lymphohistiocytic myocarditis; in the form of hyaline degeneration and necrosis of myocytes (hyalinization) and with mononuclear cells infiltration. Complete lysis of some necrosed muscle fibers and replacement by inflammatory cells were also detected in many examined cases (Fig.1E, F). Inflammatory edema with myocardial hemorrhage and vasculitis were also detected. The heart of adult animals showed mild to moderate lymphohistiocytic, non-suppurative myocarditis (Fig.1E).
Hyaline degeneration and Zenker,s necrosis of myofibers of the muscular layer of the tongue, lips, gums, omasum, abomasum, and rumen with focal myositis are usually present. The lung showed variable degrees (ranged from mild to severe) of various types of pneumonia (lymphocytic, hemorrhagic, serous, fibrinous pneumonia, and bronchopneumonia). All that forms of pneumonia were seen alone or mixed with each other's as serohemorrhagic or serofibrinous (Fig.2A, B). The liver showed mild to moderate hydropic degeneration of hepatocytes with varying degrees of coagulative necrosis and periportal inflammation (Fig.2E).
The lung was almost unaffected except in about 25% of the affected buffaloes, which showed mild pneumonia. (Fig.2C). Mild hydropic degeneration of hepatocytes (Fig.2F) and moderate lymphocytic enteritis were also observed in the affected buffaloes.
3.4. Description of selected FMDV samples assigned in 3.4. PCR analysis, Detection of FMDV by PCR and VP1 region sequencing;
PCR and RT-PCR analyzed blood and tissue samples for the presence of viral RNA. Conventional RT-PCR was employed to detect and serotyping FMD in field samples representative for different ages of both species. The PCR results for universal primer were then examined using specific primers for each serotype. All the results proved that the serotype (O) was the responsible serotype of that outbreak. Several selected positive samples were sequenced and submitted to the GenBank (Table.5).
3.5. Nucleotide and amino acid (aa) identities of VP1 region (1D gene) between isolated and other reference FMDV
The nucleotide and its deduced aa sequence alignment analysis of gene encoding for
VP1 region was performed between isolated FMDV and 30 references FMDV using blast sequence analysis program of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and MEGA6 program. The nucleotide sequence identity of FMD_EGY1_2017showed 96% with FMD_EGY3_2017, 94% with FMD_EGY4_2017and 90% identity with FMD_EGY2_2017.FMD_EGY2_2017showed 95% with FMD_EGY3_2017, and 94%, with FMD_EGY4_2017. FMD_EGY3_2017 revealed 96% nucleotide identity with FMD_EGY4_2017. The amino acid sequence identity of FMD_EGY1_2017 revealed 88% with FMD_EGY3_2017 while its aa identity with FMD_EGY4_2017and FMD_EGY2_2017 were 87% and 79%, respectively. FMD_EGY2_2017showed 84% aa identity with FMD_EGY4_2017 and 82% with FMD_EGY3_2017. FMD_EGY3_2017 revealed 91% aa identity with FMD_EGY4_2017
3.6. Alignment analysis of nucleotide and deduced amino acid sequences of VP1 region
Alignment analysis of nucleotide and its deduced amino acid sequences of isolated FMDV were performed. The complete genome for FMDV serotype O (A.N: NC_004004.1) was considered a reference strain, and nucleotide mutation, insertion, and deletion were observed. The nucleotide sequence of 4 isolated FMDV showed mutation at position 3241 (C→T), 3275 (T→ G), 3296 (A → G), 3300 (G → A), 3302 (T → C), 3335 (C → T), 3338(T → G, A, C), 3347 (T → C), 3364(A→G), 3365 (A→ C), 3402 (G → T), 3437 (A → C,T), 3470(G →A), 3516 (A→G), 3578 (T→A, C), 3579 (C →T), 3584 (G → T,C), 3587(C→ T), 3596 (A→ G). Most of these mutation points in 4 isolated FMDV were similar to that of other reference strains of Egypt 2013 and 2014. The nucleotide sequence of FMD_EGY1_2017 showed mutation at positions 3242(G A), 3287 (C →A), 3491 (G → C), 3512(A →G), 3530 (C → T), insertion at two positions 3541, & 3572. The nucleotide sequence of FMD_EGY2_2017 showed insertion at six positions 3541, 3678, 3692, 3701, 3713, and 3731. Nucleotide deletion in FMD_EGY1_2017and, FMD_EGY3_2017 and FMD_EGY4_2017 at position 3692 (Fig.3, 4). FMDV serotype O Vaccinal strain used in Egypt display several points of mutation at positions 3245, 3248, 3260, 3266, 3272, 3290, 3299, 3305, 3311, 3327, 3353,3373,3377, 3380, 3387, 3395, 3401, 3404, 3410, 3416, 3419, 3428,3432, 3434, 3449, 3452, 3456, 3461, 3464, 3482, 3500, 3517,3518, 3521, 3522, 3563, 3590, 3602. These points were somewhat similar to the old FMDV isolated from UKG2001, EGY2006, 2009, and 2010 but it was unidentical with the present study's FMDV isolates. The amino acid of 4 isolated FMDV displayed mutation at several positions includes 1107(Arginine →Glycine), 1111 (Tryptophan →Arginine), 1114 (Threonine → Alanine), 1115 (Serine, Glycine → Aspartic Acid), 1116 (Alanine, Proline → Serine), 1127 (Serine, Threonine → Proline), 1131(Cysteine → Arginine). Other points mutation at 1149, 1161, 1172, 1186, 1187, 1233, 1241, 1243, and 1247 were also noticed (Fig.5).
EGY/1/2017 display some point mutations at position 1179, 1192, 1227and 1240. Insertion also occurred in EGY/1/2017 at three positions Glutamine at position 1196 by Glycine at position 1206 and Proline at position 1240. EGY/2/2017 showed some insertion points at 1196, 1242, 1251, 1254, 1256, while EGY/4/2017 has one insertion point at 1232.
3.7. Phylogenetic analysis based on nucleotide and deduced amino acid sequences of VP1 region (1D gene)
The nucleotide sequences of the VP1 gene of 24 reference strains and four isolated FMDV sequences were analyzed using the MEGA6 program. The Phylogenetic tree showed two clusters. Cluster Ι contain two sub-clusters; the first subcluster contains O/ETH/3/96, while the second sub-cluster contains two branches the first contains the four isolated FMDV, Qaliubia/EGY/2013, EGY/24/2013, EGY/10/2014, EGY/18/2014, EGY/16/2014, 2/Giza/EGY/2014, 3/Giza/EGY/2014, Fayoum/EGY/2014 and SUD/8/2008. The second branch contains SUD/3/2008, SUD/4/2008, NIG/15/2009, NIG/5/14, NIG/4/14, NIG/6/14, NIG/3/14, NIG/1/14, NIG/9/14, and NIG/7/14. Cluster 2 has two sub-cluster; the first contains EGY/8/2006, Egy/Qaliubiya/2009, Egy/Sharquia/2009, and the second contain Egy/Menoufia/2010, Egy/Sharquia/2010, UKG/8098/2001, UKG/7675/2001, UKG/7038/2001, and EGY/3/93 vaccine (Fig. 6).