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Epidemiology of Foot and Mouth Disease in
Cattle In Pahang, Malaysia.
Jamaliah Binti Senawi
Research Masters with Training (RMT)
School of Veterinary and Biomedical Sciences
Faculty of Health Sciences
Murdoch University
Western Australia
This thesis is presented for the Research Masters (with training)
of Murdoch University
2012
ii
Declaration
I declare this thesis is my own account of my research and contains as its main
content, work which has not been previously submitted for a degree at any other
tertiary educational institution.
iii
Abstract
Foot-and-mouth disease (FMD) is one of the most contagious diseases of domestic
and wild cloven-hoofed animals. The disease has a significant negative impact on the
economy of affected countries through reduced livestock productivity and loss of
markets. In Pahang, Malaysia a severe outbreak of FMD started in December 2003,
after approximately 18 years of freedom from the disease. The FMDV strain O was
identified as the cause of this outbreak. The study reported in this thesis focused on
three areas of epidemiology of FMD and was designed to: determine the temporal
and spatial distribution and pattern of outbreaks of FMD in Pahang; identify the risk
factors associated with the occurrence of FMD in Pahang; and determine the
antibody response in local cattle following vaccination against FMD.
Although vaccination is adopted as a control measure for FMD in Pahang, the
findings of this study indicated that only half (56%) of the respondents believed in
vaccination as a preventive measure for FMD with only 37.3% of respondents
correctly explaining how the disease spreads. Unfortunately only 29% of the
respondents knew that the vaccine needed to be given at six monthly intervals and no
one knew that a second priming dose was required to be administered one month
after the primary dose. Antibody conferred after vaccination was significantly higher
in animals which had been multiply vaccinated than in animals which received their
first vaccination. There was evidence that vaccination stimulated a serological
immunity; however the immunity, in many cases, was not sufficient to protect
iv
against natural infection. In addition the NSP test indicated 7 animals (5 cows and 2
calves) were positive during the eleven month study period, although no clinical
evidence of FMD had ever been seen on the farm.
Three variables (factors) were found to be associated with FMD in Pahang after a
multivariable logistic regression analysis. The most strongly associated factor was
retaining seropositive animals in the herd (P =0.006; OR=3.62; 95% CI 1.44, 9.11).
Cattle farmers who kept other livestock were more likely (P=0.003; OR 3.2; 95% CI
1.47, 7.07) to have an infected FMD herd than owners who didn’t keep other species
of livestock. Farmers which allowed the entry of unauthorised vehicles onto their
farmland were also more likely to have an infected herd (P=0.05; OR = 2.2; 95% CI
1.0, 4.82).
The spatio-temporal distribution of FMD outbreaks in Pahang during the period from
the 16th
December 2003 to the 26th
August 2006 was assessed using a Space – Time
permutation model. This indicated there were five significant distinctive clusters
with no geographical overlap in the secondary clusters for the whole study period.
Clusters were identified in the east, west and middle of Pahang with the observed to
expected ratio of FMD outbreaks within the spatial temporal clusters being between
2.39 and 17.78. The temporal pattern of the FMD outbreaks in Pahang appeared to
be seasonal occurring during the rainy season which coincided with “Hari Raya
Korban” when many live cattle are moved throughout the country. The present study
provided valuable information for the development of an effective control and
eradication program for FMD in the state of Pahang, Malaysia.
v
TABLE OF CONTENTS
Declaration ii
Abstract iii
Table of Contents v
List of Tables ix
List of Figures x
Acknowledgements xi
Abbreviations xii
CHAPTER 1 – INTRODUCTION, BACKGROUND INFORMATION
AND LITERATURE REVIEW
1
1.1 INTRODUCTION 1
1.2 BACKGROUND INFORMATION 2
1.2.1 Malaysia and the history of Foot and Mouth Disease 2
1.2.2 History of Foot and Mouth Disease in Pahang 5
1.2.3 Cattle Management in Pahang 6
1.3 FOOT AND MOUTH DISEASE 8
1.3.1 Aetiology 8
1.3.2 Epidemiology 9
1.3.2.1 Host species 10
1.3.2.2 Transmission 11
1.3.2.3 Incubation period 13
1.3.2.4 Survival on fomites 14
1.3.2.5 Carrier state 15
1.3.3 Diagnosis 16
1.3.3.1 Clinical Diagnosis 18
1.3.3.2 Virological Diagnosis 18
1.3.3.2.1 Viral isolation 19
1.3.3.2.2 Immunological methods 19
vi
1.3.3.2.2.1 Complement Fixation Test
(CFT) and Antigen Capture
ELISA
19
1.3.3.2.2.2 Nucleic acid recognition 20
1.3.3.2.2.3 Field tests 21
1.3.3.3 Serological Diagnosis 21
1.3.3.3.1 Testing antibody to structural proteins 22
1.3.3.3.1.1 Virus neutralization test
(VNT)
22
1.3.3.3.1.2 Enzyme linked
immunosorbent assay
(ELISA)
22
1.3.3.3.2 Testing antibody to non-structural proteins 23
1.3.3.3.2.1 Agar gel Immunodiffusion
test
23
1.3.3.3.2.2 Latex agglutination 24
1.3.3.3.2.3 Immunoelectro transfer blot
analysis
24
1.3.3.3.3 Non Structural Protein (NSP) ELISA 24
1.3.4 Control of FMD 27
1.3.4.1 Vaccination 28
1.3.4.2 Movement Restrictions 30
1.3.4.3 Stamping out 30
1.3.4.4 Application of Models 32
1.3.5 Disease Distribution 33
1.4 Overview of the current study 35
CHAPTER 2 - GENERAL MATERIALS AND METHODS 36
2.1 Serological tests for measurement of antibodies against FMD 36
2.2 Test for antibody against non-structural proteins 36
2.2.1 Post- vaccination evaluation test 36
2.2.2 Statistical and Descriptive analysis 37
vii
2.3 Questionnaire 40
CHAPTER 3 - RISK FACTOR ANALYSIS ASSOCIATED WITH
FOOT AND MOUTH DISEASE IN THE STATE OF
PAHANG, MALAYSIA.
41
3.1 Introduction 41
3.2 Materials and methods 43
3.2.1 Study Area 43
3.2.2 Study design 44
3.2.3 Questionnaires 44
3.2.4 Data analysis 45
3.3 Results 45
3.3.1 Descriptive analysis 45
3.3.2 Univariable analysis 45
3.3.3 Multivariable analysis 51
3.4 Discussion 53
CHAPTER 4 - ANTIBODY RESPONSE FOLLOWING
VACCINATION AGAINST FOOT AND MOUTH
DISEASE
59
4.1 Introduction 59
4.2 Materials and methods 62
4.2.1 Vaccination and sample collection 62
4.2.2 Measurement of antibody to NSPs 62
4.2.3 Measurement of antibody on the Liquid phase blocking ELISA 64
4.2.4 Statistical analysis 64
4.3 Results 65
4.3.1 Detection of antibodies against FMDV NS-Proteins 65
4.3.2 Liquid phase blocking ELISA 70
4.4 Discussion 77
viii
CHAPTER 5 - SPATIAL AND TEMPORAL DISTRIBUTION OF
FOOT AND MOUTH DISEASE IN THE STATE OF
PAHANG, MALAYSIA 2003 – 2006.
84
5.1 Introduction 84
5.2 Materials and methods 85
5.2.1 Study area and study period 86
5.2.2 Data source and case definition 86
5.2.3 Spatiotemporal analysis 87
5.3 Results 88
5.4 Discussion 93
CHAPTER 6 - GENERAL DISCUSSION AND CONCLUSIONS 98
Appendix 1 109
Non-structural Protein (NSP) test : Ceditest procedure 109
Appendix 2 114
Liquid phase blocking ELISA test procedure 114
Appendix 3 131
Survey on the possible risk factors contributing to Foot and Mouth Disease in
the state of Pahang, Malaysia
131
REFERENCES 141
ix
LIST OF TABLES
Table 3.1 Univariable analyses of putative risk factors for foot and
mouth disease in cattle in Pahang
48
Table 3.2 Univariable analyses of potential continuous risk factors for
FMD in cattle in the state of Pahang.
51
Table 3.3 Multivariable analysis of potential risk factors for foot and
mouth disease in cattle in Pahang.
53
Table 4.1 Number of cows and calves positive to the NSP test
throughout the study
65
Table 4.2 Number of NSP positive samples in the non-revaccinated
and revaccinated cows
66
Table 4.3 Number of NSP positive animals in the non-revaccinated
and revaccinated calves
67
Table 4.4 Comparison between cow and calf groups after animals
were divided at 183 DPV into a revaccinated and a non-
revaccinated group.
67
Table 4.5 NSP results for individual cows during the study 68
Table 4.6 NSP result for individual calves during the study 69
Table 5.1 Spatio-temporal clusters detected by the spatial scan
permutation model for 342 FMD outbreak locations in the
state of Pahang from 16 December 2003 to 26 August 2006.
92
x
LIST OF FIGURES
Figure 1.1 Location of Malaysia and its FMD status 2
Figure 1.2 Cattle in an oil palm plantation 7
Figure 1.3 Different types of FMD diagnosis 17
Figure 3.1 Cattle kept together with goats 43
Figure 4.1 LPB ELISA results for serotype O in cows 71
Figure 4.2 LPB ELISA results for serotype A in revaccinated and non-
revaccinated cows.
72
Figure 4.3 LPB ELISA results for serotype Asia 1 in revaccinated and
non-revaccinated cows.
73
Figure 4.4 LPB ELISA results for serotype O in revaccinated and non-
revaccinated calves.
75
Figure 4.5 LPB ELISA results for serotype A in revaccinated and non-
revaccinated calves.
76
Figure 4.6 LPB ELISA results for serotype Asia 1 in revaccinated and
non-revaccinated calves.
77
Figure 5.1 Number of cases of FMD recorded between 16th December
2003 to 26th August 2006
89
Figure 5.2 Geographical distribution of outbreaks of FMD in the state
of Pahang that were reported to the DVS during the period
16th December 2003 to 26th August 2006. Each red dot
represents a reported outbreak.
90
Figure 5.3 Location of the spatio-temporal clusters. Cluster E is
considered the primary cluster.
93
xi
Acknowledgements
I would like to express my gratitude to the Malaysian Government for the
sponsorship and Department of Veterinary Services, Malaysia for giving me the
precious opportunity to further my study at Murdoch University, Australia.
This thesis would not been completed without the help of my supervisor Professor
Ian Robertson who has guided me with patience and encouragement.
My special thanks are given to personnel at the FMD laboratory Malaysia; Dr
Norlida Othman, Mr Daud and Mr Madzahan. Pahang State DVS staff, especially
Mr Ahmad Zainal and all the District officers and staff at PTH Ulu Lepar especially
Mdm Hajjah Joseliana for their kind assistance during my study.
I greatly appreciate my parents, children (Hakim, Anis and Alya), sisters and brother
for their unconditional love, understanding and great help in many ways throughout
my study.
I would also give my special thanks to Siti Zubaidah and Aida for their help and
encouragement and all my friends who enriched my stay in Australia especially Wan
Sofiah.
Finally I would like to dedicate this thesis to someone I love, for all the beautiful
memories and happiness he left.
xii
Abbreviations
ANOVA Analysis of Variance
AVO Assistant Veterinary Officer
CI Confidence Interval
°C Degrees Celsius
DPV Days post vaccination
DNA Deoxyribonucleic acid
EDTA Ethylenediamine tetraacetic acid
ELISA Enzyme-linked immunosorbent assay
FMD Foot and Mouth Disease
FMDV Foot and Mouth Disease Virus
H2O2 Hydrogen peroxide
ID Identification
LPBE Liquid Phase Blocking ELISA
M Molar
mM Millimolar
NSP Non Structural Protein
OR Odds ratio
OD Optical density
OIE Office International des Epizooties
OPD tablets Ortho-Phenylenediamine
PI Percentage of Inhibition
PTH Pusat Ternakan Haiwan
PBS Phosphate buffered saline
xiii
RVL Regional Veterinary Laboratory
SPSS Statistical package for the Social Sciences
μL Microlitre
nm Nanometre
WRL World reference laboratory
w/v Weight in volume
v/v Volume in volume
VIF Variable Inflation factor
χ2 Chi square
1
CHAPTER 1
INTRODUCTION, BACKGROUND INFORMATION AND
LITERATURE REVIEW
1.1 Introduction
Malaysia is a small country located in South East Asia which has a multiracial
population with unique cultural and social practices. Pahang is one of the 13 states in
Peninsular Malaysia, and the project outlined in this thesis relates to disease studies
undertaken in this state.
Although there are unique ruminant animal husbandry practices in Malaysia, the
livestock industry only makes a small contribution to the economy and currently
produces far less product (meat) than is required by the population (DVS, 2009b).
This situation is exacerbated by the presence of specific diseases, in particular Foot
and Mouth Disease (FMD). Foot and mouth disease is considered one of the most
significant threats to the livestock industry because of its impact on production,
interference with access to international markets and effect on the economy
(Kitching, 1998; James and Rushton, 2002). Currently FMD is endemic in Pahang,
although there are on-going prevention and control measures against the disease
undertaken in the state, as well as in the whole country. The control measures
adopted include vaccination, movement control and physical examination of
livestock prior to movement. Despite these efforts, numerous outbreaks occur each
year. The epidemiology of FMD has been explored extensively in developed
countries; however the situation is different for developing countries, such as
Malaysia. There is little information available on the epidemiology of FMD in the
2
livestock population in Malaysia, in particular in the state of Pahang, and the study
outlined in this thesis was undertaken to help address this deficiency.
1.2 Background Information
1.2.1 Malaysia and the history of Foot and Mouth Disease
Malaysia is comprised of two main regions, Peninsular Malaysia and Malaysian
Borneo (Sabah and Sarawak), which are separated by the South China Sea.
Neighbouring countries to Malaysia include Indonesia, Brunei, Thailand and
Singapore. Malaysia consists of 13 states and three federation territories. Pahang, the
largest state in Peninsular Malaysia, is situated on the east coast of the country
(Figure 1.1).
Figure 1.1: Location of Malaysia and its FMD status
3
Foot and mouth disease has never been reported in Malaysian Borneo (Sabah and
Sarawak). In contrast there has been a long history of FMD in Peninsular Malaysia.
The first reported occurrence of FMD in Malaysia was in the 1860‟s, however there
is little detail of the early outbreaks until an outbreak occurred in cattle in July 1909
in the districts of Kulim and Kuala Muda in the state of Kedah. In 1936 a total of 551
animals were affected by FMD in the states of Perak and Selangor (Wallace, 1936).
Towards the end of 1938 two outbreaks involving 238 cattle were recorded in Perak
(Wallace, 1939). The disease was linked to the movement of animals from
neighbouring countries, and resulted in the development of policies to control the
movement of livestock in the four northern states (Perlis, Perak, Kedah and
Kelantan). After an outbreak in Perlis in 1973, Malaysia adopted an eradication
campaign for FMD involving a slaughter policy coupled with strict sanitary
procedures. The virus responsible for the 1973 outbreak was confirmed as FMD
virus Subtype A22 by the FMD World Reference Laboratory (WRL) at Pirbright,
United Kingdom. This outbreak was quickly controlled and no further outbreaks
were reported for approximately five years (Chong, 1979).
In October 1978 another outbreak occurred in Rantau Panjang (a border town
adjacent to Golok on the Malaysian – Thai border) in the State of Kelantan and the
disease spread via the normal trading route to the district of Muar in Johor and to the
district of Ipoh in Perak. Late in 1978 another outbreak was reported in the district of
Tumpat in Kelantan and the disease subsequently spread to Perlis and Kedah. The
virus responsible for this outbreak was identified as Foot and Mouth Disease Virus
(FMDV) Subtype O1. A stamping out policy was adopted in all affected areas and
4
18,117 animals were slaughtered of which 7,511 (41.5%) were cattle
(Thuraisingham, 1977).
In January 1979 Malaysia decided to change from a stamping out policy to
vaccination in the Northern states (bordering Thailand) as social, political and
religious factors were hampering eradication (Chong, 1979). However the
vaccination policy was also unsuccessful in controlling the disease as indicated by
outbreaks in August 1980, caused by FMDV serotype O1, in the states of Kedah and
Perlis which then spread to Penang. Subsequently in July 1981 major outbreaks were
reported in three different abattoirs in the states of Selangor, Johor and Perak and in
a feedlot in Johor (Babjee, 1994).
As the incidence of FMD was increasing in the early 1980‟s it was apparent that the
policy of only vaccinating livestock in the border states was not effective.
Consequently in December 1982 a policy to vaccinate all animals on mainland
Malaysia was implemented. The effectiveness of this policy was also questioned as
more outbreaks were reported in June 1984 in Penang and Perak due to FMDV
serotype O and in Kelantan and Terengganu in June 1985 due to serotype Asia 1.
Since 1989 the policy of only vaccinating animals in the northern states of Malaysia
has been reinstated. Intensive vaccination campaigns were instigated with a bivalent
vaccine containing serotypes O and Asia 1, along with adoption of other sanitary
measures (DVS, 1996). In December 1990 an outbreak occurred in a quarantine
station and 29 cattle from Thailand were affected and subsequently destroyed (DVS,
1995). Since 1992 FMD has been seen every year in some states in Malaysia,
5
particularly in the northern states. Despite efforts to control FMD (Naheed, 2007),
the disease is currently endemic in all of Peninsular Malaysia and it is one of the
biggest factors hindering livestock production and the growth of the livestock
market.
1.2.2 History of Foot and Mouth Disease in Pahang
Clinical signs of FMD have been recognised in Pahang since at least 1917 and were
first observed in a herd owned by the Royalty (Azmie, 2006). Following the report in
1929 of FMD in the District of Raub, no cases were documented for the next 56
years. In 1986 FMD was again reported in a herd of cattle near the railway line in the
district of Jerantut (Azmie, 2006).
After 18 years of apparent disease freedom, clinical signs of FMD were again
reported in 58 cattle on the 22nd
December 2003 in the district of Pekan. The disease
was confirmed by the Kota Bahru Regional Veterinary Laboratory as FMD Serotype
O. The disease spread very rapidly due to the managerial system adopted which
allowed cattle to roam freely around villages, making disease control difficult. This
outbreak was very large and involved many neighbouring districts in the state of
Pahang. Over a two month period to the 12th
February 2004, 5,070 animals were
recorded as showing clinical signs (4,555 cattle). The worst affected district was
Pekan, contributing 77.3% of the cases. Investigation by the Department of
Veterinary Service (DVS) indicated there was illegal movement of infected buffalo
from the district of Kemaman Terengganu to Pekan. The disease then spread from
6
Pekan to Rompin, Kuantan and Jerantut. The 2004 outbreak was the largest ever
reported in Malaysia and since then the disease has become endemic in mainland
Malaysia.
1.2.3 Cattle Management in Pahang
Foot and mouth disease in Pahang is believed to be influenced by the livestock
production system practiced. There are three main types of beef cattle production
practiced in Pahang: firstly an integrated system where cattle are reared under oil
palm plantations; secondly the traditional system where a small number of cattle and
other livestock are reared in villages. These animals usually roam freely in the
villages and co-mingle with other farmer‟s animals and with different species of
animals. The animals graze freely unsupervised during the day and return to the
owner‟s place in the evening. Thirdly is a feed-lot cattle management system.
Commercial large-scale ruminant production is rare in Malaysia due to an absence of
natural grasslands, and a limit on the areas of improved pastures (Joseph, 1991). This
situation arises because of a national policy preventing the conversion of tropical
rainforest into grazing pastures for animals because of both economical and
environmental points of view. In contrast, young oil palm plantations provide a
favourable environment for cattle production by providing shade and good quality
forage with a high metabolisable energy and crude protein content (Rosli and Mokh
Nasir, 1997). At the same time the cattle act as a biological control for weeds and
their manure acts as an organic fertilizer for the oil palm trees (Ahmad, 2001) and
7
this form of raising cattle has been encouraged (Harun and Chen, 1995) (Figure 1.2).
This integrated system is practiced mainly by Federal Land Development Authority
(FELDA) settlers and oil palm plantation companies. There are 3 main types of
grazing practiced under this system; 1. The cattle are free to graze anywhere in the
oil palm plantation. This practice is similar to the traditional cattle rearing in
villages. 2. The cattle are allowed to graze in certain areas, normally defined by
fencing in the oil palm plantation. Farmers who practice this system normally have a
limited number of cattle and this number is influenced by the area of oil palm
plantation owned. 3. Rotational grazing where the cattle are restricted by electric
fencing to an area with the cattle being moved daily between areas under the
supervision of a herdsman. This system is commonly practiced by large oil palm
plantation companies and by groups of FELDA settlers who combine their oil palm
plantation areas and cattle.
Figure 1.2: Cattle in an oil palm plantation
8
1.3 Foot and Mouth Disease
1.3.1 Aetiology
Foot and Mouth Disease is caused by an Apthovirus belonging to the family
Picornaviridae (Bablanian and Grubman, 1993). It is an acid labile virus which is
unstable below a pH of 6.8, and is inactivated at a pH of less than 6.0 or more than
9.0 (OIE, 2002). The virus is a non-enveloped single-stranded positive-sense genome
RNA molecule of approximately 8500 nucleotides of positive polarity and is
surrounded by an icosahedral capsid made up of four structural proteins (Mahy,
2005). The viral RNA consists of a single open reading frame (ORF), flanked by two
highly structured non-coding regions (NCRs) that contain cis-acting structural
elements involved in viral replication and gene expression (Sáiz et al., 2002). The
viral particles are comprised of 60 copies of each of the four virus encoded capsid
proteins, VP1 (1D), VP2 (1B), VP3 (1C) and VP4 (1A) (Belsham and Martinez-
Salas, 2004). These proteins are required for viral RNA replication and consequently
are essential for infection. The capsid protein of FMD virus regulates viral
replication, protein processing and modification of host cells. Foot and mouth
disease virus, like other RNA viruses, are prone to errors during replication which
leads to the development of mutant strains (Domingo et al., 2004). These errors lead
to the virus having the ability to adapt to different factors resulting in genetic
heterogeneity resulting in difficulty in the control and eradication of the disease
(Domingo et al., 2004).
There are seven FMDV serotypes (A, O, C, Asia 1 and South African Territories
(SAT) 1, 2 and 3) with no cross protection between the serotypes. Similar to other
9
Southeast Asian countries, the serotypes isolated in Malaysia are O, A and Asia 1,
with Serotype O being the most frequently isolated type in Pahang (DVS, 2008).
1.3.2 Epidemiology
Infection with FMDV produces an acute, systemic vesicular disease. The
epidemiology of FMD is a complex interaction between host, viral and
environmental factors (Pereira, 1981; Donaldson, 2007). The viral factors that
influence disease include virulence which affects the severity of lesions, the amount
and duration of virus release, particle stability in different microenvironments and
long-term persistence. In naturally acquired infections the most common route of
virus dispersion is by direct contact with infected animals (Alexandersen and
Donaldson, 2002). The clinical outcome of the disease varies between species and is
dependent upon the infecting virus strain. It is a highly transmissible disease, and a
limited number of infective particles can initiate host infection in susceptible animals
(Sellers, 1971). Contaminated animal products, agricultural tools, vehicles (fomites)
and humans can help contribute to the mechanical dissemination of the virus. Virus
multiplication and spread also depends upon the host species and their nutritional
and immunological status and their population density (Donaldson, 1987; Amass et
al., 2004). Animal movements and contact between domestic and wild animals also
contributes to the existence and maintenance of the disease. Geographical location
can act as a barrier to the dissemination of virus or, alternatively, can promote virus
transmission when appropriate atmospheric conditions prevail (Leforban and
Gerbier, 2002).
10
1.3.2.1 Host species
Foot and Mouth Disease is a highly contagious viral disease of cloven-hoofed
animals including domesticated ruminants, pigs and more than 70 wildlife species
(Coetzer et al., 1994). The disease is characterized by fever, lameness and vesicular
lesions on the tongue, feet, muzzle/snout, and teats (Kitching, 2002a). The severity
of the disease can vary from mild or inapparent in some species to more severe in
others. Pigs and cattle suffer more severe lesions compared to sheep and goats which
develop only mild clinical signs and superficial lesions and consequently the disease
can be difficult to distinguish from other common conditions in these species
(Donaldson and Sellers, 2000). Although FMD does not result in high mortality in
adults, the disease has debilitating effects including weight loss, decreased milk
production and reduced draught power resulting in a loss of productivity for a
considerable time. Mortality, however, can be high in young animals, where the
virus can affect the cardiac muscle (Alexandersen et al., 2003a). Other domesticated
animals such as water buffalo (Bubalus bubalis) can become infected and may also
transmit the disease to other species and persistent infection in this species has
important epidemiological significance (Samara and Pinto, 1983; Barros et al., 2007;
Maroudam et al., 2008). Camelids can be infected experimentally by direct contact,
even though they are not very susceptible and do not transmit the disease to other
species (Wernery and Kaaden, 2004).
Studies in the African continent have indicated that wildlife species play a major role
in maintaining and spreading the disease to susceptible domestic animals and wild
ungulates (Hedger et al., 1980; Vosloo et al., 1996). In particular the African buffalo
11
(Syncerus caffer) is able to maintain infection for a long period of time and
disseminate the virus (Vosloo et al., 2002). Occasionally FMD can be fatal to
wildlife, as apparently occurred in South Africa in the late 19th century when large
numbers of impala (Aepyceros melampus) succumbed to the disease. A recent study
in South Africa indicated that subclinical infection of impala still occurred,
especially in an area heavily populated with African Buffalo (Vosloo et al., 2009).
Antibodies against FMD virus have been found in a range of wildlife in Africa
including buffalo, eland (Taurotragus oryx), Grant's gazelle (Nanger granti),
Thomson's gazelle (Eudorcas thomsoni), wildebeest (Connochaetes gnou), impala,
waterbuck (Kobus ellipsiprymnus), sable (Martes zibellina) and topi (Damaliscus
korrigum) (Anderson, 1981; Anderson et al., 1993). Feral pigs and wild white tail
deer (Odocoileus virginianus) are also susceptible to FMD (Ward et al., 2007).
Within the Asian region wildlife involved in FMD can include elephants, Sambar
deer (Cervus unicolour), Spotted deer/Chital (Axis axis) and Barking deer
(Muntiacus muntjak) (Barman et al., 1999). Serotype O was confirmed as affecting a
group of 30 elephants after direct contact with infected cattle and buffalo in
Kathmandu, Nepal (Pyakural et al., 1976), however serological studies in elephants
from Africa have failed to detect an antibody response to the virus (Hedger et al.,
1972).
1.3.2.2 Transmission
Transmission of FMDV is primarily from the infected animal itself, especially
during the early febrile stage (Brown, 2004), with the typical spread in a herd or
flock being 2 to 6 days for all species (Alexandersen et al., 2003c). Transmission can
include direct contact between infected and susceptible animals, air borne spread of
12
virus either through inhalation or open wounds, indirect virus carriage by personnel
and fomites and consumption of infected animal products by susceptible animals
either through water or feed (Alexandersen et al., 2003c; Schijven et al., 2005).
Direct contact is the most efficient route of transmission for most livestock. However
in cattle, air borne transmission is the most common route, unlike for pigs which
need higher doses (Donaldson et al., 1987) and are relatively resistant to air-borne
infection when compared with cattle and sheep (Alexandersen and Donaldson,
2002). Exposed humans can contribute to the transmission of FMDV through
exhalation of viral particles (Amass et al., 2003a; Amass et al., 2004) which can
persist in the human nasal passages for up to 28 hours after exposure (Sellers et al.,
1970). However the most important role for transmission by humans is through
contaminated clothing or fomites.
In pigs, goats and sheep, direct contact is considered to be the most common route of
infection (Aggarwal et al., 2002). Although pigs are less susceptible to aerosol
infection than cattle, they shed more virus per day in acute infections compared with
cattle and sheep (Donaldson et al., 1970; Sellers et al., 1970; Kitching et al., 2005).
Even though the virus has been detected in the semen of boars, the risk of sexual
transmission is considered low (Guerin and Pozzi, 2005).
During the clinical phase of FMD, the virus is present in all secretions and
excretions, and it may be excreted intermittently thereafter (Donaldson et al., 1987).
Spread of FMDV from sheep and goats to other susceptible species is more
significant in clinical or sub-clinical stages than those of carrier stages, even though
they can also act as a carrier, such as with cattle (Barnett and Cox, 1999). The virus
can also be recovered from the respiratory tract of infected animals for
13
approximately two days before clinical signs appear (Donaldson et al., 2001;
Kitching, 2002a; Orsel et al., 2009) and can persist on wool for up to 14 days
(Cottral, 1969). The movement of infected animals is considered to be the most
important factor in the spread of FMD (Robinson and Christley, 2007) particularly
with animals showing slight or no clinical signs of disease (Mansley et al., 2003),
however the excretion pattern of virus differs in different host species and between
vaccinated and non-vaccinated animals (Orsel et al., 2009).
1.3.2.3 Incubation period
The fact that the virus can be excreted during the incubation period is important in
the epidemiology of the disease (Brown, 2004). The incubation period can be highly
variable depending on host, agent and environmental factors including husbandry
management factors (Hugh-Jones, 1976). Host factors include the species of animal
and their health status; agent factors include strain, dose and route of infection; and
the environmental factors include stocking density, ventilation system, transportation
and stress due to handling (Hughes et al., 2002; Alexandersen et al., 2003b). The
incubation period within a farm is 2 to 14 days (Hugh-Jones, 1976; Alexandersen et
al., 2003c), however it can be as short as 24 hours in pigs challenged with a high
dose (Alexandersen and Donaldson, 2002; Alexandersen et al., 2003c). In a recent
study the virus was shown to be excreted 1 to 2 days after challenge in non-
vaccinated dairy cattle and piglets in contrast to 3 to 3.5 days in non-vaccinated
lambs (Orsel et al. 2009). These findings conflicted with earlier findings by
Alexandersen and Donaldson (2002) who reported that pigs and other animals, such
as sheep, normally had a shorter incubation period.
14
1.3.2.4 Survival on fomites
Foot and Mouth Disease virus is considered to be a moderately stable virus. At a pH
between 7.0 and 8.5 most strains are stable, especially at lower temperatures
(Bachrach, 1968). Unlike other picorna viruses the FMDV capsid dissociates at a pH
of 6.5 or below. It is stable at a humidity above 55 to 60% but is sensitive to heat and
desiccation (Bachrach, 1968). In contrast Bedson et al. (1927) observed a longer
period of survival on dry rather than damp hay or bran. The survival of FMDV is
also influenced by the nature of the materials as a high concentration of organic
material helps the survival of the virus (Donaldson et al., 1987).
The virus can be recovered from the blood, pharynx, vagina and rectum up to 97
hours prior to the onset of vesicular lesions. It can also persist in mammary tissue for
3 to 7 weeks after infection (Burrows et al., 1971). The virus can survive outside the
host, and potential sources of virus include excretions and secretions of infected
livestock such as saliva, semen, milk, faeces, urine, and vaginal secretions (Bedson
et al., 1927; Brown, 2004). The virus can also survive in skim milk, cream and the
pelleted cellular debris components of milk obtained from FMD infected cows after
the milk had been pasteurised at 72°C for 0.25 minutes and in cream components
after heat treatment at 93°C for 0.25 minutes (Blackwell and Hyde, 1976). The
FMDV can also survive for long periods in meat and animal products including
frozen bone marrow, lymph nodes and offal (Henderson and Brooksby, 1948; Mahy,
2005). The average period of survival of FMDV on wool at 4°C is approximately 2
months with the period of survival decreasing considerably as the temperature
increases to 18°C (McColl et al., 1995). The maximum estimated survival period of
FMDV outside the host is approximately three months in regions with daily
temperatures greater than 20°C (Bartley et al., 2002).
15
1.3.2.5 Carrier state
A carrier state of FMD is defined as an animal from which FMDV can be recovered
more than 28 days after infection (McVicar and Sutmoller, 1969; Woodbury, 1995b;
Alexandersen, 2002). Carrier animals are reported to be common in FMD-endemic
areas (Salt, 2004), however the role of carriers is not well understood and there is
controversy over their role in disease transmission.
A long-term asymptomatic persistent infection can follow the acute phase of
infection and can include vaccinated animals which have been infected and exposed
to the virus (Salt, 1993; Alexandersen, 2002; Salt, 2004). Persistent infection can last
for a variable time in different animals and can be up to 3.5 years in cattle, 9 months
in sheep, 4 months in goats but does not appear to occur in pigs (Alexandersen,
2002). The African buffalo is the only wildlife species known to be able to carry the
virus and it can do this for periods up to 5 years (Bastos et al., 2000).
The development of a carrier stage in an individual animal is not correlated with the
pre-existing antibody level. However host mechanisms, such as humoral antibody at
protective levels which restricts viral replication in the oropharyngeal tissue, may be
important in the persistence of the virus (Woodbury, 1995a). The prevalence of
carriers in a population depends on the animal species present and the immune status
of the population (Alexandersen, 2002). Consequently vaccinating animals to ensure
a high proportion of animals are immune is considered an important strategy to
control the disease when accompanied with intensive clinical surveillance for disease
in susceptible animals (Arnold et al., 2008; Schley et al., 2009). However there is no
16
clear experimental evidence indicating that vaccination can either reduce the
establishment or duration of a carrier stage in animals (Hedger, 1970).
1.3.3 Diagnosis
The rapid spread of FMD after the virus is introduced highlights the need for a quick
and accurate diagnostic technique. The disease is often initially diagnosed based on
clinical signs and therefore requires vigilance by the farming community and
veterinary profession and the infrastructure to allow early reporting of disease. The
OIE recommends confirming a diagnosis of FMD by either the isolation of the virus
or by the detection of antigen and virus-specific antibodies. The determination of the
serotype resulting in the infection is essential in order to administer emergency
vaccination with an appropriate vaccine to control the disease. However serological
diagnosis also has great importance, particularly for epidemiological purposes such
as screening for antibody before animals are moved in a prevention and control
program (Adam and Marquardt, 2002; Remond et al., 2002).
Numerous diagnostic techniques have been developed for FMD with the aim of
developing a rapid, sensitive, specific and reliable method to effectively diagnose the
disease. The diagnosis of FMD can be divided into 3 categories based on the
detection of: clinical signs, virus, or antibody to the virus (Figure 1.3).
17
Figure 1.3: Different types of FMD diagnosis
Diagnosis
Laboratory Diagnosis
Virological Diagnosis
-Based on demonstration of
antigen
Pen-side test
Viral Isolation
-Cell culture
-Calf Thyroid cells
Suckling mice
IBR - S2
Immunological methods
- CFT
- Antigen capture ELISA
Nucleic acid recognition methods
-Nucleic acid hybridization assay
- PCR
Serological Diagnosis
-Based on demonstration of antibody against
FMDV
Antibody testing to Structural
Protein
Antibody testing to Non structural
Protein
Clinical Diagnosis
-Based on clinical signs shown by the
infected animals
18
1.3.3.1 Clinical Diagnosis
Cattle and pigs infected with FMDV usually show distinctive clinical signs after an
incubation period of 2 to 8 days depending on the virulence of the virus. The disease
is characterized by fever, anorexia and the appearance of vesicles on the mucous
membranes of the mouth including the tongue, dental pad, gums and lips. On the
feet, lesions are most prominent on the bulbs of the heel, along the interdigital cleft
and to a lesser extent along the coronary bands. Lesions may also be present in the
nares and on the muzzle, udder and the teats. In milking cows, there is a sudden drop
in milk production associated with infection. Rumen and heart lesions are frequently
found at necropsy, especially in animals prior to weaning. In sheep, goats and deer,
lesions are usually not obvious and may go unnoticed making these species a
potentially dangerous source of infection (Donaldson and Sellers, 2000).
However the clinical signs of FMD cannot be distinguished from other vesicular
diseases such as vesicular stomatitis and swine vesicular disease. Furthermore in
cattle, diseases including bovine papular stomatitis, bovine mucosal disease,
infectious bovine rhinotracheitis, bovine herpes mammillitis, malignant catarrhal
fever and rinderpest induce similar clinical signs and consequently laboratory
diagnosis is essential for confirmation of the disease (Mowat et al., 1972; Anon,
2000).
1.3.3.2 Virological Diagnosis
Primary viral diagnosis of FMD is carried out on epithelial tissue or vesicular fluid
from clinical samples using specific laboratory diagnostic techniques such as
19
Complement fixation tests (CFT), Enzyme link immunosorbent assays (ELISA) or
Polymerase chain reactions (PCR) as recommended by the OIE.
1.3.3.2.1 Viral isolation
Cell culture is required to detect the presence of live virus in suitable samples. This
procedure is recommended in the OIE manual (OIE, 2006) and is widely used by
many FMD diagnostic laboratories. There are many types of cell lines used including
calf thyroid cells (De Clercq, 2003b), 2 - 7 day old suckling mice cells, IBR – S2
cells, Lamb kidney cells (Snowdon, 1966), Pig cells (Bouma et al., 2001) and baby
hamster kidney (BHK 21) (Clarke and Spier, 1980), each which have advantages and
disadvantages.
1.3.3.3.2 Immunological methods
1.3.3.3.2.1 Complement Fixation Test (CFT) and Antigen Capture ELISA
The CFT has been the test of choice for the detection and typing of the FMDV in
epithelial samples from the field and has been used in many laboratories over the last
decade (De Clercq, 2003a). It is still recommended by the OIE but has gradually
been replaced by the antigen capture ELISA due to the CFT‟s disadvantages which
are its relatively low sensitivity and the fact that it is prone to difficulty in
interpretation due to both pro- and anti- complementary activity of samples. The
sensitivity and specificity of the test is also dependent upon the animal species tested
and is not sufficiently sensitive to detect infection (antigen) in pigs (Westbury et al.,
1988c). Furthermore the CFT has more cross-reactions compared to the ELISA and
the sensitivity of the test depends on the quality and type of samples collected
(Hamblin et al., 1984) . The advantages of the ELISAs are that they are easier to
20
perform compared to the CFT, and their sensitivities are generally higher than the
CFT (Caballero et al., 1997; Smitsaart et al., 1997).
1.3.3.3.2.2 Nucleic acid recognition
The nucleic acid recognition methods are comprised of two different techniques
(nucleic acid hybridization and PCR assays) which are dependent upon detection of
the viral genome. The nucleic acid hybridization assay is no longer used as a
diagnostic tool for FMDV as it is hampered by low specificity and sensitivity
compared with the PCR, but it did prove very useful in elucidating the cellular basis
of persistent infection (Zhang and Kitching, 2001).
Detection of viral RNA by a PCR is the diagnostic method of choice and several
tests have been developed for FMDV. The PCRs are very sensitive as they require
only a small quantity of sample. The fundamental requirements for PCRs are
selection of the primers and an efficient extraction method. The primers selected
should specifically target all FMDV types and subtypes without reacting with other
unrelated viruses of the same family. Specific primers have been designed to
distinguish between each of the seven serotypes (OIE, 2004). Efficient extraction of
the FMDV RNA from field samples is a prerequisite for the successful amplification
to prepare genetic material for sequencing. Among the techniques, the fluorogenic
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is rapid, with a higher
sensitivity and specificity compared with the conventional RT-PCR. This test is
more sensitive to Types O, A, C, and Asia1 than to SAT 1 and 2 (Reid et al., 2002).
21
The RT-PCR can also be used to amplify genome fragments of FMDV in samples
other than epithelium or ruptured vesicles, including milk, serum, vesicular fluid and
oesophageal–pharyngeal (OP) samples. Reverse transcription, combined with real-
time PCR, has a better sensitivity and is quicker when compared to virus isolation
(Huang et al., 2009).
1.3.3.3.2.3 Field tests
Field tests have been developed to accommodate the need for a rapid diagnosis
which is necessary for the effective control of FMD to allow prompt action to control
an outbreak (Reid et al., 2001) and at the same time avoiding the time taken to
transport samples to the laboratory. Over the years field tests have developed from
agglutination of particles linked to a specific antibody when antigen is present, to
detection of the virus by using a portable real-time PCR instrument (King et al.,
2008). However effective preparation of the template RNA is an important
consideration for the use of this technology in the field, since the presence of tissue-
derived factors may inhibit the RT-PCR (Hearps et al., 2002). The development and
use of a rapid chromatographic strip test or pen side lateral flow device (LFD) have
been evaluated. These use simple homogenizers that have been demonstrated to be
suitable for preparing epithelial suspensions under field conditions (Ferris et al.,
2009).
1.3.3.3 Serological Diagnosis
Serological diagnosis is another important method to confirm FMD. These
techniques work by detecting an antibody response specific to FMD. They are very
22
useful for disease surveillance following an outbreak and to identify subclinical
infection in species such as sheep and goats which have inapparent clinical signs.
There are two different methods in serological diagnoses: those that detect antibody
to structural proteins and those that detect antibody to non-structural proteins of the
FMDV.
1.3.3.3.1 Testing antibody to structural proteins
1.3.3.3.1.1 Virus neutralization test (VNT)
The Virus Neutralization Test (VNT) is a serotype specific test which is recognised
as a gold standard for the diagnosis of FMD (Clavijo et al., 2004; Kitching, 2004)
and is recommended by the OIE as the prescribed test for trade (OIE, 2008a).
However it requires cell culture facilities and takes two to three days to provide
results. It is very laborious and is not a reliable test due to false positive reactions.
The VNT is also not capable of differentiating antibodies that arise from natural
infection and those induced by vaccination (Moonen et al., 2004).
1.3.3.3.1.2 Enzyme linked immunosorbent assay (ELISA)
Enzyme linked immunosorbent assays (ELISA) were developed to overcome the
disadvantages shown by the VNT and the Liquid phase blocking ELISA (LPBE) has
been adopted by many laboratories worldwide for the routine screening of FMDV.
These tests are easier to perform and correlate well with the VNT (Hamblin et al.,
1986a). The ELISAs are also considered to be more reliable than the VNT and are
useful for evaluating the immunological response of animals following infection and
23
vaccination (Hamblin et al., 1987). However they have some disadvantages
including low specificity and the lack of stability of inactivated antigens (Clavijo et
al., 2004) . There are three types of ELISA: LPBE; Solid Phase Blocking ELISA
(SPBE) which has been used on cattle for the detection of Type O virus antibodies
and has been validated as a screening test and for the detection of antibody titre
(Chenard et al., 2003); and Solid Phase Competitive ELISA (SPCE) which is more
sensitive than the VNT in samples from sheep (Paiba et al., 2004). The SPCE is also
more serotype specific than the VNT or LPBE (Mackay et al., 2001). The solid-
phase ELISAs have better specificity (SPCE: 99.1% - 99.3% compared with LPBE -
CEDITEST : 92.2%) and reproducibility than the LPBE and VNT for large scale
serological testing (Niedbalski, 2004), however they are not able to differentiate
immunity induced from natural infection with that from vaccination.
1.3.3.3.2 Testing antibody to non-structural proteins
Serological diagnosis through the testing of antibody to non-structural proteins
(NSP) is valuable in the control of FMD, particularly in endemic areas and in areas
which are free of FMD but where vaccination against the disease is practiced.
Antibody to NSP produced by infected animals can be used to differentiate natural
infection from a reaction to vaccination (Diego et al., 1997; Mackay et al., 1998;
Sørensen et al., 1998; Clavijo et al., 2004; Moonen et al., 2004).
1.3.3.3.2.1 Agar gel Immunodiffusion test
24
The agar gel immunodiffusion test is a useful tool for epidemiological surveys of
livestock, however the interpretation of results in animals that have been repeatedly
vaccinated can be confusing (McVicar and Sutmoller, 1970).
1.3.3.3.2.2 Latex agglutination
Latex Agglutination Tests (LAT) or Latex Immunoassays involve a simple slide test
and are inexpensive, when compared with other techniques (Bangs, 1988).
Furthermore there is close agreement between the CFT and the LAT (Ozdural et al.,
2002).
1.3.3.3.2.3 Immunoelectro transfer blot analysis
The Immunoelectro transfer blot analysis, along with ELISAs, can detect antibodies
against recombinant 3AB1 proteins to differentiate between infected and vaccinated
cattle (Clavijo et al., 2004). The application of the enzyme linked immunoelectro
transfer blot assay has been upgraded from defining the antibody profile of infection
to confirming suspected or positive samples (Bergmann et al., 2000). Western blot
assays using NSP 2C and 3D as antigen are useful methods for the differentiation of
infected from vaccinated pigs (Fukai et al., 2008).
1.3.3.3.4 Non-Structural Protein (NSP) ELISA
Presence of antibody against NSP 2C, 3A, 3B, 3C, 3AB and 3ABC are indicative of
infection. Among these, the polypeptide 3ABC is recognized as the most appropriate
antigen which has high immunogenicity (Bergmann et al., 2000; Robiolo et al.,
2006). Previous studies have also indicated that serum antibody specific for NSP
25
3ABC is a reliable marker of FMDV replication in vaccinated cattle (Mackay et al.,
1998; Sørensen et al., 1998). This is of great relevance for serological surveillance
purposes following an outbreak to help substantiate freedom from FMD (Paton et al.,
2006) and to distinguish between animals infected with FMD from those with
immunity arising from vaccination against FMD to allow the safe movement of
animals (Kitching, 2002b; Kweon et al., 2003). The NSP test is also used to
differentiate vaccinated from convalescent animals and to determine the carrier
status of animals (Niedbalski and Haas, 2003a; Clavijo et al., 2004). Many
diagnostic methods to differentiate infection from vaccinated animals using NSP
tests have been developed, with some being used for screening purposes and others
as confirmatory tests. The ELISA-based diagnostic techniques for NSP antibody
detection provide many advantages, such as objectivity compared with gel diffusion
tests for NSP, high sensitivity and specificity, and the capability for large-scale
screening. The NSP ELISA tests have been recommended by the OIE to be used for
serological surveillance in regions or countries that practice vaccination against
FMD and for monitoring virus circulation in the field (OIE, 2004b). The NS
Panaftosa ELISA and Western Blot technique developed by Bergmann et al. (2000)
has been accepted by the OIE as the standard test for discriminating infected from
vaccinated groups of animals.
There are many commercial ELISAs available for the NSP. Six commercially
available ELISA (NCPanaftosa-screening from PANAFTOSA; 3ABC trapping-
ELISA from IZS-Brescia; Ceditest® FMDV-NS from Cedi Diagnostics B.V.,
Lelystad, The Netherlands; SVANOVIRTM FMDV 3ABC-Ab ELISA from
Svanova, Upsala, Sweden; CHEKIT-FMD-3ABC from Bommeli Diagnostics, Bern,
26
Switzerland; and the UBI® FMDV NS ELISA from United Biomedical Inc., New
York, USA) have been compared (Brocchi et al., 2006). Five of these tests detect
antibodies to the viral non-structural polypeptide 3ABC, expressed as recombinant
antigen in different expression systems, while the UBI kit recognises antibody to a
3B synthetic peptide. The study showed that the Brescia ELISA and the Ceditest
ELISA were more sensitive in non-vaccinated cattle than vaccinated cattle within
100 days of infection, with a sensitivity approaching 100%. Whereas in vaccinated
cattle the sensitivity varied from 93.9% (Panaftosa ELISA) to 68.2% (Chekit
ELISA). In contrast the specificities for all six ELISAs in non-vaccinated and
vaccinated cattle were not significantly different (all being more than 98%) (Brocchi
et al., 2006). However receiver-operator characteristic (ROC) curve analysis of the
six NSP ELISAs showed the IZS Brescia and Ceditest had specificities of 99% and
99.5% respectively which were higher than the other tests in exposed cattle (Dekker
et al., 2008). A study undertaken in New Zealand (Kittelberger et al., 2008) with the
Ceditest FMDV NS Blocking ELISA and the Bommeli/IDEXX Checkit FMD 3
ABC indirect ELISA reported similar specificities (Brocchi et al., 2006) in cattle,
sheep and pigs. The diagnostic specificity for Bommeli/IDEXX for cattle, sheep and
pigs were 99.9, 99.7 and 99.6% respectively and for the Ceditest FMD NS 99.5, 99.7
and 99.6% respectively. The repeatability of the Ceditest FMD NS was reported to
be better than the Bommeli/IDEXX test (Kittelberger et al., 2008).
Comparative evaluations, including the above NSP ELISA kits, were conducted by a
consortium of European and American FMD reference laboratories in 2006 using
large panels of sera from cattle that had been vaccinated or vaccinated-and-infected
with different serotypes of FMDV. However in that study there were insufficient
27
samples from pigs and sheep to evaluate the tests for these species (Brocchi et al.,
2006). The use of commercial NSP ELISAs is very useful for evaluating the status of
a herd but not so for individual animals (Clavijo et al., 2004). Repeated vaccination
may also result in positive reactions in cattle due to impurities in the FMD vaccine
(Lee et al., 2006).
1.3.4 Control of FMD
Foot and mouth disease has occurred in almost two thirds of the OIE member
countries. Recent epidemics in countries such as the United Kingdom and Taiwan
have resulted in significant economic losses, however the disease remains endemic
in many developing nations (Rweyemamu and Astudillo, 2002).
Social, economic and political factors in some countries in Asia can impact on the
control of FMD (Anonymous, 2008). Although the economic status of a country has
a significant impact on the ability of a country to control FMD, other factors also
play key roles in the control of the disease. Geographical barriers can play an
important role in the control of the disease, as does implementation of legislation on
animal health and movement, the availability of expertise (both field and laboratory
staff) and adequate diagnostic laboratory facilities. Since FMD is a transboundary
animal disease, the control of it requires cooperation between neighbours at the local,
national and regional level (Lubroth et al., 2007). Based on the status of FMD, OIE
member countries are divided into FMD free countries and FMD endemic countries.
The FMD free countries are further subdivided into three categories: FMD free
without vaccination; FMD free with vaccination; and FMD free zone within the
country (OIE, 2008b).
28
1.3.4.1 Vaccination
Vaccination is one of the tools used in the control of FMD and has been studied
extensively. Vaccination has been demonstrated to limit transmission of FMDV to
in-contact susceptible animals (Doel et al., 1994; Salt et al., 1998; Cox et al., 1999).
Moreover some outbreaks of FMD that have been controlled by vaccination have not
recurred (Barteling, 2002; Leforban and Gerbier, 2002). The ability of a vaccine to
protect against the disease and prevent the spread of the virus is dependent upon the
vaccine‟s potency (Barnett and Carabin, 2002). However recent studies have
indicated that protection is also affected by the serotype, as well as the type of
adjuvant used in the vaccine (Jamal Syed et al., 2008).
Although culling has been reported to be more effective than vaccination,
combination of both practices can allow for the more rapid control of the disease
(Ferguson et al., 2001a). In countries which were previously free from FMD, the use
of emergency vaccination and culling of animals suspected to be infected or
originating from high risk areas, and restriction on animal and product movements
can result in disease eradication (Orsel et al., 2005). Other studies have demonstrated
that emergency vaccination, when implemented swiftly, will significantly reduce
clinical disease and subclinical virus replication and shedding, particularly in the
early stages of infection (Cox et al., 2007). However the significance of carrier
animals in the epidemiology of FMD is still not clearly understood (Kitching et al.,
2007) and there is a potential risk that they could subsequently lead to new
outbreaks. Some studies have demonstrated that whether an animal becomes
persistently infected is influenced by vaccination, where vaccination is able to reduce
viral replication, persistence and excretion compared to unvaccinated and clinically
29
infected sheep (Barnett et al., 2001; Parida et al., 2008). However a model
constructed by Schley et al. (2009) indicated that, although there was a potential to
increase the number of undetected carrier animals following vaccination, vaccination
offered significant benefits if administered prior to exposure to the virus. This
finding was in contrast to that of others who consider the risk of persistently infected
animals transmitting the disease is low (Cox et al., 2005).
In countries where FMD is endemic, vaccination can be used as a primary tool to
suppress the disease. Vaccination is applied to induce herd immunity leading to a
reduction in clinical infection and viral transmission (Orsel et al., 2007) and
systematic vaccination has been shown to be an effective way of controlling and
eliminating FMD from certain regions of the world, such as western Europe and
south America (Leforban and Gerbier, 2002; Saraiva, 2004). A study by Anderson et
al. (1974) indicated that vaccination was a useful tool for the eradication of FMD
from an endemic area. The crucial part of a vaccination programme is conferring
immunity to a sufficiently high proportion (>80%) of the population to prevent the
infection spreading to susceptible animals. One study indicated that goats also need
to be included in vaccination control programmes using the same schedule as for
cattle (Madhanmohan et al., 2009). Susceptible animals also must be prevented from
entering the vaccinated population. However implementation of a proper, systematic
vaccination programme as a control measure can be difficult since vaccines are
expensive, have a narrow antigenic spectrum, provide only short term immunity and
are very fragile. This results in a significant economic cost for most developing
countries where the disease is endemic (Kitching et al., 2007).
30
1.3.4.2 Movement Restrictions
Movement of infected animals is one of the most important factors in the spread of
FMD, especially in an endemic region (Cleland et al., 1996; Rweyemamu et al.,
2008b). Control of livestock movement, together with vaccination, can be effective
in the control and eradication of FMD (Anderson et al., 1974). Therefore movement
restrictions combined with effective ring vaccination, proper quarantine procedures
and establishment of a buffer zone can be applied in certain predefined areas in an
endemic country or region to create an FMD free zone. A zoning approach has been
effectively applied in many parts of the world to help control the disease and this
approach has been predicted to have a high level of success in Southeast Asia
(Edwards, 2004). However it has also been suggested that proper quarantine
procedures with vaccination, without movement restrictions, is more appropriate in
Southeast Asia because of cultural and lifestyle reasons (Sasaki, 1994a).
1.3.4.3 Stamping out
Stamping-out involves the slaughter and destruction of all infected animals and their
immediate susceptible contacts, followed by thorough cleaning and disinfection of
the affected premises. It was first adopted in the United Kingdom in 1892 and has
been used widely since (Fogedby, 1958). A stamping out policy is frequently
implemented in previously FMD free countries to ensure the disease is completely
eradicated in the event of an outbreak. This involves culling of all susceptible
animals on infected or high risk premises and strict movement restrictions
(Scudamore and Harris, 2002). Culling is considered a crucial step to regain FMD
free status from the OIE, especially for economic reasons. The decision for radial
31
culling or vaccination is normally made based on a mathematical model developed
for the local infected area such as in the 2001 outbreak in the UK (Ferguson et al.,
2001a; Keeling et al., 2001).
Some countries believe stamping out by slaughter or culling is the most effective
way to eradicate or control FMD. It is also often regarded as the cheapest and fastest
way to regain the desired freedom status. However it gives rise to ethical issues, as
well as the potential for environmental pollution and the negative psychological
effects it can have on farmers (Woods, 2004). Consequently alternative methods
have been investigated extensively. A combination of culling with emergency
vaccination to provide early protective immunity (Doel et al., 1994; Cox et al., 1999)
and reduced virus replication and excretion to limit transmission of the disease to
susceptible animals has been recommended (Cox et al., 1999; Orsel et al., 2007;
Parida et al., 2008). Furthermore the ability to detect infection in vaccinated animals
by using the NSP antibody test further supports the use of emergency vaccination as
a useful tool (Bruderer et al., 2004; El-Hakim, 2005). Therefore an optimal reactive
and responsive vaccination programme, combined with efficient culling and
restrictions on animal movements, could be effective control measures in FMD
outbreaks (Tildesley et al., 2006). Other researchers believe that establishment of a
quarantine-barrier to prevent the introduction of disease, and a veterinary and
laboratory capability to rapidly detect the disease are important (Boyle et al., 2004).
However the effectiveness of FMD control is also greatly influenced by the
geographical situation. For example a stamping out policy was successful in the
British Isles, Ireland, Scandinavia, and North America due to their favourable
geographical isolation (Sutmoller et al., 2003). The greatest disadvantage of a
stamping out policy is the extreme demand for resources including veterinarians,
32
slaughter teams, disinfection teams, heavy machinery and the permanent lost of
genetic potential (Pinto, 2004).
1.3.4.4 Application of Models
The evaluation and creation of simulation models for FMD has advanced rapidly and
models can be used to aid in the choice of control policies during an outbreak
(Morris et al., 2002). However the creation of a reliable model requires the
availability of good data. Models should be as simple as possible, transparent and
easily adapted or extended with limitations and strengths clearly identified (Thornley
and France, 2009). Some researchers consider that the strength of models lies in their
ability to evaluate a wide variety of epidemiological parameters and control options
(Tildesley et al., 2006). Many simulation models have been developed to formulate
effective control strategies, however most are based on data from FMD free
countries and optimal control policies are not always easily transferred between
countries (Tildesley and Keeling, 2008). Some simulation models have been
designed to help in the management of future outbreaks and to identify aspects
which are critical for the rapid control and eradication of the disease (Yoon et al.,
2006) while others have been used to predict the role of risk factors such as wild or
feral animals (Ward et al., 2007), distribution of airborne particles (Gloster et al.,
2007) and wind direction and velocity on airborne spread of virus between farms
(Gabier et al., 2002).
Geographical Information Systems (GIS) have been used in spatial and temporal
models. Much work has been done utilising existing historical local data (Ferguson
et al., 2001b; Keeling et al., 2001; Picado et al., 2007). Some studies have only
looked at disease occurrence patterns (Gallego et al., 2007) while others have
33
incorporated other factors, such as disease dynamics, host demography and logistical
constraints, to formulate optimal disease control (Jacquez, 2008; Shiilegdamba et al.,
2008; AlKhamis et al., 2009).
A model developed to estimate the prevalence of carriers of FMD after vaccination
indicated that the prevalence of carrier-containing herds was likely to be very low
(Arnold et al., 2008). In contrast other models have indicated an increase in the
number of carrier animals (Schley et al., 2009). More advanced models integrate
genetic data with the relative timing of infection events to compute the likely
transmission of infection between farms (Cottam et al., 2008).
1.3.5 Disease Distribution
Foot and mouth disease is a global animal disease which is considered to be a major
constraint to the international trade of livestock and animal products in infected
countries. Outbreaks of FMD have occurred in every livestock-containing region of
the world with the exception of New Zealand, and the disease is currently endemic in
all continents except for Australia, Antartica and North America (Grubman and
Baxt, 2004). Even though there are many countries free from FMD, the virus is
widely distributed in the world. In contrast the distribution of the serotypes is not
even throughout the infected regions and countries. Six of the 7 serotypes are present
in Africa (O, A, C, SAT 1, 2, 3), 4 in Asia (O, A, C and Asia 1) and only 3 in South
America (A, O, and C). Periodically infections with SAT 1 and SAT 2 occur in the
Middle East originating from Africa (Rweyemamu et al., 2008b).
34
Foot and Mouth Disease is currently endemic in 7 Southeast Asian countries:
Cambodia, Laos, Malaysia, Myanmar, parts of The Philippines, Thailand and
Vietnam. Singapore, Brunei, Indonesia, East Malaysia and the Islands of Mindanao,
Visayas, Palawan and Masbate in the Philippines are considered free without
vaccination (OIE, 2009). The most common serotype present in this region is O with
occasional outbreaks of serotype A (Sabirovic et al., 2009). The Southeast Asian
topotype is considered to be the indigenous strain and two other topotypes present
are the Pan-Asia and Cathay strains. Topotype Cathay is a pig adapted strain. The
Cathay and Pan Asia O topotypes originated from South China and crossed into
Vietnam and then spread westward into Cambodia, Laos and eventually Thailand.
Asia 1 followed the movement of livestock from Bangladesh (which imports up to 2
million head of cattle each year from India) into Myanmar and finally followed the
route of livestock movement down to Malaysia (Rweyemamu et al., 2008b). The
movement of livestock resulted in the spread of the disease from the southern part of
Thailand to the northern part of Malaysia. Clustering of outbreaks occur during the
months of October to December when there is a high demand for live cattle because
of the holding of local festivals in Malaysia (Sasaki, 1994b). A similar pattern of
outbreaks following the movement of cattle has also been recognised in the southern
regions of Cambodia and Vietnam (Sasaki, 1994b). Other than cattle movement,
contact with infected cattle at grazing/watering areas has been recognised as
important in Myanmar (Kyaw Naing Oo, personal communication 2010) and in the
Philippines spread has been reported to occur mainly through contaminated fomites
(Abila and Foreman, 2006).
35
1.4 Overview of the current study
Although FMD is endemic in peninsula Malaysia, including the state of Pahang,
there is a deficiency of knowledge about the epidemiology of the disease in Pahang.
Consequently the work reported in this thesis was developed to address this lack of
knowledge. The specific objectives of the study reported in this thesis were:
1. To determine the temporal and spatial distribution and pattern of historical
FMD outbreaks in Pahang, Malaysia
2. To identify the risk factors associated with the occurrence of FMD in Pahang
3. To study the antibody response in local cattle following vaccination against FMD.
The information gained from this study will help further the understanding of the
epidemiology of FMD in Pahang which, in turn, will help in the development of
suitable methods for controlling the disease.
In the following chapter the materials and methods adopted in this thesis are
summarised.
36
CHAPTER 2
GENERAL MATERIALS AND METHODS
2.1 Serological tests for measurement of antibodies against FMD
Whole blood from cattle was collected from the jugular vein in plain blood tubes
(BD Franklin Lakes USA). After collection the tubes were stored at a 45º angle at
room temperature for approximately one hour and then placed in a refrigerator at 4ºC
for 12 hours. The tubes were then centrifuged at 2000 rpm for 10 minutes and the
sera decanted. The sera were stored at -20°C until used in the assay for FMD.
2.1.1 Test for antibody against non-structural proteins
A commercially available NSP ELISA (Ceditest FMDV-NS ELISA manufactured
by Cedi Diagnostics, B.V., Lelystad, The Netherlands) was used to test for
antibodies to NSP‟s (Sørensen et al., 1998). The Ceditest is a blocking ELISA which
can be used for the detection of antibodies against FMDV in serum from cattle,
sheep, goats and pigs. It was used to detect antibody directed against the non-
structural 3ABC protein of FMDV as described in Appendix 1.
2.1.2 Post- vaccination evaluation test
A liquid phase blocking ELISA for detection of FMDV (Serotypes O, A and Asia 1)
antibodies was ordered from the FMD world reference laboratory (WRL), Pirbright,
United Kingdom. The test is based upon specific blocking of FMDV antigen in
liquid phase by antibodies in the test serum sample (Hamblin et al., 1986b, a). The
37
test procedure adopted was obtained from the FMD WRL, Pirbright as described in
Appendix 2.
2.2 Statistical and descriptive analysis
All recorded data in this study, such as the questionnaire data in Chapter 3 and the
percentage of inhibition data for the antibody response towards FMDV vaccination
as described in Chapter 4, were entered into a spreadsheet in Microsoft excel 2007
and exported into a statistical package (SPSS version 17 Statistics GradPack for
Windows) for analysis. Descriptive analyses were performed using the frequency
and percentage function. Percentages and their 95% confidence intervals using the
exact binomial method were calculated. Before any statistical tests were performed,
all recorded information was carefully compared with the paper questionnaires to
identify any typographical or transcription errors.
All hypothesized risk factors (categorical and continuous variables) were
individually screened for association with a history of FMD clinical infection.
Initially univariable analyses were conducted with a Chi-Square test for
independence or Fisher‟s exact test to investigate possible associations between
categorical variables and a clinical history of FMD. Results were considered to be
significant if P < 0.05. Odds ratios (OR) and their 95% confidence intervals were
also calculated (Pallant, 2004; Thrusfield, 2005). Factors with a significance level of
P < 0.2 and/or which were considered to be biologically important were offered to
the multivariable binary logistic regression model.
38
Continuous variables were tested for normality by performing a Kolmogorov-
Smirnov and Shapiro-Wilk test for normality. Skewness, kurtosis and P-P plot were
also examined. The data were considered to be normally distributed if the
significance for the Shapiro-Wilk was > 0.05, skewness was zero or close to zero;
the Kurtosis was zero or very near zero and all data points were symmetrical and
perpendicular to the diagonal line of the P-P plot (Field, 2009). The linearity of
continuous variables was also examined. The mean values of normally distributed
continuous variables were analysed for significant difference with an ANOVA.
Continuous variables which were not normally distributed and were not linear were
categorised prior to analysis. The log odds ratios for each group for these variables
were plotted against the midpoint for that group prior to inclusion in the
multivariable logistic regression model. A correlation coefficient of 0.7 or higher
was used to support the assumption of linearity (Hill and Ward, 2008). Continuous
variables with a P < 0.2 and/or which were considered to be biologically important
were then subsequently included in the multivariable binary logistic regression
model.
Potential multi-collinearity between predictor variables (close linear relationship
between two or more variables) was assessed by checking the collinearity parameters
between each independent variable through calculating a Variable Inflation Factor
(VIF). Variables were considered to have a high level of multicollinearity if the
collinearity value was greater than 0.8 and the VIF greater than 1 (Field, 2009). The
correlation between variables was assessed by calculating Pearson‟s or Spearman‟s
39
correlation coefficients, where appropriate. If the variables were not mutually
exclusive and consequently highly correlated, only one of the related variables
(selection was based on predictor variables that were more highly related to the
infection) was included in the initial model. The decision to include/exclude
variables was made on the basis of distribution and biological plausibility (Dohoo et
al., 2003).
A binary logistic regression model was built using a manual backwards elimination
process as described by Dohoo et al. (2003), with a significance level of P > 0.05 as
the criterion for removal of a variable from the model. Variables with high
collinearity (r > 0.8) were excluded from the analysis. The analysis commenced with
a full or saturated model and variables were eliminated from the model in an iterative
process. The level of significance for a factor to remain in the final model was set at
5%. The suitability of the final model was assessed based on the Hosmer and
Lemeshow, Cox and Snell R Square and Nagelkerke R Square values (Field, 2009).
The percentage of inhibition (PI) data were analysed using the repeated measures
analysis of variance (Repeated-Measures ANOVA) to describe the within random
samples variance rather than the between group variances. In this experiment the
sample members were matched according to the age of the cattle (calf and cow
groups) which had been vaccinated once or twice. The analysis for this study is
outlined in more detail in Chapter 4.
40
2.3 Questionnaire
A standardized questionnaire containing open and closed questions was developed in
English and then translated into Bahasa Melayu. It was also translated back into
English to correct any misunderstanding in the use of language before the final
version was produced (Lee et al., 1999 ). The questionnaire (Appendix 3) was then
pretested in Pahang on ten Assistant Veterinary Officers (AVO) and ten cattle
owners. The use of the questionnaire was approved by the Murdoch University
Human Ethics Committee. The questionnaire was designed to collect information
about the management, husbandry and health of cattle and to determine the
association between these factors and the presence of FMD infection. Questions
were included on the importation or purchase of cattle for breeding purposes, the
practice of exchanging cattle with other farmers, the vaccination program adopted,
the feed supplements provided to cattle, source of water, practices adopted if cattle
with positive serology were detected, farm biosecurity practices adopted, presence of
other animals on the farm, distance to the nearest farms with livestock, and the
farmer‟s knowledge about the clinical signs of FMD and the treatment and control of
the disease (Appendix 3).
41
CHAPTER 3
RISK FACTOR ANALYSIS ASSOCIATED WITH FOOT AND
MOUTH DISEASE IN THE STATE OF PAHANG, MALAYSIA.
3.1 Introduction
Pahang is one of 13 states in Malaysia and is the third largest state after Sabah and
Sarawak. It is situated in the eastern coastal region of Peninsular Malaysia and has
an area of 3,596,585 hectares and contains 11 districts and 71 subdistricts. The
physical geography can be broken down into 3 sections: highlands, rainforest and
coastal areas with rainforest accounting for approximately 2/3rd
of the land area
(Anonymous, 2007). The major economic activity is agriculture, primarily oil palm
cultivation and rubber plantations (Pahang, 2007). In 2009 the ruminant population
in Pahang was: 156,753 cattle, 19,884 buffalo, 37,712 goats and 11,395 sheep (DVS,
2009c). Cattle are commonly raised to supplement household income and for
slaughter during religious and other special occasions. This situation makes the
control of disease more challenging because of the low priority farmers place on
their animals. Many cattle owners also keep small numbers of sheep and goats
(Figure 3.1); however they do not raise pigs (DVS, 2009c). Although pig farming is
not encouraged by the state government, importation of live pigs from other states is
allowed. These pigs are transported on lorries to the local pig slaughter houses
located in the 4 districts of Kuantan, Raub, Bera and Temerloh.
In late December 2003 until 2004, Pahang experienced the largest epidemic of FMD
in the state‟s history after 18 years of freedom, as described in Chapter 1. The
epidemic was caused by FMD virus serotype O and was suspected to have been
42
introduced through the illegal movement of infected cattle from Terengganu (the
neighbouring state). This epidemic spread rapidly in the state involving many of the
districts; however no detailed epidemiological studies were undertaken to evaluate
the relative importance of potential risk factors associated with the outbreak.
Currently there is a control program for FMD in Pahang and mass vaccination was
one of the options considered in this control program, however due to the cost of
mass vaccination the program was revised. A trivalent vaccine (0, A and Asia 1) is
administered by the DVS twice a year to give at least 80% coverage in the ruminant
population across the state (DVS, 2002). The policy to control local outbreaks
involves ring vaccination with the trivalent vaccine, together with the control of
animal movements. Despite this program, the disease is endemic in the state. There
are many possible reasons for a considerable number of outbreaks occurring each
year in the face of vaccination. However a thorough understanding of the
epidemiology in the state is required to design ecologically and culturally acceptable
and appropriate control measures for this important trans-boundary animal disease.
The aim of the research outlined in this chapter was to identify management and
husbandry factors adopted in Pahang that facilitate the distribution of FMD within
the state and factors which were protective against the disease.
43
Figure 3.1: Cattle kept together with goats
3.2 Materials and methods
3.2.1 Study Area
The study was conducted from August 2008 to June 2009 and involved ten of the 11
districts in Pahang. The temperature is relatively uniform throughout the year (range
of 21°C to 32°C) and the humidity is approximately 80% throughout the year.
During the months of January to April, the weather is generally dry and warm.
Pahang has an average annual rainfall of 2,000 to 2,500 mm with May to December
being the wettest months. The ten districts included in this study were selected based
on their cattle rearing activity. Cameron Highland was the only district not included
due to the fact that no cattle rearing activity is undertaken in the district. No prior
sampling frame existed; therefore this study was constructed using the DVS Pahang
Cattle Rearing Activity Record and historical reported cases of FMD.
44
3.2.2 Study design
A case-control study was conducted based on the clinical history of FMD recorded
by the DVS together with data from cattle rearing activity records. The cattle rearing
activity record was collected from the DVS in Pahang and compared with data on the
occurrence of FMD from the DVS Pahang records. A stratified multistage random
sample of cattle herds involving 200 cattle owners (participants) was formulated.
Twenty cattle owners who owned more than 15 cattle from each of the 10 districts
were chosen (total 200 cattle owners). Ten of the owners selected from each district
had a clinical history of FMD in their farms (case group) and 10 owners had not had
a clinical history of FMD in their farms (control group). The cattle owners were
notified of the survey by telephone and a letter explaining the purpose of the
research, together with a consent form and information about the confidentiality of
the farmer‟s identity was then sent to the participants. The questionnaires had been
approved by the Human Ethics Committee at Murdoch University (Human Ethics
Permit 2008/194). One Assistant Veterinary Officer from each district was chosen to
distribute and collect the questionnaire from the selected participants. Each cattle
owner was given a letter explaining the study, confidentiality of the data and
participants, the studies objectives and the participant‟s ability to withdraw from the
survey at any time.
3.2.3 Questionnaire
The development of the questionnaire was outlined in Chapter 2.
45
3.2.4 Data analysis
The questionnaires were collected and analysed as described in Chapter 2.
3.3 Results
3.3.1 Descriptive analysis
Of the 200 farmers selected, 176 completed questionnaires but only 171 could be
used as 5 respondents had less than the minimum number of 15 cattle (all these 5
respondents were from the districts of Kuala Lipis). Completed surveys were
obtained from 93 controls and 78 cases. No questionnaires were obtained from the
district of Pekan as the key person could not be contacted. Twenty surveys were
obtained from each of the other districts except for Temerloh where four farmers did
not participate.
The result of the study indicated that there was active movement of cattle within the
state, with 98% of respondents trading cattle in the year preceding the survey. All
these respondents traded animals with other farmers in Pahang. In contrast few
respondents imported cattle from other countries (1.8%) or from other Malaysian
states (2.4%). Some (14%) respondents exchanged animals with other farmers and
25% introduced bulls from outside their herd for breeding purposes.
Approximately half (53%) of the respondents allowed their cattle herds to graze
freely and 51% of herds were grazed close (within 2 km) to other herds. People,
other than the family or workers, were allowed to enter the land where the cattle
were grazed by over half (58%) of the respondents.
46
Some of the questions in the questionnaire were designed to assess the farmer‟s
knowledge regarding the clinical signs, spread, prevention and control measures
implemented for FMD. Approximately 87% of all participants (89.6% of the case
group and 87.5% of the control group - OR = 1.23 (0.48, 3.18)) were able to describe
the clinical signs of FMD. However only 36.9% of all participants (51.6% case
group, 40.4% control group - OR = 1.57 (0.84, 2.93)) were able to explain the way
the disease spread while a larger proportion (overall 59.5%, with 54.5% and 63.5%
in the case and control groups respectively - OR = 0.69 (0.37, 1.27)) of participants
admitted that they did not have any knowledge on how the disease spread. However
there were no significant differences between control and case groups for these
factors (95% CI for OR included the value 1.0). Just over half (56%) of the
participants identified vaccination as one of the measures to control the disease,
however only 29% understood that vaccination of animals was required every 6
months. Approximately one third (32.7%) believed it (vaccination) should be
administered only once a year, 1% replied three times a year and 37% did not know
the required frequency of vaccination. The respondents also gave a wide range of the
age at which vaccination should be commenced, starting from immediately after
birth to more than one year of age, however 60% of the respondents did not know
the age cattle should be when they received their first vaccination.
3.3.2 Univariable analysis
In Tables 3.1 and 3.2 the potential risk factors for the disease and their association
with case and control farms is displayed. A total of 18 from 31 potential risk factors
47
met the criteria for inclusion into the multivariable logistic regression analysis
(indicated by asterisks in the tables). Most of these were factors relating to
biosecurity. The factors that increased the likelihood of FMD were owning other
livestock, owning goats, exchanging stock, serological reactors to FMD remaining in
the herd after testing, free grazing of pasture all the time, purchasing of new cattle
from other farms, allowing unauthorised vehicles to enter the farm, not giving feed
supplements, not practicing quarantine procedures, not fencing the area where the
herd grazed, not disinfecting tyres, vehicles or footwear of visitors prior to entering
the herd, animals not tested for FMD prior to entering the herd or before leaving the
herd and separating cattle according to their age group.
48
Table 3.1 Univariable analyses of putative risk factors for foot and mouth disease in
cattle in Pahang
Factor
% herds
positive
for FMD
Odds
Ratio
(OR)
95%
confidence
intervals
P
Other livestock owned 56.7 2.29 1.22 , 4.32 0.01*
No other livestock owned 36.4 1.0
Goats owned 59.2 2.30 1.17 , 4.55 0.015*
Goats not owned 38.7 1.0
Buffalo owned 53.3 1.53 0.69 , 3.38 0.291
Buffalo not owned 38.7 1.0
Deer owned
Deer not owned
100.0
44.3
0.44
1.0
0.37 , 0.53
0.264
Palm Kernel Cake given as a feed supplement 37.2 1.0
Palm Kernel Cake not given 52.4 1.86 3.45 , 1.01 0.047*
Tap water used for drinking water 38.5 0.75 0.32 , 1.78 0.513
Tap water not used for drinking water 45.4 1.0
River water used for drinking water 45.4 1.22 0.58 , 2.56 0.601
River water not used for drinking water 40.5 1.0
Different age groups separated 9.1 1.0
Different age groups not separated 50.0 1.0 43.48 , 2.26 0.000*
Quarantine procedures practiced 32.0 1.0
Quarantine procedures not practiced 51.3 2.23 4.51 , 1.12 0.022*
Herd area fenced 40.0 1.0
Herd area not fenced 58.1 2.08 4.22 , 1.03 0.039*
49
Factors
% herds
positive
for FMD
Odds
Ratio
(OR)
95%
Confidence
interval
P
Disinfectant used at the farm entrance
Disinfectant not used at the farm entrance
27.6
48.9
1.0
2.51 6.06 , 1.04 0.036*
Unauthorised person allowed to enter the
herd/farm
Unauthorised person not allowed to enter
herd/farm
47.4
41.4
1.28
1.0
0.69 , 2.38
0.442
Unauthorised vehicles allowed to enter the
herd/farm
Unauthorised vehicles not allowed to enter
herd/farm
50.0
34.0
1.94
1.0
0.99 , 3.83
0.052*
Farm registered with DVS Pahang
Farm not registered with DVS Pahang
47.0
36.4
1.55
1.0
0.71 , 3.41
0.271
Cattle kept in a barn and fed
Cattle not kept in a barn and fed
58.8
43.0
1.89
1.0
0.68 , 5.23 0.215
Free grazing allowed for a period of time
Free grazing not allowed
41.3
46.7
0.80
1.0
0.43 , 1.51 0.496
Free grazing practiced all the time
Free grazing not practiced all the time
50.0
36.4
1.75
1.0
0.93 , 3.30
0.082*
Herd vaccinated against FMD
Herd not vaccinated against FMD
42.2
52.5
0.66
1.0
0.32 , 1.35 0.252
Exchange livestock with other farmers
Don‟t exchange livestock
65.2
42.1
2.57
1.0
1.03 , 6.47
0.039*
50
Factors
% herds
positive
for FMD
Odds
Ratio
(OR)
95%
Confidence
interval
P
New stock brought into the herd 38.9 0.77 0.36 , 1.63 0.493
New stock not brought into the herd 45.3 1.0
New cattle purchased from friends 53.1 3.02 0.68 , 13.51 0.138
New cattle not purchased from friends 27.3 1.0
New cattle introduced into the herd
New cattle not introduced into the herd
52.0
41.5
1.53
1.0
0.78 , 2.97
0.212
Outside bull introduced into the herd 41.7 0.86 0.41 , 1.81 0.685
Outside bull not introduced into the herd 45.5 1.0
Physical inspection by veterinarian practiced
before buying new animals
48.7 0.82 0.45 , 1.57 0.547
Physical inspection by veterinarian not
practiced before buying new animals.
53.7 1.0
Blood test performed on cattle before entering
herd
Blood test not performed before cattle enter the
herd
26.53
51.33
1.0
2.92
6.10 , 1.40
0.003*
Blood test performed on cattle before leaving
the herd
Blood test not performed before cattle leave
the herd
28.07
51.42
1.0
2.71
5.41 , 1.36
0.004*
FMD seropositive animals remain in the herd
after testing
Seropositive animals removed from the herd
70.3
37.5
3.94
1.0
1.79 , 8.68
0.000*
51
Factors
% herds
positive
for FMD
Odd
Ratios
(OR)
95%
confidence
interval
P
Seropositive animals slaughtered 42.5 0.76 0.40 , 1.45 0.407
Seropositive animals not slaughtered 49.2 1.0
Seropositive animals isolated on the same farm 43.8 0.95 0.44 , 2.06 0.889
Seropositive animals not isolated on the same
farm
45.1 1.0
Table 3.2 Univariable analyses of potential continuous risk factors for FMD in cattle
in the state of Pahang.
Variable name Mean positive Mean Negative F value P value
Number of herds 2.45 1.51 14.899 0.000*
Number of cattle 123.65 125.98 0.014 0.905
Number of new cattle introduced 7.84 9.68 0.177 0.675
Number of new cattle introduced in
a year
2.05
0.76
3.414
0.066
Number of cattle sold in a year 34.6 25.67 0.334 0.564
Number of cattle vaccinated
recently
84.95
106.24
0.749
0.389
Number of vaccinations used in a
year
1.44
1.52
0.577
0.449
* : Factors included in the Logistic Regression Model
3.3.3 Multivariable analysis
52
The final model was produced using backward conditional binary logistic regression
and the results are displayed in Table 3.3. Five variables (factors) were retained in
the final model, of which three were associated with the disease and two were
protective. The most strongly associated factor with FMD was retaining seropositive
animals in the herd (P =0.006; OR=3.62; 95% CI 1.44, 9.11). Cattle farmers who
kept other livestock were more likely (P=0.003; OR 3.2; 95% CI 1.47, 7.07) to have
an infected FMD herd than owners who didn‟t keep other species of livestock.
Farmers which allowed the entry of unauthorised vehicles onto their farmland were
also more likely to have an infected herd (P=0.05; OR = 2.2; 95% CI 1.0, 4.82).
Farmers who owned only one herd were less likely (P=0.001; OR = 0.27; 95% CI
0.12, 0.60) to have an infected herd than farmers with more than one herd.
Separating a herd into different age groups also provided some protection against the
disease (P=0.02; OR = 0.15; 95% CI 0.03, 0.72).
The final model had a good fit with a chi square value for the Hosmer and
Lemeshow Test of 3.48 (P = 0.9). The Nagelkerke R square was 0.344 suggesting
that 34.4% of the variability could be explained by the final set of variables.
53
Table 3.3: Multivariable analysis of potential risk factors for foot and mouth disease
in cattle in Pahang.
Variable name B P OR 95% CI for OR
Own only one herd -1.300 0.001 0.273 0.124 0.602
Keep other livestock 1.171 0.003 3.224 1.471 7.067
Cattle separated according to age
groups
-1.926 0.018 0.146 0.029 0.722
Positive NSP animals remain in the
herd
1.285 0.006 3.615 1.435 9.111
Allow unauthorised vehicles to
enter the herd area (farm land) 0.774 0.057 2.168 0.976 4.818
Constant -0.419 0.343
3.4 Discussion
The final model contained three factors associated with infection and two protective
factors. Not surprisingly a history of positive serology to FMD was strongly
associated with clinical disease. Studies have shown that FMDV can be transmitted
from infected to susceptible animals by a variety of mechanisms including air borne
spread (Hyslop, 1965a; Alexandersen et al., 2003b; Schijven et al., 2005) with pigs
excreting the most virus followed by cattle and sheep (Donaldson et al., 1970).
However direct contact is the most common route of transmission (Donaldson et al.,
1987) and importantly the virus can be excreted during the incubation period
54
(Alexandersen, 2002; Brown, 2004; Orsel et al., 2009) and animals incubating the
disease play an important role in the dissemination of the virus during this period
(Sutmoller and Olascoaga, 2002). Therefore while a shedder remains in a herd the
risk of virus transmission to susceptible animals remains. Based on the finding that
herds that retain seropositive animals were 3.6 times more likely to be infected, it is
advisable to cull/dispose of FMD seropositive reactors as soon as possible after
identification. However in vaccinated herds, detection of NSP positive results do not
necessarily mean animals are infected and further testing is required before the
culling or disposal of seropositive animals can be recommended (Clavijo et al., 2004;
Scudamore, 2004). Cattle owners are reluctant to cull/dispose of NSP seropositive
animals probably as a result of not having a clear understanding about the disease,
particularly with respect to its spread and control. This was confirmed in the
descriptive analysis where only 37.3% of respondents were able to correctly explain
how FMD spread. Therefore further extension and education programs, to increase
the cattle owner‟s knowledge on FMD, are essential in order to help them understand
the disease and to appreciate the implementation of more suitable and effective
control programs. In Pahang there is no compensation for the culling of clinically
affected or NSP seropositive animals and consequently there is little financial
incentive to support culling (Rossides, 2002; Cassagne, 2002).
Respondents who also owned other livestock (predominantly small ruminants -
sheep and goats and buffalo - Bubalus bubalis) had increased odds of having an
infected cattle herd. Others have also highlighted the role of owning other livestock
in increasing the risk of FMD infection in a herd (Al-Majali et al., 2008). Another
study in Thailand reported that co-grazing of cattle and buffalo with other livestock
55
on a farm was commonly linked with FMD outbreaks (Cleland et al., 1995). Foot
and mouth disease can infect all cloven-hoofed animals, including domestic and wild
ruminants and pigs. The virus usually induces milder clinical signs in adult sheep
when compared with cattle or pigs, and these signs are often subtle enough to go
undetected (Alexandersen et al., 2003c). Furthermore studies have demonstrated that
the virus can be detected in the respiratory tract of sheep 1 to 2 days prior to the
appearance of clinical signs (Sellers and Parker, 1969; Donaldson et al., 2001;
Kitching, 2002a; Orsel et al., 2009). The fact that some animal species, such as sheep
and goats, may not exhibit obvious clinical signs makes the potential for spread of
infection considerable (Barnett and Cox, 1999; Alexandersen et al., 2003c). Sheep
and goats are considered to be important sources of FMDV during the first seven
days of clinical or subclinical infection (Barnett and Cox, 1999) with the intensity of
contact between animals important determinants of disease (Quan et al., 2009). Other
studies have also identified that buffalo (Bubalus bubalis) can act as a source of
infection for cattle and other animals (Gomes et al., 1997; Maroudam et al., 2008).
As with cattle, buffalo can excrete the virus prior to the development of clinical
signs. As different animal species have different incubation periods (Hugh-Jones and
Tinline, 1976; Orsel et al., 2009), the ability of the virus to be transmitted during the
incubation period increases the risk of the disease being transmitted between
different animal species (Brown, 2004).
Allowing unauthorised vehicles to enter a herd without any restrictions or
precautions, such as using tyre washers or sprays at the entrance to the farm, was
another factor associated with diseased herds in this study. It is well documented that
biosecurity measures are very important in minimising the entry and spread of the
56
disease (Amass et al., 2003b). The virus is known for its ability to survive outside
reservoir hosts on fomites (Bedson et al., 1927; Hyslop, 1965b; Brown, 2004),
especially those with a high organic matter (Donaldson and Ferris, 1975).
Consequently soil on vehicles‟ tyres has the potential to spread the virus from an
infected to a susceptible herd. Furthermore contaminated boots and clothing may
also transmit FMDV (Amass et al., 2003b; Amass et al., 2004).
The multivariable analysis showed owning only one herd was protective for
infection. Cattle owners could act as mechanical carriers for FMDV by sharing
equipment between herds, or by the movement of cattle from one herd to another.
Movement of infected animals is considered the biggest threat of entry of the disease
into a free area or herd (Abila and Foreman, 2006).
Foot and mouth disease can infect cattle at any age, but, as with other diseases, many
factors influence when disease will occur, including the immune status and level of
stress of animals and the virulence of the virus. In this study keeping different age
groups separated was shown to be protective. This finding was supported by a study
in Thailand which indicated that adult beef cattle were at higher risk of FMD
infection compared to calves less than one year old (Cleland et al., 1995) and adult
cattle and buffalo were approximately three times more likely to become a case than
were working cattle (Cleland et al., 1995). Management of livestock classes by
separating different age groups will reduce the risk of the herd from contracting the
disease from the higher risk age group.
57
Although vaccination is adopted as a control measure for FMD in Pahang, the
descriptive analysis indicated that only half (56%) of the respondents believed in
vaccination as a preventive measure for FMD. Unfortunately only 29% of the
respondents knew that the vaccine needed to be given at six monthly intervals and no
one knew that a second priming dose was required to be administered one month
after the primary dose.
Ideally the case and control herds should be differentiated by the NSP serological
test to confirm their FMD status (Bruderer et al., 2004; Clavijo et al., 2004) due to
the potential presence of carriers with the disease (Burrows, 1966; Burrows et al.,
1971; Salt, 2004). The presence of carriers allows the continued circulation of
FMDV in the cattle population even after vaccination (Alexandersen, 2002; Schley
et al., 2009), however the role of carriers in transmitting infection to susceptible
animals is not clearly understood (Woodbury, 1995b; Salt, 2004). It was not possible
to undertake serology because of time and cost constraints, therefore the status of a
herd in the current study was based solely on a history of clinical disease. Given that
the clinical signs of FMD are easily recognisable in cattle it was considered that
using the presence of clinical signs to classify case and control farms was acceptable.
Others (Barteling, 2002; Cox et al., 2007; Parida et al., 2008; Schley et al., 2009)
have highlighted the role that vaccination plays in the development of immunity and
prevention of disease outbreaks. Given the low knowledge of the benefits of
58
vaccination against FMD and the lack of awareness of the recommended vaccination
protocol in Pahang a study was developed to determine the serological response to
vaccination of Nallore cattle on a farm in Pahang. The results of this vaccination
study are reported in the following chapter.
59
CHAPTER 4
ANTIBODY RESPONSE FOLLOWING VACCINATION
AGAINST FOOT AND MOUTH DISEASE
4.1 Introduction
Foot and mouth disease (FMD) is a highly contagious viral disease which results in
significant economic impacts due to: reduced productivity in affected animals; the
cost of implementing disease control practices; and the losses resulting from
restrictions placed on the trade of livestock and their products (Kitching, 1998).
Based on benefit cost analysis a stamping out policy has generally been the preferred
control policy in countries that have previously been free of the disease (Ferguson et
al., 2001b; Keeling et al., 2001; Bates et al., 2003; Ward et al., 2009), while
vaccination and restriction of livestock movements are commonly adopted in areas
where the disease is endemic (Anderson et al., 1974; Cleland et al., 1996;
Rweyemamu et al., 2008a). In that situation vaccination is adopted to induce herd
immunity leading to a reduction in clinical infection and viral transmission (Orsel et
al., 2007). Vaccination, in combination with culling of clinically affected and
repeated NSP seropositive animals, has also been successfully adopted to control and
eradicate FMD in countries in western Europe and South America (Saraiva, 2004;
Lubroth et al., 2007). Although numerous studies have demonstrated the positive
impacts of implementing vaccination to control and eradicate FMD, many local
factors also need to be identified and considered before the practice can be adopted
in an area. In particular a benefit cost analysis is required, as is a thorough
understanding of the local epidemiological factors of the disease (Hutber et al.,
2010).
60
Vaccination has been used for many years to control FMD in Malaysia (Babjee,
1994; Hiong, 1994). The use of vaccination was initially restricted to the northern
border states of peninsular Malaysia until an outbreak in late December 2003 when it
was adopted in Pahang. Vaccination, along with movement restrictions are now key
elements in the control of FMD in Malaysia.
Following vaccination antibodies to the structural proteins of FMDV are produced
which impair the ability to differentiate vaccinated animals from those naturally
infected (Doel et al., 1994). Differentiating animals that have been naturally infected
from those vaccinated is of considerable importance because it is well established
that infected cattle and sheep frequently become carriers (Clavijo et al., 2004) and
consequently may be the source of new disease outbreaks (Sutmoller and Olascoaga,
2002). Although the infectivity of persistently infected ruminants to naïve animals is
believed to be low under most circumstances, it is not negligible. These factors lead
many parts of the world to control the disease without vaccination, for example
vaccination against FMD has been banned in the European Union since 31st
December 1991, and outbreaks have been controlled solely by stamping out.
However, the need for emergency vaccination in future outbreaks, as an adjunct to
stamping out, cannot be ruled out due to the huge economic losses incurred, adverse
psychological effects on farmers and negative effects on the environment resulting
from the slaughter and disposal of livestock.
Diagnostic techniques have been developed in an attempt to differentiate vaccinated
animals from those that are convalescent and from those that have been vaccinated
61
and subsequently become carriers (Sorensen et al., 1992; Bergmann et al., 2000;
Clavijo et al., 2004). Antibody to the NSP of FMDV, which is detected only in
naturally infected animals, has been used to differentiate between vaccinated and
infected cattle (Diego et al., 1997; Mackay et al., 1998; Sørensen et al., 1998; Clavijo
et al., 2004; Moonen et al., 2004). Vaccines which consist of partly purified
inactivated virus preparations induce antibodies principally to the structural proteins
of the virus, whereas infected animals produce antibodies to both structural and non-
structural proteins. Therefore in the current study the 3 ABC ELISA was utilised to
study the serological response to cattle vaccinated against FMD. The detection of
antibody to the polyprotein 3ABC in the 3 ABC ELISA is reported to be the most
reliable diagnostic test (Berger et al., 1990; Lubroth and Brown, 1995) because of its
high immunogenicity and its relative low concentration in infected cell lysates (Doel
et al., 1994; Bergmann et al., 2000). The ELISA, based on the detection of antibody
to NSP, has been recommended by the OIE for serological surveillance in regions or
countries that practice FMD vaccination and for monitoring the circulation of virus
in the field (OIE, 2004). On the other hand the liquid phase blocking ELISA (LP
Blocking ELISA) relies on the detection of antibodies to the structural, capsid VP1 t-
VP4 proteins of FMDV (Hamblin et al., 1986a). Antibodies to structural proteins are
induced by both vaccination and infection. The LP Blocking ELISA has been used in
endemic areas to indicate the antibody response produced after vaccination in
conjunction with the NSP ELISA (Periolo et al., 1993a; Robiolo et al., 2006).
The study outlined in this chapter describes and compares the antibody response
following vaccination against FMD. The serological response in a group of cows
which had been vaccinated yearly for more than 3 years and a group of calves which
received their initial vaccination were compared. In this study a 3ABC-ELISA and
62
an LP Blocking ELISA were used to monitor the level of protection (herd immunity)
in the population.
4.2 Materials and methods
4.2.1 Vaccination and sample collection
The study outlined in this chapter involved animals from a cow and calf unit in Pusat
Ternakan Haiwan (PTH) Ulu Lepar, Kuantan, Pahang, which is a government owned
herd of Nellore beef cattle. The farm had no clinical history of FMD. Forty-five
cows and forty-five calves were selected based on systematic random sampling
(Thrusfield, 2005) and were tested for antibody to NSP with a commercially
available NSP ELISA assay (Ceditest FMDV-NS ELISA - Cedi Diagnostics, B.V.,
Lelystad, The Netherlands). All the cows had been vaccinated annually since March
2004 and the calves were all vaccinated for the first time on the 12th
February 2008
(during this study). At the time of their first vaccination the calves were 4 to 6
months of age. The vaccine used in this study was an inactivated purified trivalent
vaccine (O, A and Asia 1) (Aftovaxpur® Merial Animal Health Ltd). Blood samples
were collected from the coccygeal vein into a plain 10 ml vacutainer (BD Franklin
Lakes USA) at 0, 14, 57, 93, 183, 212, 254 and 317 days post vaccination (DPV).
These samples included ones collected on the days of vaccination (days 0 and 183).
At 183 DPV, twenty eight cows and twenty eight calves were randomly selected
from the larger group for testing with the LP Blocking ELISA to monitor the
antibody response to vaccination. Antibody response was expressed as Percentage of
Inhibition (PI). Values greater than 50% were classified as seropositive (Naheed,
63
2008). Fourteen cows and 14 calves from this smaller group were revaccinated at
183 DPV with the same trivalent vaccine, with the remaining 14 cows and 14 calves
not being revaccinated. Sera were then collected at 183, 212, 254 and 317 DPV from
all animals. During the study all 90 cows and calves, including the 56 cows and
calves whose sera were tested with the LPBE test, were run in one paddock along
with other (untested) cows and calves.
After collection blood samples were centrifuged, sera decanted and stored at -20ºC at
the Regional Veterinary Laboratory, Kota Bahru (RVL KB) until tested.
This study had been approved by the Murdoch University Animal Ethics Committee
(R2191/08).
4.2.2 Measurement of antibody to NSPs
The antibody response to FMDV NSPs was measured with a commercially available
ELISA (Cedi FMDV-NS for the NSP 3 ABC peptides, Cedi-Diagnostics B.V., the
Netherlands). This blocking ELISA measures the competition between the test sera
and a NSP specific monoclonal antibody for the binding to the 3ABC NSP of FMDV
(Sørensen et al., 1998; Sorensen et al., 2005). The assay was performed according to
the manufacturer‟s protocol. The optical density (OD) result obtained from the assay
was expressed as PI of the monoclonal antibody binding. The PI cut off value
between a positive and negative was set at 50%. Animals with an NSP PI value more
than 50% were then not included in the analysis to monitor the LPBE level of
antibody produced from vaccination (herd immunity). The detailed NSP test
procedures are summarised in Appendix 1.
64
4.2.3 Measurement of antibody on the Liquid phase blocking ELISA
The LPB ELISA is based upon the specific blocking of FMDV antigen in a liquid
phase by antibodies present in the test serum sample (Hamblin et al., 1986b, a). The
LPB ELISA was performed according to the procedure adopted by the FMD WRL,
Pirbright, UK (Appendix 2). It was carried out to detect antibodies directed against
structural proteins of FMDV. Antibodies were expressed as PI values and a value
greater than 80% was considered protective (Westbury et al., 1988b).
4.2.4 Statistical analysis
Data were managed, collated and analysed using SPSS (SPSS version 17 Statistics
GradPack for Windows). One way repeated measures ANOVAs (analysis of
variance) were carried out to identify significant differences in the mean PI of the
LPBE for the serially collected sera. The results of the LPBE (PI) were computed
and transformed to arcsine data. The data were then explored to check for normality.
The calf group data were normally distributed, therefore the data were analysed with
a one-way repeated ANOVA. The multiple comparison Bonferroni test was used as a
post hoc test to compare differences between groups (vaccinated once and
revaccinated). The difference in the antibody level produced between the
revaccinated group and the group vaccinated only once for both cows and calves
were also determined through the use of repeated measures ANOVAs. As the cow
group data were not normally distributed, the analyses were continued using a non-
parametric test (Friedman‟s ANOVA). The mean PI values for all DPVs were ranked
and the asymptomatic Friedman‟s analysis carried out for all DPVs values.
65
The odd ratios (OR) were also calculated to identify differences within and between
groups. If a 2 x 2 table contained cells with the value 0 then 1 was added to all cells
in that table to enable calculation of OR. The OR value was considered significant if
the value of the 95% confidence intervals did not include the value 1.
4.3 Results
4.3.1 Detection of antibodies against FMDV NS-Proteins
Over the total period of the study 7 animals were identified as positive to the NSP
assay (7.8%: 95% CI; 2.2%, 13.3%) (5 cows and 2 calves) (Table 4.1). There was no
significant difference in the number of NSP positive animals between the cow and
calf groups (OR 2.7; 95% CI 0.5 – 14.6).
Table 4.1 Number of cows and calves positive to the NSP test throughout the study
Group NSP + (%: 95% CI) NSP- Odds ratios (95% CI)
Cow 5 (11.1: 1.9, 20.3) 40 (88.9%) 2.69 (0.49 - 14.64)
Calf 2 (4.4: 0.0, 10.5) 43 (93.3%) 1.0
Total 7 (7.8: 2.2, 13.3) 83 (91.1%)
Both cow and calf groups were subsequently divided into two groups at 183 DPV,
with one group receiving another dose of FMD vaccine, while the second group did
not. Results for the comparison between the groups are summarised in Tables 4.2,
4.3 and 4.4. Again there were no significant differences in the number of NSP
66
reactors in both cow and calf groups after day 183 (OR 5.2, 95% CI 0.5 – 54.1; and
OR 3.5, 95% CI 0.3, 37.5, respectively) (Table 4.4).
Overall 7 individual animals were positive at some point during the study (one cow
was positive 7 times, three animals (2 cows and 1 calf) 2 times and three animals (2
cows and 1 calf) were positive at only one point in time (Tables 4.5 and 4.6). There
were no NSP positive animals at the start of the study. Two cows became positive
at 14 DPV, 1 at 93 DPV, 1 at 183 DPV and 1 at 212 DPV (Tables 4.2 and 4.5). In
comparison the first NSP positive calf occurred at 93 DPV and the second at day
212 (Tables 4.3 and 4.6).
Table 4.2 Number of NSP positive samples* in the non-revaccinated and
revaccinated cows
DPV Number of NSP
positive samples
(non-revaccinated)
Number of NSP
positive samples
(revaccinated)
Total number
of NSP positive
samples
0 0 0 0
14 2 0 2
57 1 0 1
93 1 1 2
183 2 0 2
212 2 1 3
254 1 1 2
317 1 0 1
Total 10 3 13
*Represents 5 positive animals as the same animals were positive at multiple times
67
Table 4.3 Number of NSP positive animals* in the non-revaccinated and
revaccinated calves
DPV Number of NSP
positive samples
(non-
revaccinated)
Number of NSP
positive samples
(revaccinated)
Total number of
NSP positive
samples
0 0 0 0
14 0 0 0
57 0 0 0
93 1 0 1
183 0 0 0
212 1 0 1
254 0 0 0
317 1 0 1
Total 3 0 3
*Represents 2 positive animals as one calf was positive on two occasions
Table 4.4 Comparison between cow and calf groups after animals were divided at
183 DPV into a revaccinated and a non-revaccinated group.
NSP + NSP - Odds ratios (95% CI)
Cows
Not revaccinated 4 (28.6%) 10 (71.4%) 5.2 (0.50 – 54.05)
Revaccinated 1 (7.1%) 13 (92.9%) 1.0
Total cows 5 (17.9%) 23 (82.1%)
Calves
Not revaccinated 2 (14.3%) 12 (85.7%) 3.5 (0.32 – 37.48)*
Revaccinated 0 (0.0%) 14 (100.0%) 1.0
Total Calves 2 (7.1%) 26 (92.9%)
* 1 added to all cells to enable OR calculation
68
Table 4.5 NSP results for individual cows during the study
Days of study
Tag ID 0 14 57 93 183 212 254 317
Cows vaccinated at day 0 but NOT revaccinated at day 183
UN 8166 - - - - - - - -
UNL 1719 - + - - - - - -
UN 7352 - - - - + + - -
UNM 1842 - - - - - - - -
UNL 1748 - - - - - - - -
UNN 2184 - - - - - - - -
UNL 1642 - - - - - - - -
UNM 1773 - + + + + + + +
UNN 102 - - - - - - - -
UNM 2021 - - - - - - - -
UN 8417 - - - - - - - -
SNB 529 - - - - - - - -
UNK 1221 - - - - - - - -
UNK 1162 - - - - - - - -
Cows vaccinated at day 0 and revaccinated at day 183
UNL 1762 - - - + - - - -
UNM 5309 - - - - - + + -
UNL 1519 - - - - - - - -
UNN 6304 - - - - - - - -
UNL 1597 - - - - - - - -
UNJ 711 - - - - - - - -
UN 8550 - - - - - - - -
UNM 1792 - - - - - - - -
UNN 18 - - - - - - - -
UNK 1310 - - - - - - - -
SNB 599 - - - - - - - -
UNK 1209 - - - - - - - -
SNA 731 - - - - - - - -
UNK 1366 - - - - - - - -
69
Table 4.6 NSP result for individual calves during the study
Days of study
Tag ID 0 14 57 93 183 212 254 317
Calves vaccinated at day 0 but NOT revaccinated at day 183
UNT 252 - - - - - - - -
UNT 214 - - - + - - - -
UNT 227 - - - - - - - -
UNT 232 - - - - - - - -
UNT 235 - - - - - - - -
UNT 221 - - - - - + - +
UNT 210 - - - - - - - -
UNT 225 - - - - - - - -
UNT 229 - - - - - - - -
UNT 231 - - - - - - - -
UNT 219 - - - - - - - -
UNT 226 - - - - - - - -
UNT 234 - - - - - - - -
UNT 250 - - - - - - - -
UNT 242 - - - - - - - -
Calves vaccinated at day 0 and revaccinated at day 183
UNT 255
- - - - - - - -
UNT 244 - - - - - - - -
UNT 222 - - - - - - - -
UNT 258 - - - - - - - -
UNT 205 - - - - - - - -
UNT 216 - - - - - - - -
UNT 213 - - - - - - - -
UNT 249 - - - - - - - -
UNT 236 - - - - - - - -
UNT 254 - - - - - - - -
UNT 201 - - - - - - - -
UNT 220 - - - - - - - -
UNT 243 - - - - - - - -
UNT 224 - - - - - - - -
70
4.3.2 Liquid phase blocking ELISA
Although 28 cows and 28 calves were selected for the LPB ELISA analysis, only 21
cows (10 from the non-revaccinated group and 11 from the revaccinated group) and
20 calves (10 from the non-revaccinated group and 10 from the revaccinated group)
were included in the final analysis because these animals had been consistently
tested for FMD infection and had negative NSP results at all samplings and some of
the animals (2 cows and 6 calves) were not included because they were not sampled
at all times throughout the one year study. The objective of performing the LPB
ELISA test was to identify whether the antibody produced by the animals following
vaccination was sufficient to protect the host against FMDV infection. For the
purpose of this study a cut-off point of 80% was used to determine the protective
immunity level (Westbury et al., 1988b). The mean PI values for all serotypes in the
cow group were above the LPB ELISA cut-off point at all sampling times,
irrespective of whether the animals had been revaccinated or not. In Figures 4.1, 4.2
and 4.3 the mean PI is displayed for the three serotypes (O, A and Asia1
respectively) in animals revaccinated and those that weren‟t. The descriptive analysis
for the cow groups showed that the mean PI values were not normally distributed for
all DPVs (skewness and median values ranging from -1.0552 to 1.168 and -0.709 to
-2.340, respectively). Therefore the non-parametric Friedman ANOVA was used to
demonstrate that the PI values for cows after revaccination was significantly higher
than the value prior to vaccination (p = 0.000 and χ2 = 55.009, df 7).
71
Figure 4.1 LPB ELISA results for serotype O in cows.
For Serotype O the mean PI value was already above the mean PI cut off point
(80%) at the time of the initial vaccination and subsequently at all sampling points.
After revaccination at 183 DPV the PI values in both groups increased significantly
to 212 DPV before decreasing (χ2 = 20.985 and P < 0.05).
72
Figure 4.2 LPB ELISA results for serotype A in revaccinated and non-revaccinated
cows.
The pattern for the Serotype A (Figure 4.2) was different to that of the other
serotypes (Figures 4.1 and 4.3). The mean PI value increased significantly after
vaccination at 0 DPV; however it decreased again at 57 DPV before it increased
gradually with significantly higher PI values until 212 DPV (χ2 = 27.015 and P <
0.05). In both groups the mean PI values increased significantly after the time of
revaccination at 183 DPV with the non-revaccinated group showing a similar trend
as the revaccinated group but with higher PI values from 212 DPV.
75
80
85
90
95
100
0 14 57 93 183 212 254 317
PI
DPV
Not revaccinated
Revaccinated
Cut off point
73
Figure 4.3 LPB ELISA results for serotype Asia 1 in revaccinated and non-
revaccinated cows
The mean PI values to serotype Asia 1 increased significantly after the vaccination at
0 DPV (χ2 = 57.038; P < 0.05) (Figure 4.3). Again surprisingly the PI values in both
groups increased when the revaccinated group received its second dose before they
decreased significantly at 212 DPV (χ2 = 29.183 and P < 0.05). The response of
cows to serotype Asia 1 was similar in both groups. After revaccination the PI values
were similar until 254 DPV. At this point, the mean PI value for the non-
revaccinated group dropped dramatically, while the revaccinated group showed only
75.0
80.0
85.0
90.0
95.0
100.0
0 14 57 93 183 212 254 317
PI
DPV
Not revaccinated
Revaccinated
Cut off point
74
a small reduction. However, the mean PI values for both groups were above the
protective cut off value at all times throughout the study.
For the calf groups, the mean PI value was highest at DPV 212 in the revaccinated
group for the Serotype Asia 1 (90.2%) as shown Figure 4.6. The PI data were
normally distributed at all sampling times with skewness values between; -0.030 to -
1.151 and mean values between 0.5983 and 0.9029 (data not shown). The
significance value (0.00) for the Mauchly‟s Test of Sphericity indicated that the
assumption of the sphericity had been violated with an approximate χ2 value of
94.523. Therefore a Greenhouse-Geisser correction value was chosen (0.699). The
closer the Greenhouse-Geisser correction is to 1, the more homogenous the variance
of difference is, and hence the closer the data are to being spherical (Field, 2009).
The multivariate test statistic was also used due to the violation of sphericity. In this
analysis there was a significant change (P < 0.001) in the mean calf PI over time.
However the level of antibody was not protective at most DPVs, except for days 183
and 212 for Serotypes O and Asia 1. This indicated that the calves were susceptible
to FMD for most of the time throughout the study.
75
Figure 4.4. LPB ELISA results for serotype O in revaccinated and non-revaccinated
calves.
The mean PI values for serotype O (Figure 4.4) in the two groups of calves followed
similar patterns. Following the initial vaccination the PI increased significantly to 14
DPV and again in both groups at 183 DPV when only the revaccinated group
received a booster vaccination. However the revaccinated group had a similar mean
PI value to that of the non-revaccinated group, indicating there was no significant
effect of revaccination in the revaccinated calf group with F = 3.190; P = 0.091
Moreover, most of the PI values were below the 80% protective level throughout the
study.
0
10
20
30
40
50
60
70
80
90
100
0 14 57 93 183 212 254 317
PI
DPV
Not revaccinated
Revaccinated
Cut off point
76
Figure 4.5 LPB ELISA results for serotype A in revaccinated and non-revaccinated
calves.
The mean PI value for Serotype A (Figure 4.5) followed a different trend compared
to the other two serotypes. At only one point in time (212 DPV) did some
revaccinated calves (n = 4) have protective immunity to Serotype A. At 14 DPV
there was a significant reduction in the PI value. Subsequent to DPV 183 (time of
revaccination) the revaccinated group had a significantly higher PI value than the
non-revaccinated group until the end of the trial at day 317 when the titres were
similar. The PI value in the revaccinated group was significantly different to the non-
revaccinated group (F = 10.229; P = 0.005). However, most of the PI values were
below the 80% protective level throughout the study.
0
10
20
30
40
50
60
70
80
90
100
0 14 57 93 183 212 254 317
PI
DPV
Not revaccinated
Revaccinated
Cut off point
77
Figure 4.6 LPB ELISA results for serotype Asia 1 in revaccinated and non-
revaccinated calves.
The mean PI value for serotype Asia 1 increased sharply and significantly after the
first vaccination (Figure 4.6), however neither group failed to generate a mean
protective level until the revaccinated group received their second dose of vaccine.
Generally the two groups were not significantly different (F = 0.298; P = 0.592),
with most of the PI values below the 80% protective level throughout the study.
4.4 Discussion
The presence of NSP antibodies has been shown to be a reliable indicator of
infection with FMDV in cattle (Diego et al., 1997; Mackay et al., 1998; Niedbalski
and Haas, 2003b) although the detection of persistently infected cattle is not perfect
0
10
20
30
40
50
60
70
80
90
100
0 14 57 93 183 212 254 317
PI
DPV
Not revaccinated
Revaccinated
Cut off point
78
(Paton et al., 2006). The persistence of neutralizing antibody to FMDV in cattle has
previously been shown to be short and cattle are immune to re-infection for only
approximately 4 to 6 months (Hunter, 1996). Therefore it is important to follow the
manufacturer‟s protocol for vaccination to prevent infection. This is especially
important for the initial vaccination when calves should receive their first dose
around the age of 4 months (P1). This should be followed with a second dose (P2)
three to five weeks later to boost the antibody response. Subsequently a third dose
(P3) should be administered 4 to 6 months later. Booster vaccinations should ideally
then be given every 4 to 6 months. This protocol is recommended by manufacturers
to ensure the vaccinated animals have titres above the minimum protective level to
reduce the risk of infection. However this frequent vaccination schedule is expensive
and labour intensive and therefore is rarely adopted in the field. Consequently this
also results in many countries trying to eradicate FMD without the use of
vaccination. Furthermore a country which is classified as FMD free with vaccination
has restrictions placed on the trade of animals and animal-products, therefore most
exporting countries prefer to have the status of FMD free without vaccination.
However in South East Asia, where FMD is generally endemic, eradication is
difficult and often not practical. Therefore zoning and compartmentalization are
often adopted (Scott et al., 2005) along with vaccination to control the disease.
Consequently diagnostic tools are required to differentiate infected from vaccinated
animals. In Malaysia the Ceditest 3 ABC ELISA is used routinely to differentiate
infected from vaccinated animals. This test has a reported specificity of 99.8%
(Sørensen et al., 1998). Because of this high specificity, in the current study the
Ceditest 3 ABC ELISA was chosen to detect infection along with the LPBE to
evaluate the immune response following vaccination.
79
The LPBE demonstrated a similar pattern in both the revaccinated and the non-
revaccinated cow groups. A single dose of vaccine was adequate to confer good
immunity (mean PI value above 80% cut off value) and revaccinating cows at six
monthly intervals produced no significant advantage over the group that only
received one annual vaccination. At the beginning of the study all 90 cattle were
vaccinated at 0 DPV. As a result, it is not surprising that the mean PI value increased
at 14 DPV. The number of cattle that were positive to the NSP increased at 93 DPV
when the cow group recorded 2 NSP positive animals and the calf group 1 NSP
positive animal. This could be the reason why the mean PI of the LPB ELISA
increased at 93 DPV. The NSP positive animals could be indicative of subclinical
infection as all animals were reared in the same area for the whole study period.
Furthermore the NSP positive cattle were repeatedly positive. However since the
number of animals in this study was small and the NSP ELISA used was not able to
identify the specific serotype present and moreover no further tests were performed
in this study, conclusions cannot be drawn on the cause of the immunity conferred
pattern by the animals involved in this study. The NSP ELISA detected positive
animals in both the cow and calf groups even though this farm had never had a
clinical history of FMD (Tables 4.5 and 4.6). More NSP positive animals were
observed in the non-revaccinated cow group followed by the revaccinated cow
group. The revaccinated calf group on the other hand, showed the least number (0) of
positive NSP animals. The effect of repeated vaccination has been studied elsewhere,
and some researchers have shown that the reactivity to NSP in a revaccinated
population approaches the necessary level to confer protection after several
vaccinations (Espinoza et al., 2004; Lee et al., 2006). A study undertaken by Lee et
al. (2006) in Taiwan found 3.9% of field samples positive with the Cedi test kit,
80
indicating that the specificity was lower than that reported previously. Some
researchers also have demonstrated that cattle which received multiple vaccinations
over several years were positive to NSP (Mackay et al., 1998). In contrast other
researchers (Niedbalski and Haas, 2003b) reported that multiple vaccinations did not
affect the number positive to NSP. However no similar studies have previously been
undertaken in Pahang. In addition to that, the district of Kuantan, Pahang was
severely affected during the 2004 FMD outbreak and the disease has been considered
endemic in the district since that date. Therefore the percentage of NSP positive
animals cannot be determined based on the use of only a single test, as it is unclear if
reactors were true positive animals from natural infection, or were false-positives
including those resulting from use of a vaccine contaminated with NSP. Thus,
additional methods for conformation and epidemiological investigations are
necessary to differentiate naturally infected cattle from vaccinated animals.
Furthermore the NSP reactors could also be influenced by other factors such as the
subclinical circulation of FMDV in the cattle population. It was possible that the
positive NSP reactors observed in this study were caused by natural, subclinical
infection with FMDV based on the endemic nature of FMD in the area. The NSP
positive animals identified in this study could potentially be carriers of FMDV. Such
carriers are considered to be sources for the spread of disease to susceptible animals
as the protective immunity does not block FMD infection completely and some level
of viral replication can still occur in vaccinated cattle upon exposure to the field
virus (Alexandersen et al., 2003c). In addition Doel et al. (1994) and Mackay et al.
(1998) pointed out that cattle protected by vaccination can become transient FMD
virus carriers, with or without clinical signs, and they can also become persistently
infected without displaying any clinical signs of FMD. Therefore it is crucial for
81
farms to undertake testing with an NSP test, to implement a proper disease control
program which is tailored to the local FMD situation and to consider the culling of
NSP positive animals as soon as possible after their identification.
Historically the schedule of vaccination practiced on this farm was not carried out in
accordance with the manufacturer‟s recommendations (where P1 FMD vaccine is
followed by P2 one month later and subsequently animals are revaccinated every 6
months). However reports of the successful control of FMD by vaccination in other
countries, which also have not followed the manufacturer‟s recommended protocol,
have been made (Doel, 1999). Vaccination programs are often altered to suit the
local epidemiological features of the disease, although emphasis has traditionally
relied more on the vaccination coverage of the population (at least 80% of the
population). As for this particular farm during this study the mean PI values on the
LPB ELISA were, in general, above the seropositive cut off point (PI value : 80%)
(Westbury et al., 1988a; Robiolo et al., 2010) for the cow groups.
The LPBE is a reliable technique for the evaluation of a protective response
(Hamblin et al., 1987; Periolo et al., 1993a; Robiolo et al., 2006). Although
vaccination was commenced on this farm in 2004 it was only administered annually
for both adult and young cattle. The reason for this was not confirmed and this
situation requires further research since this study indicated only the cows had good
LPB ELISA values (above the protective level of 80% at all-time throughout the
study). Furthermore, the mean PI on the LPBE for the calf group was lower than that
for the cows and similar trends were followed for all serotypes except for serotype
A. Unlike for other serotypes, the mean PI values for serotype A in calves decreased
82
following the initial vaccination but the levels did increase after the animals were
revaccinated. The mean PI value was generally lower in the calf groups for serotypes
O and Asia 1 than for the cow groups. This probably was a result of the cows
receiving multiple vaccinations over their lifetime. Moreover no second priming
vaccination dose was given to calves a month following the first vaccination, as is
recommended by the manufacturer. This would likely contribute to the low mean PI
values for the calf groups, which were less than the protective level. Although there
were significant differences in the LPBE PI at different sampling times (DPVs)
(albeit below the protective levels), there was no significant difference between the
revaccinated and non-revaccinated groups. The failure to detect a higher titre in the
revaccinated calf group was surprising and could be due to several factors including:
1. Inappropriate vaccination protocol which omitted P2, leading to insufficient
antibody production; 2. In this study calves received their first vaccination at the age
of at least four months. At the start of the study all calves already had some LPBE PI
although no NSP positive animals were detected. The initial LPBE PI values could
be due to the presence of maternal antibody as a result of residual passive immunity
transferred from multiply immunized cows which may have a neutralising effect
towards the vaccination given (Periolo et al., 1993b). These two factors require
further study.
The protective cut off point for the LPBE is normally calculated on the titre rather
than the mean PI value (Robiolo et al., 2006), however due to time and monetary
constraints the protective cut off point was set at 80% mean PI value following the
results of a study in Thailand (Westbury et al., 1988a). Based on the 80% protective
mean LPBE cut off value, the cows appeared to be protected against FMD
83
throughout the study, in contrast calves were at risk of infection at various times
through the study. Although there were significant differences over the study in the
PI for calves, there was no significant difference between calves that were
revaccinated at 6 months of age and those that were not revaccinated. All calves in
both groups were susceptible to the disease at almost all sampling times except for
the revaccinated calf group at 212 and 254 DPV and only for serotypes O and Asia
1. None of the Serotype A mean PI values of the calf groups were above the
protective level at any sampling time. These findings indicate that the calves were at
a greater risk of infection than cows throughout the study.
Surprisingly the mean PI LPBE increased in both the revaccinated and non-
revaccinated cow groups for all serotypes after some animals were revaccinated
at183 DPV. This may have arisen from subclinical infection in the herd.
An important finding of the present study was that the antibody level conferred after
vaccination was not the same for animals which had been multiply vaccinated and
animals which received their first vaccination. Therefore it is suggested that the
vaccination practices should be tailored to the local situations based on the existing
local epidemiological factors. It is also important to adopt a vaccination monitoring
system for different FMD situations and to monitor the vaccination status of a herd.
In conclusion it was evident that vaccination stimulated serological immunity,
however the immunity in many cases was not sufficient to protect against natural
infection. Additional research is required to further understand the titre induced and
a challenge study is required to confidently conclude that the immunity induced by
vaccination is protective.
84
CHAPTER 5
SPATIAL AND TEMPORAL DISTRIBUTION OF FOOT AND
MOUTH DISEASE IN THE STATE OF PAHANG, MALAYSIA
2003 – 2006.
5.1 Introduction
Foot and mouth disease is a highly contagious febrile vesicular disease of cloven-
hoofed domesticated and wildlife species (Alexandersen et al., 2003c). The disease
results in severe economic impact because of reduced animal production and
restrictions to local and international trade (James and Rushton, 2002). Historically,
the state of Pahang has experienced several incursions of FMD caused by Serotype
O. During the initial 2003/2004 outbreak in Pahang, ring vaccination and movement
control were implemented to control the spread of the disease. Animals were
vaccinated within a 5 km radius of the outbreak and disease surveillance was
conducted within a 10 km radius of the outbreak. In order to minimise the spread of
the disease at that time, a mass vaccination campaign of the whole state was also
conducted. This campaign commenced in March 2004 using a trivalent vaccine
containing the three serotypes O, A and Asia 1. The vaccine was administrated to all
cattle, buffalo, goats and sheep older than 4 months of age. A booster vaccination
was given six months after the initial vaccination. However, despite these control
measures, further outbreaks were recorded in subsequent years (DVS, 2007).
The highly contagious nature of FMD results in clustering of cases in time and space
(AlKhamis et al., 2009). Assessment of outbreaks with respect to spatial clustering
85
and their temporal pattern is useful when planning specific prevention, surveillance
and control strategies against the disease (Shiilegdamba et al., 2008). Spatial cluster
analysis plays an important role in identifying the geographical patterns of a disease
(Jacquez, 2008). Cluster analysis can address the: (1) rapid identification of epidemic
clusters; (2) identification of confounders; and (3) generation of research hypotheses
(Carpenter, 2001). Many methods can be used to quantify disease outbreaks
including the one-dimensional scan statistic which has long been used in purely
temporal disease surveillance (Wallenstein, 1980) and spatial scan statistic which has
been used for detecting clusters in a multi-dimensional point process in a
geographical region (Kulldorff, 1997). Spatial autocorrelation methods look for the
presence of systematic patterns such as the clustering effect in the spatial distribution
of a variable (Lai et al., 2009), while temporal statistical techniques are used in many
well-defined geographical areas for monitoring and detecting sudden temporal
increases in the risk of disease, particularly for rare diseases (Bjerkedal and
Bakketeig, 1975; Radaelli, 1996). For the purpose of this study a space-time
permutation model was used to analyse the space and time of the outbreak in one
model using only outbreak data to identify the geographical areas and period of time
in which the disease is more likely to occur.
The objective of the current study was to describe the spatio-temporal patterns of
outbreaks of FMD in the state of Pahang. Findings from this study could then be
used to direct future research into the epidemiology of FMD and could serve as a
starting point for developing more effective control programs in Pahang.
5.2 Materials and methods
86
5.2.1 Study area and study period
The study area involved all eleven districts in the state of Pahang. The period of
interest in this study was from 16 December 2003 to 26 August 2006.
5.2.2 Data source and case definition
The outbreak data were obtained from the retrospective disease reporting database
for the State of Pahang Department of Veterinary Services (DVS) for the period of
study. The outbreaks were defined according to the Office International des
Epizooties (OIE) as epidemiological units (herds, farms, feedlots or where premises
could not be precisely delimited, areas with free-grazing animals) in which at least
one FMD infected animal had been detected (OIE, 2008b). When a case of FMD is
suspected, a report is issued from the District Veterinary Office to the State
Veterinary Officer. The State Veterinary Officer then organised an inspection of the
outbreak site searching for FMD-like clinical signs in livestock. The estimated
starting date is predicted based on the age of the clinical lesions. Details of the
outbreak, including the number of animals affected, the breed, species, farm address
and geographical location (latitude and longitude of the case), were recorded. Fresh
epithelial samples were collected into phosphate buffered saline (PBS) from
vesicular lesions in the oral cavity or inter-digital skin of affected animals. These
samples were then submitted to the Regional Veterinary Laboratory in Kota Bahru,
Kelantan for confirmation by the antigen detection test. Isolated serotypes were then
sent to the FMD World Reference Laboratory in Pirbright, United Kingdom for
identification.
87
The historical vaccination and surveillance data were also examined and
incorporated into the analysis. The recorded date of an outbreak used in the analysis
was based on the earliest date the cattle showed clinical signs of FMD in a
population.
5.2.3 Spatiotemporal analysis
A map of administrative units and boundaries was obtained from the DVS. Location
data were downloaded from the Global Positioning System (GPS) receiver. The
latitude and longitude data were then calculated for each case using Excel and saved
as a tab-delimited text file. The prepared text files were then imported into Quantum
GIS software version 1.0.2 – Kore (www.qgis.org/) to visualise the location of cases
on a map. The same text file was then used as the coordinate file for analysis with
spatial scan statistics.
The total number of cases for each month from 16 December 2003 to 26 August
2006 were recorded and plotted on a graph. The duration of vaccination campaigns
against FMD were superimposed on the plots to identify potential links between
cases and vaccination.
The spatio-temporal clusters for the FMD outbreaks were identified by computing
the Space-Time Permutation model of the scan statistic test using SaTScan Version
8.0 of the retrospective data of 342 locations with a total of 5335 cases in the study
period from 3rd
December to 26th
August (Kulldorff et al., 2005; Kulldorff and Inc.,
2009). The technique utilised a large number of overlapping cylinders to define the
88
scanning window, each of which was a possible candidate for an outbreak at the
geospatial coordinate of the location. The base of the cylinder represents the
geographical area of the potential outbreak (spatial) while the height represents the
number of days (temporal) with the last date always included together with a
variable number of preceding days up to the maximum number of defined days.
Scanning for clusters was done with high rates and time aggregation by day. The
maximum spatial cluster size was set to 50% of the population at risk using circular
spatial windows and the maximum cluster size was also set at 50% of the study
period (Kulldorff and Nagarwalla, 1995; Kulldorff, 1997; Kulldorff et al., 2005). The
statistical significance was evaluated using 999 Monte-Carlo simulations in
combination with the scan test to detect significant differences between the observed
and expected results. The cut-off value for the identification of significant clusters
was set at P < 0.05.
5.3 Results
The largest number of outbreaks occurred between 16th
December 2003 and 15th
January 2004. The number of outbreaks reported decreased dramatically after the
ring-vaccination program was implemented in early January 2004 (Figure 5.1). The
number of reported cases decreased further in April 2004. However they increased
gradually from early May until the middle of July 2004. Surprisingly there were no
cases reported after the middle of July 2004 until early September 2005 and after
then the number of cases fluctuated until the end of the study period.
89
Figure 5.1. Number of cases of FMD recorded between 16th
December 2003 to 26th
August 2006.
0
200
400
600
800
1000
1200
1400
16001
6/1
2/2
00
3 -
15
/01
/20
04
16
/01
/20
04
-1
5/0
2/2
00
4
16
/02
/20
04
-1
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16
/03
/20
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-1
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4
16
/04
/20
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-1
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4
16
/05
/20
04
-1
5/0
6/2
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4
16
/06
/20
04
-1
5/0
7/2
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4
16
/07
/20
04
-1
5/0
8/2
00
4
16
/08
/20
04
-1
5/0
9/2
00
4
16
/09
/20
04
-1
5/1
0/2
00
4
16
/10
/20
04
-1
5/1
1/2
00
4
16
/11
/20
04
-1
5/1
2/2
00
4
16
/12
/20
04
-1
5/0
1/2
00
5
16
/01
/20
05
-1
5/0
2/2
00
5
16
/02
/20
05
-1
5/0
3/2
00
5
16
/03
/20
05
-1
5/0
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00
5
16
/04
/20
05
-1
5/0
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00
5
16
/05
/20
05
-1
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5
16
/06
/20
05
-1
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7/2
00
5
16
/07
/20
05
-1
5/0
8/2
00
5
16
/08
/20
05
-1
5/0
9/2
00
5
16
/09
/20
05
-1
5/1
0/2
00
5
16
/10
/20
05
-1
5/1
1/2
00
5
16
/11
/20
05
-1
5/1
2/2
00
5
16
/12
/20
05
-1
5/0
1/2
00
6
16
/01
/20
06
-1
5/0
2/2
00
6
16
/02
/20
06
-1
5/0
3/2
00
6
16
/03
/20
06
-1
5/0
4/2
00
6
16
/04
/20
06
-1
5/0
5/2
00
6
16
/05
/20
06
-1
5/0
6/2
00
6
16
/06
/20
06
-1
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7/2
00
6
16
/07
/20
06
-1
5/0
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00
6
16
/08
/20
06
-1
5/0
9/2
00
6
No of cases
Ring vaccination started
Mass vaccination started
90
There were 342 outbreak locations with 5335 FMD cases recorded in the whole state
of Pahang for the period of approximately 2.75 years (16th
December 2003 to 26th
August 2006). Cases of FMD were detected in all districts in the state of Pahang
except for the Cameron Highlands (Figure 5.2).
Figure 5.2 Geographical distribution of outbreaks of FMD in the state of Pahang that
were reported to the DVS during the period 16th
December 2003 to 26th
August
2006. Each red dot represents a reported outbreak.
The Space – Time permutation model indicated there were five significant clusters
with no geographical overlap in the secondary clusters for the whole study period.
Clusters were identified in the east, west and middle of Pahang with the observed to
91
expected ratio of FMD outbreaks within the spatial temporal clusters between 2.39
and 17.78.
There were 44 location identification sites included in the primary cluster (Cluster E)
(Table 5.1). This cluster involved an area with a 50.31 km radius and occurred
between the 20th
January 2006 and the 24th
August 2006 (218 days). There were
1150 cases (P = 0.001) in this cluster. Four secondary clusters were recorded;
Cluster A involved a smaller area (35.94 km radius) within a shorter time frame of
16th
December 2003 to 25th
February 2004 (72 days). There were 187 location
identification sites in this cluster which involved 1847 cases (P = 0.001). Cluster B
involved a smaller area compared to the primary cluster and Cluster A and included
an area with a radius of 25.09 km. It involved the longest time frame compared to the
other clusters identified (15th
July 2004 to 21st September 2005 - 433 days). There
were 592 cases included in this cluster (P = 0.001). Cluster D involved only one GPS
point representing one farm, resulting in a cluster size of radius 0 km with the
shortest time frame (17th
January 2006 to 17th
January 2006 - 1 day). It included only
one location identification site with 220 cases (P = 0.001). The cluster size is
determined by the GPS point of location. If it is only one point it is indicated as
having a radius of 0 km because the statscan software recognises it as only a dot
(GPS point), where as if the clusters containing 2 or more points the cluster size is
determined by the distance from one point to others. There was no information on
the farm size to input into the program for analysis. Finally secondary Cluster C
included seven location identification sites within an area with a radius of 28.62 km.
It involved 185 cases within a time frame of 25th
September 2005 – 19th
January
2006 (117 days) (P = 0.001).
92
Clusters Centre
Coordinate District
Cluster
size km
(radius)
Time frame
Cluster
period
(days)
No of
outbreaks
No of FMD
cases
No of
observed to
expected
ratio of FMD
outbreaks
P-Value
A
3.535583N
103.454083E
Pekan
Kuantan
35.94 16/12/2003 - 25/02/2004 72 140 1847 2.39 0.001
B
3.890367N
102.593417E
Maran
Kuantan
Jerantut
25.09 15/07/2004 - 21/09/2005 433 25 592 6.41 0.001
C
3.382433N
102.809583E
Pekan Bera
Maran
28.62 25/09/2005 - 19/01/2006 117 7 185 14.22 0.001
D
4.014583N
103.156750E Kuantan 0 17/01/2006 - 17/01/2006 1 1 220 17.78 0.001
E
3.719767N
101.941917E
Raub
Temerloh
Jerantut
Lipis
50.31 20/01/2006 - 24/08/2006 218 44 1150 3.75 0.001
Table 5.1 Spatio-temporal clusters detected by the spatial scan permutation model for 342 FMD outbreak locations in the state of Pahang from
16 December 2003 to 26 August 2006.
93
Figure 5.3 Location of the spatio-temporal clusters. Cluster E is considered the
primary cluster.
5.4 Discussion
This study presents the findings of an assessment of the spatio-temporal distribution
of FMD outbreaks in the state of Pahang during the period from the 16th
December
2003 to the 26th
August 2006. The space-time permutation result showed that the
FMD outbreaks were clustered into five distinctive clusters with no geographical
overlap between the secondary clusters in space and time within the study period
(Table 5.1).
94
The results indicated that the size of the clusters (spatial) were not uniform. They
varied from one which only encompassed a single outbreak location (Cluster D) to
one with a radius of 50.31 km (Cluster E). The largest cluster was identified as the
primary cluster and involved four districts (Raub, Temerloh, Jerantut and Lipis).
However the temporal analysis indicated that this cluster was detected at the end of
the period (20th
January 2006 to 24th
August 2006). Although it involved the largest
area it included the smaller number of outbreak locations and cases compared to
cluster A which occurred at the start of the outbreak (16th
December 2003 to 25th
February 2004). Cluster A included 140 FMD outbreak locations with 1847 cases
but affected only two districts (Kuantan and Pekan). As the radius of the largest
cluster detected was more than 50 km it would suggest that a buffering zone larger
than 50 km is required to control future epidemics in the state. The control measures
should include ring vaccination with two doses of vaccine, restriction of animal
movement and pre-emptive quarantine of susceptible animals within the buffer zone
as has been suggested by others (Donaldson and Wood 2004, Yadin et al 2007). As
four of the five clusters detected in this analysis had radii wider than the surveillance
zone practiced in Pahang (10 km radius), it is likely that this surveillance zone is
insufficient to contain an outbreak of FMD. However to impose a control activity
with a radius greater than 50 km may be logistically difficult and challenging and
imposing movement control can be difficult due to the animal production systems
practiced in this state.
95
Similar to that reported in other countries the temporal pattern of the FMD outbreaks
in Pahang appeared to be seasonal. Three of the five clusters commenced in
December and January which is when school holidays end as well as in the rainy
season in the east coast of Peninsular Malaysia. “Hari Raya Korban” is celebrated in
Zulhijjah in the Muslim/Islamic calendar, and normally falls in the month of
November or December in the Gregorian calendar. During this celebration many live
cattle are moved throughout the whole country, as well as in Pahang. The majority of
people in Pahang are Muslim and the Muslims believe in sacrificing specific food
animals on this day. The fresh meat from the sacrificed animals is then distributed to
the poor. Furthermore the wedding season in Pahang, as in other parts of Malaysia,
coincides with these school holidays. Both situations are associated with a
substantial increase in the number of cattle slaughtered in Pahang. One of the
clusters started on the 25th
September 2005 and also included the months of
December and January. This cluster also coincided with another big festival in the
Muslim community (“Aidil Fitri” celebration) which usually falls in the month of
October. It is a celebration after one month of fasting and there are many parties held
within the month of Syawal in the Muslim calendar (after the fasting month). During
this month Muslims are encouraged to visit their families, relatives and friends.
Consequently there also is a tremendous amount of human movement during that
month. Furthermore when visiting relatives and friends it is traditional to exchange
food, and therefore there is an increase in the movement of animals for slaughter for
these purposes as well as increased movement of fresh meat and processed products
all of which have been shown by others to increase the risk of transmission of FMD
(Kitching, 2002a; Moutou, 2002; Mansley et al., 2003; Mahy, 2005;
Wongsathapornchai et al., 2008; Lin et al., 2009; Bressel et al., 2010).
96
There was a decrease in the number of cases of FMD after the mass-vaccination
program was implemented in March 2004 (Figure 5.1). The absence of reported
cases from August 2004 to July 2005 might reflect the effect of this mass vaccination
campaign. However the mass vaccination program was not able to be continued after
the initial round due to the high cost of vaccine administration because of the animal
husbandry system adopted where many farmers did not have proper holding yards or
facilities for the effective implementation of a herd health program. As a result the
government had to cover the cost of purchasing and administering the vaccine as
well as providing portable (transportable) holding facilities (yards and crush) to
allow the animals to be handled and vaccinated safely and efficiently. The lack of
adequate husbandry facilities makes achieving an 80% vaccine coverage
challenging. Consequently the disease was not able to be eradicated completely and
clinical cases were reported again in the month of August 2005 until the end of the
study. Furthermore local spread is an important factor in the distribution of the
disease and it is important to rapidly identify outbreaks and implement control or
stamping out practices (Gibbens and Wilesmith, 2002). However the control
program adopted by the state of Pahang does not involve the culling of infected
animals, but only the application of movement restrictions and vaccination. Although
vaccination can reduce the incidence of disease it has been found to be effective only
when combined with selective culling of clinically infected animals and NSP
positive animals (McVicar and Sutmoller, 1969; Woodbury, 1995b; Alexandersen,
2002; Saraiva, 2004; Lubroth et al., 2007).
97
This analysis can be considered the first step towards understanding the spatial
aspects of outbreaks of FMD in Pahang and highlights the need for further statistical
and empirical analyses to be conducted to ensure suitable control procedures are
implemented and subsequently evaluated. It is suggested that cluster identification
tests should be conducted with directional tests to measure the significance of the
mean directional spread of an outbreak and phylogenetic investigations undertaken
in every cluster. These will increase the local information gathered to allow planning
of a more complete and suitable control program for Pahang.
98
CHAPTER 6
GENERAL DISCUSSION AND CONCLUSIONS
The overall aim of the research reported in this thesis was to improve the
understanding of the epidemiology of FMD in Pahang. Specifically the temporal and
spatial distribution of outbreaks of FMD in Pahang during the period 2003 to 2006
were described; risk factors associated with the occurrence of FMD in Pahang were
identified; and the antibody response in Nallore cattle following vaccination against
FMDV was reported. The information gathered can be used to develop more
effective control and eradication measures for the state. Other work in Southeast
Asia has suggested that eradication of FMD offers significant economic benefits to a
country, even when no export of livestock occurs (Perry et al., 1999).
Pahang is one of the 13 states in Malaysia, therefore the findings of this study could
be useful in helping to achieve Malaysia‟s plan to become an FMD-free country
through vaccination in the year 2016 (DVS, 2009a). One of the most important
factors which determines the amount of effort provided by affected personnel in a
disease control program is the level of awareness about the relevant disease
(Garland, 1999; Leforban and Gerbier, 2002). The success of any control and
eradication program developed in Pahang is dependent upon the combined efforts of
all involved personnel including DVS staff, farmers, butchers and traders. To involve
this diverse group requires a clear understanding of the epidemiology of the disease,
knowledge about ways to control its spread, cooperation between agencies and
suitable educational and awareness campaigns.
99
The legal and illegal movement of infected animals is considered to be the most
important factor involved in the spread of FMD (Mansley et al., 2003; Robinson and
Christley, 2007), particularly in the Southeast Asian region (Abila and Foreman,
2006). Geographically Peninsular Malaysia is connected to other Southeast Asian
countries which can facilitate the spread of the disease through both international and
local movements of livestock. In this study (Chapter 3), a high percentage of cattle
trading were undertaken between farms within the state of Pahang (98%) which
confirmed the findings of a study on the movement of cattle conducted in Pahang in
2005 (Azmie et al., 2006). Although there is legislation requiring all animal
movements in Pahang to be accompanied by an animal movement permit, which
includes a veterinary health certificate, there are no clear penalties documented for
breaches to this legislation (DVS, 2004). Many studies have demonstrated that the
most effective way for the spread of FMD is through direct contact either with
clinically infected or subclinically affected animals incubating the disease
(Donaldson et al., 1987; Alexandersen, 2002; Brown, 2004; Orsel et al., 2009).
Consequently strengthening and enforcing the existing regulations is required.
In Pahang, as in all other states in Malaysia, there are special cultural and religious
festivals. For these celebrations there is a strong community preference for the
consumption/purchase of fresh meat from ruminants rather than frozen or processed
products. This results in a high demand for fresh meat and the requirement for the
daily slaughter of ruminant livestock. However the country is not self-sufficient in
meat, producing only 26.9% of the beef and 9.6% of the sheep and goat meat
required (DVS, 2009c). Consequently live cattle are imported from Thailand and
other Southeast Asian countries into Peninsular Malaysia, resulting in increased risk
100
of the importation of a transboundary animal disease such as FMD
(Wongsathapornchai et al., 2008). This situation makes disease control activities
challenging. Findings from the temporal analysis in this study (Chapter 5) indicated
that significant clusters of disease started just before and during the festival months.
During these periods there is a dramatic increase in the movement of ruminants and
in slaughter activities. It is crucial to consider this information when developing
control measures for FMD. The control measures should be based on the
establishment of effective biosecurity, surveillance and disease control programs
which suit the local situations, particularly with respect to the festive seasons.
In Chapter 5 it was also found that three of the four clusters of outbreaks progressed
for a long time, in contrast to outbreaks in some other countries such as Israel and
Palestine where outbreaks have lasted for not more than a month (AlKhamis et al.,
2009). The prolonged outbreaks with large clusters are the result of many combined
factors including the vaccination regime adopted in Pahang and the absence of a
culling policy for clinically affected and NSP positive animals. In other parts of the
world it has been demonstrated that vaccination campaigns, combined with other
zoo-sanitary control measures, have been successful in controlling the disease
(Garland, 1999; Leforban and Gerbier, 2002). The limitations of vaccination must be
considered when undertaking a control program. These include the generation of
protective immunity only to those serotypes and subtypes included in the vaccine
(Lubroth et al., 2007), difficulties in achieving a high coverage and problems with
the storage, maintenance and distribution of the vaccine to field locations. At least
80% of animals in a herd need to be vaccinated to induce protective herd immunity
(Lombard and Schermbrucker, 1993; Leforban and Gerbier, 2002). Effective
101
immunity must be considered on a herd basis and it is vital to vaccinate as many
animals as possible in a population to reduce the potential for disease in that
population (Doel, 2003). Failure to achieve an 80% vaccine coverage in a population
and maintaining a regular vaccination schedule, as recommended in Chapter 3, has a
significant negative impact on the development of protective immunity. This was
highlighted in Chapter 4 where it was found that not administering a second priming
(P2) dose to calves four weeks after the primary vaccination failed to induce high
levels of protective immunity. Furthermore an ideal immunization program should
induce both a systemic (serum) and mucosal (secretory) antibody response to protect
against clinical disease and to inhibit local virus replication and consequently virus
persistence (Barnett et al., 2004). Results reported in Chapter 3 indicated that in
Pahang most primary vaccinations were not followed by a P2. This situation was
worsened by the fact that only a few respondents understood the reason behind
vaccination and the importance of the P2 vaccination, as well as subsequent booster
vaccinations, to provide sufficient protection against FMD. The effectiveness of
vaccination in the control and eradication of FMD has been proven in other countries
(Perez et al., 2004; Saraiva, 2004; Lubroth et al., 2007). To achieve the target of
Malaysia being an FMD Free country by 2016 it is essential that there is adoption of
a second priming vaccination and booster vaccinations at 6 monthly intervals and a
high level of vaccination coverage. It is important to not just rely on vaccination but
also to improve diagnostic proficiency and capability, enforce stricter legislation
requirements, establish a surveillance network capable of providing early warning of
disease incursions/outbreaks, implement effective immediate control measures
through the coordination of several agencies, manage livestock transport
mechanisms, improve market inspection and hygiene compliance, and develop
102
public communication, education and awareness programs (Garland, 1999; McLaws
et al., 2009).
In Chapter 4 NSP positive cattle were detected on a farm which had no previous
history of clinical disease. This finding is not surprising since the farm was located
in an endemic area and FMD is known to have a persistent carrier stage (Sallt, 1993;
Alexandersen, 2002; Salt, 2004), moreover the virus can be spread by air
(Donaldson, 1987), personnel (Sellers et al., 1970; Amass et al., 2003b; Amass et al.,
2004), water, feed (Alexandersen et al., 2003c; Schijven et al., 2005) and fomites
(McColl et al., 1995; Bartley et al., 2002). Importantly sub-clinically infected
animals may spread FMDV through secretions and excretions (Bedson et al., 1927;
Blackwell and Hyde, 1976; Donaldson, 1987; Donaldson et al., 1987; Brown, 2004).
Therefore, depending on the objectives of the country, to eradicate the disease it is
important to cull clinically infected animals and those with repeatedly positive NSP
results. This recommendation is supported by the findings reported in Chapter 3
where it was demonstrated that retaining FMD seropositive animals in a farm had a
significant impact on the likelihood of a farm having the disease. The role of virus
shedders has been highlighted in other studies along with the potential for the
transmission of virus within and between herds by aerosols. Under appropriate
environmental conditions virus-laden droplets may travel vast distances whilst
maintaining infectivity (Donaldson, 1987; Alexandersen et al., 2003b; Alexandersen
et al., 2003c; Grubman and Baxt, 2004; Schijven et al., 2005). The virus may also
persist (Sallt, 1993) in the pharyngeal region of infected cattle for up to 2.5 years, in
sheep for 9 months and for up to 5 years in African buffalo. In addition, vaccinated
103
animals may become persistently infected long-term carriers without showing
evidence of clinical disease (Burrows, 1966; Alexandersen, 2002; Doel, 2003).
However the epidemiological significance of these carriers is controversial
(Alexandersen et al., 2003c). Although the amount of persisting virus is low and the
ability of carriers to transmit virus to other animals is equivocal, carriers are still
considered a potential, but unquantified risk, for the spread of infection (Thomson
and Bastos, 2004). The fear that they may occasionally initiate new outbreaks has led
to international trade rules requiring either long waiting periods for carriers to
recover from infection or the adoption of methods to remove carriers before FMD-
free status can be restored to regions that have had outbreaks (OIE, 2008b).
Consequently legislation to enable the culling of repeatedly seropositive animals,
along with a suitable compensation scheme to ensure support from local farmers, is
recommended in Malaysia. These, along with the development of more effective
educational and awareness programs to increase farmer‟s knowledge about the
disease should be encouraged, allowing a quicker and more effective control
program to be implemented (McLaws et al., 2009). A study in Australia showed that
farmers, in the face of a hypothetical outbreak, were more concerned about losses to
their individual animals than they were to losses to the livestock industry as a whole
(Chen, 2008). This may lead some farmers not to report disease or to ignore animals
with mild lesions and these may even be sold before the final disease diagnosis can
be confirmed (Chen, 2008).
In the current study (Chapter 3) most farmers had little concern about the biosecurity
of their own farm. This was probably related to the low level of awareness about the
importance of good biosecurity measures and its significance in controlling FMD, as
104
well as other diseases. Unlike in other countries where animals form part of an
important industry, cattle rearing in Pahang is mostly undertaken to supplement the
farmers‟ income. The farmers‟ priority is on their primary income activity, which
generally involves oil palm plantations. As FMD does not usually cause the death of
adult cattle, most farmers are not concerned with the disease or do not appreciate the
losses arising from infection. Therefore, efforts to change the farmers‟ perceptions
through education so they can understand the potential benefits of raising livestock
should be considered. This study has identified there is an urgent need to improve
the farmers‟ knowledge, not only on the disease but also on its economic impact.
This situation has also been identified in other countries, such as Thailand and India,
where it has been shown that more educated farmers are more likely to cooperate
with a disease control program (Saini et al., 1992). A study in the United Kingdom
also indicated that education and awareness campaigns were important in the quick
control and eradication of the disease (McLaws et al., 2009). In the current study
most participants (87%) were able to recognise the clinical signs of the disease. It is
important to educate farmers on the importance of early detection and reporting of
suspect cases to the DVS. However, for the success of a control and eradication
program for FMD in a country, identification of important local epidemiological
factors along with a cost benefit analysis should be undertaken (Hutber et al., 2010).
Although many countries have successfully controlled and eradicated FMD through
a stamping out or vaccination program, these can be challenging to implement in
countries where the disease is endemic (Garland, 1999; Scudamore and Harris, 2002;
Sutmoller et al., 2003; Lubroth et al., 2007). Clear Government policies are
important to eliminate any source of infection and to restrict the movement of
105
animals and animal products as soon as possible after disease is detected to minimise
the spread of the disease (Garland, 1999; Kahn et al., 2002). This recommendation
has been clearly supported by other studies which have concluded that the most
effective way for the spread of FMDV in ruminants is through direct contact,
particularly through the movement of affected animals. Furthermore the capability of
the virus to infect many animal species fosters rapid spread. Several authors have
recommended pre-emptive culling in combination with vaccination to control an
epidemic of FMD (Woolhouse et al., 2001; Bouma et al., 2003). The current study
demonstrated that keeping NSP seropositive animals in a herd increased the
likelihood of infection, and therefore it is recommended that clinically infected and
repeatedly NSP seropositive animals are culled. However currently there is no
legislation on the mandatory slaughter or culling of animals and there is no
compensation provided by the Government to encourage the culling of clinically
infected or seropositive animals in Malaysia (DVS, 2009a). Movement restrictions
and ring vaccination, which were imposed during the initial stages of the FMD
epidemic in Pahang, were applied to herds within a 5 km radius of new outbreaks.
This did help in controlling the disease for a short period; however subsequent
analysis of outbreaks has highlighted the need for wider control zones than those
previously implemented. By vaccinating all potentially exposed animals, sufficient
immunity should be developed to provide at the very least partial protection against
clinical disease (Perez et al., 2004).
The multivariable analysis conducted as part of this study demonstrated that farmers
with more than one species of ruminants were at a higher risk of having animals with
FMD. This was probably due to the ability of small ruminants to act as a potential
106
source of infection (Barnett and Cox, 1999; Alexandersen et al., 2003c) and these
findings supported the results of other studies (Al-Majali et al., 2008; Maddur et al.,
2009). Transmission between cattle and buffalo has been reported, both in naturally
occurring outbreaks (Dutta et al., 1983; Samara and Pinto, 1983) and experimentally
induced disease (Gomes et al., 1997; Maroudam et al., 2008). Moreover the ability of
the buffalo to be persistently infected and to act as a carrier has been confirmed
(Barros et al., 2007; Maddur et al., 2009). Small ruminants, such as sheep and goats,
can also be infected but not detected because of the absence of obvious clinical signs
(McLaws et al., 2009). These animals may result in virus circulation for an extended
period of time resulting in the spread of disease due to the common practice of
farmers in Pahang of keeping more than one species of ruminants.
Although it is very important to understand the specific reasons behind the duration
of the prolonged clusters reported in Chapter 5, this was not possible in this study
because of time constraints. This should be investigated in detail in the future. It is
important to investigate clusters of disease to identify predisposing factors for the
outbreaks so that effective disease control plans can be developed, implemented and
evaluated (Carpenter, 2001).
The majority of the important risk factors for FMD identified in Pahang were related
to a lack of biosecurity. The role of direct and indirect spread is important and the
movement of livestock, animal products and fomites were identified as important
risk factors for disease in this study. The spread of the disease through indirect
contact with fomites containing infectious viral particles, such as contaminated
vehicles, feed, or clothing of livestock personnel has previously been well
107
documented (McColl et al., 1995; Bartley et al., 2002; Amass et al., 2003b; Amass et
al., 2004). Importantly in this study, only 37.3% of farmers were able to correctly
explain how FMD is spread. This situation highlights the need for an appropriate
education campaign for personnel involved in the livestock industry. Although this
study concentrated on farmers, it is likely that similar educational programs are
warranted for other groups, including the DVS support staff, traders and butchers
(Garland, 1999).
It was suggested that in order to control FMD more effectively in an endemic area,
vaccination should be adopted in combination with movement controls together with
compulsory culling of clinically affected animals. Emergency vaccination in the face
of a severe challenge will help prevent or reduce the replication of virus and thereby
dramatically reduce the amount of virus released into the environment in the
important post-exposure period. Once vaccinated, these animals present a barrier to
the further spread of the disease. While animals exposed to infection and those that
are diseased will be eliminated, animals vaccinated for protective purposes could be
tested to confirm the absence of viral activity and, if they are free of infection,
allowed to live for the term of their productive lives. It is crucial to also get farmers
involved in the timely adoption of a vaccination program, with the first and second
vaccination given approximately one month apart, followed by booster vaccinations
at six monthly intervals. This requires input and commitment from all levels from the
DVS to the farmers. The effect of the mass vaccination program conducted
immediately after the outbreak in March 2004 was seen in 2005 with no clinical
cases reported between 15th
July 2004 and the 15th
September 2005. However
clinical disease subsequently reappeared, indicating the ineffectiveness of the FMD
108
control and eradication measures adopted in the state of Pahang. Control and
eradication of FMD either by stamping out or by vaccination is costly, however
studies have shown that vaccination is economically beneficial (James and Rushton,
2002). Stamping out has significant problems from an operational point of view and
is often not well accepted by the farming community. Due to the high cost and
implementation issues with mass vaccination, such a program needs to be replaced
by more cost-effective, strategic and carefully planned vaccination programs which
are adequately resourced and effectively administrated and supported with
appropriate legislation and penalties. Furthermore in order to achieve more than 80%
vaccination coverage in the state, full commitment by the farmers is crucial.
In conclusion this study identified important factors associated with the rapid spread
of FMD in Pahang. These included the movement of infected animals, poor
biosecurity measures and ineffective vaccination programs. It is recommended that
appropriate strategic vaccination, including the use of priming and booster
vaccinations, be adopted to control FMD in Pahang. Along with vaccination it is
recommended that legislation be developed to allow for the rapid compulsory culling
of clinical cases of FMD and suitable broad educational campaigns be developed and
implemented. Until such control measures are adopted it is likely that FMD will
remain endemic in both Pahang and Peninsular Malaysia.
109
Appendix 1
Non-structural Protein (NSP) test : Ceditest procedure
The test procedure adopted followed the manufacturer‟s test manual as described
below:
Day 1:
1.0 Incubation with test serum
80 µl ELISA buffer was dispensed into all wells
20 µl of negative control sera were dispensed into wells A1 and B1
20 µl of weak negative control sera were dispensed into wells C1 and D1
20 µl of positive control sera were dispensed into wells E1 and F1
20 µl of each sera to be tested were dispensed into the remaining wells
The test plate(s) were sealed using the enclosed plate sealers
The test plates(s) were gently shaken
The plates were incubated overnight (16-18 hours) at room temperature (20-25°C)
Day 2:
Incubation with conjugate and chromogen/substrate solution
The test plate(s) were emptied after the incubation period and the plate(s) were
washed 6 times with washing fluid. The plate(s) were tapped firmly after the last
washing.
110
100 µl of the working dilution of the conjugate were dispensed into each of the
wells.
The test plate(s) were sealed using the enclosed plate(s) sealers
The plates were incubated for 1 hour at room temperature (20-25°C)
The test plate(s) were emptied after the incubation period and the plate(s) washed 6
times with washing fluid. The plate(s) were tapped firmly after the last washing.
100 µl of chromogen/substrate solution were then dispensed into all wells
The plates were incubated for 20 minutes at room temperature (20-25°C)
100 µl of the stop solution was then added to all wells
The contents of the wells of the test plate(s) were mixed prior to measuring.
NB; The addition of the stop solution was started 20 minutes after the first
well was filled with chromogen/substrate solution. The stop solution was
added in the same order as the chromogen/substrate solution had been
dispensed.
Reading of the test plate(s) and calculating the results
The optical density (OD) of the wells was measured at 450 nm preferably within 15
minutes after colour development had been stopped.
The OD450 of wells A1 and B1 (negative control = OD max) was measured
The percentage inhibition (PI) of the control and the test sera were calculated
according to the following formula:
OD450test sample
111
PI = 100 - ----------------------------- X 100
OD450max
NB; The OD450 values of all samples were expressed as percentage inhibition (PI)
relative to the mean OD of the negative control (ODmax).
Ceditest procedure:
The test procedure adopted followed the manufacturer‟s test manual as described
below:
Day 1:
Incubation with test serum
80 µl ELISA buffer was dispensed into all wells
20 µl of negative control sera were dispensed into wells A1 and B1
20 µl of weak negative control sera were dispensed into wells C1 and D1
20 µl of positive control sera were dispensed into wells E1 and F1
20 µl of each sera to be tested were dispensed into the remaining wells
The test plate(s) were sealed using the enclosed plate sealers
The test plates(s) were gently shaked
112
The plates were incubated overnight (16-18 hours) at room temperature (20-25°C)
Day 2:
Incubation with conjugate and chromogen/substrate solution
The test plate(s) were emptied after the incubation period and the plate(s) were
washed 6 times with washing fluid. The plate(s) were tapped firmly after the last
washing.
100 µl of the working dilution of the conjugate were dispensed into each of the
wells.
The test plate(s) were sealed using the enclosed plate(s) sealers
The plates were incubated for 1 hour at room temperature (20-25°C)
The test plate(s) were emptied after the incubation period and the plate(s) were
washed 6 times with washing fluid. The plate(s) were tapped firmly after the last
washing.
100 µl of chromogen/substrate solution were then dispensed into all wells
The plates were incubated for 20 minutes at room temperature (20-25°C)
100 µl of the stop solution was then added to all wells
The contents of the wells of the test plate(s) were mixed prior to measuring.
NB; The addition of stop solution was started 20 minutes after the first well
was filled with chromogen/substrate solution. The stop solution was added in
the same order as the chromogen/substrate solution had been dispensed.
113
Reading of the test plate(s) and calculating the results
The optical density (OD) of the wells was measured at 450 nm preferably within 15
minutes after colour development had been stopped.
The OD450 of wells A1 and B1 (negative control = OD max) was measured
The percentage inhibition (PI) of the control and the test sera were calculated
according to the formula below:
OD450test sample
PI = 100 - ------------------------------- X 100
OD450max
NB; The OD450 values of all samples were expressed as percentage inhibition (PI)
relative to the mean OD of the negative control (ODmax).
114
Appendix 2
Liquid phase blocking ELISA test procedure
Reagent and sample preparation
Trapping Antibody Stocks
The freeze dried contents of a vial of each trapping rabbit antibody (FMDV
Serotypes O, A and Asia 1) were reconstituted with 0.5 ml sterile deionized
water by gentle agitation.
The stock was stored in the original vials at 1 to 8°C
The sera were further diluted 1000 fold with coating buffer to coat plates.
Only 1 vial was used at a time
Detecting Antibody Stocks
The freeze dried contents of a vial of each detecting guinea pig antibody (FMDV
Serotype O, A and Asia 1) were reconstituted with 0.5 ml sterile deionized water by
gentle agitation.
The stock was stored in the original vials at 1 to 8°C.
The sera were further diluted 100-fold with diluent Buffer B (phosphate buffered
saline) and used in the assay
Only 1 vial was used at a time
115
Anti-Species Conjugate Stocks
The stock was prepared by reconstitution of the freeze dried contents of a vial with 1
ml of sterile diluent #2 (water plus glycerol, including 0.02% merthiolate) and gently
mixed until completely dissolved.
The stock was stored in the original vials at -20°C
The conjugate was further diluted 200-fold with diluent Buffer B (phosphate
buffered saline) and used in the assay.
Only 1 vial was used at a time
Antigen Stocks
Working dilution:
O1 Manisa 1:100
A22 Mahmatli 1:100
Asia 1 Shamir 1:100
The master stock of the control antigen were stored at -80°C
Control Serum Stocks
The freeze dried contents of each positive (C++, strong antibody positive. C+,
medium antibody positive) and negative (C-, antibody negative) control were
116
reconstituted with 0.5 of sterile deionized water including 0.02% (reconstitution
diluent #1) by gentle agitation.
The stock was stored in the original vials at 1 to 8°C.
Only 1 vial was used at a time.
Chromogen Buffer
0.05M Phosphate-Citrate Buffer, pH 5.0 was obtained by dissolving one tablet in
100 ml of locally produced deionized water.
The Chromogen buffer was stored in 1 to 8°C for no longer than 1 week.
Chromogen Stock
Ortho-Phenylenediamine (OPD tablets). 1 tablet (30 mg) was dissolved per 50 ml of
0.05M phosphate-citrate buffered solution at pH 5.0 and stored at room temperature
in the dark.
Substrate Stock
3% (w/v) H2O2 (882mM). 1 hydrogen peroxidise tablet was dissolved in 10 ml of
locally produced sterile distilled/deionized water.
The stock was stored at 1 to 8°C in the dark.
Coating Buffer
117
0.05 M Carbonate/bicarbonate, pH 9.6 +/- 0.05. 1 capsule was dissolved in 100 ml of
locally produced sterile distilled/deionized water
The stock was stored at 1 to 8°C for no longer than 1 week
Diluent Buffer A (for antigen and test sera dilutions)
0.01 M Phosphate Buffered Saline, pH 7. 4 +/- 0.02 plus 0.05% (v/v) Tween 20.
5 PBS tablets were dissolved per 1 litre of locally produced distilled/deionized
water.
500µl of Tween 20 was added per litre and the solution mixed.
The diluent was stored at 4°C for no longer than 2 weeks.
Diluent Buffer B (for detecting antibody and conjugate)
0.01 M Phosphate Buffered Saline, pH 7.4 +/- 0.02 plus 0.05% (v/v) Tween 20 plus
5% (w/v) Skimmed Milk Powder
Diluent Buffer A was prepared:
0.01 M Phosphate Buffered Saline, pH 7.4 +/- 0.02 plus 0.05% (v/v) Tween 20.
500 µl of Tween 20 was added per litre and the solution mixed.
118
The diluent was stored at 4°C for no longer than 2 weeks.
On the day of testing Skimmed milk powder (5%, w/v) was added to diluent Buffer
A to make the diluent Buffer B required.
PBS tablets were dissolved per 1 litre of locally produced distilled/deionized water.
Wash Buffer
Phosphate Buffered Saline, pH 7.4 +/- 0.20
5 PBS tablets were dissolved in 1 litre of locally produced sterile distilled/deionized
water.
The wash buffer was transferred to a washing container
The wash buffer was further diluted by the addition of 4 litres of locally produced
distilled/deionized water
The wash buffer was stored at room temperature.
Stopping Solution
1.25 M Sulphuric Acid
68 ml of concentrated sulphuric acid (18 M) was slowly added to 932 ml of locally
produced distilled/deionized water. The stopping solution was stored at room
temperature.
119
Disinfectant
0.2% (w/v) citric acid solution
2 gm of citric acid was dissolved in 1 litre of locally produced distilled/deionized
water
The disinfectant was stored at room temperature.
Assay Procedure
Coating of Microplates
a. Vials containing trapping rapid stock (FMDV serotypes O, A and Asia 1) were
gently agitated
b. A working dilution of trapping antibody stock in coating buffer was prepared
at a dilution of 1:1000 (Each serotype-specific trapping antibody was diluted
separately).
The working dilution was agitated gently to ensure uniform dispersion.
All the plates were aligned correctly (with the letters on the left hand side)
50 µl of working dilution of trapping antibody was immediately dispensed into all 96
wells.
The sides of the microplates were tapped to ensure the trapping antibody was evenly
distributed over the bottom of the wells.
The microplates were covered and incubated at 1 to 8°C
120
The remainder of the aliquot of the FMDV trapping rabbit antibody stock was then
returned to 1 to 8°C
Test and Control Serum Incubation (Liquid phase)
Screening Assay
The test and control sera were gently agitated to ensure homogeneity.
A 1/16 dilution of each control and test sera in suitable dilution tubes were prepared
by the procedure below:
15 µl of an undiluted control bovine serum was added to 225 µl of Diluent Buffer A
10 µl of diluted serum was added to 150 µl of Diluent Buffer A for each test serum.
The dilutions were gently agitated to ensure homogeneity.
The test and control sera were added to the wells of the polypropylene U-bottom
microplate at a dilution of 1/16
50 µl volume of pre-diluted test and control sera were added to the wells of
polypropylene U-bottom microplate according to the plate layout #1 (Appendix 1)
50 µl of Diluent Buffer A was added to the antigen control wells
The details and position of test sera on the ELISA Data Sheet were recorded
(Appendix 3)
121
Titration Assay
Two fold dilution range (for evaluation of positive sera)
A 1/16 dilution of the control sera were prepared as in 2.1.1
A 1/8 dilution of test sera in suitable tubes or microplates were prepared by adding
20 µl of diluted serum for each test serum to 140 µl of Diluent Buffer A
For each run and for each FMDV serotype the C++ control was also titrated in the
same manner as the test serum.
50 µl of diluents Buffer A was dispensed into columns 3 – 12 for each polypropylene
U-bottom microplate.
50 µl volume of pre-diluted control sera were added to the appropriate wells
according to Plate layout #2 (Appendix 2)
50 µl of Diluent Buffer A was added to the antigen control (Ca) wells
50 µl volumes of pre-diluted test serum and the C++ control were added to the
appropriate wells of row A or E, columns 3 – 12 according to the plate layout #2.
The 100 µl contents in the well were carefully mixed by filling and emptying the
pipette tip several times, taking care not to introduce air bubbles.
50 µl of the dilution was transferred to the next row (A to B) and the contents of row
B were carefully mixed.
50 µl from row B was transferred to the next row (B to C) and the mixing procedure
was repeated.
122
50 µl from row C was transferred to row D. The dilution was carefully mixed and 50
µl of the dilution was finally discarded. This will result in a test sample dilution
series from 1/16 to 1/128 in 50 µl volumes.
The above procedure was repeated for the next test samples for row E
through to H using new pipette tips.
Details and position of test sera were recorded on the ELISA Data Sheet.
Five-fold dilution range (for evaluation of post-vaccination sera)
A 1/16 dilution of the control sera were prepared as in 2.1.1
20 µl of an undiluted control serum was added to 300 µl of Diluent Buffer A for
each control serum
60 µl volume of the pre-diluted control sera were added to the appropriate wells of
polypropylene microplates according to Plate Layout #2 (Appendix 2)
60 µl of Diluent Buffer A was added to the antigen control (Ca) wells.
1/5 dilution of each test serum (and the C++ control) were prepared by adding 20 µl
of undiluted serum to 80 µl of Diluent Buffer A.
60 µl of Diluent Buffer A was added to all columns 3 -12 in each polypropylene
microplate.
15 µl volumes of 1/5 diluted test serum (and the C++ control) were added to the
appropriate wells of row A or E, column 3 – 12 according to Plate Layout #2.
123
The 75 µl contents in the well were carefully mixed by filling and emptying the
pipette tip several times, taking care not to introduce air bubbles.
15 µl of the dilution was transferred to the next row (A to B) and the contents of row
B were carefully mixed.
15 µl from row B was transferred to the next row (B to C) and the mixing procedure
was repeated.
15 µl from row C was transferred to row D. The dilution was carefully mixed and 15
µl of the dilution was finally discarded. This resulted in a test sample dilution series
from 1/25 to 1/3125 in 60 µl volumes.
The above procedure was repeated for the next test samples for row E through H
using new pipette tips.
Details and positions of test sera were recorded on the ELISA Data Sheet.
Addition of FMDV antigen
The antigen stock was removed from the freezer and thawed.
Prior to making the working dilution in Diluent Buffer A the antigen stocks were
equilibrated to room temperature.
Working dilution of the FMDV antigen (FMDV serotype O, A and Asia 1) in
Diluent Buffer A in a volume sufficient for microplate use was prepared (5 ml per
plate plus an additional 1 ml for 5 five-fold dilution range plates).
The suggested working dilutions: O1 Manisa 1:100, A22 Mahmatli 1:100 and Asia 1
Shamir 1:100.
124
50 µl of the antigen working dilution were added to all 96 wells of the respective
polypropylene U- bottom microplate for evaluation of positive sera. All wells
contained 100 µl of a serum dilution and antigen at this stage. This resulted in a final
serum dilution of 1/32 to 1/256 in the titration assay.
60 µl of the antigen working dilution was added to all 96 wells of the respective
polypropylene U- bottom microplate for evaluation of post vaccinal sera. All wells
contained 120 µl total of a serum dilution and antigen at this stage. This resulted in a
final serum dilution of 1/50 to 1/6250 in the titration assay.
The microplates were briefly placed on an orbital shaker to ensure thorough mixing.
The polypropylene microplates were incubated at +1°C to 8°C overnight with the
seal on.
The remainder of the master stocks of the control antigens were returned to -80°C
for future use.
Transfer of Serum/Antigen Mixture to the ELISA Plate
The contents of all antibody coated microplates (NUNC Maxisorp) were discharged
by inverting the microplates using an abrupt downward hand motion into a sink and
the inverted microplates were slapped onto a lint-free absorbent towel to remove all
residual contents.
All 96 wells of the microplates were filled with wash buffer using the Handiwash.
125
The contents were discharged again after filling using the same abrupt downward
hand motion into a sink and the inverted microplates were slapped onto a lint-free
absorbent towel to remove all residual contents.
The washing procedure was repeated with two more wash cycles of filling and
emptying.
50 µl volumes of the serum/antigen mixture were immediately transferred from the
polypropylene U-bottom microplates to the appropriate wells of the different
polystyrene microplates (NUNC Maxisorp) according to the same layout as being
used for the “liquid phase” after three complete wash cycles and ensuring that no
residual contents were left in the microplates.
The microplates were sealed and placed in an orbital shaker housed in a incubator at
37°C for 1 hour with continuous shaking.
Preparation of Diluent Buffer B
0.01 M Phosphate Buffered Saline, pH 7.4 +/- 0.02 plus 0.05% (v/v) Tween 20 plus
5% (w/v) Skimmed Milk Powder
On the day of testing, the amount of Diluent Buffer B required was calculated and
prepared by adding skim milk powder to a final concentration of 5% (w/v) to the
required volume of Diluent Buffer A. (100 ml Diluent Buffer B was prepared : 5 gm
skim milk powder was added to 100 ml of Diluent Buffer A )
The pH was adjusted to pH 7.4 +/- 0.20 using 0.1 M NaOH.
126
Addition of Detecting Antibody
A 1:100 working dilution from the homologous detecting antibody stock (anti-
FMDV serotype O, A and Asia 1) in Diluent Buffer B in sufficient volume (5 ml of
working dilution per plate plus additional 1 ml) for the plates being used were
prepared immediately before the end of the serum/antigen mixture incubation for the
first plate.
The remainder of the detecting guinea pig antibody stocks were returned to 1°C to
8°C.
The microplates were removed from the incubator after one hour incubation then
washed with wash buffer as described previously.
A 50 µl volume of the working dilution of the detecting antibody (FMDV serotype
O, A and Asia 1) were added into the 96 wells of the respective microplates
immediately after washing.
The sides of the microplates were tapped to ensure that the working dilution was
evenly distributed over the bottom of each well.
The microplates were sealed and placed on an orbital plate shaker housed in an
incubator at 37°C for 1 hour with continuous shaking.
127
Addition of Conjugate
A 1:200 working dilution of the conjugate in Diluent Buffer B in sufficient volume
for all microplates (5 ml of working dilution per plate plus an additional 1 ml) were
prepared immediately before the end of the detecting antibody incubation.
Both the conjugate stock and its working dilution were agitated gently and
thoroughly.
The remainder of the conjugate stock was returned to -30 to -50°C
The microplates were removed from the incubator after one hour incubation then
washed with wash buffer as described previously.
A 50 µl volume of the working dilution of the conjugate was added into each of the
96 wells in the microplates.
The sides of the microplates were tapped to ensure that the conjugate working
dilution was evenly distributed over the bottom of each well.
The microplates were sealed and incubated at 35 to 39°C for 1 hour with continuous
shaking.
Addition of Substrate/Chromogen and Stopping Solution
The substrate/chromogen solution in a volume sufficient for the number of
microplates being run was prepared immediately before the end of the conjugate
incubation.
The final colourless substrate/chromogen solution was stored in the dark
128
A clean microplate (not coated with trapping antibody) was used as the “blanking
plate” for the photometric reading.
The microplates were removed from the incubator after one hour incubation then
washed with wash buffer as described previously.
All 96 wells of each microplate were completely flooded with wash buffer to
eliminate unreacted conjugate.
50 µl volumes of the substrate/chromogen solution were added into the 96 wells of
each microplate, starting with the first column of the “blanking plate” followed by all
96 wells of the microplates in the test run immediately after washing.
The microplates were incubated at ambient temperature for 15 minutes without plate
shaking immediately after filling the first wells.
50 µl volumes of the stopping solution (1.25 M sulphuric acid) was added
immediately starting with the first column of the “blanking plate” followed by all 96
wells of the test run after 15 minutes of substrate/chromogen incubation.
The microplates were placed briefly on the shaker to ensure even mixing. All wells
contained 50 µl of substrate/chromogen solution plus 50 µl of stopping solution at
this stage,
Measurement of Substrate Development
The “blanking plate” was placed in the carriage of the photometer and the blanking
sequence was initiated.
129
The first microplate of the test run was placed in the carriage of the photometer and
the reading sequence was initiated.
The step described in 2.2.9.2 was repeated for each microplate.
Assay performance and interpretation
Data Expression
Microplate reading was used in two data analysis.
Percent Inhibition (PI) values which were used for Quality Assurance (QA)
acceptance. These values were calculated as follows:
PI = 100 – (Replicate OD of control X 100)
___________________________
Median OD of Ca
Percentage Inhibition (PI) values were used for replicate values for test sera. These
values were calculated as follows:
PI = 100 – (Replicate OD of test serum X 100)
_____________________________
Median OD of Ca
130
Calculation and acceptance of Control Data
The data expressed in OD values and PI values for the antigen control (Ca) and the
data expressed values in three other controls (C++, C+ and C-) were used to
determined whether or not the test had been performed within acceptable limits of
variability and therefore, whether or not the test sera data may be accepted from any
given microplate.
131
Appendix 3
Survey on the possible risk factors contributing to Foot and Mouth Disease in the state
of Pahang, Malaysia
Background to the Survey
This survey is designed to identify the risk factors involved in the occurrence of Foot and
Mouth Disease (FMD) in the state of Pahang. It is believed the control of FMD in beef
cattle could be optimized by identifying and reducing the risk factors for infection which
can help reduce production losses from disease outbreaks. This study is jointly sponsored
by the Department of Veterinary Services, Malaysia and Murdoch University, Australia.
The confidentiality of any information given by respondents in this study will be
maintained throughout the course of the study.
__________________________________________________________________________
Farm No.: District: Farm Location: Date:
Instructions: Please respond by ticking or circling the appropriate answer or by
writing the answer in the allocated area.
MANAGEMENT
1. What is the total number of cattle present on your farm? ……………….
2. How many herds are run on your farm? ……………….
3. How many animals are there in one herd? …………….…
4. Is your farm registered with the State Department of Veterinary Service (DVS)?
Yes No
132
5. What breed(s) of cattle are present on your farm?
Kedah Kelantan
Nellore
Cross breed
Other: Please specify ………………
6. What type of grazing management is practiced in your herd?
Kept in barn and fed
Free grazing for a fixed time
Free grazing all the time
Other: Please Specify …………………….
7. If the cattle are able to graze, what type of grazing system(s) are adopted for the
herd?
No restriction (free area)
Systematic rotational grazing (the cattle are moved
daily to graze, electric fencing is used to confine the cattle)
The cattle are allowed to graze in particular
areas restricted by fencing.
133
8. For each of the following groups are the cattle separated according to their age
groups?
Weaned males
Heifers
Female Breeders
Bulls
Cows with calves
9. Are the cattle separated according to gender (males, females)?
Yes No
10. Are the cattle fed any feed supplement?
Yes No
11. If yes, what type of feed supplement is used?
Palm Kernel Cake (PKC)
Concentrate
Other: Please Specify ……………..
12. If a feed supplement is used, is it specially made or purchased?
Made
Purchased
Both
134
13. If the feed supplement is purchased where do you buy it from?
Feed distributor
Pet shops
Other: Please specify …………….
14. What is the source of water for the herd?
Tap water
River water
Other: Please specify …………….
15. Approximately how far is your herd from the nearest cattle herd or other
livestock, such as buffalo, goats, sheep or pigs?
<2Km
2 – 4Km
>4Km
16. Did you introduce any new animals to your farm in the last year?
Yes No
17. If yes how many animals did you introduce last year? …………….
135
18. If animals were introduced how many times did you introduce new animals to
your farm over the last year? ………………..
19. Over the last year have you introduced any bulls to your herd for breeding
purposes?
Yes No
20. If yes when was the last date of introduction? ………………..
21. Have you purchased new replacement animals in the last year?
Yes No
22. If you purchased replacement animals, how many did you purchase in the last
year? …………….…..
23. Where did you buy/obtain your replacement stock from?
From traders
From a known farm
Other: Please Specify ……………..
24. If you buy from traders, do you know where the traders get their animals from?
…………………………
25. Do you buy animals from other Malaysian states or other countries?
Yes No
26. If yes, please specify the name of the state/country?
………………………
136
27. Do you exchange livestock with friends, relatives or other people?
Yes No
28. Do you examine/check the animals before you buy them?
Yes No
29. How many times in the last year have you sold cattle?
..……………………...
30. If you sold cattle how many cattle did you sell in the last year?
……………………….
31. Is a blood screening test for Foot and Mouth Disease performed prior to
movement of cattle in and out of your herd?
Yes
No
Don‟t know
32. If you test animals against FMD, what do you do with any FMD test positive
cattle?
Culled or Slaughtered
Sent to another farm
Isolated on the same farm but confined to
an isolation area
Remain in the herd
137
BIOSECURITY
33. Do you undertake any quarantine procedures before you introduce new cattle to
your herd?
Yes No
34. If yes, please specify ………………………………………..
35. Is the herd confined to an area surrounded by a fence?
Yes No
36. Can the public or any unauthorized personnel enter the herd area?
Yes No
37. Is there a dip/spray for disinfectant at the entrance to the farm?
Yes No
38. If yes, what type of disinfectant is used? ……………………………….
39. Do you allow unauthorized vehicles to enter your farm?
Yes No
40. Are vehicles sprayed with disinfectant either on entering or leaving your farm?
Yes No
41. If you use a vehicle dip is it covered from rain and sunlight?
Yes No
138
HERD HEALTH PROGRAM
42. Are animals vaccinated against Foot and Mouth Disease (FMD) in your herd?
Yes No
43. If vaccination is used, when was your herd first vaccinated against FMD?
Date………………
44. If vaccination is used, when was the most recent vaccine against FMD given?
Date………………
45. If vaccination is used, what type (brand) of FMD Vaccine was used most
recently in your herd? ……………………
46. Do you know the strain (s) of the FMD virus in the vaccine?
Yes No
47. If yes, what FMD strain/types are included in the vaccine used
…..…………
48. How many cattle were vaccinated at the most recent vaccinations
…….……….
49. How many times your animals are vaccinated each year?
….………….
139
GENERAL
50. Has your herd previously been infected with FMD?
Yes No
51. Do you know what causes FMD?
Yes No
52. If yes please explain. ………………………………………………….
53. Do you know the clinical signs of FMD?
Yes No
54. If yes, please describe these signs. ………………………………………
55. Do you know how to control FMD?
Yes No
56. If yes, please explain how you think the disease should be controlled.
……………………………………………………….
……………………………………………………….
140
Thank you for taking your time to complete this questionnaire.
Jamaliah Senawi
Research Masters Candidate, Murdoch University/ DVS Veterinary Officer,
Supervisor
Dr Ian Robertson BVSc, PhD, MACVSc (Epidemiology)
Professor in Veterinary Epidemiology
Head of Department of Veterinary Clinical Science
School of Veterinary and Biomedical Sciences
Murdoch University
Murdoch 6150
Western Australia
AUSTRALIA
Phone 61 08 93602459 Fax 61 08 93107495
141
REFERENCES
Abila, R.C., Foreman, S., 2006. Control of foot and mouth disease in Southeast Asia.
In: Proceedings of the 11th International Symposium on Veterinary
Epidemiology and Economics. 11, 1103-1105.
Adam, K.H., Marquardt, O., 2002. Differentiation of type A, Asia1 and O foot-and-
mouth disease virus variants, amplified by the same system, by sequencing of
the capsid protein genes. Journal of Virological Methods 104, 117-123.
Aggarwal, N., Zhang, Z., Cox, S., Statham, R., Alexandersen, S., Kitching, R.P.,
Barnett, P.V., 2002. Experimental studies with foot-and-mouth disease virus,
strain O, responsible for the 2001 epidemic in the United Kingdom. Vaccine
20, 2508-2515.
Ahmad, F., 2001. Sustainable Agriculture System in Malaysia. Regional Workshop
on Integrated Plant Nutrition System (IPNS), , Development in Rural Poverty
Alleviation, . http://www.Imorganicfertilizer.com (Accessed on 14th Nov
2008), United Nations Conference Complex, Bangkok,Thailand (18-20
September 2001).
Al-Majali, A.M., Jawasreh, K., Al Nsour, A., 2008. Epidemiological studies on foot
and mouth disease and paratuberculosis in small ruminants in Tafelah and
Ma‟an, Jordan. Small Ruminant Research 78, 197-201.
Alexandersen, S., 2002. Aspects of the persistence of foot and mouth disease virus in
animals the carrier problem Microbes and Infection 4, 1099-1110.
Alexandersen, S., Donaldson, A.I., 2002. Further studies to quantify the dose of
natural aerosols of foot-and-mouth disease virus for pigs. Epidemiology and
Infection, 128, 313-323
Alexandersen, S., Kitching, R.P., Mansley, L.M., Donaldson, A., 2003a. Clinical and
laboratory investigations of five outbreaks of foot-and-mouth disease during
the 2001 epidemic in the United Kingdom. Veterinary Record 152, 489-496.
Alexandersen, S., Quan, M., Murphy, C., Knight, J., Zhang, Z., 2003b. Studies of
quantitative parameters of virus excretion and transmission in pigs and cattle
experimentally infected with foot-and-mouth disease virus. Journal of
Comparative Pathology 129, 268-282.
Alexandersen, S., Zhang, Z., Donaldson, A.I., Garland, A.J., 2003c. The
pathogenesis and diagnosis of foot and mouth disease. Journal of
Comparative Pathology.129, 268-284.
AlKhamis, M.A., Perez, A.M., Yadin, H., Knowles, N.J., 2009. Temporospatial
Clustering of Foot-and-Mouth Disease Outbreaks in Israel and Palestine,
2006–2007. Transboundary and Emerging Diseases 56, 99–107.
142
Amass, S.F., Mason, P.W., Pacheco, J.M., Miller, C.A., Ramirez, A., Clark, L.K.,
Ragland, D., Schneider, J.L., Kenyon, S.J., 2004. Procedures for preventing
transmission of foot-and-mouth disease virus (O/TAW/97) by people.
Veterinary Microbiology 103, 143-149.
Amass, S.F., Pacheco, J.M., Mason, R.W., Schneider, J.L., Alvares, R.M., Clark,
L.K., Ragland, D., 2003. Procedures for preventing the transmission of foot-
and-mouth disease virus to pigs and sheep by personnel in contact with
infected pigs. Veterinary Record 153, 137-140.
Anderson, E.C., 1981. Role of wildlife in the epidemiology of foot-and-mouth
disease in Kenya. In, Wildlife disease research and economic development.
Proceedings of a workshop held in Kabete, Kenya, (8 and 9 September
1980), Kabete, Kenya, pp. 16-18.
Anderson, E.C., Daughty, W.J., Andersen, J., 1974. The effect of repeated
vaccination in an enzootic foot and mouth disease area on the incidence of
virus carrier cattle. Journal of Hygiene 73, 229-235.
Anderson, E.C., Foggin, C., Atkinson, M., Sorensen, K.J., Madekurozwa, R.L.,
Nqindi, J., 1993. The role of wild animals other than buffalo, in the current
epidemiology of foot and mouth disease in Zimbabwe. Epidemiology and
Infection 111, 559-563.
Anonymous, 2000. Clinical Disease and Diagnosis - differential diagnosis.
http://www.fao.org/ag/aga/agah/empres/gemp/avis/a010-fmd/mod0/0212-
differential-diagnosis.html (accessed on 22nd August, 2007).
Anonymous, 2007. Profil Daerah Negari Pahang. In : Jabatan Perancang Bandar
dan Desa Negeri Pahang Darul Makmur http://jpbd.pahang.gov.my
(Accessed on 28 March 2009), Kuantan, pp. 1-29.
Anonymous, 2008. IAH celebrates 50 years as the World Reference Laboratory for
FMD. Veterinary Record, 162, 667-668.
Arnold, M.E., Paton, D.J., Ryan, E., Cox, S.J., Wilesmith, J.W., 2008. Modelling
studies to estimate the prevalence of foot-and-mouth disease carriers after
reactive vaccination. Proceedings of the Royal Society of London. Series B,
Biological Sciences 275, 107-115.
Azmie, M.Z., 2006. Gading Bersilang. Jabatan Perkhidmatan Haiwan Negeri Pahang
Kuantan.
Azmie, M.Z., Mohd, Z., Bakar, J., Abidin, A.Z., 2006. Managing animal movement
in the control of FMD. In, Veterinary Association Malaysia The Mines, Seri
Kembangan.
Babjee, S.M., 1994. History, Development and Prospects of the Animal Industry and
Veterinary Services in Malaysia. Department of Veterinary Services, Ministry
of Agriculture Malaysia Kuala Lumpur.
143
Bablanian, G.M., Grubman, M.J., 1993. Characterization of the foot-and-mouth
disease virus 3C protease expressed in Escherichia coli. Virology 197, 320-
327.
Bachrach, H.L., 1968. Foot-and-mouth disease. Annual Review of Microbiology 22,
201-244.
Bangs, L.B., 1988 Latex agglutination tests. American Clinical Laboratory News 7,
20-26.
Barman, N.N., Sarma, D.K., Das, S., Patgiri, G.P., 1999. Foot-and-mouth disease in
wild and semi-domesticated animals of the north-eastern states of India.
Indian Journal of Animal Sciences 69, 781-783.
Barnett, P.V., Carabin, H., 2002. A review of emergency foot and mouth disease
vaccine. Vaccine 20, 1505-1514.
Barnett, P.V., Cox, S.J., 1999. The Role of Small Ruminants in the Epidemiology
and Transmission of Foot-and-Mouth Disease. The Veterinary Journal 158,
6-13.
Barnett, P.V., Keel, P., Reid, S., Armstrong, R.M., Statham, R.J., Voyce, C.,
Aggarwal, N., Cox, S.J., 2004. Evidence that high potency foot-and-mouth
disease vaccine inhibits local virus replication and prevents the „carrier‟ state
in sheep. Vaccine 22, 1221-1232.
Barnett, P.V., Samuel, A.R., Statham, R.J., 2001. The suitability of the „emergency‟
foot-and-mouth disease antigens held by the International Vaccine Bank
within a global context. Vaccine 19, 2107-2117.
Barros, J.J.F., Malirat, V., Rebello, M.A., Costa, E.V., Bergmann, I.E., 2007.
Genetic variation of foot-and-mouth disease virus isolates recovered from
persistently infected water buffalo (Bubalus bubalis). Veterinary
Microbiology 120, 50-62.
Barteling, S.J., 2002. Development and performance of inactivated vaccines against
foot and mouth disease. Revue scientifique et technique (International Office
of Epizootics) 21, 577-588.
Bartley, L.M., Donnelly, R.M., Anderson, R.M., 2002. Review of foot and mouth
disease virus survival in animal excretions and on fomites. Veterinary Record
151, 667-669.
Bastos, A.D.S., Boshoff, C.I., Keet, D.F., Bengis, R.G., Thomson, G.R., 2000.
Natural transmission of foot-and-mouth disease virus between African
buffalo (Syncerus caffer) and impala (Aepyceros melampus) in the Kruger
National Park, South Africa. Epidemiology and Infection 124, 591-598.
144
Bates, T.W., Carpenter, T.E., Thurmond, M.C., 2003. Benefit-cost analysis of
vaccination and preemptive slaughter as a means of eradicating foot-and-
mouth disease. American Journal of Veterinary Research 64, 805-812.
Bedson, S.P., Maitland, H.B. and Burbury, Y.M. (1927) Further observations of foot
and mouth disease. Cited by Bartley, L.M., Donnelly, C.A. and Anderson,
R.M. (2002) Review of foot and mouth disease virus survival in animal
excretions and on fomites,151, 667-669. Journal of Comparative Pathology
and Therapeutics 40, 5-55.
Belsham, G.J., Martinez-Salas, E., 2004. Genome organisation, translation and
replication of foot and mouth disease virus RNA. In: Sobrino, F., Domingo,
E. (Eds.), Foot and mouth disease current perspectives. Horizon Bioscience,
England, pp. 19-52.
Berger, H.G., Straub, O.C., Ahl, R., Tesar, M., Marquardt, O., 1990. Identification of
foot-and-mouth disease virus replication in vaccinated cattle by antibodies to
non-structural virus proteins. Vaccine 8, 213 -216.
Bergmann, I.E., Malirat, V., Neitzert, E., Beck, E., Panizzutti, N., Sanchez, C.,
Falczuk, A., 2000. Improvement of a serodiagnostic strategy for foot-and-
mouth disease virus surveillance in cattle under systematic vaccination: a
combined system of an indirect ELISA-3ABC with an enzyme-linked
immunoelectrotransfer blot assay. Archives of Virology 145, 473-489.
Bjerkedal, T., Bakketeig, L.S., 1975. Surveillance of congenital malformations and
other condition of the newborn. International Journal of Epidemiology 4, 31-
36.
Blackwell, J.H., Hyde, J.L., 1976. Effect of heat on foot and mouth disease virus in
components of milk from FMDV infected cows. Journal of Hygiene 77, 77-
83.
Bouma, A., Eble, P., Rooij, E.v., Bianchi, A., Dekker, A., 2001. The epidemic of
foot and mouth disease in the Netharlands in 2001: Laboratory examinations
In : Report of the Session of the Research Group of the Standing Technical
Committee of EUFMD
http://www.fao.org/ag/aga/agah/eufmd/reports/rg2001im/app06.pdf.,
Research Group of the Standing Technical Committee of EUFMD. Island of
Moen, Denmark.
Bouma, A., Elbers, A.R.W., Dekker, A., Koeijer de, A., Bartels, C., Vellema, P.,
Wall van der, P., Rooji van, E.M.A., Pluimers, F.H., Jong de, M.C.M., 2003.
The Foot and Mouth Disease epidemic in The Netherlands in 2001,.
Preventive Veterinary Medicine 57, 155-166.
Boyle, D.B., Taylor, T., Cardoso, M., 2004. Implementation in Australia of
molecular diagnostic techniques for the rapid detection of foot and mouth
disease virus. Australian Veterinary Journal 42, 421-425.
145
Bressel, P.R., Shaw, D.J., Savill, N.J., Woolhouse, M.E.J., 2010. Estimating risk
factors for farm-level transmission of disease: Foot and mouth disease during
the 2001 epidemic in Great Britain. Epidemics 2, 109-115.
Brocchi, E., Bergmann, I.E., Dekker, A., Paton, D.J., Sammine, D.J., Greiner, M.,
Grazioli, S., De Simone, F., Yadin, H., Haas, Bulut, N., Malirat, V., Neitzert,
E., Goris, N., Parida, S., Sørensen, K., De Clercq, K., 2006. Comparative
evaluation of six ELISAs for the detection of antibodies to the non-structural
proteins of foot-and-mouth disease virus. Vaccine 24, 6966 - 6979.
Brown, F., 2004. Foot and mouth disease current perspectives. Horizon Bioscience
Norfolk, England.
Bruderer, U., Swamb, H., Haas, B., Visser, N., Brocchi, E., Grazioli, S.,
Esterhuysen, J.J., Vosloo, W., Forsyth, M., Aggarwal, N., Cox, S.,
Armstrong, R., Anderson, J., 2004. Differentiating infection from vaccination
in foot-and-mouth-disease: evaluation of an ELISA based on recombinant
3ABC. Veterinary Microbiology 101, 187-197.
Burrows, R., 1966. Studies on the carrier state of cattle exposed to FMDV. Journal
of Hygine Cambridge 64, 81-90.
Burrows, R., Mann, J.A., Greig, A., Chapman, W.G., Goodridge, D., 1971. The
growth and persistence of foot and mouth disease virus in the bovine
mammary gland. Journal of Hygiene 69, 307-321.
Caballero, P.H., Gonzalez, S., Orue, P.M., Vergera, N.N., 1997. Comparism of
complement fixation and ELISA for diagnosis of foot and mouth disease In :
Diagnosis and Epidemiology of Animal Diseases in Latin America. In :
Proceedings of the Final Research Co-ordination Meetings of
FAO/IAEA/SIDA Co-ordinated Research Programmes entitled
“Immunoassay Methods for the Diagnosis and Epidemiology of Animal
Diseases in Latin America”
http://www.iaea.org/programmes/nafa/d3/public/caballero-partb-1055.pdf
Carpenter, T.E., 2001. Method to investigate spatial and temporal clustering in
Veterinary Epidemiology. Preventive Veterinary Medicine 48, 303-320.
Cassagne, M.H., 2002. Managing compensation for economic losses in areas
surrounding foot and mouth disease outbreaks: the response of France. Revue
scientifique et technique (International Office of Epizootics) 21, 823-829.
Chen, S.P., 2008. Epidemiology, pathogenesis and surveillance of the pig adapted
strain of foot and mouth disease in Taiwan. Epidemiology phD thesis.
Murdoch University, Perth, p. 215.
Chenard, G., Miedema, K., Moonen, P., Schrijver, R.S., Dekker, A., 2003. A solid-
phase blocking ELISA for detection of type O foot-and-mouth disease virus
antibodies suitable for mass serology. Journal of Virological Methods 107,
89-98.
146
Chong, S.K., 1979. Control of foot and mouth disease: slaughter or vaccination.,
Annual Conference 24th Malaysian Veterinary Association. Kuala Lumpur.
Clarke, J.B., Spier, R.E., 1980. Variation in the susceptibility of BHK populations
and cloned cell lines to three strains of foot-and-mouth disease virus. .
Archive of Virology 63, 1-9.
Clavijo, A., Wright, P., Kitching, P., 2004. Developments in diagnostic techniques
for differentiating infection from vaccination in foot-and-mouth disease.
Veterinary Journal 167, 9-22.
Cleland, P.C., Baldock, F.C., Chamnanpood, P., Gleeson, L.J., 1996. Village level
risk factors for foot and mouth disease in nothern Thailand. Preventive
Veterinary Medicine 26, 253-261.
Cleland, P.C., Chamnanpood, P., Baldock, F.C., Gleeson, L.J., 1995. An
investigation of 11 outbreaks of foot and mouth disease in villages in nothern
Thailand. Preventive Veterinary Medicine 22, 293-302.
Coetzer, J.A.W., Thomson, G.R., Tustin, R.C., 1994. Foot and Mouth Disease In:
Coetzer, J.A.W., Thomsen, G.R., R.C., T., Kriek, N.P.J. (Eds.), In: Infectious
Diseases of Livestock with Special Reference to Southern Africa. Oxford
Univ Press, Cape Town, pp. 825-852.
Cottam, E.M., Baud, G.l., Wadsworth, J., Gloster, J., Mansley, L., Paton, D.J., King,
D.P., Haydon, D.T., 2008. Integrating genetic and epidemiological data to
determine transmission pathways of foot-and-mouth disease virus.
Proceedings of the Royal Society of London. Series B, Biological Sciences
275, 887-895.
Cottral, G.E., 1969. Persistence of foot-and-mouth disease virus in animals, their
products and the environment. Bulletin de l'Office International des
Epizooties 71, 549-568.
Cox, S.J., Barnett, P.V., Dani, P., Salt, J.S., 1999. Emergency vaccination of sheep
against foot-and-mouth disease: protection against disease and reduction in
contact transmission. Veterinary Research Communication 17, 1858-1867.
Cox, S.J., Parida, S., Voyce, C., Reid, S.M., Hamblin, P.A., Hutchings, G.H., Paton,
D.J., Barnett, P.V., 2007. Further evaluation of higher potency vaccines for
early protection of cattle against FMDV direct contact challenge. Vaccine 25,
7687-7695.
Cox, S.J., Voyce, C., Parida, S., Reid, S.M., Hamblin, P.A., Paton, D.J., Barnett,
P.V., 2005. Protection against direct-contact challenge following emergency
FMD vaccination of cattle and the effect on virus excretion from the
oropharynx. Vaccine 23, 1106-1113.
147
De Clercq, K., 2003b. Foot-and-Mouth disease virus detection based on a diagnostic
method or an integrated quality controlled strategy. In: Session of the
Research Group of the Standing Technical Committee of EUFMD Gerzensee,
Berne, Switzerland 16-19 September 2003.
http://www.fao.org/AG/againfo/commissions/es/documents/reports/switz/Ap
p14-Kris.pdf In: Session of the Research Group of the Standing Technical
Committee of EUFMD Berne, Switzerland.
Dekker, A., Sammin, D.J., Greiner, M., Bergmann, I.E., Paton, D., Grazioli, S.,
Clercq, d.K., Brocchi, E., 2008. Use of continuous results to compare
ELISAs for the detection of antibodies to non-structural proteins of foot-and-
mouth disease virus. Vaccine 26, 2723 - 2732.
Diego, D.M., Brocchi, E., Mackay, D., De Simone, F., 1997. The non-structural
polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in
ELISA to differentiate infected from vaccinated cattle. Archive of Virology
142, 2021 - 2033.
Doel, T.R., 1999. Optimisation of the immune response to foot-and-mouth disease
vaccines. Vaccine 17, 1767-1771.
Doel, T.R., 2003. FMD vaccines. Virus Research 91, 87-99.
Doel, T.R., Williams, L., Barnett, P.V., 1994. Emergency vaccination against foot-
and-mouth disease: Rate of development of immunity and its implications for
the carrier state. Vaccine 12, 592-600.
Dohoo, I., Martin, W., Stryhn, H., 2003. Veterinary Epidemiologic Research. AVC
Inc. Prince Edward Island, Canada.
Domingo, E., Ruiz-Jarabo, C.M., Arias, A., Garcia, A.J., Escarmis, C., 2004.
Quasispecies Dynamics and Evolution of foot and mouth disease virus. In:
Sobrino, F., Domingo, E. (Eds.), Foot and mouth disease current
perspectives. Horizon bioscience, Norfolk, England, pp. 261-304.
Donaldson, A., 2007. FMD control measures. Veterinary Record 160, 135-136.
Donaldson, A., Alexandersen, S., Sorensen, J.H., Mikkelsen, T., 2001. Relative risks
of the uncontrollable (airborne) spread of FMD by different species.
Veterinary Record 148, 602-604.
Donaldson, A.I., 1987. Investigations to determine the minimum aerosol doses of
foot-and-mouth disease virus to infect sheep and cattle In, In: Aerosols. Their
generation, behaviour and applications, Loughborough University of
Technology, 31st March-1st April 1987., pp. 121-123.
Donaldson, A.I., Ferris, N.P., 1975. The survival of foot and mouth disease virus in
open air condition. Journal of Hygiene Cambridge. 74.409-416.
148
Donaldson, A.I., Gibson, C.F., Oliver, R., Hamblin, C., Kitching, R.P., 1987.
Infection of cattle by airborne foot-and-mouth disease virus: minimal doses
with O1 and SAT 2 strains. Research in Veterinary Science 43, 339-346.
Donaldson, A.I., Herniman, K.A.J., Parker, J., Sellers, R.F., 1970. Further
investigations on the airborne excretion of foot and mouth disease virus.
Journal of Hygine 68, 557-564.
Donaldson, A.I., Sellers, R.F., 2000. Foot-and-mouth disease In: Martin, W.B.,
Aitken, I.D. (Eds.), Diseases of sheep. Blackwell Science, Oxford., United
Kingdom, pp. 254-258.
Dutta, P.K., Sarma, G., Das., S.K., 1983. Foot-and-mouth disease in 256 Indian
buffaloes. Veterinary Record 65,113-114.
DVS, 1995. Report on foot and mouth diagnostic in Malaysia Department of
Veterinary Services, Malaysia.
DVS, 1996. Foot and mouth disease diagnostic service in Malaysia. Regional
Veterinary Laboratory, Kota Bahru, pp. 1-12.
DVS, 2002. Protokol Kawalan Penyakit Haiwan Kebangsaan. Department of
Veterinary Services, Malaysia Putrajaya.
DVS, 2004. "Perintah Kawasan Kawalan Penyakit Kuku dan Mulut Negeri Pahang".
In: Malaysia, Jabatan Perhkidmatan Haiwan Negeri Pahang. (Ed.)
Depertment of Veterinary Services, Malaysia, Kuantan, Pahang, Malaysia.
DVS, 2007. Penyakit Kuku dan Mulut In: Hassan, N. (Ed.), In: Laporan Tahunan
Jabatan Perkhidmatan Haiwan Negeri Pahang. Department of Veterinary
Services Pahang, Kuantan.
DVS, 2008. Fmd laboratory data, In: Regional Veterinary Laboratory report.
Department of Veterinary Services, Malaysia, Kuantan.
DVS, 2009a. Mesyuarat Kawalan Penyakit Kebangsaan. In: Department of
Veterinary Services, M. (Ed.) Department of Veterinary Services Putrajaya.
DVS, 2009b. "Populasi ternakan di Malaysia". In: "Status Pembangunan Industri
Ternakan". Department of Veterinary Services Malaysia, Putrajaya.
Edwards, J.R., 2004. Strategy for the control of foot-and-mouth disease in Southeast
Asia (SEAFMD). Developments in Biologicals (Basel) 119, 423-431.
El-Hakim, U.A., 2005. Differentiation between foot and mouth disease virus infected
and vaccinated cattle using recent techniques. Assiut Veterinary Medical
Journal 51, 173-188.
Espinoza, A.M., Maradei, E., Mattion, N., Cadenazzi, G., Maddonni, G., Robiolo,
B., 2004. Fot and mouth disease polyvalent oil vaccines inoculated
149
repeatedly in cattle do not induced detectable antibodies to non structural
proteins when evaluated by various assays. Vaccine 23, 69-77.
Ferguson, N.M., Donnelly, C.A., Anderson, R.M., 2001a. The foot and mouth
disease epidemic in Great Britain: Pattern of spread and impact of
interventions. Science 292, 1155-1159.
Ferguson, N.M., Donnelly, C.A., Anderson, R.M., 2001b. Transmission intensity
and impact of control policies on the foot and mouth epidemic in Great
Britain. Nature 413, 542-549.
Ferris, N.P., Nordengrahn, A., Hutchings, G.H., Reid, S.M., King, D.P., Ebert, K.,
Paton, D.J., Kristersson, T., Brocchi, E., Grazioli, S., Merza, M., 2009.
Development and laboratory validation of a lateral flow device for the
detection of foot-and-mouth disease virus in clinical samples. Journal of
Virological Methods 155, 10-17.
Field, A., 2009. Discovering statistics using SPSS. SAGE Publications Ltd. London.
Fogedby, E., 1958. The control of foot-and-mouth disease in Europe. In, In: Paper
presented at the International Symposium on Virology 27th-29th June, 1958.,
p. 3.
Fukai, K., Morioka, K., Ohashi, S., Yamazoe, R., Yoshida, K., Sakamoto, K., 2008.
Differentiation of Foot-and-Mouth Disease Virus-Infected Pigs from
Vaccinated Pigs using a Western Blotting Assay Based on Baculovirus-
Expressed Nonstructural Proteins 2C and 3D. Journal of Veterinary Medicine
Science 70, 1353 - 1357.
Gabier, G., Bacro, J.N., Pouillot, R., Durand, B., Moutou, F., Chadoeuf, J., 2002. A
point pattern model of spread of foot and mouth disease. Preventive
Veterinary Medicine 56, 33-49.
Gallego, M.L., Perez, A.M., Thurmond, M.C., 2007. Temporal and spatial
distributions of foot-and-mouth disease under three different strategies of
control and eradication in Colombia (1982-2003). Veterinary Research
Communications 31, 819-834.
Garland, A.J.M., 1999. Vital elements for the successful control of foot-and-mouth
disease by vaccination. Vaccine 17, 1760-1766.
Gibbens, J.C., Wilesmith, J.W., 2002. Temporal and geographical distribution of
cases of foot and mouth disease during the early weeks of the 2001 epidemic
in Great Britain. Veterinary Record 151, 407-412.
Gloster, J., Williams, P., Doel, C., Esteves, I., Coe, H., Valarcher, J.F., 2007. Foot-
and-mouth disease – Quantification and size distribution of airborne particles
emitted by healthy and infected pigs. The Veterinary Journal 174, 42-53.
150
Gomes, I., Ramalho, A.K., Auge, D.M.P., 1997. Infectivity assays of foot and mouth
disease virus: contact transmission between cattle and buffalo (Bubalus
bubalis) in the early stage of infection. Veterinary Record 140, 43-47.
Grubman, M.J., Baxt, B., 2004. Foot-and-Mouth Disease. Clinical Microbiology
Review 17, 465-493.
Guerin, B., Pozzi, N., 2005. Viruses in boar semen: detection and clinical as well as
epidemiological consequences regarding disease transmission by artificial
insemination. Theriogenology 63, 556-572.
Hamblin, C., Armstrong, R.M., Hedger, R.S., 1984. A rapid enzyme-linked
immunosorbent assay for the detection of foot-and-mouth disease virus in
epithelial tissues. Veterinary Microbiology 9, 435 - 443.
Hamblin, C., Barnett, I.T.R., Hedger, R.S., 1986a. A new enzyme-linked
immunosorbent assay (ELISA) for the detection of antibodies against foot
and mouth diseasevirus: I. Development and method of ELISA. Journal of
immunological methods 93, 115-121.
Hamblin, C., Barnett, I.T.R., Hedger, R.S., 1986b. A new enzyme-linked
immunosorbent assay (ELISA) for the detection of antibodies against foot
and mouth diseasevirus: II. Application. Journal of immunological methods
93, 123-129.
Hamblin, C., Kitching, P., Donaldson, A., Crowther, J.R., Barnett, I.T.R., 1987.
Enzyme link immunosorbent assay (ELISA) for the detection of antibodies
against foot and mouth disease virus: III: Evaluation of antibodies after
infection and vaccination. Epidemiology and Infection 99, 733-744.
Harun, O., Chen, C.C., 1995. Better return from an integrated beef oil palm
enterprise. In, Proc. 17th. Malaysian Soc. Animal Prod. Conf., Kuala
Lumpur, pp. 30-40.
Hearps, A., Zhang, Z., Alexandersen, S., 2002. Evaluation of the portable Cepheid
SmartCycler real-time PCR machine for the rapid diagnosis of foot-and-
mouth disease. Veterinary Record 150, 625-628.
Hedger, R.S., 1970. Observation on the carrier state and related antibody titers
during an outbreak of foot and mouth disease. Journal of Hygiene 68, 53-68.
Hedger, R.S., Condy, J.B., Golding, S.M., 1972. Infection of some species of
African wild life with foot-and-mouth disease virus. Journal of Comparative
Pathology 82, 455-461.
Hedger, R.S., Condy, J.B., Gradwell, D.V., 1980. The Response of Some African
Wildlife Species to Foot and Mouth Disease Vaccination. Journal of Wildlife
Diseases 16, 431-438.
151
Henderson, W.M., Brooksby, J.B., 1948. The survival of foot and mouth disease in
meat and offal. From The Research Institute : Foot and Mouth Disease
Research committee. Pirbright Research Institute, Surrey, London, pp. 394-
402.
Hill, A.E., Ward, M.P., 2008. Logistic Regression University of Sydney, Camden
Campus, Sydney, pp. 1-31.
Hiong, G.C., 1994. Malaysia. In: Copland, J.W., Gleeson, L.J., Chamnanpood, C.
(Eds.), In: International workshop for diagnosis and epidemiology of foot
and mouth disease in Southeast Asia, Lampang, Thailand.
Huang, X., Li, Y., Zheng, C.Y., 2009. A novel single-cell quantitative real-time RT-
PCR method for quantifying foot-and-mouth disease viral RNA. Journal of
Virological Methods 155, 150-156.
Hugh-Jones, M.E., 1976. A simulation spatial model of the spread of FMD through
the primary movement of milk. Journal of Hygiene 77, 1-9.
Hugh-Jones, M.E., Tinline, R.R., 1978. Studies on the 1967-68 foot and mouth
disease epidemic: incubation period and herd serial interval. Journal of
Hygiene 77, 141-153.
Hughes, G.J., Mioulet, V., Kitching, R.P., Woolhouse, M.E.J., Alexandersen, S.,
Donaldson, A.I., 2002. Foot-and-mouth disease virus infection of sheep:
implications for diagnosis and control. Veterinary Record 150, 724-727.
Hunter, P., 1996. The performance of Southern African Territories serotypes of foot
and mouth disease antigen in oil-adjuvanted vaccine. Revue scientifique et
technique (International Office of Epizootics) 15, 913-922.
Hutber, A.M., Kitching, R.P., Fishwick, J.C., Bires, J., 2011. Foot and mouth
disease: The question of implementing vaccinal control during an epidemic.
The Veterinary Journal, 188, 18-23.
Hyslop, N., ST G., 1965a. Airborne infection with the virus of Foot and Mouth
Disease. Journal of Comparative Pathology 75, 119-126.
Hyslop, N., ST G., 1965b. Secretion of Foot and Mouth Disease Virus and antibody
in the saliva of infected and immunized cattle. Journal of Comparative
Pathology. 75, 111-117.
Jacquez, G.M., 2008. The Handbook of Geographic Information Science. Blackwell
Publishing.
Jamal Syed, M., Annemarie, B., den, B.J.v., Arjan, S., Gilles, C., Aldo, D., 2008.
Foot and mouth disease vaccine potency testing:the influence of serotype,
type of adjuvant, valency, fraction method and virus cultyre on the dose
response curve in cattle. Vaccine 26, 6317-6321.
152
James, A.D., Rushton, J., 2002. The economics of foot and mouth disease. Revue
scientifique et technique (International office of Epizootics) 21, 637-644.
Joseph, K.T., 1991. Sustaining agricultural land in Malaysia, Policies ,Prospects and
Problems. In: Abdul Aziz, S.A.K. (Ed.), Proc., Seminar on Sixth Malaysia
Plan, Agricultural Policies and Strategies, Kuala Lumpur, pp. 3-17.
Kahn, S., Geale, D.W., Kitching, P.R., Bouffard, A., Allard, D.G., Duncan, J.R.,
2002. Vaccination against foot-and-mouth disease: the implications for
Canada. Canadian Veterinary Journal 43, 349-354.
Keeling, M.J., Woolhouse, M.E.J., Shaw, D.J., Matthews, L., Topping, M.C.,
Haydon, D.T., Cornell, S.J., Kappey, J., Wilesmith, J.W., Grenfell, B.T.,
2001. Dynamic of 2001 UK epidemic: Stochastic dispersal in a heterogenous
landscape. Science 294, 813-817.
King, D.P., Dukes, J.P., Reid, S.M., Ebert, K., Shaw, A.E., Mills, C.E., Boswell, L.,
Ferris, N.P., 2008. Prospects for rapid diagnosis of foot-and-mouth disease in
the field using reverse transcriptase-PCR. Veterinary Record 162, 315-316.
Kitching, P., 2002a. Clinical variation in foot and mouth disease : cattle. Revue
scientifique et technique.(International office of Epizootics) 21, 499-504.
Kitching, P., 2002b. Identification of foot and mouth disease virus carrier and
subclinically infected animals and differentiation from vaccinated animals.
Revue scientifique et technique (International office of Epizootics) 21, 531-
538.
Kitching, P. (Ed.), 2004. Diagnosis and Control of Foot and Mouth Disease.
Horizon Bioscience, 32, Hewitts Lane, Wymondham, Norfolk NR18 0JA,
England.
Kitching, P., Hammond, J., Jeggo, M., Charleston, B., Paton, D., Rodriguez, L.,
Heckert, R., 2007. Global FMD control - is it an option? In, In: 4th
International Veterinary Vaccines and Diagnostic Conference Oslo, Norway,
25-29 June 2006., pp. 5660-5664.
Kitching, R.P., 1998. A Recent History of Foot-and-Mouth Disease. Journal of
Comparative Pathology 118, 89-108.
Kitching, R.P., Hutber, A.M., Thrusfield, M.V., 2005. A review of foot-and-mouth
disease with special consideration for the clinical and epidemiological factors
relevant to predictive modelling of the disease. The Veterinary Journal 169,
197-209.
Kittelberger, R., Mackereth, G.F., Sewell, M., Keall, J., Clough, R., Pigott, C.,
O‟Keefe, J.S., 2008. Specificity of Non Structural Protein enzyme-linked
immunosorbent assays for the detection of serum antibodies against foot-and
mouth disease virus in a target population in New Zealand. New Zealand
Veterinary Journal 56, 227-232.
153
Kulldorff, M., 1997. A Spatial scan statistic. Communication in statistic-Theory
methods. 26, 1481-1496.
Kulldorff, M., Heffernan, R., Hartman, J., Assuncao, R., Mostashari, F., 2005. A
Space–Time Permutation Scan Statistic for Disease Outbreak Detection.
PLoS Medicine 2, 216 - 224.
Kulldorff, M., Inc., I.M.S., 2009. SaTScan v 8.0 Software for the spatial and space
time scan statistics. http://www.satscan.org., (Access date;17 December
2009).
Kulldorff, M., Nagarwalla, N., 1995. Spatial disease clusters: detection and
inference. Statistics in medicine 14, 799 - 810.
Kweon, C.H., Ko, Y.J., Kim, W., II, Lee, S.Y., Nah, J.J., Lee, K.N., Sohn, H.J.,
Choi, K.S., Hyun, B.H., Kang, S.W., Joo, Y.S., Lubroth, J., 2003.
Development of a foot-and-mouth disease NSP ELISA and its comparison
with differential diagnostic methods. Vaccine 21, 1409-1414.
Lai, P.C., So, F.M., Chan, K.W., 2009. Spatial Epidemiological Approaches in
Disease Mapping and Analysis. Taylor and Francis Group LLC New York.
Lee, F., Jong, M.H., Yang, D.W., 2006. Presence of antibodies to non-structural
proteins of foot-and-mouth disease virus in repeatedly vaccinated cattle.
Veterinary Microbiology 115, 14 - 20.
Lee, J.L.M., More, S.J., Cotiwan, B.S., 1999 Problems translating a questionnaire in
a cross-cultural setting. Preventive Veterinary Medicine 41 187-194.
Leforban, Y., Gerbier, G., 2002. Review of the status of Foot and mouth disease and
approach to control/eradication in Europe and Central Asia. Revue
scientifique et technique (International office of Epizootics) 21, 477 - 492.
Lin, X.W., Chiang, C.T., Shih, T.H., Jiang, Y.N., Chou, C.C., 2009. Foot-and-Mouth
Disease Entrance Assessment Model Through Air Passenger Violations. Risk
Analysis 29, 601-612.
Lombard, M.F., Schermbrucker, C.G., 1993. Vaccine for Control of Foot-and-Mouth
Disease Worldwide: Production, Selection and Field Performance. In:
Diagnosis and Epidemiology of Foot-and-Mouth Disease in Southeast Asia.
In: Copland, J.W., Gleeson, L.J., Chanpen, C. (Eds.), ACIAR Proceedings,
Lampang, Thailand., p. 209.
Lubroth, J., Brown, F., 1995. Identification of native foot-and-mouth disease virus
non-structural protein 2C as a serological indicator to differentiate infected
from vaccinated livestock. Revue scientifique et technique. (International
office of Epizootics 59, 70 - 78.
154
Lubroth, J., Rweyemamu, M.M., Viljoen, G., Diallo, A., Dungu, B., Amanfu, W.,
2007. Veterinary vaccines and their use in developing countries. Revue
scientifique et technique. (International office of Epizootics) 26, 179 - 201.
Mackay, D.K., Bulut, A.N., Rendle, T., Davidson, F., Ferris, N.P., 2001. A solid-
phase competition ELISA for measuring antibody to foot-and-mouth disease
virus. Journal of Virological Methods 97, 33-48.
Mackay, D.K.J., Forsyth, M.A., Davies, P.R., Berlinzanij, A., Belsham, G.J., Flint,
M., Ryan, M.D., 1998. Differentiating infection from vaccination in foot-and-
mouth disease using a panel of recombinant, non-structural proteins in
ELISA. Vaccine 16, 446 - 459.
Maddur, M.S., Gopalakrishna, S., Singh, N., Suryanarayana, V.V., Gajendragad,
M.R., 2009. Immune Response and Viral Persistence in Indian Buffaloes
(Bubalus bubalis) Infected with Foot-and-Mouth Disease Virus Serotype
Asia 1. Clinical and Vaccine Immunology 16, 1832-1836.
Madhanmohan, M., Tresamol, P.V., Saseendranath, M.R., 2009. Immune Response
in Goats to Two Commercial Foot-and-Mouth Disease Vaccines and the
Assessment of Maternal Immunity in Their Kids. Transboundary and
Emerging Diseases 56, 49 - 53.
Mahy, B.W.J., 2005. Foot and mouth disease virus. Springer, New York.
Mansley, L.M., Dunlop, P.J., Whiteside, S.M., Smith, R.G.H., 2003. Early
dissemination of foot-and-mouth disease virus through sheep marketing in
February 2001. Veterinary Record 153, 43-50.
Maroudam, V., Nagendrakumar, S.B., Madhanmohan, M., Santhakumar, P.,
Thiagarajan, D., Srinivasan, V.A., 2008. Experimental transmission of foot-
and-mouth disease among Indian buffalo (Bubalus bubalis) and from buffalo
to cattle. Journal of Comparative Pathology 139, 81-85.
McColl, K.A., Westbury, H.A.K., Lewis, V.M., 1995. The persistence of foot and
mouth disease virus on wool. Australian Veterinary Journal 72, 286-292.
McLaws, M., Ribble, C., Martin, W., Wilesmith, J.W., 2009. Factors associated with
the early detection of foot-and-mouth disease during the 2001 epidemic in the
United Kingdom. Canadian Veterinary Journal 50, 53 - 60.
McVicar, J.W., Sutmoller, P., 1969. The epizootiological importance of foot-and-
mouth disease carriers II. The carrier status of cattle exposed to foot-and-
mouth disease following vaccination with an oil adjuvant inactivated virus
vaccine Archives of Virology 26, 217-224.
McVicar, J.W., Sutmoller, P., 1970. Foot and mouth disease: The agar gel diffusion
Precipitin test for antibody to virus infection - associated (via) antigen as a
tool for epizootiologic surveys. American Journal of Epidemiology 2, 273-
279.
155
Moonen, P., Linde, E.V.D., Chénard, G., Dekker, A., 2004. Comparable sensitivity
and specificity in three commercially available ELISAs to differentiate
between cattle infected with or vaccinated against foot-and-mouth disease
virus. Veterinary Microbiology 99, 93-101.
Morris, R.S., Sanson, R.L., Stern, M.W., Stevenson, M.A., Wilesmith, J.W., 2002.
Decision-support tools for foot and mouth disease control. Revue scientifique
et technique. (International office of Epizootics) 21, 557-567.
Moutou, F., 2002. Epidemiological basis useful for the control of foot-and-mouth
disease. Comparative Immunology, Microbiology and Infectious Disease 25,
321-330.
Mowat, G.N., Darbyshire, J.H., Huntley, J.F., 1972. Differentiation of a vesicular
disease of pigs in Hong Kong from foot-and-mouth disease. Veterinary
Record, 90, 618-621.
Naheed, M.H., 2007. Status of foot and mouth disease in Malaysia. In: 13 Meeting of
the OIE Sub commission of foot and mouth disease in Southeast Asia. Siem
Reep, Cambodia.
Naheed, M.H., 2008. Liquid phase blocking ELISA - Cut off point, Putrajaya,
Malaysia.
Niedbalski, W., 2004. Comparison of different ELISA methods for the detection of
antibodies against foot-and-mouth disease virus (FMDV) type O. Bulletin of
the Veterinary Institute in Pulaway 48, 5-9.
Niedbalski, W., Haas, B., 2003a. Differentiation of infection from vaccination by
detection of antibodies to the non-structural protein 3ABC of foot-and-mouth
disease virus. Bulletin of the Veterinary Institute in Pulaway 47, 51-60.
OIE, 2002. Foot and Mouth Disease ( Aetiology, Epidemiology, Diagnosis,
Prevention and Control) References Resistance to physical and chemical
action http://www.oie.int/eng/maladies/fiches/a_A010.HTM. In: OIE (Ed.)
OIE, 2004. Chapter 2.1.1. Foot and mouth disease. In: OIE Standards Commission
(Ed.), Manual of Strandards for Diagnostic Tests and vaccines. In:
Epizooties, O.I.E. (Ed.), Paris, France.
OIE, 2006. Foot and Mouth Disease In: Manual of Diagnostic Tests and Vaccines
for Terrestrial Animals 2004, Chapter 2.1.1 Updated: 15.01.2007.
http://www.oie.int/eng/normes/mmanual/A_00024.htm (accessed on April
10, 2007).
OIE, 2008. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals,
Chapter 2.1.1. Foot and Mouth Disease.
http://www.oie.int/eng/normes/mmanual/A_00024.htm (Accessed on 25th
May, 2008), Updated: 15.01.2007.
156
OIE, 2009. Foot and mouth disease, List of Foot and Mouth Disease free countries
resolution No. XVIII Recognition of the Foot and Mouth Disease Status of
Members Change in country status (Updated 20/02/2009). In: Health (Ed.),
Chapter 2.2.10. of the Terrestrial Code
http://www.oie.int/Eng/info/en_fmd.htm.
Orsel, K., Bouma, A., Dekker, A., Stegeman, J.A., De Jong, M.C.M., 2009. Foot and
mouth disease virus transmission during the incubation period of the disease
in piglets, lambs, calves and dairy cow. Preventive Veterinary Medicine 88,
158-163.
Orsel, K., de Jong, M.C.M., Boumaa, A., Stegemana, J.A., Dekker, A., 2007. The
effect of vaccination on foot and mouth disease virus transmission among
dairy cows. Vaccine 25, 327 - 335.
Orsel, K., Dekker, A., Boumaa, A., Stegemana, J.A., Jong, M.C.M., 2005.
Vaccination against foot and mouth disease reduces virus transmission in
groups of calves. Vaccine 23, 4887-4894.
Ozdural, N., Candaş, A., Cengiz, M., 2002. Diagnosis of A22 Mahmatlı and O1
Manisa FMD viruses from field samples: Development of a latex
agglutination test kit. Report of the session of the research group of the
Standing Committee of the fao-eufmd. Izmir, Turkey 17-20 September 2002
Paiba, G.A., Anderson, J., Paton, D.J., Soldan, A.W., Alexandersen, S., Corteyn, M.,
Wilsden, G., Hamblin, P., MacKay, D.K., Donaldson, A.I., 2004. Validation
of a foot-and-mouth disease antibody screening solid-phase competition
ELISA (SPCE). Journal of Virological Methods 115, 145-158.
Pallant, J., 2004. A step by step guide to data analysis using SPSS. Crows Nest,
N.S.W. Allen & Unwin Sydney.
Parida, S., Fleming, L., Oh, Y., Mahapatra, M., Hamblin, P., Gloster, J., Paton, D.J.,
2008. Emergency vaccination of sheep against foot-and-mouth disease:
Significance and detection of subsequent sub-clinical infection. Vaccine 26,
3469-3479.
Paton, D.J., Clercq, K., Greiner, M., Dekker, A., Brocchi, E., Bergmannf, I.,
Samming, D.J., Gubbins, S., Parida, S., 2006. Application of non-structural
protein antibody tests in substantiating freedom from foot-and-mouth disease
virus infection after emergency vaccination of cattle. Vaccine 24, 6503 -
6512.
Pereira, H.G., 1981. Foot-and-mouth disease. In: Gibbs, E.P.J. (Ed.), Virus Disease
of Food Animals. Academic Press Inc., London, pp. 333–363.
Perez, A.M., Ward, M.P., Carpenter, T.E., 2004. Control of a foot-and-mouth disease
epidemic in Argentina. Veterinary Record 65, 217-226.
157
Periolo, O.H., Seki, C., Grigera, P.R., Robiolo, B., Fernfindez, G., Maradei, E.,
Aloia, R.D., La Torre, J.L., 1993a. Large-scale use of liquid-phase blocking
sandwich ELISA for the evaluation of protective immunity against
aphthovirus in cattle vaccinated with oil-adjuvanted vaccines in Argentina. .
Vaccine 11, 754-760.
Perry, P.B., Kalpravidh, W., Coleman, P.G., McDermott, J.J., Randolph, T.F.,
Gleeson, L.J., 1999. The economic impact of Foot and Mouth Disease and its
control in South-East Asia: a preliminary assesment with special reference to
Thailand in the economics of animal disease control. Revue scientifique et
technique (International office of Epizootics) 18, 478-497.
Picado, A., Guitian, F.J., Pfeiffer, D.U., 2007 Space-time interaction as an indicator
of local spread during the 2001 FMD outbreak in the U.K. Preventive
Veterinary Medicine 79, 17-20.
Pinto, A.A., 2004. Foot-and-Mouth Disease in Tropical Wildlife. Annals of New
York Academic Science. 1026, 65-72.
Pyakural, S., Singh, U., Singh, N.B., 1976. An outbreak of foot-and-mouth disease in
Indian elephants (Elephas maximus). Veterinary Record 99, 28-29.
Quan, M., Murphy, C.M., Zhang, Z., Durand, S., Esteves, I., Doel, C., Alexandersen,
S., 2009. Influence of Exposure Intensity on the Efficiency and Speed of
Transmission of Foot-and-Mouth Disease. Journal of Comparative
Pathology 140, 225-237.
Radaelli, G., 1996. Detection of an unknown increase in the rate of rare event.
Journal of Applied Statistics, 23, 105-113.
Reid, S.M., Ferris, N.P., Hutchings, G.H., Clercq, K., Newman, B.J., Knowles, N.J.,
Samuel, A.R., 2001. Diagnosis of foot-and-mouth disease by RT-PCR: use of
phylogenetic data to evaluate primers for the typing of viral RNA in clinical
samples. Archives of Virology 146, 2421-2434.
Reid, S.M., Ferris, N.P., Hutchings, G.H., Zhang, Z., Belsham, G.J., Alexandersen,
S., 2002. Detection of all seven serotypes of foot-and-mouth disease virus by
real-time, fluorogenic reverse transcription polymerase chain reaction assay.
Journal of Virological Methods 105, 67-80.
Remond, M., Kaiser, C., Lebreton, F., 2002. Diagnosis and screening of foot-and-
mouth disease. Comparative Immunology, Microbiology & Infectious
Diseases 25, 309-320.
Robinson, S.E., Christley, R.M., 2007. Exploring the role of auction markets in cattle
movements within Great Britain. Preventive Veterinary Medicine 81, 21-37.
Robiolo, B., José, L., Duffyb, S., Leonb, E., Sekia, C., Adriana, T., Mattion, N.,
2010. Quantitative single serum-dilution liquid phase competitive blocking
ELISA for the assessment of herd immunity and expected protection against
158
foot-and-mouth disease virus in vaccinated cattle. Journal of Virological
Methods 166 21-27.
Robiolo, B., Seki, C., Fondevilla, N., Grigera, P., Scodeller, E., Periolo, O., Torre,
J.L., Mattion, N., 2006. Analysis of the immune response to FMDV
structural and non-structural proteins in cattle in Argentina by the combined
use of liquid phase and 3ABC-ELISA tests. Vaccine 24, 997-1008.
Rosli, A., Mokh Nasir, W.I., 1997. Environmental imteractions on livestock
integration production systems in plantations. In, In :19th. Malaysian Society
Animal Production Conference, Kuala Lumpur, Malaysia, pp. 7-12.
Rossides, S.C., 2002. A farming perspective on the 2001 foot and mouth disease
epidemic in the United Kingdom. Revue scientifique et technique.
(International office of Epizootics) 21, 831-838.
Rweyemamu, M., Roeder, P., MacKay, D., Sumption, K., Brownlie, J., Leforban, Y.,
2008a. Planning for the progressive control of foot-and-mouth disease
worldwide. Transboundary and Emerging Diseases 55, 73-87.
Rweyemamu, M., Roeder, P., Mackay, D., Sumption, K., Brownlie, J., Leforban, Y.,
Valarcher, J.F., Knowles, N.J., Saraiva, V., 2008b. Epidemiological Patterns
of Foot-and-Mouth Disease Worldwide. Transboundary and Emerging
Diseases 55, 57 - 72.
Rweyemamu, M.M., Astudillo, V.M., 2002. Global perspective for foot and mouth
disease control. Revue et scientifique technique. (International office of
Epizootics), 21, 765-773.
Sabirovic, M., Roberts, H., Hall, S., Elliott, H., Coulson, N., 2009. International
disease monitoring, October to December 2008. Veterinary Record 164, 355-
358.
Saini, S.S., Sharma, J.K., Kwatra, M.S., 1992. Assessment of some factors affecting
the prevalence of foot-and-mouth disease among traditionally managed
animal population of Punjab State. Socio-economic and animal health related
interests. Indian Journal of Animal Science. 62, 1-4.
Sáiz, M., Núñez, J.I., Jimenez-Clavero, M.A., Baranowski, E., Sobrino, F., 2002.
Foot-and-mouth disease virus: biology and prospects for disease control.
Microbes and Infection 4, 1183-1193.
Salt, J.S., 1993. The carrier state in foot and mouth disease virus. An immunological
review. British Veterinary Journal 149, 207-223.
Salt, J.S., 2004. Persistences of foot and mouth disease virus. In: Sobrino, F.,
Domingo, E. (Eds.), Foot and mouth disease current perspectives. Horizon
bioscience, England, pp. 103-143.
159
Salt, J.S., Barnett, P.V., Danit, P., Williams, L., 1998. Emergency vaccination of
pigs against foot-and-mouth disease: protection against disease and reduction
in contact transmission. Vaccine 16, 746-755.
Samara, S.I., Pinto, A.A., 1983. Detection of foot-and-mouth disease carriers among
water buffalo (Bubalus bubalis) after an outbreak of the disease in cattle.
Veterinary Record 113, 472-473.
Saraiva, V., 2004. Foot-and-Mouth Disease in the Americas Epidemiology and
Ecologic Changes Affecting Distribution. Annals New York of Academic
Science 1024, 73-79.
Sasaki, M., 1994. Patterns of National and International Livestock Movement in
Southeast Asia: Implication for a Regional Foot and Mouth Disease Control
Program. In: International Workshop 6 Sep 1993. Australian Centre for
International Agricultural Research, Lampang (Thailand), pp. 75-79.
Schijven, J., Rijs, G.B.J., Husman, A.M.R., 2005. Quantitative Risk Assessment of
FMD Virus Transmission via Water. Risk Analysis 25, 13-25.
Schley, D., Paton, D.J., Cox, S.J., Parida, S., Gubbins, S., 2009. The effect of
vaccination on undetected persistence of foot and mouth disease in cattle
herd and sheep flock. Epidemiology and infection, 137, 1494-1504.
Scott, A., Zepeda, C., Garber, L., Smith, J., Swayne, D., Rhorer, A., Kellar, J.,
Shimshony, A., Batho, H., Caporale, V., Giovannini, A., 2005. Concept of
compartmentalisation. (Ed.), In: OIE Ad hoc Group on Epidemiology. pp. 61-
67.
Scudamore, J.M., 2004. FMD – differentiating vaccinated from infected animals.
The Veterinary Journal 167, 3-4.
Scudamore, J.M., Harris, D.M., 2002. Control of foot and mouth disease: lessons
from the experience of the outbreak in Great Britain in 2001. Revue
scientifique et technique. ( International office of Epizootics) 21, 699 - 710.
Sellers, R.F., 1971. Quantitative aspects of the spread of foot-and-mouth disease.
Veterinary Bulletin 41, 431-439.
Sellers, R.F., Donaldson, A.I., Herniman, K.A.J., 1970. Inhalation, persistence,
dispersals of foot and mouth disease in man. Journal of Hygiene, Cambridge
68, 565-573.
Sellers, R.F., Parker, J., 1969. Airborne excretion of Foot and mouth disease.
Journal of Hygiene, Cambridge, 67, 671-677.
Shiilegdamba, E., Carpenter, T.E., Perez, A.M., Thurmond, M.C., 2008. Temporal-
spatial epidemiology of foot-and-mouth disease outbreaks in Mongolia,
2000-2002. Veterinary Research Communications 32, 201-207.
160
Smitsaart, E., Maradei, E., Fernandez, E., Fondevila, N., Compaired, D., 1997. Field
Assessment of the Enzyme Linked Immunosorbent Assay for Foot and
Mouth Disease Virus diagnosis and typing. In : Proceedings of the Final
Research Co-ordination Meetings of FAO/IAEA/SIDA Co-ordinated
Research Programmes entitled “Immunoassay Methods for the Diagnosis and
Epidemiology of Animal Diseases in Latin
America”http://www.iaea.org/programmes/nafa/d3/public/smitsaart-partb-
1055.pdf
Snowdon, W.A., 1966. Growth of Foot and Mouth Disease Virus in Monolayer
Cultures of Calf Thyroid Cells. Nature, 210, 1079-1080.
Sorensen, K.J., De Stricker, K., Dyrting, K.C., Grazioli, S., Haas, B., 2005.
Differentiation of foot-and-mouth disease virus infected animals from
vaccinated animals using a blocking ELISA based on baculovirus expressed
FMDV 3ABC antigen and a 3ABC monoclonal antibody. Archives of
Virology 150, 805-814.
Sorensen, K.J., Madekurozwa, R.L., Dawe, P., 1992. Foot-and-mouth disease:
detection of antibodies in cattle sera by blocking ELISA. Veterinary
Microbiology 32, 253 - 265.
Sørensen, K.J., Madsen, K.G., Madsen, E.S., Salt, J.S., Nqindi, J., Mackay, D.K.J.,
1998. Differentiation of infection from vaccination in foot-and-mouth disease
by the detection of antibodies to the non-structural proteins 3D, 3AB and
3ABC in ELISA using antigens expressed in baculovirus. Archives of
Virology 143, 1461 - 1476.
Sutmoller, P., Barteling, S.S., Olascoaga, R.C., Sumption, K.J., 2003. Control and
eradication of foot-and-mouth disease. Virus Research 91, 101-144.
Sutmoller, P., Olascoaga, R.C., 2002. Unapparent foot and mouth disease infection
(sub-clinical infections and carriers): implications for control. Revue
scientifique et technique. (International office of Epizootics) 21, 519-529.
Thomson, G.R., Bastos, A.D.S., 2004. Foot-and-mouth disease. Oxford UK.
Thornley, J.H.M., France, J., 2009. Modelling foot and mouth disease. Preventive
Veterinary Medicine 89, 139-154.
Thrusfield, M.V, 2005. Veterinary Epidemiology.3rd
Ed, Blackwell Publishing
Oxford.
Thuraisingham, S., 1977. The control and eradication of foot and mouth disease in
Malaysia. In, In: Association of Veterinary Surgeons, Malaysia and
Australian Association of Cattle Veterinarians, Kuala Lumpur, pp. 122-132.
Tildesley, M.J., Keeling, M.J., 2008. Modelling foot-and-mouth disease: a
comparison between the UK and Denmark. Preventive Veterinary Medicine
85, 107-124.
161
Tildesley, M.J., Savill, N.J., Shaw, D.J., Dearon, R., Brooks, S.P., Woolhouse,
M.E.J., Grenfell, B.T., Keeling, M.J., 2006. Optimal reactive vaccination
strategies for a foot and mouth disease outbreak in the UK. Nature 440, 83-
87.
Vosloo, W., Bastos, A.D., Kirkbride, E., Esterhuysen, J.J., Janse van Rensburg, D.,
Bengis, R.G., Keet, D.W., Thomson, G.R., 1996. Persistent infection of
African buffalo (Syncerus caffer) with SAT-type foot-and-mouth disease
viruses: rate of fixation of mutations, antigenic change and interspecies
transmission. Journal of General Virology 77, 157-167.
Vosloo, W., Boshoff, K., Dwarka, R., Bastos, A., 2002. The possible role that
buffalo played in the recent outbreak of foot and mouth disease in South
Africa. Annals New York Academy of Sciences 969, 187-190.
Vosloo, W., Thompson, P.N., Botha, B., Bengis, R.G., Thomson, G.R., 2009.
Longitudinal Study to Investigate the Role of Impala (Aepyceros melampus)
in Foot-and-Mouth Disease Maintenance in the Kruger National Park, South
Africa. Transboundary and Emerging Diseases 56, 18-30.
Wallace, W.R., 1936. Foot ans Mouth Disease situation, In: Annual report of the
veterinary department S.S. and F.M.S. for the state. In: Department of
Veterinary, M. (Ed.) Federated Malay States Government Presss. Kuala
Lumpur
Wallace, W.R., 1939. Report on the Veterinary Departments, Malaya. In:
Department of Veterinary, M. (Ed.) Federated Malay States Government
Presss, Kuala Lumpur.
Wallenstein, S., 1980. A test for detection of clustering over time. American Journal
of Epidemiology 3, 367-372.
Ward, M.P., Highfield, L.D., Vongseng, P., Garner, M.G., 2009. Simulation of foot-
and-mouth disease spread within an integrated livestock system in Texas,
USA. Preventive Veterinary Medicine 88, 286-297.
Ward, M.P., Laffan, S.W., Highfield, L.D., 2007. The potential role of wild and feral
animals as reservoirs of foot-and-mouth disease. Preventive Veterinary
Medicine 80, 9-23.
Wernery, U., Kaaden, O.R., 2004. Foot-and-mouth disease in camelids: a review.
The Veterinary Journal 168, 134-142.
Westbury, H.A., Chamnanpood, P., Tandchaitrong, S., Doughty, W.J., 1988a. Single
Dilution ELISA for Detection of Serum Antibody to Foot-and-Mouth
Disease Virus in Cattle. Veterinary Microbiology 18 273-283.
162
Westbury, H.A., Chamnanpood, P., Tandchaitrong, S., Doughty, W.J., 1988b. Single
Dilution ELISA for Detection of Serum Antibody to Foot-and-Mouth
Disease Virus in Cattle. Veterinary Microbiology 18, 273-283.
Westbury, H.A., Doughty, W.J., Forman, A.J., Tangchaitrong, S., Kongthon, A.,
1988c. A Comparison of Enzyme-linked Immunosorbent Assay,
Complement Fixation and Virus Isolation for Foot and Mouth Disease
Diagnosis. Veterinary Microbiology 17, 21-28.
Wongsathapornchai, K., Salman, M.D., Edwards, J.R., Morley, P.S., Keefe, T.J.,
Campen, H.V., Weber, S., 2008. Assessment of the likelihood of the
introduction of foot-and-mouth disease through importation of live animals
into the Malaysia-Thailand- Myanmar peninsula. American Journal of
Veterinary Research 69, 252-260.
Woodbury, E.L., 1995. A review of the possible mechanisms for the persistence of
foot and mouth disease virus. Epidemiology and Infection 114, 1-13.
Woods, A., 2004. Why slaughter? The cultural dimensions of Britain's foot and
mouth disease control policy, 1892-2001. Journal of Agricultural &
Environmental Ethics 17, 341-362.
Woolhouse, M.E.J., Chase-Topping, M., Haydon, D.T., Friar, J., Matthew, L.,
Huges, G., Shaw, D., Wilesmith, J.W., Donaldson, A., Cornell, S., Keeling,
M.J., Grenfell, B.T., 2001. Epidemiology of Foot and Mouth Disease under
control in the UK. Nature 411, 258-259.
Yoon, H., Wee, S.H., Stevenson, M.A., O‟Leary, B.D., Morris, R.S., Hwang, I.J.,
Park, C.K., Stern, M.W., 2006. Simulation analyses to evaluate alternative
control strategies for the 2002 foot-and-mouth disease outbreak in the
Republic of Korea. Preventive Veterinary Medicine 74, 212-225.
Zhang, Z.D., Kitching, R.P., 2001. The Localization of Persistent Foot and Mouth
Disease Virus in the Epithelial Cells of the Soft Palate and Pharynx. Journal
of Comparative Pathology, 124, 89-94.
