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Author/s:
Conlan, James V
Title:
Improved diagnostics and management of classical swine fever in the Lao People's
Democratic Republic
Date:
2006-08
Citation:
Conlan, J. V. (2006). Improved diagnostics and management of classical swine fever in the
Lao People's Democratic Republic, Masters Research thesis, Faculty of Veterinary Science,
The University of Melbourne.
Publication Status:
Unpublished
Persistent Link:
http://hdl.handle.net/11343/39196
File Description:
Improved diagnostics and management of classical swine fever in the Lao People's
Democratic Republic
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James V. Conlan
A thesis submitted in total fulfilment of the requirements
of the degree of Master of Science
August, 2006
School of Veterinary Science,
The University of Melbourne
Abstract
Classical Swine Fever (CSF) is a highly contagious viral disease of swine that causes major
losses to pig production. CSF virus is a member of the genus Pestivirus of the family
Flaviviridae and is closely related antigenically to other Pestiviruses, Bovine Viral Diarrhoea
(BVD) virus and Border Disease (BD) virus. In the Lao Peoples Democratic Republic (Laos),
CSF has been recognised as a disease that causes significant loss to the smallholder pig sector.
However, there exists in Laos a deficiency in fully understanding the epidemiology and impact
of CSF, together with limitations in being able to reliably detect CSF outbreaks in a timely
manner.
The research presented in this thesis is divided into three components. The first component
describes the smallholder pig sector in general and seeks to address the impact CSF has on
production. In comparison to accepted standards of international and tropical pig production,
the performance of the smallholder pig sector of Laos was quite poor. Classical Swine Fever
was found to be an important disease impacting on pig production, where the incidence of CSF
in Bolikhamxay province was 18 outbreaks per 100 village years.
The second and major component describes the development of a new diagnostic test to
improve the rapid identification of CSF outbreaks. This thesis describes the development and
assessment of a rapid and sensitive ELISA-based test for the diagnosis of CSF virus using
immunomagnetic beads (IMBs) as the solid phase. The majority of the research was conducted
in Laos where the IMB-ELISA for CSF virus was developed and compared to an antigen
capture (AC)-ELISA. Initial estimates of diagnostic specificity and sensitivity show 100 %
agreement with the AC-ELISA and the IMB-ELISA has up to 64-fold greater analytical
sensitivity. The rapid test is a highly robust and stable test format and much simpler to perform
than the AC-ELISA. The IMB-ELISA has the added advantage that the test can be read by eye,
lending it to the possibility of adaptation to a near-to-field test with minimal equipment needed.
The new test is comparable in cost to the current AC-ELISA; however it can be performed in
approximately 3 hours upon receipt of specimens compared to 1 to 2 days for the AC-ELISA.
The third and final component addresses the issue of CSF vaccine quality through the
assessment of storage temperature and vaccine shelf-life. The CSF vaccine used in Laos is a
locally produced lapinised C-strain vaccine with a recommended shelf life of 12 months when
stored at 20 C. The results presented in this thesis show the vaccine to be stable for only 4
months when stored at 20 C and stable for less than 3 months when stored at 4 C.
CSF is important in Laos and has a detrimental impact on pig production; however its presence
is not ubiquitous. With improvements in diagnostic capabilities in provincial centres through
the further development of the rapid test, outbreaks of CSF will be more readily detected
allowing control measures to be enacted. However, control measures utilising the vaccine
produced in Laos should be closely controlled to ensure the delivery of a quality product.
ii
Declaration
I hereby declare that the research presented in this thesis is comprised of only my original
work; due acknowledgement has been made in the text to all other material used. The
material has not been submitted, either whole or in part, for a degree at this or any other
university and the thesis is approximately 40, 000 words in length, exclusive of tables,
figures, bibliography and appendices.
James Conlan
August, 2006
iii
Preface
A small proportion of the research presented in this thesis was carried out prior to the
commencement of my Master of Science candidature. This research is presented in Chapter
2: Pig production and health in Laos and commenced in April 2002, approximately 16
months before my candidature commenced. The research presented in this chapter was the
result of greater than three years of surveillance work assessing the smallholder pig
production sector in Laos. Approximately two years of the survey work together with all
data analysis was completed during my candidature. Material arising from this work has not
been submitted, either whole or in part, for a degree at this or any other university and due
acknowledgement has been made in the text to all collaborations and contributors to this
section of the thesis.
iv
Acknowledgements
I wish to express my sincere thanks to my supervisors, Dr. Laurence Gleeson at CSIRO
Australian Animal Health Laboratory and Professor Colin Wilks at the School of
Veterinary Science, The University of Melbourne for their guidance and support during the
undertaking of this degree. I would also like to acknowledge the friendship and support of
Dr. Stuart Blacksell, to whom I owe a debt of gratitude for in-country guidance and counsel
(and the odd Beer Lao). I would also like to express my sincere thanks and gratitude to the
staff at the National Animal Health Centre, Department of Livestock and Fisheries,
Vientiane, Laos for their co-operation, support and friendship during my time spent in Laos
where the majority of my research was conducted. In particular, I wish to thank Dr. Syseng
Khounsy, the national project leader and the other project staff, Miss Manivanh
Phouaravanh, Miss Vilayvanh and Mr Lapin. I would also like to gratefully acknowledge
the support of the Australian Centre for International Agricultural Research (ACIAR) for
providing the project funding. In particular, I would like to thank Dr. John Copland who
had the vision to see the need for continued research into infectious diseases of livestock in
the smallholder farming sector of countries such as Laos. I would like to acknowledge the
invaluable support of the virology group and the IT and library staff at CSIRO Australian
Animal Health Laboratory.
Finally I wish to acknowledge the support of family and friends, in particular my wife,
Iwona, for her continued love and patience.
Table of contents
Abstract
Declaration
iii
Preface
iv
Acknowledgements
Table of contents
vi
Abbreviations
viii
List of tables
xi
List of figures
xii
Chapter 1
11
39
41
44
Chapter 2
45
47
2.2 Methods
48
2.3 Results
57
70
Chapter 3
77
3.1 Introduction
79
79
80
80
81
83
86
vi
Chapter 4
IMB-ELISA development
91
4.1 Introduction
93
94
97
117
Chapter 5
121
5.1 Introduction
123
123
146
Chapter 6
153
6.1 Introduction
155
157
6.3 Results
160
167
Chapter 7
7.1 Introduction
171
173
173
174
176
177
180
Bibliography
181
Appendices
Appendix I:
203
Appendix II:
207
Appendix III:
210
vii
Abbreviations
AAHL
AC-ELISA
Antigen capture-ELISA
ACIAR
AEC
BDV
BSA
BVDV
CI
confidence interval
cm
centimetre
CPE
cytopathic effect
CO2
Carbon dioxide
CSF
CSIRO
EDAC
ELISA
EMEM
FAO
FAT
FBS
FITC
Fluorescein isothiocyanate
FMD
FSG
gram
GDP
GNI
hour
viii
HEPES
Hydroxethylpiperazine-ethanesulphonic acid
HRP
H2SO4
Sulphuric acid
IgG
Immunoglobulin G
IMB
Immunomagnetic bead
Mab
Monoclonal antibody
MAF
min
minute
MLV
MPK
MS
Microsoft
NP-40
Nonidet P-40
NPLA
NS
Non-structural
NVI
OD
Optical density
OIE
ORF
PBSA
PBST
PCR
PDR
PK15
PLA
RNA
Ribonucleic acid
rpm
RT-PCR
Reverse transcriptase-PCR
SD
Standard deviation
SK6
SMP
ix
TCID
TMB
5, 5-Tetramethylbenzidine
US$
US dollars
UTR
Untranslated region
UV
Ultraviolet
VSU
VVW
microlitre
List of Tables
40
Table 2.1 Village level descriptive variables of pig production and health
in Bolikhamxay province, Laos, May 2002
59
62
Table 2.3 Number of young piglets entering the village production units in
Bolikhamxay province, Laos, 2002 to 2005
Table 5.1 Analytical sensitivity of IMB-ELISA
66
131
135
Table 5.3 Agreement between operators when IMB-ELISA test results were
determined by measuring OD450 nm spectrophotometrically
142
Table 5.4 Agreement between operators when IMB-ELISA test results were
determined by recording a visual colour change
143
143
144
144
207
xi
List of Figures
Figure 1.1 Laos in Southeast Asia
12
Figure 1.5 Map of Laos with the location of the origin of CSF virus
subgroups 2.1 and 2.2
Figure 2.1 Districts of Bolikhamxay province, Laos
23
50
54
64
65
Figure 2.5 Piglet mortality and sale patterns associated with CSF virus
outbreaks in villages in Bolikhamxay province, Laos
68
Figure 3.1 Transfer plate and cell culture plate for virus titration
82
Figure 3.2 Transfer plate and ELISA plate format for AC-ELISA
84
Figure 3.3 Control plate and serum plate format for NPLA
89
100
103
106
xii
106
Figure 4.5 Comparison of goat and rabbit polyclonal antigen capture antibody
using Lao sample 089/04 as analyte
109
Figure 4.6 Comparison of goat and rabbit polyclonal antigen capture antibody
using AAHL antigen as analyte
110
111
113
116
125
127
128
136
Figure 5.5 Incubation of samples 087/03 and 107/03 with serum before
the IMB-ELISA
137
138
139
Figure 5.8 Modified ROC plot of the diagnostic sensitivity (DSn) and
specificity (DSp) of the IMB-ELISA as a function of cut-off intervals
139
xiii
Figure 6.1 Model representing transport and storage conditions of CSF vaccine
in Laos
Figure 6.2 Flow chart of CSF vaccine storage temperature experiment
156
159
161
Figure 6.4 Serum neutralizing antibodies to CSF virus post vaccination with
C-strain vaccine batch 05/2005 at month four of the experiment
164
Figure 6.5 Serum neutralizing antibodies to CSF virus post vaccination with
C-strain vaccine batch 05/2005 at month five of the experiment
165
209
210
Figure A3. Relationship between sow/boar ratio and average litter size
210
xiv
xv
xvi
Chapter 1.
Introduction and literature review
Chapter 1
Chapter 1
Chapter 1
the burden of poverty (ADB, 2002). At the same time, many households also view poor
livestock health and poor production performance as a key contributor to poverty (ADB,
2002).
The Food and Agricultural Organisation of the United Nations (FAO, 2003b) reported that
all livestock populations showed a positive growth rate in the period 1990 to 2000. Poultry
and sheep/goat showed the greatest gains, growing at 6.0 and 4.5 % annually in this period,
respectively (FAO, 2003b). The pig population showed the slowest growth rate of all
livestock in the same period, growing at an annual rate of 0.4 % (FAO, 2003b). This slow
growth rate is reflected in Figure 1.3 below, where the national pig herd experienced the
largest population downturn in the period 1997 to 1999, decreasing by approximately 0.56
million head (MAF, 2002).
PR China
Vietnam
Myanmar
Thailand
Cambodia
Chapter 1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
3
5
6
9
8
18
10
11
1
12
Southern Region
13
Central Region
14
Northern Region
16
15
VientianeCapital
Phongsaly
LuangNamtha
Oudomxay
Bokeo
LuangPrabang
Huaphan
Xayabouly
XiengKhouang
VientianeProvince
Bolikhamxay
Khammouane
Savannakhet
Saravan
Sekong
Champassak
Attapeu
XaysombounS.R.
17
2.00
1.80
Million Head .
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
Buffalo
Cattle
Goat/Sheep
2003
2002
2001
2000
1999
1998
1997
1996
1995
1994
1993
0.00
Pig
Chapter 1
Chapter 1
high quality commercial feed which may be combined with locally produced improved feed
stuffs (Vongthilath and Blacksell, 2000). Semi-intensive farming may involve the purchase
of weaner pigs for fattening before sale (Str et al. 2002), or a farming system with both
breeders and growers. Farmers using commercial feed are, however, disadvantaged by the
relatively high cost of procuring feed compared to neighbouring countries such as Thailand
and Vietnam (Str et al. 2002).
Table 1.1 Pig populations by region and province (MAF, 2000)
Region/Province
Northern Region
554
53
Phongsaly
49
Luang Namtha
40
Odomxay
83
Bokeo
35
Luang Prabang
120
12
Huaphan
138
13
Xayabouly
89
339
33
Xieng Khouang
74
Vientiane Capital
15
Vientiane Province
65
Bolikhamxay
36
Khammouane
35
Savannakhet
94
Xaysomboun S.R.
20
144
14
Saravan
52
Sekong
23
Champassak
55
Attapeu
14
Total
1037
100
Central Region
Southern Region
Chapter 1
In 2000, total pig meat production was estimated to be 33.3 thousand metric tonnes, of
which 5 metric tonnes were exported, contributing US$ 5000 to the Lao economy (FAO,
2003b). This clearly indicates that nearly all pig meat produced was consumed locally. In
terms of agricultural output, indigenous pig meat production was ranked fourth in overall
agricultural commodities behind rice, fresh vegetables and indigenous cattle meat (FAO,
2003a).
There is only limited information available in the literature specifically addressing pig
production and reproductive performance of traditionally raised pigs in Laos. As described
above, a number of pig farming systems exist in Laos, with the majority of pigs raised by
smallholder farmers using traditional practices (Vongthilath and Blacksell, 2000).
According to a recent review of pig production in Southeast Asia, with a particular focus on
Thailand (Kunavongkrit and Heard, 2000), productivity of the pig industry varies greatly
between countries and varies according to the ratio of intensive farms to smallholder
producers. Countries with a greater proportion of pigs produced in intensive piggeries have
markedly greater productivity (Kunavongkrit and Heard, 2000). In Thailand, the ratio of
intensive to smallholder production was approximately 80:20 in 1998 compared with
Vietnam which was 20:80, and in Thailand yearly pork production per sow was more than
double that seen in Vietnam (Kunavongkrit and Heard, 2000). In Laos, the ratio of
intensive to smallholder production was approximately 10:90 in 2002 (MAF, 2002). The
reproductive performance of pure-breed sows in Thailand is, however, poor when
compared with the same breeds in Europe. A humid and hot climate, infectious disease,
poor nutrition and management practices all have a detrimental impact on productivity in
Southeast Asia (Kunavongkrit and Heard, 2000).
Recent research has shed light on the reproductive performance and productivity of pigs
produced by traditional raising practices in the Philippines (Lanada et al. 1999; Lanada et
al. 2005; Lee et al. 2005; More et al. 1999; More et al. 2005). Tropical smallholder pig
production has also been studied in the Solomon Islands (de Fredrick, 1977) and Kenya
(Wabacha et al. 2004a; Wabacha et al. 2004b). In general, the reproductive performance
Chapter 1
of sows raised in the tropical smallholder farm sector is significantly lower than the tropical
intensive farm sector, with smaller litter sizes, greater inter-farrowing interval and higher
pre-weaning piglet mortality (Lanada et al. 1999; Lanada et al. 2005; Wabacha et al.
2004b). The reproductive performance of boars in smallholder production systems has also
been demonstrated to be sub-standard. Boars in smallholder systems in the Philippines are
poorly used even if a plentiful supply of potential breeding stock exists, leading to a
decrease in sow reproductive performance and a high incidence of in-breeding (Lanada et
al. 2005). Sub-standard reproductive performance of breeding stock in smallholder systems
has largely been attributed to poor management practices, breed, poor nutrition and disease
such as parasitism (Lanada et al. 2005; Wabacha et al. 2004b). Pre-weaned and grower
pigs are also likely to perform poorly in the tropical smallholder production system due to
problems associated with nutrition, management and breed (Lee et al. 2005; More et al.
2005; Wabacha et al. 2004b).
Chapter 1
factors having a negative impact on pig production include, but are not limited to, the high
labour demand of women which makes it difficult for them to concentrate time on pig
raising (Str et al, 2002), poor market access in more remote areas (Str et al. 2002;
Vongthilath and Blacksell, 2000) and limited access to high quality veterinary attention and
effective vaccination services (Vongthilath and Blacksell, 2000).
Due to disease, in particular CSF, being an important constraint to smallholder pig
production in Laos, diagnostic capabilities and the development of improved diagnostic
tests for CSF virus will make up a significant proportion of this thesis.
10
Chapter 1
Genome
The viral genome consists of a single 12.3 kilobase (kb), positive sense, single-stranded
RNA molecule. The viral genome is made up of a 11.7 kb open reading frame (ORF)
preceded by an uncapped 5-untranslated region (UTR) approximately 360 385
nucleotides in length and followed by a 3-UTR approximately 230 nucleotides in length
and lacking a poly(A) tail (Heinz et al. 2000).
11
Chapter 1
5
UTR
NS
Structural
3OH
Non-Structural (NS)
UTR
Translation
NH2
COOH
Npro
Erns E1 E2
NS2-3
p7
NS5A
NS4A NS4B
NS5B
Proteins
The viral genome of CSF virus and other pestiviruses encodes a polyprotein that is co- and
post-translationally cleaved into four structural and eight non-structural (NS) proteins by
both host-derived and virus-encoded proteinases (Heinz et al. 2000). Figure 1.4 above
outlines the order of proteins encoded by the ORF of the pestivirus genome.
The nucleocapsid protein (denoted C) plays a structural role in the virion and has also been
postulated to play a role in the internal signal sequencing of glycoprotein translocation to
the endoplasmic reticulum (Heinz et al. 2000) and as a regulator of gene expression (Liu et
al. 1998).
The three structural envelope proteins are transcribed in the order NH2 gp44/48 gp33
gp55 COOH (Stark et al. 1990); the nomenclature has subsequently changed to Erns
(gp44/48), E1 (gp33) and E2 (gp55) (Rumenapf et al. 1993). The Erns protein, previously
denoted E0, lacks a transmembrane anchor and is shed by infected cells (Rumenapf et al.
1993), small portions however remain attached to the virion through a direct interaction
12
Chapter 1
with the E2 glycoprotein (Lazar et al. 2003). Erns has ribonuclease activity ( Hulst et al.
1994; Schneider et al. 1993) and plays a key role in virus attachment to the cell surface
(Hulst and Moormann, 1997; Weiland et al. 1999) and subsequent virus entry (Wang et al.
2004). Erns is a major stimulator of neutralising antibodies (Lin et al. 2005; Lin et al. 2004;
Weiland et al. 1992), is postulated to be involved in CSF viral pathogenesis by inducing
apoptosis of lymphocytes (Bruschke et al. 1997), and is likely to be involved in viral
persistence by preventing apoptosis of infected cells (Hulst et al. 1998). The E1 is less well
characterised but has been shown to have a transmembrane anchor (Rumenapf et al. 1993)
and is involved in virus entry into cells (Wang et al. 2004). Like Erns, the E2 glycoprotein
is a stimulator of neutralising antibodies (Weiland et al. 1990) and is the immunodominant
protein inducing a neutralising antibody response following infection (Bouma et al. 1999;
Hammond et al. 2000; Hulst et al. 1993; Konig et al. 1995; Rumenapf et al. 1991; van
Rijn et al. 1996; van Zijl et al. 1991b; Weiland et al. 1999 ). The E2 glycoprotein is a
transmembrane protein (Rumenapf et al. 1993) made up of four antigenic domains A D,
located at the N-terminus , among which B, C and D vary between isolates, whereas
domain A is conserved between different CSF virus strains (Wensvoort, 1989). E2 also
plays a key role in viral attachment (Hulst and Moormann, 1997; Weiland et al. 1999) and
entry (Wang et al. 2004) and is postulated to have epitopes that are targets for cytotoxic
lymphocytes (Ceppi et al. 2005).
The remaining proteins are non-structural, and whose functions are being increasingly
elucidated. Npro (also known as p20) has autoproteolytic activity responsible for the
cleaving of Npro and C proteins (Muyldermans et al. 1996). While Npro is not believed to be
important in viral replication (Tratschin et al. 1998), there is evidence that it is important in
viral evasion of innate host immunity (La Rocca et al. 2005; Ruggli et al. 2003). The
second NS protein, p7, whose function is yet to be elucidated, is located between E2 and
NS2 on the genome (Elbers et al. 1996) and is dispensable in virus replication in tissue
culture (Moser et al. 1999). The NS2-3 protein complex (p125) contains both helper T-cell
and cytotoxic T-lymphocyte epitopes (Armengol et al. 2002). The NS2 protein (p54) has
been found to play a role in the regulation of viral replication while not being an essential
13
Chapter 1
protein for replication to proceed (Moser et al. 1999). NS4A and NS4B follow NS3 (p80)
and cytotoxic T-lymphocyte epitopes have been identified in the NS3-NS4A domain
(Ceppi et al. 2005; Pauly et al. 1995). The final two NS proteins are NS5A and NS5B,
with NS5B shown to be an RNA-dependant RNA polymerase with specific in vitro activity
identified (Steffens et al. 1999; Xiao et al. 2002).
14
Chapter 1
Clinical features
The clinical signs of CSF virus infection have been extensively reviewed (Dahle and Liess,
1992; Moennig, 2000; Moennig et al. 2003). CSF virus infection may present with very
variable clinical signs, ranging from the most severe form that results in very high mortality
and morbidity to the most mild form that may cause few if any noticeable clinical signs.
Further, a CSF virus isolate may cause different forms of disease depending on age, breed
and immune status of the host, with young animals likely to succumb to a more severe form
(Moennig et al. 2003). Acute, chronic and prenatal forms of CSF can be distinguished
(Moennig et al. 2003). The acute form can be further subdivided into peracute, acute and
subacute depending on time to death post infection (Dahle and Liess, 1992). The clinical
signs associated with each of the three forms are described below. The common thread
associated with all forms is that animals are viraemic for at least as long as they show
clinical signs (Moennig, 2000).
Acute form
The peracute form of the disease is characterised by a rapid progression to death, usually
within five days post-infection, whereby pigs are found dead with few if any clinical signs
(Dahle and Liess, 1992). The most common form of disease seen in young pigs up to 12
weeks of age is the acute form which is characterised by death 14 to 20 days post infection
with an incubation period of 7 to 10 days (Moennig, 2000; Moennig et al. 2003). In this
15
Chapter 1
younger age bracket mortality as high as 90 % may occur, whereas disease in older animals
is generally milder (Moennig, 2000).
A range of clinical signs may be seen in a variety of combinations. Pyrexia is common with
temperatures in younger animals surpassing 40 C. Early signs may include anorexia,
lethargy, conjunctivitis, respiratory signs, huddling together and constipation with
intermittent diarrhoea (Dahle and Liess, 1992; Moennig et al. 2003). Neurological signs
are also frequently apparent and may include a staggering gait with hind leg weakness,
incoordination of movement and, less commonly, convulsions (Dahle and Liess, 1992;
Moennig et al. 2003). In white pigs, petechial or extensive haemorrhaging of the skin of
the ears, tail, abdomen and inner side of the limbs may be seen from 2 to 3 weeks post
infection and continuing until death or recovery (Dahle and Liess, 1992; Moennig et al.
2003). The subacute form of disease as described by Dahle and Liess (1992) is a less severe
form of disease with death 20 to 29 days post infection.
Chronic form
Pigs that survive the acute form of disease may suffer chronic disease (Moennig et al.
2003). By definition chronic CSF runs a course where death occurs 30 days or more post
infection (Dahle and Liess, 1992), however pigs may survive for up to 3 months post
infection and continue to shed virus from the time clinical signs first appear until death
(Moennig et al. 2003). Initial signs are similar to those of acute disease but become less
pronounced in the later stages; usually signs include intermittent fever, failure to thrive,
wasting and chronic enteritis (Moennig et al. 2003). Antibodies may be temporarily
detected, however failure to eliminate the virus leads to virus-antibody complexes that are
not detected by standard serological assay (Moennig et al. 2003).
Prenatal form
CSF virus is able to cross the placenta of infected pregnant sows and thereby infect foetuses
at all stages of pregnancy, resulting in reproductive losses (Moennig et al. 2003). The stage
in gestation and the virulence of the viral strain at the time of infection will determine the
16
Chapter 1
outcome to the foetus. Infection of sows in the early stages of pregnancy and the
subsequent trans-placental infection of foetuses may result in abortions, stillbirths,
mummifications and malformations (Moennig, 2000; Moennig et al. 2003). Infections of
sows from approximately 50 to 70 days of pregnancy can result in foetal infection leading
to the birth of persistently viraemic piglets, which may appear clinically normal (Moennig,
2000; Moennig et al. 2003). Although these viraemic piglets may appear normal at birth,
they always die from CSF several months later, with 11-month survival periods
occasionally observed (Moennig, 2000; Moennig et al. 2003). After birth, persistently
infected piglets may show poor growth, wasting and congenital tremors and shed large
amounts of virus until death, thereby acting as important reservoirs of infection (Moennig
et al. 2003). The onset of clinical signs is generally in the later stages of disease
progression, which accounts for this form of disease sometimes being referred to as late
onset CSF (Moennig et al. 2003).
Pathology
Lesions in those pigs presenting with the acute form of CSF are most commonly found in
the lymph nodes, spleen and kidneys. The lymph nodes become swollen, oedematous and
haemorrhagic. Haemorrhage in the kidney when it occurs can vary from petechial to
ecchymotic (Moennig et al. 2003). Petechiae may also be seen in the urinary bladder,
larynx, epiglottis, heart and the serosae of the abdominal and thoracic cavities (Moennig et
al. 2003). The pathological findings following the chronic course of the disease are less
typical, with haemorrhaging in organs and the serosae less common. In those animals
displaying chronic diarrhoea during the course of the disease, necrotic and ulcerative
lesions on the ileum, the ileocaecal valve and the rectum may be commonly observed
(Moennig et al. 2003).
17
Chapter 1
1.2.3 Epidemiology
Host species
The pig, both feral and domestic, is the only known natural host of CSF virus (van
Oirschot, 1999). Ruminants (cattle and sheep, deer and goats) may become infected,
however ruminants are dead-end hosts in which the infection is subclinical without virus
shedding and subsequently they are not important for transmission and maintenance
(Ribbens et al. 2004a).
Virus stability
pH and heat stability
CSF is relatively stable over a wide pH range, however below pH 4 and above pH 10 the
virus is quickly inactivated (Anonymous, 1996). The virus is heat stable, being able to
survive for 1 to 2 weeks at 37 C in blood (Anonymous, 1996). Inactivation of CSF virus
by high temperature is dependant on the medium in which it is being heated. Virus in tissue
may not be inactivated after heating to 80 C for 60 seconds (Anonymous, 1996), however
some research suggests the pasteurisation process whereby meat is heated to 71 C for 60
seconds is enough to inactivate virus (Edwards, 2000). Virus is inactivated in defibrinated
blood after heating to 68 C for 45 minutes or 69 C for 30 minutes (Anonymous, 1996;
Edwards, 2000). The virus is susceptible to rapid changes in temperature such as freezing
and thawing (Anonymous, 1996).
Animal products
CSF virus can survive in pork and processed pork products, and survival is prolonged for
months when meat is stored cool and for years when stored frozen (Anonymous, 1996).
Virus survival of 85 days in chilled pork and greater than 4 years in frozen pork has been
recorded (Edwards, 2000). CSF virus is also able to survive curing processes such as
salting, brining, smoking and drying for extended periods (Anonymous, 1996; Edwards,
2000), with survival of 17 to 188 days recorded for different smoking processes (Edwards,
2000).
18
Chapter 1
Environmental
CSF virus is rapidly inactivated by UV light; however the virus may survive for extended
periods of time under favourable conditions of temperature, humidity, presence of organic
matter and pH (Edwards, 2000).
Transmission
Ribbens et al. (2004) have extensively reviewed CSF virus transmission and for the
purposes of this thesis this review will be used as a template. CSF virus is excreted in nasal,
conjunctival and oral secretions together with the urine and faeces of infected pigs; the
natural route of infection is via the oronasal cavity (Ribbens et al. 2004a). Other
transmission routes, such as conjunctival (Ribbens et al. 2004a), genital (de Smit et al.
1999), and parenteral (Moennig, 2000; Moennig et al. 2003) have been shown.
CSF viral transmission can occur by either direct contact between pigs or by an indirect
route where there are one or more intermediate steps between primary and secondary
infection (Ribbens et al. 2004a). Direct transmission can be subsequently divided into
horizontal and vertical spread; where horizontal refers to spread by direct pig-to-pig contact
and vertical being in utero transmission between sow and offspring by trans-placental
infection (Ribbens et al. 2004a).
Horizontal transmission is most efficient in weaner pigs compared to slaughter and breeder
pigs (Ribbens et al. 2004a), with the reproductive ratio (R0) of within pen transmission
significantly greater in the weaner pigs compared to slaughter age pigs (Klinkenberg et al.
2002a; Laevens et al. 1998; Laevens et al. 1999). Klinkenberg et al. (2002a) found no
significant difference in R0s for between pen transmission for weaner and slaughter pigs
and postulated that the significant difference in within-pen R0s was due to a greater
number of contacts made between weaner pigs in a pen. Besides intra-herd transmission, an
important mode of horizontal spread is inter-herd transmission where contact between pigs
may be due to the introduction of infectious pigs into a susceptible herd or contact made at
markets (Ribbens et al. 2004a). Wild pig populations may also play an important role in
19
Chapter 1
horizontal transmission when these pigs are able to come into contact with domestic pigs in
non-closed raising systems (Ribbens et al. 2004a). Vertical transmission, whereby piglets
may become infected in utero has been discussed in some detail in the above section
outlining clinical forms of CSF. This infection route is very important in CSF epidemiology
as it can lead to persistence within a herd and facilitate spread between herds as piglets may
be born clinically normal ( Ribbens et al. 2004a; Terpstra, 1987).
Indirect transmission comprises all other modes of transmission and involves an
intermediary step between the infectious primary host and the susceptible secondary host.
Ribbens et al. (2004) describe a classification system in which indirect transmission routes
are defined as either distance independent or distance dependant. Distance independent
modes of transmission include mechanical spread of the virus through the use of infected
semen in artificial insemination, excretions and secretions in livestock transport vehicles
used to transport infected pigs, mechanical transfer of virus on the clothing and/or footwear
of livestock handlers and the feeding of infected pork products to susceptible pigs in swill
(Ribbens et al. 2004a). As an example, pens housing infected pigs that were not cleaned
following depopulation contained sufficient virus after a 10 hour withholding period to
cause disease in susceptible pigs placed in the same pens (Ribbens et al. 2004b).
Transmission of virus to a secondary herd commonly occurs within a 1 km vicinity of the
primary infected herd (Ribbens et al. 2004a). Some of the underlying distant dependent
mechanisms responsible for these neighbourhood infections include aerosol transmission
and mechanical transfer by arthropod vectors and small animals, however birds are not
thought to play an important role in transmission (Ribbens et al. 2004a). Airborne
transmission has been experimentally shown (Laevens et al. 1998; Laevens et al. 1999),
however the distance over which airborne transmission can occur remains conjecture
(Ribbens et al. 2004a) and when it does occur it is likely to be within a holding (Paton and
Greiser-Wilke, 2003). Factors such as climate and geography together with virus virulence
are likely to play important roles in the distance aerosol transmission may occur (Ribbens et
20
Chapter 1
al. 2004a). This said, the precise mechanisms by which virus spreads between
neighbouring holdings are poorly defined (Paton and Greiser-Wilke, 2003).
Wild pig populations play an important role in virus transmission to domestic (commercial)
pig herds in a number of ways. Transmission can occur through the direct route as
explained above in non-closed domestic herds, but also through indirect routes. Indirect
transmission can occur where offal from wild boar is fed to domestic pigs, through the
feeding of silage originating from areas populated by infected wild boar and through the
mechanical transfer of virus by hunters and vehicles used to transport infected carcasses
(Ribbens et al. 2004a).
World distribution
CSF virus has a world wide distribution with Canada and the United States of America,
Australia and New Zealand and some Northern European countries able to remain free
from disease with a fully susceptible pig population (Edwards et al. 2000). Africa has
predominantly remained free of CSF virus with Madagascar the exception (Edwards et al.
2000), however more recently Mauritius (outbreaks in 2000 to 2002) and South Africa
(outbreaks in 2005) have experienced CSF virus incursions (OIE, 2005). In the East and
Southeast Asian region CSF virus is endemic with Japan the exception (Edwards et al.
2000). Japan last reported an outbreak of disease in 1992 (OIE, 2005), however an
eradication campaign commenced in 1996 to eliminate CSF virus from the Japanese islands
(Edwards et al. 2000). CSF virus infections were detected on one Japanese farm in 2004
and subsequently traced to the use of an illegal live vaccine (Promed, 2005). CSF virus has
been reported throughout all regions of Laos (Blacksell et al.2004a; Blacksell et al. 2005;
Vongthilath & Blacksell, 2000) and is endemic in all bordering countries, Thailand,
Vietnam and Myanmar (Edwards et al. 2000), present in Cambodia (OIE, 2005) and has a
wide distribution in PR China (Tu et al. 2001). In Europe, European Union member
countries such as The Netherlands, Germany, Belgium, France, Luxembourg, Italy and
Spain commonly experience periodic outbreaks which are frequently linked to swill feeding
(Edwards et al. 2000). In recent years outbreaks have occurred in Germany (2004), France
21
Chapter 1
(2004), Italy and Luxembourg (2003), Spain (2002), Great Britain (2000), The Netherlands
(1998) and Belgium (1997) (OIE, 2005). In Central and Eastern Europe, a non-vaccination
policy exists in only a few countries (Edwards et al. 2000) with recent outbreaks occurring
in Russia and Bulgaria (2005) (Promed, 2005), Slovakia (2004), Slovenia (2004), Serbia
and Montenegro (2004), Romania (2004), The Former Yugoslavian Republic of Macedonia
(2004), Bulgaria (2004), Bosnia and Herzegovina (2004) and Croatia (2002) (OIE, 2005).
In the Americas, Canada and the United States of America are free of CSF virus with nonvaccination (Edwards et al. 2000). Central and South America are endemically infected
with Belize, Panama, Chile and Uruguay the exceptions (Edwards et al. 2000). Several
states of Brazil are free of disease (Edwards et al. 2000), however, an outbreak outside the
disease free zone in Ceara State was reported in mid 2004 (Promed, 2005).
Molecular epidemiology
Based on phylogenetic analysis of the E2 glycoprotein and NS5B genes and the 5 UTR,
CSF viruses are able to be classified into 3 groups (1, 2 and 3) each with 3 or 4 subgroups
(1.1, 1.2, 1.3; 2.1, 2.2, 2.3; 3.1, 3.2, 3.3 and 3.4) (Paton et al. 2000c). Recent CSF virus
isolates from Europe belong to group 2, whereas up until 1970 all isolates belonged to
group 1, most of which were subgroup 1.2 (Paton et al. 2000c). Only group 1 viruses have
been reported in the Americas, but all 3 groups and subgroups have been reported in the
Asian region (Paton et al. 2000c). In Laos, only groups 2.1 and 2.2 have been reported and
these can be demarcated along geographical lines, with subgroup 2.1 predominantly found
in the north-central region and 2.2 in the south-central region (Blacksell et al. 2005;
Blacksell et al. 2004a).
22
Chapter 1
Subgroup 2.1
Subgroup 2.2
Figure 1.5 Map of Laos with the location of the origin of CSF virus
subgroups 2.1 and 2.2 (Source: Blacksell et al. 2004a)
23
Chapter 1
24
Chapter 1
Laboratory diagnosis
A definitive diagnosis of a CSF outbreak must be confirmed by laboratory tests of
suspected cases and/or herds by one of several methods: isolation and detection of virus,
detection of viral antigen, the demonstration of a rise in the titre of viral antibody or the
detection of specific viral RNA (van Oirschot, 1999). When developing diagnostic
capability for CSF virus, care must be taken in selecting the most appropriate diagnostic
technique for the laboratory and the environment in which samples will be collected. The
choice of laboratory technique to be employed in the diagnostic process is greatly
influenced by the conditions under which samples are collected and the quality of the
samples received at the laboratory. This is particularly important in a least developed and
tropical country setting where delays in sample transport to the laboratory may be common
(Blacksell et al. 2004b). At present, there are no rapid pen-side diagnostic tests available
for the diagnosis of CSF in the field. All current methodologies are reliant on laboratory
facilities (Paton and Greiser-Wilke, 2003). Risatti et al. (2003) have described a portable
real-time RT-PCR methodology; however this requires a mobile laboratory set up to
facilitate RNA extraction and the subsequent PCR amplification.
25
Chapter 1
26
Chapter 1
27
Chapter 1
28
Chapter 1
CSF virus from other pestiviruses include nested RT-PCR using species specific secondary
primer sets and size differentiation by gel electrophoresis (Katz et al. 1993; Sandvik et al.
1997), restriction enzyme digestion of PCR products and subsequent size differentiation by
gel electrophoresis (Parchariyanon et al. 2000; Vilcek and Belak, 1998; Zaberezhny et al.
1999), RT-PCR-ELISA using DIG-labelled primers and biotin labelled, species specific
probes (Barlic-Maganja and Grom, 2001) and recently, the use of species specific
fluorogenic-probe hydrolysis in real time RT-PCR (McGoldrick et al. 1998; McGoldrick et
al. 1999; Risatti et al. 2003). Real time RT-PCR affords significant increases in diagnostic
sensitivity when compared with virus isolation (Risatti et al. 2005). Conventional RT-PCR
has been found to be 100 % sensitive compared to virus isolation using a nested
amplification round and greater than 80 % sensitive using a single amplification round
(Harding et al. 1996). RT-PCR has greater analytical sensitivity than other diagnostic tests.
When performance was assessed using samples collected sequentially from experimentally
infected live animals, a nested RT-PCR detected virus on average 2 3 days earlier after
infection than virus isolation and 3 4 days earlier than antigen capture ELISA (Dewulf et
al. 2004a). The amplification capabilities of RT-PCR allowed detection of a much smaller
amount of analyte compared to virus isolation and other tests (McGoldrick et al. 1999;
Paton et al. 2000a). There exists little difference between the analytical sensitivity of
nested RT-PCR and real-time PCR (McGoldrick et al. 1999; Paton et al. 2000a).
McGoldrick and others (1999) found that a single tube closed system nested RT-PCR
method resulted in a reduction in contamination due to a decrease in manipulations.
Recently the inter-laboratory and inter-method variation of RT-PCR has been assessed to
determine the level of agreement between laboratories and protocols (Paton et al. 2000a;
Paton et al. 2000b). From these studies it was concluded that standardisation of RT-PCR
protocols resulted in more consistent sensitivity, however contamination due to poor
technique remained an issue even after standardisation of protocols. While RT-PCR is a
very sensitive and specific test for the diagnosis of CSF virus, it is not suited to all
conditions or all laboratories. Blacksell and others (2004b) found that sample degradation
at ambient temperature in a tropical environment had a detrimental impact on test
performance.
29
Chapter 1
30
Chapter 1
31
Chapter 1
1.2.5 Vaccination
Inactivated vaccines
Inactivated vaccines were commonly used from the 1930s up to 1970 before the
development of safe and potent live attenuated vaccines. These inactivated vaccines were
prepared from crystal violet and formalin-inactivated virus preparations but due to the
development of poor and short term immune responses, they were considered inefficient
(de Smit, 2000). The use of detergent-split tissue culture propagated CSF virus preparations
delivered in combination with oil or Quil A adjuvants showed great promise, with
demonstrated protection against challenge with a highly virulent strain 12 days after
vaccination (Dalsgaard and Overby, 1976). However, the development and availability of
high quality live attenuated vaccines likely discouraged further development of detergent
split vaccines (de Smit, 2000).
32
Chapter 1
organs has lost favour due to the need to maintain a large rabbit colony and the relative
inflexibility of the system, where shortages during epidemics can occur (Terpstra et al.
1990). Laos continues to produce CSF vaccine by inoculating rabbits with a C-strain virus
and harvesting the spleens for vaccine production (Jetterur, 1998).
The GPE vaccine strain was developed by serially passaging the ALD strain through a
range of cell lines at 30 C, while the Thiverval strain was developed by serial passage of
the Alfort strain more than 170 times in cell culture at 29 30 C (van Oirschot, 2003).
Both the Thiverval and GPE strain can be distinguished from their parent strains and field
virus through the identification of in vitro markers (van Oirschot, 2003).
Subunit vaccines
Subunit vaccines, like vectored vaccines described below, have the advantage that it is
possible to discriminate between animals that have had natural infection and those that have
been vaccinated by using an antibody test employing an Erns antigen ( de Smit, 2000;
Langedijk et al. 2001; van Oirschot, 2003). The vaccines have been dubbed DIVA
vaccines; differentiating infected from vaccinated individuals. These vaccines have
enormous advantage for countries demonstrating freedom from disease or countries
attempting to eradicate CSF virus, where the use of traditional live vaccines may conceal
inapparent infections that would otherwise be detected in the absence of vaccination.
Hulst et al. (1993) described a baculovirus expression system in insect cells for the
production of an E2 glycoprotein lacking the transmembrane region (note that the
terminology has changed and the glycoprotein was denoted E1 when this paper was
published; van Rijn et al.(1996), Bouma et al.(1999) and Moormann et al.(2000) and van
Oirschot, 2003 refer to this glycoprotein as E2). The expressed glycoprotein when
harvested from cell culture supernatant and subsequently purified, elicited a protective
immune response in pigs after being administered with a double water-oil adjuvant. Others
have used a similar methodology to further develop E2 subunit vaccines (Bouma et al.
1999; Moormann et al. 2000; van Rijn et al. 1996). Van Rijn and others (1996) also found
33
Chapter 1
that E2 subunit vaccines, which lacked either the highly conserved antigenic unit A or the
non-conserved B/C antigenic unit, were able to elicit an immune response that protected
against lethal challenge.
34
Chapter 1
Plasmid DNA vaccines expressing the E2 protein have been developed and while great
promise has been shown, mixed results have been achieved. Clinical protection against
lethal challenge, where no clinical signs were observed, has been achieved after multiple
vaccinations with a DNA vaccine expressing the entire E2 gene (Ganges et al. 2005).
Protection against lethal challenge has also been achieved using multiple vaccinations with
DNA expressing the entire E2 gene, however with elevated rectal temperatures in a number
of pigs post-challenge (Andrew et al. 2000). Protection without the development of clinical
signs was however achieved when pigs were inoculated with a large dose (1000 g) of
plasmid DNA administered intramuscularly (Andrew et al. 2000). A DNA vaccine
expressing the E2 gene lacking the transmembrane portion was able to confer immunity
and incomplete clinical protection with elevated rectal temperatures observed post
challenge (Markowska-Daniel et al. 2001). Interestingly, Markowska-Daniel et al.(2001)
found a similar DNA vaccine, which contained the entire E2 sequence, failed to confer
protective immunity. The co-delivery of E2 gene DNA together with porcine interleukin
(IL)-18 and CD154 genes via a plasmid resulted in an earlier appearance of neutralising
antibodies as compared to the E2 gene segment alone, less B-cell deficiency post challenge
and protected pigs against a lethal challenge (Wienhold et al. 2005)
Vaccines based on a series of peptides derived from the E2 N-terminal B/C domain
conjugated to bovine serum albumin have been developed and confer protection against
virus challenge using either a Freunds or aluminium adjuvant (Dong et al. 2002; Dong et
al. 2005). Like subunit and vectored vaccines described above, peptide and DNA vaccines
have potential as DIVA vaccines (Dong et al. 2002; Dong et al. 2005; Ganges et al.
2005).
Recently, the development of transgenic plants expressing the E2 glycoprotein of CSF virus
has been described (Legocki et al. 2005). Expression of the E2 glycoprotein in alfalfa and
orally administered to mice two times at 30 day intervals resulted in a rise in E2 specific
IgA and IgG antibody titre in faeces and serum respectively (Legocki et al. 2005). While
35
Chapter 1
much work remains to be done, this initial research shows promise for the novel production
and delivery of CSF vaccine and could become an important future control tool.
36
Chapter 1
NPLA, were greater than or equal to 12.5 and less than 25 then these pigs were able to
survive a lethal challenge whilst remaining viraemic and able to transmit virus. Pigs with
antibody titres greater than or equal to 25 and less than 50 displayed some clinical signs
without evidence of transmission. Antibody titres greater than or equal to 50 resulted in
complete protection with no virus transmission or clinical signs (Terpstra and Wensvoort,
1988). Vaccination with 32 g of baculovirus expressed E2 subunit vaccine on the other
hand conferred protection against lethal challenge if the serum antibody titre measured by
NPLA was greater than or equal to 50 (Bouma et al. 1999). Titres below 50 resulted in a
majority of challenged pigs succumbing to a lethal progression of disease. This difference
is supported by the finding that the E2 glycoprotein is not a major T-cell stimulator and is
not involved to a great extent in cellular immunity (Kimman et al. 1993). Protection
conferred by lower titres after C-strain vaccination indicates that mechanisms other than
neutralising antibodies are also playing a role.
37
Chapter 1
infections when used in combination with other control strategies such as hunting
(Moennig et al. 2003).
38
Chapter 1
39
Chapter 1
addition to separation from complex samples, incubation times with reagents are reduced
(Kijek et al. 2000; Lim et al. 1998; Tam and Lim, 2003; Yan et al. 2004) and the
concentration of analyte results in greater analytical sensitivity (Chapman and Ashton,
2003; Gilpatrick et al. 2000; Kijek et al. 2000; Yan et al. 2004). As a consequence, IMBs
have been used in the development of portable and rapid test kits ( Ndhlovu et al. 1995;
Tam and Lim, 2003).
Table 1.2 Summary of immunomagnetic bead detection systems
Detection System
Analyte
Authors
PCR/ RT-PCR
Norwalk-like virus
Norwalk virus
Cryptosporidium parvum
Plasmodium falciparum
Shigella sonnei
Bordetella pertussis
Group A Rotavirus
Salmonella sp.
Staphylococcus sp.
Staphylococcus sp.
Escherichia coli
Schistosoma haematobium
Atrazine (pesticide)
Chemiluminescence or
Staphylococcal enterotoxin B
electrochemiluminescence
Trichinellosis antibodies
ELISA
Colorimetric beads
Trichinella spiralis
40
Chapter 1
Activity 1: To develop, evaluate and implement a simple, rapid diagnostic test for CSF
The present diagnostic test for CSF is an antigen trapping ELISA that is suitable for use on
fresh field specimens. However, the application of this ELISA requires a suitably equipped
laboratory and trained personnel. Transport of specimens from the field to the central
laboratory continues to be a problem, and causes delays in the field workers providing an
answer to farmers. A robust and simple test will make specific laboratory diagnosis more
readily available and improve the service to farmers. The principle to be explored is to use
a magnetic bead to separate the antigen away from the crude sample suspension and to then
detect this antigen using an indicator system. In the case of a near-to-field test it is
necessary that the system does not require the operator to prepare any reagents and that the
reagents used are relatively stable in the environment where they will be applied.
41
Chapter 1
Activity 2: To establish and validate a system to apply locally produced CSF vaccine in Lao
villages
The current project (AS1/1994/038) is conducting active disease surveillance in a group of
8 pilot villages in Bolikhamxay province. These villages will be introduced to CSF
vaccination using the standard extension approach of the provincial animal health officers.
Serological monitoring will also be undertaken from these villages to monitor the antibody
status of the herd. The uptake of vaccination will be assessed and constraints on
implementation evaluated.
The project will develop a HACCP type approach to the implementation of a CSF
vaccination program through the district animal health system. To assist understanding of
the critical control points, studies will be conducted on the thermostability of the locally
produced CSF vaccine and the project will devise a procedure to evaluate vaccine potency.
In addition, the project will evaluate approaches to quality control of the vaccination
program. Husbandry and hygiene standards for pigs vary in Lao villages and it is very
likely that management practices have an impact on maternal antibody transfer. If possible,
animals in different management systems will be sampled to determine if there is
management related variability in maternal antibody transfer. Data will be analysed to
develop a strategy for CSF vaccination that is tailored to the needs of the village producers,
the vaccine properties and the capability and capacity of the DLF.
Activity 3: To measure the impact of the CSF vaccine program in the village pig production
system
A methodology will be developed to measure the socio-economic impact of CSF
vaccination. Used in conjunction with the continued monitoring of disease incidence and
production data, a comprehensive means of assessing the impact of a CSF vaccination
program will be achieved.
42
Chapter 1
Activity 5: To communicate project findings to extension staff and animal health and
production scientists in national, regional and international networks
An important component of the project will be the use of regular feedback sessions in
participating villages to understand the progress of the work and to ascertain farmers
responses to the activities that are being undertaken. Project outputs will be disseminated
nationally through a workshop and scientific outputs from the work will be reported in the
international literature.
43
Chapter 1
1. To describe the performance of the smallholder pig sector and develop a better
understanding of the importance of CSF to pig production in this sector.
What are the performance characteristics of the Lao smallholder pig sector in terms of
reproductive output and performance?
What are the limiting factors for effective pig production in the smallholder sector?
2. To develop, validate and implement a simple, rapid and portable diagnostic test for the
diagnosis of CSF.
What are the optimum conditions under which immunomagnetic bead technology can
be applied to CSF diagnostics?
What is the relative diagnostic performance of the newly developed test format?
3. Determine the stability of the locally produced CSF vaccine under different temperature
storage conditions.
Can the locally produced C-strain vaccine be stored for long periods of time at 4 C and
maintain a high level of immunogenicity?
Under optimal storage conditions of 20 C, how long is the locally produced C-strain
vaccine stable for?
44
Chapter 2
Chapter 2.
Pig production and health in Laos
45
Chapter 2
46
Chapter 2
47
Chapter 2
management practices undertaken and the results of the longitudinal study to examine
productivity and health of the smallholder pig raising system.
2.2 Methods
The research presented in this chapter was carried out by a team of personnel. The author
participated in all facets of the research, however the dominant roles were:
(i) Questionnaire design and trial run. Translation in to Lao language was performed by
Dr. Syseng Khounsy (Lao project leader)
(ii) Baseline survey of the first eight villages recruited into the survey in conjunction with
Dr. Syseng Khounsy and district and provincial livestock officers
(iii) Training of field staff in data collection and management at the start of the survey in
May 2002
(iv) Training of field staff in sample collection (with Dr. Syseng Khounsy)
(v) Data quality checking and entry into MS Excel spreadsheets from 2002 2004, data
entry was subsequently carried out by Lao project staff
(vi) All data analysis
All other work was coordinated and/or conducted by Dr. Syseng Khounsy with support
from the Australian project leader, Dr. Laurence Gleeson.
48
Chapter 2
season and is a mix of low-land and up-land agricultural areas with some elevated country
near the northern and north-western district border. Bolikhamxay province farmers raise
approximately 36 thousand pigs or 3 % of the national herd (MAF, 2000), almost entirely
in the smallholder sector (MAF, 2002). A second province, Xieng Khouang, bordering
Vietnam in the north-east of Laos (refer to Figures 1.1 and 1.2) was also selected for
inclusion in pig production and health surveillance. Two districts, Paek and Nong Het, were
selected in collaboration with an AusAID funded livestock nutrition project entitled
Forages and Livestock Systems Project. Xieng Khouang province farmers raise
approximately 74 thousand pigs or 7 % of the national herd (MAF, 2000).
49
Chapter 2
Vietnam
Bolikhan
Thaphabath
Viengthong
Paksan
Pakading
Khamkeuth
Thailand
Bolikhamxay province
50
Chapter 2
A baseline survey was also conducted at the initial meeting to gain an understanding of the
main production systems in place in each village, trading practices, nutrition and
husbandry, disease control and proximity to other villages, markets, schools and major
transport routes. These surveys were conducted in a group where half the village pig raisers
were asked to meet in the morning and the other half in the afternoon to minimise drawing
too heavily on the village agricultural labour force. The questionnaire was conducted by the
Lao national project leader and the answers were recorded after the group had come to a
consensus. Data was entered into a Microsoft (MS) Excel spreadsheet and descriptive
characteristics were extracted for presentation in this thesis.
51
Chapter 2
ensure any problems or issues were addressed early and all staff involved in the survey
were comfortable and confident in collecting and processing information.
52
Chapter 2
The data collected in Xieng Khouang province from January to September 2005 was of a
poor standard and the only information that could be reliably used was average litter size.
This will serve as the only reproductive performance indicator for this province.
53
Chapter 2
Province:
Reporting Officer:
Certified by:
10-12
7-9
4-6
Boars
Breeding Pigs
Sows
10-12
7-9
4-6
0-3
Boars
Breeding Pigs
0-3
Sows
No. Live
Born Piglets
10-12
7-9
4-6
0-3
Boars
Sows
Breeding Pigs
Litters
No.
Producing
sows
Disease Reports
(yes/no)
Total
Figure 2.2 (a) Questionnaire for the collection of village pig health and production information
54
Disease
Reports
Village name:
Sample
Submit
No. Farmers
per group
Farmer Group
Number
Date:
Chapter 2
Village name:
District:
Province:
Reporting Officer:
Certified by:
Total
Figure 2.2 (b) Questionnaire for the collection of village pig in-take and off-take from the village production unit
55
Feral Pigs
10-12
7-9
4-6
0-3
Boars
Procurements in this
reporting period came
from where?
Breeding Pigs
Sows
10-12
7-9
4-6
0-3
Boars
Breeding Pigs
Sows
10-12
7-9
4-6
Home Consumption
0-3
Boars
Sows
Breeding Pigs
Were boars
hired from
group
Farmer Group
Number
Date:
Chapter 2
Incidence =
Number of outbreaks
x 100
Number of village years
For those villages where outbreaks were confirmed in the laboratory, further analysis was
conducted to describe the piglet mortalities associated with these outbreaks in comparison
to baseline mortality levels and animal movement in the village. To achieve this end, the
baseline mortality was calculated as the average number of piglet mortalities each month in
the survey and animal movement was represented as total pig sales in and out of the village
production unit.
56
Chapter 2
2.3 Results
2.3.1 Baseline surveys
The village level descriptive variables of pig production and health for the eight villages
enrolled into the production and health survey in May 2002 are presented in Table 2.1
below. The majority of pigs were purchased by farmers from other farmers in the same
village or from near-by villages. Only one village had pig producers who commonly
purchased pigs from a middleman, where a middleman was a livestock buyer from a nearby village or town who supplied a vehicle for transport of stock to market or other villages.
A middleman trader was much more important for the sale of pigs. All villages reported
that the middleman was the major receiver of pigs sold. Two villages reported near-by
villages as major destinations for sale and only one village reported that some pigs are sold
direct to market.
All villages reported that low quality feed stuffs such as rice bran, household scraps, waste
from alcohol production and cut-and-carry weeds and grasses constituted the main source
of nutrition for their pigs. No villages were using improved feeds such as corn or cassava at
the time the survey was conducted. Two villages reported that a small number of farmers
provided supplementary feed for breeders, growers and piglets. In all villages, water supply
was restricted and was mixed with the feed, where no separate watering trough was
provided. The median time taken tending pigs each day was two hours with a range of one
to four hours, where the majority of time taken was for the collection and preparation of cut
and carry grasses and weeds.
Farmers raising pigs indicated a genuine fear of disease as shown by the majority of
villages (7/8) responding to occurrence of disease by selling sick pigs and apparently
healthy piglets. Farmers from three of the seven villages who reported selling pigs
responded that in an outbreak situation sick pigs and apparently healthy pigs were sold at
reduced prices. Farmers from half of the villages responded that they themselves
implemented quarantine and animal movement restrictions when they had sick pigs in an
attempt to prevent pigs belonging to the same household becoming sick. On the other hand,
57
Chapter 2
farmers from less than half of the villages reported that they implemented animal
movement restrictions and quarantine when other farmers in the village had sick pigs. In all
cases where animal movement restrictions were implemented, small piglets that were able
to escape from pens remained free to roam. Several villages reported that farmers used
antibiotics to treat sick pigs or prevent apparently healthy pigs from becoming sick. The
most common antibiotic administered was referred to as Oxy, which was oxytetracycline
and purchased from the VVW. Four of the eight villages surveyed reported vaccination
against CSF, but only two of these villages reported that greater than 50 % of pigs were
vaccinated.
All villages raised the indigenous Moo laat pig as the main breed with a majority of
villages (7/8) also having some farmers raising a small number of exotic/Moo Laat cross
breed pigs. The most common of the exotic crosses was with Large White. Most piglets
were weaned after eight weeks of age and one village reported that piglets were not weaned
until the sows resented suckling.
58
Chapter 2
Table 2.1 Village level descriptive variables of pig production and health in Bolikhamxay
province, May 2002
Proportion of
villages
Median
(range)
Marketing
Distance to market (km)
7.5 (4 - 10)
Middleman
1/8
of purchase
Within village
8/8
Nearby village
8/8
Market
0/8
Middleman
8/8
Within village
0/8
Nearby village
2/8
Market
1/8
Rice bran
8/8
Household scraps
8/8
8/8
8/8
Cassava
0/8
Corn
0/8
Breeders
2/8
Growers
2/8
Pre-weaned
2/8
Restricted
8/8
Unrestricted
0/8
Supplementary feeding
Water availability
Time tending pigs per day
(hours)
2 (1 - 4)
59
Chapter 2
7/8
sick pigs
7/8
4/8
1/8
Administer antibiotics
3/8
7/8
3/8
Administer antibiotics
4/8
4/8
Production
Pig breeds
8/8
7/8
1-8
0/8
>8
7/8
1/8
60
Chapter 2
61
Chapter 2
Table 2.2 Indicators of reproductive performance for smallholder sows in Bolikhamxay province, Laos, 2002 to 2005.
Village
District
Months in
Survey
Median monthly
sow population
(95% CI)
Median monthly
boar population
(95% CI)
Number
of litters
Average
litter size
Sow/Boar
ratio
Pre-weaning
mortality (%)
Houana
Bolikhan
41
42 (39, 45)
1 (1, 2)
227
5.3
42.0
6.3
Phonsavath
41
83 (80, 90)
1 (0, 2)
159
5.6
83.0
2.1
Nalong
41
95 (90, 100)
5 (4, 6)
271
6.7
19.0
3.1
Nampa
41
25 (22, 26)
2 (2, 2)
96
6.1
12.5
15.0
Phonthong
19
51 (49, 55)
1 (1, 1)
62
6.0
51.0
15.3
Hatpho
19
26 (25, 30)
1 (0, 1)
48
6.7
26.0
0.3
Songkhonmai
19
14 (12, 18)
1 (1, 1)
26
6.4
14.0
1.2
Na-o
19
5 (4, 6)
0 (0, 1)
7.3
0.0
41
10 (9, 12)
1 (1, 1)
32
6.4
10.0
8.3
Borthoun
41
39 (35, 41)
1 (1, 1)
125
5.7
39.0
1.1
Nabone
41
20 (18, 22)
2 (1, 2)
31
5.9
10.0
2.2
Hadxaykham
41
43 (35, 47)
3 (2, 3)
78
5.9
14.3
11.9
Donsai
19
6 (5, 6)
1 (1, 1)
5.5
6.0
0.0
Phonsai
19
26 (20, 28)
1 (1, 1)
26
5.0
26.0
0.0
Namkhou
19
25 (23, 28)
0 (0, 0)
24
5.7
0.0
Namdeua
19
51 (46, 51)
2 (2, 2)
63
6.1
25.5
0.0
Paksa
Pakading
62
Chapter 2
63
Chapter 2
100
a)
80
All Villages
60
Bolikhan District
40
Pakading District
20
0
Deaths
Sales
Home Con.
100
b)
80
60
40
20
0
Sows
Boars
0-3
4-6
7-9
10-12
64
Chapter 2
100
80
All villages
60
Bolikhan District
40
Pakading District
20
0
Sows
Boars
0-3
4-6
7-9
10-12
65
Chapter 2
Table 2.3 Number of young piglets entering the village production units in Bolikhamxay province, Laos, 2002 - 2005
Median monthly
sow population
(95% CI)
Median
monthly
number of
farmers
Piglets Born
Number of 0 - 3 month
old piglets purchased
Village
District
Months in
Survey
Houana
Bolikhan
41
42 (39, 45)
57
1213
32
Phonsavath
41
83 (80, 90)
107
891
23
Nalong
41
95 (90, 100)
103
1819
92
Nampa
41
25 (22, 26)
32
587
40
Phonthong
19
51 (49, 55)
50
373
Hatpho
19
26 (25, 30)
40
319
Songkhonmai
19
14 (12, 18)
27
165
49
Na-o
19
5 (4, 6)
17
51
83
41
10 (9, 12)
26
205
73
Borthoun
41
39 (35, 41)
46
714
38
Nabone
41
20 (18, 22)
18
183
11
Hadxaykham
41
43 (35, 47)
51
462
178
Donsai
19
6 (5, 6)
16
11
732
Phonsai
19
26 (20, 28)
39
131
50
Namkhou
19
25 (23, 28)
32
137
37
Namdeua
19
51 (46, 51)
59
383
42
Paksa
Pakading
66
Chapter 2
67
Chapter 2
100
50
80
60
30
40
20
20
0
Total sales
40
0 - 3 mo deaths
60
30
40
20
10
20
10
May 04
Jun 04
Jul 04
Aug 04
Sep 04
Oct 04
Nov 04
Dec 04
Jan 05
Feb 05
Mar 05
Apr 05
50
300
100
C. Nalong
40
30
30
40
20
10
20
10
20
50
0
40
(1.9 6.4)
60
150
100
80
Total sales
200
50
D. Nampa
0 - 3 mo deaths
(1.2 3.5)
May 04
Jun 04
Jul 04
Aug 04
Sep 04
Oct 04
Nov 04
Dec 04
Jan 05
Feb 05
Mar 05
Apr 05
Total sales
250
40
(0.4 1.5)
Figure 2.5 A D Piglet mortality and sale patterns associated with CSF virus
outbreaks in villages in Bolikhamxay province, Laos. Solid line represents monthly
mortality and the grey bars represent total monthly pig sales. The average monthly
mortality SD is in parenthesis. Arrows represent when samples were first
collected that tested positive for CSF virus antigen
68
0 - 3 mo deaths
(3.0 6.6)
May 04
Jun 04
Jul 04
Aug 04
Sep 04
Oct 04
Nov 04
Dec 04
Jan 05
Feb 05
Mar 05
Apr 05
Total sales
80
50
B. Phonsavath
0 - 3 mo deaths
A. Phonthong
Feb 04
Mar 04
Apr 04
May 04
Jun 04
Jul 04
Aug 04
Sep 04
Oct 04
Nov 04
Dec 04
Jan 05
100
Chapter 2
200
50
100
40
80
50
80
20
40
0
40
60
30
40
20
10
20
10
0 - 3 mo deaths
30
(1.9 6.4)
Jan 03
Feb 03
Mar 03
Apr 03
May 03
Jun 03
Jul 03
Aug 03
Sep 03
Oct 03
Nov 03
Dec 03
120
Total sales
(0.2 0.8)
May 03
Jun 03
Jul 03
Aug 03
Sep 03
Oct 03
Nov 03
Dec 03
Jan 04
Feb 04
Mar 04
Apr 04
100
50
G. Houana
(1.6 5.7)
40
30
40
20
20
10
Jan 03
Feb 03
Mar 03
Apr 03
May 03
Jun 03
Jul 03
Aug 03
Sep 03
Oct 03
Nov 03
Dec 03
60
0 - 3 mo deaths
80
Total sales
Total sales
F. Nampa
0 - 3 mo deaths
E. Borthoun
160
Figure 2.5 E G Piglet mortality and sale patterns associated with CSF virus
outbreaks in villages in Bolikhamxay province, Laos. Solid line represents monthly
mortality and the grey bars represent total monthly pig sales. The average monthly
mortality SD is in parenthesis. Arrows represent when samples were collected and
tested positive for CSF virus antigen
69
Chapter 2
70
Chapter 2
71
Chapter 2
The number of litters-per-sow-per-year in Laos does not compare favourably with the
tropical commercial sector, which averages greater than two litters-per-sow-per-year
(Kunavongkrit and Heard, 2000), however a similar result was seen in Kenya where the
number of litters per-sow-per-year was one (Wabacha et al. 2004b).
The final measure of sow reproductive performance was the mortality rate of unweaned
piglets. The median rate in the villages studied in Bolikhamxay province was low at 2 % ,
compared to a median of 11.1 % in a Philippines study (Lanada et al. 2005). However, the
results of the study described in this chapter are likely biased as a majority of farmers
reported that young pigs would be sold if disease events occurred in a village or if pigs
became sick.
The results of this study provide evidence that boar access was an important limiting factor
to smallholder pig production. The median sow: boar ratio of 22:1 observed in this study
did not compare well with a 4.1:1 ratio in the Philippines (Lanada et al. 2005). A low
sow/boar ratio does not however necessarily translate into improved reproductive
performance. In this study no significant relationship could be demonstrated, indicating that
access to boars and the servicing of sows is more complex than simply having a large
number of boars in the village. Likewise, the large number of boars in the Philippines study
did not correspond with an increase in the number of sows serviced (Lanada et al. 2005).
Access to boars was found to be a complex issue and was limited by such things as
proximity within a village, a pig raisers knowledge and understanding of reproductive
cycles (including oestrus detection and boar training) and access to boars (if hired) if and
when oestrus was detected (Lanada et al. 2005). These issues of complexity and boar
access also likely exist in the smallholder sector of Laos, contributing to overall poor sow
reproductive performance. As a consequence of poor boar access, inbreeding was also
highly probable, subsequently affecting downstream elements of pig production if
undesirable traits were recessively inherited.
72
Chapter 2
The results of off-take and in-take are in contrast to the agricultural census data collected in
1998-1999 (MAF, 2000), which stated that the majority of pigs in Laos were in the 3-9
month age bracket. The results of this study suggest that a majority of pigs in Bolikhamxay
province leave the village production unit by the age of three months. Without data from
other provinces, particularly in the north of the country where the majority of pigs are
produced, it is difficult to speculate as to why this might occur and if the findings of this
study are representative of the entire country.
The results of this study suggest that the dominant form of pig production in the survey
group are farrow-finisher farmers each with a small number of sows and where the
finishing age was approximately three months of age. A number of farmers in each village
also raise only growers and some villages practice predominantly this form of production.
During a follow up interview with farmers in several villages, approximately 12 months
into the survey by the Lao project leader, Dr. Syseng Khounsy, the reasons for the early
departure of pigs from the production unit were determined. The main reason was nutrition,
where high costs in terms of labour and provision of high quality feed were unable to be
met by smallholder producers in these villages. Secondly, fear of disease was a contributing
factor to the early sale of pigs.
The potential for economic gain would be greatly increased if smallholder producers could
afford to hold pigs for a longer period of time and sell at a greater weight. Village piglets at
three months of age weigh approximately 10 kg compared to approximately 20 25 kg at
six months of age (Dr. Syseng Khounsy, personal communication). To enable farmers to
hold pigs for a longer period of time, a number of factors would need to be improved.
Firstly, nutrition would need to be improved, where farmers have access to higher quality
feed stuffs such as cassava, corn, maize and/or legumes such as Stylosanthes guianensis.
Keoboualapheth and Mickled (2003) found Lao indigenous breed pigs to have superior
growth rates when Stylosanthes guianensis was incorporated into their feed, as compared to
traditional feed stuffs such as rice bran. Improved nutrition should, however, be combined
with improved management practices of water provision and treatment of parasites to
73
Chapter 2
facilitate improved weight gain. Secondly, improved control of diseases such as CSF would
greatly reduce mortalities. But equally as important, improved disease control would
provide farmers with added confidence that their stock will not succumb to a severe disease
event. One way of achieving such a goal would be the implementation of effective
vaccination programs together with controlled introduction of new stock into villages.
74
Chapter 2
opportunity to sell sick or exposed pigs and so skew CSF mortality estimates (Blacksell
(2001). Results presented in this chapter support this contention as sales tended to increase
when a disease event was recognised, so that farmers losses were minimised. In several
villages pig sales rose sharply at the time or immediately after CSF virus was detected. In
Phonthong village, while CSF could not be directly related to the peak in mortalities, risk
aversion was clearly evident as reflected by the sharp down turn in deaths corresponding
with a sharp upturn in sales.
The sale of pigs during outbreaks of CSF would be expected to facilitate the spread of CSF
virus to surrounding villages. Based on the findings of this study and on previous
serological prevalence studies (Blacksell, 2001), it is proposed that CSF virus is endemic in
pockets of the country facilitated by localised trading of infected pigs and pork products.
This mode of disease transmission and spread has been well recognised (Ribbens et al.
2004) and incursions into non-endemic areas would then sporadically occur as a
consequence of introducing infected pigs or pork products into susceptible populations.
More detailed epidemiological studies using serology and detailed outbreak investigation
are needed to clearly demonstrate this proposal, and if clearly demonstrated, may provide a
useful means of using targeted vaccination strategies to control CSF in Laos.
Conclusions
Smallholder pig production in Bolikhamxay province, Laos, was found to be poor in
comparison to accepted standards in the tropical commercial pig sector. The performance
indicators in Laos are however comparable to the smallholder pig production sectors of
other tropical countries. Key factors such as nutrition, management, breed and disease play
a critical role in reducing the potential production output. Infectious diseases of swine,
including CSF, influence trade behaviour as farmers seek to realise the economic potential
of sick pigs or pigs they believe will become sick. The incidence of CSF in Bolikhan
district was substantially greater than that observed in Pakading district; however no clear
difference in the production output from the smallholder pig sector of the two districts
could be demonstrated.
75
Chapter 2
76
Chapter 3
Chapter 3.
General materials and methods
77
Chapter 3
78
Chapter 3
3.1 Introduction
This chapter describes the general materials and methods of standard procedures used for
the detection of live virus and viral antigen and for the detection of serum antibodies to
CSF virus. Specific methods and materials used for the development and assessment of the
IMB-ELISA are described in the individual chapters outlining particular experiments.
79
Chapter 3
80
Chapter 3
filtered (0.45 M, Sartorius AG, Germany), viral isolation was achieved by PLA whereby 1
part filtered homogenate was mixed with 9 parts freshly prepared PK15 cells in growth
medium at a cell concentration of 2 x 105 cells/mL. One millilitre of virus/cell suspension
was then inoculated into 4 wells of a 24 well tissue culture plate (Nunc, Denmark). Four
wells received uninfected cells as negative controls. The plates were incubated for 3 4
days at 37 C and 5 % CO2 in a humidified incubator before staining the cells for the
presence of replicating CSF virus by PLA as described above.
81
Chapter 3
L of freshly prepared cells added to all wells as described in the previous paragraph. One
row of uninoculated cell controls was included on each plate and the plates incubated and
stained according to the PLA described previously and the TCID50 end point was
calculated.
a.
S1
S2
S3
S4
S5
S6
10
10
10
10
10
10
100.5
100.5
100.5
100.5
100.5
100.5
101.0
101.0
101.0
101.0
101.0
101.0
101.5
101.5
101.5
101.5
101.5
101.5
102.0
102.0
102.0
102.0
102.0
102.0
102.5
102.5
102.5
102.5
102.5
102.5
103.0
103.0
103.0
103.0
103.0
103.0
10
11
12
10
11
12
b.
S1
1
S2
3
S3
7
10
10
10
10
10
10
10
10
10
10
10
100
100.5
100.5
100.5
100.5
100.5
100.5
100.5
100.5
100.5
100.5
100.5
100.5
101.0
101.0
101.0
101.0
101.0
101.0
101.0
101.0
101.0
101.0
101.0
101.0
101.5
101.5
101.5
101.5
101.5
101.5
101.5
101.5
101.5
101.5
101.5
101.5
102.0
102.0
102.0
102.0
102.0
102.0
102.0
102.0
102.0
102.0
102.0
102.0
102.5
102.5
102.5
102.5
102.5
102.5
102.5
102.5
102.5
102.5
102.5
102.5
103.0
103.0
103.0
103.0
103.0
103.0
103.0
103.0
103.0
103.0
103.0
103.0
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
CC
Figure 3.1 a) Transfer plate and b) cell culture plate. S1: sample 1; S2: sample 2 etc; Cc,
uninoculated cell control
82
Chapter 3
Sample preparation
Spleen and occasionally lymph node and tonsil tissue samples were processed into a 20 %
(w/v) homogenate in 1 % NP40 (v/v) in PBSA using a mortar and pestle and transferred to
a centrifuge tube. The homogenised samples were allowed to stand at room temperature for
2 h with mixing every 10 20 min by vortex. Following incubation the tissue preparation
was centrifuged at 2000 rpm for 10 min to pellet tissue debris. The supernatant was
collected and tested undiluted in the AC-ELISA. Processed samples were kept at -85 C for
long term storage.
Test format
The AC-ELISA utilised two microtitre plates, the first (referred to as the transfer plate) was
a 96-well U-bottom untreated polystyrene microtitre plate (Nunc, Denmark) used to
incubate the monoclonal antibody with the undiluted sample and controls. The second
(referred to as the ELISA plate) was a 96-well flat-bottom polystyrene MaxisorpTM
microtitre plate (Nunc, Denmark) used to capture the viral antigen by coating with goat
anti-pestivirus polyclonal antibody.
83
Chapter 3
The transfer plate was first blocked by the addition of 200 L/well of blocking buffer A
(Appendix I) and incubated overnight at 4 C in a humidified sealed plastic container. The
ELISA plate was coated with 100 L/well anti-pestivirus polyclonal antibody diluted
1:4000 in carbonate buffer (Appendix I) and incubated overnight as per transfer plate.
Following the overnight incubation, the ELISA plate was washed 3 times with PBST
(Appendix I) and potential non-specific binding sites blocked by the addition of 200
L/well of blocking buffer A and incubated stationary for 60 min at 37 C. The transfer
plate was also washed 3 times with PBST and 100 L of each test sample, positive and
negative control samples and reagent control (PBST) were added to two appropriate wells
(refer to plate format below). One hundred microlitres per well of NS3 pestivirus group
reactive monoclonal antibody (MAb) diluted 1:10 in PBST was added to columns 1, 3 and
5 of the plate and 100 L/well of negative control MAb diluted 1:10 in PBST was added to
columns 2, 4 and 6 (refer to plate format below, Figure 3.2). The transfer plate was
incubated stationary for 1 h at 37 C in a humidified incubator.
C++
C++
10
10
C++
C++
11
11
C+
C+
12
12
C+
C+
13
13
C-
C-
14
14
C-
C-
15
15
Cc
Cc
16
16
Cc
Cc
10
11
12
84
Chapter 3
10
11
12
C++
C++
C++
C++
10
10
10
10
C++
C++
C++
C++
11
11
11
11
C+
C+
C+
C+
12
12
12
12
C+
C+
C+
C+
13
13
13
13
C-
C-
C-
C-
14
14
14
14
C-
C-
C-
C-
15
15
15
15
Cc
Cc
Cc
Cc
16
16
16
16
Cc
Cc
Cc
Cc
At the completion of the ELISA plate blocking step, the plate was left to stand at room
temperature until the transfer plate incubation was complete. Then the ELISA plate was
washed 3 times with PBST and 95 L volumes of the sample/MAb mixtures transferred in
duplicate from the transfer plate to the appropriate wells (refer to plate format, Figure 3.2).
The ELISA plate was then incubated stationary for 90 min at 37 C in a humidified
incubator. At the completion of the incubation, the plate was washed 5 times and reblocked with the addition of 100 L/well of blocking buffer B and incubated for 30 min at
37 C with shaking. Following blocking incubation, the plate was washed 5 times and 100
L/well of rabbit anti-mouse IgG HRP conjugate (Zymed, USA) diluted 1:2000 in
conjugate dilution buffer (Appendix I) was added to all wells. At the completion of the
incubation the plate was washed 5 times and 100 L/well of activated TMB substrate
(Appendix I) was added and incubated at room temperature for 10 minutes. The reaction
was stopped after 10 min with the addition of 100 L/well of 1M H2SO4 and the optical
density read at 450 nm (OD450nm) on a microplate reader (Labsystems, Finland).
85
Chapter 3
The results were interpreted by calculating a signal to noise ratio (S/N) for each sample and
controls using the following equation:
S/N Ratio =
As recommended by Shannon et al. (1993), the following interpretation was made for each
sample: a S/N ratio greater than 2.00 was considered positive for the presence of CSF viral
antigen; a S/N ratio between 1.50 and 1.99 was considered doubtful and the test repeated
and a S/N ratio less than 1.50 was considered negative for the presence of CSF viral
antigen. In addition to the recommendations made by Shannon et al. (1993), upper and
lower control limits were placed on the raw optical density readout data to serve as an
additional control measure to minimise the likelihood of false positive results. Samples
with an optical density of less than or equal to 0.15 were considered negative even if the
signal to noise ratio was greater than or equal to 2.00. This cut-off limit was established as
3 times the standard deviation of 10 runs of four replicates of the negative control, 40
replicates in total.
86
Chapter 3
Blood samples were collected in the absence of anticoagulant and the serum allowed to
separate at 4 C before collection. If samples were sent to the laboratory as separated blood,
centrifugation at 1000 x g for 15 min was used to clarify the serum fraction. Serum was
collected into a sterile 2 mL tube and the complement was inactivated by heating at 56 C
for 30 min. Heat inactivated positive and negative control serums were supplied by CSIRO
AAHL. All test and control sera were stored at 20 C until tested.
The virus used in the NPLA was isolated from Khammuanne province in the central region
of Laos in September 1998 and was designated Lao/Kham225. Before use in the NPLA the
isolate had undergone two passages through pigs and two passages through cell culture,
once each through SK6 and PK15. Lao/Kham225 was genotyped as 2.2 (Blacksell et al.
2004a; Blacksell et al. 2005) and found to be a pathogenic virus causing an acute course of
disease (Blacksell, 2001). Strain Alfort/187 has been recommended for use by the European
Union (EU diagnostic manual for CSF diagnosis 2002 2nd draft) and has been used as the
standard virus for neutralising assays by a number of researchers (Clavijo et al. 2001;
Terzic et al. 2003). Like Lao/Kham225, Alfort/187 is a highly virulent strain causing an
acute course of disease (Floegel-Niesmann et al. 2003). Due to the relative lack of
biohazard control at the diagnostic laboratory in Vientiane, a local strain of CSF virus was
used to minimise the risk of introducing a new CSF genotype into the Lao pig population.
Alfort/187 belongs to genogroup 1.1 (Floegel-Niesmann et al. 2003) while only genotypes
2.1 and 2.2 have been identified in Laos (Blacksell et al. 2005; Blacksell et al. 2004a).
87
Chapter 3
Test Method
Negative control and test serum were serially diluted two-fold from 1:4 to 1:32 and positive
control serum was serially diluted two-fold from 1:20 to 1:2560 in duplicate. All dilutions
were carried out in growth medium to a final volume of 50 L (refer to plate format below,
Figure 3.3).
The standard virus stock was diluted in growth medium to a final virus concentration of
approximately 200 TCID50 / 50 L. Fifty microlitres of diluted virus was dispensed to all
wells containing test and control serum. A back titration of the standard working dilution of
virus was set up to ensure a virus concentration of 30 300 TCID50 / 50 L was included in
each well (OIE, 2004). The working dilution of virus was serially diluted ten-fold by
transferring 40 L of the working dilution to 360 L of growth medium for a 10-1 dilution.
After mixing and changing pipette tip, 40 L of the 10-1 dilution was transferred to 360 L
of growth medium for a 10-2 dilution and the process repeated once more for a final dilution
of 10-3. Fifty microlitres of each dilution was added in quadruplicate to 50 L of growth
medium in accordance with the plate format below. One hundred microlitres of growth
medium was added to each of eight wells (refer to plate format below, Figure 3.3) for the
cell controls.
For the neutralisation step, the plates were gently mixed and incubated for 1 h at 37 C and
5 % CO2 in a humidified incubator. Following the incubation period 100 L of freshly
prepared PK15 cells were added to all wells at a concentration of 2 x 105 cells/mL. The
plates were incubated for 3 to 4 days at 37 C and 5 % CO2 in a humidified incubator and
the cells checked daily to ensure healthy growth. Any serum toxicity or poor growth was
recorded for later interpretation of results. Following this incubation period the cells were
fixed and stained according to the PLA described previously. Neutralisation titres were
expressed as the reciprocal of the highest serum dilution that prevented viral growth in 50
% of two replicate wells. Test sera with a neutralisation titre greater than 1:32 were retested
and diluted to a final concentration of 1:512 to determine the end point titre of virus
neutralisation.
88
Chapter 3
a.
1
C+
C+
C-
C-
120
1:20
1:4
1:4
1:40
1:40
1:8
1:80
1:80
1:160
10
11
12
Wd
Wd
S1 1:4
S1 1:4
S2 1:4
S2 1:4
S3 1:4
S3 1:4
1:8
Wd
Wd
1:8
1:8
1:8
1:8
1:8
1:8
1:16
1:16
10-1
10-1
1:16
1:16
1:16
1:16
1:16
1:16
1:160
1:32
1:32
10-1
10-1
1:32
1:32
1:32
1:32
1:32
1:32
1:320
1:320
Cc
Cc
10-2
10-2
S4 1:4
S4 1:4
S5 1:4
S5 1:4
S6 1:4
S6 1:4
1:640
1:640
Cc
Cc
10-2
10-2
1:8
1:8
1:8
1:8
1:8
1:8
1:1280
1:1280
Cc
Cc
10-3
10-3
1:16
1:16
1:16
1:16
1:16
1:16
1:2560
1:2560
Cc
Cc
10-3
10-3
1:32
1:32
1:32
1:32
1:32
1:32
10
11
12
S7 1:4
S7 1:4
S8 1:4
S8 1:4
S9 1:4
S9 1:4
S10 1:4
S10 1:4
S11 1:4
S11 1:4
S12 1:4
S12 1:4
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
S13 1:4
S13 1:4
S14 1:4
S14 1:4
S15 1:4
S15 1:4
S16 1:4
S16 1:4
S17 1:4
S17 1:4
S18 1:4
S18 1:4
b.
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:8
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:16
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
1:32
Figure 3.3 a) Control plate and b) Serum plate. C+, positive control serum; C-, negative
control serum; Wd, Working dilution of virus; Cc, Cell control; S1, Serum sample 1; S2,
Serum sample 2 etc
89
Chapter 4
90
Chapter 4
Chapter 4.
IMB-ELISA development
91
Chapter 4
92
Chapter 4
4.1 Introduction
The definitive diagnosis of CSF is reliant on the identification of virus in diagnostic
specimens. The methods generally employed are either virus isolation in tissue culture with
subsequent identification using virus specific antibodies, antigen detection by ELISA, FAT
and PCR detection of viral RNA. In Laos, accurate laboratory diagnosis is especially
important as CSF virus is endemic and the skills needed to make a presumptive clinical
diagnosis are somewhat lacking (Blacksell, 2001). Blacksell (2001) also described a
number of important constraints to the maintenance of effective disease diagnosis; first and
foremost was minimal infrastructure for specimen collection and submission. Other factors
hindering an effective diagnostic service included lack of facilities for sample storage, lack
of financial support for field and laboratory activities, lack of continued interest shown by
district and provincial livestock officers and the redeployment of trained staff to other
government sectors. The AC-ELISA is recognised as a robust diagnostic methodology and
as such was deemed the most appropriate technology to be introduced to Laos for CSF
diagnosis (Blacksell, 2001). To off-set the constraints encountered in collecting, storing and
transporting specimens in a tropical country with poor infrastructure, sensitive, relatively
inexpensive, robust and more rapid diagnostic tests are required. This chapter describes the
development of a robust, cheap, rapid and potentially portable diagnostic test for CSF virus.
The test described utilised carboxyl modified polystyrene paramagnetic particles
(immunomagnetic beads {IMB}) to capture viral antigen onto a solid surface followed by
an ELISA detection procedure.
This chapter describes the steps taken to assess and optimise the development of the IMBELISA. In short, reaction conditions were optimised to covalently attach polyclonal capture
antibody to the surface of the IMBs so as to maximise antigen binding capacity. Two
polyclonal antibodies were assessed in the course of assay development; an anti-pestivirus
antibody raised in a goat at CSIRO AAHL, Australia and an anti-CSF virus antibody raised
in rabbits at the National Animal Health Centre, Laos. Optimisation of antibody attachment
to the beads was followed by optimisation of ELISA reaction conditions that included bead
93
Chapter 4
volume per reaction, monoclonal antibody concentration, conjugate concentration and the
reaction times.
In order to present an easily followed progression of steps taken to develop and optimise
the IMB-ELISA described in this chapter, a general materials and methods section
describing the reagents and materials used will be followed by a section entitled Methods
and results for the development of IMB-ELISA. This section will contain methods and
results for each stage of test development.
4.2.2 Antibodies
Polyclonal trapping antibodies
Anti-pestivirus goat polyclonal antibody was produced and supplied by the viral
diagnostics group of the CSIRO AAHL, Australia. Hyperimmune goat serum was purified
by caprylic acid/ammonium sulphate precipitation (see below), reconstituted in PBSA and
supplied ready for use. Storage was at 20 C in 100 L aliquots.
Rabbit polyclonal antibody against CSF virus was produced at the Lao National Animal
Health Centres (NAHC) Disease Diagnostics Laboratory. Five rabbits were
intramuscularly inoculated four times with 1 mL of CSF C-strain vaccine at fortnightly
intervals (day 0, 14, 28 and 42). The vaccine used to inoculate the rabbits was produced as
a lyophilised rabbit spleen homogenate at the Lao National Vaccine Institute from rabbits
infected with a C-strain CSF virus and reconstituted with sterile distilled water. The rabbits
were euthanased and bled out four weeks after the final inoculation. The serum fraction was
94
Chapter 4
collected and stored at 20 C. The titre of anti-CSF virus antibody in each rabbit was
determined by the NPLA described above (Chapter 3).
High titre rabbit sera were pooled and the immunoglobulin fraction purified by caprylic
acid/ammonium sulphate precipitation. Briefly, serum was diluted with four volumes of 60
mM acetate buffer, pH 4, and the final pH adjusted to 4.5 with 1N NaOH. Caprylic acid
(Sigma, USA) was added drop-wise to a final concentration of 25 L/mL of diluted serum
and mixed thoroughly at room temperature for 30 min. Precipitated albumin and other nonIgG proteins were removed by centrifugation at 10,000 x g for 30 min and the fines
removed by passing the supernatant through a cheese cloth filter. The supernatant was
mixed thoroughly with 1 part 10x PBS to 9 parts supernatant and the pH adjusted to 7.4
with 1N NaOH. The supernatant was cooled to 4 C before the addition of 0.277g/mL
ammonium sulphate (BDH Chemicals, USA) to give 45 % saturation. The sample was
stirred for a further 30 min at 4 C after which the precipitated IgG was pelleted by
centrifugation at 5000 x g for 15 min. The supernatant was discarded and the IgG pellet
resuspended in PBSA to one-tenth the original volume of serum. The resuspended IgG
fraction was then dialysed against 100 volumes of PBSA overnight before heating to 55 C
for 20 min and a final centrifugation at 5000 x g for 15 min. The purified fraction was
stored in aliquots at 20 C.
The concentration of total protein in the purified fraction of goat and rabbit polyclonal
capture antibody was estimated using the Biorad Protein Assay kit with gamma-globulin as
the protein standard (Biorad, USA) according to the suppliers recommendations. Briefly,
the gamma-globulin standard, supplied dehydrated and reconstituted to a concentration of
1.43 mg/mL, was diluted in deionised reverse osmosis (DiRO) water to concentrations of
0.50, 0.25, 0.10 and 0.05 mg/mL to enable the construction of a standard curve. Each
polyclonal antibody preparation was diluted 1:50, 1:100, 1:150 and 1:200 in DiRO water
immediately prior to protein estimation. Ten microliters of each dilution of the samples and
gamma-globulin standard was added in quadruplicate to a Maxisorp ELISA plate (Nunc,
Denmark). The concentrated dye reagent was diluted 1:5 in DiRO water and filtered. Two
95
Chapter 4
hundred microliters of diluted dye was added to all 10 L dilutions of sample and standard
and left at room temperature for 10 min. Following incubation the optical densities were
read at 570 nm in a Biorad 680 series plate reader. MS Excel was used to construct a
standard curve from which the estimates of polyclonal antibody protein concentrations
were determined from the slope of a linear curve of best fit.
Monoclonal antibody
Monoclonal antibody (Mab) 24/10 specific for the E2 protein of CSF virus was produced
from a hybridoma cell line grown in vitro at CSIRO AAHL and used as tissue culture
supernatant without purification in the IMB-ELISA test development. Mab 24/10 was first
produced at the Federal Research Centre for Virus Diseases of Animals, Tubingen,
Germany using the Thiverval vaccine strain (Weiland et al. 1990). Mab 24/10 was further
described and found to be broadly reactive with different CSF virus isolates from Europe
and also laboratory and vaccine strains (Kosmidou et al. 1995). No cross reactivity with
nine isolates of BVD virus was evident, indicating a high degree of specificity for the E2
protein of CSF virus (Kosmidou et al. 1995).
96
Chapter 4
described in Chapter 3. The laboratory numbers of the samples pooled were 005/05, 006/05
and 007/05. This second antigen was stored at 80 C in small aliquots. This antigen will
be referred to as Lao antigen. CSF negative antigen was prepared similarly to Lao antigen
as a 5 % w/v spleen homogenate using spleens collected from imported breed pigs and
confirmed negative by AC-ELISA.
97
Chapter 4
was used with the constant amount of goat polyclonal antibody in each reaction estimated
to be a sub-optimal concentration. Each reaction contained 185 g/mL antibody (1:400
dilution), 5 mM sodium acetate buffer pH 5 (Appendix I), 1 % w/v IMBs, DiRO water and
EDAC at a final concentration of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mM. All ingredients were
combined in each of six tubes excluding EDAC and mixed thoroughly by vortex. Tubes
were incubated with agitation on a microplate shaker at room temperature for 20 min at a
speed sufficient to prevent settling of IMBs. After 20 min incubation, the appropriate
amount of EDAC was added to each tube and the contents thoroughly mixed by vortex. The
tubes were then agitated on a microplate shaker for 2 h at room temperature to prevent
settling of IMBs and periodically mixed by vortex. Following this incubation, the tubes
were placed in a magnet for 2 min to take beads out of suspension. The supernatant was
removed and 500 L of phosphate buffered saline (PBSA) containing 0.2 % bovine serum
albumin (BSA, Sigma Aldrich, USA) was added to each tube to wash the IMBs. The IMBs
were mixed by vortex and again placed in the magnet for 1 min. The wash fluid was
removed and 248 L of PBS containing 0.2 % BSA was added to the beads to block any
exposed protein binding sites. The tubes were mixed by vortex and shaken at room
temperature as before. After 1 h the IMBs were taken out of suspension by placing in a
magnet. The blocking solution was removed and the IMBs were washed 3 times as above
using 500 L 0.2 % BSA PBSA. Following the final wash and removal of wash solution,
248 L of 0.2 % BSA PBSA was added to the IMBs for a final working concentration of
0.5 % w/v IMBs coated with polyclonal capture antibody. Coated beads were stored for up
to one week at 4 C until used in ELISA.
To determine the optimum concentration of EDAC that maximised antibody capture
activity, the coated IMBs were used in an ELISA as follows. Twenty microlitres of IMBs
for each EDAC concentration were added to 100 L AAHL antigen in duplicate and 100
L of negative antigen in a single tube, mixed by vortex and incubated at 37 C with
shaking to prevent settling of the IMBs. After 1 h the tubes were placed on a magnet for 30
sec and the supernatant removed. The IMBs were washed by adding 200 L PBST to the
IMBs and mixing by vortex. The tubes were placed in a magnet for the 30 sec and the wash
98
Chapter 4
removed. This wash step was repeated a further 2 times. After the third wash 100 L of
Mab 24/10 diluted 1:100 in PBST containing 0.1 % BSA was added to all tubes and mixed
by vortex. The tubes were then incubated at 37 C with shaking for 1 h. The beads were
again washed three times as previously described and 100 L of HRP conjugated antibody
diluted 1:2000 in PBST with 0.1 % BSA was added. The tubes were then incubated at 37
C with shaking for 1 h. The beads were again washed three times as above and transferred
to a new tube on the third wash. The final wash was removed and 50 L of activated TMB
substrate was added. The tubes were mixed by vortex and then every 2 min for 10 min.
After 10 min the reaction was stopped by the addition of 50 L 1N H2SO4. The supernatant
was removed after magnetic separation of the IMBs and transferred to an ELISA plate and
the OD450nm measured in a microplate reader with blanking on air. The average triplicate
OD450nm results were normalised by subtracting the negative control OD450nm. The optimum
EDAC concentration was visualised graphically by plotting concentration on the x-axis
against OD450nm on the y-axis using MS Excel.
Results
The results obtained using different concentrations of EDAC for the antibody/IMB
coupling reactions are presented in Figure 4.1. A peak was observed at a concentration of 1
mM with a clear difference demonstrated between 0.5 mM and 1.0 mM. The suboptimal
concentration of 0.5 mM resulted in inefficient antibody binding leading to a decrease in
antigen capture. At concentrations greater than 1.0 mM the suboptimal antibody activity
can be attributed to an inhibitory effect on antibody/antigen interactions (Seradyn, 1994).
At all EDAC concentrations, no difference in background noise, as represented by the
negative control, was demonstrated. The optimal concentration of EDAC was set at 1.0
mM, which is consistent with the optimal concentrations recommended by Seradyn
(Seradyn, 1994).
99
Chapter 4
1.20
1.00
OD450 nm
0.80
0.60
0.40
0.20
0.00
0.5
1.0
2.0
3.0
4.0
5.0
100
Chapter 4
initial estimation. These concentrations were equivalent to reciprocal dilutions of 400, 200,
133, 100 and 80, respectively. Based on the protein estimation using the Biorad kit of 73.6
mg/mL, these dilutions corresponded to concentrations of 185, 370, 555, 740 and 925
g/mL. Rabbit polyclonal antibody was titrated with a final reciprocal dilution per reaction
of 200, 150, 100 and 50. Following protein estimation using the Biorad kit, these dilutions
corresponded to final concentrations of 111, 148, 223, and 446 g/mL.
Each reaction contained antibody (diluted in DiRO water), 5 mM sodium acetate buffer pH
5 (Appendix I), 1 % w/v IMBs, made up to volume with DiRO water and 1 mM EDAC. All
ingredients were combined in a 2 mL screw cap microfuge tube except EDAC and mixed
thoroughly by vortex. Tubes were agitated on a microplate shaker at room temperature for
20 min. Following this incubation, EDAC was added to each tube to a concentration of 1
mM; the tubes were mixed by vortex and then agitated on a microplate shaker for 2 h at
room temperature. Following incubation, the tubes were placed in a magnet for 2 min to
take beads out of suspension. The supernatant was removed and 500 L PBSA containing 1
% fish skin gelatine (FSG, Sigma Aldrich, USA) and 0.02 % sodium azide was added to
each tube to block any exposed protein binding sites. The tubes were mixed by vortex and
shaken at room temperature for 1 h. Following the blocking incubation, the beads were
taken out of suspension by placing in a magnet for 1 min. The blocking solution was
removed and the beads were washed three times in 500 L of PBSA. Following the final
wash and removal of wash solution, 497 L of PBSA containing 0.2 % FSG and 0.02 %
sodium azide was added to the beads for a final working concentration of 0.5 % w/v beads
coated with polyclonal capture antibody. Coated beads were stored at 4 C until used in
ELISA.
The optimum concentration of polyclonal antibody used in the IMB/antibody coupling
reaction was determined by ELISA as follows. Twenty five microlitres of IMBs for each
dilution of antibody were added to 100 L AAHL antigen in duplicate and 100 L of
negative antigen in a single tube and mixed by vortex. The tubes were then incubated at 37
C with shaking to prevent settling of the beads. After 1 h the supernatant was removed
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Chapter 4
following magnetic separation of IMBs for 30 sec and the beads washed once with 200 L
PBST. One hundred microlitres of Mab 24/10 diluted 1:20 in PBST containing 0.1 % FSG
and 5 % normal rabbit serum was added to all tubes and mixed by vortex. The tubes were
then incubated at 37 C with shaking for 1 h and washed once with PBST. One hundred
microlitres of HRP-conjugated antibody diluted 1:3000 in PBST with 0.1 % FSG and 5 %
normal rabbit serum was added. Normal rabbit serum was incorporated into dilution buffers
to minimise background resulting from non-specific reactivity. The tubes were then
incubated at 37 C with shaking for 1 h. The IMBs were then washed three times in PBST
and transferred to a new tube on the third wash. The final wash was removed and 50 L of
activated TMB substrate was added. The tubes were mixed by vortex and then every 2 min
for 5 min. After 5 min the reaction was stopped by the addition of 50 L 1M H2SO4. The
supernatant was removed after magnetic separation and transferred to an ELISA plate;
OD450nm was measured with blanking on air. The average duplicate OD450nm results were
normalised by subtracting the negative control OD450nm. The optimum coating
concentration for each antibody was visualised graphically by plotting concentration on the
x-axis against OD450nm on the y-axis using MS Excel.
Results
Figure 4.2 illustrates the relationship between the coating concentration of polyclonal
antibody and antigen capture activity in the ELISA. A dilution of 1:200 of the goat
polyclonal antibody gave an OD reading equivalent to the least dilute preparation of
antibody and was not in the region of the curve where activity was descending. This
dilution was chosen as the IMB coating concentration and corresponded to a total protein
concentration of 370 g/mL. For the rabbit polyclonal antibody, a dilution of 1:100 gave an
OD reading equivalent to the least dilute preparation and was not in the region of the curve
where antibody activity was descending. This dilution of rabbit polyclonal antibody was
chosen as the IMB coating concentration and corresponded to a total protein concentration
of 223 g/mL.
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Chapter 4
1.60
a)
0.40
OD450 nm
0.80
1.20
0.80
b)
0.40
0.00
1:80
1:100
1:133
1:200
1:400
OD450 nm
1.60
1.20
0.00
1:50
1:100
1:150
1:200
Figure 4.2 Optimisation of (a) goat and (b) rabbit polyclonal antibody coating
concentration. Dashed line represents OD of negative control and error bars
indicate 1xSD from duplicate mean.
103
Chapter 4
104
Chapter 4
third wash 50 L of TMB substrate was added and stopped after 5 min as previously
described. The supernatants were transferred to an ELISA plate and the OD450nm measured
with blanking on air.
For the optimisation of Mab 24/10, 25 L of IMBs coated with either rabbit or goat
polyclonal antibody were added to 100 L AAHL antigen in duplicate and 100 L of CSF
negative antigen in a single tube for each dilution of Mab 24/10. The tubes were mixed by
vortex and shaken at 37 C for 1 h followed by washing once with 200 L PBST. Mab
24/10 was diluted 1:25, 1:50, 1:75 and 1:100 in PBST and 100 L was added to the
respective tubes. The beads were mixed by vortex and shaken at 37 C for 1 h and washed
once with PBST following the incubation. One hundred microlitres of HRP-conjugated
antibody diluted 1:3000 in diluent, as described above, was added to all tubes. The IMBs
were mixed and shaken at 37 C for 1 h and then washed three times. On the third wash the
beads were transferred to a new tube. The colour was developed with TMB substrate and
OD450nm measured as previously described.
Results
The results of the titrations of anti-mouse HRP-conjugated antibody and Mab 24/10 in the
IMB-ELISA are presented in Figure 4.3 and 4.4 respectively. Both polyclonal capture
antibody coated IMBs were assessed and produced similar results. HRP-conjugated
antibody was optimised first in the presence of excess Mab 24/10. Using IMBs coated with
either goat or rabbit polyclonal capture antibody, a dilution of 1:3000 gave an OD reading
equivalent to the least dilute preparation of HRP-conjugated antibody and was not in the
region of the curve where the OD was descending (at dilutions of 1:4000 and 1:5000). This
dilution was selected as the HRP-conjugated antibody dilution to be used in future assays.
Using IMBs coated with goat polyclonal capture antibody to assess the optimal Mab 24/ 10
dilution, a peak OD was observed at a dilution of 1:50. When beads coated with rabbit
polyclonal antibody were assessed, a dilution of 1:50 resulted in OD equivalent to 1:25
which was the least dilute preparation of Mab 24/10. The OD decreased with dilutions of
105
Chapter 4
both 1:75 and 1:100. A Mab 24/10 dilution of 1:50 was selected as the dilution to be used
in future assays.
1.60
1.20
a)
0.40
OD450nm
OD450 nm
1.20
0.80
0.80
b)
0.40
0.00
0.00
1:2000
1:3000
1:4000
1:2000
1:5000
1:3000
1:4000
1:5000
Dilution Factor
Dilution Factor
1.50
1.50
1.00
1.00
a)
0.50
OD450nm
OD450 nm
b)
0.50
0.00
0.00
1:25
1:50
1:75
Dilution Factor
1:100
1:25
1:50
1:75
1:100
Dilution Factor
Figure 4.4 Optimisation of Mab 24/10 dilution in IMB-ELISA using beads coated
with (a) goat and (b) rabbit polyclonal antibody. Dashed line represents OD of
negative control and error bars indicate 1xSD from duplicate mean.
106
Chapter 4
107
Chapter 4
with a S/N ratio of 2.80. The spleens were prepared as 5 % w/v homogenates in 1 % NP40
as described previously. The homogenates were diluted 5-fold from 5 % to 0.04 % w/v in 1
% NP40 PBSA and tested in duplicate in the IMB-ELISA, with duplicates of positive and
negative controls included.
Antigen capture efficiency of the two antibodies were visualised graphically by plotting
incubation time on the x-axis against OD450nm on the y-axis using MS Excel. Significance
of difference between the two polyclonal antibodies was demonstrated at a 99 %
confidence interval where there was no overlap of three standard deviations from the
duplicate mean.
Results
The results obtained using beads coated with either rabbit or goat polyclonal antibody to
trap CSF virus antigen are presented in Figures 4.5, 4.6 and 4.7. Initially comparison was
made using spleen, tonsil and kidney tissue from a locally infected pig titrated from 5 % to
0.2 % w/v (Figure 4.5). Goat polyclonal antibody was able to capture viral antigen more
efficiently than the rabbit polyclonal antibody resulting in significantly higher OD values in
tissue suspensions of 1 and 0.2 % for spleen; 5, 1 and 0.2 % for tonsil tissue and 5 and 1 %
for kidney tissue. When AAHL antigen titrated from 1:4 to 1:256 was used as the analyte,
the opposite was observed. Rabbit polyclonal antibody was able to capture antigen more
efficiently than the goat polyclonal antibody resulting in higher OD values at all dilutions
except 1:256 (Figure 4.6).
108
Chapter 4
a)
2.00
OD450nm
1.50
1.00
0.50
0.00
5%
1%
0.2 %
b)
2.00
OD450nm
1.50
1.00
0.50
0.00
5%
1%
0.2 %
OD450nm
0.75
c)
0.50
0.25
0.00
5%
1%
0.2 %
109
Chapter 4
2.00
OD450nm
1.50
1.00
0.50
0.00
1:4
1:16
1:64
1:256
Dilution Factor
The rabbit polyclonal antibody captured viral antigen from the AAHL antigen extract with
greater efficiency, whereas the goat polyclonal antibody captured viral antigen from the
Lao isolate, 089/04, with greater efficiency. As a result, three geographically distinct
isolates of CSF virus from Laos were prepared and tested in the IMB-ELISA. The results
are presented in Figure 4.7 and clearly show that viral antigen capture using the goat
polyclonal antibody resulted in higher OD values than the rabbit polyclonal antibody on
most occasions. A significant difference between the two antibodies was demonstrated at
high tissue concentrations (5 and 1 % w/v) for samples 028/04 and 087/04, where the goat
polyclonal antibody out performed the rabbit polyclonal antibody. No significant difference
was evident in these samples when diluted out to 0.2 and 0.04 % w/v tissue suspension.
Antigen capture using goat polyclonal antibody resulted in higher OD values at all
concentrations of tissue suspension for sample 088/04 from Houaphan province.
110
Chapter 4
2.00
a)
OD450 nm
1.60
1.20
0.80
0.40
0.00
5.00
1.00
0.20
0.04
OD450 nm
1.20
b)
0.80
0.40
0.00
5.00
1.00
0.20
0.04
2.00
c)
OD450 nm
1.60
1.20
0.80
0.40
0.00
5.00
1.00
0.20
0.04
111
Chapter 4
Results
The relationship between the volume of beads used in each reaction and the amount of
antigen captured is illustrated in Figure 4.8. Using a Mab 24/10 dilution of 1:50 and a HRPconjugated antibody dilution of 1:3000 in the IMB-ELISA, a 25 L volume of coated IMBs
resulted in the same OD as 40 L of IMBs per reaction. Based on this knowledge and a
need to maintain a high level of sensitivity at minimal cost, 25 L was selected as the
volume of coated beads to be included in each test.
112
Chapter 4
2.00
OD450nm
1.60
1.20
0.80
0.40
0.00
10
15
20
25
30
35
40
4.3.8 Time course experiments to optimise incubation times for antigen capture, Mab
24/10, conjugate and TMB substrate
Method
Using the standard test reagents and methods described above, the time for incubation at
each stage of the test was optimised. For antigen capture, the sample/bead incubation time
was determined by reacting 25 L of beads coated with the goat polyclonal capture
antibody with 100 L of Lao antigen in duplicate and with the negative antigen in a single
tube for each time point. The tubes were mixed by vortex and shaken at 37 C for 15, 30,
45 or 60 min. At each time point, two tubes containing positive and one containing negative
samples were removed from the shaker and washed once with 200 L of PBST to remove
antigen. Immediately 100 L of Mab 24/10 diluted 1:50 in 0.5 % FSG, 5 % glycerol and
0.1 % Proclin 300 in PBST was added to the three tubes, mixed by vortex and shaken at 37
C for 60 min. This step was repeated for the tubes at each time point. At the completion of
each Mab 24/10 incubation, the beads were washed with 200 L of PBST and 100 L of
113
Chapter 4
HRP-conjugated antibody diluted 1:3000 in the same diluent as the Mab 24/10 was added,
the beads mixed by vortex and shaken at 37 C for 1 h. The IMBs were then washed three
times with PBST and transferred to a new tube on the third wash. The beads were left at
room temperature in the final wash until all incubations were complete. Following the
completion of all HRP-conjugated antibody incubations and the subsequent washes, the
final wash was removed and 50 L of TMB substrate was added to all tubes. The tubes
were mixed by vortex and the reactions stopped after 5 min with the addition of 1 M
H2SO4. The supernatant was transferred to an ELISA plate and the OD450nm measured as
previously described.
The Mab 24/10 incubation time was optimised using the same methodology as described
above with a 30 min sample incubation time and a 1 h HRP-conjugated antibody
incubation. Likewise, the HRP-conjugated antibody incubation time was optimised with a
30 min incubation of both the sample and Mab 24/10. The optimal TMB substrate
incubation time was determined using 30 min incubations of sample, Mab 24/10 and HRPconjugated antibody. TMB substrate times assessed were 2.5, 5, 7.5 and 10 min
incubations, where the reaction was stopped after each time point and the OD450nm
measured at the completion of the 10 min incubation. After stopping at 2.5, 5 and 7.5 min,
the supernatant was transferred to an ELISA plate and protected from light to minimise
fading. The results were normalised by subtracting the negative control OD450nm from the
test OD450nm. The optimum incubation times were visualised graphically by plotting
incubation time on the x-axis against OD450nm on the y-axis using MS Excel. Significance
of difference between incubation times was demonstrated at a 99 % confidence interval
where there was no overlap of three standard deviations from the duplicate mean.
114
Chapter 4
Results
The results obtained using different incubation times for sample, Mab 24/10, HRPconjugated antibody and the TMB substrate are presented in Figures 4.9. The optimal
incubation time for each sample, Mab 24/10 and HRP-conjugated antibody was
demonstrated to be 30 min. The sample was incubated with IMBs for 15, 30, 45 or 60 min
and the results show that no significant difference in the detection of antigen was observed.
However, a large SD was seen with only a 15 min incubation period. When Mab 24/10 was
incubated at different times a significant difference in antigen detection was observed
between 15 and 45 min, but not between 15 and 30 min or 30 and 45 min. At different
HRP-conjugate incubation times no difference in antigen detection was demonstrated.
Taking a conservative approach, 30 min incubation times were selected. However, if time
becomes a crucial factor, 15 min incubation times could be adopted with little or no loss of
sensitivity. TMB substrate incubation times of 2.5, 5, 7.5 and 10 min were assessed. After
2.5 min incubation a high degree of variation was observed due to a delay in mixing the
beads following the addition of TMB substrate. No significant difference between 5, 7.5
and 10 min incubations could be demonstrated. A TMB substrate incubation time of 5 min
was selected as the optimum incubation time.
115
Chapter 4
OD450nm
2.00
1.50
1.00
a)
0.50
0.00
15
30
45
60
OD450nm
2.00
1.50
1.00
b)
0.50
0.00
15
30
45
60
OD450nm
2.00
1.50
1.00
c)
0.50
0.00
15
30
45
60
2.50
OD450nm
2.00
1.50
1.00
d)
0.50
0.00
2.50
5.00
7.50
10.00
116
Chapter 4
Optimisation of reagents
The optimisation of test reagent concentrations and reaction conditions are an important
part of test development. In the case of developing an enzyme immunoassay utilising
microparticles with a carboxyl modified surface, the concentration of EDAC used to enable
the covalent bond between antibody and bead was a critical step. At high concentrations,
EDAC has been shown to inhibit antibody activity (Seradyn, 1994). The results of this
study have clearly shown this to be the case. At concentrations above 1 mM, antibody
reactivity decreased, and at concentrations of 3 and 4 mM, antibody reactivity was highly
variable as demonstrated by large standard deviations from the duplicate mean. An EDAC
117
Chapter 4
concentration of 1mM resulted in the greatest antibody reactivity; as such this concentration
was used in all subsequent coating reactions.
The IMB-ELISA described in this chapter utilised a polyclonal antibody to recognise and
capture CSF viral antigen in tissue homogenate. Two polyclonal antibodies were assessed
for their ability to capture viral antigen. One a pestivirus group reactive polyclonal antibody
raised in goats and the other a polyclonal antibody raised in rabbits against a CSF C-strain
virus. Both the goat and rabbit polyclonal antibodies were semi-purified globulin fractions
of total serum protein, which may explain the need for much higher antibody
concentrations than recommended by the manufacturer of the IMBs.
118
Chapter 4
al. 2004a; Pan et al. 2005; Tu et al. 2001), the goat polyclonal antibody was selected as
the capture antibody to be used in the IMB-ELISA.
119
Chapter 4
In terms of cost, the gain in extra surface area was met with an increase in cost. However,
this extra cost was offset by the fact that four wells per sample are required in the ACELISA whereas a sample in the IMB-ELISA can be tested in duplicate. The cost of
biological and chemical reagents was similar for both the AC-ELISA and IMB-ELISA per
sample.
120
Chapter 5
Chapter 5.
Assessment and validation of IMB-ELISA
121
Chapter 5
122
Chapter 5
123
Chapter 5
5.2.2 Repeatability
Method
To determine precision estimates of the IMB-ELISA and to ensure that the coupling of
antibody to the IMBs was not introducing unacceptable levels of test variation from one
batch to the next, repeatability of results was assessed between batches of antibody-coated
IMBs. One hundred microlitre aliquots of Lao antigen, as a strong positive control, and
CSF negative spleen homogenate was frozen and stored at 85 C before use in the assay
so as to eliminate the variation generated through the freeze-thaw process. Four replicates
of each control were tested in the IMB-ELISA at each run, where each run used a different
batch of IMBs. The mean, standard deviation and coefficient of variation were calculated
for each set of replicates to determine a measure of intra-batch variance. The mean,
standard deviation and coefficient of variation was also calculated on all replicates to
measure inter-batch variation.
Results
The repeatability of the IMB-ELISA was assessed over 15 runs with new lots of IMBs
coated at each run. The results obtained from the repeatability experiment are summarised
in Figure 5.1 below. The mean corrected OD for four replicates over 15 runs (60 replicates
in total) was 1.64 with a standard deviation from the mean of 0.14. These results
corresponded to a coefficient of variation of 8.75. The dashed lines in Figure 5.1 indicate
one, two and three standard deviations from the mean. Ten of the 15 runs resulted in an OD
falling within one standard deviation of the overall mean. Five fell within two standard
deviations of the mean and no runs resulted in ODs greater than two standard deviations.
Intra-batch variation is represented in Figure 5.1 as the error bars of each run. The error
bars indicate 1 standard deviation from the run mean and the median coefficient of
variation of each of the 15 runs was 3.48 with a range of 1.21 to 7.23.
The mean OD of the negative control for the four replicates over 15 runs (60 replicates)
was 0.07, with a standard deviation of 0.03 and range of 0.03 to 0.14.
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Chapter 5
2.25
OD450 nm
2.00
1.75
1.50
1.25
1.00
1
10
11
12
13
14
15
Run Number
5.2.3 Stability
Method
The stability of the capture antibody coated IMBs used in the IMB-ELISA was assessed
along side the stability of Mab 24/10 and HRP-conjugated antibody diluted to working
concentration. Working stocks of reagents and IMBs were stored at 4 C in diluent or
storage buffer containing Proclin 300 as a preservative. The TMB substrate used at each
time point was made up fresh immediately prior to use in the ELISA. Aliquots of Lao
antigen and CSF negative spleen homogenate were prepared and stored as above (Section
5.2.2)
Four replicates of each control antigen were tested in the ELISA using IMBs coated with
goat polyclonal capture antibody at week 0. Horse radish peroxidase-conjugated antibody
and Mab 24/10 were pre-diluted at 1:3000 and 1:50, respectively in diluent buffer
containing 0.5 % w/v FSG, 5 % v/v glycerol and 0.1 % Proclin 300 as preservative. Four
replicates of each control were tested at week 0, 1, 2, 4, 6, 8 and 10 using the same batch of
coated IMBs and pre-diluted reagents (treatment 1) or reagents diluted on the day of the test
125
Chapter 5
(treatment 2). The mean and standard deviations were calculated for each set of replicates
to provide a measure of the stability of the IMB bound capture antibody, Mab 24/10 and
HRP-conjugated antibody. The stability of the IMB bound capture antibody was visualised
by plotting the results from weeks 0, 1, 2, 4, 6, 8 and 10 on the x-axis against OD450nm on
the y-axis using Mab 24/10 and HRP-conjugated antibody diluted on the day of the test.
The stability of the pre-diluted Mab 24/10 and HRP-conjugated antibody were visualised
by plotting the results from weeks 0, 1, 2, 4, 6, 8 and 10 on the x-axis against the
percentage decrease in OD450nm on the y-axis. The percentage decrease was calculated
using the following formula:
1
OD450nm
% Decrease = 100 - 2OD
x 100
450nm
where 1OD450nm equalled the OD using Mab 24/10 and HRP-conjugated antibody prediluted at week 0 and 2OD450nm equalled the OD using Mab 24/10 and HRP-conjugated
antibody diluted on the same day the test was performed.
At week 10 post dilution of reagents, an IMB-ELISA was performed to access the
individual stability of the pre-diluted Mab 24/10 and HRP-conjugated antibody. Reactions
were set up in duplicate using newly coated IMBs and Lao antigen to test the following
combination of reagents. The IMB-ELISA was performed as previously described. The
decrease in activity was calculated using the formula outlined above and the results plotted
on a bar graph.
126
Chapter 5
Mab 24/10*
HRP-conjugated antibody
Pre-diluted
Pre-diluted
New
Pre-diluted
Pre-diluted
New
New
New
Results
The results of the experiment to assess the stability of the reagents used in the IMB-ELISA
when stored at 4 C are summarised in Figures 5.2 and 5.3. Figure 5.2 demonstrates that the
IMB bound capture antibody remains stable for at least 10 weeks, with no observed
difference between the OD at week 0 and week 10. Using Mab 24/10 and HRP-conjugated
antibody pre-diluted in diluent buffer at week 0, a steady decline in activity was observed
when compared to reagents prepared on the day of the test for each time point. After 10
weeks the activity had decreased by greater than 31 % (Figure 5.3 a). The cause of this
declining activity was demonstrated to be due to a loss of HRP-conjugated antibody
activity (Figure 5.3 b). Ten week old Mab 24/10 diluted 1:50 in diluent buffer maintained
activity equal to Mab 24/10 diluted on the same day of the test.
2.25
OD450 nm
2.00
1.75
1.50
1.25
1.00
0
10
Chapter 5
50
a)
40
30
31
20
18
10
0
10
10
10
25
22
b)
20
20
15
10
5
Conj-10 wo
Conj-10 wo
Conj-new
Conj-new
Mab-10 wo
Mab-new
Mab-10 wo
Mab-new
Reagent combination
128
Chapter 5
450nm
X 100
The results of the two ELISA methods were compared to each other and also to the amount
of virus in each tissue sample as determined by the peroxidase-linked assay (described in
Chapter 3).
129
Chapter 5
Results
The analytical sensitivity of the IMB-ELISA was demonstrated to be greater than that of
the AC-ELISA. The results of the analytical sensitivity of both the IMB-ELISA and ACELISA are summarised in Table 5.1 below. By AC-ELISA, the limit of detection of viral
antigen in spleen tissue was 7.08 1.1 x 105 TCID50/100 L (n=2) of infectious virus
compared to 2.21 0.37 x 104 TCID50/100 L (n=2) when tested using the IMB-ELISA; a
32-fold increase in analytical sensitivity. In the case of tonsil tissue, the AC-ELISA was
able to detect 1.58 x 105 TCID50/100 L (n=1) of infectious virus compared to 2.22 0.35
x 103 TCID50/100 L (n=2) by IMB-ELISA; a greater than 64-fold increase in analytical
sensitivity. Viral antigen from infected kidney tissue could only be detected by IMBELISA in sample 089/04. The limit of detection was found to be 2.81 x 104 TCID50/100 L
of infectious virus. Both ELISA procedures were able to detect fewer units of infectious
virus in tonsil tissue as compared to spleen and kidney tissue. In the case of submission
089/04, an approximately 10-fold increase in analytical sensitivity for tonsil tissue was
observed in comparison to spleen and kidney tissue when tested by the IMB-ELISA. For
the same submission, a 5-fold increase in analytical sensitivity was observed in tonsil tissue
compared to spleen tissue when tested by the AC-ELISA.
130
Chapter 5
Table 5.1 (a) Analytical sensitivity of IMB-ELISA (5 % w/v homogenate of submission 089/04)
Tissue type (Virus titre)
Dilution
AC-ELISA
(S/N Ratio)
IMB-ELISA
(% Pos)
TCID50/100 L
089/04 Spleen
1:1
Pos (5.1)
nt
3.16 x 106
(106.5 TCID50/100 L)
1:2
Pos (3.3)
Pos (93)
1.58 x 106
1:4
Pos (2.1)
Pos (81)
7.91 x 105
1:8
n (1.8)
Pos (80)
3.95 x 105
1:16
n (1.5)
Pos (61)
1.98 x 105
1:32
n (1.4)
Pos (38)
9.88 x 104
1:64
n (1.3)
Pos (31)
4.94 x 104
1:128
n (1.4)
Pos (17)
2.47 x 104
1:256
nt
n (9)
1.24 x 104
1:512
nt
n (6)
6.18 x 103
089/04 Tonsil
1:1
Pos (3.2)
Pos (84)
3.16 x 105
(105.5 TCID50/100 L)
1:2
Pos (2.0)
Pos (79)
1.58 x 105
1:4
n (1.6)
Pos (70)
7.91 x 104
1:8
n (1.1)
Pos (56)
3.95 x 104
1:16
n (1.3)
Pos (40)
1.98 x 104
1:32
n (1.2)
Pos (25)
9.88 x 103
1:64
n (1.1)
Pos (16)
4.94 x 103
1:128
n (1.3)
Pos (12)
2.47 x 103
1:256
nt
n (8)
1.24 x 103
089/04 Kidney
1:1
n (1.1)
Pos (21)
5.62 x 104
(104.75 TCID50/100 L)
1:2
n (1.1)
Pos (13)
2.81 x 104
1:4
n (1.1)
n (8)
1.41 x 104
1:8
n (1.1)
n (5)
7.03 x 103
1:16
n (1.3)
n (3)
3.52 x 103
1:32
nt
n (2)
1.76 x 103
1:64
nt
n (2)
8.79 x 102
1:128
nt
n (2)
4.39 x 102
131
Chapter 5
Table 5.1 (b) Analytical sensitivity of IMB-ELISA (5 % w/v homogenate of submission 091/04)
Tissue type (Virus titre)
Dilution
AC-ELISA
(S/N Ratio)
IMB-ELISA
(% Pos)
TCID50/100 L
091/04 Spleen
1:1
Pos (8.4)
nt
1.00 x 107
(107 TCID50/100 L)
1:2
Pos (8.5)
Pos (105)
5.00 x 106
1:4
Pos (6.5)
Pos (102)
2.50 x 106
1:8
Pos (4.2)
Pos (103)
1.25 x 106
1:16
Pos (2.8)
Pos (91)
6.25 x 105
1:32
n (1.8)
Pos (73)
3.13 x 105
1:64
n (1.6)
Pos (54)
1.56 x 105
1:128
n (1.6)
Pos (37)
7.81 x 104
1:256
nt
Pos (23)
3.91 x 104
1:512
nt
Pos (14)
1.95 x 104
091/04 Tonsil
1:1
n (1.7)
Pos (59)
3.16 x 104
(104.5 TCID50/100 L)
1:2
n (1.5)
Pos (39)
1.58 x 104
1:4
n (1.5)
Pos (26)
7.91 x 103
1:8
n (1.4)
Pos (17)
3.95 x 103
1:16
n (1.3)
Pos (11)
1.98 x 103
1:32
n (1.3)
n (7)
9.88 x 102
1:64
n (1.3)
n (5)
4.94 x 102
1:128
nt
n (5)
2.47 x 102
1:256
nt
n (5)
1.24 x 102
091/04 Kidney
1:1
1.78 x 103
(103.25 TCID50/100 L)
1:2
8.89 x 102
1:4
4.45 x 102
1:8
2.22 x 102
1:16
1.11 x 102
1:32
nt
0.56 x 102
1:64
nt
0.28 x 102
1:128
nt
0.14 x 102
132
Chapter 5
133
Chapter 5
homogenate. Assays were also set up without added serum. The samples were mixed by
vortex and incubated at room temperature for 20 min. Twenty five microlitres of coated
IMBs were then added to the samples and the IMB-ELISA performed.
As a result of the above experiment (refer to results section below), an additional step was
included in the protocol. Twenty five microlitres of sample homogenate prepared for
testing was added to 75 L of sample diluent buffer (which contained PBST plus 10 %
normal goat serum with 0.1 % Proclin 300 added as a preservative). The sample was
allowed to incubate in the presence of sample diluent for 20 min at room temperature
before the addition of 25 L of antibody coated IMBs. The IMB-ELISA was then
performed. The 43 negative and 15 positive samples tested using the initial protocol were
re-tested. The remaining diagnostic specimens were tested using this revised protocol and
the results calculated according to the percent positivity equation given above.
The cut-off was expressed in two ways and the relative diagnostic sensitivity and
specificity were compared using both methods. Diagnostic sensitivity (DSn) and specificity
(DSp) were calculated using the following equation (Jacobson, 1998).
IMB-ELISA
AC-ELISA
Positive Negative
Positive
Negative
A
D
x 100 and DSp =
x 100
(A+C)
(D+B)
The first cut-off was set as the mean percent positivity of the AC-ELISA negative samples
plus three standard deviations. The second was determined by producing a scatter plot in
MS Excel with the AC-ELISA S/N ratio on the y-axis and the IMB-ELISA result on the x-
134
Chapter 5
axis. To confirm the visual inspection of the scatter plot, modified receiver-operator curves
(ROC) (Jacobson, 1998) were constructed with intervals of test result cut-offs on the x-axis
plotted against diagnostic sensitivity and specificity on the y-axis.
Results
Using the initial test methodology without the inclusion of a pre-incubation step with
normal goat serum, the mean percent positivity of the 43 CSF negative samples which
tested negative by AC-ELISA was six with a standard deviation of 14 and range of 0 to 73
%. By setting the cut-off at the mean plus three standard deviations, the cut-off was 49. The
results obtained from the 58 samples tested are summarised in the 2x2 table below (Table
5.2). The relative diagnostic sensitivity and specificity were 87 % (95 % CI: 58-98 %) and
95 % (95 % CI: 83-99 %), respectively. Similarly, the positive and negative predictive
values were 87 % (95 % CI: 58-98 %) and 95 % (95 % CI: 83-99 %), respectively, with an
accuracy of 93 % (95 %CI: 82-98 %).
Table 5.2 AC-ELISA versus IMB-ELISA without a pretest incubation of sample with normal pig or goat serum.
IMB-ELISA cut-off set at 49 % positivity.
IMB-ELISA
AC-ELISA
Pos
Neg
Pos
13
15
Neg
41
43
15
43
58
The greatest concern from these preliminary samples was the spread of high OD450nm in the
IMB-ELISA for samples testing negative by AC-ELISA (Figure 5.4). The ability to
distinguish negative and positive results by eye is highly dependent on a clear distinction
between the two. This is not possible if negative samples produce a strong colour change
that is clearly visible by eye. Two samples negative by AC-ELISA but producing high
readouts in the IMB-ELISA, 087/03 and 107/03 (Figure 5.4), were tested by virus isolation
135
Chapter 5
to ensure that the increased analytical sensitivity of the IMB-ELISA (refer to previous
section) was not detecting an AC-ELISA false negative result. Both samples were found to
be negative by virus isolation, with no infectious virus able to be detected.
The results obtained with pre-incubation of samples 087/03 and 107/03 with serum are
presented in Figure 5.5. Both samples were freshly prepared from spleen tissue stored at
minus 20 C rather than using stored homogenate. As can be seen in Figure 5.5, sample
107/03 returned a very low OD450nm without a pre-test incubation with normal goat or pig
serum, indicating that the initial sample preparation was probably contaminated with CSF
virus antigen. For sample 087/03 however, the change in OD450nm following incubation
with either pig or goat serum was substantial.
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
-10
107/03
10
20
30
40
50
60
70
80
087/03
90
100
110
IMB-ELISA (% Positivity)
136
Chapter 5
OD450nm
1.80
1.60
10% NGS
1.40
10% NPS
1.20
No Serum
1.00
0.80
0.60
0.40
0.20
0.00
C++
087/03
107/03
C-
Sample
One hundred and twenty three negative and 39 positive samples by AC-ELISA were tested
in the IMB-ELISA using the modified methodology incorporating incubation of the
samples with sample diluent containing 10 % normal goat serum for 20 min before the
addition of IMBs. The effect of the pre-test incubation with serum is apparent in Figure 5.6,
where a tight clustering of negative samples can be seen. The mean percent positivity of the
AC-ELISA negative samples was zero with a standard deviation of 2.05. By setting the cutoff as the mean plus three standard deviations, the cut-off was 7 % positivity. Thirty nine
AC-ELISA positive samples were also assessed by IMB-ELISA. Using a cut-off of 7 %,
the IMB-ELISA had a relative diagnostic sensitivity and specificity of 100 and 98 %
respectively. As the IMB-ELISA results are somewhat skewed (Figure 5.7), a modified
receiver-operator curve (ROC) (Jacobson, 1998) was constructed to determine the optimal
cut-off (Figure 5.8). With a cut-off of 10 % positivity in the IMB-ELISA the relative
diagnostic sensitivity and specificity were both 100 % (95 % CI: 89-100 % and 96-100 %,
respectively). At 5 % positivity the relative diagnostic sensitivity and specificity were 100
137
Chapter 5
% and 88 % respectively (95 % CI: 89-100 % and 81-93 %, respectively). Based upon these
results, samples were deemed to be positive for the presence of CSF virus antigen if the
percent positivity was equal to or greater than 10. The sample was suspected to have CSF
virus antigens and subjected to re-testing if the percent positivity was greater than or equal
to five and less than 10.
The percent positivity of samples 087/03 and 107/03 when re-tested using the modified
methodology were observed to be 8 and 1 % respectively, falling below the cut-off of 10 %.
However, in Figure 5.5 above the percent positivity of sample 087/03 was found to be 15 %
which would be considered a positive result. This indicates that some variability of the final
result (i.e. positive or negative) could exist for those samples with a percent positivity close
to the cut-off.
40
38
36
34
32
30
28
26
24
22
20
18
16
14
12
10
8
6
4
2
0
-10
10
20
30
40
50
60
70
80
90
100 110
IMB-ELISA (% Positivty)
138
Chapter 5
50
Frequency
40
30
20
10
0
-3
-2
-1
% Positivity in IMB-ELISA
100
DSp
80
DSn
60
40
20
110
100
90
80
70
60
50
40
30
20
10
0
0
Diagnostic sensitivity
and specificity
120
Intervals
of normalised
IMB-ELISA
test results
Intervals
of IMB-ELISA
test results
139
Chapter 5
140
Chapter 5
of CSF and Foot and Mouth Disease. Operator 3 was a new graduate from the National
University of Laos, Faculty of Agriculture, with limited experience (approximately one
year) in ELISA procedures. Prior to inter-operator assessment, three training sessions were
conducted to familiarise Operators 2 and 3 with the IMB-ELISA and ensure they were both
comfortable and confident in performing the procedure on their own.
Results of each sample were entered into a Microsoft Excel spreadsheet to match coded
numbers with sample number at the completion of each operators 40 samples. The
agreement of operators was assessed by kappa statistic analysis (Smith, 2006). The kappa
statistic of agreement was calculated using the following formula:
Operator Y
Positive
Negative
Positive
(a)
(b)
Negative
(c)
(d)
Operator X
a+d
Observed agreement = a + b + c + d
(a + b) x (a + c)
Expected (chance) agreement for (a) = a + b + c + d
Expected (chance) agreement for (d) =
(c + d) x (b + d)
a + b + c +d
Kappa statistic was also used to calculate the level of agreement between determining a
result by measuring the OD450nm and recording a visual colour change for each operator.
141
Chapter 5
The relative diagnostic sensitivity and specificity for each method of determining a result
were calculated using the equations shown in Section 5.2.5 above. The interpretation of the
kappa values were made according to the scale summarised by Smith (2006). In short,
kappa values of 0.41-0.60, 0.61-0.80, 0.81-0.99 and 1.00 correspond to strength of
agreement described as moderate, substantial, almost perfect and perfect, respectively.
Results
The inter-operator agreement results are summarised in Tables 5.3 and 5.4 below. When
results were determined by measuring the OD450nm in an ELISA plate reader, agreement
was highest between Operators 1 and 2 and lowest between operators 1 and 3. Two out of
forty samples tested were different for Operators 1 and 2, five differences between
operators 1 and 3 and five differences between Operators 2 and 3. Refer to Appendix II for
greater detail of inter-operator results.
Table 5.3 Agreement between operators when
IMB-ELISA test results were determined by
measuring OD450 nm spectrophotometrically
OP1
OP2
OP3
OP1
OP2
0.90
OP3
0.74
0.74
The agreement of results between operators was improved when the results were
determined by recording a noticeable colour change (resulting by eye), the highest level of
agreement was between Operators 1 and 2 and the lowest between Operators 2 and 3. Only
one difference was observed between Operators 1 and 2, four differences between
Operators 1 and 3 and five differences between Operators 2 and 3.
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Chapter 5
OP1
OP2
OP3
OP1
OP2
0.95
OP3
0.80
0.75
Kappa statistic was also used to calculate the agreement between the result by eye and the
result determined by measuring the OD450nm for each operator. The results for each operator
are summarised in Table 5.5. Only one difference between results was observed for
Operator 1, two differences for Operator 2 and four differences for Operator 3. Agreement
levels were considered almost perfect for Operators 1 and 2 and for Operator 3 the level of
agreement was substantial.
0.95
OP2
0.90
OP3
0.79
The relative diagnostic sensitivity and specificity were also determined for each operators
results in comparison with the AC-ELISA as the reference standard. Table 5.6 shows the
results comparing the results of the IMB-ELISA with the AC-ELISA when the results were
determined by recording a visual colour change and Table 5.7 when results were
determined by measuring the OD450nm. It appears from the small number of samples tested
143
Chapter 5
that the relative diagnostic specificity suffered somewhat when the IMB-ELISA was read
by eye, whereas there was no difference in relative diagnostic sensitivity for the two
methods of determining a result in the IMB-ELISA.
Table 5.6 Comparison of IMB-ELISA with the ACELISA results when IMB-ELISA was read by eye (DSn,
diagnostic sensitivity; DSp, diagnostic specificity)
OP1
OP2
OP3
OP1
OP2
OP3
144
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Results
The mean OD of the negative control over 15 runs was 0.07 with a standard deviation of
0.03. The UCL was set as 0.13 and the LCL at 0.01.
The mean OD of the positive control over 15 runs was 1.64 with a standard deviation of
0.14. The UCL was set at 1.92 and the LCL at 1.36
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Chapter 5
Repeatability
The first performance characteristic assessed was the repeatability of the IMB-ELISA. This
performance indicator was considered important to show that the test was not inherently
variable, so that operators would have confidence in the results produced during routine use
of the assay. Assessment of repeatability was accomplished by first evaluating the results of
four replicates of negative and positive control samples tested using 15 different batches of
IMBs coated with goat polyclonal capture antibody (inter-batch variation). The coefficient
of variation was determined to be 8.75 % and on no occasion did the mean OD of the 15
runs return a result greater than two standard deviations from the overall mean. When using
OD as a preliminary measure of repeatability, Jacobson (1998) recommended that
coefficients of variation less than 20 % are sufficient, indicating that the repeatability of the
IMB-ELISA was well within accepted performance limits.
The second measure of repeatability was the evaluation of intra-batch variation, that is, a
measure of the variation seen between replicates using the same batch of IMBs. Once
again, the coefficients of variation within runs of the test were found to be low, the median
146
Chapter 5
coefficient of variation was 3.48. These results were considered indicative of a high degree
of repeatability.
While these initial estimates of repeatability show a great deal of promise, results obtained
during the determination of diagnostic cut-offs (Section 5.2.5) point to the need for further
repeatability assessment. Sample 087/03, negative by AC-ELISA, was found to be either
just below or just above the cut-off of 10 % positivity in repeat runs of the IMB-ELISA.
Further repeatability assessment should include a range of samples close to the diagnostic
cut-off together with a number of negative and positive samples; where the results are
expressed as percent positivity in comparison to the strong positive control.
Stability of reagents
The antibody/IMB complex was found to be very stable, with no observed decrease in test
OD after being stored at 4 C for 10 weeks. These results indicate that no antibody
degradation had taken place and the covalent bond between the antibody and IMB was
stable. When antibodies were passively adsorbed onto IMBs without a reactive surface,
Lim et al. (1998) found the IMB/antibody complex to be stable for at least nine months.
The antibody/IMB complex described in this thesis was made via the activation of a
carboxyl modified IMB surface that provides a greater degree of binding strength than
IMBs relying on passive adsorption. Therefore the interaction between antibody and IMB
would not be expected to affect the long term sensitivity of the test using a single batch of
coated IMBs. Rather the long term sensitivity of the IMB-ELISA using a single batch of
IMBs will be limited by the antigen binding capacity of the antibody, whereby reduced
antibody degradation will result in a longer shelf life. While degradation was not evident
after 10 weeks storage, assessment of stability over a 6 12 month period would provide an
excellent estimate of potential shelf-life. This will become particularly important for the
test to be packaged into a kit format.
Other reagents assessed for stability were Mab 24/10 and HRP-conjugated antibody, which
were diluted to a working concentration. Mab 24/10 was found to be stable in the
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homemade storage buffer developed during the course of this research; however the HRPconjugated antibody was not. The lack of stability of the HRP-conjugated antibody will be
an issue that needs to be resolved prior to the packaging of the test into a kit format.
Stabilisation buffers for the storage of enzyme-conjugated antibodies at working
concentrations are commercially available. Such a buffer will need to be assessed in the
IMB-ELISA assay, and the length of time for which the HRP-conjugated antibody is stable
will need to be determined. Furthermore, the chromogen-substrate used in the development
and assessment of the IMB-ELISA was TMB-hydrogen peroxide that was made up fresh
immediately prior to addition to IMBs. Made up in this manner the TMB-substrate is stable
for a short period of time and could not be used in a provincial laboratory. As with HRPconjugated antibody stabilisation buffer, stable activated TMB is commercially available
with a shelf life of approximately 12 months and will need to be assessed in the IMBELISA format.
Analytical sensitivity
Analytical sensitivity is defined as the smallest amount of analyte that can be detected by a
given assay (Jacobson, 1998). The assessment of analytical sensitivity revealed the IMBELISA to be able to detect a smaller amount of viral antigen than the AC-ELISA. The
IMB-ELISA was 32-fold more sensitive than the AC-ELISA for spleen samples and in the
region of 64-fold more sensitive for preparations of tonsil sample. Only the IMB-ELISA
was able to detect viral antigen from one of the kidney samples tested. McGoldrick et al.
(1999) observed that virus isolation was in the order of 10- to 100-fold more analytically
sensitive than commercial AC-ELISA and nested RT-PCR 10- to 1000-fold more
analytically sensitive than virus isolation. Without a direct comparison to virus isolation
and RT-PCR, it is only possible to say that the IMB-ELISA has greater analytical
sensitivity than the AC-ELISA and probably less than virus isolation and RT-PCR.
However, there may be circumstances when, due to poor sample handling, viral infectivity
is lost yet viral antigen may still be detectable in the IMB-ELISA. Such a potential benefit
is of importance for diagnostic testing in least developed countries with poor infrastructure.
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An increase in analytical sensitivity correlates with a tests ability to detect CSF virus early
in the course of disease progression (Dewulf et al. 2004a; Kaden et al. 1999). The
significance of the IMB-ELISAs greater analytical sensitivity could be in being able to
detect virus earlier after infection compared to the AC-ELISA. This argument is however
limited by the fact that the Dewulf et al. (2004a) and Kaden et al. (1999) studies were
performed on blood. The ability of the IMB-ELISA to detect viral antigen in blood has not
yet been assessed. Even if the IMB-ELISA was found to be able to detect virus in blood
earlier after infection, this would be unlikely to improve the control of CSF in Laos due to
constraints in identifying potential outbreaks and then collecting blood samples. The real
potential of the greater analytical sensitivity could be realised if this was shown to translate
to greater diagnostic sensitivity in detecting low virulent strains that grow to lower titres in
organs.
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The diagnostic cut-off of the IMB-ELISA was evaluated using a panel of 162 positive and
negative samples whose result had been predetermined by AC-ELISA as the reference
standard. Initial tests using the optimised method described in Chapter 4, resulted in very
high absorbance values for AC-ELISA negative samples and a small number of false
positives were seen even if the cut-off was set very high. The problem of high absorbance
values would make the reading of the test by eye difficult when developed into a test
suitable for use in the field. To overcome this initial problem of high absorbance values, the
test samples were pre-incubated with normal pig or goat serum to adsorb out possible
interfering molecules. The most likely of which was considered to be Rheumatoid factor,
an IgM class of circulating antibodies commonly found in healthy mammals (Crowther,
1995). Incubation of test samples with a diluent containing 10 % normal goat or pig serum
greatly reduced background noise and all false positive results were eliminated. Following
the screening of all 162 test samples the diagnostic cut-off was set at 10 % positivity, where
the OD of test samples were expressed as a percentage of the high positive control included
in each run of the test. At this cut-off, all samples negative by AC-ELISA were negative by
IMB-ELISA and likewise for samples testing positive by AC-ELISA.
The diagnostic cut-off has been established using only a single batch of strong positive
control to measure the percent positivity of test samples. Based on the repeatability
experiments using this same strong positive control, the upper and lower control limits of
absorbance were determined to be 1.92 and 1.36 with a mean absorbance over 15 runs of
1.64. For a new batch of strong positive control to be introduced as the standard for
determining the percent positivity of test samples, it would need to be assessed and shown
to provide like results to the current batch. Alternatively, other methods of determining a
result, such as signal to noise ratio, could be explored to minimise variation of test results
between batches of strong positive controls. Signal to noise ratio is reliant on a comparison
with a representative negative control sample, which potentially could be easier to control
than a standard strong positive control.
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151
Chapter 5
even less experienced. Due to the high level of subjectivity involved in reading a colour
change, the result that was somewhat unexpected was the level of agreement observed
when the test was read by eye. The inter-operator agreement when read by eye was
numerically higher than the agreement observed when absorbance was measured. The level
of agreement between Operator 1 and 2 was almost perfect at 0.95 and between Operators 1
and 3 and Operators 2 and 3 the level of agreement was substantial with kappa values of
0.80 and 0.75, respectively. These results provide confidence that this format could be
adapted to a near-to-field based assay.
One of the key results to come out of this evaluation of robustness was the observation that
the inter-operator variation had little impact on the diagnostic sensitivity of the test
compared to the AC-ELISA. Furthermore, the diagnostic specificity was only slightly
affected. These findings were encouraging even though only a small number of samples
have been assessed. As mentioned earlier further research needs to be conducted to fully
assess the repeatability of the test particularly for those samples with percent positivity
close to the diagnostic cut-off. The results of this additional research will have relevance to
inter-operator agreement, where assessment of test repeatability and accuracy for individual
operators will need to be an ongoing process.
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Chapter 6
Chapter 6.
Assessment of storage temperature on
lapinised C-strain vaccine produced
in Laos
153
Chapter 6
154
Chapter 6
155
Chapter 6
question addressed was can the locally produced C-strain vaccine be stored for long
periods of time at 4 C and maintain a high level of immunogenicity? With little
information available to support the current shelf life estimates used by the NVI, the second
research question addressed was under optimal storage conditions of 20 C, how long is
the locally produced C-strain vaccine stable? To address these questions, nave pigs were
vaccinated and their serological response monitored by NPLA. No virus challenge work
was carried out due to the relative lack of biosecurity at the National Animal Health Centre.
Temperature (C)
20
Transport
10
VILLAGE
Transport
0
Transport
-10
PAFO
-20
NVI
DAFO
VSU
Time
Figure 6.1 Model representing transport and storage conditions of CSF vaccine in
Laos. NVI, National Vaccine Institute; VSU, Veterinary Supply Unit; PAFO,
Provincial Agriculture and Forestry Office; DAFO, District Agriculture and
Forestry Office
156
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157
Chapter 6
158
Chapter 6
Month of Experiment
6 pigs purchased
Group 1 (wk 2)
0.5
1
9 pigs purchased
Group 2 (wk 6)
1.5
2
10 pigs purchased
Group 3 (wk 10)
3.5
4
10 pigs purchased
Group 5 (wk 18)
2.5
3
10 pigs purchased
Group 4 (wk 14)
Storage at 4 C commences
4.5
5
Chapter 6
6.3 Results
6.3.1 Vaccine storage
The vaccine stored at 20 C was maintained at a constant temperature with little
fluctuation for the duration of the experiment. The average temperature was 18.28 C with
a standard deviation of 1.28 and a range of 11.00 to 21.00 C. The spike at 11.00 C
was for only one data point measured at 20 min intervals, the temperature quickly returned
to between 15 and 20 C. The vaccine stored at 4 C was likewise maintained at a
constant temperature with very little fluctuation for the duration of the experiment. The
average temperature over the course of the experiment was 4.34 C with a standard
deviation of 0.73 and range of 2.50 to 9.00 C. The spike at 9.00 C was for only one data
point measured at 20 min intervals and the temperature rapidly returned to below 8.00 C
within 20 min.
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Reciprocal of serum
dilution
48
40
32
24
16
8
0
0
10
14
21
28
35
161
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162
Chapter 6
163
Chapter 6
48
40
32
a)
24
16
8
0
0
10
14
21
28
48
40
32
b)
24
16
8
0
0
10
14
21
28
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Chapter 6
48
40
32
a)
24
16
8
0
10
14
21
28
48
40
32
b)
24
16
8
0
0
10
14
21
28
165
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166
Chapter 6
167
Chapter 6
assessment (Figure 6.3). All pigs developed a neutralising antibody response greater than or
equal to 32 by day 35 post vaccination with a median titre of 44. On day 28 post
vaccination 3/4 pigs had developed an antibody titre greater than or equal to 32 and one pig
had a titre of 22. These results are consistent with those of Terzic et al. (2003). Neutralising
antibodies first appeared at day 10 in one pig and 3/4 pigs on day 14, with titres gradually
increasing up to day 35.
However, when the vaccine had been stored for five months under conditions that were
ideal as recommended by the manufacturer (stored at 20 C with essentially no significant
temperature fluctuation), it failed to elicit a serological response that would afford
protection against CSF virus challenge. This represents a shelf life far shorter than that
recommended by the manufacturer, approximately four months compared to 12 months.
The Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (OIE, 2004) states
that a modified live virus vaccine for CSF must maintain its immunogenicity for at least as
long as the shelf-life stated by the manufacturer. In saying this however, it must be noted
that only one batch of vaccine was assessed. A greater number of batches need to be
assessed to determine a statistically significant estimate of vaccine shelf-life.
When vaccine was stored at 4 C for three months it failed to elicit a serological response in
vaccinated pigs that would afford protection against CSF virus challenge. The principle aim
of this chapter was to determine if the vaccine produced in Laos could be stored at 4 C for
prolonged periods of time and remain immunogenic. While no significant difference could
be demonstrated between the two storage temperatures at month 4 using the Fisher exact
test, a clear difference could be demonstrated using relative risk (risk ratio) as a measure of
the likelihood of developing a CSF neutralising antibody response by day 28. At month 4
of the experiment, vaccine stored at 20 C was three-fold more likely to result in the
development of an effective immune response when compared to vaccine stored at 4 C.
These results demonstrate that the vaccine can not be stored at 4 C in the long term and
remain immunogenic.
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A number of factors, or combination of factors could have caused the poor vaccine
performance observed during this study. Post-production handling of the vaccine was likely
one of the problems contributing to poor vaccine quality. The process of labelling vaccine
vials involved removing the vaccine from storage at 20 C and holding at room
temperature until tubes were dry enough for the label to adhere (personal observation). The
vials were then labelled and returned to the storage freezer. This process can take up to half
a day and ambient room temperature in a tropical country may reach +30 C. The reasons
given for this process were cost saving, such that only viable batches of vaccine were
labelled and secondly that labels interfered with the freeze-drying equipment.
Another important factor that could have contributed to the poor performance of the
vaccine was infection with an immunosuppressive disease agent affecting response to the
vaccine. Infection with an immunosuppressive disease agent has been shown to negatively
impact on antibody response following vaccination (Holland et al. 2003 and Li and Yang,
2003). A number of pigs died in the last two cohorts without a diagnosis being made, the
possibility of infection with an immunosuppressive agent cannot be ruled out.
During the course of this research, two vaccine freezers in Bolikhamxay province (one new
refrigerator with an internal freezer compartment where vaccine was stored and one old
freezer that was approximately 30 years old) were tested to determine the temperature
range at which vaccine was stored at the provincial level. The average temperatures of the
freezers over one month were 10.78 1.12 C and 10.46 1.13 C, respectively (data not
shown). Under these field conditions the shelf life of the vaccine would be even less than
the four months determined during this research.
It is well recognised that CSF virus is susceptible to temperature fluctuations such as the
process of freezing and thawing (Anonymous, 1996). In Laos, the process of vaccine
delivery to provincial and district agricultural offices results in one or multiple freeze-thaw
events taking place before the vaccine reaches the pig. Previous research has shown that
problems with vaccine delivery exist, a serological survey in Vientiane Capital in
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Chapter 6
1997/1998 demonstrated that only 26 % of vaccinated pigs had antibodies to CSF virus
(Blacksell, 2001). Under the current delivery processes there appears little likelihood that
pig producers, particularly those in the smallholder sector, will gain benefit from
participating in a vaccination program.
To navigate through the constraints of delivering a quality vaccine, a great deal of planning
will be required on the part of the Lao animal health service. Vaccine delivery to the point
of use should ideally be from the VSU without storage at district and provincial offices if
the issue of freeze-thaw cannot be circumvented. This strategy will be met with logistical
and financial constraints; however small-scale targeted delivery would provide greater
benefit than the use of impotent C-strain vaccine. The chief limitation of the research
presented in this chapter was the failure to determine the stability of vaccine in the two
months following storage at 4 C. If the vaccine were found to be stable for at least a month
at 4 C, short term storage at provincial and district officers would be a possibility. For this
reason, the experiment should be repeated to determine the short term duration of stability
when the vaccine is stored at 4 C.
Conclusions
In order to assess the potential of removing the freeze-thaw cycles inherent in the current
transport and storage process in Laos, vaccine stability at 4 C was assessed over a four
month period. The vaccine was found to be stable for less than three months under these
storage conditions.
In Laos, the locally produced lapinised C-strain vaccine has a prescribed shelf life of one
year when stored at 20 C. The research presented in this chapter clearly shows that under
optimal storage conditions the vaccine shelf-life is not greater than four months. This
problem is compounded at the provincial level where vaccine was found to be stored at
approximately 10 C.
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Chapter 7
Chapter 7.
General discussion
171
Chapter 7
172
Chapter 7
7.1 Introduction
This thesis presents information that adds to the body of knowledge about the CSF situation
in Laos. The introduction section described three central aims that would contribute to the
understanding of CSF in Laos and provide new tools and strategies for the control and
management of outbreaks if and when they occur. These aims were as follows:
1. To describe the performance of the smallholder pig sector and develop a better
understanding of the importance of CSF to pig production in this sector.
2. To develop, validate and implement a simple, rapid and portable diagnostic test for the
diagnosis of CSF.
3. To determine the stability of the locally produced CSF vaccine under different
temperature storage conditions.
The issues of CSF in Laos have been addressed from these three different perspectives. The
following discussion and conclusions provide a linkage between the results of the three
different components that separately contribute to improved diagnosis and management of
CSF in Laos. Also discussed are suggestions for future research.
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The research presented in this thesis demonstrates that disease has a negative impact on
production and trade in a village, with farmers selling off sick pigs when a disease event is
recognised in order to maximise profits from a valuable yet vulnerable asset. This practice
is not necessarily limited to CSF; disease in general severely impedes production potential.
The exodus of potentially infected stock from a village during an outbreak, however, only
serves to exacerbate the problem. The introduction of infected stock into a susceptible
population has been recognised as a major mode of CSF virus transmission; as has the
distribution of pork products containing CSF virus (Ribbens et al. 2004). In relation to
disease events, the trading patterns described in this thesis play a critical role in the
transmission and maintenance of CSF virus in the smallholder pig population of Laos.
Also of importance is the practice of not growing out pigs beyond the age of approximately
three months due to fear of disease and the constraints of poor nutrition. This practice
results in considerable lost production. If disease control were to be achieved, resulting in
improved confidence of pig owners to grow their pigs to a larger size, production could
potentially be increased without necessarily increasing pig numbers.
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sample storage, lack of continued interest shown by field staff and redeployment of trained
staff (Blacksell, 2001). In addition to these constraints, turn-around time can be up to and
greater than one month in some cases. The reasons for these delays are prolonged sample
transport time and the cost of ELISA plates and reagents for the laboratory off-setting the
need for urgency. Generally, a test run will be performed when enough samples have been
submitted to the laboratory to minimise wastage. Combined with these two factors is the
length of time it takes to perform the AC-ELISA; under normal conditions in Laos this test
takes a minimum of two days to perform. At present there are no simple, rapid, accurate
and inexpensive tests available that can be performed on-site or near-to-field.
Research has been presented that describes a cheap, simple, rapid, robust and sensitive test
for the diagnosis of CSF. The current format of the test can not be performed in the field or
in a provincial office/laboratory. However, the test has been developed and validated to
such an extent that the next phase of adaptation to a kit format can proceed. Such
developments would provide a valuable tool to veterinary and livestock officers in Laos. It
is anticipated that turn-around time would be greatly reduced if tests were to be performed
near-to-field. Many of the constraints to effective diagnosis discussed above and elsewhere
in this thesis could be reduced by taking reliable diagnostic services to provincial centres
rather then attempting to transport clinical samples to Vientiane. With such a tool, the
spread of and maintenance of CSF virus in the Lao pig population could be monitored with
greater accuracy.
One key constraint to effective diagnosis of CSF is participation by farmers and their
understanding of the diagnostic process and how it may benefit disease management.
Without effective disease management coinciding with improved diagnostic services,
farmer participation levels in the process will not increase. The greatest benefit will come
from the continued development of the rapid IMB-ELISA if the results of testing are used
to coordinate an effective response. Without such a response farmers will not benefit and
interest in the test at all levels will wane. It will be important for the test to be brought into
the mainstream of CSF diagnosis in Laos so that the investment in this work results in a
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Chapter 7
sustainable benefit. It is the authors great fear that this test will end up on a shelf
somewhere in a storeroom of the National Animal Health Centre.
176
Chapter 7
IMB-ELISA development
The IMB-ELISA presented in this thesis has been shown to be a cheap, simple, rapid,
robust and potentially portable format for the diagnosis of CSF. However, a number of
knowledge gaps exist that need to be filled by conducting further research. In order to show
that the test is highly specific and the monoclonal antibody used is not cross-reactive with
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other pestiviruses, a panel of BVD viruses and BD viruses should be screened. Conversely,
to show that the test is broadly reactive with all groups of CSF virus, a panel of viruses
spanning all groups should be tested to assess the assays potential to be applied universally
for CSF diagnosis. While it is not likely that BVD and BD viruses would cause
complications with CSF diagnosis in Laos, more widespread acceptance of the test would
follow from this sort of validation of specificity.
As mentioned in the discussion sections of Chapters 4 and 5, a number of areas need further
work to fully validate and evaluate the IMB-ELISA. The robustness of the test needs
further evaluation using a wider range of samples that should be tested using the percent
positivity against the strong positive control as the test variable. The IMB-ELISA should
also be assessed for its usefulness in capturing viral antigen from blood; from the white cell
fraction (buffy coat), from whole blood and from serum. Detection of antigen in the buffy
coat fraction would not be expected to pose a problem as this has been shown to be possible
using the AC-ELISA. However, antigen detection from the buffy coat in the field would not
be possible as laboratory equipment is required. Detection of antigen from whole blood or
serum would be useful in the field if the test was shown to be sensitive enough to achieve
this end. Also, to evaluate fully the diagnostic sensitivity and specificity of the test, with the
AC-ELISA as the reference standard, a second set of clinical samples needs to be screened
The IMB-ELISA in its current form can be simplified and modified for adaptation into a
near-to-field test. A number of areas need special attention for this to be achieved. As
mentioned in Chapter 5, conjugated-antibody stabilisation buffer and stabilised TMB
substrate need to be fully assessed to eliminate the need for reagent dilutions near-to-field.
The next major challenge will be adapting the sample preparation procedure into a simple
test format. At present, the tissue homogenate is clarified by centrifugation, which will not
be possible in the field. A number of methods could be explored; initially, microfuge tube
pestles could be examined for their potential in grinding the tissue in a 1.5 mL microfuge
tube with the required volume of 1 % NP40 solution. The resulting homogenate could be
clarified sufficiently by allowing the tissue to settle out of solution. Secondly, the use of
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Chapter 7
metal ball bearings or something similar could be used in a larger tube to sufficiently break
up the tissue sample with the required volume of 1 % NP40. As before, the homogenate
could then be clarified by settling. Ideally, to minimise any problems with the separation of
the magnetic beads from the homogenate, a small, portable and inexpensive microcentrifuge could be used to clarify the homogenate.
The next step in developing the test will involve the standardisation of volumes dispensed
from dropper bottles used to supply reagents. This step will need to be carried out in
conjunction with the development of a means to remove small amounts of reagent from the
IMBs following wash steps. It will be particularly important to remove all traces of
unbound HRP-conjugated antibody to reduce the chance of generating false positive results.
Incubation times and temperature need to be assessed and validated. All incubations have
been carried out at 37 C; this will not always be possible near-to-field, particularly if the
test is performed in an air-conditioned provincial office/laboratory. Ndhlovu et al. (1995)
found no significant difference between incubation temperatures of 18, 30 and 37 C using
a similar test format to that presented in this thesis.
Of critical importance will be the standardisation of the end result to ensure different users
are returning the same result for the same sample; that is, to ensure the test is robust in the
kit format. As it stands, the test is read by eye in the microfuge tube. This method can be
troublesome for samples returning low positive results as microfuge tubes are generally
slightly opaque. To aid the user in determining a result after the 5 min incubation with
TMB substrate, the IMBs could be pulled out of suspension by magnetic force and the
supernatant spotted onto a piece of white absorbent paper. There would then need to be a
standard colour chart to accompany the kit with which to compare the test sample and
controls.
179
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180
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202
Appendix
9.5 mL
N,N-dimethyl formamide
0.5 mL
3-amino-9-ethylcarbazole (AEC)
4 mg
30 % H2O2
5 L
4.5 mL
Sterile PBSA
95.5 mL
Proclin 300
100 L
Store at 4 C
1.5 g
2.93 g
Make up to 1 litre with distilled TC water and adjust the pH to 9.6 using 10M NaOH
203
Appendix
Citrate/Acetate buffer
Sodium acetate (anhydrous)
8.2 g
DiRO H2O
100 mL
Dissolve sodium acetate in approximately 90 mL DiRO H2O and adjust to pH 5.9 with 1M
citric acid. Make up volume to 100 mL with DiRO H2O
1.15 g
250 mL
Dissolve sodium acetate in 240 mL sterile DiRO H2O and adjust pH to 5 with concentrated
acetic acid. Bring final volume up to 250 mL with sterile DiRO H2O
2.8 mL
PBST
235 mL
Glycerol
12.5 mL
Proclin 300
250 L
Store at 4 C
204
Appendix
50 mL
50 mL
100x Amphotericin B
5 mL
100x pen/strep
5 mL
7% NaHCO3
5 mL
1M HEPES
1.25 mL
100x L-Glutamine
5 mL
1 % NP40 PBSA
NP40
2 mL
PBSA
198 mL
10x PBSA
NaCl
80 g
KCl
2g
Na2HPO4 (anhydrous)
11.5 g
KH2PO4
2g
Make up to 1 L with DiRO H2O water and dilute 10 for working dilution of 1x
5 mL
10x PBSA
1L
9L
205
Appendix
180 mL
20 mL
Proclin 300
200 L
Store at 4 C
56 mL
DiRO H2O
944 mL
1125 L
Sterile PBSA
99 mL
Proclin 300
100 L
Store at 4 C
0.101 g
DMSO
10 mL
TMB substrate
DiROH2O
7.2 mL
0.8 mL
TMB stock
80 L
30 % H2O2
1 L
206
Appendix
Operator 2
Operator 3
Sample
Number
AC-ELISA
Result
Previous
IMB-ELISA
Result (% P)
By eye
%P
Result
By eye
%P
Result
By eye
%P
Result
003/03
n (1)
010/03
073/03
110/03
010/04
025/04
032/04
040/04
045/04
050/04
073/04
079/04
080/04
084/04
109/04
127/04
024/02
032/02
035/02
049/02
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n (1)
n (-1)
n (0)
n (0)
n (5)
n (1)
n (-1)
n (0)
n (-1)
n (-1)
n (2)
n (-1)
n (0)
n (-2)
n (-1)
n (-1)
n (-1)
n (-1)
n (1)
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
3
0
1
0
0
0
1
0
1
0
7
0
0
0
0
1
0
0
0
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
p
n
n
n
n
n
n
n
n
1
0
0
3
4
1
0
0
0
0
7
0
2
0
0
4
0
2
0
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
p
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
1
35
0
0
3
0
0
2
3
0
6
0
0
0
0
3
0
0
0
n
p
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
n
207
Appendix
Operator 2
Operator 3
Sample
Number
AC-ELISA
Result
Previous
IMB-ELISA
Result (% P)
By eye
%P
Result
By eye
%P
Result
By eye
%P
Result
061/03
n (8)
14
062/03
p (16)
23
26
19
079/03
p (22)
13
17
17
122/03
p (20)
20
24
21
034/04
n (5)
047/04
n (7)
11
11
119/04
n (9)
11
086/04
n (4)
11
087/04
p (17)
11
11
002/03
n (5)
041/02
p (11)
11
10
10
066/03
p (104)
102
100
101
070/03
p (88)
74
81
67
074/03
p (37)
54
29
42
105/03
p (88)
106
93
85
109/03
p (106)
102
111
99
033/04
p (37)
32
43
36
107/04
p (36)
21
23
28
117/04
p (86)
80
70
53
120/04
p (92)
105
84
82
208
Sample
Figure A1. Graphical representation of operator agreement when IMB-ELISA result was expressed as percent positivity. Error bars
indicate 20 % of Operator 1 result.
209
120/04
117/04
107/04
033/04
109/03
105/03
074/03
070/03
066/03
041/02
002/03
087/04
086/04
119/04
047/04
034/04
122/03
079/03
062/03
061/03
049/02
035/02
032/02
024/02
127/04
109/04
084/04
080/04
079/04
073/04
050/04
100
045/04
040/04
032/04
025/04
010/04
110/03
073/03
010/03
003/03
Percent positivity
Appendix
140
120
Operator 1
Operator 2
80
Operator 3
60
40
20
Appendix
2.0
R2 = 0.0011
1.6
1.2
0.8
0.4
0.0
0
20
40
60
80
100
Sow/Boar Ratio
10
R2 = 0.0614
8
6
4
2
0
0
20
40
60
80
100
Sow/boar ratio
Figure A3. Relationship between sow/boar ratio and average litter size
210
Appendix
211