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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
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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
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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
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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
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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
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7. REFERENCES
.............................................................................................................................
100
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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
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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2
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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3
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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4
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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5
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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6
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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7
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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8
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
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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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10
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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11
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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12
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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13
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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14
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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15
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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16
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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17
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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18
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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19
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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20
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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21
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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22
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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23
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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24
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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30
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