Pathological and genetic characterization of foot and mouth disease viruses collected from cattle and water buffalo in Egypt

Foot-and-mouth disease (FMD), a highly contagious viral disease caused by FMD virus (FMDV) that threatens Egypt’s livestock industry. FMDV causes severe economic losses in the livestock, with restriction of international trade from endemic regions. Surveillance for FMDV serotypes circulating in Egypt is urgently needed to assess the epidemiological situation in the country. FMD outbreaks reported in Egypt in between December 2016 and January-March 2017. A cross-sectional study was conducted to identify the FMDV serotypes responsible for the outbreaks and to collect information on the virus’s morphopathological effects. Postmortem tissue and clinical samples (oral swabs, vesicular fluids from ruptured vesicles, and blood) were collected from recently deceased and infected animals. Pathological examination revealed classical FMD lesions as vesicular and erosive lesions on epithelial tissues with non-suppurative lymphoplasmacytic myocarditis. Phylogenetic and sequencing analyses demonstrated that FMDV serotype O, EA-3 topotype, VP1 is the prevalent serotype responsible for the pathological alterations and the high mortality in young calves, adult cattle, and water buffalo. The outcomes indicate continuous mutations in the circulating FMDV, which result in the occasional failure of vaccination. Based on these findings, extensive continuous monitoring and serotyping of the existing circulating FMDV isolates and regular vaccination with reevaluation of the currently used vaccine in Egypt are recommended to prevent the recurrence of such outbreaks.


Introduction
FMD is an acute, highly contagious transboundary disease of domestic and wild cloven-hoofed animals, resulting in severe economic damage to livestock industries [1][2][3].FMDV outbreaks have been reported recently in many worldwide areas, such as Nigeria [4], Egypt [5,6], Ethiopia [7], and other FMD-free countries, including the United Kingdom [8].FMD is an enzootic disease, and FMDV belongs to the aphthovirus genus, Picornaviradae family, and has seven serotypes (A, O C, Asia-1 and South African Territories (SAT) 1, SAT2, SAT3), with distinct genetic, antigenic and immunologic features [9].FMD genome is linear, non-segmented single-stranded RNA with approximately 8500 nucleotide bases long, surrounded by an icosahedral capsid consisting of four structural proteins VP1, VP2, VP3, and VP4 [4,7,10].Due to the presence of serotype-specific amino acids, VP1 nucleotide sequencing aids in the determination of variation, geographical circulation, genetic correlations, and differentiations among the various variables of FMDV serotypes [6,[11][12][13].The disease is characterized by a high morbidity and mortality rate in young animals and low mortalities in adults (approximately 5%) [14].The high mortality rate in young animals due to cardiac degeneration and necrosis is estimated to exceed 50% [14,15].
Variable clinical signs were seen in the affected animals.Nevertheless, most of these signs were vesicular lesions on the oral cavity, feet, tongue, snout, teats, loss of appetite, fever, lameness, and drop in milk production [3,[16][17][18].Several outbreaks occurred in many countries, either the endemic settings (such as Africa, Asia, and South America) or even the places that are free from FMDV (e.g., Korea, Japan, Netherlands, United Kingdom, and France) [19][20][21].Recently, during the last few years, Egyptian farm animals have been overwhelmed with FMD outbreaks despite applying the vaccination regime in these farms, leading to higher economic losses seasonally.O, A, and SAT2 were the most frequently reported prevalent serotypes in Egypt [6,18,[22][23][24].Infection or vaccination with a specific serotype does not provide crossprotection against other serotypes, as the above-mentioned seven serotypes have a broad spectrum of antigenically distinct subtypes because of the high mutation rate [25][26][27].Therefore, continuous surveillance of the circulating FMDV serotypes is urgently required to identify the most suitable vaccine candidate [28] to control FMD.Hence, the current study aimed to genetically characterize the FMDV strains responsible for the outbreaks during 2016-2017, in addition to genetically correlating the isolated serotype with the recently isolated FMDV strains during the last outbreaks in Egypt.Moreover, documentation of the up-to-date morphopathological pictures resulted in high mortality in the affected cattle and buffalo.

Study areas
This study was conducted in El-Beheira provinces in Egypt (Fig 1) where FMDV outbreaks occurred between the end of 2016 and the beginning of 2017.Despite vaccination, veterinarians and owners have observed high mortality rates in calves as well as high mortality rates in cattle and buffalo older than two years.A comprehensive cross-sectional study was conducted to collect samples from a variety of diseased animals suspected to be infected by FMDV.The samples were collected from different clinically infected herds (these herds were FMDV vaccinated).Diseased animals were carefully examined, and the clinical samples (vesicular fluids, blood, serum, and oral swabs) were submitted to the Pathology and Clinical Pathology Lab at the Faculty of Veterinary Medicine, Damanhur University, for diagnosis and investigation.

Animals and clinical signs
The study was performed on five (5) FMDV vaccinated herds (one buffalo, one cattle, three mixed containing cattle and buffalo), as well as eight (8) FMD diseased and dead animals / herd were collected.A total representative of 40 FMD-positive infected animals from five herds were included in that study.Diseased animals displayed severe salivations, depression, loss of appetite, and vesicular lesions in the mouth, tongue, udder, and foot.

Gross pathology and postmortem examination
Postmortem (PM) examinations were conducted immediately on recently deceased animals, according to animal welfare abattoirs and the Research Ethics Committee of Faculty of Veterinary Medicine, Damanhour University, Egypt (DMU-VETMED-PATH-2019-/0145).PM tissues from (the mouth, tongue, foot, rumen, reticulum, omasum, abomasum, liver, lung, intestine, and heart) (n = 170, 4 to 5 PM tissues from the above-mentioned organs/from each dead animal) were collected from recently dead animals during necropsy.Collected samples were washed (2-3 min under tap water), cleaned, labeled, and preserved in a 10% neutral buffered formalin for further histopathological examinations [29].

Samples collection and preservations
Vesicular fluid (from freshly ruptured vesicles and unruptured vesicles) (n = 31) and oral swab samples (n = 18) were collected in specific tubes containing 50% glycerol and 0.04 M phosphate buffer [30].The collected samples were labeled and kept in ice box containers during transportation to the regional labs at the faculty of Veterinary Medicine, Damanhur University/ Egypt, where they were preserved at -80˚C until processing [31].Conversely, blood samples from suspected animals (5-10 ml blood/ each animal) were collected from the jugular vein on anticoagulant tubes containing EDTA and RNA later and stored at -20˚C until RNA extraction.

Virus isolation
Epithelial tissue samples of unruptured and recently ruptured vesicles and oral swab samples were cultured in BHK-21.Cell line(The kindly cell line supplied by Animal Health Research Institute, Virology department, Dokki, Giza).Briefly, tissue samples were ground in sterile sand, pestle, and mortar containing tissue culture medium containing specific antibiotics (penicillin, polymyxin B sulfate, neomycin sulfate, and Mycostatin) [32].Afterward, the tissue samples homogenate were centrifuged at 2000 rpm for 15 min for clarification and then filtered through membrane filter paper with a pore size of 0.22 μm.Subsequently, the prepared samples were incubated with Confluent monolayer cell cultures, 10% fetal calf serum, and minimum essential medium (MEM).Normal non-infected cells served as control.0.2-0.4mL filtered epithelium samples were inoculated to (BHK-21) to investigate the cytopathic effects (CPE) monitored for 24-72 hours according to [7,31,33].Cultured samples that did not show CPE were frozen at -70˚C and reinfected onto BHK-21 cells for a second trial.Positive samples were also examined by the RT-PCR technique.

Histopathology
Fixed specimens in 10% neutral buffered formalin were routinely processed through dehydration in ascending grades of ethanol, cleared in xylene, and embedded in paraffin blocks.Paraffin sections were prepared at 4-5μm thickness on glass slides.The sections were then stained with hematoxylin & eosin and examined using the light microscope [29].Based on previously described grading systems, the severity of gross and histopathological lesions between calves and adults was evaluated [34][35][36][37].

RNA extraction and cDNA synthesis
Total RNA extraction from different samples (vesicular fluid, oral swabs, and blood) was performed using a high pure Qiagen (All Prep1 DNA/RNA Mini kit, Germany), according to the manufacturer's instructions.The extracted samples were then reverse-transcribed according to the manufacturer's instructions using HiSenScript™ RH (-) cDNA Synthesis kit (NtRONBiotechnology, Korea) according to the manufacturer's instructions.The obtained cDNA was used for Conventional PCR and RT-PCR using specified primers (Table 1) to amplify FMDV serotypes.

Conventional PCR analysis
Each cDNA was used as a template for PCR amplification of the 5'UTR of the FMD virus genome using 1F and 1R universal primer for all FMDV serotypes (Table 2), according to [38].All PCR reaction was carried out using an Eppendorf thermal cycler (SENsQUEsT labcycler).The PCR amplification was performed in a 25 μL volume containing 4 μL DNA, 2 μL dNTP, 1 μL of each primer (10μmol), 2.5μL 10× Ex Taq buffer, 0.25 μL Ex Taq polymerase (Takara, Kyoto, Japan), and 14 μL RNA, DNA free water.The PCR condition for 1F and 1R universal primer was 94˚C for 5 min, one cycle; 94˚C for 1 min, 55˚C for 1 min, followed by 35cycles at 72˚C for 2 min with a final extension for 7 min, one cycle.Initially, All suspected samples were tested utilizing a universal primer.Subsequently, positive samples were tested again using serotype-specific RT-PCR (Table 2) by amplifying 1D (VP1region), which is the most variable region of the genome among the seven serotypes utilizing serotype-specific primer [12].The PCR condition for A-1C562/ EUR-2B52R and C-1C536/ EUR-2B52R primer were set to 94˚C for 1 min, one cycle; 62˚C for 1 min, followed by 35cycles at 72˚C for 2 min with final extension for 7 min, one cycle.Slight modifications were applied to PCR conditions for other primer sets for detecting other serotypes.As the PCR condition for O-1C283F/ EUR-2B52, O-1C244F / EUR-2B52R primer sets were 94˚C for 30 seconds, one cycle; 3 to 5 cycles at 60˚C for 1 min, followed by 35cycles at 72˚C for 2 min with a final extension for 7 min, one cycle.All primers used were supplied by Sigma, Aldrich, Japan.

DNA purification, sequencing and sequence analysis
According to manufacturer instructions, PCR amplicons were purified and prepared for sequencing using Qiagen1 gel extraction kits.
The purified PCR product of the VP1 positive sample was sequenced using its gene-specific primer by 3-500 Genetic analysis, AB applied Biosystem, at colors laboratory, Elmaadi, Cairo, Egypt, EG11431.
The obtained nucleotide sequences of FMDV-positive samples were edited using the sequence scanner software program (http://www.appliedbiosystems.com).The edited sequence was computationally compared with other FMDVs for homology and phylogenetic analysis using Mega 6 program software (www.megasoftware.net/).The phylogenetic trees were generated using the neighbor-joining (N-J) tree method, and the liability of internal branches was assessed by 1000 bootstrap replication.The reference sequences of the FMDV VP1 gene were retrieved from the GeneBank database, and their accession numbers are listed in S1 Table.

Clinical signs expressed by FMDV-infected animals
In cattle, clinical signs varied slightly with age; in adult cattle (over two years), fever ranged from (39˚C-40˚C) and persisted for 3-4 days with anorexia.However, in calves, under six months of age, fever reached up to 41˚C in some cases, and some animals were suddenly found dead without previous clinical signs.Severe salivation (Fig 2A) with vesicular lesions in the mouth and tongue were seen.Hemorrhage of the oral commissure with subsequently development of ulceration in the upper lips, tongue tips, dental pad, and upper third of the tongue (Fig 2B -2C).In some severely affected cases, the lesions of the foot accompanied by a claw detachment.These lesions were visible on the feet of adult animals older than two years and calves older than six months but not on the feet of animals younger than six months.Animals suffering from severe ulceration of the digits exhibited obvious lameness.Conversely, clinical signs in buffalo were mild to moderate, mouth lesions were mild in the form of small shallow vesicles with scanty fluid.The foot lesions were absent in calves under six months of age, while the calves aged (6 months to 2 years) and adults (over than two years) showed only mild vesicles and erosion mainly at the bulb of the heal, so lameness was unclear.Prior to death, certain animals exhibited cardiac arrhythmia, followed by dyspnea and grunting.
According to the history of the owners, and veterinarians, the mortality rate was extremely high, particularly among young animals less than six months old, where it reached 100 percent for both cattle and buffalo calves.In addition ahigh fatality rate were recorded also in cattle and buffalo older than six months.

Virus isolation and characterization
Positive epithelial tissues and oral swabs samples collected from calves, cattle, and buffalo during 2016-2017 FMDV outbreaks demonstrated a CPE characterized by swelling, rounding, clumping, granulation, or detachment of monolayer cells from the culture flasks surface, followed by rabid destruction of (BHK-21) cell cultures within 48 to 96 hrs post-inoculation in tissue cultures, indicating the presence of an infectious virus.Samples that did not develop CPE after two passages in cell culture were considered virus-free, and all labeled samples that belonged to these samples were eliminated from the study.

Gross pathology
The mouths and interdigital spaces of the adult cattle and buffalo exhibited numerous blisters, erosions, and ulcerations.Vesicles, irregular-shaped erosions/ulcers of variable size, were typically found on the torus lingua and anterior third of the tongue and more frequently on the gingiva, lip, and dental pad.Hyperemia, ulceration, heel bulb detachment, and sole separation were also identified.Calves exhibited moderate to the severe oral cavity, omasum, abomasum, and rumen erosions and ulcers.As the disease progressed rapidly and the animal died, no foot lesions were detected.All dead calves exhibited myocardial hemorrhage with yellowish-gray streaks in the myocardium (Fig 2D -2F).In addition, similar ulcers were observed in the abomasum and ruminal pillars (Fig 2G).Myocardial hemorrhage (petechial and ecchymotic) with various degrees of myocardial necrosis were also observed.

Histopathological findings
The histopathological changes of the cornified epithelial tissues of (mouth, tongue, coronary bands, omasum, abomasum, and rumen) were characterized by hydropic degeneration with increased cytoplasmic eosinophilia, micro-abscess formation, hyperkeratosis and subsequent mononuclear cell and granulocyte infiltration in the cells of the stratum spinosum.Intraepithelial bullae and vesicular formations were clearly visible (Fig 3A).Severe lesions exhibited erosions, Zenker , s necrosis, and neutrophilic and eosinophilic infiltration of the underlying epithelium (Fig 3B and 3C).Lesions of cornified epithelial tissues (oral cavity and gastrointestinal mucosa (GIT)) were moderate to severe in young calves of cattleand buffalo, adult cattle and buffalo.
The heart was the most severely affected organ and may be the leading cause of death in young calves under six months.Non-suppurative myocarditis, in the form of hyaline degeneration and necrosis of myocytes (hyalinization) with mononuclear cell infiltrations, were the main observed pathological lesions in the heart.Moderate non-suppurative myocarditis was seen in the animals aged six months (Fig 3E).Nevertheless, the calves under six months of age in cattle and buffalo suffered from severe myocarditis and died as a result.In severe cases of myocarditis, necrosed muscle fibers are completely lysed and replaced by a large number of inflammatory cells (Fig 3F).Inflammatory edema with myocardial hemorrhage and vasculitis were also detected.Mild non-suppurative myocarditis (Fig 3D ) was not common except in one exceptional case in buffalo aged lower than 2 years and exhpited severe pneumonia.
Hyaline degeneration and coagulative necrosis of myofibers of the muscular layer of the heart (Fig 4A and 4B), tongue, lips, gums, omasum, abomasum, and rumen with focal myositis were also detected.
The lung displayed varying degrees (ranging from mild to severe) of different types of pneumonia (lymphocytic, hemorrhagic, serous, fibrinous, and bronchopneumonia).All that forms of pneumonia were seen alone or mixed with each other's as serohemorrhagic or serofibrinous (Fig 4D -4F).Moderate to severe lung lesions were seen in adult cattle and calves under two years (except the buffalo calves aged 6 moths-2 years) with mild lesions.However, no lesions were detected in the lung of adult buffalo (Fig 4C).
Sporadic cases of examined buffalo and cattle displayed lymphocytic enteritis in the form of leukocytic cell infiltrations of the intestinal villi (Fig 5C and 5D).The liver showed mild to moderate hydropic degeneration of hepatocytes with varying degrees of coagulative necrosis and periportal inflammation (Fig 5A and 5B).

Detection of FMDV by PCR and VP1 region sequencing
Blood and tissue samples extracts were tested using RT-PCR for the presence of viral RNA.Conventional RT-PCR was utilized to detect and serotype FMD in field samples representative of different ages of both species.The PCR results for the universal primer were then examined using specific primers for each serotype.All the results demonstrated that serotype (O) was the responsible serotype for that outbreak.Four samples were sequenced at Elmaadi, laboratory, Cairo, Egypt, EG11431 for genetic characterization of the partial VP1 region, and the nucleotide sequences were submitted to GenBank (Table 2) under accession numbers (LC384395, LC384396, LC384397, LC384398).

Nucleotide and amino acid (aa) identities of VP1 region (1D gene) between isolated and another reference FMDV
The nucleotide and its deduced aa sequence alignment analysis of gene encoding for VP1 region were performed between isolated FMDV and 30 references FMDV using the blast sequence analysis program of NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and MEGA6 program.The identity of the four isolates' nucleotide sequences ranged from 90% to 96%, with the lowest identity between FMD_EGY1_2017 and FMD_EGY2_2017 and the highest identity between FMD_EGY3_2017 and FMD_EGY4_2017.The identity of the four isolates' amino acid sequences ranged from 79% to 91%.The highest identity was between FMD_EGY3_2017 and FMD_EGY4_2017 at 91%, and the lowest identity was between FMD_EGY1_2017 and FMD_EGY2_2017 at 79%.

Phylogenetic analysis based on nucleotide and deduced amino acid sequences of VP1 region (1D gene)
The nucleotide sequences of the VP1 gene of 35 reference strains and four isolated FMDV sequences were analyzed using the MEGA6 program.The Phylogenetic tree showed two clusters.Cluster 1 contain two sub-clusters; the first sub-cluster contains two branches, the first contains four isolated

Discussion
FMD has been a highly contagious endemic viral disease affecting cloven-hoofed animals in Egypt since the 1950s [39,40].Multiple FMD outbreaks occur annually despite the efforts of the Egyptian government to control and eradicate the virus, and newly emerged lineages of the O, A, and SAT2 serotypes have been identified in Egypt [17,18,41].Despite the mandatory application of locally-produced vaccines containing A, O, and SAT2 lineages, FMD outbreaks continue to recur in Egyptian farms [18,[42][43][44], with a severe, devastating economic impact on the livestock performance and production in Egypt [17,41,44].The highly contagious nature of the disease and its significant adverse economic impact on the livestock industry motivated us to conduct a comprehensive overview of FMD's pathological alterations in young calves, adult cattle, and buffalo during 2016-2017 outbreak in Egypt, which resulted in high mortality rates not only in young livestock but also in adults.In addition, we recorded the isolated serotype responsible for that outbreak and compared our results with those of recent studies.
In the current study, vesicle formations at various epithelial sites, including the oral cavity, feet, teats, and ruminal pillars, were observed, followed by erosion and ulcers that often extended during the illness stage.These lesions are similar to the classical form of FMD [18,45,46].Foot lesions ranged from moderate to severe in calves and adult animals.However, no foot lesions were detected in calves aged 1-6 months due to the rapid progression of the disease; the infected calves died prior to the development of foot lesions.According to the owners of the animal herds and veterinarians, the mortality rate in this study was 100% for calves under six months old in both cattle and buffalo, as reported in previous studies [3,[47][48][49].This finding was due to viral tropism in the cardiac muscles, resulting in severe myocarditis [41,47].Therefore, a high calf mortality rate was anticipated in our study.In contrast to previous FMD studies, which indicated that FMD did not result in high mortality rates in adult animals [15], the high mortality rates in adult buffalo and cattle were unexpected.Buffalo act as reservoirs for specific serotypes; if infected by FMDV, they develop mild or no clinical signs [40,50].However, serotypes A and O were under control by the bivalent vaccine (A&O) used in Egypt, as recorded previously [44].A newly emerging strain belonging to serotype O, the Europe-South America topotype (EURO-SA) lineage, was recorded recently in Egypt and resulted in severe economic losses [18].Furthermore, the SAT2 serotype resulted in significant mortalities in young and adult cattle and buffalo during the 2012/2013 outbreak in Egypt [22].This result suggests higher virulence and aggressiveness of the FMDV field circulating serotype, which may be reached Egypt via movements and importation of animals from FMD-infected areas with continuous viral mutations leading to severe histopathological lesions scoring, especially in the hearts and lungs of infected cattle and buffalo.
Heart lesions presented here as myocardial degeneration, Zenker's necrosis, non-suppurative myocarditis, and intense mononuclear cell infiltration in young and infected adult animals.The lesions have varying degrees of severity [51,52].Consistent with the myotropic nature of FMDV [47], identical lesions of non-suppurative myositis were also detected in the examined tongue, cheeks, omasum, abomasum, and ruminal muscles.Serofibrinous, serohemorrhagic, and bronchopneumonia were frequently observed, particularly in one-month-totwo-year-old calves.Serofibrinous, serohemorrhagic, and bronchopneumonia were frequently observed, especially in calves aged one month to 2 years [53].In a few cases, mild-to-moderate hepatic lesions manifested as hepatocellular degeneration and congestion of blood sinusoids, similar to those described by [24,54].The virus's extra-epithelial tropism may be triggered by hepatic degeneration and necrosis.
In our study, conventional RT-PCR was used to detect FMDV using a universal primer [38].The decrease in the positive rate of the total collected samples may be due to the unstable nature of FMDV RNA and the detection limit of this primer set [55].Of the seven immunologically different serotypes, A, C, SAT1, 2, 3 (Southern African Territories) and Asia1 [56] circulating worldwide, only serotypes O, A, and SAT2 are prevalent in Egypt [22,43,57], while types O, A, SAT1, SAT2, and SAT3 circulate in Africa [58].In our study, only serotype O, EA-3 topotype was detected using serotype-specific primers.Serotype O is the most prevalent serotype worldwide [30], alone or with other serotypes, and is responsible for several outbreaks in Egypt [18,42,59] and other countries, such as Argentina [60], Sri Lanka [61], India [62] and neighboring countries as Palestine, Libya [3,18,63] and Sudan [59,64].Isolation of FMDV serotype O, EA-3 topotype from Egyptian farms during this FMDV outbreak, however controlling this serotype (O) and serotype A by vaccination in Egypt [44], indicating that the FMDV constitutes an endemic severe health problem for the livestock industry in Egypt [18].
The phylogenetic results obtained from 636 nucleotides of VP1 completely match the obtained when 2208 nucleotides of the complete P1 polyprotein (the genomic region encode all four structural proteins VP1-4) [65].FMDV serotypes are usually affected by spontaneous mutation points in the VP1 region [69], representing the capsid's most variable part and containing the leading immunogenic site [70,71].Nucleotide sequencing of the VP1 region and phylogenetic analysis has been used extensively to determine the relationships between field isolates; therefore, nucleotide sequencing of the VP1 region and phylogenetic analysis has been used to determine the relationships between our field isolates and other recently isolated Egyptian isolates, and other isolates from some African countries as well as the vaccinal strain used for serotype O in Egypt.The alignment and phylogenetic analyses of the present four isolates revealed that; the new four isolates are closely related to each other by 95% nucleotide identity and to the other reference strains as Egypt 2013 [72], 2014 [73], SUD/8/2008 [74], and Nigeria isolates [75,76], their identities were 96%, 95%, 91%, 89% respectively.All previously mentioned strains belonged to the EA-3 topotype [67].In the same scene, the present four isolates were distant from other Egyptian strains 2006, 2009, 2010, 2022, united kingdom strain 2001, and the vaccinal strains used in Egypt (O1 Manisa, pan Asian II (EGY/2010), O/TUR/5/ 2009, Al-Sharquia 72, O/KEN/77/78), all these strains belong to ME-SA topotype, as previously recorded [59].The presence of some previously isolated strains within a separate subclade in the phylogenetic tree further enforces our conclusion that the present isolates of FMDV have increased virulence against bovine calves, which could be related to several point mutations in VP1 gene regions of the RNA genome of FMDV [17].
The increased virulence strains among cattle and buffalo in Egypt could be attributed to the introduction of new viral strains through uncontrolled transboundary movements of animals [18,64,77].The sequence and phylogenetic analyses revealed the presence of older isolates and vaccinal strains in separate clusters and subclades far away from the present isolate and recently isolated reference strain, which was grouped in one cluster.Moreover, the presence of sequence divergence between the recent and older isolates and a vaccinal strain far away from the present isolates in the phylogenetic tree could explain the insufficient protection of vaccinated animals against infection with the recent strain.Similar conclusions have been reported by Carrillo [65], who mentioned that the topotype classification system has significant value for vaccine selection.
Finally, we suggest that this increased mortalities in calves, young adults, and adult animals of cattle and buffalo in the last crises of FMD can be attributed to the increased virulence and/ or point mutations of the RNA genome circulating strains among animals in Egypt.
Eventually, these findings suggest extensive surveillance and serotyping of the existing field isolates, as well as routine vaccination and reevaluation of the current vaccine in Egypt.Therefore, it is preferable to use vaccines containing the current field strains of FMDV to limit the recurrence of these outbreaks.

Fig 2 .
Fig 2. Clinical and gross pathological lesions in calves, cattle and buffalo of FMDV naturally infected animals.(a) Heavy salivation of infected cattle.(b) Hemorrhagic area of sub mucosa of oral commissure of a calf (arrows).(c) Ulcer formation of dental pad with secondary bacterial infection.(d) Marked whitish necrosed areas all over the heart (black star).(e) A longitudinal section in the heart ventricle and interventricular septum, showing yellowish to whitish streaking of myocardial necrosis (black star)"tiger heart".(f) cardial hemorrhage alover the heart atrium, ventricles and papillary muscles (arrows).(g) Focal ulcerative lesions of ruminal pillars (arrows).https://doi.org/10.1371/journal.pone.0291970.g002

Fig 3 .
Fig 3. Histopathological lesions in FMDV naturally infected animals.(a) Vesicles formations (arrows) with elevation of superficial epithelium in the stratified squamous epithelium of dermis, H&E, X200.(b) Vesicular stomatitis of the dental pad of infected calf characterized by leukocytic infiltrations of mucosal and submucosal layer of cornified epithelial tissue (arrows), H&E, X200.(c) Zenker , s necrosis (arrows) of muscular layers of stratified squamus epithelim with edema inbetween (arrowheads) of cattle, H&E, X200.(d) Mild myocarditis of the myocardium of buffalo with mild lymphocytic cell aggregations (arrows), H&E, X400.(e) Moderate myocarditis of the myocardium of buffalo calf (less than 2 Y) with moderate lymphocytic cell aggregations (arrows) and mild myocardial muscle necrosis (arrowheads), H&E, X400.(f) Severe non-suppurative myocarditis of heart of calf with a significant number of lymphocytic cell aggregations (arrows) with complete lysis of necrosed muscle fibers (arrowheads) and replacement of this muscles by a large number of inflammatory cells (arrows).The black star, referring to the myocardial muscle that still normal not necrosed, H&E, X400.https://doi.org/10.1371/journal.pone.0291970.g003