Benha University

Faculty of Veterinary Medicine Animal medicine Department

Studies on Foot and Mouth Disease Virus type O1,A in Sheep

Thesis presented By

Amr Ismail Hassan B.V.Sc Cairo University (2001)

M.V.Sc 2007, Infectious Diseases, Benha University 2007

Under supervision of

Prof. Mohammed Hassanin Ebeid

Professor of Infectious Diseases, Faculty of Veterinary Medicine,Moshtohor Benha University

Prof. Faisal Khalil Hamoda

Professor of Infectious Diseases, Chairman of Animal Medicine Dept.Faculty of Vet. Medicine, Moshtohor, Benha University

Prof. Adel Mohamed Hassan Azab

Chief Researcher, Veterinary serum and Vaccine Research Institute, Abbassia

A Thesis Submitted to

Benha University For the degree of

PhD of Veterinary Medical Science (Infectious Diseases)

2011

Acknowledgment I wish first to thank forever ALLAH for helping me to complete this

work and giving me every thing I need. I would like to take this opportunity to express my cardial

gratitude and deepest thanks for Prof. Dr. Mohamed Hassanin Ebeid, Professor of Infectious Diseases, Faculty of Veterinary Medicine, Benha University, for his valuable help and encouragement giving to me during this work.

I wish to express my deepest appreciation and sincere gratitude to Prof. Dr. Faisal Hamoda, Professor of Infectious Diseases and Head of Animal Medicine Department Faculty of Veterinary Medicine, Benha University and Prof. Dr. Adel Mohamed Hassan Azab and Prof. Dr. Laila Ismail EL-Shahawy, Chief Researcher, Foot and Mouth Disease Department, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo, for their suggestion and supervision of this work. Also I would like to express my deepest thanks for Pro. Dr. Abd El-Moneim Mohamed Mostafa Professor of Infectious Diseases , Benha Universty.

My great appreciation to the late Prof. Dr. Sinan El-Nakashly, Prof. Dr. Magdy Abdel Atty, Prof. Dr. Hosam Gamal El-Din, Prof. Dr. Abu Baker Aggour, Dr. Samir Mohammed Ali and Dr. Wael Mossad for their assistance to complete the plan of this work.

Also Special thanks for Dr. Safi El-Dean Mahdy, Dr. Ehab El-Sayed, Dr. Mohammed Gamil, Dr. Akram Zakarya , Dr. Assem Abou Bakr and Dr. Ahmed Fathy.

My sincere recognition to all the staff members of Foot and Mouth Disease Department, Veterinary Serum and Vaccine Research Institute, Abbassia, Cairo,

and all staff members of Animal medicine Department, Benha University.

Amr Ismail

List of contents

1. INTRODUCTION ............................................................................................................................ 1

2. LITERATURE ................................................................................................................................. 3

2.1. FOOT AND MOUTH DISEASE ......................................................................................................... 3 2.2. FMD VIRUS ................................................................................................................................. 3 2.3. TYPING AND SUB TYPING OF FMD VIRUS ...................................................................................... 4 2.4. EPIDEMIOLOGY OF FOOT AND MOUTH DISEASE........................................................................... 5

2.4.1. Foot and mouth disease in Egypt ........................................................................................ 5 2.4.2. Prevalence of Foot and mouth disease in the world ............................................................ 7

2.5. TRANSMISSION OF FMD .............................................................................................................. 9 2.6. PATHOGENESIS OF FMDV ......................................................................................................... 11 2.7. FOOT AND MOUTH DISEASE IN SHEEP ........................................................................................ 13 2.8. PERSISTENCE OF FMD .............................................................................................................. 15 2.9. ANTIGENIC COMPONENTS OF FMD VIRUS .................................................................................. 18

2.9.1. Structural proteins............................................................................................................ 18 2.9.2. Non- structural proteins (NSP)......................................................................................... 19

2.9.2.1. Virus Infection Associated Antigen (VIA) .............................................................................. 19 2.10. INACTIVATION OF FMDV........................................................................................................... 21 2.11. IMMUNITY AGAINST FOOT AND MOUTH DISEASE ........................................................................ 21

2.11.1. Active Immunity ............................................................................................................... 21 2.11.1.1. Immunity after infection ........................................................................................................ 21 2.11.1.2. Immunity after Vaccination ................................................................................................... 24

2.12. ISOLATION AND IDENTIFICATION OF THE VIRUS .......................................................................... 27 2.13. SERUM NEUTRALIZATION TEST (SNT) ........................................................................................ 27 2.14. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) .................................................................... 29 2.15. 3ABC-ENZYME LINKED IMMUNOSORBENT ASSAY (3ABC-ELISA) ............................................... 33 2.16. POLYMERASE CHAIN REACTION .................................................................................................. 35

3. MATERIAL AND METHODS ...................................................................................................... 40

3.1. MATERIALS ................................................................................................................................ 40 3.1.1. Animals ............................................................................................................................ 40

3.1.1.1. Sheep .................................................................................................................................... 40 3.1.1.2. Unweaned baby mice ............................................................................................................. 40

3.1.2. Sheep Serum samples ....................................................................................................... 40 3.1.3. Epithelial tissue samples................................................................................................... 43 3.1.4. Oesophageal pharyngeal (op) fluids ................................................................................. 43 3.1.5. Reference virus................................................................................................................. 43 3.1.6. Tissue cultures (established cell lines) .............................................................................. 43

3.1.7. Chemical reagents ............................................................................................................ 44 3.1.7.1. Media .................................................................................................................................... 44

3.1.7.1.1. Growth medium ........................................................................................................ 44 3.1.7.1.2. Maintenance medium ............................................................................................... 44

3.1.7.2. Bovine serum ......................................................................................................................... 44 3.1.7.3. Trypsin .................................................................................................................................. 44 3.1.7.4. Sodium bicarbonate solution ................................................................................................. 44 3.1.7.5. Neomycin .............................................................................................................................. 44 3.1.7.6. Nystatine (antifungal) ............................................................................................................ 45 3.1.7.7. Tween 20 ............................................................................................................................... 45 3.1.7.8. Crystal Violet Stain ................................................................................................................ 45

3.1.8. Reagents used in ELISA ................................................................................................... 45 3.1.8.1. Coating Buffer ....................................................................................................................... 45 3.1.8.2. Phosphate Buffer Saline (PBS)and Bovine Albumin.............................................................. 46 3.1.8.3. Washing Buffer ..................................................................................................................... 46 3.1.8.4. Phosphate Citrate Buffer ....................................................................................................... 46 3.1.8.5. Substrate ............................................................................................................................... 47 3.1.8.6. Stopping Solution .................................................................................................................. 47 3.1.8.7. Conjugate .............................................................................................................................. 47 3.1.8.8. Tween 20 ............................................................................................................................... 47 3.1.8.9. Blocking buffer...................................................................................................................... 47

3.1.9. Prio Check FMDV Non Strctural protein ......................................................................... 47 3.1.10. Nucleic acid recognition reagents and kits ....................................................................... 48

3.1.10.1. Total RNA purification kit ..................................................................................................... 48 3.1.10.2. RNA extraction reagents ........................................................................................................ 49 3.1.10.3. RT-PCR kit ............................................................................................................................ 49 3.1.10.4. PCR kit .................................................................................................................................. 50 3.1.10.5. Primers .................................................................................................................................. 50

3.1.11. Materials used for detection of RT-PCR & PCR products ................................................ 51 3.1.11.1. Tris Acetate EDTA (TAE) gel electrophoresis buffer (40X) .................................................... 51 3.1.11.2. Loading Buffer ...................................................................................................................... 51 3.1.11.3. Agarose ................................................................................................................................. 51 3.1.11.4. Ethidium bromide (Eth Br) .................................................................................................... 51 3.1.11.5. Nucleic acid markers ............................................................................................................. 52

3.1.12. Equipment and supplies ................................................................................................... 52 3.1.12.1. Biological safety cabinet ........................................................................................................ 52 3.1.12.2. Cooling centrifuge ................................................................................................................. 52 3.1.12.3. Vortex mixer .......................................................................................................................... 52 3.1.12.4. Water bath ............................................................................................................................. 52 3.1.12.5. Single and multichannel pipettors (microtitre pipette) ............................................................ 52 3.1.12.6. Disposable syringe filters ....................................................................................................... 53 3.1.12.7. Equipment for cell cultures .................................................................................................... 53 3.1.12.8. Inverted light microscope ....................................................................................................... 53 3.1.12.9. Microtubes with attached cap (Microfuge tubes) .................................................................... 53

3.1.12.10. Thermocycler ......................................................................................................................... 53 3.1.12.11. Electrophoresis unit (set) ....................................................................................................... 54 3.1.12.12. Power supply ......................................................................................................................... 54 3.1.12.13. Transilluminator, UV ............................................................................................................ 54 3.1.12.14. Gel Documentation and Analysis system ................................................................................ 54 3.1.12.15. Other equipment .................................................................................................................... 54

a. Refrigerators and Freezers ................................................................. 54 b. Incubators ........................................................................................ 54

3.2. METHODS .................................................................................................................................. 55 3.2.1. Protocol of sheep vaccination ........................................................................................... 55 3.2.2. Preparation of serum samples .......................................................................................... 55 3.2.3. Oesophageal pharyngeal(op) fluids .................................................................................. 55 3.2.4. Preparation of FMD virus ................................................................................................ 55 3.2.5. Baby mice ......................................................................................................................... 56 3.2.6. Serological tests ................................................................................................................ 56

3.2.6.1. Enzyme linked ImmunoSorbent Assay (ELISA) ..................................................................... 56 3.2.6.1.1. Preparation of ELISA antigen .................................................................................. 56 3.2.6.1.2. Titration of the conjugate ......................................................................................... 56 3.2.6.1.3. Testing of the serum samples using indirect ELISA .................................................. 57

a- Coating ....................................................................................... 57 b- Blocking ..................................................................................... 57 c- Serum dilutions .......................................................................... 57 d- Addition of the conjugate ............................................................ 57 e- Addition of the substrate ............................................................. 57 f- Addition of the stopping solutions ............................................... 57 g- Interpretation of the results ........................................................ 57

3.2.6.2. Serum Neutralization Test (SNT) ........................................................................................... 58 3.2.6.2.1. Staining the SNT microplates used in the test Procedures ......................................... 58

3.2.6.3. PrioCHECK FMDV NS test ................................................................................................... 59 a- Day 1 .......................................................................................... 59 b - Day 2 .......................................................................................... 60

3.2.6.3.1. Interpretation of the Percentage Inhibition ............................................................... 60 3.2.7. Molecular detection of FMDV by RT-PCR and PCR........................................................ 61

3.2.7.1. FMD-RNAextraction-(isolation) ............................................................................................ 61 3.2.7.2. One-step reverse transcriptase-polymerase chain reaction (RT PCR) ..................................... 62 3.2.7.3. Polymerase chain reaction (PCR) .......................................................................................... 62 3.2.7.4. Agarose Gel Electrophoresis of PCR Products ....................................................................... 62

4. RESULTS ....................................................................................................................................... 64

4.1. ISOLATION AND IDENTIFICATION OF FOOT AND MOUTH DISEASE VIRUS FROM FIELD SAMPLES..... 64 4.2. REVERSE TRANSCRIPCATION CHAIN REACTION POLYMERASE (R.T PCR) ................................... 66

5. DISCUSSION ................................................................................................................................. 92

6. SUMMARY .................................................................................................................................... 98

7. REFERENCES ............................................................................................................................. 100

Abbreviations and Symbols

µL Microliter BHK21 Baby Hamster Kidney cells clone 21 CFT Complement Fixation Test CPE Cytopathic Effect D.D.W Double Distilled Water DPI days post infection ELISA Enzyme Linked Immunosorbant Assay FMD Foot and Mouth Disease FMDV Foot and Mouth Disease Virus I/P Intra Peritoneal MEM Minimum Essential Media MLD50 Mice lethal dose 50 nm Nanometer. NSPs Non Structural proteins OD Optical density OIE Office International des Epizootie OP Oesophageol Pharyngeal fluid OPD OrthoPhyneyleneDiamine PBS Phosphate buffer saline PD50 Protective Dose 50 RIP Radio-Immuno Precipitation RNA Ribonucleic acid rpm Revolution per minute RT- PCR Reverse Transcriptase Polymerase Chain Reaction SC Sedimentation coefficient SAT South Africa Territories SNT Serum Neutralization Test TCID50 Fifty tissue culture infective dose µL Microliter UV Ultra Violet VIA Virus Infection Associated Antigen VNT Virus Neutralization test VP Virus protein VSVRI Veterinary Serum and Vaccine Research Institute WPI Week post infection WRL World Reference Laboratory of foot and mouth disease

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1. Introduction

Foot and Mouth Disease is difficult to control because of it's highly contagious nature, it's ability to infect different domestic and wild life hosts and it's causation by multiple non-cross-protective virus serotypes, (Rodrieguez and Gurbman 2009).

The occurrence of FMD continues to largely reflect economic prospectively

with countries having eradicated the disease and countries struggling or unable to do so. Geographic isolation can favor FMD eradication. Movement of live animal still constitutes the greatest risk for spread of FMD, followed by trade in animal products.FMD virus continues to evolve, giving rise to new strains that cause periodic upsurges in the number of cases and increase the risk of spread into new areas (Knowles et al., 2007).

FMD is considered enzootic in Egypt and many outbreaks have

recurrently occurred involving most governorates (Mousa etal., 1976; Daoud et al., 1988; and EL-Nakashly et al., 1996). The main causative serotypes are O1 & A (Abd El-Rahman et al., 2006)

Routine prophylactic vaccination has been conducted with locally produced

bivalent inactivated serotypes O1 and A Egypt 2006 vaccine. Outbreaks from serotype O1 was in 2000 and 2006, and other serotypes have not been reported since 1972 when serotype A occurred (Aidaros 2002).

On January 2006 clinical cases of FMD were first recognized in cattle farm

and widely spread in Egypt and the virus was isolated and identified by FMD Department and institute for Animal Health United Kingdom (Knowles et al., 2007).

Diagnosis of FMD is based on clinical signs followed by confirmation by

laboratory tests (Giridharan 2005). RT-PCR assays considers an alternative or complementary to classical serological and viral isolation method due to their higher sensitivity, speed and the fact that the handling of infectious virus is not required (Saiz et al 2003)

Recently, There are increase application of tests that detect foot and mouth

disease virus antibodies to non- capsid proteins (NCP) to assess past or present FMD infection/circulation irrespective of vaccination.

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So, the objective of this study for: Isolation and identification of Foot and mouth disease from field cases.

Reverse Transcripcation Polymerase Chain Reaction (R-T PCR) for

isolated O1 strain. Prevelence of FMDV in different governorate of Egypt using SNT and

ELISA. Detection of NSP antibodies in sera of sheep proved to be positive by

ELISA and SNT in different governorates.

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2. Literature

2.1. Foot and Mouth Disease

Foot and mouth disease is the most important livestock disease in the world in terms of economic impact. The reason is the ability of disease to cause losses of production, also due to hindering on the trade of animal both locally and internationally, and restrictions on the movement of people which affect the tourism sector, James and Rushton (2002). Foot-and mouth disease virus continues to exist and evolve, thus posing a serious threat to the livestock industry worldwide Mohapatra et al. (2009b), It is also highly infectious and economically devastating disease of livestock, Rodriguez and Gurbman (2009).

2.2. FMD virus

Brown (1973) stated that FMD virus is a member of the family Picornaviridae.

Rueckert and Wimmer (1984) stated that FMDV is a member of genus

Aphthovirus of family Picornaviridae. Picorna viral RNA genomes encode of viral polyprotein precursor, which is processed into the P1 region, containing the capsid proteins VP1, VP2, VP3 and VP4 and the P2 and P3 regions which contain the non-structural proteins.

Franki et al. (1991) classified FMDV as belonged to genus Aphthovirus,

within family Picornaviridae which also includes Enterovirus, Cardiovirus, Rhinovirus and hepatovirus.

Belsham (1993) classified FMDV as a single strand positive sense RNA

virus that belong to the genus aphthovirus in the family Picornaviridae. Kitching (1994) revealed that the FMDV is one of the animal viruses which

are rapidly producing pathogenic mutants. These mutants can spread quickly throughout affected geographical regions leading to severe economic losses.

ICTV (2000) mentioned that FMD was caused by seven types of foot and

mouth disease in the genus Aphtovirus, family Picornaviridae.

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Domingo et al. (2002) reported that the FMDV is an aphthovirus of the family Picornaviridae. FMDV is non-enveloped particles of icosahedral symmetry.

Musser (2004) mentioned that FMD is caused by RNA virus of the genus

Aphthovirus; 7 immunological distinct serotypes of the virus have been identified. Susceptible species are mainly cattle, sheep, goats, pigs, bison and deer. All body fluids of infected animals can contain the virus and are considered infective.

Buenz and Howe (2006) found that members of the picornavirus family,

including poliovirus and foot-and-mouth disease virus, are widespread pathogens of humans and domestic animals.

Nick et al.(2007) the disease is highly contagious and combined with high

antigenic diversity of the virus makes FMD difficult to control

2.3. Typing and sub typing of FMD virus

Valee and carre (1926) named the classical O, A and C types of FMD virus according of the isolation site (O) being isolated from Oise Valley in France (A) from Allemange (Germeny) and (C) for the third isolated type.

Brooksby (1958) added the so-called exotic types of FMD virus which

involved South Africa Territories (SAT1, SAT2, SAT3) and Asia 1 isolated from Asian countries.

Bachrach (1968) mentioned that seven classified immunological types of

FMDV O, A, C, South African Territory types (SAT1, SAT2, SAT3) and an Asian type designated Asia 1.

Callis et al. (1968) showed that the immunity against one type did not protect

against infection by other types. It was indicated that subtype differentiation could be based on complete or partial lack of cross protection between FMDV strains.

Pereira (1977) identified a total of 65 subtypes of FMD virus classified as A

32, O 11, C 5, SAT1 7, SAT2 4, SAT3 3 and Asia1 3. Anon (1978) mentioned that there was no cross immunity between types but

partial immunity only between subtypes within the same type.

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Abu El-Zein and Crowther (1980) reported that serological difference

between some subtypes of FMDV was significant enough to recognize each of them apart with safe.

Aggarwal et al. (2002) mentioned that the International Vaccine Bank

(IVB) for FMD at Pirbright holds quantities of seven strains of inactivated FMDV antigen over liquid nitrogen, ready for immediate formulation into vaccine if required. These will protect against the viruses of serotypes that are most likely to threaten the livestock of the UK or other IVB member countries (i.e. serotypes A, O, C and Asia1).

Brown (2002) reported that the extent of the antigenic diversity is such that

animals, whose level of immunity is waning, as for example several months after vaccination, may be susceptible to infection with viruses within the same serotype, although still immune to infection with the virus from which the vaccine had been made.

Domingo et al. (2002) cited that FMD virus is the prototype member of the

Aphthovirus genus of the family Picornaviridae. The virus exists in the form of seven different serotypes A , O, C, SAT1, SAT2, SAT3 and Asia1. But a large number of subtypes have involved within each serotype.

Mason et al. (2002) mentioned that during the last 12 years a strain of

FMDV serotype O, named as PanAsia, has spread from India throughout southern Asia and the Middle East. During 2000, this virus strain caused outbreaks in the Republic of Korea, Japan, Russia (Primorsky Territory), Mangolia and South Africa.

Knowels and Samuel, (2003) reported that Infection or vaccination with

one serotype dose not conferm protection against other serotype

2.4. Epidemiology of Foot and Mouth Disease

2.4.1. Foot and mouth disease in Egypt Zahran (1961) mentioned that different types of FMD virus (SAT2, O and

A) were identified in Egypt, type A and SAT2 were the main causes of outbreaks during 1953, 1958 and 1960. Type ‘O’ virus was the most prevalent in setting up the disease.

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Awad et al. (1984) reported that field survey on the epizootiology of FMD in Egypt revealed that the percentage of antibodies against the virus infection associated antigen (VIA) in the sera of naturally infected cattle was 88.8% while in contact cattle was 50%. In buffaloes, it was 80% in naturally infected animals and 30.7% in contact ones. The sera of immunized cattle under field conditions were negative to the VIA antibodies. The percentage of VIA antibodies in sera of investigated animals allover Egypt was 13.5%, 18.36% and 15.4% for cattle, buffaloes and sheep respectively.

Moussa et al. (1984) cited that FMD took an enzootic form in Egypt. The

disease appeared each year and attacked susceptible animal causing losses in milk and meat production and sometimes death of young animal.

Omar et al. (1985) recorded in February 1980, an outbreak of FMD in

lactating animals in a village near Alexandria. The isolated FMD Virus was confirmed to be (O).

Daoud et al. (1988) identified a high prevalence of FMD among different

animal species (cattle, buffaloes and sheep) in different provinces in Egypt during 1987 outbreak. The isolated virus was confirmed to be type (O).

Kitching (1990) recorded since 1950, attention was drawn to the importance

of FMD in Egypt after occurrence of several outbreaks and subtype (O1) was the most prevalent isolated strain of FMD virus.

El-Nakashly et al. (1996) isolated FMDV type O1 /93/Egypt in 1993 and

the strain was typed as O1. OIE (2000) Published about outbreak of FMD in Fayoum governorate in

Egypt in September (2000) typed as O1 and cattle and sheep were affected. This indicates that the virus is still being actively transmitted within livestock.

Aidaros (2002) cited that the occurrence of FMD serotype SAT2, A and O

were last reported in Egypt in year 1950, 1972, and 2000, respectively while type O was the only virus isolated.

OIE (2006) published that the Egyptian authorities had reported several

outbreaks of FMD in cattle and buffaloes. The outbreaks were located in eight governorates of Egypt.

Abdel-Rahman et al. (2006) reported that an outbreak of FMD started in

bulls imported and kept in quarantine station at Al-Ismailia Governorate, then spread among local cattle, buffaloes and dairy farms in most governorates in

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upper and Lower Egypt with 100% morbidity and high mortality rates reach to 80% in newly born calves. The virus isolated from imported and local animals was identified in Egypt as serotype A.

Farag et al. (2006) observed that clinical signs of foot and mouth disease

(FMD) among bulls imported from Ethiopia into a quarantine station at Al-Ismailia Governorate. FMD-WRL reported that the recovered type A/EGY/1/2006 virus was antigenically related to serotype A FMD isolated from Ethiopia, Kenya, Yemen and Saudi Arabia. The recovered type A FMDV re-isolated from local indigenous cattle, buffaloes and dairy animals, fattening bull and backyard allover 10 governorates in upper and Lower Egypt. 100% morbidity, 80% mortality in the newly born calves and 50% losses in milk were recorded in the affected dairy farms. Also, 50% losses were estimated in meat production of fattening bulls.

Ghoneim et al. (2010) cited that Egypt is endemic with two FMDV serotype

(O&A) and the outbreaks still reported since 2006 till now.

2.4.2. Prevalence of Foot and mouth disease in the world Callis et al. (1968) reported that FMDV has been detected in almost

countries of Asia such as Kuwait, Israel, Iraq, Saudi Arabia, Oman, Yemen, United Arab Emirates, Iran, Jordan, Pakistan and India.

Ramarao and Rao (1988) found that the four types of FMDV (O, A, C and

Asia 1) have been isolated from various part of India. Samuel et al. (1990) stated that FMDV serotype 'O' continued to be isolated

from outbreaks in Middle East during the period 1981-1988. Dehoux and Hounsou (1992) gave a brief account on the FMD epidemic of

Borgou Department between November 1990 and April 1991. Morbidity rates of 80-100% were observed in affected cattle herds. Antibodies to types A, O and SAT2 were demonstrated.

Kitching (1998) cited recent outbreaks of FMD in 1996 in the European

countries namely: Bulgaria, Greece, Turkey, Albania and Macedonia. Leforban (1999) recorded outbreaks of FMD type A in 1996 in Macedonia,

Albania and Yugoslavia.

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Huang et al. (2000) demonstrated that since March 1997 two strains of FMDV have found their way to Taiwan, causing severe outbreaks in pigs and in Chinese yellow cattle.

Benveniti et al. (2001) declared that FMD is one of the most dangerous

diseases of cloven-hoofed animals and is a constant threat in the Middle East and other regions throughout the world despite intensive vaccination programs.

EU FMD Meeting (2001) concluded that outbreak of FMD type O was

confirmed in UK, this outbreak was caused by FMD strain that was responsible for the outbreak in Japan.

Gibbens et al. (2001) confirmed FMD was confirmed in Great Britain. A

major epidemic developed, which peaked around 50 cases a day in late March, declining to under 10 a day by May. By mid-July, 1849 cases had been detected. The main control measures employed were livestock movement restrictions and the rapid slaughter of infected and exposed livestock.

Blanco et al. (2002) reported that during 1999, 11 outbreaks of FMD were

declared in the east and central part of Morocco. All the FMD cases reported were in cattle.

Davis (2002) mentioned that types O, A, and C are the strains that have been

identified in Europe and South America while O, A and ASIA1 are common through Asia. SAT strain 1 and 2 found throughout Africa while SAT 3 is confined to southern Africa. The strain found in the Middle East includes A, O, Asia 1 and SAT 1. FMD is endemic in much of Africa, Asia and parts of South America.

EU FMD Meeting (2002) reported that in Turkey, a total of 29 outbreaks

have been reported, 16 due to type 'O' 11 due to type 'A' and 2 due to type 'Asia1'.

Joe et al. (2002) illustrated that the Republic of Korea has been free for 66

years prior to the introduction of the virus and had recently suspended imports of pork products from neighboring Japan owing to a reported FMD outbreak in that country. On March 2000 a suspected vesicular disease in cattle was reported and confirmed as FMD by the national veterinary research and Quarantine service of the Republic of Korea.

Mason et al. (2002) analyzed the relationship between FMD type O viruses

belonging to the Pan Asia strain. They revealed that all portions of the genomes of these isolates are highly conserved and provided confirmation of close

9

relationship between the viruses responsible for South Africa and UK outbreaks.

Sakamoto et al. (2002) reported that 4 outbreaks of FMD occurred from

March to May 2000 in Miyazaki and Hokkaido prefectures, Japan. FMD virus isolated was achieved by sampling probaing materials from Japanese Black cattle. The FMD was identified as type O by ELISA for antigen detection and nucleotide sequence encoding the VP1 was determined.

Anderson et al. (2003) detected antibody to FMDV serotypes Asia1 and C

during the UK epidemic in 2001. Francois et al. (2004) recorded FMD outbreaks in 62 countries in Africa,

Europe, Middle East, Southern Asia and South-East Asia from the beginning of 2003 and up to Sept. 2004. All other reported outbreaks occurred in countries in which FMD is endemic.

Paiba et al. (2004) isolated FMD type O during the UK 2001 outbreak. Sammin et al. (2004) reported that two different serotypes SAT1 and SAT2

were involved in FMD outbreaks in Zimbabwe in 2003/2004. Bhattacharya et al. (2005) cited that in the state of West Bengal, India,

1,082 FMD outbreaks were reported in the 18 years from 1985 to 2002. Of the prevalent four serotypes, O type FMD virus accounted for the most outbreaks (67%), followed by Asia-1 virus type (15%) and A virus type (14%).

Ryan et al. (2008) recorded that a case of foot-and-mouth disease (FMD) on

a cattle farm in Normandy, Surrey, was confirmed on Friday August 3, 2007, the first case in the UK since 2001.

Hoang (2009) stated that in 2008 FMDV types O and A were reported to be

the prevalent and circulating serotypes causing the endemic outbreaks every month throughout 2008 and the first 2 months of 2009, while FMDV types O and Asia 1 were prevalent in 2007 in Vietnam.

2.5. Transmission of FMD

Burrows (1966) reported that the virus is released from infected animals in blood, milk, pharynx and vagina for variable periods before clinical signs appear on infected animals.

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Sellers and Parker (1969) mentioned that FMDV was transmitted principally through aerosols and by direct contact with infected animals.

Henderson (1970) recorded that birds contaminated air and wild animals

constituted an uncontrollable source of FMDV infection. McVicar and Sutmoller (1971) reported that milk and milk products could

also play an important role in transmission of FMDV. Gloster et al. (1981) mentioned that FMD could be occurred by airborne.

The virus could be carried for at least 60 kilometers. The two main modes of infection with the disease were inhalation and ingestion under field conditions, an inhalation being the more likely route in cattle.

Moussa et al. (1984) mentioned that rodents may play a role in the

mechanical or biological transmission of FMDV to susceptible cattle. Callis (1996) reported that FMDV could potentially be spread in semen,

food products and by fomites. Bastos et al. (1999) suggested sexual transmission of FMD from carrier

buffalo bulls to domestic cows. Hutber and kitching (2000) said that transmission of FMDV by aerosol

spread can occur over considerable distance, however this is less effective in hot and dry environmental conditions.

Holzhauer et al. (2001) assumed that the disease probably entered the

Netherlands through subclinically infected fattening calves imported from Ireland in late February 2001 that spread the infection to goats housed adjacently.

Alexandersen et al. (2002) mentioned that the contagious nature of FMD is

a reflection of a number of factors, including the wide host-range of the virus, the amount of infectivity excreted by affected animals, the low doses required to initiate infection and many routes of infection.

Dekker et al. (2002) reported that FMD was likely introduced in the

Netherlands by calves imported from Ireland via an FMDV- contaminated resting point in Mayenne, France. The clinical sings of FMD were reported in goats 3 weeks after arrival of the calves.

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Donaldson and Alexandersen (2002) said that in case of cattle, other important sources of virus that can cause heavy contamination of the environment are saliva, vesicular fluid, epithelium, milk and faeces.

Amass et al. (2003) cited that people could act as mechanical vectors of

FMDV when they move from infected to susceptible animals. Hand washing and changes of clothing were sufficient to reduce the dose of FMDV on people handling.

Uppal (2004) mentioned that small ruminant have been responsible for

epidemic of FMD in cattle in Greece in 1994. Sanson (2005) reported that People, vehicles, livestock and other items can

travel off pastoral livestock farms in New Zealand to other farms either directly or via saleyards over extensive distances. This has implications for the potential spread of infectious diseases such as FMD.

Gloster J. et al. (2008) mentioned that foot and mouth disease virus may

spread by direct contact between animals or via fomites as well as through airborne transmission.

Petrez et al. (2008) documented that the disease was spread by transmission

of virus through direct contact between animals and by indirect contact with fomites containing infectious virus particles, such as contaminated vehicles, feed,or clothing of livestock personnel.

Quan et al., (2009) stated that a strong correlation exists between dose (i.e.

infectiousness of source and intensity of contact) and length of incubation period, severity of clinical disease and efficiency of spread of FMD.

2.6. Pathogenesis of FMDV

Wisniewski (1962) failed to isolate the virus from muscle and heart of

infected cattle, two days after slaughter. The virus was found in the liver 5 days post infection and in the spinal cord and hip lymphnodes 9 days post infection.

Cottral et al. (1963) showed that cattle can be infected with FMDV by

tongue inoculation. The virus rapidily multiplies in the epithelial cells leading to a sever systemic infection.

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Muntiu et al. (1970) concluded that FMDV could be detected in the blood stream of infected cattle up to seven days post infection. Viraemia in these cattle was observed over 4 days period and on the same day generalized lesions appeared.

Sellers (1971) reported that inoculation of tongue epithelium was the most

sensitive method for initiating infection in cattle. There was a considerable variation in the amount of virus required from each strain to initiate infection. It may be worth while to mention that a minimum infective dose in cattle must contain 10000 virus particles.

Davis (2002) mentioned that FMDV is excreted during viraemia for some

days, thereafter as serum antibody develops viraemia decreases, and the animal ceases to be infectious as the lesion heal.

De Clereq (2002) mentioned that FMD is a very contagious disease because

small dose of virus is infectious, a large amount of virus can be excreted, and there are several routes of infection and excretion. Virus excretion starts 24-48 hours before the onset of clinical signs and declines with the appearance of circulating FMD specific antibody at around 4 to 5 days after infection. Preferred samples for virus detection are the epithelium, vesicular fluid, oesophagopharyngeal fluid probangs, hearinized blood and milk.

Kitching and Hughes (2002) cited the local replication of FMD virus

occurs at the site of entry, in the mucosa of respiratory tract or at a skin or mucous membrane abrasion. The virus then spread throughout the body favoring epithelial tissue in the adult and heart muscle in the juvenile. Lytic changes in the cells of the stratum spinosum and consequent edema give rise to the characteristic vesicles and accumulation of granulocytes, and in the developing myocardium of young animals, to a lympho-histocytic myocadidits.

Sung (2002) recovered FMDV from oesophageal-pharyngeal fluid from both

dairy sheep and dairy cattle, artificially inoculation with 104.6 TCID50 O/Taiwan/99 strain in tongue and feet tissue, four days after inoculation.

Pacheco et al (2008) mentioned that After aerosol exposure of cattle FMDV

first replicates in the pharynx. In 24–48 h the virus invades the blood stream and shortly thereafter lesions appear in the mouth and feet of susceptible animals. Viremia usually disappears after 3–4 days but virus replicates to very high titers (>8 log 10 infectious units per ml) at lesions sites and is shed in the air and body fluids. Between 5 and 10 days after their appearance, lesions resolve and virus is no longer found at the lesion sites and can only be recovered from pharyngeal fluid and tissues.

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Sellers and Gloster (2008) reveled that in cattle, FMD has been

experimentally reproduced by exposing animals to virus via direct or indirect contact with infected animals, via injection by various routes, by intra-tracheal aerosol infection, by intra-pulmonary implantation, or via respired aerosol.

Juan et al. (2010) suggests that early in FMDV infection of cattle,

replication occurs in the upper respiratory tract within respiratory associated lymphoid tissue.

2.7. Foot and Mouth Disease in Sheep

Shahan (1962) stated that cattle, swine, goats, and sheep are the most commonly affected species, although other ruminants and cloven-footed animals may contact the disease as well.

McVicar and Sutmoller (1968) reported that 3 groups of sheep and goats

were experimentally exposed to FMD virus by different routes including intranasal instillation and intradermolingual injection. These animals were kept and treated with type specific antiserum to prevent carrier state. The results revealed that these animals still acts as a carrier indicating that sheep and goats has a very important role in the spread of the disease.

Kukharov et al. (1973) studied the isolation of FMD virus from cattle and

sheep at various times after recovery from infection. They found that the isolated strains from cattle have a lowered pathogenicity for cattle and Guinea pigs while strains recovered from sheep retained their full pathogenicity for this species. All the isolated viruses were found to be highly pathogenic for pigs.

Forman et al. (1974) reported that FMD carrier state in three species of deer

in UK. Sellers and Gloster (1980) found that the main route of infection was

airborne infection in cattle and sheep up to 20 Km. In some outbreaks movement of the people and vehicles play a role in spreading infection. They added that sheep acts as a source of infection for other species so the ring vaccination of sheep is effective in limiting spread of the disease.

Sharma et al. (1981) studied the patterns of infection and morbidity in

sheep and goats exposed to foot and mouth disease, both naturally and under experimental conditions; they investigated the infection by the presence of

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viraemia, virus in the pharyngeo-oesophageal region and serum neutralizing antibodies. The authors also recorded that in case of field outbreaks there was greater gap between observed morbidity and actual infection. They concluded that sheep and goats play an important role in the spreading of infection during epizootic of FMD.

Rahman et al. (1988) reported a natural case of FMD in an Indian elephant.

The isolated virus was typed Asia1, it was possibly indirectly transmitted through an outbreak of FMD Asia1 in cattle and buffaloes of the district.

Shawkat et al. (1989) isolated FMD virus type O1 from sheep during 1987

outbreak in Egypt. They studied the pathogenicity of the isolate and concluded that sheep can play a role in the epidemiology of FMD in Egypt.

Fondevila et al. (1995) studied that llamas are resistant to FMD infection,

and they play a minor role in transmitting the virus to domestic livestock. Barnett and Cox (1999) recorded the epidemiological role played by sheep

and goat in transmitting the disease due to the unapparent nature of the disease among those hosts as well as their ability to become carriers representing a reservoir for further infection and spread of the disease.

Ganter et al. (2001) mentioned that sheep and goats might be carriers, so

they play an important role in the epidemiology and transmission of FMD. Shipping and trade with live sheep and goats is much more common world wide than in other FMD susceptible species. Lack of registration and individual identification signs (ear tag) of sheep and goat herd may result in incomplete control measurement under FMD conditions.

Bronsvoot et al. (2002) reported that sheep play important role in

epidemiology and transmition of FMD. Kitching and Hughes (2002) recorded that serotype (O) FMD virus has

been recovered from over 90 % of the positive samples from sheep submitted to the World Reference Laboratory for FMD, Pirbright, U.K. In East Africa where outbreak due to serotype O, A, C, SAT1 and SAT2 are common, predominantly serotype (O) virus was identified in clinically affected sheep and goats.

Hughes et al. (2002a) revealed that the optimal dose of FMDV to infect

sheep and for producing in-contact transmission is about 104 TCID50.

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Hughes et al. (2002b) mentioned that lesions of FMD may fall to develop in approximately 25% of infected sheep; a further 20% may develop only a single observable lesion.

Amas et al. (2003) found that contact sheep with infected pigs had

developed gross lesions consistent with FMD by 5DPI. Georgiev et al. (2004) cited that Foot and mouth disease have transmitted to

sheep in which infection is frequently sub-clinical Laila et al. (2004) reported that sheep play an important role in the

epidemiology and transmission of FMD. Moreover, FMD is suspected to have transmitted to sheep in which infection is frequently sub-clinical. So, it's of importance to identify animals which have been exposed to the virus and have developed antibodies. Such animals may become carriers and thus be a potential source of new outbreaks.

2.8. Persistence of FMD

Burrows (1966) found that the frequency of recovery of FMDV in esophageal-pharyngeal fluid (OP) samples taken from convalescent cattle after clinical infection was 9-26 weeks and infectivity of the isolated virus was 1-2.4 log10/ml. He also added that the FMDV was recovered from cattle 14-196 days after infection. The chief sites of virus multiplication were the dorsal surface of the soft palate and the pharynx.

Burrows (1968) demonstrated that goats and sheep develop persisted

infection. McVicar and Sutmoller (1968) found that about 50% of a group of goats

and sheep exposed to an infected steer were FMD carriers 4 weeks later. The carrier state may last up to 12 months in some animals.

Sutmoller et al. (1968) observed that virus multiplication was established in

the pharynx of immunized cattle in spite of the presence of high serum antibody titre.

McVicar and Sutmoller (1972) reported that 88 out of 91 (97%) goats

exposed to FMDV became infected and 92% of the infected ones had demonstrated viraemia. They also added that all goats showed viraemia when placed in contact with infected cattle.

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Kukharov et al. (1973) recorded examination of pharyngeal mucous of

infected cattle and sheep, the virus persisted for at least 271 and 106 days after convalescent, respectively.

Sharma (1979) inoculated 6 sheep with FMDV type "O" (104TCID50/ml)

subcutaneously, intranasaly and intraderolingualy. The animals were viraemic 24 hours after inoculation, and viraemia lasted for 32 to 68 hours.

Arafa (1980) reported that in experimentally infected goats with FMDV, the

duration of FMDV excretion from OP fluid was 4-6 weeks post inoculation. Sharma et al. (1981) stated that 11 out of 19 infected sheep (58%) and 17

out of 20 infected goats (85%) showed the clinical FMD in a work of experimental infection. In field outbreak, (22%) and (17%) of infected sheep and goats, respectively, showed the clinical lesions. The results showed that sub clinical infection of sheep and goats could have an important role in the spread of infection during epidemics.

Ginaru et al. (1986) cited that young buffaloes were infected with FMD by

exposing them to previously infected buffaloes of same age. In acute stages of infection with SAT1 and SAT2 viruses, clear foot lesions developed in most of the buffaloes. During the 1st week following infection, FMDV was found in blood 1-4 days post infection (dpi), in nasal secretion 26 dpi, in saliva 1-6 dpi at a titre of 1.3-4.3 log10 MLD50/ml, and in faeces 1 dpi at a titre of 1.4 log10 MLD50/gm. They added that FMDV could be detected in nasal secretion or saliva of 3 buffaloes up to 4 weeks pi.

Martin et al. (1987) assigned the term “carrier “only to animals that are able

to disseminate infection. Pay (1988) pointed out that the duration of FMD carrier state in sheep and

goats lasted for up to 9 months. Witmann (1990) indicated that FMDV infection could cause a long lasting

virus carrier state in the oesophageal-pharyngeal (OP) region of cattle, sheep, goats, African buffaloes, wildebeest and kudu. Virus could be recovered from OP fluids with low titres for several months up to more than 2 years. During this time, phases of positive virus recovery were interrupted by negative phase. The number of virus carriers decreased as time progresses. The virus carrier state was always accompanied by FMDV antibodies in serum and OP fluid. Vaccinated animals also became virus carriers after FMDV infection, to the same extent as unvaccinated animals. More over experimental contact

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transmissions of carrier virus to cattle, sheep and goats had failed. Only buffaloes transmit carrier virus to the own species and perhaps to cattle. Nevertheless, virus carriers represent a natural reservoir of FMDV in infected areas and a potential source of antigenically altered virus mutants take place in the animals during the carrier state.

Salt (1993) referred to animals in which FMDV persists in the oesophageal-

pharyngeal region for more than 4 weeks after infection as “carrier ". Yadin and Cloudia (1995) suggested that carrier goats and sheep played

insignificant role in disease transmission. Farag et al. (1998) pointed out that the comparative molecular studies

carried out in FMD WRL (Pirbright, London, UK) revealed closer antigenic relationship of the serotype "O" carrier strain (selected from 21 serotype "O" carrier strain isolated from goats and sheep) to the type "O" viruses that caused outbreaks in the neighboring dairy herds. These results reinforce the evidence that the infected goats and sheep would transmit the virus to the neighboring dairy farms.

Barnett and Cox (1999) concluded that sheep and goats were most likely to

be involved in the transmission of FMDV during the early stages of either clinical or subclinical FMD infection, rather than when they are carriers, and the period of great risk of transmission was up to 7 days after contact with the infection.

OIE annual status (2000) reported that pigs did not become carriers. The

carrier state in cattle was 6 months but it may last up to 3 years, while in African buffalo was 5 years, while in sheep and goats were few months.

Zhang and Kitching (2001) indicated that virus persisted in the basal layer

cells of the pharyngeal epithelium, particularly of the dorsal soft palate. Alexandersen et al. (2002) found that sheep experimentally infected with

the UK 2001 strain showed virus excretion. Firstly, a highly infectious period of around 7 to 8 days, secondly, a period of 1 to 3 days when trace amount of viral RNA were recovered in nasal and rectal swabs; thirdly, a carrier state involving 50% of the sheep.

De Clercq (2002) pointed out that sheep and goats could harbor the FMDV

for up to 9 months after infection, they added that some breeds of cattle could carry the virus for at least 3 years while African buffalo were considered to be life long carriers.

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Kitching (2002b) cited that the establishment of the carrier state and the

duration of this stage did not only depend on the host species and its breed but also on the strain serotype of FMDV. Also the quantity of virus present in the pharynx of carrier animals could vary considerably over time and the successful recovery of virus would depend on this and other factors, such as the subsequent handling of the sample and the skill of the operator.

Doel (2003) mentioned that the carrier state in FMD appears to last up to 6-9

months in sheep and goats and up to several years in extreme cases in cattle.

2.9. Antigenic components of FMD virus

2.9.1. Structural proteins Bachrach et al. (1963) mentioned that the virus had 140 S sedimentation

rate of FMDV with or without a small amount of 14 S protein degradation products.

Graves et al. (1968) reported that the empty capsid (RNA free) non

infectious with 75 S sedimentation coefficient the antigenicity of it closely related to both 12 S and 140 S components.

Talbot and Brown (1972) mentioned that FMDV consisted of RNA and

protein subunits, which consisted of three relatively large polypeptides (VP1, VP2, VP3) and a smaller polypeptide (VP4). It was found that the molecular weights of them were 34, 30, 26 and 15.5 X106 Dalton, respectively.

Cartwright et al. (1980) recorded that the complete FMD virus infected

particle sedimentation constant lies between 140 S induced the formation of type specific precipitation, complement fixing and neutralizing antibodies in cattle and guinea pigs.

Rueckert and Wimmer (1984) reported that FMDV is a member of the

genus Aphthovirus of the family Picornaviridae. Picornaviral RNA genome encodes a viral polyprotein precursor, which is processed into the P1 region containing the capsid proteins VP1, VP2, VP3, VP4 and P2 and P3 region, which contains the nonstructural proteins.

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Broekhijsen et al. (1985) found that the capsid polypeptide VP1 of FMDV is capable to elicit neutralizing antibodies. VP1 is known to contain an antigenic determination in the region of amino acids 200-312.

Suroval et al. (1987) recorded that the peptides obtained from sequence

130-160 of VP1 protein were capable of inducing neutralizing antibodies which protect rabbits and guinea pigs from infection mean while sequence 144- 159, 141- 152, 141- 148 and 148- 159 were inactive.

Fox et al. (1989) indicated that amino acid sequence RGD (Arginine-

Glycine- Aspartic acid), at position 145 to 147 and amino acid from C. terminal region of VP1 (positions 203 to 213) contributed to the cell attachment site on FMD virus for BHK cells.

Acharya et al. (1990) mentioned that the FMD genome consists of a single

stranded positive sense RNA molecule that is polyadenylated at 3 ends and terminates at the 5 end with a small covalently attached VP9.They added that the icosahedral capsid WAS composed of 60 copies of each of the four protein of which VP1-3 were partly exposed at the surface the smallest capsid protein VP4 is interland.

Belsham (1993) found that after translation, the polyprotein is cleaved into

four primary cleavage products: namely the amino terminal L-protease, which cleaves at its own carboxyl terminus, P1-2A, the precursor of the capsid proteins, 2BC, and P3, which is cleaved to make the replicative or NSPs 3A, 3B, 3C and 3D (the RNA-dependant-RNA polymerase).

Jackson et al. (2003) concluded that for FMDV, the major structure

proteins, VP1-3 are smaller than their counterparts in other Picornaviruses, especially so VP1, each having a molecular weight of approximately 24000 dalton. The capsid is both thinner with average thickness 33 A0 and smoother, than other Picornaviruses. It also lacks the remarkable surface features such as the pits and canyons described for Picornaviruses.

2.9.2. Non- structural proteins (NSP) 2.9.2.1. Virus Infection Associated Antigen (VIA)

Cowan and Graves (1966) named Virus Infection Associated (VIA) antigen which distinct from the recognized 140 S particle of FMD virus and produce as a result of virus multiplication but not constitute as part of virus moiety.

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McVicar and Sutmoller (1970) found that Virus Infection Associated Antigen (VIA) antibodies occur only in sera of infected animals and not in sera of immunized animals with inactivated virus vaccine.

Garland et al. (1981) stated that repeated vaccination had been shown to

give rise to VIA antibody.

Villinger et al. (1989) stated that cattle develop antibodies to VIA antigen mainly following the replication of FMDV. They also develop an indirect ELISA for the identification of VIA antibodies in animal sera.

O’Donnell et al. (1997) found that VIA (virus-infection-associated antigen)

or NSP 3D was present in both tissue cultures from which vaccine was prepared and in the viral particle. However, in general no VIAA antibodies were detectable following the initial vaccination, but were not unusual in the sera of animals, which had been given multiple vaccinations.

Brocchi et al. (1998) mentioned that the detection of antibody to non-

structural (NS) proteins of FMD virus has been used to identify past or present infection.

Kitching (2002) reported that the period of time after infection that 2C antibodies may be detected was 12 months while the 3ABC antibodies persist for longer period. The severity of the infection was likely to be the major influence on the levels and the subsequent duration of detection of the NS protein antibodies.

Mason et al. (2003) recorded that Picorna virus proteins derived from the P2

and P3 regions of the genome participate in RNA replication and structural protein folding and assembly. The authors added that the P2 portion of the Picorna virus polyprotein could be processed into three mature polypeptides, 2A, 2B and 2C. They also stated that the non-structural proteins 2C and 3A have membrane-binding properties, 2B enhance membrane permeability and block protein pathways, 3B are required for replication, 3C proteinase (Cpro) performed most of the cleavages of the viral polyprotein and some host cell proteins and 3D polymerase catalyzed the elongation of the nascent RNA chains.

Laila et al. (2004) found that the most reliable single NSP indicator was the

poly-protein 3ABC, antibodies to which appear to provide conclusive evidence of previous infection, whether or not the animal had been vaccinated. Therefore an ELISA detecting antibodies against the non-structural proteins of FMDV detected not only infected animals but also discriminates between infected and

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vaccinated animals. The FMDV NS detects antibodies directed against the non-structural 3ABC protein of FMDV. The ELISA detects FMDV infected animals independent of the fact that the animal is vaccinated or not. ELISA could be used to test serum samples of cattle, sheep, goats and pigs.

2.10. Inactivation of FMDV

Brown et al. (1963a) concluded that formaline alter the structure of virus and leave residual virus leading to low confidence in formaldehyde treatment of biological products. Laboratories changed to Aziridine which is used now as an inactivator.

Goel and Rai (1985) reported the inactivation of FMDV type O (subtype

O1, O5 and O6) of Indian origin using AEI occurred after 6-12 hours, while inactivation using formaline and heat was incomplete in 48 hours.

Radlett (1989) found that formaline inactivated FMD vaccine may contain

infective virus, the aziridine (AEI) was used successfully for inactivation but its toxicity was high and specialized plan was needed for its manufacture.

Omar et al. (1990) compared between formaline inactivated and BEI

inactivated FMD vaccines. They found that FMD vaccine inactivated with BEI was better in its quality and potency than formaline inactivated one.

Barteling, Cassim (2004) recorded that using Binary Ethyleneimine and

Formaldehyde would be a very fast and safe way for inactivation of foot and mouth disease Virus and enteroviruses.

Nuanualsuwan S et al. (2008) used UV inactivation of foot and mouth virus

in suspension.

2.11. Immunity against Foot and Mouth Disease

2.11.1. Active Immunity

2.11.1.1. Immunity after infection Shahan (1962) revealed that cattle recovered from infection with one type of

FMD virus were immunized to any nature infection to the same type for one to three years but if challenged occur by intradermolingual inoculation of

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homologous virus during few month only primary vesicle develop in the mouth and not progress to a generalized infection.

Dellers and Hyde (1964) detected 60 hours post inoculation virus

neutralizing antibodies, which persisted for at least 147 days from sheep Infected with FMDV. Initial peak titres occurred by the 10th day.

Gomes et al. (1972) recorded neutralizing antibodies persisted for 18

months in convalescent cattle. McVicar and Sutmoller (1974) stated that carrier animals maintain higher

neutralizing antibody titres to FMDV than convalescent cattle, experimentally infected with FMDV subtype ‘O1’.

Fernandez et al. (1975) detected antibodies against virus infection

associated (VIA) antigen of FMD virus and presence indicated previous exposure to FMD virus.

Lobo et al. (1975) cited that serum contained high titres against VIA

obtained from different types of virus A27 and O1 Vallee. The positive response to VIA antigen of naturally infected cattle was 88% two months after infection. They added that repeated vaccination has no influence on VIA antibodies.

Anderson et al. (1976) reported that goats, which were infected by

inoculation with FMDV, developed serum-neutralizing antibody that reached a peak titre at 14 day and there after declined slowly.

Moussa et al. (1976) mentioned that duration of antibody response in FMD

experimentally infected cattle lasted for a period of 40 weeks. The maximum antibody titre was reached at 10 weeks post infection (p.i.) followed by steady reduction in titre to the 4th month p.i.

Sobko et al. (1976) detected VIA antigen antibodies in the sera of cattle

recovered from FMD infection. Such antibodies were absent from non-infected cattle and cattle immunized with inactivated vaccine.

Graves et al. (1977) described VIA antigen as non-capsid virus particle

produced during virus replication and similar to virus RNA polymerase. They added also that it might be obtained as a by- product following the replication of the virus.

Matsumato et al. (1978) mentioned that the serum neutralization antibodies

rose to high titres within 7 to 10 days after infection of cattle with type ‘O’

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FMD virus. The level remained high for 4 months; while the virus could be isolated from oesophageal pharyngeal fluid (O.P.) up to 4 weeks post inoculation.

Sharma (1978) detected the antibodies against VIA antigen as early as 8

days and remained detectable for 105 days in infected sheep. Antibody to VIA antigen could not be detected in vaccinated sheep.

Pinto and Hedger (1978) revealed that the VIA antigen test was more

sensitive than the virus isolation for demonstrating infection. Salt (1993) mentioned that infection of susceptible cattle with FMDV results

in rapid rise in serum antibody, which could be detected from around 4 days post-infection. This early antibody was largely IgM, and IgG1 was detectable at 7-10 days post-infection and is highly serotype-specific. Serum antibody levels peaked at 28 days and remain at protective titres for prolonged periods up to 4.5 years in cattle and for life in mice.

Lubroth and Brown (1995) found that the presence of antibodies to protein

2C and to a lesser extent to the polypeptide 3ABC could be used to differentiate the potential carrier convalescent animal from the vaccinated one. Antibodies to 2C could be detected in cattle up to 365 days after infection.

Mackay et al. (1998) concluded that the polyprotein 3ABC was the most

reliable single indicator of infection in both bovine and porcine sera. The immune response to 3ABC appeared early after infection and antibody to 3ABC could be detected for longer than antibody to any other NSP.

Malirat et al. (1998) detected Anti-3ABC antibodies in experimentally

infected animals up to 560 and 742 days post infection. Sorensen et al. (1998) reported detectable antibodies to 3AB and 3ABC in

sheep after 14 days post-infection and to 3D after 22 days post-infection. The same animals were positive to structural proteins by LPB ELISA on day 8 post-infection.

Shen et al. (1999) demonstrated anti-3B antibodies in sera of convalescent

cattle and swine up to 364 and 301 days, respectively. King (2002) argued that the main mechanism of neutralization of

Picornaviruses (such as FMDV) was interference with viral attachment to the host cell. In the case of FMDV, one of the main targets of neutralizing antibodies was the ‘G-H loop’ on viral protein VP1.

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Ciara Murohy et al. (2002) found that both humoral and cellular responses

were induced as a result of infection with FMDV.

2.11.1.2. Immunity after Vaccination

Mackowiak (1970) studied the suitable technique for assessing the immunity of sheep, these methods are titration of antibodies after vaccination, calculation of an index of protection and the presence or absence of viraemia. He showed that sheep could be successfully vaccinated against FMD using the 1/3 normal cattle dose of a vaccine of good antigenic quality. The resulting immunity lasted for 5-7 months after a primary vaccination and for 12 months after revaccination, and it was recommended that sheep were vaccinated twice in the first year and then annually.

Witmann et al. (1970) concluded that the highest titres of neutralizing

antibodies were achieved after subcutaneous inoculation than that of intramuscular injection of the vaccine even if 108.1 TCID50/dose were applied by intramuscular inoculation.

Muntiu et al. (1971) showed that the duration of immunity of FMD vaccine

varies with breed, age, sex and condition of the vaccinated animal. Wisniewski et al. (1971) mentioned that the adult cattle vaccinated with

conventional FMD vaccine had 1.55, 1.05, 0.94 and 0.84 serum neutralizing indices at 1, 2, 3, 4 months post vaccination respectively.

Muntiu et al. (1974) mentioned that the normal dose of monovalent ‘O’

FMD vaccine contained 10 PD50 protected adult cattle for eight months. Moussa et al. (1974b) reported that the average of serum antibody titre in

Egyptian cattle vaccinated with a locally prepared FMD vaccine was 1.36 at 21 days post vaccination.

Ibrahim et al. (1977) established that Egyptian buffaloes, vaccinated with

locally prepared conventional FMD vaccine, developed serum antibody titres as early as 7 days post vaccination and an average genomic mean of peak neutralization antibody titres of 2.1 log10 at 14 to 21 days.

Abu-EL-Zein and Crowther (1978) concluded that both neutralizing test

and ELISA were the most valuable serological method for measuring the protective antibodies.

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Sharma (1978) mentioned that the antibody response of vaccinated sheep was similar to that for infected sheep but the antibody to virus infectious associated antigen (VIA) could not be detected in vaccinated sheep.

El-Mikkawi (1980) vaccinated Egyptian cattle and buffaloes with locally

prepared conventional FMD vaccine containing 0.03 ml Guinea pig PD50, and found that the neutralizing antibody titre achieved were so high and reached 2.07 log10 neutralizing titres up to 15 weeks post vaccination.

Sutmoller and Vieira (1980) found that cattle with neutralizing antibody

titre above 1.64 had high chance for protection while titres 1.8 and 1.32 were difficult to interpret in term of protection at challenge.

Student (1980) reported that sheep aged 3 to 4 years old inoculated with

inactivated monovalent FMD vaccine, had neutralizing antibodies were first detectable in the serum at 7 days post vaccination. Antibody titres (average 1: 9.3) were determined by SNT, 1:8.2 by titration in baby mice, 1: 6.8 by color test and 1: 7.02 by CFT based on 50% haemolysisrespectively. Antibodies were detectable in sheep sera for up to 4 months after a single vaccination and for up to 6 months when a second dose of vaccine was given 28 days from the first vaccination.

Abu El-Zein and Crowther (1981) revealed that IgG level was sharply

increased after vaccination with FMD monovalent vaccine and reached constant level at about 35 days post vaccination.

Falchsel et al. (1982) studied the relationship between neutralizing antibody

titre and the relative frequency of occurence of FMD among cattle as examined by probit analysis. Optimum results were achieved by an initial immunization for calves at 5-6 months of age, with a second inoculation 3 months later. Adult cattle required vaccination every other year to maintain 90% of them immune and/or protected.

Pay et al. (1983) found that the vaccination of cattle with inactivated FMDV

elicits a relatively short- lived protection serum response, which lasts only 3-6 months after single vaccination.

Sharma and Murty (1984) reported that in 8 sheep infected with a local

ovine strain of type O aphthovirus, by inoculation into the tongue or foot, neutralizing antibody appeared after 4-6 days with peak titres at 12-18 days, CF antibody and precipitins appeared 16th day and persisted for at least 15 weeks. The indirect FAT was positive between 6 days and at least 15 weeks.

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Pay and Hingly (1987) stated that higher log SN50 value (2.14) was required

for type ‘O’ vaccine to equate with 50 % protection of cattle than was required for type ‘A’ (1.17) and type ‘C’ (1.41) vaccines. They added also that before 1977 the PA50 value for type ‘O’ vaccine strain was only 1.34 and an antigenic shift was the cause for the large differences between that value and the current PA50 value.

DeClereq et al. (1987) concluded that revaccination of young calves was

effective even with FMD vaccine which was different from the primary vaccine used.

Felfe et al. (1990) studied the seasonal variations of FMD vaccination in

cattle. FMD immunization from February to July proved to be more effective than those performed in the rest of the year. This is confirmed by the formation of higher antibody titres within 14 days post vaccination.

Uluturk et al. (1990) noted that the serum neutralizing index of 57 cattle

given monovalent vaccine against FMD type ‘O’ was 1.76 and 54 animals (94%) resisted the challenge.

Cleland et al. (1994) cited on a vaccination program of 6-8 months old

cattle and buffaloes in a two groups at different times with a trivalent FMD vaccine (type O, A and Asia1). Group 1 at 0 and 180 days, group 2 at 0, 30 and 180 days, the antibody titrers were measured by SNT. Group 2 had significantly higher mean titres and percentage of protected animals (defined as animals with log reciprocal SNT of >1.5) to all 3 serotypes at day 60. At day 180 group 2 had significantly higher mean titres to serotype O and A, but not to Asia1 and there were no difference between the groups in the percentage of animals protected against any of the serotype.

Archetti et al. (1995) showed that FMDV infected cattle regularly mount an antibody response in oesopharyngeal fluids in contrast to vaccinated cattle. Antibodies could be revealed by specific kinetic ELISA. Cattle vaccinated once seldom showed a mucosal antibody response, which could only be detected by a total IgA specific ELISA generally allowed an early detection of FMDV infected cattle. In particular it proved to be more sensitive than the usual indirect, antigen trapping ELISA in experiments on saliva methods.

Fatthia (2003) found that immune response of vaccinated goats with

Alhydragel and DOE Montanide ISA 206 vaccines persisted for 20 and 36 weeks post challenge, respectively.

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Cox et al., (2008) cited that immunization with a single shot of vaccine containing high antigen payload will protect cattle from clinical disease at 6 months post vaccination and a boost may be unnecessary.

2.12. Isolation and identification of the virus

Roeder and Smith (1987) stated that samples could be checked for the presence of FMD antigen by an indirect sandwich ELISA.

House and House (1989) and Ahl et al. (1996) reported that homogenized

and clarified suspensions of samples must be inoculated into sensitive cell culture, such as primary foetal lamb kidney cells, bovine thyroid cells (BTY), baby hamster kidney (BHK) to isolate and grow virus.

Reid et al. (2001) mentioned that polymerase chain reaction (PCR) could be

used to detect and typing the FMD viral genome.

2.13. Serum Neutralization Test (SNT)

Capstick et al. (1959) employed several types of cell cultures in the neutralization test antibodies developed by infecting cattle or Guinea pigs with FMDV appeared after one week post inoculation .The titre of antibody increased at 3rd week of infection.

Graves (1960) used the so-called color test, which depends on colormetric

reading of the serum neutralization results. Witmann (1965) mentioned the neutralization test for the differentiation

between types and subtypes of FMDV, where unweaned mice were the laboratory host of the test.

Babini (1966) used SNT for investigation of efficiency of vaccines and

duration of immunity. Graves et al. (1972) found that there was good correlation of immunity with

the serum neutralizing antibody titre and thus the degree of protection at the time of challenge. This relation was definitely influenced by subtype of the challenge virus compared with that used in the vaccine.

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Wisniewski et al. (1974) showed that determination of serum neutralization antibody titres at the time of challenge indicated a correlation between antibody titres and resistance of infection.

Matsumato et al. (1978) reported that serum-neutralizing antibodies rose to

a high titre within 7 to 10 days after infection with FMDV type 'O'. This level continued for 4 months.

Arbelaez et al. (1979) indicated that the micro-neutralization test was very

efficient for the evaluation of antibody levels in bovine sera. El-Mikkawi (1980) vaccinated Egyptian cattle with a locally prepared

conventional vaccine. The neutralizing antibody titres persist up to 15 weeks post vaccination.

Sutmoller and Vieira (1980) found that cattle having neutralizing antibody

titres in excess of 64 would seem to indicate a high level of protection, while titres within the range of 1.8 to 1.32 are particularly different to interpret in terms of protection upon challenge with virulent FMDV.

Abu El-Zein and Crowther (1981) cited that neutralizing test was the most

valuable serological test for measuring protective antibodies. De Simone et al. (1981) reported that the antibody titres of sera from cattle

used in the potency testing of FMD vaccines were assayed both by SNT and by ELISA. The results of the two tests were in good agreement for the sera from vaccinated cattle taken at 21 days post vaccination.

Gaschutz et al. (1986) used SNT to assay the neutralizing antibodies of sera

from cattle of different ages in the Federal Republic of Germany against FMDV. The antibody titres of sera increased with the increase in number of vaccinations of animals.

De Clereq et al. (1987) carried out a serological survey in cattle herds by the

micro-neutralization. In most cases the negative herds were composed of a majority of young un-vaccinated animals or of animals vaccinated once that one year previously.

Bengelsdroff (1989) showed that the SNT proved to be a very suitable aid

for judging the immunological relations between the various strains.

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Uluturk et al. (1990) noticed that the serum neutralizing index of 57 cattle given a monovalent vaccine against FMD type O was 1.76 and 54 animals (94%) resisted the challenge with virulent virus.

Dhanalakshm and Krishnaswamy (1992) used BHK21 and IBRS2 in

micro-neutralization test for detecting FMD antibodies in 50 healthy cattle aged 2.5-3 years. The animals were vaccinated using a polyvalent vaccine containing FMDV strains: (O, A2, C and Asia1). This method was found sensitive and accurate for monitoring the immune status of vaccinated animals.

Nair and Sen (1992) monitored the antibody responses of sheep vaccinated

with an aluminum hydroxide gel and oil adjuvated FMD vaccine by using SNT and ELISA.

OIE (2000) reported that virus neutralization test is used as FMDV serotype

specific serological test. The quantitative VN microtest of FMD antibody was performed with IB-RS-2, BHK-21, lamb or pig kidney cells in flat-bottomed tissue culture grade microtitre plates. At the World Reference Laboratory (WRL), a titre of 1/45 or more of the final serum dilution in the serum/virus mixture was regarded as positive. Titres 1/16 to 1/32 were considered to be doubtful, and further serum samples are requested for testing. A titre of 1/8 or less was considered to be negative.

Armstrong et al. (2002) mentioned that SNT was considered definitive in

determining the antibody status of live stock. Chung et al. (2002) used SNT for detection of antibody titre with sera

collected from vaccinated pigs after 2.5 years post outbreak in Taiwan 1997. Paiba et al. (2004) cited that using the O1 UK 2001 FMDV in the virus

neutralization test with samples representitive of the uninfected Great Britain sheep population, the test specificity was 100% at a cut-off point of 1/45.

2.14. Enzyme linked immunosorbent assay (ELISA)

Sutmoller and Cowman (1974) employed three immuno-peroxidase techniques (direct, indirect and peroxidase, anti-peroxidase) for detection of FMDV antigen in bovine Kidney cell cultures. They concluded that specificity and technical simplicity of such methods made them useful in the detection of FMDV in infected cell and tissues.

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Crowther and Abu El-Zein (1979) concluded that ELISA test was successful in the specific detection of FMDV from infected tissue culture or epithelial tissue. The ELISA compared with CFT, being more sensitive and unaffected by anti-complementary factors.

Abu El-Zein and Crowther (1980) by using solid phase ELISA found

immunological activity detected from 4th day post vaccination and levels remained low until 28th day. High antibody titre was detected at 7th day post challenge with increase of (5-10) fold more than the pre-challenged animals. All the cattle (at 21st day) were protected with relatively low levels of protective antibodies detected.

Rai and Lahiri (1981) mentioned that the use of an indirect ELISA was

efficient in detecting FMDV in cell culture fluids, mouse carcasses and cattle tongue epithelium. The technique was also recommended for detecting serum antibody titres.

Hamblin et al. (1986) developed liquid phase blocking ELISA for

quantification of antibodies against FMDV, which might replace the VNT. It is a rapid and relatively simple to perform, reagents, economically and results recorded within 24 hours.

Have (1987) mentioned that ELISA was a rapid and convenient method for

large scale application. The results showed that the technique is more sensitive than CFT and SNT but not entirely type specific.

Holl and Sulk (1988) detected FMD specific antibodies in serum samples

from vaccinated and convalescent cattle by means of ELISA and SNT. A linear correlation between the two tests was obtained with a correlation coefficient of 0.79 for individual values and 0.80% for mean values.

Olver et al. (1988) collected epithelial samples from lesions in the mouth

and feet of calves experimentally infected with FMD type 'O1'. The authors assayed the presence of FMD viral antigens using a double antibody sandwich ELISA and CFT. The titer of infective virus in each sample was also determined. The antigen was detected by ELISA in 70% of mouth samples and 92% of samples from the feet. The CFT was less sensitive and demonstrated antigen in 44% of mouth and 85% of feet samples.

Westbury et al. (1988) found that single dilution blocking ELISA was

sensitive, specific and gave reproducible results. The technique had a potential test quickly and efficiently a large number of sera.

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Alonso et al. (1990) claimed that ELISA had the same specificity of agar gel immunodiffusion test but was more efficient in detecting small amount of VIA. The method was more satisfactory in the prevention, control and eradication of FMD.

Christensen and Kreider (1992) found that there are correlations between

the neutralizing antibody titre with antibody concentration as determined by ELISA.

Sorensen et al. (1992) developed a blocking ELISA for the detection of

antibodies against FMDV (types SAT1, SAT2 and SAT3) and for the quantification of antibodies on a single dilution of serum. It was a resource saving and proved to be a reliable and precise method for the assessment of antibody levels.

Srinivasan et al. (1992) compared between CFT and indirect sandwich

ELISA in typing O72 FMDV samples including, epithelial tissue and tissue culture supernatants. By ELISA 76.38% of samples were typed whereas by CFT 62.5% were positive. They concluded that ELISA is more sensitive and economical.

Hafez et al. (1993) used indirect sandwich ELISA for local diagnosis of

FMD in the Kingdom of Saudi Arabia. Testing epithelial tissues and/or vesicular fluids, it was possible to carry out serotyping of FMDV before its isolation in cell culture.

Periolo et al. (1993) tested serum samples were collected from vaccinated

with commercial quadrivalent oil vaccines and from un-vaccinated control animals from the FMDV free area of Argentina, using the liquid phase blocking sandwich ELISA (LP-ELISA) test. they was concluded that the use of the LP-ELISA test, a rapid and reliable evaluation of efficacy of FMD commercial vaccines as well as for the assessment of the immunological status of cattle in FMD free and enzootic regions of South America.

Steamer et al. (1993) used ELISA for measuring the combined effect of

concentration and affinity, giving an estimate of the overall biological activity of specific antibodies.

Blacksell et al. (1994) prepared antisera at the central laboratory in Thailand

against the endemic serotypes O, A and Asia1of FMDV. These antisera were used in ELISA for the detection and serotyping of FMDV antigens. ELISA readings of 0.01 optical density (OD) units were considered as a negative result.

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Niedbalski et al. (1994) studied the response of calves against the trivalent FMD vaccine. They used the liquid phase ELISA for detection of antibodies in sera of the non-vaccinated calves in early stages.

Radostitis et al. (1995) found that a rapid ELISA test