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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Unpublished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
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Improved diagnostics and management of classical swine fever in the Lao People's
Democratic Republic
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Improved Diagnostics & Management

of Classical Swine Fever in the
Lao Peoples Democratic Republic

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

Introduction and literature review

1.1 Pig production and health in Laos

1.2 Classical Swine Fever Virus

11

1.3 Immunmagnetic bead technologies and diagnostic application

39

1.4 Project outline and perspective

41

1.5 Research objectives and hypothesis

44

Chapter 2

Pig production and health in Laos

45

2.1 Introduction and research design

47

2.2 Methods

48

2.3 Results

57

2.4 Discussion and conclusions

70

Chapter 3

General materials and methods

77

3.1 Introduction

79

3.2 General cell culture

79

3.3 Peroxidase linked assay

80

3.4 Virus isolation

80

3.5 Virus titration

81

3.6 Antigen capture-ELISA

83

3.7 Neutralising peroxidase linked assay

86

vi

Chapter 4

IMB-ELISA development

91

4.1 Introduction

93

4.2 General materials and methods

94

4.3 Methods and results for the development of IMB-ELISA

97

4.4 Discussion and conclusions

117

Chapter 5

Assessment and validation of IMB-ELISA

121

5.1 Introduction

123

5.2 Methods and results

123

5.3 Discussion and conclusions

146

Chapter 6

Assessment of storage temperature on lapinised C-strain

vaccine produced in Laos

153

6.1 Introduction

155

6.2 Material and methods

157

6.3 Results

160

6.4 Discussion and conclusions

167

Chapter 7

General discussion and conclusions

7.1 Introduction

171
173

7.2 Smallholder pig production and CSF as a contributor

to poor performance

173

7.3 The importance of improved CSF diagnostics

174

7.4 Vaccination as a control strategy

176

7.5 Future research

177

7.6 Concluding remarks

180

Bibliography

181

Appendices
Appendix I:

Reagents and solutions

203

Appendix II:

Inter-operator agreement results

207

Appendix III:

Reproductive performance correlations

210

vii

Abbreviations
AAHL

Australian Animal Health Laboratory

AC-ELISA

Antigen capture-ELISA

ACIAR

Australian Centre for International Agricultural Research

AEC

amino ethyl carbazole

BDV

Border disease virus

BSA

Bovine serum albumin

BVDV

Bovine viral diarrhoea virus

CI

confidence interval

cm

centimetre

CPE

cytopathic effect

CO2

Carbon dioxide

CSF

Classical Swine Fever

CSIRO

Commonwealth Scientific and Industrial Research Organisation

CTB-ELISA Complex trapping blocking-ELISA

DiRO

Deionised reverse osmosis

EDAC

1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride

ELISA

Enzyme linked immunosorbent assay

EMEM

Earles minimum essential medium

FAO

Food and Agriculture Organisation of the United Nations

FAT

Fluorescent antibody test

FBS

Foetal bovine serum

FITC

Fluorescein isothiocyanate

FMD

Foot and mouth disease

FSG

Fish skin gelatin

gram

GDP

Gross domestic product

GNI

Gross national income

hour

viii

HEPES

Hydroxethylpiperazine-ethanesulphonic acid

HRP

Horse radish peroxidase

H2SO4

Sulphuric acid

IgG

Immunoglobulin G

IMB

Immunomagnetic bead

Mab

Monoclonal antibody

MAF

Ministry of Agriculture and Forestry

min

minute

MLV

Modified live vaccine

MPK

Minipig kidney cell line

MS

Microsoft

NP-40

Nonidet P-40

NPLA

Neutralising peroxidase linked assay

NS

Non-structural

NVI

National Vaccine Institute

OD

Optical density

OIE

Office International des Epizooties (World Health Organisation for Animals)

ORF

Open reading frame

PBSA

Phosphate buffered saline (A)

PBST

Phosphate bufferedsaline with Tween 20

PCR

Polymerase chain reaction

PDR

Peoples Democratic Republic

PK15

Porcine kidney cell line

PLA

Peroxidase linked assay

RNA

Ribonucleic acid

rpm

revolutions per minute

RT-PCR

Reverse transcriptase-PCR

SD

Standard deviation

SK6

Swine kidney cell line

SMP

Skim milk powder

ix

TCID

Tissue culture infectious dose

TMB

5, 5-Tetramethylbenzidine

US$

US dollars

UTR

Untranslated region

UV

Ultraviolet

VSU

Vaccine supply unit

VVW

Village veterinary worker

microlitre

List of Tables

Table 1.1 Pig population by region and province

Table 1.2 Summary of immunomagnetic bead detection systems

40

Table 2.1 Village level descriptive variables of pig production and health
in Bolikhamxay province, Laos, May 2002

59

Table 2.2 Indicators of reproductive performance for smallholder sows in

Bolikhamxay province, Laos, 2002 to 2005

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

Table 5.2 AC-ELISA verses IMB-ELISA without a pre-test incubation

of sample with normal pig or goat serum

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

Table 5.5 Agreement between IMB-ELISA results determined by recording

a visual colour change and measuring the OD450 nm

143

Table 5.6 Comparison of IMB-ELISA with the AC-ELISA results when

IMB-ELISA was read by eye

144

Table 5.7 Comparison of IMB-ELISA with the AC-ELISA results when

IMB-ELISA result was determined from OD450nm

144

Table AI. Raw data for inter-operator agreement assessment

207

xi

List of Figures
Figure 1.1 Laos in Southeast Asia

Figure 1.2 Provinces and regions of Laos

Figure 1.3 Livestock population trends in Laos, 1993 to 2003

Figure 1.4 Schematic diagram of the pestivirus genome and the

corresponding translated proteins

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

Figure 2.2 Questionnaire for the collection of village pig

health and production information

54

Figure 2.3 a) Pig production off-take by off-take category; and

b) production off-take by age class

64

Figure 2.4 Proportion of purchases into the village production units

by age class

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

Figure 4.1 Optimisation of EDAC concentration

100

Figure 4.2 Optimisation of goat and rabbit polyclonal antibody

coating concentration
Figure 4.3 Optimisation of HRP-conjugated antibody dilution in IMB-ELISA

103
106

xii

Figure 4.4 Optimisation of Mab 24/10 dilution in IMB-ELISA

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

Figure 4.7 Comparison of goat and rabbit polyclonal antigen capture

antibody using diagnostic spleen specimens a) 028/04,
Luang Prabang; b) 087/04, Bolikhamxay; and c) 088/04, Houaphan

111

Figure 4.8 Optimisation of bead volume used in each reaction

113

Figure 4.9 Optimisation of incubation times

116

Figure 5.1 Repeatability of IMB-ELISA

125

Figure 5.2 Stability of polyclonal capture antibody bound to IMBs

127

Figure 5.3 Stability of pre-diluted reagents in IMB-ELISA stored at 4 C

128

Figure 5.4 IMB-ELISA verses AC-ELISA without pre-incubation of

sample with serum

136

Figure 5.5 Incubation of samples 087/03 and 107/03 with serum before
the IMB-ELISA

137

Figure 5.6 IMB-ELISA verses AC-ELISA with a pre-incubation of sample with

10 % normal goat serum (NGS) before the addition of IMBs

138

Figure 5.7 Frequency distribution of IMB-ELISA results for samples

negative by AC-ELISA

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

Figure 6.3 Serum neutralising antibodies to CSF virus post vaccination

with C-strain vaccine batch 05/2005

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

Figure A1. Graphical representation of operator agreement when IMB-ELISA

result was expressed as percent positivity

209

Figure A2. Relationship between sow/boar ratio and litter-per-sow-per-year

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

1.1 Pig production & health in Laos

1.1.1 Country overview and livestock sector brief
The Lao Peoples Democratic Republic (Laos) is the only landlocked country in Southeast
Asia and is situated between the Peoples Republic of China to the north, Myanmar to the
northwest, Thailand to the west, Vietnam to the east and Cambodia to the south (Figure
1.1). The country is approximately 230,800 km2 in area, of which 18,360 km2 or 8.0 % of
total land area is considered agricultural land. The amount of agricultural land per 100
people is 34.8 hectares or 45.5 hectares for every 100 people involved in agricultural
production (FAO, 2003b). The country is divided into 18 provinces, 142 districts and has a
total of 12,458 villages (Figure 1.2; WHO, 2002).
Laos has a predominantly rural based human population of 5.5 million with a density of
22.9 persons per square kilometre and an average annual growth rate of 2.3 % (FAO,
2003b; World Bank, 2004). In 2000, 81 % or 4.2 million people lived in rural areas and 2
million people were economically active in the agricultural sector (FAO, 2003b). Of the
0.80 million households in Laos in 1999, 0.67 million (83 %) were considered agricultural
holdings, of which 94 % were village smallholder producers using traditional practices
(MAF, 2000).
In 2002, Laos had a total gross domestic product (GDP) of 1.7 billion US dollars (US$) and
a gross national income (GNI) per capita of US$ 310. The significance of the agricultural
sector to Laos is highlighted by the contribution made to the GDP, contributing 50.9 %
followed by services and industry contributing 25.7 % and 23.4 %, respectively of total
GDP (World Bank, 2004). In 2000, the livestock sector contributed 9.2 % to total GDP
(FAO, 2003b).
The Agricultural Census of 1998/1999 (MAF, 2000) reported that 95 % of livestock in the
country was produced by smallholder farmers and 89 % of all agricultural households raise
at least one type of livestock; whether it be cattle, buffalo, pigs, goats or poultry. Many
households view livestock acquisition and improved livestock health as a means to reduce

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

Figure 1.1 Laos in Southeast Asia

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

Figure 1.2 Provinces and regions of Laos (WHO, 2002)

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

Figure 1.3 Livestock population trends in Laos, 1993 to 2003 (MAF,

2004)
5

Chapter 1

1.1.2 Pig Production

Four indigenous pig breeds are recognised in Laos, Moo Chid, Moo Laat, Moo Daeng
and Moo Nonghaet (Vongthilath and Blacksell, 2000), most of which are high fat,
swaybacked Asian breeds (Str et al. 2002) and are black in colour with the exception of
Moo Daeng (which literally means red pig). Landrace and Duroc are the most popular and
common of the imported breeds raised in Laos (Vongthilath and Blacksell, 2000). While
indigenous pig breeds raised by traditional practices far out number imported breeds
(Vongthilath and Blacksell, 2000), commercial farming using non-traditional practices is on
the increase, with semi-intensive farming concentrated in the central provinces of Vientiane
Capital and Savannakhet (MAF, 2002). Indigenous pig production using traditional farming
practices accounted for approximately 89 % of total production in 2002 (MAF, 2002)
compared to an estimated 96 % in 1999 (Vongthilath and Blacksell, 2000). Pig raising
density using traditional practices varies somewhat by region and is most prevalent in the
north of the country; greater than 60 % of agricultural holdings in the northern provinces
raise pigs, compared to 30 60 % of agricultural holdings in most central and southern
provinces (Table 1.1; MAF, 2000).
Vongthilath and Blacksell (2000) describe three pig raising systems in Laos: smallholder or
traditional farming system, small family business and semi-intensive. The typical
smallholder farm raises 1-3 pigs that are left to roam free in the village and in some cases
are penned. These pigs are fed a variety of feed stuffs, ranging from rice bran, corn,
cassava, waste from village rice whiskey production, edible grasses or weeds, household
food scraps (Vongthilath and Blacksell, 2000), banana stem, taro and yams (Str et al.
2002). This form of production is very labour intensive and food collection can take up to 2
to 3 hours each day in some areas (Str et al. 2002). Smallholder pig farming is generally
the responsibility of women in all regions of Laos (ADB, 2002). The small family business
system generally raises 3 to 6 indigenous, imported or cross breed pigs in a penned system
in the backyard or village environment. Pigs raised in this manner are managed and fed
with higher quality feed (Vongthilath and Blacksell, 2000). Semi-intensive pig farming
systems raise large numbers of imported breed pigs in a managed farm situation and are fed

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

1999 Pig Population ('000)

Proportion of Total (%)

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).

1.1.3 Pig production constraints

Disease has been identified as the major constraint to pig production in Laos by a number
of authors (Str et al. 2002; Vongthilath and Blacksell, 2000). Classical swine fever (CSF)
is recognised as the infectious disease of pigs that has the greatest importance to pig
production (Str et al. 2002), and likely accounts for a large number of pig deaths in all
production systems (Vongthilath and Blacksell, 2000). Other viral diseases of swine and
bacterial diseases such as salmonellosis (Salmonella choleraesuis) and erysipelas
(Erysipelothrix rhusiopathiae) are likely to have a large impact, as does infestation with the
parasite Ascaris suum, which results in retarded growth and production losses (Vongthilath
and Blacksell, 2000). Poor nutrition is also a major constraint to pig production in Laos
(Str et al. 2002; Vongthilath and Blacksell, 2000), and contributes to poor resistance,
which in turn exacerbates the problems of infectious disease (Str et al. 2002). Poor
nutrition is the result of feeding poor quality feed and the lack of availability of higher
quality feeds such as broken and cracked rice, cassava and maize (Str et al. 2002;
Vongthilath and Blacksell, 2000). The high cost of quality commercial feed limits its use
predominantly to semi-intensive pig farmers (Vongthilath and Blacksell, 2000). Other

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.

1.1.4 Diagnostic capabilities for the detection CSF virus in Laos

At present, the only reliable diagnostic test used for the detection and diagnosis of CSF
virus in Laos is the antigen capture (AC)-ELISA described by Shannon et al. (1993) with
some minor modifications made to the original protocol (Blacksell, 2001). More details of
this test are described below under diagnostic methodologies for CSF virus. Diagnostic
samples are collected during disease outbreaks by district and provincial livestock officers
who are part of a diagnostic sample submission network coordinated by National
Veterinary staff (Blacksell, 2001; Vongthilath and Blacksell, 2000). Following collection,
samples are submitted to the National Animal Health Centre in Vientiane by road, air
freight or express mail service and are usually in transit for less than one week depending
on the origin (Blacksell, 2001). The detection of outbreaks (or suspected outbreaks) in Laos
is passive, with detection reliant on the pig owners notifying district or provincial livestock
officers (Blacksell, 2001). Inherent problems exist in using a passive system of disease
notification, with underreporting and slow reporting likely to occur (Cameron, 2000). In
Laos, the submission network is highly dependent on disease and diagnostic service
awareness at the village, district and provincial level together with the provision of funds to
reimburse fuel and shipping costs (Blacksell, 2001). Blacksell (2001) identified a number
of constraints to the network; namely financial sustainability, maintaining interest of district
and provincial livestock officers and the redeployment of trained staff to other government
sectors.

10

Chapter 1

1.2 Classical swine fever virus

1.2.1 Viral Properties
Taxonomy
Classical swine fever (CSF) virus, otherwise known as hog cholera virus, is a member of
the Pestivirus genus of the family Flaviviridae. Other members of the Pestivirus genus
include border disease (BD) virus, bovine viral diarrhoea virus 1 (BVDV-1), bovine viral
diarrhoea virus 2 (BVDV-2) and a tentatively classified species isolated from giraffe (Heinz
et al. 2000; Pringle, 1999). All members of the genus are antigenically related and crossreactive epitopes are present on all pestiviruses. Demarcation of species within the genus
has been carried out using nucleotide homology analysis, cross neutralisation properties and
host specificity (Heinz et al. 2000).

Viral structure & morphology

Pestivirus virions are spherical in shape and 40-60 nm in diameter. Pestiviruses are
enveloped viruses with 10 12 nm ring-like subunits on the envelope surface. Virions have
a buoyant density in sucrose of 1.10 1.15 g/cm3 and a sedimentation coefficient (S20W) of
140 150 S. Virus infectivity remains relatively stable over a broad temperature range but
becomes increasingly less stable at temperatures above 40 C. Solvents and detergents
rapidly inactivate viral particles (Heinz et al. 2000).

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

~ 4000 amino acid polyprotein

Co- and post-translational
processing

Npro

Erns E1 E2

NS2-3
p7

NS5A
NS4A NS4B

NS5B

Figure 1.4 Schematic diagram of the pestivirus genome and the

corresponding translated proteins (Source: Heinz et al. 2000)

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).

1.2.2 Nature of the disease caused by CSF virus infection

Pathogenesis
The principal mode of entry in the pig under natural conditions is the oronasal route
although other possible routes of entry include the conjunctival and genital mucous
membranes and via skin abrasions (van Oirschot, 1999). The primary site of viral
replication is the tonsil and can be demonstrated as early as 7 hours post exposure (Stewart,
1981; van Oirschot, 1999). The initial infection of the epithelial cells of the tonsil spreads
to surrounding lymphoreticular tissue and virus is subsequently transferred to the lymph
nodes draining the tonsillar region via lymphatic vessels (van Oirschot, 1999). Following
replication in these lymph nodes, the virus enters the peripheral blood resulting in rapid
replication and growth to high titres in the spleen, bone marrow, visceral lymph nodes and
lymphoid structures lining the small intestine such as Peyers patches and lymphoid
follicles. A high level of viraemia is seen as a result of growth in lymphoid structures and
circulating leukocytes and mononuclear cells (van Oirschot, 1999). The presence of virus
has also been shown in the epithelial cells of the pharyngeal mucosa, gastrointestinal tract,
kidney, urinary bladder, gall bladder, bile duct, pancreas, salivary glands, nictitating
membrane, uterus, adrenal gland and thyroid (Stewart, 1981). Evidence of virus has also
been found in the male reproductive tract (Choi and Chae, 2002; Choi and Chae, 2003a),
ovaries (Choi and Chae, 2003b), renal glomeruli (Choi and Chae, 2003c) and thymus
(Sanchez-Cordon et al. 2002). While almost all pigs die of acute CSF, the mechanism
responsible for death remains unclear, however the severe disturbance of the circulatory
system seems to be the most likely cause (van Oirschot, 1999). The initial phase of chronic
infection resembles the acute course albeit at a much slower rate of spread and lower virus
titres in serum and organs (van Oirschot, 1999).

14

Chapter 1

Leukopenia, particularly depletion of B-lymphocytes, helper T-cells and cytotoxic T-cells,

is a classic feature of CSF in the early stages of disease (Summerfield et al. 2001).
Thrombocytopenia is also a typical characteristic of CSF with large numbers of platelets
lost in the first instance as a result of massive activation and aggregation, with subsequent
phagocytosis of platelets by resident macrophages in the spleen and liver (Bautista et al.
2002). This initial thrombocytopenia is likely to be exacerbated by impaired
megakaryocytopoiesis, as suggested by the appearance of megakaryocyte lesions in the
bone marrow (Gomez-Villamandos et al. 2003).

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

1.2.4 Diagnostic techniques

Clinical diagnosis
It is difficult to make a confident diagnosis of CSF based solely on clinical findings. The
clinical signs and lesions (section 1.2.2 above) vary depending on the virulence of the
causative virus strain (Floegel-Niesmann et al. 2003; Pearson, 1992) and may also vary
between individual animals infected by a single strain of virus (Floegel-Niesmann et al.
2003). In fact, Floegel-Niesmann et al. (2003) found that a single strain of virus could show
varying degrees of virulence in experimentally infected pigs, indicating that host factors
play a key role in the development of clinical signs and lesions. Clinical diagnosis is further
complicated by inter-clinician variation. Accuracy of clinical diagnosis varies depending on
whether post-mortem findings and clinical signs are considered together or separately and
on whether CSF virus is known to exist in a particular region (Elbers et al. 2004). During
the 1997 1998 outbreak of CSF in The Netherlands, it was found that diagnosis based on
either clinical signs or post mortem lesions had only moderate sensitivity and specificity
even under optimised conditions (Elbers et al. 2002; Elbers et al. 2003).
It is therefore necessary to subject suspicions of CSF infection to further laboratory
investigation (Pearson, 1992; van Oirschot, 1999) to distinguish between CSF and other
diseases such as African swine fever, porcine reproductive and respiratory syndrome,
salmonellosis, erysipelas, pasteurellosis, streptococcosis, Haemophilus suis infection (van
Oirschot, 1999), Aujeszkys disease, viral encephalomyelitis, poisoning by agents such as
warfarin (Anonymous, 1996) and newly emerging pig diseases such as porcine dermatitis
and nephropathy syndrome (Paton and Greiser-Wilke, 2003) and porcine multi-systemic
wasting disease.
When CSF is suspected in a herd the likelihood of correctly identifying the disease in the
laboratory depends on the number of animals sampled for further investigation and on the
veterinarians choice of animal, based on clinical observations (Bouma et al. 2001). Bouma
and others (2001) found that if four or five animals were selected for sampling during the
epidemic in The Netherlands in 1997 1998, then the probability of correctly identifying

24

Chapter 1

an outbreak increased to greater than 95 %, compared to 60 % if only one animal was

selected. The appropriate samples for maximising sensitivity of detecting CSF virus in the
laboratory are discussed in the section outlining laboratory diagnosis below.

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.

Detection of viral antigen

Detection of viral antigen is achieved by either immunofluorescence staining of frozen
tissue sections, by the capture and detection of antigen by enzyme linked immunosorbent
assay (ELISA) (OIE, 2004; Shannon et al. 1993) or by immunohistochemical detection of
viral antigen in paraffin-embedded tissues (de las Mulas et al. 1997). Immunofluorescence
staining or the fluorescent antibody test (FAT) using tissue impression smears of
mandibular and parotid lymph nodes, kidneys, lungs, mandibular and parotid salivary
glands, lacrimal glands and spleen from experimentally infected pigs has been described
(Aiken et al. 1964). An improved direct FAT using cryostat sections has now become the

25

Chapter 1

standard method of detecting CSF viral antigen by immunofluorescence staining (OIE,

2004; Pearson, 1992). Direct FAT is the test of choice for detecting viral antigen in tissue
samples (van Oirschot, 1999) due to its sensitivity and the speed of achieving a test result
(de Smit, 2000). The tissue of choice for early detection is the tonsil. However, the spleen,
kidney, mandibular lymph node and the distal portion of the ileum are appropriate samples
(de Smit, 2000; Pearson, 1992). Pan and others (1993) have also found that the skin, tongue
and brain are suitable organs for CSF diagnosis by FAT. Cryostat sections are fixed in
acetone and washed before staining directly with fluorescein isothiocyanate (FITC)
conjugated to anti-CSF virus polyclonal immunoglobulin and examined by fluorescence
microscopy. Positive results are subjected to further scrutiny using a panel of speciesspecific monoclonal antibodies conjugated to horse radish peroxidase to differentiate
between CSF, BVD and BD viruses (OIE, 2004; Pearson, 1992).
Immunohistochemical detection of CSF virus in formalin-fixed and paraffin wax embedded
tissue sections has been achieved using a monoclonal antibody avidin-biotin-peroxidase
complex (ABC) methodology (de las Mulas et al. 1997). The monoclonal antibody
employed in this study was specific for the E2 glycoprotein of CSF virus and has been
shown to be non-reactive with other pestivirus E2 glycoproteins (de las Mulas et al. 1997).
This feature gives the test the advantage that secondary screening of positive results is not
necessary. Another advantage of this test format is the preservation of histological detail;
disadvantages however include a prolonged test period compared to the direct FAT and the
fact that formalin fixation prevents infectious virus isolation and subsequent virus
characterisation (de Smit, 2000).
Methodologies to capture and detect viral antigen from tissue and blood of infected pigs by
ELISA have been developed and employ a sandwich format using polyclonal and
monoclonal antibodies (Clavijo et al. 1998; Shannon et al. 1993). In both ELISA formats
one antibody is used to capture viral antigen followed by detection using the second
antibody; a horse radish peroxidase conjugated tertiary antibody is then used to detect
bound secondary antibody by chromogen colour development.

26

Chapter 1

The antigen-capture ELISA (AC-ELISA) developed by Clavijo et al.(1998) gave a

specificity of greater than 98 % and sensitivity greater than 91 % when compared with both
virus isolation and RT-PCR on tissue samples collected from infected pigs. Detection of
antigen by ELISA is however less sensitive when used to detect antigen in blood collected
from live animals at different stages of infection (Dewulf et al. 2004a). Dewulf and others
(2004) found that detection of virus by AC-ELISA was at a significantly later stage of
infection than virus isolation or RT-PCR; this finding is in agreement with other studies
(Kaden et al. 1999). However, the Dewulf et al. (2004) study found that only 75 % of
infected pigs were eventually detected by AC-ELISA on blood compared with 100 % of
those tested in the Kaden et al.(1999) study.
The AC-ELISA is primarily suited for use in screening (de Smit, 2000; Dewulf et al.
2004a; Paton and Greiser-Wilke, 2003) due to its relatively low cost and the test format is
suited to being scaled up to rapidly test a large number of samples. However, the antigencapture ELISA is not generally used as the sole means of making a laboratory diagnosis
and negative results need to be followed up using more sensitive detection methods
(Dewulf et al. 2004a). Antigen-capture ELISA kits are commercially available and the
performance of a number of these kits has been assessed (Depner et al. 1995; Kaden et al.
1999).

Virus isolation and detection

In vitro isolation and subsequent detection of CSF virus is laborious and time consuming
and requires reliable tissue culture facilities and results may not be known for several days
after samples are processed (de Smit, 2000; OIE, 2004). However, the technique is
considered the most sensitive method for the detection of CSF virus in clinical samples (de
Smit, 2000; Pearson, 1992; van Oirschot, 1999). Virus isolation is achieved by inoculating
a 10 20 % clarified tissue homogenate or freeze thawed whole blood fraction onto a nonconfluent monolayer of porcine kidney (PK15) cells or other suitable cells such as SK6
(OIE, 2004). Following a 1 hour adsorption period the inoculum is replaced with growth
medium and the cells incubated. After 3 or 4 days the cells are fixed and antigen detection

27

Chapter 1

methods are used to identify CSF virus. Immunofluorescence or immunoperoxidase

staining techniques with monoclonal or polyclonal antibodies are generally used to achieve
this end (OIE, 2004; Pearson, 1992; van Oirschot, 1999). The use of polyclonal primary
antibodies can however lead to false positives if another pestivirus caused the infection, and
further discriminatory testing is required (van Oirschot, 1999).
Tonsil is the tissue of choice from dead animals. Alternatively spleen, kidney and lymph
nodes are suitable organs for the isolation of CSF virus (OIE, 2004; Pearson, 1992). Whole
blood is the preferred option for the isolation of virus from live animals because it has been
shown to be more sensitive than isolation from leukocyte fractions in the earlier stages of
the disease ( de Smit, 2000; OIE, 2004). However, one recent study failed to detect any
difference in sensitivity using whole blood or leukocyte fraction (Dewulf et al. 2004a). For
simplicity and sensitivity, a freeze-thawed fraction of whole blood is recommended (OIE,
2004).

Detection of viral RNA by RT-PCR & real time RT-PCR

With improvements in polymerase chain (PCR) technology and advances in methodologies,
the detection of viral RNA as a diagnostic tool has now largely surpassed the more
traditional procedures such as virus isolation and FAT. And with the advent of recent
research assessing the diagnostic performance of reverse transcriptase-PCR (RT-PCR) in
various forms, the true diagnostic potential of molecular based detection methodologies
have been shown to live up to the promise of being able to rapidly detect CSF virus
sensitively and specifically in the early stages of infection (Aguero et al. 2004; Risatti et al.
2005). Real-time RT-PCR has the potential to be adapted to a portable field laboratory test
(Risatti et al. 2003).
RT-PCR procedures have been developed using primers specific for the 5-UTR (BarlicMaganja and Grom, 2001; Katz et al. 1993), Npro, C, Erns (Barlic-Maganja and Grom,
2001), E2 (Katz et al. 1993; Vilcek et al. 1996), NS5B (Vilcek et al. 1996), and the 3UTR (Vilcek et al. 1999) regions of the genome. Methods used to specifically differentiate

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

Detection of virus specific serum antibodies

Detection of antibodies to CSF virus has limited scope in diagnosis, particularly if a focus
is on the early detection of virus in a herd. This is due to CSF virus being
immunosuppressive, resulting in a delay in the appearance of detectable serum antibodies
specific for CSF virus (Paton and Greiser-Wilke, 2003). Dewulf et al. (2004a) found that
virus-specific antibodies could be detected in the serum of infected pigs on average 7 8
days after CSF virus could be detected by virus isolation from whole blood, and
approximately 10 days after detection by nested RT-PCR; however antibodies may not
appear until 4 weeks after infection (Pearson, 1992). Serological diagnosis does however
serve a purpose in detecting inapparent infections in herds where low virulent strains of
virus are circulating, making clinical detection of infected pigs difficult (de Smit, 2000).
Serological testing also has diagnostic application for surveillance in an apparently disease
free region or in conjunction with a CSF virus eradication program (Pearson, 1992).
The detection of serum antibodies is best achieved by virus neutralisation or ELISA (OIE,
2004; Pearson, 1992). Antibody detection tests that fail to discriminate between antibodies
to CSF virus and those to other pestiviruses, such as the agar gel diffusion precipitin
(AGDP) test, complement fixation, indirect fluorescent antibody technique and the indirect
peroxidase antibody technique, are no longer in favour.
Virus neutralisation and the subsequent detection of non-neutralised virus can be achieved
by either immunofluorescence or peroxidase linked staining. They are referred to,
respectively as the fluorescent antibody virus neutralisation test (FAVN) or neutralising
peroxidase linked assay (NPLA) (OIE, 2004; Pearson, 1992). The NPLA was first
described by Jensen (1981) with modifications made by Terpstra et al. (1984). In brief,
dilutions of complement-inactivated serum are incubated with virus and neutralisation
allowed to occur for one hour at 37 C in the presence of carbon dioxide. PK15 cells or
other suitable cells such as SK6 are then added to the virus/antibody mix. The cells are
grown for 2 4 days and then fixed before specific staining for CSF virus. The NPLA
employs an anti-CSF virus polyclonal antibody (or monoclonal antibody) to detect non-

30

Chapter 1

neutralised virus followed by incubation with a horse radish peroxidase conjugated

antibody. Detection of virus is finally achieved by reacting cells with an insoluble
chromogen substrate such as 3-amino-9-ethyl carbazole hydrogen peroxidase and
reading by eye and microscopy. The FAVN test employs an anti-CSF virus polyclonal
antibody conjugated to FITC, with fluorescence examined by microscopy. The detection of
virus by either method is indicative of no neutralising antibodies being present at a specific
dilution and the neutralisation titre is expressed as the reciprocal of the highest serum
dilution that prevents viral growth in 50 % of replicate wells (OIE, 2004).
Detection of serum antibodies to CSF virus by ELISA is a much more rapid test when
compared to neutralisation and is able to be scaled up for the rapid processing of large
numbers of samples (Wensvoort et al. 1988). An antibody detection ELISA for the specific
detection of antibodies to CSF virus was first developed by Wensvoort et al. (1988) and
utilised CSF virus-specific monoclonal antibodies in a complex-trapping-blocking (CTB)
ELISA format. This test was further developed and modified by Colijn et al. (1996) using
monoclonal antibodies raised against a recombinant CSF virus E2-antigen. This modified
test showed 99 % specificity and sensitivity when compared to the CTB-ELISA format
described by Wensvoort et al.(1988), however with a simpler and more rapid one-step
format making the test more favourable for scaling up and automation (Colijn et al. 1997).
A competition ELISA utilising a truncated E2 recombinant protein has also been developed
(Clavijo et al. 2001), as has an ELISA using a CSF virus Erns peptide for use as a
companion test for E2 subunit vaccines (Langedijk et al. 2001; Moormann et al. 2000) to
allow discrimination between vaccinated and previously infected animals.

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).

Live attenuated vaccines

Live attenuated vaccines, also known as modified-live vaccines (MLV), have been derived
from wild-type CSF virus (de Smit, 2000). The most common and effective MLVs
currently available are those based on the Chinese (C) strain, the Japanese guinea-pigexaltation-negative (GPE ) strain derived from the virulent ALD strain, the cell culture
adapted Thiverval strain derived from the virulent Alfort strain (de Smit, 2000; van
Oirschot, 2003), the PAV-250 strain (de Smit, 2000) and the CS and LK vaccine strains
developed in Russia (Vlasova et al. 2003; Zaberezhny et al. 1999).
There are a number of different C-strain CSF viruses currently used to produce vaccine
(van Oirschot, 2003) with the likely origin in Taiwan, where the C-strain virus was
developed from the Rovac strain after greater than 800 serial passages of live virus through
rabbits (Terpstra et al. 1990). Traditionally C-strain vaccine was produced from the organs
of rabbits inoculated with a working seed (Terpstra et al. 1990). The C-strain virus has
subsequently been adapted to cell culture systems for large scale production of vaccine
using the swine kidney cell line SK-6 (Terpstra et al. 1990) or minipig kidney (MPK) cells
(Ferrari, 1992; Rivero et al. 1988). The production of C-strain vaccine batches from rabbit

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.

Live vector vaccines

A number of live vectored vaccines carrying and expressing the E2 protein of CSF virus
have been developed. An early vectored vaccine developed by inserting the immunogenic
E2 protein gene into a recombinant pseudorabies virus provided challenge protection from
both CSF and pseudorabies viruses (van Zijl et al. 1991a), (refer to note above; this
glycoprotein insert was also denoted E1 at the time of publishing). Vaccinia virus also
proved useful for the delivery of CSF virus structural proteins, with the recombinant virus
inducing production of neutralising antibodies in mice and pigs and protection against
lethal challenge in pigs (Rumenapf et al. 1991). More recently, porcine adenovirus
(Hammond et al. 2000) and swinepox virus (Hahn et al. 2001) have been used as a vector
for CSF virus vaccine delivery with an E2 protein insert. Porcine adenovirus has been
found to be a stable vector and provides both a strong and persistent immunity together
with protection against lethal challenge when administered by either the oral or subcutaneous route (Hammond et al. 2000; Hammond et al. 2001; Hammond et al. 2003).

Novel vaccine development

Technological advances in molecular biology have enabled virus attenuation to be achieved
through the excision of particular genes or gene segments. Partial and complete E2 protein
deletion mutants produced from a cDNA clone of Alfort/187 strain resulted in the
production of non-infectious CSF virus replicon particles that show potential as orally
administered live attenuated vaccines (Maurer et al. 2005). Likewise, chimeric CSF viruses
that have had gene segments exchanged with the analogous BVD virus gene segment have
been developed and confer protective immunity (de Smit et al. 2001b; van Gennip et al.
2000).

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.

Vaccine induced immunity & efficacy of C-strain vaccines

Following a single vaccination with C-strain virus, neutralising antibodies typically first
appear at 2 3 weeks (and as early as 1 week) post vaccination and gradually increase up to
4 to 12 weeks (Dahle and Liess, 1995; Terpstra et al. 1990; Terzic et al. 2003). Protection
against lethal challenge is complete at around 7 days post vaccination (de Smit et al.
2001a; Terpstra et al. 1990). More recently however C-strain vaccination has been
demonstrated to confer complete protection to pigs challenged on the same day as
vaccination and these challenged pigs were unable to transmit virus to pigs vaccinated at
the same time (Dewulf et al. 2004b). This early protection is not attributed to neutralising
antibodies (Suradhat et al. 2001), rather, it is attributed to cellular immunity with
interferon-gamma secreting cells specific for CSF virus present in the peripheral blood as
early as 6 days post vaccination (van Oirschot, 2003). Sterile immunity, where no
significant rise in antibody titre is seen after challenge with virulent CSF virus, has been
reported between 1 and 4 weeks post vaccination (Dahle and Liess, 1995). Other research
has however demonstrated a significant rise in titre following challenge (Terpstra and
Wensvoort, 1988; Terpstra et al. 1990). C-strain vaccine induced antibodies may persist for
many years in some pigs and disappear in others; vaccinal protection from infection has
however been shown to last for at least 6 18 months and maybe even life long (van
Oirschot, 2003). The C-strain vaccine produced in Laos has been shown to elicit an
immune response in which antibodies could be detected by CTB-ELISA for at least 9
months post vaccination (Dr Syseng Khounsy, personal communication).
The absence of neutralising antibodies following vaccination is not necessarily indicative of
a lack of protection against infection (van Oirschot, 2003), however a number of
researchers have demonstrated a correlation between vaccine induced antibody levels at the
time of challenge and protection against lethal infection. Terpstra and Wensvoort (1988)
found that if serum neutralising antibody titres against C-strain virus, as measured by

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.

Use of C-strain vaccination in control programs

Vaccination is commonly employed in endemic countries to avoid pig losses to CSF,
however the use of live attenuated vaccines will limit the use of serology in detecting
circulating field virus (Moennig, 2000). Use of prophylactic vaccination in a systematic and
consistent manner in countries with endemic CSF is however a good starting point to
establish disease control and subsequently introduce an eradication program (Moennig,
2000). C-strain vaccine has been used extensively to control CSF virus in Southeast Asia.
In Laos in particular, C-strain vaccine produced from rabbit spleens is not currently applied
in a consistent and systematic manner, with less than 8 % of households who raise pigs
vaccinating against CSF in 1998/1999 (MAF, 2000). Problems exist in the maintenance of
the cold-chain and delivery of vaccine to the pig, with village pig producers who vaccinate
observing little benefit from vaccination (Gibson and Wilkie, 2000). In contrast, Brazil has
successfully used C-strain vaccination in combination with zoning, slaughter policy and
strict animal movement controls to eliminate CSF virus from several states (Roehe, 1998).
In Germany, C-strain vaccine delivered orally using baits has been used experimentally to
vaccinate wild boar with some success (Kaden et al. 2000) and may limit endemic

37

Chapter 1

infections when used in combination with other control strategies such as hunting
(Moennig et al. 2003).

Factors effecting vaccination success

A number of factors may influence the success of vaccination at both the animal and herd
level. The influence of maternally derived antibodies is well recognised, with antibodies
passed from sow to piglet in colostrum reported to persist for greater than 7 weeks
(Vandeputte et al. 2001) and this may prevent vaccine response for up to 9 to 10 weeks
(Terpstra and Wensvoort, 1987). Maternally derived antibodies have been shown to
negatively affect vaccine uptake by piglets for both live attenuated vaccines (Vandeputte et
al. 2001; Suradhat and Damrongwatanapokin, 2003) and subunit vaccines (Klinkenberg et
al. 2002b). Other factors influencing vaccination success are cold chain failure in tropical
countries (Gibson and Wilke, 2000) and infection of pigs with other agents with
immunosuppressive properties such as Trypanosoma evansi (Holland et al. 2003).
Infection with porcine reproductive and respiratory syndrome (PRRS) virus prior to
vaccination has also been found to significantly reduce antibody response (Li and Yang,
2003). Vaccination programs have also inadvertently caused outbreaks where vaccination
procedures facilitated the spread of virus from congenitally infected litters to susceptible
piglets (Terpstra and Robijns, 1977).

38

Chapter 1

1.3 Immunomagnetic bead technologies and diagnostic application

Immunomagnetic beads (IMBs) provide a versatile solid phase for the capture and
separation of analyte from a solution or diagnostic specimen enabling the subsequent
detection of analyte by a variety of techniques. As such, IMBs have been used extensively
in the detection of bacterial agents of infectious disease and their toxins, including
Salmonella sp., Escherichia coli, Shigella sp., Staphylococcus sp., Bordetella pertussis, and
Bacillus stearothermophilus spores. Similarly, IMBs have been used in separation and
detection of viruses such as Norwalk and Norwalk-like virus and group A rotaviruses, and
for the detection of Plasmodium falciparum and Cryptosporidium parvum oocysts.
Immunomagnetic bead technology has also been applied to the detection of antibodies to
Salmonella sp. and Trichinella spiralis and for the detection of environmental
contaminates. Refer to Table 1.2 below for references and scope of IMB use.
Immunomagnetic bead technology has found its greatest use in the separation and
subsequent detection of analyte in environmental samples (Blake and Weimer, 1997; Deng
et al. 1997; Grinde et al. 1995; Myrmel et al. 2000; Urdahl et al. 2002) and in the food
industry for the detection of bacterial contamination, such as on beef carcasses (Bennett et
al. 1996; Fu et al. 2005; Gehring et al. 2004). However, they have been commonly used
for the detection of disease agents in clinical samples such as bodily fluid and faeces
(Bruno et al. 1996; Islam et al. 1993; Seesod et al. 1997).
The use of IMBs in combination with immunoassays were first described in the 1970s
(Guesdon and Avrameas, 1977; Smith and Gehle, 1977). More recently however a number
of other techniques have been employed to detect the captured and separated analyte. Table
1.2 below provides a summary of the different methods used once IMB separation has been
achieved. The principal benefits of using IMBs as the solid phase include the separation of
analyte from complex biological or environmental samples that may contain inhibitors or
contaminates that affect subsequent detection (Deng et al. 1997; Fu et al. 2005; Gilpatrick
et al. 2000; Seesod et al. 1997). This is particularly important for nucleic acid detection
where PCR inhibitors in samples can lead to the generation of false negative results. In

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

Escherichia coli O157:H7

(Fu et al. 2005)

Norwalk-like virus

(Myrmel et al. 2000)

Norwalk virus

(Gilpatrick et al. 2000)

Cryptosporidium parvum

(Deng et al. 1997)

Plasmodium falciparum

(Seesod et al. 1997)

Shigella sonnei

(Achi-Berglund and Lindberg, 1996)

Bordetella pertussis

(Stark et al. 1996)

Group A Rotavirus

(Grinde et al. 1995)

Salmonella sp.

(Mansfield and Forsythe, 2000)

Staphylococcus sp.

(Yazdankhah et al. 1999)

Staphylococcus sp.

(Yazdankhah et al. 1998)

Escherichia coli

(Bennett et al. 1996)

Schistosoma haematobium

(Ndhlovu et al. 1995, 1996)

Atrazine (pesticide)

(Hayes et al. 1996)

Escherichia coli O157:H7

(Gehring et al. 2004)

Chemiluminescence or

Staphylococcal enterotoxin B

(Kijek et al. 2000)

electrochemiluminescence

Biotoxoids and bacteria

(Gatto-Menking et al. 1995)

Trichinellosis antibodies

(Ko and Yeung, 1992)

Salmonella typhi antibodies

(Tam and Lim, 2003)

ELISA

Colorimetric beads

(Lim et al. 1998)

Salmonella enteritidis

(Lim and Ko, 1990)

Trichinella spiralis

(Lim and Ko, 1990)

40

Chapter 1

1.4 Project outline and perspective

The research and results described and discussed in this thesis were assembled during the
course of a project funded by the Australian Centre for International Agricultural Research
(ACIAR) based in Canberra, Australia, Project Number AS1/2003/001. The organisation
commissioned to administer the project was the Commonwealth Scientific and Industrial
Research Organisation (CSIRO), Division of Livestock Industries based at the Australian
Animal Health Laboratory (AAHL) in Geelong, Australia. The project entitled
Management of classical swine fever and foot and mouth disease at the village level in
Laos commenced in July 2003 and is due to run until June 2006. As might be ascertained
from the project title, the central objective of the project was to improve the management
and control of CSF and foot and mouth disease (FMD) in village livestock systems in Laos
with a particular focus on pigs. The activities undertaken to achieve this objective are listed
below. Note that the following descriptions are summaries taken from the original project
document pertaining to ACIAR project AS1/2003/001 written and submitted by Dr.
Laurence Gleeson. Only activities 1, 2, 4 and 5 relate directly to this thesis, with the main
attention devoted to activity 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 4: To continue to monitor the epidemiology of FMD and CSF in Laos

The project will continue to monitor pig health in eight pilot project villages in
Bolikhamxay province where monthly production and health data are collected, the number
of villages will be gradually expanded and disease outbreaks will be investigated. Routine
CSF diagnostic testing of samples submitted from other provinces and districts will
continue. In the case of FMD epidemiology, laboratory diagnostics and typing will
continue, key field samples will be submitted to reference laboratories for molecular
epidemiological investigation. Purposive monitoring of recent FMD outbreak areas will
also take place to gain a better understanding of viral persistence in Laos.

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

1.5 Research objectives and research questions

This thesis is made up of three broad objectives based on the AS1/2003/001 project
activities described above. The central research questions related to each objective are 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.

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?

How does disease influence production?

What is the significance of CSF in the smallholder pig 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 are the performance characteristics of the newly developed test?

What is the relative diagnostic performance of the newly developed test format?

Can immunomagnetic bead technology be applied to rapid diagnostics?

Can immunomagnetic bead technology be applied to field-based diagnostics?

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

2.1 Introduction and research design

The research and results presented in this chapter were carried out under the umbrella of
two ACIAR funded projects (AS1/94/38 and AS1/2003/01). Project AS1/94/38
commenced in July 1997 with the intention of running until December 1999. The project
was extended to December 2001 following a successful independent review. This initial
phase of the project concentrated on two infectious diseases deemed to be of significant
importance to the livestock sector. The specific aims and objectives of this phase of the
project can be found elsewhere (Blacksell, 2001). The project was again extended for a
further 12 months to December 2002 and again for 6 months to June 2003. These
subsequent extensions were not the result of external reviews of the project. After a second
independent review of the project in January 2003, a second phase of the project was
developed and a new project, AS1/2003/01 commenced in July 2003 and is due to run for
three years to June 2006. The results presented in this chapter arose from research
conducted in the second and third extensions of project AS1/94/38 (December 2001 to June
2003) and project AS1/2003/01 (July 2003 to present). Refer to Chapter 1 for a detailed
description of the specific aims and objectives of project AS1/2003/01. The author
commenced work on project AS1/94/38 in April 2001, work relevant to this chapter
commenced in May 2002.
To enable a better understanding of the epidemiology of CSF virus and the development of
sustainable interventions in smallholder pig raising systems in Laos, basic information
relating to management, production and health of the smallholder pig raising system was
required. A baseline study was set up and conducted in two districts in the central province
of Bolikhamxay initially to characterise the pig production system in the selected study
sites. This was further developed into a longitudinal study to provide estimates of the
parameters of reproductive performance of sows, the health and productivity of grower and
pre-weaning pigs and to understand better the input and output trends of the smallholder pig
production system. This chapter describes the methodologies used, a descriptive overview
of the smallholder pig raising system in the central province of Bolikhamxay, the

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.

2.2.1 Study area

Both the baseline and longitudinal studies were conducted in two districts of the central
province of Bolikhamxay province and those villages recruited for the baseline study were
also recruited into the longitudinal study. The districts selected were Pakading district in the
east-south-east of the province and Bolikhan district in the central north of the province
(Figure 2.1). Pakading district has the countrys major sealed road and north-south transport
route transecting it and is made up of low-lying flood plains of the Mekong River with
some elevated ranges in the north east towards the Vietnamese border. Bolikhan district on
the other hand is accessed via an unsealed secondary road that is difficult to pass in the wet

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).

2.2.2 Selection and recruitment of villages and pig raisers

The province of Bolikhamxay was purposively selected due to its proximity by road to
Vientiane, the capital of Laos, where visits to conduct surveys, provide training, investigate
disease outbreaks and follow-up anomalies in survey data could be carried out in one or
two days by road. Districts were chosen after consultation with the provincial livestock and
fisheries departmental chief and villages were selected following meetings with provincial
and district livestock officers. The selection criteria that were set out for village selection
included: (a) pig production was an important aspect of maintaining livelihood; (b) pig
production contributed significantly to the agricultural economy of the village; (c) the
livestock officers had a strong working rapport with the village administration; and (d) the
villages selected were accessible during the wet season (June to October). Eight villages
were recruited into the production and health survey in May 2002, four from each district.
A further eight villages were recruited into the survey in March 2004. In Xieng Khouang
province, four villages from each district were selected and recruited in the surveillance
program in January 2005.

49

Chapter 2

Vietnam

Bolikhan
Thaphabath

Viengthong

Paksan
Pakading

Khamkeuth

Thailand
Bolikhamxay province

Figure 2.1 Districts of Bolikhamxay province, Laos (WHO, 2002)

2.2.3 Recruitment of pig raisers and baseline survey

The survey was initiated at a village meeting with those pig raisers able to attend, subject to
other work commitments. At this meeting, all pig raisers were recruited to provide monthly
production and health data to a designated data collection officer; in many cases this was
the village veterinary worker (VVW) or in a small number of cases the village chief
(administrative head). In all villages participating in the survey the number of households
raising pigs would vary from month to month depending on each households production
system; whether it involved having breeders and growers or just growers. The
responsibility of keeping up to date with new pig raisers and the subsequent collection of
production data was left to the data collection officer.

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.

2.2.4 Pig production and health questionnaire design and training

The questionnaires used in this survey are shown in Figure 2.2. All farmers in a village
were grouped into ten similar sized groups based on location within the village as
determined by the data collector so as to streamline and make data collection easier. An
added benefit was the ability to cross check anomalies in data. All groups were assigned a
number and the number of farmers per group was recorded. Production and health data for
each village were then added together and the production and health statistics for each
village were recorded in a MS Excel spreadsheet. A one-day training session was held with
district and provincial livestock officers prior to the commencement of the survey. The
chief aim of the training session was to ensure all sections of the questionnaires were
understood and to clarify any translation errors that would potentially lead to incorrect data
being collected. Following this training session, provincial and district livestock officers
accompanied project staff to participating villages where training sessions were held with
VVWs and village chiefs.
The VVW or chief of each village compiled pig production and health information over the
course of a month and the provincial and district livestock officers visited villages once per
month to collect data. Survey data were then checked by the provincial officer and sent to
project staff in Vientiane where it was re-checked and entered into a MS Excel spreadsheet.
Project staff provided follow-up input after the first and second months of the survey to

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.

2.2.5 Reproductive performance

Three animal level indicators of sow reproductive performance were used in this survey:
the number of live born piglets at farrowing; an estimate of the number of litters per sow
per year and an estimate of the pre-weaning piglet mortality. Individual pigs were not
tagged and followed up to determine the number of live born piglets per sow and the
number of litters per year. Rather, the number of piglets live born in a given month was
recorded for each village as was the number of sows that farrowed (refer to Figures 2.2).
The average litter size per village was determined by dividing the total number of live born
piglets by the total number of farrowing sows for the entire survey period. No indicator of
precision, such as standard deviation, could be extracted from the data as individual litter
sizes were not recorded. The number of litters per sow per year was calculated as an
estimate only as individual sows were not followed over time and no reliable information
pertaining to how long sows remain in the production system could be extracted. The
estimate was calculated using the following formula:
Total number of litters x 12
Litters per sow per year = Median monthly sow population x Number of months in survey
Pre-weaning mortality estimates were calculated as a percentage of the total number of live
born piglets that died before the age of three months. In all villages weaning was not
managed by actively separating the piglets from the sow, and in most villages piglets were
reported to cease suckling at greater than eight weeks of age.
One herd level indicator affecting sow reproductive performance was measured to gain a
better understanding of the management strategy employed to service sows; this indicator
being the sow/boar ratio. The ratio was calculated by dividing the median monthly sow
population over the entire survey period by the median monthly boar population.

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.

2.2.6 Off-take and in-take trends

Off-take and in-take data were collected with the production unit assigned at the village
level. Off-take refers to pigs leaving the production unit by either death, home slaughter for
consumption or sale out of the village. In-take into the production unit refers to those pigs
entering the production unit by either purchase from outside the village or birth.
All pig deaths in each village were recorded at the end of each month into their respective
age class. Pigs dying from disease are often consumed by the owners family and to prevent
confusion between home consumption and deaths, only those pigs specifically slaughtered
for home consumption were recorded in this off-take category. Figure 2.2 shows sales
information collected at the farmer level, where sales could be either within or outside of
the village production unit. Likewise, the total number of purchases could be from inside or
outside the village; however the number of pigs purchased from outside the village was
recorded. In order to determine the actual number of sales out of the production unit, the
number of pigs procured from inside the village was subtracted from the total number of
sales. Off-take data were collated over the whole survey period for each district to
determine from which animal class and off-take category the greatest number of pigs left
the production unit.

53

Chapter 2

Pig Population and Health Record

District:

Province:

Reporting Officer:

Certified by:

VVW Active (yes/no):

10-12

7-9

Young Pigs in age class (mths)

4-6

Boars

Breeding Pigs

Sows

10-12

7-9

4-6

Young Pigs in age class (mths)

0-3

Boars

Breeding Pigs

Deaths in this reporting period

0-3

Sicknesses in this reporting period

Sows

No. Live
Born Piglets

10-12

7-9

4-6

Young Pigs in age class (mths)

0-3

Boars

Sows

Breeding Pigs

Litters

No.
Producing
sows

Current Pig Population

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

Reporting period (months):

No. Farmers
per group

Date of previous report:

Farmer Group
Number

Date:

Chapter 2

Monthly In-take and Off-take Record

Reporting period (months):

Village name:

District:

Province:

Reporting Officer:

Certified by:

VVW Active (yes/no):

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

Young Pigs in age class (mths)

0-3

Boars

Procurements in this
reporting period came
from where?

Breeding Pigs

Sows

10-12

7-9

4-6

Young Pigs in age class (mths)

0-3

Boars

Breeding Pigs

Sows

10-12

7-9

4-6

Home Consumption

Procurements in this reporting period

Young Pigs in age class (mths)

0-3

Boars

Sows

Breeding Pigs

Were boars
hired from

Live sales in this reporting period

group

No. Farmers per

Date of previous report:

Farmer Group
Number

Date:

Chapter 2

2.2.7 CSF incidence and associated mortalities

During the course of the survey, farmers in all villages submitted spleen samples to the
National Animal Health Laboratory in Vientiane from pigs suspected to have died from an
infectious disease, or from a selection of pigs. These samples were tested for the presence
of CSF antigen by AC-ELISA (described in the next chapter). It was not possible to
describe incidence of CSF at the pig level as data pertaining to the total number of pigs
infected was not able to be collected. As a result, incidence (or rate of outbreaks) was
calculated and expressed at the village level as the number of outbreaks per 100 village
years. The total number of villages participating in the survey was sixteen, with eight
villages participating for 41 months and eight villages for 19 months. This equates to a total
observation period of 480 village months or 40 village years for all 16 villages or 20 village
years for each district. The incidence of CSF in the cohort of 16 villages was calculated
with the 95 % confidence interval (CI) using the program EpiCalc (2000). The formula
used to calculate incidence was:

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)

Most common method

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

Cut and carry grasses

8/8

Rice whiskey waste

8/8

Cassava

0/8

Corn

0/8

Breeders

2/8

Growers

2/8

Pre-weaned

2/8

Restricted

8/8

Unrestricted

0/8

Most common method of sale

Feeding and water availability

Main feed source

Supplementary feeding

Water availability
Time tending pigs per day
(hours)

2 (1 - 4)

59

Chapter 2

Pig disease and health

Response to having

Sell sick pigs

7/8

sick pigs

Sell healthy piglets

7/8

Quarantine sick pigs/restrict

movement

4/8

Slaughter sick pigs

1/8

Administer antibiotics

3/8

Response to having other

Sell healthy piglets

7/8

sick pigs in the village

Restrict movement of pigs

3/8

Administer antibiotics

4/8

Vaccination for CSF

4/8

Production
Pig breeds

Age at weaning (weeks old)

Predominantly 'Moo laat'

8/8

Some cross breed pigs

7/8

1-8

0/8

>8

7/8

Until sow resents

1/8

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Chapter 2

2.3.2 Reproductive performance

In Bolikhamxay province, 1,277 sows farrowed in the study period commencing in May
2002 with 7,644 live piglets born at an average litter size of six. As mentioned previously,
no range or indicator of precision could be extracted from the information collected during
the study period. The reproductive performance indicators for these farrowing events are
summarised in Table 2.2 for each village. The median litter size for the 16 villages in
Bolikhamxay province was 5.9 (range: 5.0 to 7.3). In Xieng Khouang province, 231 sows
farrowed in the study period commencing in January 2005 with 1,024 live piglets born at
an average litter size of 4.4. The median number of litters-per-sow-per-year for the 16
villages in Bolikhamxay province was 0.8 (range: 0.2 to 1.6) which approximates to two
litters every 18 months. In Bolikhan district the median number of litters-per-sow-per-year
was 1.0 (range: 0.6 to 1.6) compared to 0.6 (range: 0.2 to 0.9) in Pakading district. The
estimate of pre-weaning mortality was quite varied with a median of 2 % (range: 0 to 15.3).
Similarly, the sow/boar ratio was quite varied with a median of 22 sows to one boar (range:
6 to 83).
In order to determine if a correlation existed between the sow/boar ratio and the number of
litters per-sow-per-year, a scatter plot was constructed in MS Excel and the R2 value
calculated from the linear line of best fit. No significant correlation was demonstrated
between litters-per-sow-per-year and the sow/boar ratio in each village (R2 = 0.0011).
Similarly, no significant correlation was demonstrated between the sow/boar ratio and the
average litter size per village (R2 = 0.0614). The scatter plots constructed to examine the
relationship between sow/boar ratio and sow reproductive performance can be found in
Appendix III.

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

Litters per sow per

year (95% CI)

Sow/Boar
ratio

Pre-weaning
mortality (%)

Houana

Bolikhan

41

42 (39, 45)

1 (1, 2)

227

5.3

1.6 (1.5, 1.7)

42.0

6.3

Phonsavath

41

83 (80, 90)

1 (0, 2)

159

5.6

0.6 (0.5, 0.7)

83.0

2.1

Nalong

41

95 (90, 100)

5 (4, 6)

271

6.7

0.8 (0.8, 0.9)

19.0

3.1

Nampa

41

25 (22, 26)

2 (2, 2)

96

6.1

1.1 (1.1, 1.3)

12.5

15.0

Phonthong

19

51 (49, 55)

1 (1, 1)

62

6.0

0.8 (0.7, 0.8)

51.0

15.3

Hatpho

19

26 (25, 30)

1 (0, 1)

48

6.7

1.2 (1.0, 1.2)

26.0

0.3

Songkhonmai

19

14 (12, 18)

1 (1, 1)

26

6.4

1.2 (0.9, 1.4)

14.0

1.2

Na-o

19

5 (4, 6)

0 (0, 1)

7.3

0.9 (0.7, 1.1)

0.0

41

10 (9, 12)

1 (1, 1)

32

6.4

0.9 (0.8, 1.0)

10.0

8.3

Borthoun

41

39 (35, 41)

1 (1, 1)

125

5.7

0.9 (0.9, 1.0)

39.0

1.1

Nabone

41

20 (18, 22)

2 (1, 2)

31

5.9

0.5 (0.4, 0.5)

10.0

2.2

Hadxaykham

41

43 (35, 47)

3 (2, 3)

78

5.9

0.5 (0.5, 0.7)

14.3

11.9

Donsai

19

6 (5, 6)

1 (1, 1)

5.5

0.2 (0.2, 0.3)

6.0

0.0

Phonsai

19

26 (20, 28)

1 (1, 1)

26

5.0

0.6 (0.6, 0.8)

26.0

0.0

Namkhou

19

25 (23, 28)

0 (0, 0)

24

5.7

0.6 (0.5, 0.7)

0.0

Namdeua

19

51 (46, 51)

2 (2, 2)

63

6.1

0.8 (0.8, 0.9)

25.5

0.0

Paksa

Pakading

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Chapter 2

2.3.3 Off-take and in-take from the village production unit

The off-take patterns for the village production units are presented in Figure 2.3.
In total, 10,931 pigs left the 16 village production units. Of which 9,173 (84 %) left through
sales out of the production unit, 466 (4 %) through deaths and 1,292 (12 %) through home
slaughter for consumption. Seventeen percent of off-take in Bolikhan district was due to
home slaughter compared to only 4 % in Pakading district. The majority of pigs, 8,270 (76
%), left the production system in the 0 3 month age group with little difference between
the two districts, 77 and 74 % for Bolikhan and Pakading, respectively (Figure 2.3).
Seventy six percent of all sales out of the production unit were piglets from the 0 3 month
old age group, 12 % from the 4 6 month age group and 6 % from the sow population.
In-take to the village production unit was comprised of live births and purchases from
outside the village. In the study period, a total of 7,644 pigs (76 %) entered the production
unit of the 16 villages by way of birth and a total of 2,365 (24 %) by way of purchase. Sixty
three percent of all purchases into the villages were in the 0 3 month age group, followed
by 16 and 13 % for 4 9 month old pigs and sows, respectively. The purchases into the
village production units are summarised in Figure 2.4 below. In-take patterns varied
between villages and these are summarised in Table 2.3. All but two villages had a majority
of pigs entering the production unit by way of births. Donsai village (Table 2.3) produced
only a very small number of pigs with 11 piglets born from 6 sows, compared to the
purchase of 732 piglets 0 3 months old.
A majority of villages had a combination of farmers who used farrow-to-weaner or farrowto-finisher production methods and farmers who relied solely on a grower operation. The
latter operation was demonstrated by the number of farmers being greater than the number
of sows in all but two villages (Table 2.3).

63

Proportion of total production .

off-take (%)

Chapter 2

100

a)

80

All Villages

60

Bolikhan District
40

Pakading District

20
0
Deaths

Sales

Home Con.

Production output category

Proportion of total production .

off-take (%)

100

b)

80
60
40
20
0
Sows

Boars

0-3

4-6

7-9

10-12

Animal classification by age (months).

Figure 2.3 a) Pig production off-take by off-take category;

and b) production off-take by age class. Home con. refers to
home consumption.

64

Proportion of total purchases into .

production unit (%)

Chapter 2

100
80
All villages

60

Bolikhan District
40

Pakading District

20
0
Sows

Boars

0-3

4-6

7-9

10-12

Animal classification by age (months)

Figure 2.4 Proportion of purchases into the village

production units by age class

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

2.3.4 CSF incidence and associated piglet mortality

During the total observation period of 40 village years, a total of seven outbreaks were
observed in six different villages. One village, Nampa in Bolikhan district, experienced two
outbreaks in the study period in June 2003 and August 2004. The incidence of CSF for all
16 villages during the observation period was determined to be 18 outbreaks per 100 village
years (95 % CI: 4 to 28). The incidence of CSF in each district was, however quite
different. Incidence of CSF in Bolikhan district was 30 outbreaks per 100 village years (95
% CI: 6 to 54) and in Pakading district 5 outbreaks per 100 village years (95 % CI: 0 to 15).
Piglet mortality and movement patterns associated with the seven outbreaks are graphically
illustrated in Figure 2.5 below. The average monthly mortality standard deviation in the 0
3 month age group is displayed in parenthesis under the village name in each graph. Only
two out of the seven outbreaks had higher than normal mortality in the 0 3 month age
group at the time CSF virus was detected. Nampa and Houana villages (Figures 2.5 G and
F) had much higher mortality than the monthly mean around the time samples were
collected and confirmed positive for CSF virus antigen (as represented by the arrow). As
presented in Figure 2.5 A, Phonthong village experienced high piglet mortality in the
months June and July; however samples were not submitted for testing until November and
December 2004. These samples subsequently tested positive for CSF virus antigen. It was
not possible to ascertain if CSF virus persisted in the village for the four months between
the period of high mortality and sample submission. Low mortality was evident in all other
villages where CSF virus was found to be present. In Phonsavath, Nalong and Nampa
villages (Figures 2.5 B, C and F), sale of pigs peaked immediately after or at the time CSF
virus was detected. In Nalong village alone, almost 250 pigs were sold in the same month
that piglet mortality peaked; 199 piglets aged 0 3 months and 33 sows were sold. The
majority of pigs sold in these three villages were in the 0 3 month age group. In
Phonthong village, this phenomenon was also evident as a response to a disease event. In
August 2004, a dramatic decrease in piglet mortalities corresponded with a sharp increase
in pig sales.

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

2.4 Discussion and conclusions

Baseline survey: a descriptive overview of smallholder pig production
Pig farmers in the eight villages participating in the baseline survey followed a traditional
form of production as described by Vongthilath and Blacksell (2000). The predominant
breed raised was the indigenous breed Moo Laat pig with only a small number of exotic
cross breed pigs raised in each village. The quality of feed used was generally very poor
with few farmers able to provide supplementary higher quality feed stuffs. There was also a
deficiency in the supply of water to pigs as water supply was restricted for penned animals
and provided by mixing with food. Those pigs unpenned or small enough to get out of pens
were likely to access water lying in puddles; however during the dry hot season water
availability for unpenned pigs would also be restricted. Dehydration would be expected to
exacerbate the problem of using poor quality feed, affecting productivity and reproductive
performance.
The use of routine veterinary services and medicines was limited to a small number of
farmers. Farmers did not report the routine use of anthelmintics and the use of antibiotics
such as oxy (oxytetracycline) appeared to be based on a hope-for-the-best treatment
strategy rather being based on a sound reasoning of the most likely disease agent. The use
of oxy as a treatment strategy would not influence the outcome of viral infectious diseases
such as CSF, and there was no indication that this distinction was clearly understood. As
has been described previously (Blacksell, 2001; MAF, 2000), vaccination coverage for CSF
at the time of the survey was found to be low.
Marketing of pigs in Pakading and Bolikhan districts appeared to be mainly localised with
the majority of farmers preferring to purchase pigs locally from within their own village or
from villages close-by. Sale of pigs was, however predominantly through middlemen
traders. The distances that these traders operated could not be determined from the data
collected during the baseline surveys.

70

Chapter 2

Production and health survey

Data collected during the longitudinal study was highly reliant on recall by the owner. As
such, the monthly collection times were assumed to be frequent enough to ensure the
collection of good quality information. Pig farmers in all surveyed villages raised only a
small number of pigs which aided the recall of production and health related information.
Of concern however was the ability of pig farmers to recall the correct age of the pigs in
their keep as farrowing dates were not recorded; thus increasing the likelihood of error in
the recording of age for animals close to an age-bracket cut off.
In comparison with both commercial and smallholder tropical pig production, the
reproductive performance of sows in the smallholder production sector described in this
study was low. The average number of pigs per litter in Bolikhamxay province was 6.0 and
in Xieng Khouang province 4.4. This compares unfavourably with the smallholder sector in
the Philippines and Kenya where median live born litter sizes of 8.0 (Lanada et al. 2005)
and 9.0 (Wabacha et al. 2004b) were observed, respectively. Average litter sizes were not
reported in these two studies. The average number of live born pigs per litter in the
commercial sector in Thailand ranged from 8.9 to 9.3 (Kunavongkrit and Heard, 2000).
Breed likely plays an important role in the discrepancies seen between the litter sizes in
Bolikhamxay province when compared to those in the smallholder sector of Kenya and the
Philippines. The predominant breed in the smallholder farms studied in both Kenya and the
Philippines were native/exotic cross-breed pigs (Lanada et al. 2005; Wabacha et al.
2004a). The nutritional status of the sow is also an important factor affecting litter size and
likely has a strong influence on the small litter sizes observed in Laos. The average litter
size in Bolikhamxay province however compares well with other tropical native breed pig
production systems in the Solomon Islands, with average litter sizes of 5.5 (de Fredrick,
1977) and Papua New Guinea, with average litter sizes ranging from 3.6 to 6.4 (Hide,
2003). The average litter sizes observed in Xieng Khouang province compare well with
those observed in highland provinces of Papua New Guinea where litter sizes ranged from
3.6 to 4.8 (Hide, 2003).

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.

Incidence of CSF and associated mortalities

Previous research has suggested that the overall serological prevalence of CSF virus in the
Lao smallholder pig population is quite low, with 8.9 % of pigs in four provinces positive
for the presence of antibodies against CSF virus (Blacksell, 2001). This same research
however demonstrated variation between districts in these four provinces. In nine districts
of Vientiane Capital, serological prevalence as determined by active surveillance ranged
from 0 to 27 % in the unvaccinated pig population. In four districts surveyed in
Savannakhet province, the range observed was 4 to 17 % in unvaccinated pigs (Blacksell,
2001). The research presented in this chapter supports these findings. A substantial
difference in CSF incidence at the village level was demonstrated between Pakading and
Bolikhan districts. The incidence of CSF in Bolikhan district during the observation period
was 30 outbreaks per 100 village years compared to five outbreaks per 100 village years for
Pakading district. This translates to outbreaks in villages in Bolikhan district occurring
approximately every three to four years, whereas the findings of this research suggest
villages in Pakading District will experience outbreaks approximately every 20 years.
The mortalities associated with CSF outbreaks were also found to be quite varied, and
overall, quite low. Research reported by Blacksell (2001) observed low mortality rates
associated with known CSF outbreaks, with only 7.1 % of village pigs dying at the time
samples were submitted to the laboratory for testing. Blacksell (2001) concluded that
overall recorded mortalities were low because samples and mortality figures were
submitted at the start of an outbreak. Earlier work conducted in Lao indicated that native
breed pigs were equally susceptible to CSF as improved breed pigs but that the disease
process was of longer duration in native breed pigs. This could provide farmers with more

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.

3.2 General Cell Culture

The cell line of choice for the growth and subsequent identification of CSF virus is the
continuous pig kidney cell line designated PK15 or other pig cell lines with similar
sensitivity to CSF virus infection, such as SK6 (OIE, 2004).
Cell cultures of PK15 were provided by the cell culture department of the CSIRO-AAHL
and transported to the project laboratory in Laos in growth medium (Appendix I) at ambient
temperature. Cells were grown in closed cap tissue culture flasks (Nunc, Denmark) in
growth medium containing minimum essential medium with Earles salts (EMEM) (Gibco,
USA), 10 % foetal bovine serum (FBS) (Trace Biosciences, Australia), 2 mM L-glutamine
(Gibco, USA), 2.5 mM hydroxethylpiperazine-ethanesulphonic acid (HEPES), 0.07 %
sodium bicarbonate, 200 IU/mL penicillin, 100 g/mL streptomycin and 2.5 g/mL
amphotericin B (Gibco, USA) and grown at 37 C in a humidified incubator. The FBS as
supplied by Trace Biosciences was provided certified free of potentially interfering
adventitious pestiviruses and immunoglobulins to pestiviruses. Confluent monolayers of
PK15 cells grown in tissue culture flasks were dissociated with 0.05 % trypsin (Gibco,
USA) for 5 to 10 minutes at 37 C in a humidified incubator to detach cells from the flask
growth surface. Growth medium was added to inactivate trypsin and the cells were counted
in a haemocytometer by taking 100 L of cell suspension and mixing with 100 L 0.2 %
trypan blue; the remaining cells were pelleted by slow centrifugation at 1500 rpm for 5
minutes. The supernatant containing trypsin was removed and the cells resuspended in
growth medium to an appropriate concentration. Cells were used for virus isolation, virus
titration, NPLA or passed and stored at -85 C.

79

Chapter 3

3.3 Peroxidase Linked Assay (PLA)

CSF virus growth in PK15 cells does not result in a cytopathic effect (CPE) so
immunological staining is utilised to demonstrate the presence of viral antigens; the
fluorescent antibody test (FAT) and peroxidase linked assay (PLA) employ antibodies
conjugated to fluorescein isothiocyanate (FITC) or horse radish peroxidase (HRP),
respectively to achieve this. Due to the low technology environment of Laos, the PLA
technique was deemed appropriate as it can be read by eye and checked under a low power
inverted microscope. The PLA was carried out by first removing growth medium from
infected cells in a Class II biological safety cabinet followed by one wash with phosphate
buffered saline (PBSA). The cells were then fixed for 20 minutes with 10 % formaldehyde
and 0.1 % Nonidet P-40 (NP40; v/v) in PBSA followed by a 3-cycle wash with PBSA.
Visualisation of the cells was achieved by the addition of CSF E2 specific monoclonal
antibody 24/10 (Mab 24/10) diluted 1:400 in PBSA containing 1 % skim milk powder
(SMP) to all wells and incubated for 1 h at 37 C in a humidified incubator. Following
incubation, the plates were washed 3 times with PBSA Tween 20 (PBST) and rabbit antimouse HRP-conjugated antibody (Zymed, USA) diluted 1:2000 in PBSA containing 1 %
SMP was added to all wells. The plates were incubated for 1 h at 37 C in a humidified
incubator. Following incubation, the plates were washed 4 times with PBST and freshly
prepared 3-amino-9-ethylcarbazole (AEC) with hydrogen peroxide (Appendix I) was added
to all wells. Staining was allowed to proceed for 20 30 minutes before the reaction was
stopped by washing the plates with tap water. The water was removed and the plates read
by eye and checked by inverted microscope.

3.4 Virus Isolation

A small piece of spleen, tonsil or kidney tissue (approximately 1 2 g) stored in PBS
buffered glycerol (1:1 ratio) was ground by mortar and pestle and further homogenised in 5
10 mL of PBSA or tissue culture growth medium to make a 10 20 % (w/v) suspension.
The homogenised tissue was held at room temperature for 1 h with occasional mixing by
vortex before the suspension was clarified by centrifugation at 5000 rpm for 15 min. The
supernatant was collected and stored at -85 C until tested. After the homogenate was

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.

3.5 Virus titration

To determine the virus titre in cell culture supernatant for use in the NPLA, supernatant was
titrated in the PLA to determine the concentration of virus measured as 50 % end point of
tissue culture infectious doses (TCID50). Sterile 1.5 mL microfuge tubes were used for virus
titration and a tissue culture grade plate (Nunc, Denmark) was used for PLA. Cell culture
supernatant was titrated in growth medium in a log10 dilution series starting at 10-1 through
to 10-8 in microfuge tubes and 50 L volumes transferred to 10 wells of a sterile tissue
culture plate row. One hundred microlitres of freshly prepared cells in growth medium were
added to all wells at a concentration of 2 x 104 cells/mL. Two columns of uninoculated cell
controls were included on each plate. The plates were incubated for 3 4 days at 37 C and
5 % CO2 in a humidified incubator before staining cells for the presence of replicating CSF
virus by the PLA. The TCID50 end point was calculated using methods described by Reed
and Muench (1938).
For the determination of virus titre in tissue samples, 10 % w/v homogenates of tissue were
prepared according to virus isolation described above. Virus was titrated in growth medium
in 96-well round bottom plates (transfer plate; Nunc, Denmark) and transferred to a 96well, round bottom plate. To row A of the transfer plate, 165 L of tissue homogenate was
added to 165 L of growth medium for a 1:2 dilution (5 % w/v tissue suspension) and will
be referred to as 100 dilution (refer to plate format below). The tissue homogenate was
titrated down the plate from 100 to 103 in log100.5 steps to determine the virus end point.
Fifty microlitre volumes were then transferred to 4 wells of the tissue culture plate and 100

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

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Chapter 3

3.6 Antigen Capture-ELISA

The CSF AC-ELISA is an assay to detect the presence of CSF viral antigen in the tissue
and blood of swine. The assay is a sandwich ELISA utilising a pestivirus group reactive
polyclonal antibody immobilised on a solid surface (the ELISA plate) to capture viral
antigen in tissue or leukocyte preparations. A pestivirus group reactive monoclonal
antibody specific for the NS3 protein is reacted with the sample homogenate prior to
capture. The subsequent detection of the trapped antigen is achieved by the addition of an
anti-mouse HRP-conjugated antibody and the colour developed by the addition of 5, 5
tetramethylbenzidine (TMB) substrate. The test is essentially the same as that developed by
Shannon et al. (1993) and modified by Fuqing et al. (2000) and Blacksell (2001).

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.

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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

Figure 3.2a Transfer plate format

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

Figure 3.2b ELISA plate format

C++, strong positive control; C+, mid-positive control; C-, negative control; Cc, PBS control
NS3 specific Mab

Negative control Mab

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

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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 =

Mean OD450nm using positive Mab

Mean OD450nm using negative Mab

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.

3.7 Neutralising Peroxidase Linked Assay (NPLA)

The neutralising peroxidase linked assay (NPLA) was used as a gold standard test for the
detection of neutralising antibodies in pig serum after natural infection and following
vaccination with the locally produced lapinised C-strain live virus vaccine. The method is
based on that outlined in OIE (2004) with some minor modifications. A standard amount of
CSF virus containing approximately 200 TCID50 / 50 L was incubated with test serum and
then allowed to infect PK15 cells. The replicating virus in the PK15 cells was detected by
immunoperoxidase staining. Those sera with antibodies to CSF neutralised the virus
preventing viral attachment and replication; subsequent staining did not occur in those
wells where the virus had been neutralised. The NPLA was carried out in a 96-well flat
bottom tissue culture plate (Nunc, Denmark).

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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.

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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

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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

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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 General materials and methods

4.2.1 Microparticles
The test utilised 4.0 4.5 m carboxyl modified IMBs supplied as a 2.5 % (w/v)
suspension in storage buffer by Spherotech, Inc. (USA), product number CM-40-10. The
surface area of 4.35 m magnetic beads is equal to approximately 1.321 x 104 cm2/g
(Seradyn, 1994).

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

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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

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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).

Horse-radish-peroxidase conjugated antibody

Antibody to mouse IgG, raised in goat and conjugated to HRP, was supplied by Dako
Cytomation, Denmark. Catalogue number P 0447.

4.2.3 Standard CSF Virus Antigen

Two standard CSF virus antigen extracts were utilised in the development of the IMBELISA. An homogenate of pooled infected tissues from pigs infected with the Weybridge
strain made up as a 20 % w/v suspension in 1 % NP40 PBSA and gamma irradiated was
initially used diluted 1:4. This homogenate was produced at CSIRO AAHL, Australia and
transported to Laos in 1997 and stored at 20 C for a number of years and then stored at
80 C. This antigen will be referred to as AAHL antigen. The second CSF virus antigen
used was a pooled spleen homogenate from pigs infected in Laos by a local strain and
prepared as a 5 % w/v suspension in 1 % NP40 PBSA according to sample preparation

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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.

4.3 Methods and results for the development of IMB-ELISA

4.3.1 Polyclonal antibody total protein estimation
The total protein of the goat derived polyclonal antibody was estimated to be 73.6 3.1
mg/mL against a gamma globulin standard. The estimated total protein concentration of the
rabbit derived polyclonal antibody was 22.3 1.4 mg/mL.

4.3.2 Optimisation of Polyclonal Antibody Coupling

Method
The manufacturer of the IMBs (Spherotech, Inc., USA) recommended a coupling reaction
containing protein at a concentration of 125 g/mL, 4.5 m IMBs diluted to a concentration
of 1.25 % w/v and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC)
added to a final concentration of 6.5 mM. Seradyn (USA), another manufacturer and
supplier of microparticles, recommended a coupling reaction with IMBs diluted to a
concentration of 1 % w/v and using an EDAC concentration approximately one tenth of
that recommended by Spherotech, Inc (Seradyn, 1994). Seradyn, who describe optimisation
protocols using antibodies for use in enzyme immunoassays, recommended titrating EDAC
and antibody concentrations in the coupling reaction due to EDAC having an inhibitory
effect on antibody activity at high concentrations (Seradyn, 1994). As the optimised protein
coupling conditions made by Spherotech, Inc. referred to avidin, a non-antibody protein,
the decision was made to follow those recommendations made by Seradyn (Seradyn, 1994).
Antibody was coupled to IMBs in 2 mL screw cap microfuge tubes with rubber O-rings and
using two LifeSep magnets (Dexter Magnetic Technologies, USA) designed to hold a
microfuge tube. For the optimisation of EDAC concentration a reaction volume of 125 L

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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

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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).

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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

EDAC concentration (mM)

Figure 4.1 Optimisation of EDAC concentration. Dashed line

represents OD of negative control and error bars indicate 1xSD
from duplicate mean

4.3.3 Optimisation of antibody concentration

Method
Following the optimisation of the EDAC concentration used to covalently bind the capture
antibody to the IMBs, the antibody concentration used in the coupling reaction that elicited
optimal capture activity was determined as follows. Goat and rabbit polyclonal antibodies
were each titrated and coated to IMBs in a reaction volume of 250 L. For the goat antipestivirus polyclonal antibody, the protein concentration was initially determined using a
BCA protein estimation kit (Pierce, USA) that used a BSA standard. The Biorad protein
estimation kit described in Section 4.2.2 above was not used until after this section had
been completed as no spectrophotometric filter with a wavelength of 570 nm was available
in Laos at the time. The initial estimate was 40 mg/mL based on the BCA protein
estimation kit with an albumin protein standard. Dilutions of the goat polyclonal antibody
were made to 100, 200, 300, 400 and 500 g/mL of antibody per reaction based on this

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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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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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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

Goat Polyclonal Antibody Dilution

OD450 nm

1.60
1.20

0.00
1:50

1:100

1:150

1:200

Rabbit Polyclonal Antibody Dilution

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.

4.3.4 Standard coupling reaction

Once the EDAC and antibody concentrations were optimised, the following standard
coupling reaction was used. Two hundred microlitres of 2.5 % w/v IMBs were combined
with 50 L of 50 mM sodium acetate buffer, either 5 L of rabbit polyclonal antibody or
2.5 L of goat polyclonal antibody and made up to 490 L with DiRO water in a 2 mL
screw cap tube. The reaction mixture was briefly mixed by vortex and rotated at 20 rpm for
20 min at room temperature. Note that with larger volumes good mixing was not achieved
using a plate shaker. Following the 20 min incubation, 10 L of freshly made 52 mM
EDAC was added, mixed by vortex and rotated for 2 h at room temperature. After 2 h the
tubes were placed in the magnet for 2 min and the supernatant was discarded. One millilitre
of blocking buffer containing FSG in PBSA was added and mixed by vortex. Blocking was
allowed to proceed for 1 h at room temperature by rotation. Following the blocking step,
the tubes were placed on the magnet for 1 min and the blocking buffer removed. The beads
were washed three times with 1 mL PBSA and finally resuspended in 990 L of storage
buffer containing FSG in PBSA for a working stock of 0.5 % w/v coated beads.
Note that the blocking and storage buffers changed slightly as the research progressed.
Initially the blocking buffer contained BSA instead of FSG as this was all that was

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available in stock in Bangkok, Thailand. FSG was recommended by Seradyn, USA

(Seradyn, 1994) as the protein of choice for blocking to minimise the potential for crossreactivity with other antibodies used in an ELISA. BSA was used while FSG was in transit
from the USA. The new blocking buffer contained 1 % FSG in PBSA with 0.02 % sodium
azide as preservative and the storage buffer contained 0.2 % FSG in the same buffer and
preservative. At a later stage this changed slightly to 2 % FSG in PBSA with 0.1 % Proclin
300 (Sigma Aldrich, USA) as a preservative for the blocking buffer and 0.5 % FSG in
PBSA and Proclin 300 as the storage buffer.

4.3.5 Optimisation of Mab 24/10 and conjugate dilutions

Method
The optimal dilution of Mab 24/10 and HRP-conjugated antibody was determined by
titrating each in an IMB-ELISA using beads coated with each of goat or rabbit polyclonal
antibody. A checker board format was not used as optimisation and development was
carried out in 1.5 mL microfuge tubes. To perform a checker board titration a large number
of tubes was needed leading to increased error rates. To perform this form of titration a 96well plate format with a corresponding magnet would have been deemed necessary.
For the optimisation of HRP-conjugated antibody, 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 conjugate. The tubes were
mixed by vortex and shaken at 37 C. After 1 h incubation, the IMBs were washed once
with 200 L PBST followed by the addition to all tubes of 100 L of Mab 24/10 diluted
1:25 in PBST. A 1:25 dilution of Mab 24/10 was estimated to be in excess for 25 L IMBs.
The beads were mixed by vortex and shaken at 37 C for 1 h and washed once with PBST
following the incubation. Conjugate was diluted 1:2000, 1:3000, 1:4000 and 1:5000 in
diluent buffer containing 5 % normal rabbit serum and 0.1 % FSG in PBST. One hundred
microlitres of each dilution was added to the respective tubes and mixed by vortex. The
tubes were then shaken for 1 h at 37 C after which the IMBs were washed three times in
200 L of PBST and transferred to a new tube on the third wash. After the removal of the

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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

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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

Figure 4.3 Optimisation of HRP-conjugated antibody 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.

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.

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4.3.6 Comparison of IMBs coated with goat or rabbit polyclonal antibody

Method
Comparative assessment of the two polyclonal capture antibodies was achieved by
screening a panel of different infected spleen samples obtained locally in Laos (confirmed
infected by AC-ELISA) together with the AAHL antigen. Initially, a locally derived
sample, designated laboratory number 089/04, which was found to be positive for CSF
virus by both AC-ELISA and virus isolation (refer to Chapter 3 above) was used. Spleen,
tonsil and kidney tissue from 089/04 were prepared as a 5 % w/v homogenate in 1 % NP40
PBSA and clarified by centrifugation (refer to Chapter 3). A five-fold dilution series to 0.2
% w/v was prepared in 1 % NP40 PBSA and assessed by IMB-ELISA with beads coated
with goat or rabbit polyclonal antibody. Twenty five microlitres of IMBs were added to 100
L of each sample dilution and negative control antigen in duplicate and then mixed by
vortex. The tubes were shaken at 37 C for 1 h and the beads washed once. One hundred
microlitres of Mab 24/10 diluted 1:50 in PBST was added to each tube, mixed and
incubated at 37 C. After 1 h the beads were washed and 100 L of HRP-conjugated
antibody diluted 1:3000 in diluent was added to each tube and mixed by vortex. The IMBs
were mixed and shaken at 37 C for 1 h and then washed 3 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. A comparison of the two capture antibodies was
also carried out using the AAHL antigen diluted 1:4, 1:16 and 1:64 using the same IMBELISA protocol.
Following this initial assessment of the two capture antibodies, a further three samples
submitted from geographically different regions of Laos were tested. The spleen samples
were all submitted in 2004 and came from Luang Prabang province in the central north,
(sample ID: 028/04), the central province of Bolikhamxay (sample ID: 087/04) and from
Houaphan province in the north east bordering Vietnam (sample ID: 088/04). All samples
returned a positive result in the AC-ELISA and were not tested by virus isolation due a
limited amount of spleen tissue submitted. Samples 028/04 and 088/04 were strong
positives with S/N ratios of 9.67 and 13.43 respectively. Sample 087/04 was a low positive

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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).

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a)

2.00

OD450nm

1.50
1.00
0.50
0.00
5%

1%

0.2 %

Tissue concentration (w/v)

b)

2.00

OD450nm

1.50
1.00
0.50
0.00
5%

1%

0.2 %

Tissue concentration (w/v)

OD450nm

0.75

c)

0.50

0.25

0.00
5%

1%

0.2 %

Tissue concentration (w/v)

Figure 4.5 Comparison of goat () and rabbit () polyclonal

antigen capture antibody using Lao sample 089/04 as analyte
where a) Spleen, b) tonsil and c) kidney. Error bars indicate
3xSD.

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2.00

OD450nm

1.50

1.00

0.50

0.00
1:4

1:16

1:64

1:256

Dilution Factor

Figure 4.6 Comparison of goat () and rabbit ()

polyclonal antigen capture antibody using AAHL
antigen as analyte. Error bars indicate 3xSD.

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.

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2.00

a)

OD450 nm

1.60
1.20
0.80
0.40
0.00
5.00

1.00

0.20

0.04

Tissue Concentraion (% w/v)

OD450 nm

1.20

b)

0.80

0.40

0.00
5.00

1.00

0.20

0.04

Tissue Concentraion (% w/v)

2.00

c)

OD450 nm

1.60
1.20
0.80
0.40
0.00
5.00

1.00

0.20

0.04

Tissue Concentration (%w/v)

Figure 4.7 Comparison of goat () and rabbit () polyclonal

antigen capture antibody using diagnostic spleen specimens a)
028/04, Luang Prabang; b) 087/04, Bolikhamxay; and c)
088/04, Houaphan. Error bars indicate 3xSD.

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4.3.7 Optimisation of bead volume in IMB-ELISA

Method
Reaction conditions thus far were optimised using 25 L of beads where the final IMB
concentration was 0.1 % w/v. To determine whether this concentration of beads was
optimal an ELISA was set up where 10, 15, 20, 25, 30, 35 and 40 L of beads coated with
goat polyclonal antibody were added to 100 L of Lao antigen in duplicate and 100 L of
negative antigen in a single tube. The final bead concentrations were 0.045, 0.065, 0.083,
0.100, 0.115, 0.130 and 0.143 % w/v respectively. The tubes were mixed by vortex and
shaken at 37 C for 1 h. The beads were washed once and 100 L of Mab 24/10 diluted
1:50 in diluent buffer containing 0.5 % FSG and 0.1 % Proclin 300 in PBST was added.
The tubes were again mixed and shaken at 37 C for 1 h. The wash was repeated and HRPconjugated antibody diluted 1in 3000 in the same diluent buffer was added. 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. The results were normalised by subtracting the negative control
OD450nm from the test OD450nm. The optimum bead volume was visualised graphically by
plotting bead volume on the x-axis against OD450nm on the y-axis using MS Excel.

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.

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2.00

OD450nm

1.60
1.20
0.80
0.40
0.00
10

15

20

25

30

35

40

Bead Volume Added (L)

Figure 4.8 Optimisation of bead volume used in each reaction. Dashed

line represents OD of negative control and error bars indicate 1xSD
from duplicate mean.

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

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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.

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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.

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OD450nm

2.00
1.50
1.00

a)

0.50
0.00
15

30

45

60

Incubation time (min)

OD450nm

2.00
1.50
1.00

b)

0.50
0.00
15

30

45

60

Incubation time (min)

OD450nm

2.00
1.50
1.00

c)

0.50
0.00
15

30

45

60

Incubation time (min)

2.50

OD450nm

2.00
1.50
1.00

d)

0.50
0.00
2.50

5.00

7.50

10.00

Incubation Time (min)

Figure 4.9 Optimisation of incubation times a) sample; b) Mab 24/10; c)

HRP-conjugated antibody and d) TMB substrate. Dashed line represents OD
of negative control and error bars indicate 3xSD from duplicate mean.

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4.4 Discussion and Conclusions

Increased surface area
This chapter describes the development and optimisation of a new ELISA method
employing IMBs as the solid surface for the capture of CSF viral antigen. All antigen
capture-ELISAs described in the literature for the detection of CSF viral antigen in
diagnostic specimens utilise a 96-well microplate as the solid surface interface for the
ELISA reactions to take place (Clavijo et al. 1998; Depner et al. 1995; Kaden et al. 1999;
Shannon et al. 1993). The IMBs used in this ELISA afford a greater surface area for the
binding of capture antibody and subsequent capture of antigen. Individual wells of a 96well microplate have a maximum surface area of 2.7 cm2/well using a volume of 400 L
(Nunc, 2005). In the AC-ELISA described in this thesis, only 100 L of polyclonal
antibody was used to coat the well reducing the total binding surface area to less than 1
cm2. The surface area of the immunomagnetic beads used in this study is approximately
1.321 x 104 cm2/g, which is equal to 1.65 cm2/25 L of a 0.5 % w/v suspension (Seradyn,
1994). Therefore, the utilised surface area in individual wells of the microplate was almost
half that of the IMBs per reaction. An added advantage of the IMBs used in this study was
the carboxyl modified surface that allowed antibody molecules to be covalently bound such
that a majority of antigen binding sites were functional. This is in contrast to a microplate
that relies on passive binding to the solid surface.

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

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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.

Selection of antigen capture antibody

The AAHL antigen extract was made from pigs infected with the Weybridge strain of CSF
virus, a strain that has a high level of sequence homology with a C-strain vaccine virus used
in China (Tu et al. 2001). Both the Weybridge strain and the C-strain vaccine virus belong
to subgroup 1.1 based on sequence data generated from the major neutralising epitope
region of the E2 protein (Tu et al. 2001). This would suggest that a polyclonal population
of antibodies against the immunodominant E2 protein of a C-strain virus would have a high
degree of avidity for the Weybridge strain. This was indeed found to be the case. The
polyclonal antibody produced in rabbits against the C-strain vaccine virus used in Laos had
a high degree of avidity for the AAHL antigen as demonstrated by capture efficiency in the
IMB-ELISA. Conversely, the polyclonal antibody raised in goats with pestivirus group
reactivity captured AAHL antigen less efficiently, as demonstrated by a lower OD in the
IMB-ELISA compared to the rabbit polyclonal antibody.
The CSF virus strains circulating in Laos belong to subgroups 2.1 and 2.2 (Blacksell et al.
2005; Blacksell et al. 2004a). The rabbit polyclonal antibody against the C-strain vaccine
virus captured antigen from Lao isolates less efficiently than did the pestivirus group
reactive goat polyclonal antibody. Based on these findings and that group 2 viruses are now
the predominant strains circulating in the Asian region (Blacksell et al. 2005; Blacksell et

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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.

Specificity and sensitivity

In this IMB-ELISA, the specific recognition of the captured antigen was reliant on the
unique specificity of a monoclonal antibody, Mab 24/10. This monoclonal antibody has
been shown to have a high level of specificity for a broad range of CSF virus isolates with
no cross-reactivity evident when reacted with other pestiviruses (Kosmidou et al. 1995).
Using the format described in this chapter, Mab 24/10 has been shown to have reactivity
with CSF viruses from sub-group 1.1 (Weybridge strain) and group 2 viruses (Lao isolates).
Unfortunately, during the development of the IMB-ELISA, no other CSF virus strains were
assessed. Nor was the degree of specificity examined using isolates of BVD virus and BD
virus in the test. These additional samples were not tested due to a lack of availability at the
National Animal Health Centre and constraints to importing virus isolates, even if
inactivated.

Improved reaction times

The extra surface area per reaction afforded for the capture of antigen and the fact that the
IMBs are spread though the reaction mixture during the incubation has resulted in a
reduction in the incubation times required compared to the AC-ELISA. Incubation times of
IMBs with test sample, Mab 24/10 and conjugate were optimal at 30 min each, with little
impact on final absorbance if reduced to 15 min. The incubation time with TMB substrate
was optimal at 5 min meaning the total time to perform the test was less than 3 h if the
sample preparation time was included. When compared to the AC-ELISA format, which
can take up to 1 to 2 days to perform, the IMB-ELISA was more rapid. In addition to the
improvement in turn-a-round time, the format of the IMB-ELISA was found to be much
simpler, as the AC-ELISA described by Shannon et al. (1993) requires manipulations in a
transfer plate followed by further manipulations in the ELISA plate.

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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.

Conclusions and general comments

The IMB-ELISA described in this chapter has been fully developed with conditions and
reagents optimised. The test is simpler to perform than the AC-ELISA and the turn-a-round
time to produce a result is far shorter, 3 hours compared with 1 to 2 days. The test is
comparable in cost to the AC-ELISA which is far cheaper than tissue culture or PCR based
diagnostic methods.

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Chapter 5.
Assessment and validation of IMB-ELISA

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5.1 Introduction and research design

The previous chapter describes the development and optimisation of test parameters for an
IMB-ELISA for the detection of CSF virus antigen. This chapter explores the next phase of
test development, namely the assessment and validation of test parameters and
performance, the establishment of diagnostic criteria and estimates of diagnostic sensitivity
and specificity. The final phase of test development explored in this thesis is an assessment
of inter-operator agreement using Kappa statistics and an assessment of the potential for the
test to be adapted to a kit format without the need for a microplate reader.
As in Chapter 4, the methods and results of each stage of test assessment and validation
will be reported together in each section.

5.2 Methods and results

5.2.1 IMB-ELISA
The antigen, antibodies and beads used in this chapter are the same as those described in
the previous chapter. The standard coupling reaction using goat polyclonal capture antibody
is the same as that described earlier (Section 4.2.6) using 2 % FSG and 0.1 % Proclin 300
in PBSA as blocking buffer and 0.5 % FSG and 0.1 % Proclin 300 in PBSA as storage
buffer. Twenty-five microlitres of IMBs per reaction were used for all assessment and
validation experiments with 30 min incubations for IMB/sample, Mab 24/10 and HRPconjugated antibody and 5 min incubation with TMB substrate. The diluent buffer used for
the dilution of Mab 24/10 and HRP-conjugated antibody was 5 % glycerol with 0.5 % FSG
and 0.1 % Proclin 300 in PBST (Appendix 1). Since the end result of the development of
this IMB-ELISA was for use in a field environment where all reagents are pre-diluted to a
working concentration, the diluent buffer above was developed to assess its potential for
providing a stable environment for both Mab 24/10 and HRP-conjugated antibody diluted
to a working concentration. All dilutions were made on the day of the test except for those
experiments explicitly testing the stability of pre-diluted Mab 24/10 and HRP-conjugated
antibody.

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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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2.25

OD450 nm

2.00
1.75
1.50
1.25
1.00
1

10

11

12

13

14

15

Run Number

Figure 5.1 Repeatability of IMB-ELISA. Solid horizontal line indicates

overall mean of the 15 runs. Dashed lines indicate 1, 2 and 3 SD from the
mean. Error bars indicate 1 SD from the mean OD of each run.

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

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(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.

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Mab 24/10*

HRP-conjugated antibody

Pre-diluted

Pre-diluted

New

Pre-diluted

Pre-diluted

New

New

New

*New, diluted on day of test; Pre-diluted, diluted at week 0

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

Weeks post coupling antibody to IMBs

Figure 5.2 Stability of polyclonal capture antibody bound to

IMBs. Error bars indicate 1xSD of quadruplicate mean.
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Percentage decrease in activity

Chapter 5

50

a)

40
30

31

20
18

10
0

10

10

10

Percentage decrease in activity

Weeks post preparation

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

Figure 5.3 Stability of pre-diluted reagents in IMB-ELISA

stored at 4 C. a) Loss of activity using 10 week old (wo) prediluted Mab 24/10 and HRP-conjugated antibody and b) Loss
of activity using a combination of Mab 24/10 and HRPconjugated antibody (conj) diluted on day of test (new) or 10
weeks old (wo).

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5.2.4 Analytical sensitivity

Method
Diagnostic specimens from submissions 089/04 and 091/04 (one animal per submission)
were used to compare the analytical sensitivity of the IMB-ELISA with the AC-ELISA.
Five percent weight per volume homogenates of spleen, tonsil and kidney were prepared in
1 % NP40 PBSA and diluted two-fold in either PBST plus 5 % normal goat serum (ACELISA) or PBST plus 0.5 % FSG (IMB-ELISA) to minimise background noise in the
ELISAs. The AC-ELISA was performed according to the method described in Chapter 3
where samples of spleen, tonsil and kidney, individually diluted 1:1 to 1:128, were tested in
duplicate. Positive and negative controls were included on each plate as was a control
consisting of PBST plus 5 % normal goat serum. The IMB-ELISA was performed
according to the method previously described where samples of spleen diluted from 1:2 to
1:512, tonsil diluted 1:1 to 1:256 and kidney diluted 1:1 to 1:128 were tested in duplicate.
Lao antigen and CSF negative antigen were tested in quadruplicate in each run of the IMBELISA together with PBST plus 0.5 % FSG in duplicate to measure the amount of
background noise in diluent. The results of the IMB-ELISA were expressed as the percent
positivity of the strong positive control and calculated using the following formula:
Sample OD
- Negative Control OD450nm
% Positivity = Positive Control450nm
OD
- Negative Control OD
450nm

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).

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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.

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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

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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

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5.2.5 Establishing diagnostic cut-off

Method
To determine the diagnostic cut-off and to gain an initial estimate the diagnostic sensitivity
and specificity of the newly developed IMB-ELISA, spleen samples submitted during the
period 2001 2004 to the National Animal Health Centre laboratory in Vientiane, Laos
were tested in both the AC-ELISA and IMB-ELISA. Spleen specimens collected as part of
routine diagnostic testing were stored at 85 C as 20 % w/v homogenates in 1 % NP40
PBSA. Samples were re-tested by AC-ELISA to confirm the result and then divided into
those that were positive and those that were negative by AC-ELISA. In total 123 ACELISA negative and 39 AC-ELISA positive spleen samples were available for comparative
tests.
In the first instance 43 CSF negative and 15 CSF positive spleen samples were tested in the
IMB-ELISA. The samples were thawed and diluted 1:4 in PBST by adding 25 L of
sample to 75 L of PBST. Twenty five microlitres of goat polyclonal antibody coated
IMBs were added to duplicate samples and the test performed as described in Section 5.2.1.
In each run of the IMB-ELISA, a strong positive control (Lao antigen) was tested in
duplicate as was a CSF negative spleen homogenate. The results of the test samples were
expressed as a percentage of the strong positive control OD (% positivity) and calculated
using the equation above (Section 5.2.4).
The results obtained were compared to the AC-ELISA results. A number of samples
negative by AC-ELISA were found to react very strongly in the IMB-ELISA; as such the
cause of this reactivity was investigated by virus isolation. Two of these samples 087/03
and 107/03 were found to be negative by virus isolation but appeared positive by IMBELISA. These samples were further investigated by adding normal goat or pig serum to the
samples prior to the addition of IMBs to determine if there were non-specific reactants that
might be adsorbed out. In duplicate, 10 L of normal goat or pig sera were added to 65 L
of PBST and 25 L of sample 087/03 or 107/03. Ten microlitres of normal goat or pig
serum was added to 90 L of Lao antigen as a positive control and CSF negative

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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

Where relative DSn =

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-

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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

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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

AC-ELISA (S/N ratio)

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)

Figure 5.4 IMB-ELISA verses AC-ELISA without pre-incubation of sample

with serum. Dashed horizontal line represents cut-off for AC-ELISA. Dashed
vertical line represents tentative IMB-ELISA cut-off of 49 %.

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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

Figure 5.5 Incubation of samples 087/03 and 107/03 with serum

before the IMB-ELISA. Normal goat serum (NGS) and normal pig
serum (NPS) were incubated with the samples and controls (C++,
strong positive control and C-, negative control). Error bars indicate
3xSD from duplicate mean.

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

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% 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.

AC-ELISA (S/N Ratio)

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)

Figure 5.6 IMB-ELISA versus AC-ELISA with a pre-incubation of

sample with 10 % normal goat serum (NGS) before the addition of IMBs.
In total, 123 CSF negative and 39 CSF positive samples tested by ACELISA were run in the IMB-ELISA.

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50

Frequency

40
30
20
10
0
-3

-2

-1

% Positivity in IMB-ELISA

Figure 5.7 Frequency distribution of IMB-ELISA results for

samples negative by AC-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

Figure 5.8 Modified ROC plot of the diagnostic sensitivity (DSn)

and specificity (DSp) (y-axis) of the IMB-ELISA as a function of
cut-off intervals (x-axis). Diagnostic sensitivity and specificity were
measured against the AC-ELISA as a reference standard

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5.2.6 Inter-operator agreement and preliminary assessment of reading test by eye

Method
From the 162 samples assessed to determine the cut-off between a positive and negative
result, the samples were categorised as (i) negative, (ii) low positive and close-to-cut-off
negatives and (iii) strong and mid positives. Category (i) consisted of 117 samples with
percent positivity less than four (less than two standard deviations from the mean). From
category (i), twenty were randomly selected using a random number generator function in
MS Excel. Low positives and close-to-cut-off negatives were classified as those samples
returning a percent positivity in the IMB-ELISA greater than or equal to 10 and less than 25
and greater than or equal to four and less than 10, respectively. All samples in this category
were selected for assessment of operator agreement as there were only 11 samples falling
into this category. For category (iii) samples, nine strong or mid positives were randomly
selected from a total of 34 with a percent positivity of greater than or equal to 25 %.
Each of three operators assessed the same samples independently and the samples were
given a different identification number for each operator to minimise inadvertent bias. All
operators prepared their own batch of IMBs coated with capture antibody, and this one
batch was used to test all 40 samples. Samples were prepared in duplicate and stored at 85
C in 100 L aliquots with a randomised number that could be linked back to original test
results for each operator. The IMB-ELISA was conducted with the inclusion of duplicate
positive and negative controls. At the completion of the IMB-ELISA and before the
addition of H2SO4, each operator scored samples as positive or negative based on visual
appearance of a green colour. Following reporting the result by eye, the reaction was
stopped by adding 1M H2SO4 and the supernatant was transferred to an ELISA plate and
the OD450nm measured.
Operator 1 was the author of this thesis, who was considered experienced in IMB-ELISA
methodology. Operator 2 and 3 were laboratory technicians working in the Lao National
Animal Health Laboratory. Operator 2 had been working in the laboratory for greater than
10 years and for the past six years has been performing ELISA procedures for the diagnosis

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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

Expected (chance) agreement overall =

(expected (a)) + (expected (d))

a + b +c + d

Kappa (agreement beyond chance) =

Observed agreement - Expected agreement

1 - Expected agreement

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.

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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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Table 5.4 Agreement between operators when

IMB-ELISA test results were determined by
recording a visual colour change
Kappa

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.

Table 5.5 Agreement between IMB-ELISA

results determined by recording a visual
colour change and measuring the OD450 nm
Eye v's OD450nm
OP1

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

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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

IMB-ELISA (eye) V's AC-ELISA

DSn
DSp
100
88
100
85
93
85

Table 5.7 Comparison of IMB-ELISA with the

AC-ELISA results when IMB-ELISA result was
determined from OD450nm

OP1
OP2
OP3

IMB-ELISA (OD450nm) V's AC-ELISA

DSn
DSp
100
92
100
92
93
92

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5.2.7 Establishment of upper and lower control limits

Method
Lao antigen was used to assess the repeatability of the IMB-ELISA and was also used in all
tests as the strong positive control, from which the percent positivity was calculated.
Likewise, the negative control used in the assessment of repeatability, CSF negative spleen,
was also used as the negative control in all IMB-ELISA tests described in this chapter. The
upper and lower control limits (UCL and LCL) were set as two standard deviations from
the mean of 15 runs of the IMB-ELISA using the same data set used to assess the
repeatability in Section 5.2.2.

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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5.3 Discussion and Conclusions

Introduction
The validation of an assay is an important process in the development of new diagnostic
methods and for their subsequent implementation in laboratories and disease control
programs. A validated test should consistently provide results that identify diagnostic
specimens as either positive or negative and accurately predict the infection status of an
animal from which the specimen was collected (Jacobson, 1998). Chapter 4 was primarily
concerned with the development of the IMB-ELISA method through optimisation of
standard reagents and protocols. The second stage in test development and validation is the
assessment of test parameters and the determination of performance characteristics of the
assay. The final stage of assay development is the continued monitoring of assay
performance during routine use of the newly standardised test. The results presented in this
chapter describe the second stage of test development.

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

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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.

Establishing a diagnostic cut-off

Typically, virus isolation has been considered the diagnostic gold standard for CSF (de
Smit, 2000; Pearson, 1992), however recent research suggests that real-time RT-PCR has
greater diagnostic sensitivity (Risatti et al. 2005). In this evaluation of the IMB-ELISA to
determine the diagnostic cut-off, the AC-ELISA was used as the reference standard due to
the limitations of the diagnostic laboratory and facilities in Laos. Tissue culture and RTPCR facilities are in place; however neither tissue culture nor RT-PCR was used for routine
detection of CSF virus for a number of reasons. First and foremost, the technical skills of
local laboratory staff are not at a sufficiently high standard to maintain a quality tissue
culture or PCR system. Secondly, cost and a relatively low number of samples deter
maintaining a fully functional tissue culture and PCR facility. Lastly, samples can on
occasion take up to a week to arrive at the laboratory thus limiting the scope of diagnostic
tests which detect either live virus or intact RNA. Blacksell et al. (2004b) demonstrated a
significant negative impact on RT-PCR detection using samples transported at ambient
tropical temperatures. Therefore, from the outset, the determination of diagnostic cut-off
was limited by the reference test.

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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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Diagnostic sensitivity and specificity estimates

As described above, the determination of diagnostic sensitivity and specificity of the IMBELISA was also limited by the reference standard. The diagnostic sensitivity and specificity
of an AC-ELISA method has been shown to be 91 and 98 %, respectively in comparison to
virus isolation and RT-PCR (Clavijo et al. 1998). The results of this study show the initial
estimates of IMB-ELISA diagnostic sensitivity and specificity to be equivalent to the ACELISA format described in Chapter 3. At a cut-off of 10 % positivity of the strong positive
control, the IMB-ELISA was 100 % sensitive and specific in comparison to the AC-ELISA
(n = 162). These results provide an initial estimate of diagnostic sensitivity and specificity
only, as the samples used to determine the cut-off were also used to determine this initial
estimate. The same test samples used to determine the cut-off of a newly developed ELISA
may only be used to compare diagnostic sensitivity and specificity with other assays
provided the other assays were not used in the selection of individual samples (Wright et al.
1993). As such, to determine a more accurate measure of the diagnostic sensitivity and
specificity of the IMB-ELISA in comparison to the AC-ELISA, a new panel of negative
and positive samples will need to be tested in a blinded fashion to avoid inadvertent bias.

Inter-operator agreement and reading result by eye

Technician or operator error is generally considered the greatest source of variation for a
majority of assays and the level of agreement between operators is deemed a measure of an
assays relative robustness (Jacobson, 1998). In this evaluation of IMB-ELISA robustness,
the level of agreement of results observed between operators in the laboratory in Lao
ranged from substantial to almost perfect. When the results were determined by measuring
absorbance, the level of agreement between the two most experienced laboratory staff was
almost perfect according to the criteria outlined by Smith (2006), with a kappa value of 0.9.
Only two differences were observed, both of which occurred with samples close to the
diagnostic cut-off. The level of agreement between the least experienced staff member
(Operator 3) and the other two operators was rated as substantial, with kappa values of
0.74. This finding was not wholly unexpected. However, care must be taken to ensure
adequate training if the test is to be transferred to provincial laboratories where staff are

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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.

Conclusions and general comments

From the results presented in this chapter it is evident that the IMB-ELISA developed
during the course of this research project is highly repeatable and the capture antibody/IMB
complex is very stable for at least 10 weeks. The test is likely to be sufficiently robust as
demonstrated by strong agreement between different operators and the diagnostic
performance of the test is at least as good as the AC-ELISA. Furthermore, the increased
level of analytical sensitivity compared to the AC-ELISA lends the test to the possibility of
being better able to detect outbreaks of low virulent field virus. The evaluation of reading
the test by eye shows the test has potential to be adapted to a near-to-field test for the
detection of CSF virus.

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Chapter 6.
Assessment of storage temperature on
lapinised C-strain vaccine produced
in Laos

153

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Chapter 6

6.1 Introduction and research design

The management and control of CSF virus outbreaks in an endemic environment is highly
dependent on early diagnosis and the use of an effective vaccine. In Laos, a locally
produced lapinised C-strain vaccine is used to control CSF; however high levels of vaccine
failure have been reported, as indicated by vaccination not resulting in the development of a
protective antibody response (Blacksell, 2001). Vaccination failure has been attributed to
the delivery of poor quality vaccine as a result of cold-chain failure and poor administration
of vaccine to inadequately restrained animals (Blacksell, 2001; Gibson and Wilkie, 2000).
The issue of vaccine failure is further compounded by poor farmer participation in
vaccination programs (Blacksell, 2001; MAF, 2000).
The CSF vaccine is produced at the National Vaccine Institute (NVI) as a freeze dried
rabbit spleen homogenate and stored at 20 C before transport on ice to the Veterinary
Supply Unit (VSU). The VSU was set up as a joint initiative of the Lao government and a
now completed European Union Livestock Project, with responsibility for supplying
stakeholders with a range of veterinary supplies including vaccine. Once at the VSU, the
CSF vaccine is again stored at 20 C before being transported on ice to provincial
livestock offices. Once at the provincial office the vaccine may either be sold directly to
farmers or VVWs or transported again to district livestock offices. The vaccine is again
stored before being sold on to farmers and VVWs. Storage temperatures at provincial and
district offices are quite varied depending on the freezer used. Figure 6.1 represents a model
of predicted vaccine storage and delivery conditions from production to end-user, where the
storage temperatures at the provincial and district offices are estimates based on freezer
types widely used. At present, the NVI recommends that the vaccine be stored at 20 C
and has a shelf life of 12 months from the date of production (Dr. Syseng Khounsy,
Personal communication).
The key factor to be addressed by the research described in this chapter is the effect of
storage temperature on vaccine efficacy. With storage at 4 C the freeze-thaw cycles
experienced under the current supply chains could be eliminated. Hence, the first research

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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

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6.2 Materials and Methods

6.2.1 Vaccine
The C-strain vaccine was produced at the NVI as a lyophilised rabbit spleen homogenate
using their standard protocol for vaccine production and supplied in a rubber-stoppered vial
(FAO vaccine production manual). The vaccine vials from batch number 05/2004 were
transported from the NVI to the NAHC laboratory on ice and stored at 20 C in a chest
freezer. To minimise temperature fluctuations generated by opening the freezer lid the
vaccine vials were stored inside a secondary foam container. The temperature inside the
foam container was monitored using a temperature logger (Thermocron, OnSolution,
Australia) to record temperature at 20 min intervals throughout the course of the
experiment. One month after receiving the vaccine from the NVI, a proportion of vaccine
batch 05/2004 was placed at 4 C and the temperature monitored.
Immediately prior to administering the vaccine to pigs, the lyophilised homogenate was
reconstituted in 10 mL of sterile distilled water (supplied by the NVI ) at room temperature
and 1 mL administered intramuscularly to each pig.

6.2.2 Experimental pigs

The pigs used in all experimental work were indigenous Lao breed pigs purchased from a
nearby market at approximately 6 8 weeks of age and earmarked. The pigs were housed
under low level biological containment and were treated for internal parasites with pyrantel
(500 mg per piglet). All pigs were given the same feed, a commercially produced high
quality feed mixed with rice bran. Pigs were able to access water at all times. The pens
housing the pigs were sheltered from the weather and were cleaned once per day to remove
faeces and other waste material. Pig handling was carried out by Lao project staff, under
the veterinary supervision of Dr. Syseng Khounsy.

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6.2.3 Experimental design

To assess the efficacy of the vaccine procured from the NVI at the commencement of the
experiment, six pigs were purchased and managed as above. All pigs were housed in the
same pen and blood was collected to test serum for the presence of anti-CSF virus
antibodies by NPLA. All pigs were allowed to acclimatise for 14 days before four of the six
pigs were selected and vaccinated with a single dose of C-strain vaccine batch number
05/2004. Blood for serum was collected on days 10, 14, 21, 28 and 35 post vaccination.
Serum was tested for neutralising antibodies against CSF virus by NPLA.
A second group of pigs were brought into the laboratory facility at week 6 of the
experiment. Nine pigs were purchased and bled to determine their serological status and
then allowed to acclimatise for a further two weeks. The pigs were randomly allotted to two
lots of four pigs and housed in separate pens; the remaining pig was a non-vaccinated
control. After the acclimatisation period, each lot of pigs was vaccinated with either
vaccine stored at 20 C (two months storage) or vaccine stored at 4 C (one month
storage) at month 2 of the experiment. Blood was collected on days 0, 10, 14, 21 and 28
post vaccination. After the final bleed the pigs were removed to make room for subsequent
experiments.
A third group of pigs were brought into the laboratory facility at week 10 of the
experiment, 10 pigs were purchased, treated and left to acclimatise as described above. The
pigs were allotted to separate lots as above and vaccinated with either vaccine stored at 20
C (three months storage) or vaccine stored at 4 C (two month storage) at month 3 of the
experiment. The two control pigs were not vaccinated. Blood was collected on days 0, 10,
14, 21 and 28 and the pigs removed after the final bleed. This process was repeated at
weeks 14 and 18 whereby pigs were brought into the research facility two weeks prior to
being vaccinated at months 4 and 5 of the experiment. Refer to Figure 6.2 for detailed flow
diagram of the experimental timeline. Vaccination of pigs and the collection of blood were
carried out by Lao project staff, Dr. Syseng Khounsy and Mr. Lapin.

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Month of Experiment

6 pigs purchased
Group 1 (wk 2)

Vaccine batch 05/2004 procured

Group 1 pigs vaccinated
Vaccine stored at 20 C

0.5
1
9 pigs purchased
Group 2 (wk 6)

1.5
2

10 pigs purchased
Group 3 (wk 10)

Group 3 pigs vaccinated

3.5
4

10 pigs purchased
Group 5 (wk 18)

Group 2 pigs vaccinated

2.5
3

10 pigs purchased
Group 4 (wk 14)

Storage at 4 C commences

Group 4 pigs vaccinated

4.5
5

Group 5 pigs vaccinated

Figure 6.2 Flow chart of CSF vaccine storage temperature experiment

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6.2.4 Serology and analysis

Following the bleeding of the pigs, the clotted blood was left to separate and serum was
collected and stored at 20 C. Serum neutralising antibodies were detected by NPLA and
serum with titres greater than or equal to 12.5 were considered positive for protective levels
of neutralising antibody (refer to Section 1.2.5, subsection Vaccine induced immunity and
efficacy of C-strain vaccine). For epidemiological purposes, Terpstra and Wensvoort (1988)
recommend that titres less than 32 are inadequate as virus transmission is still possible. For
this reason, antibody responses were scored on day 28 and were considered positive if titres
greater than or equal to 32 were detected (Terpstra and Wensvoort, 1988).
Data were analysed by comparing both treatment groups (4 C and 20 C) at each month
using the non-parametric one-tailed Fisher exact test together with risk ratio as a measure of
likelihood of providing antibody levels greater than or equal to 32 twenty-eight days postvaccination using the program EpiCalc (2000). To determine the shelf life of vaccine stored
at 20 C, antibody titres 28 days following vaccination were compared to the pre-trial
assessment results using the Fisher exact test described above.

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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6.3.2 Pre-trial assessment of vaccine quality

All pigs were negative for the presence of antibodies to CSF virus on the day of
vaccination. All vaccinated pigs were positive for CSF virus neutralising antibodies on day
35 post vaccination with a median serum neutralising titre of 44 (range: 32 to 44).
Neutralising antibodies were first detected on day 10 in one pig, three pigs on day 14 and
all four pigs on day 21 (Figure 6.3). A positive neutralising antibody response was not seen
until day 28 post vaccination. Control pigs receiving no vaccine remained negative for the
presence of virus neutralising antibodies for the duration of the experiment.

Reciprocal of serum
dilution

48
40
32
24
16
8
0
0

10

14

21

28

35

Days post vaccination

Figure 6.3 Serum neutralising antibodies to CSF virus post

vaccination with C-strain vaccine batch 05/2005. Symbols
correspond to individual vaccinated pigs. Solid horizontal bars
indicate median antibody titre. Dashed lines represent titres of
12.5 and 32.

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6.3.3 Mortalities and anomalies

During the acclimatisation period of pigs purchased at week 6 and week 10, a number of
pigs became ill and showed clinical signs consistent with CSF. In the group of pigs brought
into the laboratory facility at week 6, one pig was euthanased and samples collected for
testing by AC-ELISA. The spleen and tonsil samples returned a strong positive result for
the presence of CSF virus antigen. The remaining eight pigs were euthanased with one
other pig returning a positive result. The pens were decontaminated and left for two weeks
before the purchase of the Group 3 pigs. One of the pigs brought into the laboratory facility
at week 10 also showed clinical signs consistent with CSF, this pig was euthanased before
vaccination and CSF virus antigen was detected in its tissues. The pens were once again
depopulated and decontaminated. The fourth group of pigs were brought into the laboratory
facility at week 14 following a two week withholding period. One of the 10 pigs in Group 4
died five days before the due vaccination date, however CSF viral antigen was not detected
in its tissues by AC-ELISA. All surviving pigs remained healthy and no CSF virus
neutralising antibodies were detected at day 14 or day 0 of vaccination. The fifth group of
pigs were brought into the laboratory facility at week 18. Two of the pigs died, one prior to
vaccination and one pig on day 10 post vaccination with vaccine stored at 20 C. Spleen
tissue from both pigs tested negative in the AC-ELISA and no CSF neutralising antibodies
were detected on day 14 or day 0 of vaccination. All surviving pigs remained healthy.

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6.3.4 Vaccine storage at 4 C versus 20 C

Because of the inadvertent presence of CSF infection in the pigs brought into the laboratory
facility at weeks 6 and 10, results are only available for pigs vaccinated at months 4 and 5.
This corresponded to vaccine stored at 4 C for 3 and 4 months, respectively.
Of the Group 4 pigs vaccinated at month 4 of the experiment, three of the four pigs
vaccinated with vaccine stored at 20 C had strong neutralising antibody titres 28 days
post vaccination. Neutralising antibodies first appeared at day 10 post vaccination in one
pig (titre: 16), three pigs on day 21 (median: 32) and three pigs on day 28 (median: 44). For
the pig that returned negative results for the presence of CSF virus neutralising antibodies
at day 0 to 28, the serum collected on all days post vaccination did not produce typical cell
infection patterns in the NPLA. On all days and even at serum dilutions of 32, only a small
number of cell clusters across the well became infected with virus. One of the four pigs
vaccinated with vaccine stored at 4 C had a strong neutralising antibody titre at day 28
post vaccination (titre: 32). The three remaining pigs vaccinated with vaccine stored at 4 C
did not develop protective levels of CSF virus neutralising antibodies by day 28.
No significant difference between the vaccine storage temperatures could be demonstrated
at month 4 (Fisher exact test; p = 0.243). However, vaccine stored at 20 C was three
times more likely to produce neutralising antibodies to a titre of 32 or greater (Risk Ratio =
3, 95 % CI: 0.5 17.95). The control pig receiving no vaccine remained negative for the
presence of virus neutralising antibodies for the duration of the experiment. The NPLA
results of the pigs vaccinated at month 4 of the experiment are presented in Figure 6.4.
Of the Group 5 pigs vaccinated at month 5 of the experiment, one of the four pigs
vaccinated with vaccine stored at 20 C died as described in section 6.3.3. The three
remaining pigs did not have protective levels of CSF virus neutralising antibodies by day
28 post vaccination. Similarly, all four pigs vaccinated with vaccine stored at 4 C did not
have protective levels of neutralising antibody by day 28 post vaccination.

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No significant difference could be demonstrated between the two vaccine storage

temperatures at month 5 (Fisher exact test; p = 1.000). The NPLA results of the pigs
vaccinated at month 5 of the experiment are presented in Figure 6.5.

Reciprocal of serum dilution

48
40
32

a)

24
16
8
0
0

10

14

21

28

Days post vaccination

Reciprocal of serum dilution

48
40
32

b)

24
16
8
0
0

10

14

21

28

Days post vaccination

Figure 6.4 Serum neutralising antibodies to CSF virus post vaccination

with C-strain vaccine batch 05/2005 at month four of the experiment.
a) Vaccine stored at 20 C and b) Vaccine stored at 4 C.
Symbols correspond to individual vaccinated pigs. Solid horizontal bars
indicate median antibody titre and dashed lines represent titres of 12.5 and
32

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Reciprocal of serum dilution

48
40
32

a)

24
16
8
0

10

14

21

28

Days post vaccination

Reciprocal of serum dilution

48
40
32

b)

24
16
8
0
0

10

14

21

28

Days post vaccination

Figure 6.5 Serum neutralising antibodies to CSF virus post vaccination

with C-strain vaccine batch 05/2005 at month five of the experiment.
a) Vaccine stored at 20 C and b) Vaccine stored at 4 C.
Symbols correspond to individual vaccinated pigs.Solid horizontal bars
indicate median antibody titre and dashed lines represent titres of 12.5 and
32

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6.3.5 Estimated shelf life of vaccine stored at 20 C

To estimate the shelf life of the vaccine stored at 20 C, the response of pigs vaccinated at
month 4 and 5 were compared to the results obtained in the pre-trial assessment of the
vaccine using Fisher exact test. In the pre-trial assessment, three out of four vaccinated pigs
had positive neutralising antibody titres at day 28 post vaccination. Three of the four pigs
vaccinated at month 4 had positive neutralising antibody titres and one pig failed to respond
to the vaccine. No significant difference could be demonstrated between new vaccine and
vaccine stored at 20 C for four months (Fisher exact test, p = 0.571). No pigs vaccinated
with vaccine stored at 20 C for five months were positive for the presence of neutralising
antibodies 28 days post vaccination. No significant difference between new vaccine and
vaccine stored for five months (Fisher exact test, p = 0.114) could be demonstrated due to
small sample numbers; however a clear distinction between the two groups was evident.
Based on these results the estimated shelf-life of the vaccine should be no greater than four
months when stored under optimal conditions.

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6.4 Discussion and Conclusions

Ideally, the immunogenicity of the lapinised C-strain vaccine produced in Laos would have
been assessed by challenging vaccinated pigs seven days after vaccination according to the
OIE standards for assessing vaccine quality (OIE, 2004). Because of the constraint of not
working in a biologically secure environment the decision was made to assess the vaccines
stability by measuring its ability to elicit an immune response. The lack of biological
security was evident in the second and third group of pigs, both of which succumbed to
CSF infection.
The absence of neutralising antibodies following vaccination with a C-strain vaccine is not
necessarily indicative of a lack of protection (van Oirschot, 2003), however Terpstra and
Wensvoort (1988) demonstrated that a correlation existed between vaccine induced
antibody titre and protection against viral challenge. Terpstra and Wensvoort (1988)
showed that pigs with antibody titres less than 12.5 developed clinical signs, were able to
transmit virus and 3/11 pigs developed a lethal course of infection. Pigs with antibody titres
greater than or equal to 12.5 and less than 25 were protected against a lethal course of
infection but a proportion of pigs were still able to develop clinical signs and transmit virus
(Terpstra and Wensvoort, 1988). Antibody titres greater than or equal to 25 but less than 50
prevented a lethal course of infection, however a proportion of pigs still developed clinical
signs and shed virus, but transmission was not detected (Terpstra and Wensvoort, 1988).
Terpstra and Wensvoort (1988) concluded that for epidemiological purposes, C-strain
vaccine-induced antibody titres less than 32 were inadequate. Neutralising antibodies
following vaccination typically appear 2 3 weeks post vaccination and increase up to 4
12 weeks (Dahle and Liess, 1995; Terpstra et al. 1990; Terzic et al. 2003). Terzic and
others (2003) found that 90 % of pigs vaccinated with a tissue culture-produced C-strain
vaccine had antibody titres greater than or equal to 16 at day 28 post vaccination; and 40 %
had titres greater than or equal to 32.
The results of this research show clearly that the batch of the vaccine assessed in this study
elicited an adequate neutralising antibody response as demonstrated by the initial

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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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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.
General discussion

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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.

7.2 Smallholder pig production and CSF as a contributor to poor

performance
As stated in the introduction to this thesis, a number of authors have asserted that disease,
in particular CSF, is a major contributor to the poor performance of the smallholder pig
sector, and possibly even the major constraint to pig production in Laos. The results
presented in this thesis provide support to this assertion. However, the research does not
provide an answer to the question of whether CSF is the major constraint to smallholder pig
production. The most likely scenario is that CSF together with other diseases, and in
combination with a variety of other factors such as nutrition, management and breed,
culminates in overall poor performance of the smallholder pig sector.

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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.

7.3 The importance of improved CSF diagnostics

The choice of technique to be employed for the diagnosis of CSF must be well considered.
In general, the diagnostic methods used include RT-PCR, virus isolation, FAT and ACELISA (Van Oirschot, 1999). However, these methods have been developed for use under
strictly controlled conditions in well equipped laboratories. Factors such as sensitivity and
specificity of a particular test must be considered together with other equally important
factors. These factors include the conditions under which samples are collected, sample
storage capabilities and the duration of transit. The cost of performing a test and the turnaround time in processing a sample are also important factors that must be considered. In
Laos, the test currently used for CSF diagnosis is the AC-ELISA developed by Shannon et
al. (1993) and adapted to the laboratory in Laos by Blacksell (2001). A number of
constraints to the diagnosis of CSF in Laos have been previously identified. These include
minimal infrastructure for collection and submission of specimens, financial sustainability,

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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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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.

7.4 Vaccination as a control strategy

At present, vaccination is the only control measure undertaken to prevent the occurrence of
CSF in Laos. The current vaccination policies in place in Laos and the push from
development organisations to place the responsibility of vaccination into the hands of
smallholder producers is a high risk proposition with a substantial likelihood of failure.
Issues with vaccine delivery and the problems of vaccine stability discussed in this thesis
leave only a very small window of opportunity to deliver vaccine to the pig. Poor levels of
education, particularly in the area of infectious disease, mean that farmers are unaware of
what the term quality vaccine means, and are unsure of the potential for increased
production output if a quality vaccine were to be used. At the same time many provincial
and district livestock officers are unaware of vaccine quality control and quality assurance
measures needed to deliver a quality vaccine. As such, the small window of opportunity left
for the delivery of quality vaccine is not likely to be taken.
Blacksell (2001) pointed out that vaccination should only be considered one component of
an effective disease control program. Other measures including management of animals
entering a village and the introduction of strict controls on live animal movement and the
sale of animal products should be equally important components. While these suggestions
have great merit, the system currently in place in many villages means that these initiatives
would be slow to be implemented. For vaccination to play a significant role in the control
of CSF in Laos, its place in the control strategy should be well considered and vaccine
delivery well planned. It is the authors contention that the claimed shelf-life of the vaccine
be reduced substantially pending further assessment of vaccine stability and the
responsibility of vaccine delivery taken out of the hands of VVWs and farmers. The NVI
should be strongly encouraged to make smaller batches of vaccine on a more regular basis
and recall vaccine once expired. This would increase the unit cost per dose of vaccine;
however, this should not deter their pursuit of providing a quality product.

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7.5 Future research

Smallholder pig production and the impact of CSF
A good deal of research is required to understand better the smallholder pig production
sector in Laos. First and foremost, further research is required to understand better the
limitations to holding pigs to a finishing age that will maximise profits to the producers. A
high priority is also the development of strategies to improve nutrition and management.
An AusAID funded Forages and Livestock Systems project is currently working towards
this goal with very promising outcomes for pig producers; where labour costs are reduced
in conjunction with the supply of high quality feed stuffs. As in Kenya and the Philippines,
the impact of parasite infestation on the smallholder pig production unit needs to be further
explored with control measures implemented. It is highly likely that improved management
and nutrition as mentioned above would have a positive impact on this problem.
In terms of CSF and the smallholder pig sector, research is required to understand better the
epidemiology of CSF virus. Research directed at understanding the role of trade and in
particular middlemen traders in the movement of CSF virus between villages is required in
conjunction with more detailed investigation of outbreaks to assess the movement of pigs
out of a village once CSF has been identified. Furthermore, serological studies along the
lines of those described by Blacksell (2001) would provide valuable information with
regards to areas with greatest serological prevalence. Serological surveillance should
coincide with studies of disease incidence to identify those locations where virus is
circulating. The results of more detailed epidemiological research would enable the
development of targeted vaccination programs based on knowledge of where the virus is
and where it is likely moving to.

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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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

Chapter 7

Vaccination as a control measure

The obvious suggestion for future research in the area of vaccination is the development of
more stable vaccines able to withstand the rigours of the Lao environment. Such vaccines
would need to be thermostable to tolerate the tropical conditions, but ideally would also be
able to withstand frequent temperature fluctuations. The use of vectored vaccines such as
that described by Hammond et al. (2000; 2001; 2003) could be explored to determine if the
virus vector is less labile than CSF virus under Lao conditions. Other opportunities exist in
the development of thermostable inactivated vaccines. The detergent split vaccine
described by Dalsgaard and Overby (1976) showed great promise and continued research
into its effectiveness was probably discontinued due to the introduction of highly effective
modified live vaccines (de Smit, 2000). Further development of effective thermostable
vaccines would, however, require a substantial financial commitment from a vaccine
research and development organisation. The need to provide a cheap vaccine to enhance
affordability for the Lao farmer and the relatively small market means there would be little
chance of making a return on their investment.
The most feasible area of research would be the development and implementation of
targeted vaccination programs administered by national veterinary staff, combined with the
development of thorough vaccine quality control protocols. Initially, the program would be
small scale and selection of targeted areas based on sound epidemiological investigation of
CSF incidence and prevalence. The relative success of the program could be assessed with
continued monitoring of epidemiological indicators.

7.6 Concluding remarks

Classical swine fever is an important disease of pigs in many parts of the world, and no
more so than in Laos, a developing country with a smallholder farming sector in need of
support. It is hoped that the information presented in this thesis will provide veterinary staff
in Laos a means to better manage and control CSF so that direct benefit may be afforded to
the Lao pig farmer.

180

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202

Appendix

Appendix I: Reagents and solutions

AEC plus substrate
50 mM sodium acetate buffer (pH 5.0)

9.5 mL

N,N-dimethyl formamide

0.5 mL

3-amino-9-ethylcarbazole (AEC)

4 mg

30 % H2O2

5 L

Blocking Buffer for IMB antibody coupling (2% FSG in PBSA)

45 % Fish Skin Gelatin

4.5 mL

Sterile PBSA

95.5 mL

Proclin 300

100 L

Store at 4 C

Blocking Solution A (for AC-ELISA)

Carbonate buffer
5% Skim Milk Powder

Blocking Solution B (for AC-ELISA)

PBSA
5% SMP
5% Normal Goat Serum

Carbonate Buffer (CB)

Sodium carbonate

1.5 g

Sodium hydrogen carbonate

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

Conjugate Buffer for AC-ELISA

PBST
1% gelatin
1% skim milk powder
5% normal goat serum

Coupling Buffer for IMB-ELISA (50 mM Sodium acetate pH 5) 10x concentrated

Sodium acetate (anhydrous)

1.15 g

Sterile DiRO H2O

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

Conjugate and Mab Diluent Buffer for IMB-ELISA(0.5 % FSG in PBST)

45 % Fish Skin Gelatin

2.8 mL

PBST

235 mL

Glycerol

12.5 mL

Proclin 300

250 L

Store at 4 C

204

Appendix

Growth Media (GM)

10x EMEM concentrate

50 mL

Foetal bovine serum

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

Made up to 500 mL with sterile DiRO water

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

PBST (PBSA + 0.05 % Tween 20)

Tween 20

5 mL

10x PBSA

1L

DiRO H2O water

9L

205

Appendix

Sample diluent buffer for IMB-ELISA (10 % NGS in PBST)

PBST

180 mL

Normal goat serum (NGS)

20 mL

Proclin 300

200 L

Store at 4 C

Stopping solution (2N H2SO4)

Concentrated H2SO4

56 mL

DiRO H2O

944 mL

Storage Buffer for IMB-ELISA (0.5 % FSG in PBSA)

45 % Fish Skin Gelatin

1125 L

Sterile PBSA

99 mL

Proclin 300

100 L

Store at 4 C

TMB stock (42mM)

TMB

0.101 g

DMSO

10 mL

Store at 4 C away from light

TMB substrate
DiROH2O

7.2 mL

Citrate acetate buffer

0.8 mL

TMB stock

80 L

30 % H2O2

1 L

Make up immediately before use

206

Appendix

Appendix II: Inter-operator results

Table AI. Raw data for inter-operator agreement assessment
Operator 1

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

Table AI continued. Raw data for inter-operator agreement assessment

Operator 1

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

Appendix III: Reproductive performance correlations

Litters per sow per year .

2.0

R2 = 0.0011

1.6
1.2
0.8
0.4
0.0
0

20

40

60

80

100

Sow/Boar Ratio

Figure A2. Relationship between sow/boar ratio and litter-per-sow-per-year

Average litter size .

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