Investigation of Campylobacter jejuni and Campylobacter

Investigation of Campylobacter jejuni and

Campylobacter coli colonisation of commercial

free-range chickens

Pongthorn Pumtang-on

Doctor of Veterinary Medicine (DVM)

Master of Science (MSc)

Submitted to Charles Sturt University in fulfilment of the requirements

for the degree of Doctor of Philosophy

School of Biomedical Sciences

Faculty of Science

August, 2019

II

Table of contents

Certificate of Authorship........................................................................ IX

Acknowledgement .................................................................................... X

List of Tables .................................................................................... XI

List of Figures ................................................................................. XIII

List of Abbreviations ......................................................................... XVIII

Presentations and Publications ........................................................... XXI

Ethics Approval ................................................................................ XXII

Abstract ............................................................................... XXIII

Chapter 1 A review of Literature ............................................................. 1

1.1 Introduction .................................................................................. 1

1.2 Campylobacter spp. classification .................................................. 4

1.3 Impact of Campylobacter infections and Socio-economic cost ..... 4

1.4 Epidemiology of human Campylobacter infections ....................... 5

1.4.1 Surveillance and outbreaks in developed countries .............. 6

1.4.2 Surveillance and outbreaks in developing countries ............. 9

1.5 Epidemiology of Campylobacter in chickens ................................. 9

1.5.1 Prevalence of Campylobacter spp. in chicken products ....... 12

1.5.2 Prevalence of Campylobacter spp. in chicken flocks ............ 13

1.6 Campylobacter infections and immune responses in humans and

chickens ................................................................................ 14

1.6.1 Human Campylobacter spp. infections and immune

responses ......................................................................... 15

1.6.2 Campylobacter spp. colonisation in chickens and immune

responses ......................................................................... 18

1.7 Routes of Campylobacter transmission in chickens .................... 22

1.8 Prevention of Campylobacter colonisation in chicken farms ...... 25

1.9 Vaccine approaches ..................................................................... 26

1.9.1 Killed Whole-Campylobacter Cell Vaccine (WCV) ............. 27

III

1.9.2 Subunit and DNA vaccines ................................................... 30

1.9.3 Live attenuated vaccines ...................................................... 44

1.9.4 Development of a viral vectored vaccine against

Campylobacter ......................................................................... 53

1.10 Objectives and aims of this study ............................................... 56

Chapter 2 Campylobacter colonisation and transmission among

commercial free-range broiler farms in New South Wales, Australia .. 58

2.1 Introduction ................................................................................ 58

2.2 Materials and methods ................................................................ 60

2.2.1 Free-range meat chicken production ................................... 60

2.2.2 Free-range broiler farm practices ........................................ 60

2.2.3 Farm information and farm codes ....................................... 61

2.2.4 Determination of sample size ............................................... 65

2.2.5 Sample collection .................................................................. 66

2.2.6 Campylobacter spp. isolation ................................................ 68

2.2.7 Campylobacter jejuni and Campylobacter coli identification 70

2.2.8 Stock culture preparation and DNA extraction .................. 70

2.2.9 Campylobacter jejuni and Campylobacter coli confirmation

by PCR ......................................................................... 71

2.2.10 Genotyping ......................................................................... 73

2.2.11 DNA sequencing analysis ..................................................... 75

2.3 Results ................................................................................ 75

2.3.1 Isolation of Campylobacter jejuni and Campylobacter coli

from breeder farms ......................................................................... 76

2.3.2 Isolation of Campylobacter jejuni and Campylobacter coli

from broiler farms ......................................................................... 77

2.3.3 Genetic diversity of Campylobacter jejuni and Campylobacter

coli ......................................................................... 78

IV

2.3.4 Dynamics of Campylobacter colonisation in broiler flocks

(between flocks and the experiments) ............................................. 94

2.3.5 Similarity of Campylobacter jejuni and Campylobacter coli

isolates from breeders and their progeny (broilers) ..................... 105

2.4 Discussion .............................................................................. 108

Chapter 3 Identification and characterisation of Campylobacter genes ...

.................................................................................. 119

3.1 Introduction .............................................................................. 119

3.2 Materials and Methods ............................................................. 123

3.2.1 Campylobacter strains and culture conditions ................... 124

3.2.2 Genomic DNA extraction ................................................... 124

3.2.3 Campylobacter gene detection............................................. 124

3.2.4 Cloning, sequencing, and expression of Campylobacter jejuni

genes ....................................................................... 129

3.3 Results .............................................................................. 138

3.3.1 Gradient PCR analysis ....................................................... 138

3.3.2 Detection of katA, cadF, peb1A, cjaA, omp18, and flpA genes

in C. jejuni and C. coli isolates representing flaA-HRM clusters . 139

3.3.3 Nucleotide sequence and amino acid sequence analysis .... 140

3.3.4 Screening of transformed E. coli cells containing the ligated

pET SUMO plasmid ...................................................................... 149

3.3.5 Confirmation of the ligated pET SUMO plasmids ............ 152

3.3.6 Protein expression of pET SUMO carrying katA, peb1A,

cjaA, and cadF ....................................................................... 153

3.4 Discussion .............................................................................. 158

Chapter 4 Expression of Campylobacter genes and HVT vector vaccine

preparation .................................................................................. 165

4.1 Introduction .............................................................................. 165

4.2 Materials and Methods ............................................................. 167

V

4.2.1 Gene expression using the pcDNA™ 3.1 D/V5-His-TOPO®

vector ....................................................................... 167

4.2.2 Construction of recombinant pEGFP-C1 harbouring katA,

peb1A, cjaA, and cadF ................................................................... 174

4.2.3 Preparations of HVT virus and CEF ................................. 180

4.3 Results .............................................................................. 184

4.3.1 5´-CACCATG-overhanging insert gene amplicons for

directional cloning ....................................................................... 184

4.3.2 Screening of transformed E. coli cells harbouring the

recombinant TOPO plasmids ........................................................ 186

4.3.3 Restriction enzyme analysis of recombinant TOPO plasmids

....................................................................... 188

4.3.4 Sequence analysis of recombinant TOPO plasmids .......... 190

4.3.5 Eukaryotic expression of Campylobacter polypeptides ..... 193

4.3.6 Screening of the transformed E. coli containing the

recombinant pEGFP-C1 plasmids ................................................ 194

4.3.7 Analysis of the recombinant pEGFP-C1 containing the genes

....................................................................... 203

4.3.8 Evaluation of Campylobacter polypeptide expression as

EGFP fusions ....................................................................... 205

4.3.9 Western blot analyses ......................................................... 207

4.3.10 mRNA analysis ................................................................... 208

4.3.11 TCID50 analysis ................................................................. 209

4.3.12 Evaluation of HVT infections ............................................. 211

4.4 Discussion .............................................................................. 213

Chapter 5 General discussion ............................................................... 219

5.1 General aims and experimental chapter summaries................ 219

5.2 Major findings and limitations ................................................. 220

5.3 Future directions ....................................................................... 229

References .................................................................................. 232

VI

Appendices .................................................................................. 279

Appendix 1: Raw data of the notification rate of human gastroenteritis in

Australia from 2002 and 2018 ............................................................. 279

Appendix 2.1: MALDI-TOF protocol ........................................... 280

Appendix 2.2: Summary of clustering Campylobacter jejuni and

Campylobacter coli isolates on breeder farms based on MALDI-TOF,

PCR, flaA-HRM analysis and flaA amplicon sequencing ..................... 280

Appendix 2.2.1 A: Clustering of Campylobacter jejuni isolates from

BD–A ....................................................................... 280

Appendix 2.2.1 B: Clustering of Campylobacter coli isolates from

BD–A ....................................................................... 284


BD–B ....................................................................... 286


BD–B ....................................................................... 288


BD–C ....................................................................... 290


BD–C ....................................................................... 292


BD–F ....................................................................... 294


BD–F ....................................................................... 297


BD–G ....................................................................... 299


BD–G ....................................................................... 302


Campylobacter coli isolates from all broiler farms in experiments 1 and 2

based on MALDI-TOF, PCR, flaA-HRM analysis and flaA sequencing303

VII


free-range broiler farm 1 (FB1) in experiment 1 (Exp.1) .................. 303

Appendix 2.3.1 B: Clustering of Campylobacter jejuni isolates of


Appendix 2.3.1 C: Clustering of Campylobacter coli isolates of free-

range broiler farm 1 (FB1) in experiment 2 (Exp.2) ......................... 313





Appendix 2.3.2 C: Clustering of Campylobacter jejuni isolates from






Appendix 2.3.3 C: Clustering of Campylobacter jejuni isolates from


Appendix 3.1: Analysis of fliD primers and gradient temperature PCR ....

.................................................................................. 338

Appendix 3.2: PCR analysis of Campylobacter antigenic gene detection .

.................................................................................. 342

Appendix 3.3: Nucleotide sequence analysis ..................................... 347

Appendix 3.3.1: Nucleotide sequence of katA amplicons ................. 347

Appendix 3.3.2: Nucleotide sequence of cadF amplicons ................ 372

Appendix 3.3.3: Nucleotide sequence of peb1A amplicons .............. 391

Appendix 3.3.4: Nucleotide sequence of cjaA amplicons ................. 405

Appendix 3.4: The alignment of subsequence amino acids ................ 437

Appendix 3.4.1: KatA amino acid .................................................... 437

Appendix 3.4.2: CadF amino acid .................................................... 447

VIII

Appendix 3.4.3: Peb1A amino acid .................................................. 456

Appendix 3.4.4: CjaA amino acid .................................................... 462

Appendix 3.5: Nucleotide sequence analysis from pET SUMO ......... 468

Appendix 3.5.1: Nucleotide sequence analysis of pET SUMO-katA. 468

Appendix 3.5.2: Nucleotide sequence analysis of pET SUMO-cadF 472

Appendix 3.5.3: Nucleotide sequence analysis of pET SUMO-peb1A ....

.............................................................................. 477

Appendix 3.5.4: Nucleotide sequence analysis of pET SUMO-cjaA. 481

Appendix 3.6.: The alignment analysis of subsequent amino acids of the

ligated pET SUMO contained cadF or peb1A ......................................... 485

Appendix 3.6.1: The alignment analysis of subsequent amino acids

between pET SUMO-cadF and the original cadF gene ........................ 486

Appendix 3.6.2: The alignment analysis of subsequent amino acids

between pET SUMO-peb1A and the original peb1A gene .................... 487

Appendix 4.1: DNA sequencing analysis of the recombinant pEGFP-C1

plasmids .................................................................................. 489

Appendix 4.1.1: Nucleotide analysis of pEGFP-C1-katA plasmid .... 489

Appendix 4.1.2: Nucleotide analysis of pEGFP-C1-cadF plasmid ... 491

Appendix 4.1.3: The nucleotide analysis of pEGFP-C1-peb1A plasmid .

.............................................................................. 494

Appendix 4.1.4: The nucleotide analysis of pEGFP-C1-cjaA plasmid ....

.............................................................................. 497

Appendix 4.2: Maintenance media used for Vero and RK-13 (rabbit

kidney-13) cells .................................................................................. 500

Certificate of Authorship

Certificate of Authorship

I hereby declare that this submission is my own work and to the best of my knowledge and belief, understand that it contains no material previously published or written by another person, nor material which to a substantial extent has been accepted for the award of any other degree or diploma at Charles Sturt University or any other educational institution, except where due acknowledgement is made in the thesis [or dissertation, as appropriate]. Any contribution made to the research by colleagues with whom I have worked at Charles Sturt University or elsewhere during my candidature is fully acknowledged.

I agree that this thesis be accessible for the purpose of study and research in accordance with normal conditions established by the Executive Director, Library Services, Charles Sturt University or nominee, for the care, loan and reproduction of thesis, subject to confidentiality provisions as approved by the University.

Name

Date

jPongthorn Pumtang-on

joB/08/2019

IX

X

Acknowledgement

This thesis is indebted to many people for their support, advice, and

encouragement. Firstly, I would like to express my sincere gratitude to Dr

Thiru Vanniasinkam who is my principal supervisor, for her leading me to

take the journey to the PhD. Her professional guidance and warm support

steered me in the right direction and pace to gain confidence and complete

this study.

I must also offer my heartful thanks to Professor Timothy Mahony for his

supporting me to carry out experimental procedures at the Queensland

Alliance for Agriculture and Food Innovation (QAAFI). He did not only

provide me with very positive feedback and brilliant advice but also

encouraged me when I faced challenges in laboratory procedures and writing.

Professor Rodney Hill is another very important person for my PhD study.

He facilitated my study plan and helped me to move forward. I deeply

appreciate this wonderful supervisor and Head of School.

I would also like to acknowledge the technical support and assistance

received at the National Life Sciences Hub (NALSH), the Avian Laboratory

and the QAAFI, with special thanks to Ashleigh Van Oosterum, Therese

Moon, Lynn Matthews, Dr Toni Pavic, Dr Jeremy Chenu, Dr Elizabeth

Fowler, Sandy Jarrett, Dr Bing Zhang, and Dr Rebecca Ambrose.

Last but not least, my family have supported me emotionally, physically and

financially over the years. Many thanks to my superb parents, elder sister, and

partner. I am so grateful to have these irreplaceable people in my life. I might

have given up this PhD journey if without their understanding, acceptance

and support.

I am glad that I did not give up. And now I have even more courage and

confidence to move forward.

Thank everybody I made it.

XI

List of Tables

Table 1.1: Prevalence of Campylobacter contamination in broiler carcasses,

retail poultry meat and by-products among countries ................................ 12

Table 1.2: Prevalence of Campylobacter colonisation in broiler flocks

among countries ....................................................................................... 14

Table 1.3: Summary of studies of anti-Campylobacter jejuni vaccines

(killed vaccine) evaluated in animal models .............................................. 28

Table 1.4: Summary of studies of anti-Campylobacter jejuni vaccines

(subunit and DNA vaccines) evaluated in animal models .......................... 33

Table 1.5: Summary of studies of anti-Campylobacter jejuni vaccines (live

vector vaccine) evaluated in animal models .............................................. 47

Table 2.1: Summary of breeder farms and the supplied free-range broiler

sheds from the experiments 1 and 2 in this study....................................... 64

Table 2.2: The list of input parameters for sample size calculation ........... 65

Table 2.3: Sample types and total number(s) collected for Campylobacter

spp. isolation on breeder and broiler sheds over the course of this study.... 68

Table 2.4: Oligonucleotide primers used for identification of

Campylobacter spp., Campylobacter jejuni, and Campylobacter coli ........ 72

Table 2.5: Isolation rates of Campylobacter jejuni and Campylobacter coli

identified in faecal samples from breeder sheds ........................................ 77

Table 2.6: Summary of the isolation of Campylobacter jejuni and

Campylobacter coli from samples collected from broiler farms. ................ 78

Table 2.7: Clustering of Campylobacter jejuni isolates from breeder farms

and free-range broiler sheds using High Resolution Melt Polymerase Chain

Reaction targeting flaA gene (flaA-HRM PCR) analysis and flaA sequencing

................................................................................................................. 80

Table 2.8: Clustering of Campylobacter coli isolates from breeder farms

and free-range broiler sheds using High Resolution Melt Polymerase Chain


................................................................................................................. 83

Table 2.9: Classification of Campylobacter jejuni and Campylobacter coli

clusters isolated from breeder farms .......................................................... 88

Table 2.10: Classification of selected isolates of representative

Campylobacter jejuni and Campylobacter coli genotypes from broiler

farms, based on flaA-HRM clusters, flaA allele no. and MLST.................. 93

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Table 3.1: Information of Campylobacter genes used in Chapter 3 ......... 121

Table 3.2: Oligonucleotide primers used for the detection of genes in

Campylobacter jejuni and Campylobacter coli and summary of the

estimated sizes of the PCR product ......................................................... 126

Table 3.3: Summary of oligonucleotides of the gene primers used for

bacterial antigen expression .................................................................... 131

Table 3.4: The ligation reaction for pET SUMO vector and PCR amplicons

............................................................................................................... 132

Table 3.5: Information of restriction enzymes and buffer used ............... 135

Table 3.6: Oligonucleotide primer pairs used for DNA sequencing of the

pET SUMO plasmid containing Campylobacter genes............................ 135

Table 3.7: Summary of gradient PCR results using Campylobacter jejuni

and Campylobacter coli reference strains ................................................ 139

Table 3.8: PCR analysis of Campylobacter gene detections, using all

Campylobacter jejuni and Campylobacter coli isolates that represents the

flaA-HRM clusters identified from the breeder and broiler farms ............ 140

Table 4.1: Oligonucleotide primers used for gene amplification and

expression vector cloning ....................................................................... 168

Table 4.2: Cloning reaction for the TOPO® vector and gene amplicons .. 169


plasmid containing Campylobacter genes and the recombinant pEGFP-C1

plasmids ................................................................................................. 171

Table 4.4: Cloning reaction for the pEGFP-C1 vector and Campylobacter

ORF fragments ....................................................................................... 175

Table 4.5: Oligonucleotide primers and probes used for a duplex qPCR . 183

Table 4.6: Analysis of Ct values of each HVT dilution from a duplex qPCR

............................................................................................................... 210

Table 4.7: Appearance of CPE on the replicates of each dilution of HVT-

CEF ........................................................................................................ 211

XIII

List of Figures

Figure 1.1: Notification rates of bacterial foodborne disease in Australia

between 2002 and 2018. ............................................................................ 8

Figure 1.2: Mechanisms of C. jejuni infections and immune responses.

Source: Man (2011), Reuse License Number: 4756290941203,

authorised by Springer Nature............................................................... 17

Figure 2.1: Diagrams of free-range broiler sheds and their parent

breeder farms in the experiments 1 and 2. ............................................ 62

Figure 2.2: Schematic diagram of the dynamics of C. jejuni and C. coli

clusters identified on free-range broiler farm 1 (FB1) in the

experiments 1 and 2 ................................................................................ 96

Figure 2.3A: Schematic diagram of the dynamics of C. jejuni and C. coli

clusters identified on free-range broiler farm 2 (FB2) in the experiment

1 ............................................................................................................... 99

Figure 2.3B: Schematic diagram of the dynamics of C. jejuni and C. coli


2 ............................................................................................................. 100

Figure 2.4A: Schematic diagram of the dynamics of C. jejuni and C. coli


1 ............................................................................................................. 103

Figure 2.4B: Schematic diagram of the dynamics of C. jejuni and C. coli


2 ............................................................................................................. 104

Figure 2.5: Schematic diagram of similarity of C. jejuni and C. coli

clusters between breeder farms and their progeny in the experiments 1

(A) and 2 (B).......................................................................................... 107

Figure 3.1: Example of alignment analyses of the nucleotide sequences

and subsequent amino acid sequences generated from the katA

amplicon of the selected C. jejuni and C. coli clusters. ........................ 142


and subsequent amino acid sequences generated from the cadF


XIV


and subsequent amino acid sequences generated from the peb1A


Figure 3.4 Example of alignment analyses of the nucleotide sequences

and subsequent amino acid sequences generated from the cjaA


Figure 3.5: Example of agarose gel electrophoresis of the katA amplicon

generated from the pET SUMO plasmid contained katA using whole

cells from the transformed One Shot® Mach1™-T1 competent E. coli

colonies as DNA template in PCR reactions. ....................................... 149

Figure 3.6: Example of agarose gel electrophoresis of the cadF amplicon

generated from the pET SUMO plasmid contained cadF using whole



Figure 3.7: Example of agarose gel electrophoresis of the peb1A

amplicon generated from the pET SUMO plasmid contained peb1A

using whole cells from the transformed One Shot® Mach1™-T1

competent E. coli colonies as DNA template in PCR reactions. .......... 151

Figure 3.8: Example of agarose gel electrophoresis of the cjaA amplicon

generated from the pET SUMO plasmid contained cjaA using whole



Figure 3.9: Agarose gel electrophoresis of the digestion of pET SUMO

clones after digestion with HindIII and BamHI-HF (for the katA ORF)

or XhoI and BamHI-HF (for the cadF, peb1A and cjaA ORFs). ......... 152

Figure 3.10: Western blot analysis of the soluble protein fraction of

BL21 (DE3) E. coli cells containing pET SUMO/CAT (control), pET

SUMO-katA, and pET SUMO-cjaA plasmids at 0 h (T0) and 6 h (T6)

with and without after IPTG induction. .............................................. 155

Figure 3.11: Western blot analysis of the soluble protein fraction of

BL21 (DE3) E. coli cells containing the pET SUMO/CAT (control), pET

SUMO-cadF, and pET SUMO-peb1A plasmids at 0 h (T0) and 6 h (T6)

with and without after IPTG induction. .............................................. 157

Figure 4.1: Schematic representation of the BamHI-HF and XhoI

restriction sites located on the recombinant TOPO vector containing

XV

each inserted PCR amplicon from the gene of interest (green colour).

............................................................................................................... 171

Figure 4.2: Agarose gel electrophoresis of the PCR products containing

the katA and cadF ORFs used for cloning into the TOPO plasmid

vector. .................................................................................................... 185

Figure 4.3: Agarose gel electrophoresis of the PCR product containing

the cjaA ORF used for cloning into the TOPO plasmid vector. .......... 185


the peb1A ORF used for cloning into the TOPO plasmid vector. ....... 186

Figure 4.5: Example of agarose gel electrophoresis of the katA ORF

PCR products using whole cells from transformed One Shot® TOP10

chemically competent E. coli colonies as the DNA template. .............. 187

Figure 4.6: Example of agarose gel electrophoresis of the cadF ORF



Figure 4.7: Example of agarose gel electrophoresis of the peb1A ORF



Figure 4.8: Example of agarose gel electrophoresis of the cjaA ORF



Figure 4.9: Agarose gel electrophoresis analysis of the TOPO plasmids

after double digestion with BamHI-HF and XhoI and the original PCR

used in the cloning process. .................................................................. 189

Figure 4.10: Agarose gel electrophoresis of insert cadF ORF of cloned

TOPO plasmids after double digestion using BamHI-HF and XhoI and

the cadF PCR amplicon used in the cloning process. .......................... 190

Figure 4.11: Example of sequence alignment of the pcDNA3T-katA-1

compared with the original PCR amplicon and the TOPO vector alone.

............................................................................................................... 191

Figure 4.12: Example of sequence alignment of the pcDNA3T-cadF-4


............................................................................................................... 191

XVI

Figure 4.13: Example of sequence alignment of the pcDNA3T-peb1A-1


............................................................................................................... 192

Figure 4.14: Example of sequence alignment of the pcDNA3T-cjaA-1


............................................................................................................... 192

Figure 4.15: SDS-PAGE analysis of total proteins from the RK-13 cells

and the recombinant TOPO plasmids containing katA, cjaA, peb1A, or

cadF. ...................................................................................................... 193

Figure 4.16: The Western blot analysis of total cell protein extracts

from RK-13 cells transfected with plasmids encoding ORFS for katA,

cjaA, peb1A, and cadF. .......................................................................... 194

Figure 4.17: Example of agarose gel electrophoresis of PCR products

for the katA ORF fragment using whole cells from the transformed One

Shot® TOP10 E. coli colonies as a DNA template. ............................... 196


for the cadF ORF fragment using whole cells from the transformed One



for the peb1A ORF fragment using whole cells from the transformed

One Shot® TOP10 E. coli colonies as a DNA template. ....................... 200


for the cjaA ORF fragment using whole cells from the transformed One


Figure 4.21: Example of agarose gel electrophoresis of the inserted katA

ORF after HindIII and BamHI-HF digestion of the recombinant

pEGFP-C1 plasmids. ............................................................................ 203

Figure 4.22: Example of agarose electrophoresis of the inserted cadF


pEGFP-C1 plasmids. ............................................................................ 204

Figure 4.23: Example of agarose gel electrophoresis of the inserted

peb1A ORF after HindIII and BamHI-HF digestion of the recombinant

pEGFP-C1 plasmids. ............................................................................ 204

XVII

Figure 4.24: Example of agarose gel electrophoresis of the inserted cjaA


pEGFP-C1 plasmids. ............................................................................ 205

Figure 4.25: Transfection analysis of the recombinant pEGFP-C1

containing katA, cadF, peb1A, or cjaA ORFs in Vero cells visualised

under a fluorescent microscope with the 10 X objectives of at 48 h after

transfection. .......................................................................................... 206

Figure 4.26: Western blot analyses of VERO cell extracts from cells

transfected with pEGFPC1, pEGFPC1-KatA, pEGFPC1-CjaA,

pEGFPC1-Peb1A, and pEGFPC1-CadF expression with the exposure

time of 10 sec. ........................................................................................ 208

Figure 4.27: Agarose gel electrophoresis of the PCR amplicons

generated by PCR using from Vero cells transfected with pEGFP-C1,

pEGFP-C1-KatA, pEGFP-C1-CadF, pEGFP-C1-Peb1A, or pEGFP-C1-

CjaA. ..................................................................................................... 209

Figure 4.28: Quantification data for Cycling A. Orange for HVT

dilutions. ................................................................................................ 209

Figure 4.29: Samples of CPE lesions in CEF cells infected with HVT

and non-infected CEF cells were evaluated using an inverted

microscope at 7 days post-infection...................................................... 211

Figure 4.30 : Microscopic analysis of infected CEF cells with different

MOIs of HVT using an inverted microscope at 1 day after infection. 212


MOIs of HVT using an inverted microscope at 2 days post-infection. 213

Figure 4.32: Microscopic analysis of infected CEF cells with different

MOIs of HVT using an inverted microscope at 3 days post-infection. 213

XVIII

List of Abbreviations

ACMF Australian Chicken Meat Federation

CadF Campylobacter adhesin fibronectin

CCs Clonal Complexes

CDC Centres for Disease Control and Prevention

CDT Cytolethal Distending Toxin

CiaB Campylobacter invasion antigen B

CjaA Campylobacter antigen A

DC Dendritic cells

DNA Deoxyribonucleic acid

ECDC European Centre for Disease Prevention and Control

EFSA European Food Safety Authority

EU European Union

FAO Food and Agricultural Organization of the United Nations

FlaA Flagellin

FliD Flagella cap protein

FlpA Fibronectin-like protein A

FREPA Free Range Egg & Poultry Australia

GBS Guillain-Barrè syndrome

GC Guanine – cytosine

h hour(s)

HRM High-Resolution Melt

IL Interleukin

ISO International Organization for Standardisation

KatA Catalase protein

XIX

Kb Kilobase pairs

kDa Kilodalton

km kilometres

LPS lipopolysaccharide

LT E. coli heat-labile toxin

LTR Toll-like receptor

MALDI-TOF Matrix-assisted laser desorption ionisation time-of-flight

mCCDA Modified charcoal-cefoperazone-deoxycholate agar

MI Michigan

min minute(s)

MLST Multilocus sequence typing

MOI Multiplicity of infection

MOMP Major Outer Membrane Protein

mRNA messenger Ribosomal ribonucleic acid

NCBI National Center for Biotechnology Information, USA

NNDSS National Notifiable Diseases Surveillance System

NZ New Zealand

NZFSA New Zealand Food Safety Authority

NSW New South Wales

OIE Office International des Epizooties or World Organisation for

Animal Health

Omp18 Outer membrane protein 18

ORFs Open reading frames

PCR Polymerase chain reaction

PFGE Pulsed-field gel electrophoresis

XX

PorA Porin A protein

QLD Queensland

qPCR Quantitative polymerase chain reaction

RFLP Restriction fragment length polymorphism

rpm revolutions per minute

rRNA Ribosomal ribonucleic acid

sec seconds

spp. Species (multiple)

ST Sequence type

UK United Kingdom

USA United States of America

WCV Whole-Campylobacter cell vaccine

WHO World Health Organisation

SA South Australia

VIC Victoria

XXI

Presentations and Publications

Conference proceedings

Pumtang-on, P., Mahony, T. J., Hill, & Vanniasinkam, T. Campylobacter

transmission in Australian free-range broiler flocks. Australian Society for

Microbiology Annual Scientific Meeting 2018, Brisbane, Australia. Jul. 1-4,

2018

Pumtang-on, P., Mahony, T. J., Hill, R., Pavic, A., Chenu, J., &

Vanniasinkam, T. Campylobacter transmission in commercial poultry flocks

in Australia. In Proceedings of the Sixty-Sixth Western Poultry Disease

Conference: Facing the challenges for disease control in the current poultry

industry (pp. 159-161), Sacramento, USA, Mar. 20-22. 2017

Pumtang-on, P., Mahony, T. J., Hill, & Vanniasinkam, T. Antimicrobial

susceptibility of Campylobacter species in Australian commercial chicken

flocks. Australian Society for Microbiology Annual Scientific Meeting 2016,

Perth, Australia. Jul. 3-6, 2016.

Peer-reviewed publication

Pumtang-on, P., Mahony, T. J., Hill, R. A., Pavic, A, & Vanniasinkam, T.

(2020). Investigation of Campylobacter colonization in three Australian

commercial free-range broiler farms. Poultry Science. To be submitted in

April 2020.

XXII

Ethics Approval

All experiments with animals in this thesis were approved by the Charles Sturt

University Animal Care and Ethics Committee (Protocol number 15/057).

XXIII

Abstract

Campylobacter spp. are a leading cause of human gastroenteritis worldwide.

Most infections are caused by C. jejuni, followed by C. coli. Chickens are

considered a natural reservoir of Campylobacter spp. with most outbreaks

associated with the consumption of poultry products contaminated with these

bacteria at slaughter. Changing consumer awareness of issues associated with

animal welfare and well-being is driving a move away from intensive poultry

production to free-range systems. As a consequence of this recent shift, there

is a need for greater understanding of the epidemiology of C. jejuni and C.

coli colonisation and genetic diversity in relation to meat production on free-

range poultry farms in Australia. Currently, there is limited information on

this, and no specific strategies are applied on free-range farms to prevent

Campylobacter colonisation of poultry. This study aimed to address these

important knowledge gaps by investigating C. jejuni and C. coli colonisation

of chickens in commercial free-range broiler farms in New South Wales,

Australia through targeted isolation of C. jejuni and C. coli from chicken

faeces. Potential sources of C. jejuni and C. coli on farms were also

investigated by culturing these bacteria from samples taken from the

production environment. The genetic relatedness of isolates was assessed to

evaluate modes of transmission.

Fresh chicken faecal/caecal droppings (n=1,265) and environmental samples

(n=471) were collected from 18 free-range broiler flocks at weekly intervals

for three weeks after placement. Faecal/caecal droppings (n=120) were also

collected from the five breeder farms which supplied the broiler chicks.

Samples were used for Campylobacter isolation using standard methods (ISO

10272:2006). A combination of MALDI-TOF and PCR methods was used to

identify and speciate the C. jejuni and C. coli isolates. The C. jejuni and C.

coli isolates were genotyped with a flaA-HRM PCR assay to evaluate genetic

diversity within and between the sampled flocks. These data were also used

to evaluate potential sources of the C. jejuni and C. coli genotypes isolated

from chickens. C. jejuni and C. coli genes homologous which encode antigens

known to induce immune responses that significantly reduce Campylobacter

colonisation of chickens, were characterised by PCR amplification and DNA

sequencing.

XXIV

Campylobacter spp. were isolated from 526 (28%) samples in this study.

Forty-one and 26 flaA-HRM genotypes were identified for the C. jejuni

(n=406) and C. coli (n=145) isolates, respectively. C. jejuni and C. coli were

isolated from the production environment prior to chick placement. C. jejuni

and C. coli were first detected in free-range broiler faeces as early as 15 and

10 days of rearing, respectively. Typically, once a few broiler chicks in the

flock were positive for C. jejuni or C. coli, all sampled broilers within the

same flock were later found to be colonised with multiple genotypes of C.

jejuni and/or C. coli within one week. Very few C. jejuni and C. coli flaA-

HRM genotypes (n=3) were shared between free-range broiler chicks and

their parental breeder flocks.

Four genes, katA, cadF, peb1A and cjaA, encoding protective antigens were

found to be present in the genomes of the dominant C. jejuni and C. coli flaA-

HRM genotypes identified in this study. These conserved genes were

expressed in both prokaryotic and eukaryotic systems (Escherichia coli cells

and Vero cells). Different levels of protein expression in each system were

observed for each antigen. In E. coli cells, the expression of KatA was highest,

while Peb1A expression was lowest. In contrast, the expression of KatA was

the low and Peb1A was high in Vero cells.

The results of the current study have enhanced the understanding of the

timing, potential sources, and genetic diversity of Campylobacter

colonisation in free-range broiler farms. There was minimal evidence to

indicate the spread of Campylobacter by vertical transmission between layers

and broiler chickens. Rather, the results suggested some birds initially

acquired Campylobacter spp. from the production environment soon after

placement. Subsequently, horizontal transmission was the major route of

colonisation, leading to the rapid spread of Campylobacter within the free-

range broiler flocks in this study.

The results of this study suggest that any intervention in the commercial free-

range chicken meat production industry to prevent Campylobacter

transmission, such as enhanced biosecurity measures, would need to be

implemented early in the broiler growth stage, at the farm level, to be

effective. Vaccination was identified as a potential future control method, as

genes encoding antigens known to provide significant protection from

XXV

colonisation were characterised and shown to have high sequence identity, in

the isolates from this study. These antigens could underpin the future

development of a multivalent vaccine for C. jejuni and C. coli.

1

Chapter 1 A review of Literature

1.1 Introduction

Zoonotic Campylobacter infections linked to contaminated poultry products

are important causes of foodborne illnesses worldwide (CDC, 2010;

European Centre for Disease Prevention and Control [ECDC], 2010; WHO,

2012). In Australia, it has been one of the most common notified foodborne

infections (Liu et al., 2009; NNDSS, 2015). Campylobacter jejuni (C. jejuni)

followed by Campylobacter coli (C. coli) have been most frequently reported

as two common aetiological agents of human enteric infections (Gurtler et al.,

2005; Taylor et al., 2013; Weinberger et al., 2013).

Most outbreaks of Campylobacter induced gastrointestinal disease are

attributed to the consumption of contaminated poultry products (Kosa et al.,

2015; Mazick et al., 2006; NNDSS, 2019; O'Leary et al., 2009; Parry et al.,

2012; Stafford et al., 2007; Tompkins et al., 2013). Previous studies have

reported that C. jejuni isolated from chickens at slaughter and human patients

were genetically related (Kovanen et al., 2016; Sheppard et al., 2009). Hence,

food products originated from chickens are considered a major cause of

human campylobacteriosis (Black et al., 2006b; EFSA, 2014; Mughini Gras

et al., 2012; Sears et al., 2011; Wingstrand et al., 2006). Moreover, some other

foods, such as milk and water have been reported as sources of

Campylobacter contamination leading to human infections (Davis et al.,

2016; Heuvelink et al., 2009; Jakopanec et al., 2008).

To date, various interventions have shown effective results in the reduction

of Campylobacter contamination of carcasses such as UV radiation, the

combination of steam and ultrasonic treatment, acid treatment, and freezing

have been developed and integrated into chicken meat production systems

(Birk et al., 2010; Isohanni & Lyhs, 2009; Maziero & de Oliveira, 2010;

Musavian et al., 2014). However, none of these approaches has eliminated

Campylobacter contamination in retail products. Moreover, it has been

estimated that a reduction of C. jejuni loads in the intestines of chickens by

2–3 log10 Colony Forming Unit per gram (CFU/g) of caecal contents could

decrease the incidence of human campylobacteriosis more than 75%

(Romero-Barrios et al., 2013; Rosenquist et al., 2003). Similarly, a study by

2

Sears et al. (2011) has shown that the significant reduction of human

campylobacteriosis was related to the interventions aimed to reduce levels of

Campylobacter at chicken farms in New Zealand. Therefore, a reduction of

Campylobacter colonisation in chicken flocks could be one of the most

effective strategies to prevent the foodborne Campylobacter infection in

humans (EFSA, 2011).

Various interventions reported from overseas with the purpose of controlling

Campylobacter spp. colonisation have been developed and investigated at

farm-level such as biosecurity, feed additives, bacteriocin administration,

bacteriophages, probiotics and chicken genetic selection (Bailey et al., 2018;

Connerton et al., 2011; Ghareeb et al., 2012; Smith et al., 2016; Solis de los

Santos et al., 2009; Stern et al., 2008; Wagenaar et al., 2006). Even though

these strategies have shown significant reductions in the number of

Campylobacter excreted from the intestines of chickens, no effective

intervention has been approved to prevent the colonisation of this pathogen

at commercial farm-level. Recently, vaccines against Campylobacter spp.

colonisation have been developed and their efficacy evaluated as described in

section 1.9. This could be an alternative potential intervention in commercial

chicken farms due to concerns about public health and animal welfare.

However, no commercial vaccine is available for commercial chicken farms

at this moment.

In order to implement effective strategies to prevent Campylobacter spp.

colonisation of chickens, understanding the colonisation and transmission

patterns of Campylobacter spp. is necessary. Most recent studies

investigating Campylobacter spp. transmission within chickens on

commercial intensive farms have been conducted overseas and have resulted

in evidence to support the importance of horizontal transmission in

Campylobacter colonisation in chicken farms, whereas, no evidence of

vertical transmission was reported (Callicott et al., 2006; Ellis-Iversen et al.,

2012; Fonseca et al., 2006; Ingresa-Capaccioni et al., 2016; Messens et al.,

2009; O'Mahony et al., 2011; Sahin, Kobalka, et al., 2003). Even though some

studies found the same C. jejuni or C. coli isolated from breeders and their

progeny, the vertical transmission was not confirmed (Cox, Stern, et al.,

2002b; Idris et al., 2006).

3

Currently, the direction of chicken farming systems has gradually moved

forward to the adoption of free-range meat chicken production systems due

to consumer perceptions. In Australia, the trend of chicken meat consumption

has increased over recent years (Wong et al., 2015). On this note, free-range

broiler production has been expanding in Australia due to the increasing

preference of Australian consumers (Singh & Cowieson, 2013) based on the

perceptions of better welfare and meat quality, compared to those from the

intensive raring system (Brown et al., 2008). It is believed that horizontal

transmission is an important pathway in Campylobacter spp. colonisation of

free-range chickens since chickens are exposed to an outdoor environment

during the period of rearing, until slaughter. A consequence of this

management system is that free-range chickens may have more opportunities

to contract Campylobacter from their expanded access to the environment

(Nather et al., 2009). However, Campylobacter spp. colonisation and

transmission have been rarely investigated in free-range chicken farms. Of

further note, the presence of Campylobacter varied based on farm practices

(Smith et al., 2016), climate condition (EFSA, 2010) and geographic location

(Bi et al., 2008). Thus, applying international findings may not provide

effective strategies toward Campylobacter elimination in chicken meat

production systems in Australia.

In Australia, the transmission of Campylobacter spp. has not been studied in

free-range chicken farms. However, some studies have reported the

environment including drinking water, darkling beetles and litter as sources

of Campylobacter colonisation in intensive broiler flocks (Miflin et al., 2001;

Shanker et al., 1990).

Therefore, understanding of Campylobacter transmission in commercial free-

range chickens of Australia would assist in developing more effective

controls of Campylobacter colonisation in the commercial free-range chicken

farms to ensure product integrity. This chapter reviews general information

about Campylobacter spp., epidemiology of Campylobacter spp. in humans

and chickens, and controls of Campylobacter spp. colonisation in chicken

farms.

4

1.2 Campylobacter spp. classification

Campylobacter spp. are members of the family Campylobacteraceae. The

genus of Campylobacter includes 17 species and 6 subspecies (Silva et al.,

2011). Campylobacter spp. are gram-negative, non-spore forming bacteria.

They are mainly spiral-shaped, S-shaped, rod-shaped bacteria (Pead, 1979)

with a size of 0.2-0.5 μm length and a width of 0.2-0.9 μm (Pielsticker et al.,

2012). They have a polar flagellum at one or both ends (Balaban &

Hendrixson, 2011; OIE, 2008).

Most Campylobacter species prefer a micro-aerobic atmosphere (containing

3-10% oxygen) for growth (Haines et al., 2011). Some other species favour

an anaerobic environment (containing little or no oxygen) in spite of being

able to grow under micro-aerobic conditions as well (WHO, 2011). Because

Campylobacter spp. are intolerant to oxygen and dryness (Koene et al., 2004),

being left outside of the host gut can result in rapid death of the bacteria.

The temperature suitable for Campylobacter growth is 30-45°C, with an

optimum of 42°C (OIE, 2008; van Vliet & Ketley, 2001). According to the

preferred temperature for growth, Campylobacter are divided as non-

thermophiles (<37°C) and thermophiles (37-42°C). The survival rate at room

temperature (22 ± 2 °C) is poor. Campylobacter can survive for a short period

of time at a refrigeration temperature but die below 0°C (Maziero & de

Oliveira, 2010). Most Campylobacter spp. are heat sensitive and the cells are

destroyed at temperatures above 48°C. The optimum pH for Campylobacter

growth is 6.5-7.5. They cannot grow in culture media below pH 5 (Shaheen

et al., 2007).

C. jejuni is frequently reported as a major cause of human enteric infections,

followed by C. coli (Gurtler et al., 2005; Taylor et al., 2013; Weinberger et

al., 2013). C. jejuni can be divided into two subspecies, C. jejuni subsp. jejuni

and C. jejuni subsp. doylei (On, 2001). C. jejuni subspecies jejuni were more

commonly isolated than subspecies C. jejuni doylei (OIE, 2008).

1.3 Impact of Campylobacter infections and Socio-economic cost

Human campylobacteriosis has a significant impact on socio-economic costs

in many countries due to the impacts on public health (Buzby et al., 1997;

5

EFSA, 2011, 2014; Hall et al., 2005; Hoffmann et al., 2012; Kirk et al., 2008).

For example, Campylobacter infection involved in more than eight hundred

thousand cases was responsible for $1.7-1.9 billion in the USA (Hoffmann et

al., 2012; Scharff, 2012). Of these losses, $ 0.2-1.8 billion was annually

associated with Campylobacter-related Guillain-Barré syndrome (GBS)

(Buzby et al., 1997). It has been estimated the number of human

Campylobacter infections in the 27 European Union Member States (EU-27)

was approximately 9 million cases per annum (determined in the years

between 2005-2009) with a socio-economic cost of 2.4 billion Euro per year

(EFSA, 2011). Tam and O'Brien (2016) estimated that the annual healthcare

costs in the United Kingdom (UK) for Campylobacter foodborne disease and

Campylobacter‐related GBS were £50 and £1.26 million, respectively. In

2011, the illness costs related to Campylobacter spp. infections have been

estimated at €76 million per year in The Netherlands (Mangen et al., 2015).

In Switzerland, the healthcare costs related to Campylobacter infection were

estimated at €29–45 million per year (Schmutz et al., 2017; Schmutz et al.,

2016). In Australia, it has been estimated that approximately 5.4 million cases

of foodborne illness occurred per year which costs $1.2 billion Australian

Dollars (AUD) to the national economy annually (Hall et al., 2005; Kirk et

al., 2008). The notification rate of human campylobacteriosis has been the

leading notified bacterial foodborne infection over decades (NNDSS, 2015;

OzFoodNet, 2015), as shown in Figure 1.1 and Appendix 1.

1.4 Epidemiology of human Campylobacter infections

Campylobacter infection is an important cause of human gastroenteritis

(CDC, 2010; European Centre for Disease Prevention and Control [ECDC],

2010; WHO, 2012), especially in diarrhoea prevalent among children and

travellers (Allos, 2001).Campylobacter spp. infection has become one of the

most common causes of human gastroenteritis in both developed and

developing countries (CDC, 2010; WHO, 2012). The consumption of

contaminated chicken products with improper food preparation has been

associated with several outbreaks (Bergsma et al., 2007; CDC, 2013; Kosa et

al., 2015; Merritt et al., 2011; Mylius et al., 2007; Sheppard et al., 2009;

Stafford et al., 2007; Wei et al., 2015; Yoda & Uchimura, 2006; Yu et al.,

6

2010). Similarly, a study by Willis and Murray (1997) reported that more than

90% of clinical cases reported a history of consuming retail broiler meat.

Moreover, other transmissions included waterborne, contact with animals and

international travel have also been reported (Bless et al., 2014; Clark et al.,

2003; Evans et al., 2003; Jakopanec et al., 2008). C. jejuni and C. coli are

responsible for most symptomatic cases in humans (Gurtler et al., 2005;

Taylor et al., 2013; Weinberger et al., 2013).

1.4.1 Surveillance and outbreaks in developed countries

Campylobacter related gastroenteritis in humans has been reported as a

sporadic disease in developed countries (Effler et al., 2001; MacDonald et al.,

2015) since they have implemented better strategies in order to investigate

foodborne diseases such as national surveillance programmes and advanced

diagnostic systems and elimination-controls (EFSA, 2012, 2015; Schielke et

al., 2014). The incidence of human Campylobacter infections was related to

seasonality (EFSA, 2010, 2014; Jore et al., 2010; Patrick et al., 2004; Taylor

et al., 2013), geographic locations (Schielke et al., 2014), and age, and

population diversity (Nichols et al., 2012).

A higher incidence of human campylobacteriosis was found in summer than

in winter (EFSA, 2010; Huang et al., 2015; RefreÂgier-Petton et al., 2001;

Taylor et al., 2013). Young children (under 4 years) were most likely affected

by Campylobacter infections (OzFoodNet, 2010; Weinberger et al., 2013),

followed by young adults (20-29 years old) (Schielke et al., 2014). On the

other hand, children residing in rural areas were more likely to have sustained

Campylobacter infections (Schielke et al., 2014).

In the USA, Campylobacter infection was the second leading cause of

bacterial diarrhoea after Salmonella infection (Scallan et al., 2011). Over the

past two decades, the incidence of human Campylobacter infections

decreased from 23.6 (Samuel et al., 2004) to 13.8 cases per 100,000

population (Crim et al., 2014). Most outbreaks were associated with handling

and consuming contaminated foods such as chicken livers (CDC, 2013, 2015;

Department for Environment Food and Rural Affairs, 2013), dairy milk

(CDC, 2013; Heuvelink et al., 2009) and poultry meat (Taylor et al., 2013).

7

In the EU, this foodborne disease has become the most frequently reported

gastrointestinal bacterial disease since 2005 (EFSA, 2012, 2014, 2015). In

2012, more than 2,200 cases were confirmed as human campylobacteriosis

with the notification rate of 55.49 cases per 100,000 population (EFSA,

2014). In comparison between 2010 and 2013, the trend in notified human

campylobacteriosis cases in the EU has increased from 48.6 to 64.8 per

100,000 population, whereas, the fatality rate has decreased from 0.22 to 0.03

per 100,000 population (EFSA, 2012, 2015). As a member of the EU, the

incidence in Germany was as high as 80 cases per 100,000 population

(Schielke et al., 2014). In Switzerland, human Campylobacter infection is one

of the most common zoonotic foodborne infections with the notification rate

of 105 cases per 100,000 population (Schmutz et al., 2017; Schmutz et al.,

2016). Moreover, the notified rate of human Campylobacter infection has

been reported in some other developed countries as well. For example, a 3-

fold increase in the incidence rate of Campylobacter infections from 31.0 to

91.0 cases per 100,000 population within 10 years in Israel (Weinberger et

al., 2013). More than 2,000 cases of human Campylobacter infections have

been reported every year in Japan with the estimated notification rate of 100

cases per 100,000 population (Haruna et al., 2012; Kumagai et al., 2015).

In Australia, human campylobacteriosis has been commonly reported in most

states except New South Wales (Liu et al., 2009). The notification rate of

human Campylobacter infection has increased annually from 77.4 to 130.5

cases per 100,000 population between 2002 and 2018 (NNDSS, 2015, 2019;

OzFoodNet, 2010, 2015) as shown in Figure 1.1 and Appendix 1. It has been

estimated that the number of Campylobacter-related foodborne disease cases

was 277,000 per year (Stafford et al., 2007). A study by Dalton et al. (2004)

reported that 19 % of foodborne outbreaks in Australia between 1995 and

2000 had unknown aetiologies. While, it has been estimated that the

consumption of either cooked or uncooked chicken meat led to 30% of human

campylobacteriosis cases (Black et al., 2006a; Stafford et al., 2007).

8

Figure 1.1: Notification rates of bacterial foodborne disease in Australia between 2002 and 2018.

The graph shows that human campylobacteriosis has been the leading cause in Australia and the notification rate has increased over time

from 77.4 to 130.5 per 100,000 population. This chart is modified from Australia's notifiable diseases status, NNDSS annual report 1991-2018

(NNDSS, 2015; OzFoodNet, 2010, 2015) and http://www9.health.gov.au/cda/source/rpt_2.cfm.

http://www9.health.gov.au/cda/source/rpt_2.cfm

9

1.4.2 Surveillance and outbreaks in developing countries

Public health surveys at a national level are rarely conducted in developing

countries due to limited availability of funding and technology (Coker et al.,

2002; Meeyam et al., 2004; Zaidi et al., 2008). The estimated incidence rates

of foodborne diseases in these countries were generally based on the

laboratory outcomes of diarrhoea surveillance (Coker et al., 2002). The

species that were most often investigated included Salmonella spp.,

Escherichia coli, Vibrio spp. and Shigella spp. (Patricia & Azanza, 2006) but

not Campylobacter spp. Consequently, information about the epidemiology

of Campylobacter infections is sparse for these types of countries. The

prevalence of human Campylobacter infections was generally lower than

10% in developing countries such as Thailand (Meeyam et al., 2004),

Tanzania (Deogratias et al., 2014) and Uganda (Mshana et al., 2009).

In developing countries, Campylobacter infections were most often caused

by C. jejuni and were seen among children under 5 years of age (Adekunle et

al., 2009; Deogratias et al., 2014; van Vliet & Ketley, 2001). While, in

Nigeria, approximately 0.5% of children sustaining diarrhoea were identified

with C. coli infection (Adekunle et al., 2009). Paediatric death resulting from

Campylobacter infections was limited (WHO, 2011), although the serious

consequences rarely occurred in adults (Coker et al., 2002). Most outbreaks

were associated with poor sanitation, contact with animals, and/or human-to-

human transmission (Adekunle et al., 2009; Coker et al., 2002). Seasonality

has been reported as a risk factor of Campylobacter infections in some

developing countries (Rahimi et al., 2010; van Vliet & Ketley, 2001).

1.5 Epidemiology of Campylobacter in chickens

Chickens are considered as a natural host for Campylobacter spp. since the

microorganisms colonise the intestines of chickens without any clinical signs

(Dhillon et al., 2006; Wingstrand et al., 2006). C. jejuni isolated from human

Campylobacter infections and chickens were genetically related based on

molecular genotyping (Sheppard et al., 2009), and thus, chicken meat and

products could be considered as the main source of human

campylobacteriosis (Wingstrand et al., 2006). The detection of

10

Campylobacter spp. from chickens and products vary among countries due to

geographic differences (Bi et al., 2008), different farming systems (Hald et

al., 2015) and climate conditions (Jore et al., 2010; Jorgensen et al., 2011;

Kovats et al., 2005; O'Mahony et al., 2011; Patrick et al., 2004). In addition,

differences in the monitoring programmes, the type of samples collected, and

isolation methods used in studies may have influenced Campylobacter spp.

detection levels. The detection rates of Campylobacter spp. from the cloacal

swabs, faeces, and caecal contents were not statistically different when the

direct plating method on blood-free Modified Charcoal Cefoperazone

Deoxycholate agar (mCCDA) agar without pre-enrichment was used to

isolate these bacteria (Ingresa-Capaccioni et al., 2015). The enrichment of

boot swabs, caecal droppings and faecal samples prior to isolation did not

have a significant effect (difference) on Campylobacter detection (Vidal et

al., 2013). Samples from the environment such as air, feed, soils and litters

have also been examined to investigate the source of Campylobacter

infections in chicken farms in several studies and resulted in the environment

being identified as a potential source of Campylobacter in chickens

(Schroeder et al., 2014; Zhang et al., 2017). The types of selective media and

enrichment broth used have also affected the efficiency of Campylobacter

isolation and detection. For example, samples enriched with Exeter broth had

a higher sensitivity than the direct plating method for detecting

Campylobacter (Rodgers et al., 2017). In the same study, the Exeter broth

containing polymyxin B enhanced the detection of C. jejuni, whereas the

Bolton broth promoted C. coli detection (Rodgers et al., 2017). Although the

Preston Broth improved the recovery of stressed Campylobacter better than

Bolton broth and CampyFood Broth (CFB), there was no significant

difference compared with using the direct plating method (Ugarte-Ruiz et al.,

2015). Furthermore, the mCCDA agar was more sensitive than Skirrow’s agar

for Campylobacter detection (Bi et al., 2012). The selective chromogenic

medium CASA performed better isolation and detection of Campylobacter

than Campyfood agar (CFA) and mCCDA agars (Ugarte-Ruiz et al., 2015).

In addition, various methods including the culture-based (direct plating)

methods, PCR and immunoenzymatic assays have been developed and

evaluated for Campylobacter detections. The sensitivity and specificity of

those techniques varied. For C. jejuni and C. coli detection, the PCR had a

11

higher sensitivity than the immunoenzymatic and direct plating methods,

whereas the speciation of immunoenzymatic method was higher than the PCR

and direct plating methods (Zaghloul et al., 2012). The detection of C. jejuni

and C. coli using the direct plating method was less sensitive than that of PCR

in previous studies (Arnold et al., 2015; Bessede, Delcamp, et al., 2011; Singh

et al., 2011). In contrast, Lund et al. (2004) reported that the direct plating

technique with enrichment samples made no significant difference in the

detection of Campylobacter in chicken faecal samples, compared with a

quantitative reverse transcription PCR (RT-qPCR) assay. Of further note, the

different surveillance programs have been implemented for the detection of

Campylobacter among countries. In the 27 EU countries and Australia, the

surveillance programs focused on the incidence of Campylobacter in humans,

animals, and food, and Campylobacter detection was conducted mainly using

the conventional bacterial culture methods (ISO, 2006), followed by PCR

assays (EFSA, 2015; OzFoodNet, 2015). While in New Zealand, the

surveillance program utilising a molecular-based method (e.g. MLST) was

used not only to detect specific Campylobacter genotypes in clinical cases but

also to trace and identify the source of the infections which led to a 50%

reduction in the incidence of campylobacteriosis (Muellner et al., 2013).

Thus, comparing information on Campylobacter spp detection from one

study to another requires cautious evaluation of how the data was generated.

Recently, the direction of chicken farming has gradually moved forward

towards the free-range meat chicken production system due to consumer

perceptions of improved animal welfare and meat quality, compared to that

of the intensive system (Brown et al., 2008). Consequently, the demand for

free-range chicken products has increased in many countries such as the USA,

UK and France (Miele, 2011; Naald & Cameron, 2011; Sumner et al., 2011;

Walley et al., 2015). In Australia, the per capita consumption rate of chicken

meat (kg/person/year) has increased in Australia over past decades

(Australian Bureau of Agricultural and Resource Economics and Sciences-

ABARES, 2017, 2018; Wong et al., 2015) and the demand of free-range

chicken meat and the number of free-range chicken farms have also rapidly

increased in Australia as well (Erian & Phillips, 2017; Singh & Cowieson,

2013). However, the epidemiological information of Campylobacter spp. in

12

free-range chicken flocks is limited. Therefore, it is important to understand

the epidemiology of C. jejuni and C. coli in chickens on the free-range farms

and chicken products.

1.5.1 Prevalence of Campylobacter spp. in chicken products

Increased carriage of Campylobacter by poultry would likely lead to the

occurrence of outbreaks since Campylobacter spp. from chickens could

contaminate carcasses and products during processing at abattoirs (Herman

et al., 2003). Williams and Oyarzabal (2012) have suggested that chicken

products including skinless and boneless meats are particularly vulnerable to

Campylobacter contamination. The prevalence of Campylobacter

contamination varies among countries ranging between 51 and 93 %. Of

these, chicken products produced in Australia were found to have the highest

prevalence compared with other countries (Table 1.1). Seasonality and retail

types influence the level of Campylobacter contamination in retail chicken

meat and products. A study from Huang et al. (2015) has ported that a greater

contamination level of Campylobacter spp. in chicken carcasses was found in

the wet market, compared to supermarkets and the summer had the highest

incidence.

Table 1.1: Prevalence of Campylobacter contamination in broiler carcasses,

retail poultry meat and by-products among countries

Country Prevalence Reference

Australia 93% King and Adams (2008)

EU 75.8 EFSA (2010)

United Kingdom 87% Powell et al. (2012)

France 56% Denis et al. (2001)

Japan 60% Suzuki and Yamamoto (2009)

France 76% Guyard-Nicodeme et al. (2015)

Poland 87% Wieczorek et al. (2012)

Turkey 67% Pamuk and Akgun (2009)

Iran 56% Rahimi et al. (2010)

Trinidad 84% Rodrigo et al. (2005)

China 56% Huang et al. (2015)

Thailand 51% Chokboonmongkol et al. (2013)

13

1.5.2 Prevalence of Campylobacter spp. in chicken flocks

The detection of Campylobacter spp. in chickens varies depending upon

farming practices (Smith et al., 2016), farming systems (Hald et al., 2015),

climatic conditions (Jonsson et al., 2012; Jore et al., 2010; Jorgensen et al.,

2011; Kovats et al., 2005; O'Mahony et al., 2011; Patrick et al., 2004) and

geographical location (Bi et al., 2008). Most studies have been conducted in

conventional intensive farming systems. The detection rates of

Campylobacter spp. also varied among countries, ranging from 11 and 80%

(Table 1.2). In Australia, a recent survey conducted in the State of Western

Australia has shown that the prevalence of Campylobacter spp. in broiler

flocks was 64.4% (FSANZ, 2010), and this was lower than in some countries

such as France, United Kingdom, Spain, Trinidad, and Brazil (Table 1.2).

However, the information on prevalence in other Australian States is limited.

Based on the farm systems, a previous study suggested that closed-house farm

systems can prevent or delay Campylobacter spp. colonisation in chicken

flocks (Huat et al., 2010). Moreover, the effect of climate/seasonality on

Campylobacter spp. showed higher survival rates on rainy days compared

with sunny days (Hansson et al., 2007). Furthermore, a higher temperature is

related to a higher incidence of Campylobacter infections in both humans and

broiler flocks (Patrick et al., 2004). In contrast, a study by Berrang et al.

(2015) has disagreed with the seasonal effect on the prevalence of

Campylobacter in chicken flocks.

14

Table 1.2: Prevalence of Campylobacter colonisation in broiler flocks among

countries

Country Prevalence Reference

France 79% Powell et al. (2012)

Australia 64.4% FSANZ (2010)

United

Kingdom 76% Ingresa-Capaccioni et al. (2015)

Spain 71% Denis et al. (2001)

Latvia 56% Kovalenko et al. (2013)

Denmark 52% Hald et al. (2015)

Canada 50% Thibodeau et al. (2011)

Japan 47% Haruna et al. (2012)

Germany 44% Nather et al. (2009)

Belgium 39%

Herman et al. (2003); Messens et al.

(2009)

Iceland 27% Guerin et al. (2008)

Brazil 82% Kuana et al. (2008)

Trinidad 80% Rodrigo et al. (2005)

Turkey 17% Acik et al. (2013)

Thailand 11% Chokboonmongkol et al. (2013)

1.6 Campylobacter infections and immune responses in humans and

chickens

Campylobacter infection is a multifactorial process mediated by interactions

among bacteria, host epithelial cells, and the host immune responses (Aguilar

et al., 2014). However, the specifics of pathogenesis are not well understood

(Epps et al., 2013). The ability of Campylobacter colonisation varied due to

host factors (Pielsticker et al., 2012) and consequently resulted in the different

invasiveness, adherence and pro-inflammatory responses (Aguilar et al.,

2014). It has been demonstrated that C. jejuni equally invades human

epithelial cells and chicken epithelial cells (Byrne et al., 2007; Smith et al.,

2005). In contrast, Larson et al. (2008) have found that C. jejuni was less

invasive in LMH chicken hepatocellular carcinoma epithelial cells than in

INT 407 human embryonic epithelial cells. The levels of cytokine production

in human and chicken cells were also distinct after infection with C. jejuni

(Larson et al., 2008). Janssen et al. (2008) suggested the invasion of C. jejuni

is a potential factor which induced pro-inflammatory responses in the human

intestinal epithelial cells and led to blood-containing and inflammatory

15

diarrhoea. Because mechanisms of Campylobacter infections differed

between humans and chickens, a larger number of bacteria could invade

human epithelial cells (Young et al., 2007). Of further note, invasion,

adherence and immune responses also varied among chickens (Beery et al.,

1988; Larson et al., 2008; Li et al., 2008). Genetic diversity among bacterial

strains could be a variable contributing to different infection mechanisms and

this factor also affected the innate and specific immunity at the very early

phase of colonisation (Pielsticker et al., 2012). Hence, a better understanding

of Campylobacter pathogenic mechanisms could help to identify risk factors

for foodborne infection (Janssen et al., 2008).

1.6.1 Human Campylobacter spp. infections and immune responses

Campylobacter spp. are highly infectious pathogens with a low infectious

dose, causing human illnesses. Studies have demonstrated that ingestion of

C. jejuni between 500 and 800 microorganisms could develop into human

illness within a few days (Black et al., 1988; Robinson, 1981). Campylobacter

infection is not clinically distinguishable from other foodborne diseases. It is

generally self-limited causing diarrhoea (frequently bloody), abdominal

cramp, fever, nausea and vomiting (Allos, 2001). However, it can be related

to the development of reactive arthritis (Hannu et al., 2002; Pope, Krizova, et

al., 2007) and GBS (Buzby et al., 1997; Islam et al., 2012; McCarthy &

Giesecke, 2001; Nachamkin et al., 1998). The occurrence of Campylobacter

infection is generally sporadic or endemic and more prevalent in children and

young adults (van Vliet & Ketley, 2001; Zilbauer et al., 2008).

The colonisation mechanisms of human C. jejuni infections are not well

understood; however, some identified mechanisms have been previously

reviewed (Dasti et al., 2010; Wassenaar & Blaser, 1999). In vitro studies have

demonstrated that the mechanisms involving virulence factors include

motility (Guerry, 2007), chemotaxis (Korolik, 2019), adhesion and invasion

(Rubinchik et al., 2012), multidrug and bile resistance (Lin et al., 2003), iron

transport and regulation (Palyada et al., 2004), toxin production (Florin &

Antillon, 1992), and oxidative and nitrosative stress defence (Palyada et al.,

2009).

16

It has been shown that the microorganisms pass through the lumen of the human

intestine and mucosa layer of intestinal epithelial cells (IECs) after ingestion

(Watson & Galań, 2008; Young et al., 2007). The microorganisms start

colonising and replicating in lower parts of the intestines (Janssen et al.,

2008). Once Campylobacter spp. adhere IECs, it can be taken up and survives

in cytoplasmic vacuoles (Buelow et al., 2011). Some Campylobacter spp. can

translocate across the intestinal epithelium through a paracellular pathway

(Figure 1.2) (Man, 2011). Polar flagella of Campylobacter spp. attached to

host cells through flagellum-microvillus interactions in vitro (Konkel et al.,

2004; Man et al., 2010). The non-flagellated end is attached to the

neighbouring microvilli and cell-cell junction and breaks down the epithelial

barrier (Man et al., 2010).

Based on molecular mechanisms of C. jejuni infections, the interactions

between host intestinal epithelial cells and the microorganism and immune

responses occur at a crucial stage of disease development. Campylobacter can

adhere to and invade human intestinal epithelial cells with a process

dependent on or independent of a polar flagellum in vitro (Byrne et al., 2007;

Everest et al., 1992; Man et al., 2010). Some other C. jejuni proteins such as

fibronectin-binding protein CadF (Konkel et al., 1997; Monteville et al.,

2003), Peb1A (Pei & Blaser, 1993), fibronectin-like protein A (FlpA)

(Konkel et al., 2010), glycoprotein encoding protein encoded by Cj1496c

gene (Kakuda & DiRita, 2006) and O-linked glycan outer membrane proteins

(Mahdavi et al., 2014) have been suggested to adhere to and/or invade host

cells in vitro. C. jejuni can invade human intestinal epithelial cells in a

microtubule-dependent, actin-independent, microfilament- and caveolin-

dependent manner (Byrne et al., 2007; Oelschlaeger et al., 1993) or use of

Campylobacter invasion antigen B protein (ciaB) (Konkel, Kim, et al., 1999)

and survive within intestinal epithelial cells by avoiding delivery to

lysosomes (Watson & Galań, 2008). In vitro studies have demonstrated that

C. jejuni, C. coli and C. concisus invaded CaCo-2 cells via flagella binding

(Everest et al., 1992; Man et al., 2010). In addition to flagella-related

mechanisms, Campylobacter spp. use transcellular invasion to enter the

intercellular junctional space and cross the epithelium through a paracellular

route (Backert et al., 2013; Man et al., 2010). After Campylobacter spp.

17

penetrate intestinal epithelial cells, they can invade other organs via the

bloodstream as shown in Figure 1.2.

The increased human immune responses including innate, humoral and cell-

mediated immunities have been detected in humans infected by C. jejuni (van

Vliet & Ketley, 2001); however, the exact mechanism of the interactions

between human immune cells and C. jejuni infections are still unclear (Young

et al., 2007). Regarding immune responses during infection, Ó Croinin and

Backert (2012) have suggested that the invasion of C. jejuni affects the

change of intestinal epithelial cells and induces cytokine production and

resulted in inflammation. Numerous studies have reported the potential of C.

jejuni to enhance various points of initial immune responses in vitro. Hu et al.

(2006) has demonstrated that C. jejuni internalised and induced the

maturation of DC cells through lipooligosaccharide between 2 to 24 h after

inoculation in vitro and resulted in increased productions of NF-KB and innate

pro-inflammatory responses including IL-1, IL-6, IL-8, IL-10, gamma

interferon, tumour necrosis factor-alpha (TNF-α), and adaptive immunity IL-

12 by stimulating Th1 cells. In contrast, C. jejuni flagellin was unable to

Figure 1.2: Mechanisms of C. jejuni infections and immune responses.

Source: Man (2011), Reuse License Number: 4756290941203, authorised

by Springer Nature.

18

stimulate cytokine productions since it was poorly activated by TLR5 in

human intestinal epithelial cells (Watson & Galan, 2005). Other in vitro

studies have shown that C. jejuni infected human intestinal epithelial cells

produced pro-inflammatory responses and cytokines such as Interleukin-1 β

(IL-1 β), Interleukin-6 (IL-6), Interleukin-8 (IL-8) and intracellular nitric

oxide synthase as well (Smith et al., 2005). Similarly, studies have supported

that IL-8 and tumour necrosis factor-alpha (TNF-α) were produced in C.

jejuni-infected human intestinal tissue cultures and cell lines through Toll-

like receptor (LTR) signalling (Borrmann et al., 2007; MacCallum et al.,

2006; Watson & Galan, 2005; Zheng et al., 2008). In addition, IL-8 induced

dendritic cells (DC), macrophages and neutrophils were stimulated to high

levels of production of pro-inflammatory response proteins and cytokines

against C. jejuni (Sturm et al., 2005). Cytokines are relevant to attracting

leukocytes such as neutrophils and macrophages to the infected site

(MacCallum et al., 2006). It has been suggested that the stimulation of pro-

inflammatory cytokines could be associated with intestinal inflammation and

disease pathology (Jones et al., 2003). This is an important function in

diarrhoea and infection clearance (Zilbauer et al., 2008). Although gene

expression of interleukin-1 α (IL1α), IL-1β, IL6, IL-8, CXCL2, and CCL20

were considered as strong pro-inflammatory responses in human epithelial

cells after Campylobacter infection in vitro (Aguilar et al., 2014; Hu et al.,

2006; Jones et al., 2003), one of these studies showed that there was lower

expression of these genes and lower invasion levels in different cell cultures

especially those of animal origin (Aguilar et al., 2014).

1.6.2 Campylobacter spp. colonisation in chickens and immune

responses

Campylobacter spp. colonise the intestines of birds and other warm-blooded

animals (Carrique-Mas et al., 2014; El-Adawy et al., 2012; Gressler et al.,

2012; Kwan et al., 2008; Polzler et al., 2018; Workman et al., 2005).

Campylobacter spp. infection can cause enteritis and reproductive problems

in sheep and cattle (Gressler et al., 2012; Truyers et al., 2014), whereas, this

microorganism colonises the intestines of chickens without any clinical signs

(Beery et al., 1988; Hendrixson & DiRita, 2004; Newell & Fearnley, 2003).

19

Chickens are considered as natural hosts for Campylobacter spp. (Hermans

et al., 2011; Newell & Fearnley, 2003; Wingstrand et al., 2006). By contrast,

some recent findings suggested C. jejuni induces immune responses, affects

behaviours of chickens and possibly may be harmful to chickens with

pathogenic lesions (Colles et al., 2016; Connell et al., 2012; Humphrey et al.,

2014; Smith et al., 2008).

At farms, Campylobacter spp. commonly colonise chickens by 3 weeks of

age and then rapidly spread to other chickens within a flock and the

environment via faecal/caecal excretions (Ingresa-Capaccioni et al., 2015;

Kalupahana et al., 2013; Messens et al., 2009; Miflin et al., 2001; van Gerwe

et al., 2009). The detection rate peaks at the end of rearing (Ingresa-

Capaccioni et al., 2015). Existence of maternal immunity (Sahin, Luo, et al.,

2003; Sahin et al., 2001) and gut flora composition (Newell & Fearnley,

2003) could delay the detection of Campylobacter spp. in chicken flocks. On

the other hand, Stern et al. (1988) indicated that young chickens (1-3 days

post-hatch) were colonised by inoculation of a small number of C. jejuni (as

few as 35 CFU under experimental conditions.

Chickens take up Campylobacter spp. via the oral route, with the bacteria

moving through the intestine and colonising the caecum within 24 h (Coward

et al., 2008; Hendrixson & DiRita, 2004). The microorganism mainly

colonises the mucus overlying the epithelial cells of lower intestines,

especially the caecal mucosal crypts that are considered the primary site of

colonisation (Beery et al., 1988; Hendrixson & DiRita, 2004; Newell &

Fearnley, 2003). Campylobacter spp. proliferate with a high density of

bacterial loads of 108 to 109 CFU/g of caecal content (Sahin et al., 2002;

Thibodeau et al., 2011). However, Campylobacter may be isolated from some

internal organs such as liver and spleen (Knudsen et al., 2006; Lamb-Rosteski

et al., 2008; Williams et al., 2013) and induce lesions (Lemos et al., 2015;

Pielsticker et al., 2012), suggesting a possibility of C. jejuni invasion within

chickens. This agreement with an in vitro study conducted by Lamb-Rosteski

et al. (2008) who have reported that C. jejuni attached to and invaded

nontumorigenic canine intestinal epithelial cells via disrupted tight junctional

claudin-4, and resulted in increased transepithelial permeability. Li et al.

(2008) demonstrated that C. jejuni showed the capability of invasion into

20

chicken embryo intestinal cells (CEICs) in vitro, even though invasion

gradually decreased within 24 h.

Over past decades, several molecular studies aiming to investigate how C.

jejuni colonise and survive in chickens using in vivo and in vitro studies and

have suggested that pathogenesis of C. jejuni colonisation was associated

with bacterial virulence, host responses. However, the interactions between

chickens’ immune responses and C. jejuni colonisation, and mechanisms of

colonisation are not well understood. Campylobacter spp. have multiple

virulence factors involving pathogenicity as described in section 1.6.1. Of

these virulence factors, adhesion is a crucial step in colonisation and

infection. Several Campylobacter adhesin proteins play a role in adherence to

chicken epithelial cells as well as influence a significant role in colonisation

in chickens. For example, Campylobacter adhesins, such as CadF, Peb1A and

Flp, have been identified as important factors of Campylobacter colonisation

by promoting the interaction of this microorganism and host cells (Flanagan

et al., 2009; Konkel et al., 2010; Monteville et al., 2003). The CadF encoded

by cadF gene plays an important role in the binding of Campylobacter to

fibronectin (Fn) of host intestinal epithelial cells (Konkel et al., 1997; Konkel,

Gray, et al., 1999). The fibronectin-like protein A (Flp) encoded by flp gene

is another putative adhesin protein involved in the colonisation of C. jejuni

by binding to the Fn of host cells (Konkel et al., 2010). Campylobacter protein

A (CapA) encoded by the capA gene has been implicated in adhesion to

chicken epithelial cells (Flanagan et al., 2009). The Peb1A protein encoded

by peb1A gene is another factor involved in Campylobacter colonisation via

adherence and invasion of host cells (Ó Croinin & Backert, 2012; Oh et al.,

2017; Pei & Blaser, 1993; Pei et al., 1998; Pei et al., 1991). This gene encodes

a periplasmic binding protein (PEB1) which is similar to glutamine and

histidine-binding proteins from ABC transporter systems which are essential

for Campylobacter growth on dicarboxylic amino acid substrates (Leon-

Kempis Mdel et al., 2006).

Groups of outer membrane proteins (OMPs) are associated with adhesion and

invasion as well of Campylobacter spp. For example, the major outer

membrane protein (OMP) encoded by omp18 gene, is associated with the

maintenance of bacterial cell walls (Godlewska et al., 2009). The cjaA gene

21

encodes for the solute-binding protein (CjaA), which is a component of the

ABC transport system (Muller et al., 2005; Wyszynska et al., 2008). The fliD

gene encodes for a flagella cap protein (FliD) which is an essential element

in the assembly of the functional flagella and is a crucial factor for

colonisation by binding to host epithelial cells (Freitag et al., 2017). Catalase

encoded by katA gene is also involved in the oxidative stress defence which

is induced by oxygen exposure and converts hydrogen peroxide to water and

dioxygen (Garenaux et al., 2008; Palyada et al., 2009). Day et al. (2000)

suggested that the Campylobacter catalase is essential for the persistence and

growth of C. jejuni in macrophages. Moreover, other virulence genes have

been associated with colonisation in chicken gut (in vivo) such as Cj1496c

encoding glycoprotein (Kakuda & DiRita, 2006), docA encoding a

periplasmic cytochrome C peroxidase, docB encoding a methyl-accepting

chemotaxis protein, docC encoding another methyl-accepting chemotaxis

protein (Hendrixson & DiRita, 2004), pldA encoding a protein for

phospholipase function and dnaJ encoding heat shock protein (Ziprin et al.,

2001). In addition, Woodall et al. (2005) suggested that electron transport

regulation and metabolic pathways are alternative pathways which are

important during C. jejuni colonisation in chicks.

In terms of interactions between immune responses of chickens and C. jejuni

colonisation, it has remained unclear how C. jejuni triggers immune responses

in chickens (Lin, 2009). It is believed that C. jejuni colonises the intestine by

adhesion and invasion (Smith et al., 2005). Larson et al. (2008) reported that

C. jejuni attached to the intestinal epithelial cells lining the glandular crypts

in vivo but these invasive and pathogenic properties were not found.

However, some mild to strong inflammation of intestines were observed in

vivo (Humphrey et al., 2014), even this may not be associated with lack of

pathology in vivo as suggested by Smith et al. (2005). Immune responses such

as mucosa immunity and systemic immune response were triggered to fight

against Campylobacter in vivo (Humphrey et al., 2014). Some researchers

reported that C. jejuni did not attach via microvilli in vivo (Beery et al., 1988).

While others indicated that C. jejuni could invade and attach to epithelial cells

and stimulate inflammatory responses from macrophages and epithelial cells

(Byrne et al., 2007; Li et al., 2008; Newell & Fearnley, 2003). The interaction

22

between C. jejuni and intestinal cells stimulated formation of pro-

inflammatory cytokines such as chicken IL-8 orthologues (chCXCLi2 and

chCXCLi1), chemotaxis, IL-1β, IL-6 and inducible nitric oxide secretions

and induced heterophil migration in vitro (Larson et al., 2008; Li et al., 2008;

Smith et al., 2008). Like Salmonella infection, the expression of Toll-like

receptor (TLR) 4 and TLR21 genes were detected in chickens after challenge

with C. jejuni, whereas, that of TLR5 and TLR15 genes were not detected

(Shaughnessy et al., 2009). Similarly, cytokines including IL-1β, IL-6, IL-4,

IL-17A, interferon (IFN)-γ and anti-inflammatory IL-10 and transforming

growth factor (TGF)-β4 significantly increased after challenge with C. jejuni

in vivo (Reid et al., 2016). This indicated that C. jejuni can stimulate chicken

innate immune responses (Young et al., 2007). Interestingly, increasing TGF-

β4 was found in infected chickens (Reid et al., 2016) but not in infected cells

(Li et al., 2008). This suggests that in vivo studies may induce more

proinflammatory cytokines. Pielsticker et al. (2012) suggested that

Campylobacter colonisation in the chicken gut hardly induced any changes

in the number of CD4+ and CD8α+ T cells in a group of intraepithelial

lymphocytes (IELs). By contrast, it has been suggested that C. jejuni can

stimulate T cell-mediated activity after chickens were inoculated with C.

jejuni (Shaughnessy et al., 2011). However, the strength of Campylobacter-

specific antibody responses varied among chicken breeds since T helper

lymphocytes or Th (17) and Il-10 regulation were breed-dependent in vivo

(Humphrey et al., 2014; Reid et al., 2016). Of further note, C. jejuni not only

induced proinflammatory cytokines during infection in vitro, but it also

expresses some virulence genes such as ciaB, dnaJ and racR as well (Li et

al., 2008). This implies that expressions of some virulence genes by C. jejuni

could be related to the induction of immune responses.

1.7 Routes of Campylobacter transmission in chickens

At farms, Campylobacter spp. are commonly isolated from chickens after 14

days of age, and rapidly spread to the environment and other chickens in the

same flock, and thus, the chicken flock were positive with this bacteria within

a week of first detection (Ingresa-Capaccioni et al., 2015; Ingresa-Capaccioni

23

et al., 2016; Kalupahana et al., 2013; Messens et al., 2009; Miflin et al., 2001;

Thomrongsuwannakij et al., 2017; van Gerwe et al., 2009).

It has been suggested that chickens could acquire Campylobacter spp. from

the environment (horizontal transmission) (Cox et al., 2012; Ellis-Iversen et

al., 2012; Messens et al., 2009) and/or chicken parent flocks (vertical

transmission) (Cox, Stern, Musgrove, et al., 2002; Hiett et al., 2013; Idris et

al., 2006; Rossi et al., 2012). Epidemiological studies have demonstrated that

the horizontal transmission from the environment is an important source of

Campylobacter spp. primary colonisation in intensive chicken farms (Cox et

al., 2012; Ellis-Iversen et al., 2012; Messens et al., 2009; Workman et al.,

2008). Potential sources include the shed entrance, the anteroom (Ellis-

Iversen et al., 2012), litter (Newell & Fearnley, 2003), animal feed, drinking

water (Cox et al., 2012; Ellis-Iversen et al., 2012; Messens et al., 2009; Perez-

Boto et al., 2010), darkling beetles (Miflin et al., 2001), flies (Bahrndorff et

al., 2013), footwear (Cox et al., 2012), and other animals on the farm such as

cattle, dogs, rodents or invasive wild animals (Ellis-Iversen et al., 2012;

Workman et al., 2008).

In contrast, Campylobacter transmission from the parent chickens to their

progeny (vertical transmission) has remained controversial (Cox et al., 2012;

Marin et al., 2015; O'Mahony et al., 2011). This is based upon isolation of

Campylobacter spp. from various sites of the chicken reproductive system

including isthmus, magnum, shell gland, vagina, cloaca (Buhr et al., 2002;

Hannah et al., 2011) and semen (Buhr et al., 2005; Cox, Stern, Wilson, et al.,

2002). These bacteria have also been detected on paper tray liners of hatched

chicks descendent from Campylobacter positive breeder flocks (Byrd et al.,

2007), eggshells (Messelhausser et al., 2011), hatchery fluff (Hiett et al.,

2002) and caecal contents of day-old-chicks (Marin et al., 2015). In addition,

there is some evidence to show that Campylobacter positive hens passed the

bacteria on to their eggs, embryos (Hiett et al., 2013; Rossi et al., 2012), and

internal organs (Idris et al., 2006) as well as caecal contents of their one-day-

old chicks (Marin et al., 2015). On the other hand, some researchers have

argued that vertical transmission rarely occurs (Fonseca et al., 2006; Ingresa-

Capaccioni et al., 2016; Sahin, Kobalka, et al., 2003) or never occurs

(Callicott et al., 2008; Fonseca et al., 2006; O'Mahony et al., 2011; Shanker

24

et al., 1986) based on Campylobacter isolation with conventional methods. A

few studies have reported that Campylobacter can be recovered from hatchery

tray liners with low prevalence at 0.75% by culture methods (Byrd et al.,

2007). These could not clearly elucidate the occurrence of vertical

transmission. Some studies have reported that the identical strains of C. coli

and C. jejuni were isolated from breeders and their offspring (Cox, Stern, et

al., 2002a; Idris et al., 2006). Marin et al. (2015) reported that Campylobacter

spp. were detected in samples from Day-Old-Chick by real-time PCR.

Despite some studies showing evidence of potential vertical transmission,

whether or not vertical transmission occurs remains unknown.

Such information on current epidemiological studies, several risk factors

affecting Campylobacter spp. colonisation and transmission have been

primarily investigated in conventional intensive poultry production systems

but not the free-range farming system. Moreover, most studies on

Campylobacter transmission in poultry have been conducted overseas and

this may be less relevant to Australia. In Australia, some studies have

suggested that horizontal transmission via drinking water, darkling beetles

(Miflin et al., 2001) and litters (Shanker et al., 1990) play an important role

in intensive broiler flocks. In addition, there is no reported evidence to support

the occurrence of vertical transmission (Miflin et al., 2001; Shanker et al.,

1986; Shanker et al., 1990).

The most effective routes of C. jejuni and C. coli transmission in free-range

chicken flocks remains poorly understood. It is generally believed that

horizontal transmission plays a crucial role in Campylobacter spp.

colonisation of free-range broiler flocks since the chickens freely roam in the

environment outside of the barn, suggesting they may have multiple

exposures to these microorganisms from multiple sources in the environment

(Nather et al., 2009). However, there is limited information available on C.

jejuni and C. coli colonisation and transmission on free-range chicken farms

in Australia. Understanding the C. jejuni and C. coli colonisation and

transmission in free-range broiler farms is essential for the development of

effective intervention strategies to control the Campylobacter in this

expanding production system. Consequently, more effective control of

25

Campylobacter is warranted on the commercial free-range chicken farms to

ensure product integrity.

1.8 Prevention of Campylobacter colonisation in chicken farms

Contaminated chicken meat and chicken products were responsible for 30%

of Campylobacter infection in humans (Stafford et al., 2007). Numerous

strategies have been implemented to eliminate Campylobacter spp.

contamination on chicken carcasses in abattoirs (Allen et al., 2008; Magrinyà

et al., 2015; Musavian et al., 2014; Nair et al., 2014; Rasschaert et al., 2013;

Sharma et al., 2012). However, the prevalence of chicken products and

carcasses contaminated with Campylobacter spp. and the incidence of human

campylobacteriosis remain high as described in sections 1.4 and 1.5.1.

Moreover, it has been estimated that a reduction of C. jejuni count by 2–3

log10 CFU/g in chicken intestines could lead to a decline of human

campylobacteriosis by at least 75% (Romero-Barrios et al., 2013; Rosenquist

et al., 2003). Therefore, the control of Campylobacter spp. colonisation in

chicken at the farm level could be one of the most effective strategies to

reduce human Campylobacter spp. infections (EFSA, 2011; Friesema et al.,

2012; Van de Giessen et al., 1998; Wagenaar et al., 2006).

To date, various interventions with the purpose of controlling Campylobacter

spp. colonisation have been developed and investigated at intensive chicken

farming systems including reduction of exposure to environmental and on-

site pathogens (Newell et al., 2011; Wagenaar et al., 2006), administration of

antimicrobial or probiotic agents (Connerton et al., 2011; Ghareeb et al.,

2012; Janez & Loc-Carrillo, 2013; Lin, 2009; Loc Carrillo et al., 2005; Stern

et al., 2008), and application of bacteriophage (Loc Carrillo et al., 2005).

Reduction of exposure to environmental and on-site pathogens is critical to

farm biosecurity (Friesema et al., 2012; Ridley et al., 2011; Wagenaar et al.,

2006). Outcomes of the biosecurity methodologies could be difficult to assess

because pathways of Campylobacter transmission are not clearly understood

(Cox et al., 2012). Even though the probiotic administration prevented

Campylobacter colonisation by competitive exclusion and bacteriocin

production, this approach was effective only when the concentration of C.

26

jejuni infection is low (Stern et al., 2008). Antimicrobial resistance of bacteria

including Campylobacter is related to the prophylactic use of antibiotics in

animals (Moore et al., 2006). This implies antibiotic prophylaxis is not an

ideal approach to prevent Campylobacter colonisation in chickens (Gallay et

al., 2007; Lütticken et al., 2007). Of further note, a resistance to bacteriophage

also occurred after the beneficial virus was administered to chickens (Loc

Carrillo et al., 2005). Clearly, these interventions have yet to be effectively

proven in sustainably preventing Campylobacter colonisation of chicken

(Friesema et al., 2012; Gallay et al., 2007; Lin, 2009; Loc Carrillo et al., 2005;

Lütticken et al., 2007; Ridley et al., 2011; Robyn et al., 2015; Wagenaar et

al., 2006).

In addition, the level of Campylobacter spp. at farm-level varies depending

upon farming practices (Smith et al., 2016), climatic conditions (EFSA, 2010;

Jonsson et al., 2012) and geographical location (Bi et al., 2008). Therefore,

the same control interventions may not be effective if applied to other farms.

Therefore, it is important to understand the potential factors promoting the

colonisation by Campylobacter spp. of chickens in order to develop the most

effective intervention to prevent Campylobacter colonisation in chicken

farms.

1.9 Vaccine approaches

An effective vaccine against Campylobacter colonisation is one intervention

with considerable promise to improve the prevention of Campylobacter

colonisation in chickens. Vaccination is generally considered as an effective

tool to control infectious diseases in humans and animals (Lütticken et al.,

2007). In the past, bacterial vaccines used in animals, have mainly been based

on killed whole-cell vaccines or attenuated vaccines (Heldens et al., 2008).

Advanced progress in immunology, biochemistry, molecular biology,

proteomics and genomics has led to new strategies in vaccine developments

(Nascimento & Leite, 2012). Over past decades, various anti Campylobacter

vaccines containing several C. jejuni antigens have been investigated for their

immunogenicity and used in vaccine development in many forms including

killed-whole Campylobacter cells (Table 1.3), subunit vaccine (Table 1.4)

27

and recombinant based vector vaccines (Table 1.5). Despite years of research

on vaccines against Campylobacter, no commercially available vaccine can

prevent Campylobacter infection in chickens.

1.9.1 Killed Whole-Campylobacter Cell Vaccine (WCV)

WCVs demonstrated advantages including cost-effectiveness, safety,

induction of high immune responses especially for humoral and mucosal

immunity (Baqar, Bourgeois, et al., 1995; Prokhorova et al., 2006; Scott,

1997). The WCVs in most studies have been derived from C. jejuni 81-176

strain which was isolated from humans and has been used in studies and

evaluated for vaccine efficacy. Several types of WCVs have been used on

various animals to reduce Campylobacter colonisation via different routes of

administration (Table 1.3). Rhesus Monkeys orally administrated high doses

of WCVs alone and the combination of WCVs and E. coli heat-labile toxin

(LT) showed immune responses (IgA and IgG) and T-cell proliferation but

showed no significant effects on clinical signs (such as diarrhoea) (Baqar,

Bourgeois, et al., 1995). Similarly, mice administered high doses of WCVs

and E. coli heat-labile toxin (LT) also responded with high levels of induced

secretory immunoglobulin A (sIgA), and immunoglobulin G (IgG) responses

in serum and providing protection after challenge (Baqar, Applebee, et al.,

1995). In the ferret model, oral administration of WCVs with and without LT

led to high IgG responses and enhanced 80-100% homologous protection in

addition to partial cross-protection (Burr et al., 2005). Commercial broiler

chickens orally vaccinated with whole-killed cells of C. jejuni showed WCVs

enhanced humoral mediated immunity especially sIgA and 50% protection in

terms of Campylobacter colonisation at 50 days of age following challenge

with the homologous strain (Rice et al., 1997). Nevertheless, some

disadvantages of WCVs have been observed. Multiple doses of WCVs are

necessary (Walker, 2005). WCVs only partially reduced Campylobacter

colonisation (Burr et al., 2005; de Zoete et al., 2007).

28

Table 1.3: Summary of studies of anti-Campylobacter jejuni vaccines (killed vaccine) evaluated in animal models

Type

of

vaccine

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain Adjuvant

Immune

responses

Vaccine

efficacy

Whole-

cell

C.

jejuni

81-176

LT 1 BALB/c

Mice Oral

Challenge

study

C. jejuni

81-176

Induced specific

IgG and

secretory IgA

80% protection

Baqar,

Applebee, et al.

(1995)

Whole-

cell

C.

jejuni

81-176

LT 1 Rhesus

Monkey Oral

Infection

study Nil

Induced specific

IgG and

secretory IgA

N/A

Baqar,

Bourgeois, et

al. (1995)

Whole-

cell

C.

jejuni

81-176

LT 192G Ferret Oral Challenge

study

C. jejuni

81-176 or

C. jejuni

CGL7

Induced specific

IgG

80-100%

protections

with

homologous

and 40-89%

with partial

cross-protection

Burr et al.

(2005)

Note: 1E. coli heat-labile toxin (LT)

29

Table 1.3: Summary of studies of anti-Campylobacter jejuni vaccines (killed vaccine) evaluated in animal models (cont’)

Type

of

vaccine

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain Adjuvant

Immune

responses

Vaccine

efficacy

Whole-

cell

C.

jejuni

F1BCB

No

adjuvant

Commercial

broiler

Chicken

(Paterson

A/C)

Oral Challenge

study

C. jejuni

F1BCB

Induced

secretory IgA

3.6 log10

reduction

colonisation in

caecal

contents

Rice et al.

(1997)

Whole-

cell

C.

jejuni

F1BCB

25 µg LT

1

Commercial

broiler

Chicken

(Paterson

A/C)

Oral Challenge

study

C. jejuni

F1BCB

Induced

secretory IgA

1.1 log10

reduction

colonisation in

caecal

contents

Rice et al.

(1997)

Whole-

cell

C.

jejuni

F1BCB

50 µg of

LT 1

commercial

broiler

Chicken

(Paterson

A/C)

Oral Challenge

study

C. jejuni

F1BCB

Induced

secretory IgA

1.3 log10

reduction

colonisation in

caecal

contents

Rice et al.

(1997)

Note: 1E. coli heat-labile toxin (LT)

30

1.9.2 Subunit and DNA vaccines

Subunit vaccines, as the name suggestions contain only specific antigens or

epitopes of the pathogen of interest, formulated to stimulate immune

responses. Various proteinaceous, polypeptide, and DNA antigens have been

developed for anti-C. jejuni subunit and DNA vaccines which are summarised

in Table 1.4.

In mice, intranasal administration of a FlaA flagellin subunit vaccine with the

maltose-binding protein (MBP) or MBP-FlaA without the adjuvant LTR192G

induced serum IgG and sIgA responses with 84% efficacy against

Campylobacter colonisation (Lee et al., 1999). C. jejuni colonisation was

completely eliminated when mice were immunised with a high dose of MBP-

FlaA vaccine containing the adjuvant LTR192G via the intranasal route (Lee et

al., 1999).

Capsular polysaccharide (CPS) of C. jejuni has been used as an antigen

candidate against C. jejuni colonisation in various animal models. A study by

Bertolo et al. (2013) showed that mice subcutaneously administered with the

CPS of C. jejuni serotype HS15 (strain PG2887) conjugated with diphtheria

toxin mutant protein CRM197 (CPSHS15–CRM197) vaccine developed anti-

CPSHS15 antibodies. Monteiro et al. (2009) showed that subcutaneous

administrations of the CPS from two C. jejuni strains (81-176 and CG8486)

conjugated with CRM197 vaccines (CPS81-176- CRM197 and CPS CG8486-

CRM197) in mice not only elicited long-lasting immune responses but also

significantly reduced a homologous C. jejuni strain colonisation after

challenge. In monkeys (Aotus nancymaae), CPS81-176- CRM197 vaccine

provided full protection against diarrhoea, when C. jejuni 81-176 was

introduced through the orogastric pathway (Monteiro et al., 2009).

Capsular polysaccharide (CPS) of C. jejuni strain 81-176 conjugated with

CRM197 vaccine facilitated a 0.64 log10 CFU/g reduction in C. jejuni

colonisation of commercial Ross chickens after challenge with the same

strain, but maternal immunity may be a factor that could interfere with

serological responses (Hodgins et al., 2015). However, the predominant CPSs

in the circulating strains of the population of interest would need to be

31

considered in vaccine development due to the diversity of CPS structures

(Bertolo et al., 2013).

Intramuscular vaccination of specific-pathogen-free (SPF) Cornish Cross

broiler chickens with subunit vaccines containing recombinant CadF, FlaA,

FlpA, polypeptides or a CadF-FlaA-FlpA fusion protein elicited immune

responses and significantly reduced C. jejuni colonisation, whereas a

formulation containing Campylobacter multidrug efflux pumps protein

(CmeC) did not affect colonisation (Neal-McKinney et al., 2014). A study by

Zeng et al. (2010) has shown that use of 200 µg of CmeC subunit vaccine

formulated with 70 µg of modified E. coli heat-labile enterotoxin (mucosal

adjuvant LT-R192G; mLT) elicited IgG responses but failed to provide

protection against Campylobacter colonisation after challenge with the same

C. jejuni strain in chickens. Chickens subcutaneously immunised with subunit

vaccine containing FliD or SodB protein elicited immune responses and

significantly reduced C. jejuni colonisation in caecal contents, whereas,

subunit vaccines with FspA, CjaA, and CmeC failed to do so (Chintoan-Uta

et al., 2015; Chintoan-Uta et al., 2016).

Nanoparticle (NP) encapsulated outer membrane proteins (OMP) of C. jejuni

and/or only OMPs themselves administrated via subcutaneous injection

generated highly protective antibodies which effectively reduced C. jejuni

colonisation in caecal contents (Annamalai et al., 2013). Nevertheless, these

vaccines failed to elicit an immune response when being orally administrated

(Annamalai et al., 2013). Of further note was a C. jejuni prototype vaccine,

composed of a recombinant glutathione S-transferase (GST) fused to the

PorA polypeptide. Mice orally administrated with this type of vaccine and

adjuvant mLT demonstrated robust immune responses (IgG, IgM and IgA) in

serum and intestinal lavage samples (Islam et al., 2010). The level of

protection against heterologous C. jejuni colonisation varied from 29-42% in

a strain-dependent manner (Islam et al., 2010). In addition, Widders et al.

(1998) demonstrated that vaccine administration of WCV and FlaA protein

via intraperitoneal injection of commercial chickens elicited immune

responses and significantly reduced caecal colonisation (1.91 log10 reduction)

at 7 days after challenge with a homologous strain. In contrast, the same

immunogens administered intraperitoneally, following an oral boost, did not

32

show a significant reduction of C. jejuni colonisation in caeca. A recent study

by Liu et al. (2019) who developed and evaluated a prototype vaccine

containing CfrA and CmeC DNA, immunised via the in ovo route with and

without adjuvant (neutral lipid; incomplete Freund’s adjuvant) reported

insignificant immune responses and failure of protection of C. jejuni

colonisation were observed after challenge with a homologous strain.

Thus, subunit vaccines can be considered candidates to control

Campylobacter, since they generally elicit a high immune response.

However, the limitations of subunit vaccines have been observed. Typically,

multiple doses and/or adjuvant combinations are required since single doses

do not induce robust immune responses (Baxter, 2007; Lee et al., 1999;

Newell, 2001). While a multidose vaccine may prove useful in longer-lived

populations of chickens (e.g. layer and breeder flocks), they would have less

application in shorter-lived broiler flocks. Moreover, as studies have shown

that chickens are colonised early in the production cycle, to prevent

colonisation the opportunity to induce protective immune responses prior to

this may be limited. To date, administration routes commonly used in poultry

industries, including intranasal and oral routes, have not been able to prevent

Campylobacter colonisation (Meeusen et al., 2007; Zeng et al., 2010). To

avoid these complications, factors such as immunogenicity of antigens,

administration routes and dosage should be taken into account because these

can influence immune responses and Campylobacter colonisation outcomes.

33

Table 1.4: Summary of studies of anti-Campylobacter jejuni vaccines (subunit and DNA vaccines) evaluated in animal models

Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

rCadF,

rFlaA, rFlpA

C.

jejuni

F38011

GST 1

or 6X

HIS2

Montanide

ISA 70

VG

SPF

Cornish

cross

broilers

Intramuscular Challenge

study

C. jejuni

F38011

Induced

specific

IgY

4 to 7 log10

CFU/g of

reduction in

caecal

contents

Neal-

McKinney

et al.

(2014)

rCmeC

C.

jejuni

F38011

GST 1

or 6X

HIS2

Montanide

ISA 70

VG

SPF

Cornish

cross

broilers


study

C. jejuni

F38011

Induced

specific

IgY

Failure of the

significant

reduction in

caecal

contents

Neal-

McKinney

et al.

(2014)

Recombinant

CadF-FlaA-

FlpA

C.

jejuni

F38011

GST 1

or 6X

HIS2

Montanide

ISA 70

VG

SPF

Cornish

cross

broilers


study

C. jejuni

F38011

Induced

specific

IgY

3.7 log10

CFU/g of

reduction in

caecal

contents

Neal-

McKinney

et al.

(2014)

Note: 1 GST - N-terminal glutathione S-transferase, 2 6X HIS – C-terminal hexa-histidine

34

Table 1.4: Summary of studies of anti-Campylobacter jejuni vaccines (subunit and DNA vaccines) evaluated in animal models (cont’)

Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

Capsular

polysacchar

ide (CPS)

C.

jejuni

81-

176

Diphther

ia toxoid

CRM197

CpG or

Addavax

Ross

308

broiler

chicken

Subcutaneous Challenge

study

C. jejuni

81-176

Induced

specific

IgG

0.64 log10

CFU/g of

reduction in

caecal

contents

Hodgins et

al. (2015)

Whole-

killed cells

and Fla

protein

C.

jejuni

isolate

#v2

– Montanide

Comme

rcial

broiler

chicken

Intraperitoneal Challenge

study

C. jejuni

#V2

Induced

specific

IgG

1.91 log10

CFU/g of

reduction in

caecal

contents

Widders et

al. (1998)

Whole-

killed cells

and Fla

protein

C.

jejuni

isolate

#v2

– Montanide

Comme

rcial

broiler

chicken

Intraperitoneal

and oral

Challenge

study

C. jejuni

#V2

Induced

specific

IgG

Insignificant

reduction in

caecal

contents

Widders et

al. (1998)

35


Antigen(s)

Vaccine

Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

FliD

C.

jejuni

M1

GST 1 TiterMax

Gold

Specific-

pathogen

free White

Leghorn

Chicken


study

C. jejuni

M1

Induced

specific

IgY

2.0 log10

CFU/g of

reduction in

caecal

contents

Chintoan-

Uta et al.

(2016)

FspA

C.

jejuni

M1

GST 1 TiterMax

Gold

SPF White

Leghorn

Chicken


study

C. jejuni

M1

Induced

specific

IgY

Failure of

the

significant

reduction in

caecal

contents

Chintoan-

Uta et al.

(2016)

CjaA

C.

jejuni

M1

GST 1 TiterMax

Gold

SPF White

Leghorn

Chicken


study

C. jejuni

M1

Induced

specific

IgY

Failure of

the

significant

reduction in

caecal

contents

Chintoan-

Uta et al.

(2016)

Note: 1 GST - N-terminal glutathione S-transferase

36


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

SodB

C.

jejuni

M1

GST 1 TiterMax

Gold

SPF

White

Leghorn

Chickens


study

C. jejuni

M1

Induced

specific

IgY but not

secretory

IgA

1.3 log10

CFU/g of

reduction in

caecal

contents

Chintoan-

Uta et al.

(2015)

CjaA

C.

jejuni

M1

GST 1 TiterMax

Gold

SPF

White

Leghorn

Chickens


study

C. jejuni

M1

Induced

specific

IgY but not

secretory

IgA

Failure of the

significant

reduction in

caecal

contents

Chintoan-

Uta et al.

(2015)

CmeC

C.

jejuni

NCTC

11168

N-

terminal

Histidin

e-tagged

mucosal

adjuvant

LT-

R192G

Broiler

chickens Oral

Challenge

study

C. jejuni

NCTC

11168

Induced

specific

IgG

Failure of the

significant

reduction in

caecal

contents

Zeng et al.

(2010)


37


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

CmeC

C.

jejuni

NCTC

11168

N-

terminal

Histidine-

tagged

mucosal

adjuvant

LT-R192G

or Freund’s

incomplete

adjuvant

Broiler

chickens

Oral or

subcutaneous

Challenge

study

C. jejuni

NCTC

11168

Induced

specific

IgG but

not

secretory

IgA

Failure of

the

significant

reduction

in caecal

contents

Zeng et al.

(2010)

FlaA

C.

jejuni

ALM-

80

pCAGGS Chitosan

White

Leghorn

Chicken

Intranasal Challenge

study

C. jejuni

ALM-80

Induced

specific

IgG and

secretory

IgA

2 to 3 log10

CFU/g of

reduction

in caecal

contents

Huang et

al. (2010)

PorA

C.

jejuni

C31

GST 1 LT R192G BALB/c

Mice Oral

Challenge

study

C. jejuni

strains C31,

48 (O:19),

75 (O:3) and

111 (O:1,44)

Induced

specific

IgG, IgM

and

secretory

IgA

29-42 % of

disease

protection

Islam et

al. (2010)


38


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein

Adjuva

nt

Immune

responses

Vaccine

efficacy

Capsular

polysacchar

ide (CPS)

C.

jejuni

81-176

CRM197 3 N/A BALB/c

Mice


study

C. jejuni

81-176

Induced

specific IgG,

IgM and

secretory IgA

Significantl

y reduced

illness signs

Monteiro et

al. (2009)

CPS

C.

jejuni

CG848

6

CRM197 3 N/A

BALB/c

Mice Subcutaneous

Challenge

study

C. jejuni

CG8486

Induced

specific IgG,

IgM and

secretory IgA

Significantl

y reduced

illness signs

Monteiro et

al. (2009)

CPS

C.

jejuni

81-176

CRM197 3

Ringer’s

solution

and

combine

d with

alum

Monkey Subcutaneous Challenge

study

C. jejuni

81-176

Induced

specific IgG,

IgM and

secretory IgA

No illness

signs

Monteiro et

al. (2009)

CPS

C.

jejuni

ATCC4

442

CRM197 3

Alhydro

gel

BALB/c

Mice Subcutaneous

Infection

study N/A

Specific

antibodies

were detected

on

immunoblot

N/A Bertolo et

al. (2013)

Note: 3 Diphtheria toxin mutants

39


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

FlaA C. coli

VC 176 MBP 4

LT

R192G

BALB/c

Mice Intranasal

Challenge

study

C. jejuni

81-176

Induced

specific

IgG and

secretory

IgA

81.1 % of

disease

protection

and 84.0%

of reduction

in intestinal

colonisation,

respectively

with a dose

of 50 mg of

MBP-FlaA

and

LTR192G.

Lee et al.

(1999)

FlaA C. jejuni

81-176 – CpG

Ross

PM3

chickens


study

C. jejuni

81-176

Induced

specific

IgY but

not

secretory

IgA

No

significant

reduction in

caecal

contents

Meunier

et al.

(2018)

Note: 4 Maltose-binding protein

40


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

FlaA

C.

jejuni

81-176

–

CpG and

Montainde

ISA70

Ross

PM3

chickens


study

C. jejuni

81-176

Induced

specific

IgY but

not

secretory

IgA

No

significant

reduction

in caecal

contents

Meunier

et al.

(2018)

FlaA

C.

jejuni

81-176

–

CpG and

Montainde

ISA70

SPF

Leghorn

chickens


study

C. jejuni

81-176

Induced

specific

IgY but

not

secretory

IgA

8 log10

CFU/g of

reduction

in caecal

contents

Meunier

et al.

(2018)

rCjaA

C.

jejuni

M1

His-

tagged TiterMax®

SPF

Light

Sussex

Chicken


study

C. jejuni

M1

Induced

specific

IgY

1.91 and

2.3 log10

CFU/g of

reduction

in caecal

contents

Buckley

et al.

(2010)

41


Antigen(s)

Vaccine

Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

rCjaA

C.

jejuni

M1

His-

tagged TiterMax®

SPF

Light

Sussex

Chicken

s


study

C. jejuni

M1

Induced

specific

IgY

1.57 and 3.03

log10 CFU/g of

reduction in

caecal contents

Buckley et

al. (2010)

CjaA and

CjaD

C.

jejuni

NCTC

11168

GEM 5 – Chicken

s

Oral and

Subcutaneous

Challenge

study

C. jejuni

12/2

Induced

specific

IgG

Failure of the

significant

reduction in

caecal contents

Kobiereck

a,

Wyszynsk

a, et al.

(2016)

CjaA and

CjaD

C.

jejuni

NCTC

11168

GEM 5

or

liopsom

e

– Chicken

s In ovo

Challenge

study

C. jejuni

12/2

Induced

specific

IgG and

secretory

IgA

1 (with GEM)

and 2 (with

liposome)

log10 CFU/g of

reduction in

caecal contents

Kobiereck

a,

Wyszynsk

a, et al.

(2016)

Note: 5 Gram-positive Enhancer Matrix

42


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

Outer

membrane

vesicles

(OMVs)

C. jejuni 81-

176 – – Chickens In ovo

Challenge

study

C. jejuni

12

Induced

specific

IgG and

secretory

IgA

2 log10

CFU/g of

reduction

in caecal

contents

Godlewska

et al. (2016)

OMVs and

CjaA

C. jejuni 81-

176 – – Chickens In ovo

Challenge

study

C. jejuni

12

Induced

specific

IgY and

secretory

IgA

1 log10

CFU/g of

reduction

in caecal

contents

Godlewska

et al. (2016)

N-glycan C. jejuni

NCTC11168

ToxC

and HIS Freund

SPF

Leghorn

chickens


study

C. jejuni

81-176

Induced

specific

IgY

4 to 7

log10

CFU/g of

reduction

in caecal

contents

Nothaft et

al. (2016)

43


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain

Tagged

protein Adjuvant

Immune

responses

Vaccine

efficacy

cfrA and

CmeC

C.

jejuni

NCTC

11168

N-

terminal

Histidin

e-

tagged

–

Cobb

500

broiler

breeder

hens

In ovo Challenge

study

C. jejuni

NCTC

11168

Insignificantl

y induced

specific IgG

and secretory

IgA

Failure of the

significant

reduction in

caecal contents

Liu et al.

(2019)

cfrA and

CmeC

C.

jejuni

NCTC

11168

N-

terminal

Histidin

e-

tagged

neutral

lipid

(incomple

te

Freund’s

adjuvant)

Cobb

500

broiler

breeder

hens

In ovo Challenge

study

C. jejuni

NCTC

11168

Insignificantl

y induced

specific IgG

and secretory

IgA

Failure of the

significant

reduction in

caecal contents

Liu et al.

(2019)

Outer

membrane

proteins

(OMP)

C.

jejuni

81-176

Nanopa

rticle

(NP)

N/A Chicke

ns Subcutaneous

Challenge

study

C. jejuni

81-176

Induced

specific IgY

and secretory

IgA

8 log10 CFU/g

of reduction in

caecal contents

Annamala

i et al.

(2013)

OMP

C.

jejuni

81-176

Nanopa

rticle

(NP)

N/A Chicke

ns Oral

Challenge

study

C. jejuni

81-176

Induced

specific IgY

and secretory

IgA

1.5 to 3 log10

CFU/g of

reduction in

caecal contents

Annamala

i et al.

(2013)

44

1.9.3 Live attenuated vaccines

Live-attenuated vaccines can induce prolonged humoral and cellular

immunity. Traditional live attenuated vaccines have been successfully used

to protect against various avian infectious diseases such as Mycoplasma

gallisepticum infections (Javed et al., 2005; Papazisi et al., 2002),

salmonellosis (Babu et al., 2003; Pei et al., 2014), Newcastle disease

(Corbanie et al., 2007; Rauw et al., 2009), Infectious Bronchitis disease

(Deville et al., 2012; Geerligs et al., 2011), and coccidiosis (Price, 2012).

However, a vaccine for Campylobacter using this technology has not been

successful due to this pathogen’s genomic and phenotypic instability (Ridley,

Toszeghy, et al., 2008).

A type of attenuated Campylobacter vaccine has been used to prevent the

colonisation of a homologous Campylobacter strain in rabbits (Guerry et al.,

1994). However, the administration of the live-attenuated vaccine can result

in unfavourable consequences such as reversion to virulence and poor

stability (Baxter, 2007). These factors made further development of a live-

attenuated C. jejuni vaccine quite difficult (Albert, 2014). Therefore, live

recombinant vector vaccines have been developed as a safer alternative for

the control of infectious diseases (Nascimento & Leite, 2012). Live

recombinant vector vaccines are able to stimulate an immune response similar

to that caused by natural infections (Nascimento & Leite, 2012).

Consequently, live recombinant vector vaccine contributed to better

immunization (Ndi et al., 2013).

Currently, several studies have developed various live-attenuated vector

vaccines expressing various antigens and have evaluated vaccine efficacies.

However, there were inconsistent outcomes depending on vector types, route

of administrations, challenge strains, and animal models (Table 1.5). For

example, an oral vaccine against Eimeria tenella infection in chickens was

used to deliver the Campylobacter CjaA antigen. This vaccine regimen

provided protective immune responses, significantly reducing the

colonisation of vaccinated chickens approximately 1 log10 CFU/g at 42 days

of age or 14 days post-challenge with C. jejuni (Clark et al., 2012). Likewise,

oral vaccination of SPF Light Sussex chickens with an attenuated S.

Typhimurium expressing CjaA protein fusion to the C-terminus of tetanus

45

toxin (TetC) showed strong immune responses (IgY and IgA) and a

significant reduction (1.4 log10) in C. jejuni colonisation following challenge

with a homologous strain (Buckley et al., 2010). By contrast, an earlier study

by Wyszynska et al. (2004) has demonstrated that chickens orally immunised

with non-virulent Salmonella vector vaccine expressing CjaA showed IgG

and mucosal IgA responses in serum and 6 log10 CFU/g reduction in caecal

content after challenge with heterologous C. jejuni strains. In contrast,

Laniewski et al. (2014) utilised S. Typhimurium ᵡ9718 strain expressing CjaA

to induce immune responses in chickens; however, it was an insignificant

reduction of C. jejuni colonisation after challenge with a different C. jejuni

strain.

A study by Layton et al. (2011) has shown that an oral vaccination of broiler

chickens (Cobb-500) with a live-attenuated Salmonella Enteritidis (S.

Enteritidis) expressing Omp18 (CjaD), CjaA, or ACE393 elicited the

production of high IgG and IgA titres and showed a significant decline in C.

jejuni colonisation after challenge with a mixture of C. jejuni strains; of these

antigens, Omp18 showed the best efficacy (4.8 log10 reductions). By contrast,

chickens orally immunised with Avirulent ∆crp ∆cya S. enterica sv.

Typhimurium (S. Typhimurium) strain χ3987 expressing Omp18 (cjaD)

showed strong immune responses but no significant reduction in C. jejuni

colonisation from caecal content after challenge with heterologous C. jejuni

strain (Laniewski et al., 2012).

Saxena, John, et al. (2013) has shown that chickens immunised with

attenuated ∆aroA S. Typhimurium expressing C. jejuni CjaA CadF, CiaB,

Cj1496 polypeptides, or recombinant CjaA-CadF-CiaB-Cj1496 fusion

polypeptide via the oral route elicited immune responses but showed low

reductions of C. jejuni colonisation, as measured in the caecal contents (1-2

log10 CFU/g). Orally immunised mice, using an attenuated S. Typhimurium

vector vaccine carrying the C. jejuni PEB1 minus gene (PEB1-ss), showed

significant induction of serum IgG response and protection against C. jejuni

colonisation was not observed (Sizemore et al., 2006).

Kobierecka, Olech, et al. (2016) reported that orally immunised Hy-line

chickens with Lactococcus lactis (L. lactis) expressing either rCjaAD or

rCjaA elicited both IgY and IgA responses but did not significantly reduce C.

46

jejuni colonisation after challenge with a heterologous C. jejuni strain. The

intramuscular vaccination of SPF Leghorn chickens using live E. coli cells

harbouring C. jejuni N-glycan elicited a strong immune response (IgY) and a

significant reduction in colonisation (6 log10) at 7 days post-immunisation

following challenge with a heterologous C. jejuni strain (Nothaft et al., 2016).

Live attenuated bacteria can be good candidates for recombinant vector

vaccine development because of ease of manipulation for administration (da

Silva et al., 2014), induction of mucosal immune system (Cortes-Perez et al.,

2007) and low production costs (Nascimento & Leite, 2012). On the other

hand, some potentially deleterious consequences need to be considered. Some

attenuated bacterial vector vaccines may show reactogenicity and/or a

potential of reversion to virulence in chickens (Kuttappan et al., 2013; Medina

& Guzmań, 2001) or may be rapidly cleared from hosts (Nothaft et al., 2016).

This could result in inadequate immune responses (Kuttappan et al., 2013).

Mutations of virulence factors in the attenuation of the recombinant bacterial

vector of interest may delay antigen production, resulting in poor immune

responses (Pei et al., 2014). In addition, strong immune responses resulting

from live attenuated bacterial-based vector vaccines may not be associated

with Campylobacter colonisation (Sizemore et al., 2006). Pre-existing

immunity against live bacterial vaccine vectors antigens could prevent

successful eliciting of immune responses to the vectored antigen(s) (Saxena,

Van, et al., 2013).

47

Table 1.5: Summary of studies of anti-Campylobacter jejuni vaccines (live vector vaccine) evaluated in animal models

Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes

Reference Strain Vector

Immune

responses

Vaccine

efficacy

CjaA C. jejuni Eimeria tenella

White

Leghorn

Chickens

Oral Challenge

study

C. jejuni

02M6380

Increased

antibodies

1 log10 CFU/g

of reduction in

caecal contents

Clark et

al. (2012)

CjaA C. jejuni Eimeria tenella

White

Leghorn

Chickens

Oral Challenge

study

C. jejuni

02M6380

Increased

antibodies

1 log10 CFU/g

of reduction in

caecal contents

Clark et

al. (2012)

CjaA C. jejuni

M1

Live-attenuated

∆aroA/

AspaS/∆ssaU S.

Typhimurium

SPF

Light

Sussex

Chickens

Oral Challenge

study

C. jejuni

M1

Induced

specific IgY

and biliary

IgA

1.38 to 1.42

log10 CFU/g of

reduction in

caecal contents

Buckley

et al.

(2010)

TetC-CjaA C. jejuni

M1

Live- Live-

attenuated ∆aroA/

AspaS/∆ssaU S.

Typhimurium

SPF

Light

Sussex

Chickens

Oral Challenge

study

C. jejuni

M1

Induced

specific IgY

1.85 log10

CFU/g of

reduction in

caecal contents

Buckley

et al.

(2010)

48

Table 1.5: Summary of studies of anti-Campylobacter jejuni vaccines (live vector vaccine) evaluated in animal models (cont’)

Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes


Immune

responses

Vaccine

efficacy

GlnH

C.

jejuni

M1

Live-

attenuated

∆aroA/

AspaS/∆ssaU

S.

Typhimurium

SPF

Light

Sussex

Chickens

Oral Challenge

study

C. jejuni

M1

Induced

specific

IgY

No significant

reduction in

caecal

contents

Buckley et

al. (2010)

ChuA

C.

jejuni

M1

Live-

attenuated

∆aroA/

AspaS/∆ssaU

S.

Typhimurium

SPF

Light

Sussex

Chickens

Oral Challenge

study

C. jejuni

M1

Induced

specific

IgY

No significant

reduction in

caecal

contents

Buckley et

al. (2010)

Peb1A

C.

jejuni

M1

Live-

attenuated

∆aroA/

AspaS/∆ssaU

S.

Typhimurium

SPF

Light

Sussex

Chickens

Oral Challenge

study

C. jejuni

M1

Induced

specific

IgY

1.64 log10

CFU/g of

reduction in

caecal

contents

Buckley et

al. (2010)

49


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes


Immune

responses Vaccine efficacy

PEB minus

C.

jejuni

81-176

Live-

attenuated

∆phoP/QS.

Typhimurium

BALB/c

Mice Oral

Challenge

study

C. jejuni

81-176

and C.

jejuni

MGN

4735

Induced

specific IgG

No disease

protection

Sizemore

et al.

(2006)

CjaA

C.

jejuni

81-176

S.

Typhimurium

ᵡ9718

Chickens Oral Challenge

study

C. jejuni

wild-type

Wr1

Induced

specific IgY

and secretory

IgA

No significant

reduction in

caecal contents

Laniewski

et al.

(2014)

Cj0113

(Omp18/CjaD)

C.

jejuni

Live-

attenuated S.

Enteritidis

∆aroA and/or

∆htrA

Broiler

chickens

(Cobb-

500)

Oral Challenge

study

C. jejuni

PHLCJ1,

2, and 3

Induced

specific IgG

and secretory

IgA

4.8 log10 CFU/g

of reduction in

caecal contents

Layton et

al. (2011)

Cj0982c

(CjaA)

C.

jejuni

Live-

attenuated S.

Enteritidis

∆aroA and/or

∆htrA

Broiler

chickens

(Cobb-

500)

Oral Challenge

study

C. jejuni

PHLCJ1,

2, and 3

Induced

specific IgG

and secretory

IgA

1 log10 CFU/g of

reduction in

caecal contents

Layton et

al. (2011)

50


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes


Immune

responses

Vaccine

efficacy

Cj0420

(ACE393)

C.

jejuni

Live-

attenuated S.

Enteritidis

∆aroA and/or

∆htrA

Broiler

chickens

(Cobb-

500)

Oral Challenge

study

C. jejuni

PHLCJ1, 2,

and 3

Induced

specific

IgG and

secretory

IgA

2 log10

CFU/g of

reduction in

caecal

contents

Layton et

al. (2011)

Cj0113

(CjaD)

C. coli

72Dz/9

2

Avirulent

∆crp ∆cya S.

Typhimurium

χ3987

Commerci

al broiler

chickens

Oral Challenge

study

wild-type C.

jejuni 12

Induced

specific

IgG and

secretory

IgA

No

significant

reduction in

caecal

contents

Laniewski

et al.

(2012)

CjaA

C.

jejuni

72Dz/9

2

Avirulent S.

Typhimurium

χ3987

Commerci

al broiler

chickens

Oral Challenge

study

wild type

heterologous

C.

jejuni/pUOA

18

Induced

specific

IgG and

secretory

IgA

6 log10

CFU/g of

reduction in

caecal

contents

Wyszynsk

a et al.

(2004)

51


Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes


Immune

responses

Vaccine

efficacy

CjaA C. jejuni

Attenuated

∆aroA S.

Typhimurium


study

C. jejuni

81116 N/A

1.5 log10 CFU/g

of reduction in

caecal contents

Saxena,

John, et al.

(2013)

cadF C. jejuni

Attenuated

∆aroA S.

Typhimurium


study

C. jejuni

81116 N/A

1.5 log10 CFU/g

of reduction in

caecal contents

Saxena,

John, et al.

(2013)

ciaB C. jejuni

Attenuated

∆aroA S.

Typhimurium


study

C. jejuni

81116 N/A

1 log10 CFU/g of

reduction in

caecal contents

Saxena,

John, et al.

(2013)

cj1496 C. jejuni

Attenuated

∆aroA S.

Typhimurium


study

C. jejuni

81116 N/A

1 log10 CFU/g of

reduction in

caecal contents

Saxena,

John, et al.

(2013)

Note: N/A; Non-applicable

52


Note: N/A; Non-applicable

Antigen(s)

Vaccine Animal

model

Route of

administration

Experiment

type

Challenge

strain

Outcomes


Immune

responses

Vaccine

efficacy

CjaA-cadF-

ciaB-cj1496

C.

jejuni

Attenuated

∆aroA S.

Typhimuri

um


study

C. jejuni

81116 N/A

2 log10 CFU/g

of reduction in

caecal contents

Saxena,

John, et al.

(2013)

rCjaAD

C.

jejuni

81-176

L. lactis

IL1403

Hy-line

chickens Oral

Challenge

study

C. jejuni

12/2

Induced specific

IgG and

secretory IgA

1 log10 CFU/g

of reduction in

caecal contents

Kobierecka,

Olech, et al.

(2016)

rCjaAD

cytoplasm

C.

jejuni

81-176

L. lactis

IL1403

Hy-line

chickens Oral

Challenge

study

C. jejuni

12/2

Induced specific

IgG and

secretory IgA

Failure of the

significant

reduction in

caecal contents

Kobierecka,

Olech, et al.

(2016)

CjaA

C.

jejuni

81-176

L. lactis

IL1403

Hy-line

chickens Oral

Challenge

study

C. jejuni

12/2

Induced specific

IgG and

secretory IgA

Failure of the

significant

reduction in

caecal contents

Kobierecka,

Olech, et al.

(2016)

N-glycan

(glycosylate)

C.

jejuni

NCTC

11168

E. coli

SPF

Leghorn

chickens

Oral Challenge

study

C. jejuni

81-176

Induced specific

IgY

6 to 8 log10

CFU/g of

reduction in

caecal contents

Nothaft et

al. (2016)

53

1.9.4 Development of a viral vectored vaccine against Campylobacter

Important considerations in Campylobacter vaccine development for

commercial chickens are concerns of public health and animal welfare.

Vaccine development against Campylobacter is challenging in commercial

chicken farms, particular broiler chickens since the commercial chickens are

commonly slaughtered between 42–56 days of age depending on when they

reach market weight (Animal Liberation NSW, 2019). To provide a practical

and effective solution for use on commercial farms, Campylobacter vaccines

need to induce rapid immune responses at a young age and significantly

reduce caecal colonisation within the lifespan of broiler chickens.

Vaccine efficacy in young chicks may be affected by several factors. A recent

study by Lacharme-Lora et al. (2017) has reported that the antibodies of the

chicks provided adequate functions from approximately 6 weeks of age. This

suggests that the antibody-mediated immunity may not eliminate C. jejuni

and C. coli from the intestines before the slaughter of the commercial broiler

chicken, which typically occurs between 42-56 days. The persistence of

protective maternal immunity which generally remains in commercial chicks

until 2–3 weeks of age has been associated with the delay of Campylobacter

colonisation in chickens (Cawthraw & Newell, 2010; Laniewski et al., 2012;

Rice et al., 1997; Sahin, Luo, et al., 2003; Wyszynska et al., 2004). These

factors may have contributed to the inconsistent results observed with the

previously described vaccine delivery strategies which primarily induced

antibody-mediated immune responses. Hence, a vaccine that rapidly induces

a strong immune response, especially cell-mediated immune response, may

solve these problems and provide more consistent protection from

Campylobacter colonisation in commercial broiler chickens.

Accordingly, a viral vector vaccine especially the cell-associated form of the

virus could provide a solution, since these types of vaccines can elicit both

humoral and cellular immunity, provide protection at early challenge and less

interference by pre-existing immunity compared to the cell-free form (Baron

et al., 2018; Dey et al., 2017; Gerdts et al., 2006; Ingrao et al., 2017; Prasad,

1978; Santra et al., 2005; Witter & Burmester, 1979). Recombinant viral

vector-based vaccines have been used in animals especially chickens in order

to control viral infections and intracellular bacterial infections such as

54

recombinant fowlpox virus (rFPV) vector-based vaccine harbouring avian

influenza virus antigens (Qiao et al., 2009), adenovirus vector carrying the

avian influenza virus HA antigen (Ramos (Ramos et al., 2011), and

adenovirus vector carrying antigen from Listeria monocytogenes (Jensen et

al., 2013).

In addition to these viral vectors, herpesvirus of turkeys (HVT) is one of the

most potent delivery vectors for vaccines. HVT comprises a large genome

which can be inserted with a large foreign DNA (Ross, 1998; Sadigh et al.,

2018). It has been extensively generated as a cell-associated viral vector

vaccine and commercially used to prevent various chicken diseases such as

Chlamydia psittaci (Liu et al., 2015), infectious bursal disease (Roh et al.,

2016; Tsukamoto et al., 1999), Newcastle disease (El Khantour et al., 2017),

avian influenza (Kapczynski et al., 2015), and infectious laryngotracheitis

(Esaki et al., 2013; Vagnozzi et al., 2012). The recombinant HVT vector

vaccine is known to be safe and the cell-associated form of the virus is less

sensitive to maternal immunity (Baron et al., 2018; Dey et al., 2017; Ingrao

et al., 2017). Moreover, Li et al. (2011) reported that HVT-based vectors

expressing antigens that were constructed using an infectious copy of the viral

genome maintained as a bacterial artificial chromosome. The constructed

vectors were shown to be very effective in both in vitro and in vivo

experiments (Li et al., 2011). Recombinant HVT vector vaccines can provide

a long-lasting protective immunity with a single administration (Tsukamoto

et al., 2002). HVT vector expressing the inserted viral antigens, administrated

in ovo, rapidly elicited strong immune responses and provided strong

protection against diseases after early challenge by7 days of age (Gimeno et

al., 2016; Zhang & Sharma, 2001). Gimeno et al. (2015) reported that

administration of HVT in ovo induced high levels of CD45+, CD45+MHC-

I+, CD3+MHC-II+, CD3+, CD4+, and CD4+CD82 in spleen cells from day-

old-chicks. Immunised one-day-old -chicks with recombinant HVT via eye

drop and subcutaneous routes showed significant high levels of immune

responses and provided strong protection against diseases after challenge by

4 weeks of age (Sedeik et al., 2019) These suggest that immunised chicks

with HVT vector may have some protective immune responses at hatch and

at the time when they are going to be exposed to specific pathogens. In

55

addition, the HVT vector has been delivered on commercial scales via in ovo

and subcutaneous vaccinations with significantly high immune responses

(Prandini et al., 2016; Roh et al., 2016). For these reasons, HVT has potential

as a vector candidate use in preventing Campylobacter colonisation in

chicken farms. The construction of a HVT vector harbouring conserved

Campylobacter genes will be of interest for further study.

56

1.10 Objectives and aims of this study

Currently, limited information is available on the onset of C. jejuni and C.

coli colonisation of Australian free-range chicken farms, their genetic

diversity, or the degree of conservation of genes encoding protective antigens.

Therefore, the objectives of this study were to address the knowledge gap in

relation to C. jejuni and C. coli colonisation of free-range broilers and their

candidate antigens which may be amenable for use in a live viral vectored

vaccine to prevent Campylobacter colonisation of poultry. The study

objectives will be accomplished by addressing the following hypotheses and

research aims:

Hypothesis 1:

C. jejuni and C. coli colonise chickens in the first few weeks of age.

Study aim:

1. Determine the timing of C. jejuni and C. coli colonisation of chickens

through the isolation of these species from samples collected from

free-range broiler farms during the rearing cycle.

Hypothesis 2:

Colonisation of C. jejuni and C. coli in chickens in a commercial free-range

farm environment may occur via horizontal and/or vertical transmission.

Study Aim:

1. Determine the key mode(s) of transmission by determining genetic

diversity and the potential sources of C. jejuni and C. coli which

colonise chickens on free-range broiler farms using flaA-HRM

analysis.

Hypothesis 3:

Conserved genes encoding known immunogenic antigens could potentially

be used in developing a multivalent vaccine for C. jejuni and C. coli.

57

Study Aims:

1. Determine if the genes encoding known protective antigens are

conserved and shared between C. jejuni and C. coli isolated from

chicken farms using PCR assays.

2. Evaluate and characterise the over-expression of conserved C. jejuni

and C. coli antigens in prokaryotic and eukaryotic systems to identify

candidate genes for future use in a multivalent viral vector delivery

system.

58

Chapter 2 Campylobacter colonisation and transmission among

commercial free-range broiler farms in New South Wales, Australia

2.1 Introduction

Zoonotic Campylobacter species especially C. jejuni and C. coli are

frequently identified as major causes of human enteric infections (CDC,

2010; EFSA, 2015; European Centre for Disease Prevention and Control

[ECDC], 2010); NNDSS (2015); (WHO, 2012). Most outbreaks are attributed

to the consumption of contaminated poultry products (Sears et al., 2011;

Wagenaar et al., 2013). Chickens are a major source of human Campylobacter

infections and they can be colonised by 2-3 weeks of rearing (Friis et al.,

2010; Ingresa-Capaccioni et al., 2015; Ingresa-Capaccioni et al., 2016;

Kalupahana et al., 2013; Messens et al., 2009; Miflin et al., 2001;

Prachantasena et al., 2016; Thomrongsuwannakij et al., 2017). It has been

estimated that reducing Campylobacter loads in chicken intestines by two to

three orders of magnitude could lead to a decline of human


et al., 2003). Thus, control of Campylobacter colonisation in chicken at farm-

level is one of the most effective strategies to reduce the incidence of human

Campylobacter infections (EFSA, 2011).

In commercial intensive poultry farms, the horizontal transmission from the

environment is an important source of Campylobacter spp. colonisation

(Ellis-Iversen et al., 2012; Messens et al., 2009). It is believed that horizontal

transmission route is more crucial to in the colonisation of free-range broiler

farms since these chickens roam outside the shed, hence potentially being

exposed to these microorganisms multiple times and from different

environmental sources (Nather et al., 2009). However, information on the

Campylobacter spp. transmission in free-range broilers is limited, whereas,

the number of free-range farms and consumer demands for free-range

chickens have increased (Miele, 2011; Naald & Cameron, 2011; Singh &

Cowieson, 2013; Sumner et al., 2011; Walley et al., 2015). Although a

previous study conducted in Australia reported on the distribution of C. jejuni

genotypes across Australian broiler farms including intensive and free-range

farms (Templeton, 2014), however the epidemiology of Campylobacter spp.

59

colonisation in free-range broiler farms, particularly regarding the bacterial

transmission has not been addressed.

Currently, molecular genotyping is commonly used to investigate the source

of infections and genetic populations of pathogens in many epidemiological

investigations. Various methods such as Pulsed-field gel electrophoresis

(PFGE), Restriction fragment length polymorphism (RFLP) and Multilocus

Sequence Typing (MLST) have been extensively used to discriminate

Campylobacter spp. isolates in many studies (Bakhshi et al., 2016; Eberle &

Kiess, 2012; Ge et al., 2006; Gomes et al., 2016; Kamei et al., 2014; Kittl et

al., 2013; Nebola & Steinhauserova, 2006; Nielsen et al., 2010;

Noormohamed & Fakhr, 2014; Posch et al., 2006; Stone et al., 2013).

However, these methods are time-consuming, labour-intensive and expensive

(Eberle & Kiess, 2012; Frasao et al., 2017; Levesque et al., 2008;

Noormohamed & Fakhr, 2014; Tabit, 2016; Wassenaar & Newell, 2000).

The High Resolution Melt Polymerase Chain Reaction method (HRM-PCR)

has recently been suggested as it is a rapid, discriminatory and cost-effective

tool that can be alternatively used to discriminate C. jejuni and C. coli

(Banowary et al., 2015; Hoseinpour et al., 2017). Also, the flaA gene, which

is one of several potential genes, has been suggested as an informative

epidemiologic marker due to its hypervariable and conserved gene among

Campylobacter spp. (Meinersmann et al., 1997; Wassenaar & Newell, 2000).

It has been used for Campylobacter spp. genotyping in epidemiological

studies (Gomes et al., 2016; Hiett et al., 2007; Petersen & On, 2000; Singh &

Kwon, 2013). Recently, the combination of HRM-PCR targeting flaA gene

(flaA-HRM PCR) has been developed by Merchant-Patel et al. (2010) and

resulted in high discrimination for C. jejuni and C. coli genotypes.

Therefore, the objective of this chapter was to use molecular approaches to

improve the understanding of the dynamics of C. jejuni and C. coli

colonisation, potential sources of transmission as well as their genetic

diversities in commercial free-range broiler farms. To achieve these, the flaA-

HRM PCR was used to discriminate C. jejuni and C. coli isolates from various

sources on commercial free-range broiler and breeder farms. Then the

outcomes from flaA-HRM PCR were supported with the flaA amplicon

60

analysis, The MLST was only used to support the C. jejuni and C. coli

genotypes identified from broiler farms.

2.2 Materials and methods

2.2.1 Free-range meat chicken production

Fertilized eggs from breeder farms were incubated under standard controlled

environment for 21 days in a commercial hatchery located in northern NSW.

After hatching, the broiler chicks were transported to commercial broiler

farms. At a commercial free-range broiler farm, all broiler chicks were reared

in closed sheds (flocked area) for 21 days. Then, the chickens were free to

roam in a fenced outdoor environment (free-range area) through shed flaps

during daytime for approximately 42-56 days until achieving market weight.

This is defined as the free-range system (Free Range Egg & Poultry Australia

- FREPA, 2012). The numbers of chickens located at the breeder farms,

hatched at the hatchery and transported to the broiler farms were not available

for this study, since the information was deemed to be confidential by the

commercial company. The stock density of free-range chicken (inside the

shed) ranging between 28 and 34 kg/m2 (Australian Chicken Meat

Federation-ACMF, 2018b). A flock was defined as the entire group of

chickens that were housed in the same shed.

2.2.2 Free-range broiler farm practices

Three free-range broiler farms belonged to the same owner and the same farm

practice was applied. The all-in-all-out management system was operated on

all chicken farms which meant that all chickens of each farm were completely

depopulated within the same period, left empty for one week, and then

restocked simultaneously with a new batch of chicks in the same period. The

infrastructures of the shed, equipment, and the environment were cleaned and

disinfected during the empty period. Chickens were reared on three free-range

broiler farms in this study. Even though antibiotics can be used to treat sick

chickens in free-range farming systems (Australian Chicken Meat

Federation-ACMF, 2018b), antibiotics had not been used on these farms from

61

at least 2 years prior to this study to the completion of data collection,

according to the farm records on antimicrobial use.

During the rearing period, the same person accessed all chicken sheds within

the same farm, whereas, the farm manager accessed all shed on all farms in

this study. Wearing overalls, putting on a headdress, and change of boots were

required before entering the farms. Boot dips containing disinfectant and

hands sanitation with 70% alcohol were required before entering the flocked

area and these were provided in the anteroom of each shed. Shed boots were

sanitized before use. The disinfectant for boot dips was changed daily. Wood

shaving was used as litter and changed every cycle of farm production.

2.2.3 Farm information and farm codes

Eleven farms (eight breeder and three free-range broiler farms) were included

in this study (Figure 2.1). All farms were part of an integrated poultry

production company based in New South Wales (NSW), Australia which has

requested anonymity for commercial reasons.

2.2.3.1 Breeder farms

All breeder farms that supplied Ross chicks to the broiler farms were selected

for sample collection in this study (Figure 2.1 and Table 2.1). The eight

breeder farms were designated BD–A to BD–H. Five (BD–B, BD–C, BD–D,

BD–G, and BD–H) and three (BD–A, BD–E, and BD–F) were located in

NSW and Queensland (QLD), respectively.

62

Figure 2.1: Diagrams of free-range broiler sheds and their parent breeder farms in the experiments 1 and 2.

Eight breeder farms supplied broiler chicks to three free-range farms (18 sheds). Three breeder farms were completely depopulated

during sampling on 7 days after broiler chick placement as indicated a .

63

2.2.3.2 Free-range broiler farms

All three broiler farms (designated FB1, FB2, and FB3) were in the same

vicinity (approximately 800 metres apart) with 60 km away from Sydney,

NSW. The number of birds was between 11,880 and 15,390 birds per flock

in this study (Table 2.1).

This study was conducted with two similar experiments (designated Exp.1

and Exp.2) over two production cycles of free-range broiler farms (from May

to August 2016). The Exp.1 and Exp. 2 were conducted in free-range broiler

farm production cycles I and II, respectively (Figure 2.1). For both

experiments, one shed from each broiler farm was selected as the target shed

(designated T), focusing on the transmission (Table 2.1). The sheds on either

side of the target shed were appointed as adjacent sheds (designated A1 and

A2) and used to examine the transmission between sheds (Table 2.1). The

same sheds of each farm were subsequentially selected in the next production

cycle of free-range broiler farms.

The free-range broiler shed codes of this study were abbreviated as follows:

the farm–the shed–experiment. For example, in experiment 1 (Exp.1), the

target shed (T) of free-range broiler farm 1 (FB1) was coded as FB1–T–Exp.1

and its adjacent sheds were coded as FB1–A1–Exp.1 and FB1–A2–Exp.1.

For the experiment 2 of the same farm, the target broiler shed was coded as

FB1–T–Exp.2 and its adjacent sheds were coded as FB1–A1–Exp.2 and FB1–

A2–Exp.2. These codes were applied for free-range broiler farms 2 (FB2) and

3 (FB3) as well. All farm codes are described in Table 2.1 and Figure 2.1.

64

Table 2.1: Summary of breeder farms and the supplied free-range broiler sheds from the experiments 1 and 2 in this study

Experiment Broiler farm Breeder farm a

(weeks of age) Farm Shed Shed size

(m × m)

Free-range size

(m × m)

Chickens

(n)

Shed code

1

1

Adjacent1 12.1 × 95.2 17 × 65 14,670 FB1–A1–Exp.1 BD–C (47)

Target 12.1 × 95.2 17 × 65 14,670 FB1–T–Exp.1 BD–Db

Adjacent2 12.1 × 95.2 17 × 65 15,390 FB1–A2–Exp.1 BD–Db

2

Adjacent1 12.1 × 95.2 17 × 65 15,030 FB2–A1–Exp.1 BD–A (65)

Target 12.1 × 95.2 17 × 65 15,030 FB2–T–Exp.1 BD–A (65)

Adjacent2 12.1 × 95.2 17 × 65 14,850 FB2–A2–Exp.1 BD–A (65)

3


Target 12.1 × 69.4 17 × 50 11,980 FB3–T–Exp.1 BD–B (61) and BD–C (47)


2

1

Adjacent1 12.1 × 95.2 17 × 65 15, 480 FB1–A1–Exp.2 BD–F (55) and BD–Eb

Target 12.1 × 95.2 17 × 65 14,760 FB1–T–Exp.2 BD–Eb

Adjacent2 12.1 × 95.2 17 × 65 14,760 FB1–A2–Exp.2 BD–F (55)

2

Adjacent1 12.1 × 95.2 17 × 65 14,670 FB2–A1–Exp.2 BD–F (55)

Target 12.1 × 95.2 17 × 65 14,670 FB2–T–Exp.2 BD–F (55)

Adjacent2 12.1 × 95.2 17 × 65 15,390 FB2–A2–Exp.2 BD–F (55) and BD–Eb

3

Adjacent1 12.1 × 69.4 17 × 50 11,880 FB3–A1–Exp.2 BD–Hb

Target 12.1 × 69.4 17 × 50 11,880 FB3–T–Exp.2 BD–Hb

Adjacent2 12.1 × 73.2 17 × 65 14,850 FB3–A2–Exp.2 BD–G (57) and BD–Hb Note: a indicates the breeder flock ages when the samples were collected at breeder farms; b indicates the depopulated breeder farms; Adjacent sheds are either side of the target shed; Adjacent1 is on the left side of the target shed; Adjacent2 is on the right side of the target shed

65

2.2.4 Determination of sample size

A standard sample calculation tool, the Epitools programme (AusVet Animal

Health Services) was used to calculate the sample size for demonstration of

freedom (detection of disease) in a finite population via

http://epitools.ausvet.com.au/content.php?page=FreedomFinitePop&Populat

ion (accessed 02/04/2016). Based upon discussions with industry/scientific

experts at a commercial company (informed by data from a previous study

(Chenu, 2014)), the estimated prevalence of Campylobacter spp. was set at

10% to ensure detection within a focus flock (target shed) at the early stage

of rearing. The Epitools program’s input parameters are shown in Table 2.2.

Based upon these parameters, the required sample size was calculated to be

34 faecal/caecal samples from each shed.

Table 2.2: The list of input parameters for sample size calculation

Parameters Input number

Population size (for finite populations) 14,000

Test sensitivity 0.9

Desired herd sensitivity (Confidence level) 0.95

Estimated prevalence 0.1

Due to practical issues on commercial farms, 35 faecal/caecal droppings were

collected in the target sheds (focusing on the transmission) and it was only

possible to collect ten faecal/caecal samples from each adjacent shed. With

these sample sizes, 35 and 10 faecal/caecal samples were allowed to detect

Campylobacter spp. when the prevalence of disease detections were 9.6% and

35%, respectively in a free-range broiler flock (N > 10,000) at a young age.

As for breeder farm, only five faecal/caecal samples per shed were obtained

by the industry partner. Based on a previous study, the prevalence of

Campylobacter in breeders ranged from 78% to 86% in the age groups of 48

and 60 weeks (Ingresa-Capaccion et al., 2016). In this study, the youngest

group of breeders was 47 weeks of age, and thus this sample size of the

breeder farm (five samples per shed) was appropriated to detect this

microorganism at the prevalence of 67% within a breeder flock (N ≥ 5,000).

Each breeder farm consisted of four or six breeder sheds and resulted in 20 or

30 faecal/caecal samples, respectively.

66

2.2.5 Sample collection

Each bird generally excreted faecal/caecal droppings more than once a day

and this was considered as a potential limitation of individual bird sampling

in large populations. To overcome this limitation, the sheds selected were

divided nominally into 16 equal zones, and 2-3 fresh voided faecal/caecal

samples were collected from each zone in this study. Freshly voided faecal

and caecal excretions were immediately taken after observed excretion from

individual chickens from different zones of each shed on the breeder and free-

range broiler farms. These samples were defined as faecal samples in this

study. The researcher (P.P.) was at the farms to witness the defecation

moment of individual birds and immediately collect the fresh faecal/caecal

samples accordingly. The environment was only sampled from free-range

broiler farms. Overall, 1856 samples were collected from breeder farms

(n=120) and broiler farms (n=1736) in this study. Faecal samples were

collected from both breeder farms (n=120) and broiler flocks (n=1265),

whereas, the environmental samples (n=471) were exclusively collected from

the broiler farm.

2.2.5.1 Samples collected from breeder farms

The original plan was to obtain fresh faecal samples from the breeder farms

21 days before the linked broilers were placed at farms. However, due to

logistical issues, it was not possible to obtain these samples. Fresh faecal

samples were obtained 7 days after the linked broiler chickens were placed

on the farms. Consequently, farms BD–D, BD–E, and BD–H were completely

depopulated at the time of sample collection and thus, no samples were

available from those three farms. Samples from a total of 24 sheds from five

farms were included in this study.

A total of 120 faecal samples from breeder farms (5 samples per shed) were

randomly taken by using Amies swabs containing charcoal transport medium

(Copan Italia, Brescia, Italy) (Table 2.3). All faecal swab samples were

transferred in insulated boxes containing ice packs and transported to the

Birling avian laboratories for processing within 24 h.

67

2.2.5.2 Samples collected from broilers

Each free-range broiler farm was sampled before chick placement (Day 0),

the date of chick placement (Day 1 or 3), and then weekly sampled until the

first detection of Campylobacter spp. in a target shed (Table 2.3). For

logistical reasons, the day of sample collection from the various broiler farms

varied by three or fewer days. During each visit, samples of faeces and the

environment were collected from each broiler shed as described in Table 2.3.

All samples were transferred in insulated boxes and transported to the Birling

avian laboratories for processing within 1-2 h.

Faecal samples were randomly collected from chickens of each shed as soon

as possible after observing faecal/caecal excretion using Amies swabs

containing charcoal transport medium (Copan Italia) on the day of chick

placement (Day 1 or 3) and a sterile faecal container with a spoon (Techno

Plas, St Marys, SA, Australia) on Weeks 1 (Day 8 or 10), 2 (Day 15 or 17),

and 3 (Day 22 or 24). An Amies swab and an integrated spoon of the faecal

container were used to collect the fresh brown matter of faeces by picking

from the top to the middle part of faeces (avoiding urate and floor

contamination); the swabs were then individually placed in a labelled

container. Additional samples were also obtained from the environment such

as shed wall (swabbing a 100-cm2 area each side), water and feed pans, and

boots (shed boots and farm boots) using Amies swabs containing charcoal

transport medium (Copan Italia) or Amies swabs (Copan Italia). Other

environmental samples, floor samples (flocked area and anteroom) were

collected using sterile tampons (Libra regular; Svenska Cellulosa

Aktiebolaget, Springvale, VIC, Australia) moistened with sterile buffered

peptone water (Acumedia; Neogen Corporation, Lansing, MI, USA). The

floor within the flocked area was divided into two equal sections by the length

of the shed (front and back floors) and drag swabbing them on the floor (a

zigzag pattern across the length of the shed by following the water pipelines

(5 lines). The floor sampled from the anteroom was obtained by drag

swabbing across the perimeter and centre of the room. Soil samples from

outside of the shed (free-range area) were obtained by drag swabbing a moist

sterile tampon (Libra regular, Australia) along the outside perimeter of the

shed (1 metre from the shed wall). All swabs were placed separately in sterile

68

plastic bags. All water samples collected had a volume of 250 mL. Drinking

water samples were collected from drinkers in three to six areas of each shed

with a sterile plastic bottle (Techno Plas). Water from the main tank and

puddles were collected in sterile plastic bottles. Fresh rodent faeces (dark in

colour, soft and moist textures, and spindle-shaped) and insects (darkling

beetles and flies) were collected from the anteroom of each shed in sterile

plastic bags.

Table 2.3: Sample types and total number(s) collected for Campylobacter

spp. isolation on breeder and broiler sheds over the course of this study

Samples Sample collection at time points (per shed)

Day 0 Day 1

or 3

Day 8

or 10

Day 15

or 17

Day 22

or 24

Faecal samples a – – 5 – –

Faecal samples b – 35 35 35 35

Faecal samples c – 10 10 10 10

Walls b 2 2 2 2 2

Floors b 2 2 2 2 2

Anteroom b 1 1 1 1 1

Feed pans b 2 2 2 2 2

Water pans b 2 2 2 2 2

Shed boots b 1 1 1 1 1

Drinking water b 1 1 1 1 1

Main tank water b, d 1 1 1 1 1

Farm boots b 1 1 1 1 1

Free-range area b, c 1 1 1 1 1

Puddle b, d 1 1 1 1 1

Insects b, d (darkling

beetles and flies)

1 1 1 1 1

Rodent faeces d 1 1 1 1 1

Note: a Samples from breeder farm (sample per shed), b Samples from target broiler shed, c Sample from

the adjacent broiler shed, d Opportunistic sampling from a broiler farm

2.2.6 Campylobacter spp. isolation

All samples were processed following the standard ISO 10272:2006 method

for Campylobacter spp. isolation (ISO, 2006) with some modifications.

Briefly, the Campylobacter selective agar including Campylobacter

69

(charcoal) agar (bioMérieux, Marcy l’Etoile, France), Skirrow’s agar

(bioMérieux), and Campy Food Agar (CFA) (bioMérieux) were used as

selective media for promoting Campylobacter spp. growth in this study as

previously described (Karmali et al., 1986; Ugarte-Ruiz et al., 2012; Vaz et

al., 2014).

All individual faecal samples (0.5-2 g) and fresh rodent faeces (0.3 g)

collected from farms were resuspended in sterile phosphate-buffered saline

(PBS) at a 1:1 (w/v) ratio (e.g. 1 g of faecal material to 1 mL PBS). Then, a

disposable inoculating loop was used to sample (10 µL) each faecal

suspension and directly streak it onto selective media. All swabbed samples

such as walls, floors, shed boots, farm boots. feed pans, water pans, anteroom,

and the free-range area (soil) were pre-enriched in Bolton broth (Oxoid,

Cambridge, UK) containing Bolton broth selective supplement (Oxoid) with

a ratio of 1:10 (weight per volume; w/v). Insects (one fly and one darkling

beetle) were macerated and pre-enriched in the Bolton broth (Oxoid) as

described above. Water samples (250 mL per sample) such as drinking water,

tank water and puddles were filtered onto a membrane with 47 mm-diameter

and pore size of 0.45 µm-pore -size (Merck Millipore, Burlington, MA,

USA). The membranes were then pre-enriched in Bolton broth (Oxoid) as

described above. For Campylobacter isolation, C. jejuni ATCC 29428 and C.

coli ATCC 33559 were used as positive controls, whereas E. coli ATCC

11775 was used as a negative control.

All streaked plates and enriched samples were incubated for 48 h at 42 °C

under a microaerobic environment generated using a BD GasPakTM EZ

container system (Becton Dickinson Microbiology, North Ryde, NSW,

Australia). All enriched samples were screened for Campylobacter spp.

detection using the VIDAS® Campylobacter assay (BioMérieux), according

to the manufacturer’s instructions. For all Campylobacter-positive broth

samples, one loopful of a disposable inoculating loop (10 µL) of each positive

enrichment broth was streaked onto the selective agar plates and incubated

under a microaerobic environment as described earlier.

Up to 5 presumptive colonies showing typical morphological characteristics

of Campylobacter spp. were identified as C. jejuni and C. coli using Matrix-

assisted laser desorption ionisation time-of-flight (MALDI-TOF) (VITEK®

70

MS; BioMerieux) as described in section 2.2.6 and Polymerase Chain

Reaction (PCR) method (section 2.2.8).

2.2.7 Campylobacter jejuni and Campylobacter coli identification

The MALDI-TOF (VITEK® MS; BioMérieux) method was used to identify

C. jejuni and C. coli by picking the edge of every single colony for the

assessment following the manufacturer’s instructions (Appendix 2.1).

2.2.8 Stock culture preparation and DNA extraction

The same single colony, obtained from section 2.2.7, was processed for stock

culture and DNA extraction.

2.2.8.1 Stock culture

The same single colony of C. jejuni and C. coli from section 2.2.7 was directly

streaked onto the Sheep Blood Agar plate (BioMérieux) and then incubated

as described above. After the incubation, a half plate of the bacterial growth

was collected and made up in an aliquot as a stock culture by mixing in the

FBP Campylobacter growth medium [0.025% sodium pyruvate (w/v),

0.025% sodium metabisulphite (w/v),0.025% ferrous sulphate (w/v)] with

15% glycerol (Gorman & Adley, 2004), and subsequently stored at -80°C

until required.

2.2.8.2 Genomic DNA extraction

C. jejuni and C. coli isolates from section 2.2.8.1 were harvested and used for

genomic DNA extraction using PrepMan® Ultra Sample Preparation (Applied

Biosystems, Australia) according to the manufacturer’s instructions. DNA

samples were stored at 4°C until required.

71

2.2.9 Campylobacter jejuni and Campylobacter coli confirmation by

PCR

One isolate of C. jejuni and C. coli from each culturable sample which was

identified from MALDI-TOF (section 2.2.7) was selected and verified with a

conventional PCR method. The reactions were performed in a BIO-RAD

S1000TM Thermal Cycler (BIO-RAD, Australia). PCR primers (Table 2.4)

and reactions were conducted according to Devi (2019). All isolates were

initially tested to detect the 16s rRNA gene (Campylobacter genus) and were

further examined to detect mapA (C. jejuni) and IpxA (C. coli) genes. Each

PCR reaction volume was 25 µL containing 2 U Platinum Taq polymerase

(Invitrogen, Carlsbad, CA, USA), 1 × Green PCR Rxn Buffer- MgCl2

(Invitrogen), 1.5 mM MgCl2 (Invitrogen), 0.2 mM of dNTPs mixed

(Invitrogen), 0.2 µM 16s rRNA gene primers (Integrated DNA Technologies,

Singapore) or a mixture of primers of 0.2 µM IpxA and 0.2 µM mapA

(Integrated DNA Technologies, Singapore), RNase-free water (to a final

volume of up to 24 µl) and 1 µL of DNA template (10-30 ng).

The PCR cycling conditions were activation of Platinum Taq polymerase at

94oC for 2 min, one cycle, followed by 40 cycles of denaturation at 94oC for

10 sec, annealing at 60oC for 20 sec and extension at 72oC for 30 sec, and

elongation at 72°C for 5 min.

The PCR products were analysed using agarose gel electrophoresis at 80 V

for 40 min in 1.5% (w/v) agarose gel stained with Midori Green Advanced

DNA stain (Nippon Genetics Europe GmbH, Germany) in 1× Tris-acetate-

EDTA (TAE) buffer (40 mM Tris-HCl pH 7.6, 20 mM acetic acid, 1 mM

EDTA). The PCR product was visualised using a Gel DocTM XR+ imaging

system (Bio-Rad, Australia) with Gel Green software (Bio-Rad, Australia).

The sizes of PCR products were compared with a standard molecular weight

marker (1-kb ladder, New England Biolabs, Ipswich, MA, USA). C. jejuni

ATCC 49943 and C. coli ATCC 33559 were used as positive controls for each

PCR reaction. RNAase water was used as non-DNA template control. All

PCR amplicons were purified (section 2.2.11) and commercially sequenced

using dideoxynucleotide technology by the Australian Equine Genetics

Research Centre (AEGRC) at the University of Queensland (Brisbane,

Australia).

72

Table 2.4: Oligonucleotide primers used for identification of Campylobacter spp., Campylobacter jejuni, and Campylobacter coli

Group or Species Gene Sequence 5′ to 3′ Amplicon size (bp)

Campylobacter flaA Forward: GGA TTT CGT ATT AAC ACA AAT GGT GC

Reverse: CAA GWC CTG TTC CWA CTG AAG

639

Campylobacter 16S rRNA Forward: CGT GCT ACA ATG GCA TAT ACA ATG A

Reverse: CGA TTC CGG CTT CAT GCT C

113

C. jejuni mapA Forward: CAC TTT AGA CAC TGG TAT TGC TTT G

Reverse: GAT CGT TAT TGT CAA GCA CAA CTA TTC

191

C. coli lpxA Forward: GAT GAT GTT GTT ATT GAG GCT TAT G

Reverse: GAA AGT ATT CTC GCC CCT TG

92

73

2.2.10 Genotyping

All genomic DNA samples of C. jejuni and C. coli isolates from section

2.2.8.2 were assessed for genotyping using High Resolution Melting PCR

targeting the flaA gene (flaA-HRM PCR). Representative DNA samples for

each HRM profile (between 1 and 20 amplicons) were further analysed with

flaA amplicon sequencing flaA sequencing. Following this, one isolate from

each flaA genotypes of C. jejuni and C. coli isolated from broiler farms were

characterised by Multilocus Sequence Typing (MLST).

2.2.10.1 flaA-HRM PCR

One isolate of C. jejuni and C. coli from all culturable samples was selected

and performed with flaA-HRM PCR for genotyping which was slightly

modified from those previously described by Merchant-Patel et al. (2010).

Briefly, each flaA-HRM PCR reaction (20 µL) contained 1 ×Type-it HRM

PCR kit (Qiagen), 6.6 µl of RNase-free water or MillQ water, 0.7 µM of flaA

primers (Sigma-Aldrich, St. Louis, MO, the United States) and 2 µL of DNA

template.

The flaA-HRM PCR was performed in a Rotor-Gene Q thermal cycler

(Qiagen). The real-time PCR conditions were as follows: initial denature at

95°C for 5 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 15 sec

and 72°C for 30 sec. The flaA-HRM PCR protocol included 0.1°C increments

for each step and was ramped between 77°C and 85°C. All isolates were

analysed in triplicate. The HRM melting curves and HRM normalised graphs

were created using the QIAGEN Rotor Q Series software version 2.3.1

(Qiagen).

2.2.10.2 flaA amplicon sequencing

The flaA amplicon sequencing was used to support the results from flaA-

HRM PCR in this study. The representative flaA amplicons from designated

HRM groups were commercially sequenced using the Sanger sequencing

method (Australian Genomic Research Facility, Sydney, NSW, Australia).

The flaA nucleotide sequences were analysed as described in section 2.2.11.

74

2.2.10.3 Multilocus sequence type (MLST)

DNA fragments of seven housekeeping genes were selected and amplified by

PCR according to a previously published method (Dingle et al., 2001). All

PCR products were commercially sent for DNA sequencing as described

above and further analysed for the nucleotide sequences as described in

section 2.2.11.

2.2.10.4 Clustering analysis

The discrimination and characterisation of C. jejuni and C. coli isolates were

determined using flaA-HRM PCR analysis. The flaA amplicon sequences

have supported the outcomes of flaA-HRM PCR. The MLST was used to

support the results of flaA-HRM PCR and flaA amplicon sequencing of C.

jejuni and C. coli genotyped from broiler farms only.

All flaA-HRM data were distinguished using the Rotor-Gene ScreenClust

HRM software (version 1.10.1.2), but the triplicates of the same isolate

showed different HRM groups identified (data not shown). Therefore, all

flaA-HRM data obtained from the QIAGEN Rotor Q Series software was

used to differentiate C. jejuni and C. coli genotypes based on the difference

in peak(s) of melting temperature (Tm; °C) and curve shape(s) of flaA-HRM

data. The evaluation of the same flaA-HRM profile was calculated using

minimal differentiation power of HRM-PCR. The same genotype was

determined using the combinations of similar shape of the HRM curve

pattern, Tm, and the flaA allele number. In this study, the difference of the

mean on average of melting temperature (Tm) ± SD of the C. jejuni with flaA

allele 12,16a (ST-257) was used as a cut off value to determine the difference

of flaA-HRM profiles for the same genotype.

All C. jejuni and C. coli genotypes identified from the flaA-HRM analysis

were assigned arbitrary cluster numbers of each species. Then, all

representative samples of each HRM profile were verified for genetic

variation using flaA sequencing analysis and obtained the flaA peptide allele

and nucleotide numbers as described in section 2.2.11. However, some

different flaA sequences had the same flaA peptide allele number and

nucleotide number. Thus, in this study, all different flaA sequences from the

75

same flaA allele were manually determined as different genotypes by adding

subscript alphabet after flaA allele number to support the flaA-HRM analysis

as shown in Appendix 2.2 and 2.3.

2.2.11 DNA sequencing analysis

The PCR products obtained in the flaA-HRM-PCR and MLST (Section

2.2.10) were sequenced as described below.

Prior to sequencing, all PCR amplicons (10 µL) were purified with the

ExoSAP-IT system (USB Corporation, Cleveland, Ohio, USA) according to

the manufacturer’s instructions. The purified PCR product (21 – 40 ng) was

mixed with relevant primer (forward or reverse amplification primer) at a

final concentration of 0.8 µM in a 12 µL reaction. The flaA amplicons were

commercially sequenced using the Sanger sequencing method at the

Australian Genomic Research Facility, Sydney, Australia (AGRF).

The nucleotide sequence alignment was performed using BioEdit Sequence

Alignment Editor (version 7.2.5). The flaA peptide allele number and

nucleotide number were identified by interrogation of Campylobacter flaA

database for each isolate via http://pubmlst.org/Campylobacter (accessed

17/04/2017). Allele number, Sequence types (STs) and Clonal complexes

(CCs) were determined based upon the Campylobacter MLST database

comparisons from https://pubmlst.org/Campylobacter/ (accessed

15/08/2017).

2.3 Results

Overall, Campylobacter spp. were cultured from 526 of the 1856 samples

(28%) collected. Of these, 465 samples (88.4%) were isolated from faecal

samples obtained from the breeder (n=118) and free-range broiler farms

(n=347). The remaining 61 samples (11.6%) were isolated from the

environment of free-range broiler farms. Based on the outcomes of MALDI-

TOF, 384 and 117 samples were identified as C. jejuni and C. coli,

respectively, and the remaining 25 contained both. By contrast, 381 and 120

samples were identified as C. jejuni and C. coli, respectively, and the

remaining 25 contained both, based on PCR reactions. Therefore, 406 C.

76

jejuni and 145 C. coli isolates identified from all 526 culturable samples were

assessed for genotyping with flaA-HRM PCR.

2.3.1 Isolation of Campylobacter jejuni and Campylobacter coli from

breeder farms

All five breeder farms were positive for Campylobacter spp. which were

cultured from 118 (98%) of the 120 faecal samples collected from the five

breeder farms by culture method (Table 2.5). The detection rate for

Campylobacter spp. isolated from breeder farms was 98.33 % on average

ranging from 80 to 100% among breeder sheds (Table 2.5). Based upon

MALDI-TOF, 70 and 30 were identified as C. jejuni and C. coli, respectively,

and 18 contained both. Five C. jejuni isolates from 5 samples (BD–B, n=2;

BD–C, n=1; and BD–BF, n=2) identified from MALDI-TOF were later

identified as C. coli by PCR (Appendix 2.2). Six additional isolates of C.

jejuni from the same samples (as re-culturable) identified by MALDI-TOF

were tested with PCR and they were identified as C. coli. Two C. coli isolates

from 2 samples (BD–A, n=1 and BD–BF, n=1) identified from MALDI-TOF

were later identified as C. jejuni by PCR (Appendix 2.2). Six additional

isolates of C. coli from the same samples (as re-culturable) identified by

MALDI-TOF were tested with PCR and they were identified as C. jejuni.

MALDI-TOF was used to putatively identify C. jejuni and C. coli isolates,

whereas the PCR assay was used to designate isolates as either C. jejuni or C.

coli. The reason why a PCR method was used to speciate C. jejuni and C. coli

in this study is that a previous study using the species-specific primers of C.

jejuni and C. coli has reported reliable results for confirming C. jejuni and C.

coli (Devi, 2019). Therefore, of the 118 culturable samples, 67 and 33

belonged to C. jejuni and C. coli, respectively, and the remaining 18 contained

both C. jejuni and C. coli in this study (Table 2.5). Consequently. 85 C. jejuni

and 51 C. coli isolates identified from all 118 culturable samples were

selected for flaA-HRM PCR. Based on the experiment, C. jejuni was the most

frequently isolated species in most breeder farms in both experiments: Exp.1:

BD–A and BD–C and Exp.2: BD–F and BD–G (Table 2.5).

77

Table 2.5: Isolation rates of Campylobacter jejuni and Campylobacter coli

identified in faecal samples from breeder sheds

Farm Flock Samples Campylobacter species

identified*

Tested Positive % C.

jejuni

C.

coli

C. jejuni

and C. coli

A

1

2

3

4

5

5

5

5

5

5

5

5

5

5

5

100.0

100.0

100.0

100.0

100.0

2

3

2

4

3

2

2

1

–

1

1

–

2

1

1

B

1

2

3

4

5

5

5

5

5

5

4

5

100.0

100.0

80.0

100.0

2

3

1

2

3

1

3

2

–

1

–

1

C

1

2

3

4

5

5

5

5

5

5

5

5

100.0

100.0

100.0

100.0

3

2

1

4

2

1

4

1

–

2

–

–

F

1

2

3

4

5

6

5

5

5

5

5

5

5

5

5

5

5

5

100.0

100.0

100.0

100.0

100.0

100.0

1

3

2

2

3

3

2

–

2

1

2

1

2

2

1

2

–

1

G

4

5

6

7

8

5

5

5

5

5

5

5

5

4

5

100.0

100.0

100.0

80.0

100.0

5

5

4

3

5

–

–

–

1

–

–

–

1

–

–

Total 24 120 118 98.3 67 33 18 Note: * C. jejuni and C. coli were identified with a conventional PCR assay

2.3.2 Isolation of Campylobacter jejuni and Campylobacter coli from

broiler farms

Overall, Campylobacter spp. were isolated from 17 of 18 broiler sheds,

whereas, one shed (FB3–A2–Exp.1) was culture negative for Campylobacter

spp. (Table 2.6). Among the Campylobacter positive sheds, nine were

positive for either C. jejuni or C. coli, whereas the remaining eight were

positive for both species (Table 2.6).

Campylobacter spp. were cultured from 408 (23.5%) of the 1,736 samples

which 347 and 61 samples were from faecal and environmental samples,

respectively. The analyses of MALDI-TOF and PCR showed the same

outcomes for the identification of isolates at the species level (Appendix 2.3).

78

Of the 408 culturable samples, C. jejuni and C. coli were identified from 314

(77.0%) and 87 (21.3%) samples, respectively, and seven samples (1.7%)

were positive for both (Table 2.6). Consequently, 321 C. jejuni and 94 C. coli

isolates identified in each culturable sample from these free-range broiler

farms were selected for flaA-HRM PCR analysis. C. jejuni was the most

frequently isolated species in 14 sheds of both Exp.1 and Exp.2, whereas, C.

coli was the most frequently isolated species in three sheds of Exp.1 (Table

2.6).

Table 2.6: Summary of the isolation of Campylobacter jejuni and

Campylobacter coli from samples collected from broiler farms.

Shed

Samples Campylobacter species

identified

Tested Positive % C.

jejuni

C.

coli

C. jejuni

and C. coli

FB1–A1–Exp.1 45 11 24.4 11 0 0

FB1–T–Exp.1 213 42 19.7 42 0 0

FB1–A2–Exp.1 45 20 44.4 20 0 0

FB2–A1–Exp.1 45 11 24.4 10 1 0

FB2–T–Exp.1 211 45 21.3 34 9 2

FB2–A2–Exp.1 45 12 26.7 0 12 0

FB3–A1–Exp.1 34 8 23.5 0 8 0

FB3–T–Exp.1 161 46 28.6 1 45 0

FB3–A2–Exp.1 34 0 0.0 0 0 0

FB1–A1–Exp.2 45 16 35.6 8 6 2

FB1–T–Exp.2 214 42 19.6 42 0 0

FB1–A2–Exp.2 45 21 46.7 20 1 0

FB2–A1–Exp.2 45 11 24.4 11 0 0

FB2–T–Exp.2 210 45 21.4 45 0 0

FB2–A2–Exp.2 45 11 24.4 11 0 0

FB3–A1–Exp.2 45 12 26.7 10 1 1

FB3–T–Exp.2 209 43 20.6 40 3 0

FB3–A2–Exp.2 45 12 26.7 9 1 2

Total 1736 408 23.5 314 87 7

2.3.3 Genetic diversity of Campylobacter jejuni and Campylobacter coli

The C. jejuni and C. coli isolates were characterised with a flaA-HRM PCR

assay and determining the nucleotide sequences of the flaA amplicons. The

isolates with the same genotype were determined by grouping isolates with

similarly shaped HRM curve profiles and on the basis of the amplicon melting

temperature (Tm). In this study, C. jejuni flaA-HRM cluster 27 was used to

79

determine the variation of Tm within the same genotype. The results showed

that a difference in Tm of ± 0.5°C was used as a cut-off value to determine

the same genotype (Appendix 2.3.1 B and Appendix 2.3.2 C).

All C. jejuni (n=406) and C. coli (n=145) isolates identified from the 526

culturable samples were categorized into 41 (Table 2.7) and 25 (Table 2.8)

flaA-HRM clusters, respectively. For C. jejuni, 32 and 6 flaA-HRM clusters

were found in the breeder and the broiler farms, respectively, and the

remaining three flaA-HRM clusters (clusters 5, 6, and 26) were identified in

both. Among the 26 C. coli flaA-HRM clusters, 21 and 2 flaA-HRM clusters

were identified exclusively in breeder or broiler farms, respectively. The

remaining three flaA-HRM clusters (clusters 3, 5, and 13) were common to

both breeder and broiler farms.

80

Table 2.7: Clustering of Campylobacter jejuni isolates from breeder farms and free-range broiler sheds using High Resolution Melt Polymerase Chain


flaA-HRM profile

(Cluster)

flaA allele

(Peptide, Nucleotide)

Breeder farm(s)

(number of isolates)

Free-range broiler shed(s)


Total number

of isolate(s)

1 4, 57 – FB1–A1–Exp.1 (10), FB1–T–Exp.1 (16),

and FB3–T–Exp.2 (36) 62

2 11, 14

– FB1–A1–Exp.1 (1), FB1–T–Exp.1 (26),

FB1–A2–Exp.1 (20), FB2–A1–Exp.1 (1),

and FB2–T–Exp.1 (14)

62

3 20, 208 – FB2–A1–Exp.1 (9) and FB2–T–Exp.1 (21) 30

4 20, 18a BD–F (1) – 1

5 20, 18b BD–C (3) and BD–F (2) FB2–T–Exp.1 (1) 6

6 9, 239a BD–F (2)

FB1–A1–Exp.2 (1), FB2–A1–Exp.2 (4),

FB2–T–Exp.2 (1), FB2–A2–Exp.2 (10),

FB3–T–Exp.1 (1), FB3–A1–Exp.2 (11),

FB3–T–Exp.2 (1), and FB3–A2–Exp.2 (10)

41

7 9, 239b BD–F (2) – 2

8 125, 419 BD–A (1), BD–B (3) and BD–C

(2) – 6

9 8a BD–G (1) – 1

10 8b BD–B (4) and BD–F (5) – 9

11 1a BD–B (1) – 1

12 1b BD–C (2) – 2

81


Reaction targeting flaA gene (flaA-HRM PCR) analysis and flaA sequencing (cont’)

flaA-HRM profile

(Cluster)

flaA allele


Breeder farm(s)




Total number of

isolate(s)

13 1, 56 BD–B (1) – 1

14 1, 34a BD–A (2) – 2

15 1, 34b BD–C (1) – 1

16 1, 34c BD–B (1) and BD–C (2) – 3

17 11a BD–G (2) – 2

18 11b BD–G (1) – 1

19 11c BD–C (1) – 1

20 3, 106 BD–C (1) – 1

21 1, 36a BD–G (5) – 5

22 1, 36b BD–A (9) – 9

23 1, 467a BD–A (3) – 3

24 1, 467b BD–F (1) – 1

25 33, 222 BD–A (3) – 3

26 1, 105 BD–A (1) and BD–F (1) FB2–T–Exp.2 (1), FB3–T–Exp.2

(2), and FB3–A2–Exp.2 (1) 6

27 12, 16a

–

FB1–A1–Exp.2 (9), FB1–T–Exp.2

(42), FB1–A2–Exp.2 (20), FB2–

A1–Exp.2 (7), FB2–T–Exp.2 (40),

FB2–A2–Exp.2 (1), and FB3–T–

Exp.2 (1)

120

82



flaA-HRM profile

(Cluster)

flaA allele


Breeder farm(s)




Total number of

isolate(s)

28 257, 1033 – FB2–T–Exp.2 (1) 1

29 27, 2 – FB2–T–Exp.2 (2) 2

30 2, 612 BD–G (4) – 4

31 1, 32a BD–G (5) – 5

32 1, 32b BD–G (1) – 1

33 11, 30a BD–G (2) – 2

34 8, 67 BD–G (1) – 1

35 5 BD–G (1) – 1

36 1, 8a BD–F (2) – 2

37 1c BD–F (1) – 1

38 10, 28a BD–F (1) – 1

39 2, 54 BD–F (1) – 1

40 5, 5a BD–F (1) – 1

41 15 BD–F (1) – 1

83

Table 2.8: Clustering of Campylobacter coli isolates from breeder farms and free-range broiler sheds using High Resolution Melt Polymerase Chain


flaA-HRM profile

(Cluster)

flaA allele


Breeder farm(s)




Total number

of isolate(s)

1 1, 769 – FB2–A1–Exp.1 (1) 1

2 97, 256 – FB2–T–Exp.1 (5), FB2–A2–Exp.1 (3), and

FB3–T–Exp.2 (1) 9

3 11, 30b BD–A (2), BD–B (1), BD–C (1)

and BD–G (2)

FB2–T–Exp.1 (6), FB2–A2–Exp.1 (9),

FB3–A1–Exp.1 (7), and FB1–A1–Exp.2 (7) 35

4 1, 36c BD–A (1) – 1

5 1, 36d BD–A (2)

FB3–A1–Exp.1 (1), FB3–T–Exp.1 (45),

FB1–A2–Exp.2 (1), FB3–A1–Exp.2 (2),

FB3–T–Exp.2 (2), and FB3–A2–Exp.2 (3)

56

6 21, 13 BD–A (4), BD–B (1) and BD–C

(7)

– 12

7 1d BD–B (1) – 1

8 1e BD–C (1) – 1

9 11d BD–B (1) – 1

10 11e BD–B (1) – 1

11 1, 34d BD–B (1) – 1

12 1, 22 BD–B (1) – 1

13 12, 16b BD–B (1) and BD–F (2) FB1–A1–Exp.2 (1) 4

14 8c BD–C (1) – 1

84

Table 2.8: Clustering of Campylobacter coli isolates from breeder farms and free-range broiler sheds using High Resolution Melt Polymerase Chain


flaA-HRM profile

(Cluster)

flaA allele


Breeder farm(s)




Total number of

isolate(s)

15 8d BD–B (2) – 2

16 9, 239c BD–B (1) – 1

17 1, 467c BD–A (2) – 2

18 1, 467d BD–F (1) – 1

19 1, 467e BD–F (4) – 4

19 10, 28b BD–F (4) – 4

20 New BD–F (1) – 1

21 1, 8b BD–F (1) – 1

22 20, 18c BD–F (1) – 1

23 4 BD–F (1) – 1

24 5, 5b BD–F (1) – 1

25 33 BD–F (1) – 1

85

2.3.3.1 Genetic diversity of Campylobacter jejuni and Campylobacter coli

in breeder farms (Farms BD–A, BD–B, BD–C, BD–F, and BD–G)

Overall, more than one flaA-HRM cluster of C. jejuni and C. coli were

identified in most breeder farms, except farm BD–G where one genotype of

C. coli was identified (Table 2.9). All C. jejuni and most C. coli isolates from

breeder farms were divided into the same numbers of genotypes using either

flaA-HRM PCR analysis or flaA sequencing (Table 2.9).

All C. jejuni isolates (n=85) isolated from breeder farms were classified into

35 flaA-clusters, consistent with flaA amplicon sequencing (Tables 2.6 and

2.8). Among these 35 clusters, 12 and 20 clusters were identified in Exp.1 and

Exp.2, respectively, and the remaining three were found in both experiments.

C. jejuni clusters 10 (n=9) and 22 (n=9) were the most frequently isolated

genotypes among the breeder farm (Table 2.7).

By contrast, all C. coli isolates (n=51) among the breeder farms were assigned

to 23 flaA-HRM clusters (n=12) with only one dominant cluster and 22

smaller groups of clusters (Table 2.8). Of these, 12 and 8 clusters were

identified in Exp.1 and Exp.2, respectively, and three were isolated from both

experiments. Most flaA-HRM clusters of C. coli were related to flaA amplicon

sequencing, except for cluster 19. Cluster 19 had eight isolates (isolate no.

1967, 1999, 2004, 2022, 2036, 2052, 2058, and 2087) from one farm (BD–

F). These isolates showed a similar HRM profile, but they had two different

flaA amplicon sequences as shown in Table 2.8 and Appendix 2.2.5 B. C. coli

cluster 6 (n=12) was the most frequently identified, followed by C. coli cluster

3 (n=6). The remaining 22 clusters were less frequently detected (Table 2.8).

BD–A: Six and five distinct flaA-HRM clusters were identified in C. jejuni

(n=19) and C. coli (n=11) isolates, respectively (Table 2.9). Six flaA-HRM

clusters of C. jejuni comprised clusters 8 (flaA allele 125, 419), 14 (flaA allele

1, 34a), 22 (flaA allele 1, 36b), 23 (flaA allele 1, 467a), 25 (flaA allele 33,

222), and 26 (flaA allele 1, 105) as shown in Table 2.7 and Appendix 2.2.1 A.

Cluster 22 was the most common genotype in farm D (n=9), followed by

clusters 23 (n=3) and 25 (n=3). The remaining three clusters, 8 (n=1), 14

(n=2), and 26 (n=1), were less frequently identified. For C. coli, five flaA-

HRM clusters consisted of clusters 3 (flaA allele 11, 30b), 4 (flaA allele 1,

86

36c), 5 (flaA allele 1, 36d), 6 (flaA allele 21, 13), and 17 (flaA allele 1, 467)

as shown in Table 2.8 and Appendix 2.2.1 B. Among these, the cluster 6 (n=4)

was the most common genotype. While clusters 3 (n=2), 4 (n=1), 5 (n=2), and

17 (n=2) were less frequent genotypes.

BD–B: Five and ten distinct flaA-HRM clusters were identified in C. jejuni

(n=10) and C. coli (n=11) isolates, respectively (Table 2.9). Five flaA-HRM

clusters of C. jejuni were clusters 8 (flaA allele 125, 419), 10 (flaA allele 8b),

11 (flaA allele 1a), 13 (flaA allele 1, 56), and 16 (flaA allele 1, 34c) as

described in Table 2.7 and Appendix 2.2.2 A. The C. jejuni clusters 8 (n=3)

and 10 (n=4) were the most common genotypes, while other genotypes were

less frequently identified (Appendix 2.2.2 A). In comparison, 10 flaA-HRM

clusters of C. coli were clusters 3 (flaA allele 11, 30b), 6 (flaA allele 21, 13),

7 (flaA allele 1d), 9 (flaA allele 11d), 10 (flaA allele 11e), 11 (flaA allele 1,

34d), 12 (flaA allele 1, 22), 13 (flaA allele 12, 16b), 15 (flaA allele 8d), and

16 (flaA allele 9, 239c) as described in Table 2.8 and Appendix 2.2.2 B. Of

these 10 clusters, cluster 15 was detected in two samples, while other clusters

were represented by one isolate each.

BD–C: Seven and four distinct flaA-HRM clusters were identified in C. jejuni

(n=12) and C. coli (n=10) isolates, respectively (Table 2.9). Seven flaA-HRM

clusters of C. jejuni were clusters 5 (flaA allele 20, 18b), 8 (flaA allele 125,

419), 12 (flaA allele 1b), 15 (flaA allele 1, 34b), 16 (flaA allele 1, 34c), 19

(flaA allele 11c), and 20 (flaA allele 3, 106) as described in Table 2.7 and

Appendix 2.2.3 A. Cluster 5 (n=3) was the most frequent genotype, followed

by clusters 8 (n=2), 12 (n=2), and 16 (n=2). Whilst, clusters 19 (n=1) and 20

(n=1) were separately isolated from one sample. In comparison, four flaA-

HRM clusters of C. coli were clusters 3 (flaA allele 11, 30b), 6 (flaA allele

21, 13), 8 (flaA allele 1e), and 14 (flaA allele 8c) as described in Table 2.8

and Appendix 2.2.3 B. C. coli cluster 6 (n=7; flaA allele 21, 13) was the most

common genotype, whereas the remaining clusters were represented by single

isolates.

BD–F: Seven and four distinct flaA-HRM clusters were identified in C. jejuni

(n=21) and C. coli (n=17) isolates, respectively (Table 2.9). 13 different flaA-

HRM clusters of C. jejuni were clusters 4 (flaA allele 20, 18a), 5 (flaA allele

20, 18b), 6 (flaA allele 9, 239a), 7 (flaA allele 9, 239b), 10 (flaA allele 8b), 24

87

(flaA allele 1, 467b), 26 (flaA allele 1, 105), 36 (flaA allele 1, 8a), 37 (flaA

allele 1c), 38 (flaA allele 10, 28a), 39 (flaA allele 2 ,54), 40 (flaA allele 5,5a),

and 41 (flaA allele 5) as described in Table 2.7 and Appendix 2.2.5 A. The C.

jejuni cluster 10 (n=5) was the most common genotype. In comparison, nine

flaA-HRM clusters of C. coli were clusters 13 (flaA allele 12,16b), 18 (flaA

allele 1, 467d), 19 (flaA allele 1, 467e and flaA allele 10, 28), 21 (unassigned

flaA allele), 22 (flaA allele 1, 8b), 23 (flaA allele 201, 18c), 24 (flaA allele 4),

25 (flaA allele 5, 5b), and 26 (flaA allele 33). By contrast, 10 different flaA

amplicon sequences were identified among nine clusters as described in Table

2.8 and Appendix 2.2.5 B. The C. coli cluster 19 (eight isolates) had a similar

HRM containing two different flaA sequences and was the most frequent

genotype with four isolates of each flaA type (flaA allele 1, 467e, n=4; and

flaA allele 10, 28, n=4). Following this, cluster 13 was found in 2 samples.

The remaining clusters —clusters 18, 21, 22, 23, 24, 25 and 26— were

represented by single isolates.

BD–G: Ten and one distinct flaA-HRM clusters were identified in C. jejuni

(n=23) and C. coli (n=1) isolates, respectively (Table 2.9). All C. jejuni

isolates (n = 23) from this farm were classified into 10 flaA-HRM clusters:

clusters 9 (flaA allele 8a), 17 (flaA allele 11a), 18 (flaA allele 11b), 21 (flaA

allele 1, 36a), 30 (flaA allele 2, 612), 31 (flaA allele 1, 32a), 32 (flaA allele 1,

32b), 33 (flaA allele 11, 30a), 34 (flaA allele 8, 67), and 35 (flaA allele 5) as

described in Table 2.7 and Appendix 2.2.4 A. C. jejuni clusters 21, 30 and 31

were most common genotypes. In contrast, only one flaA-HRM cluster of C.

coli was identified in two isolates and it was classified into cluster 3 (flaA

allele 11, 30b) (Table 2.8 and Appendix 2.2.4 B).

88

Table 2.9: Classification of Campylobacter jejuni and Campylobacter coli

clusters isolated from breeder farms

Farm Species No. of

isolates

No. of clusters

flaA-HRM

profile(s)

flaA sequence

type(s)

BD–A C. jejuni 19 6 6

C. coli 11 5 5

BD–B C. jejuni 10 5 5

C. coli 11 10 10

BD–C C. jejuni 12 7 7

C. coli 10 4 4

BD–F C. jejuni 21 13 13

C. coli 17 9 10

BD–G C. jejuni 23 10 10

C. coli 2 1 1

2.3.3.2 Genetic diversity of Campylobacter jejuni and Campylobacter coli

in free-range broiler farms (FB1, FB2, and FB3)

All C. jejuni (n=231) and C. coli (n=94) isolates from 17 broiler sheds were

distinguished into nine and five flaA-HRM clusters, respectively, consistent

with flaA sequencing (Tables 2.6 and 2.7). Among nine C. jejuni flaA- HRM

clusters, three (clusters 2, 3, and 5) and four (clusters 26, 27, 28, and 29) were

identified in Exp.1 and Exp.2, respectively, and the remaining two (clusters 1

and 6) were found in both. By contrast, one C. coli flaA-HRM cluster was

exclusively isolated from Exp.1 (cluster 1) and one exclusively from Exp.2

(cluster 13). The remaining three C. coli flaA- HRM clusters (clusters 2, 3,

and 5) were isolated from both experiments.

The most frequently isolated flaA- HRM clusters of C. jejuni and C. coli

varied between the broiler sheds and the experiments (Exp.1 and Exp.2). A

few flaA-HRM clusters were detected in some broiler sheds in both

experiments. Some C. jejuni and C. coli flaA- HRM clusters showed a wide

distribution among broiler sheds but were not the most frequently isolated

flaA- HRM clusters. Five C. jejuni flaA-HRM clusters (clusters 1, 2, 3, 6 and

27) and two C. coli clusters (clusters 3 and 5) were identified as the most

frequent genotype in different broiler farms between experiments (Tables 2.6

and 2.7).

89

The C. jejuni cluster 27 was found in the seven sheds of Exp.2 (Table 2.7),

and it was the most frequently isolated flaA-HRM cluster in five broiler sheds

(FB1–A1–Exp.2, FB1–T–Exp.2, FB1–A2–Exp.2, FB2–A1–Exp.2, and FB2–

T–Exp.2). Following this, C. jejuni cluster 6 was isolated from samples of

eight broiler sheds from both Exp.1 and Exp.2 (Table 2.7), and it was the most

frequently isolated flaA-HRM cluster only in three broiler sheds (FB2–A2–

Exp.2, FB3–A1–Exp.2, and FB3–A2–Exp.2). C. jejuni cluster 1 was

identified in three broiler sheds from both experiments (Table 2.7) and it was

the most frequent genotype in two sheds (FB1–A1–Exp.1 and FB3–T–

Exp.2). The flaA-HRM clusters 2 and 3 of C. jejuni were isolated only from

Exp.1 (Table 2.7). C. jejuni cluster 2 was the most frequently isolated flaA-

HRM cluster in two of five sheds identified (FB1–T–Exp.1 and FB1–A1–

Exp.1). C. jejuni cluster 3 was not only detected in FB2–A1–Exp.1 and FB2–

T–Exp.1 but was also the most frequently isolated flaA- HRM cluster of these

two sheds.

In comparison, two C. coli flaA-HRM clusters — clusters 3 and 5 — were the

most frequently isolated flaA-HRM clusters, particularly in Exp.1 (Table 2.8).

The C. coli cluster 3, isolated from four broiler sheds, was the most frequently

isolated flaA-HRM cluster in FB2–A2–Exp.1 and FB3–A1–Exp.1. While the

C. coli cluster 5 was found in five broiler sheds and it was only the most

frequently isolated flaA-HRM cluster in F32–T–Exp.1 (Table 2.8).

Free-range broiler farm 1 (FB1): a total of 73 C. jejuni isolates were

identified in Exp.1, and they were grouped into two flaA-HRM clusters:

clusters 1 (flaA allele 4, 57) and 2 (flaA allele 11, 14) as described in Appendix

2.3.1 A. Both C. jejuni flaA-HRM clusters 1 and 2 were isolated from FB1–

A1–Exp.1 and FB1–T–Exp.1, whereas, only the C. jejuni flaA-HRM cluster

2 was additionally isolated from FB1–A2–Exp.1. The C. jejuni cluster 1 was

the most frequently isolated flaA-HRM cluster in FB1–A1–Exp.1 (n=10).

While the C. jejuni cluster 2 was the most frequently isolated flaA-HRM

cluster in FB1–T–Exp.1 (n=26) and FB1–A2–Exp.1 (n=20).

In Exp.2, both C. jejuni and C. coli were isolated from FB1–A1–Exp.2 and

FB1–A2–Exp.2, while C. jejuni was the only one species in FB1–T–Exp.1.

Seventy-two C. jejuni isolates were grouped into two flaA-HRM clusters:

clusters 6 (flaA allele 9,239a) and 27 (flaA allele 12, 16a) as described in

90

Appendix 2.3.1 B. The C. jejuni cluster 27 was the most frequently isolated

flaA-HRM cluster among FB1–A1–Exp.2 (n=9), FB1–T–Exp.2 (n=42) and

FB1–A2–Exp.2 (n=20). The C. jejuni flaA-HRM cluster 6 was isolated from

one sample of FB1–A1–Exp.2. In contrast, C. coli was not the most frequent

flaA-HRM clusters on this farm. Nine C. coli isolates resulted in 3 flaA-HRM

clusters: clusters 3 (flaA allele 11,30b), 5 (flaA allele 11,16b), and 13 (flaA

allele 1,36d) as described in Appendix 2.3.1 C. The C. coli cluster 3 was the

only flaA-HRM cluster isolated from FB1–A1–Exp.2 (n=7). C. coli clusters

12 (n=1) and 13 (n=1) were isolated from FB1–T–Exp.2 and FB1–A2–Exp.2,

respectively.

Free-range broiler farm 2 (FB2): Forty-six C. jejuni isolates were identified

in Exp.1 from FB2–A1–Exp.1 (n=10) and FB2–T–Exp.1 (n=36), and were

grouped into three flaA-HRM clusters: clusters 2 (flaA allele 11, 14), 3 (flaA

allele 20, 208), and 5 (flaA allele 20, 18b) as described in Appendix 2.3.2 A.

The C. jejuni cluster 3 was the most frequently isolated flaA-HRM cluster

(FB2–A1–Exp.1, n=9; and FB2–T–Exp.1, n=21). Following this, the C. jejuni

cluster 2 was less common (FB2–A1–Exp.1; n=1 and FB2–T–Exp.1, n=14).

C. jejuni cluster 5 was isolated from one sample of FB2–T–Exp.1. Moreover,

twenty-four C. coli isolates were identified in FB2–T–Exp.1 (n = 11), FB2–

A1–Exp.1 (n=1), and FB2–A2–Exp.1 ( n=12). These isolates were grouped

into three flaA-HRM clusters: clusters 1 (flaA allele 1, 769), 2 (flaA allele 97,

256), and 3 (flaA allele 11, 30b) as described in Appendix 2.3.2 B. The C. coli

clusters 2 (n=8) and 3 (n=15) were the most frequently isolated flaA-HRM

cluster in FB2–T–Exp.1 and FB2–A2–Exp.1. In contrast, C. coli cluster 1

(n=1) was less common and it was isolated only from the FB2–A1–Exp.1.

In Exp.2, sixty-seven C. jejuni isolates were recovered from FB2–A1–Exp.2

(n=11), FB2–T–Exp.2 (n=40), and FB2–A2–Exp.2 (n=11) and these were

grouped into 5 flaA-HRM clusters: clusters 6 (flaA allele 9,239a), 26 (flaA

allele 1,105), 27 (flaA allele 12, 16a), 28 (flaA allele 257, 1033), and 29 (flaA

allele 27, 2) as described in Appendix 2.3.2 C. The C jejuni cluster 27 was

the most frequently isolated flaA-HRM cluster in FB2–A1–Exp.2 (n=7) and

FB2–T–Exp.2 (n=40), whereas it was only isolated from one sample in FB2–

A2–Exp.2 (n=1). The C. jejuni cluster 6 was the most frequently isolated flaA-

HRM cluster in FB2–A2–Exp.2 (n=10), whereas it was less common in FB2–

91

A1–Exp.2 (n=4) and FB2–T–Exp.2 (n=1). The remaining three flaA-HRM

clusters (clusters 26, 28, and 29) were only isolated from a few samples from

FB2–T–Exp.2.

Free-range broiler farm 3 (FB3): one C. jejuni isolate was identified in

Exp.1 from FB3–T–Exp.1 and assigned to flaA-HRM cluster 6 (flaA allele

9,239a) as described in Appendix 2.3.3 A. By contrast, C. coli (n=51) was the

majority species isolated from FB3–A1–Exp.1 (n=8) and FB3–T–Exp.1

(n=45) in the same experiment. All fifty-three isolates were grouped into two

flaA-HRM clusters: clusters 3 (flaA allele 11,30b) and 5 (flaA allele 1,36d) as

described in Appendix 2.3.3 B. The C. coli cluster 3 was the most frequently

flaA-HRM cluster in FB3–A1–Exp.1 (n=7). The C. coli cluster 5 was the most

frequently flaA-HRM cluster in FB3–T–Exp.1 (n=45), whereas it was only

isolated from one sample of FB3–T–Exp.1 (n=1).

In Exp.2, sixty-two C. jejuni isolates were identified in FB3–A1–Exp.2

(n=11), FB3–T–Exp.2 (n=40) and FB3–A2–Exp.2 (n=10). These isolates

were grouped into four flaA-HRM clusters: clusters 1 (flaA allele 4, 57), 6

(flaA allele 9,239a), 26 (flaA allele 1,105), and 27 (flaA allele 12,16a) as

described in Appendix 2.3.3 C. The C. jejuni cluster 1 was the most frequently

isolated flaA-HRM cluster in FB3–T–Exp.2 (n=36). The C. jejuni cluster 6

was the most frequently isolated flaA-HRM cluster in FB3–A1–Exp.2 (n=11)

and FB3–A2–Exp.2 (n=10), whereas it was a minimal frequently isolated

flaA-HRM cluster in FB3–T–Exp.2 (n=1). The C. jejuni cluster 26 was a less

frequently flaA-HRM cluster in FB3–T–Exp.2 (n=2) and FB3-A2-Exp.2

(n=1). The C. jejuni cluster 27 was isolated from one sample of FB3–T–

Exp.2. Moreover, eight C. coli isolates were isolated from FB3–T–Exp.2

(n=2), FB3–T–Exp.2 (n=3), and FB3–A2–Exp.2 (n=3) and these were

grouped into two flaA-HRM clusters: clusters 2 (flaA allele 97, 256) and 5

(flaA allele 1, 36d) as described in Appendix 2.3.3 D. The C. coli cluster 5

was isolated from all sheds (FB3–A1–Exp.2, n=2; FB3–T–Exp.2, n=2; and

FB3–A2–Exp.2, n=3), whereas the C. coli cluster 2 was isolated from FB3–

T–Exp.2 (n=1) only.

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2.3.3.3 Clustering of Campylobacter jejuni and Campylobacter coli from

free-range broiler farms with flaA-HRM clusters, flaA allele number and

MLST

Representative C. jejuni (n=10) and C. coli (n=5) isolates representing flaA-

HRM clusters obtained from the broiler farms were further characterised by

MLST analysis. The genotyping of C. jejuni and C. coli isolates using MLST

and flaA-HRM analyses resulted in similar groupings (Table 2.10). On the

basis of MLST analysis, nine sequence types (ST) were identified in among

C. jejuni isolates (Table 2.10). Six of these could be assigned to recognised

five clonal complexes (CCs) included ST-353 complex (the C. jejuni flaA-

HRM cluster 1), ST-354 complex (the C. jejuni flaA-HRM cluster 5), ST-45

complex (the C. jejuni flaA-HRM clusters 6 and 29), ST-206 complex (the C.

jejuni flaA-HRM cluster 26), and ST-257 complex (the C. jejuni flaA-HRM

cluster 27). Of these, the C. jejuni ST-257 complex (the C. jejuni flaA-HRM

cluster 27) was the most frequent genotype in five broiler sheds (Table 2.7,

Appendices 2.3.1 B and 2.3.2 C). Following this, the C. jejuni ST-45 complex

(the C. jejuni flaA-HRM clusters 6 and 29) was the most common genotype

in three broiler sheds (Table 2.7, Appendices 2.3.2 C and 2.3.3 C). Three C.

jejuni flaA-HRM clusters (clusters 1, 2, and 3) individually were the most

frequent genotypes in two broiler sheds (Table 2.7, Appendices 2.3.1 A and

2.3.2 A). While the remaining three C. jejuni flaA-HRM clusters (clusters 2,

3, and 28) were new CCs.

As for C. coli, five flaA-HRM clusters were determined using flaA allelic

number and MLST showed similar results (Table 2.10). All five C. coli

clusters were identified as belonging to two different STs and three new STs.

Two STs (ST-860 and ST-966) from C. coli flaA-HRM clusters 1 and 2

seemed to belong to the same CC (ST-828 complex), whereas the remaining

three new genotypes (flaA-HRM clusters 3, 5, and 13) could not be assigned

to any STs and CCs. Among these new genotypes, C. coli clusters 3 and 5

were the most frequent genotypes in two and one sheds, respectively (Table

2.8, Appendices 2.3.2 B and 2.3.3 A).

93

Table 2.10: Classification of selected isolates of representative Campylobacter jejuni and Campylobacter coli genotypes from broiler farms, based on

flaA-HRM clusters, flaA allele no. and MLST

Species flaA-HRM

Cluster

flaA allele no. MLST house-keeping genes allele no. Sequence Type

(ST)

Clonal Complex

(CC) Peptide Nucleotide asp gln glt gly pgm tkt unc

C. jejuni 1 4 57 181 17 5 10 11 3 6 4896 ST-353 complex

C. jejuni 2 11 14 166 2 27 10 151 3 1 7323 New

C. jejuni 3 20 208 8 2 2 212 309 253 147 2083 New

C. jejuni 5 20 18b 74 10 2 2 11 12 6 528 ST-354 complex

C. jejuni 6 9 239a 4 7 10 4 42 51 1 583 ST-45 complex


C. jejuni 27 12 16a 9 2 4 62 4 5 6 257 ST-257 complex

C. jejuni 28 257 1033 1 165 5 91 261 7 1 6998 New


C. coli 1 1 769 33 39 30 79 113 47 17 860 ST-828 complex

C. coli 2 97 256 33 39 30 79 112 47 17 966 ST-828 complex

C. coli 3 11 30b 33 39 122 79 188 47 79 New New

C. coli 5 1 36d 33 39 30 79 113 43 79 New New

C. coli 13 12 16b 33 39 30 79 188 47 79 New New

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2.3.4 Dynamics of Campylobacter colonisation in broiler flocks

(between flocks and the experiments)

Most broiler sheds showed similar patterns of C. jejuni and/or C. coli

colonisation between farms and the experiments. The same C. jejuni and/or

C. coli flaA-HRM clusters were identified in the environment and the faecal

samples in most broiler sheds (11 sheds) at the same time approximately 3

weeks after placement. While some similar C. jejuni and/or C. coli flaA-HRM

clusters were identified in the environment of three sheds (FB2–T–Exp.1,

FB2–A2–Exp.1, and FB3–A1–Exp.2) before these bacteria were isolated

from chicken faeces. In addition, some C. jejuni and/or C. coli flaA-HRM

clusters were exclusively discovered among different sheds, different farms,

and the experiments.

Free-range Broiler Farm 1 (FB1): All C. jejuni isolates (n=73) identified in

Exp.1 belonged to flaA-HRM clusters 1 and 2 (Table 2.11 and Appendix 2.3.1

A). The dynamics of C. jejuni colonisation are shown in Figure 2.2. the C.

jejuni flaA-HRM cluster 2 was first isolated from faecal samples (n=10) in

FB1–A2–Exp.1 on Day 15. One week later, Day 22, this cluster was

recovered from faecal samples of all sheds (FB1–A1–Exp.1, n=1; FB1–T–

Exp.1, n=23; and FB1–A2–Exp.1, n=10), farm boots and the environment of

FB1–T–Exp.1 (drinking water and free-range area). Moreover, the C. jejuni

flaA-HRM cluster 1 was also identified on Day 22 in faecal samples from

FB1–A1–Exp.1 (n=9) and FB1–T–Exp.1 (n=12) as well as the free-range area

of FB1–A1–Exp.1 and the environment of FB1–T–Exp.1 (floors, wall, and

shed boots).

In Exp.2, both C. jejuni and C. coli were identified in FB1–A1–Exp.2 and

FB1–A2–Exp.2, whereas C. jejuni was only found in FB1–T–Exp.2. The

dynamics of C. jejuni and C. coli colonisation are shown in Figure 2.2. All C.

jejuni isolates (n=72) belonged to flaA-HRM clusters 6 and 27 (Table 2.11

and Appendix 2.3.1 B). The C. jejuni cluster 27 was isolated from ten faecal

samples of FB1–A2–Exp.2 for the first time on Day 15. One week later, Day

22, this cluster was found among the sheds and the environment, such as

faecal samples (FB1–A1–Exp.2, n=8; FB1–T–Exp.2, n=35; and FB1–A2–

Exp.2, n=10) and samples from the free-range area of FB1–A1–Exp.2 and

FB1–T–Exp.2, farm boots, and the internal environment of FB1–T–Exp.2

95

(anteroom, floors, wall, shed boots). However, the C. jejuni flaA-HRM cluster

6 was isolated from one faecal sample from FB1–A1–Exp.2 on Day 22. All

nine C. coli isolates were assigned to flaA-HRM clusters 3, 5, and 13 (Table

2.11 and Appendix 2.3.1 B). C. coli flaA-HRM clusters 3 and 13 were isolated

from FB1–A1–Exp.2 on Day 22. The C. coli flaA-HRM cluster 3 was first

isolated from five faecal samples on Day 15. Then, some faecal samples of

the same shed were positive for the C. coli flaA-HRM clusters 3 (n=2) and 13

(n=1) on Day 22. Moreover, C. coli flaA-HRM cluster 5 was isolated from

the free-range area of FB1–A2–Exp.2 on Day 15.

96

Figure 2.2: Schematic diagram of the dynamics of C. jejuni and C. coli clusters identified on free-range broiler farm 1 (FB1) in the

experiments 1 and 2

97

Free-range Broiler Farm 2 (FB2): The dynamics of C. jejuni and C. coli

colonisation of Exp.1 are shown in Figure 2.3A. C. jejuni and C. coli were

isolated from FB2–A1–Exp.1 and FB2–T–Exp.1, whereas only C. coli was

isolated from FB2–A2–Exp.1 All C. jejuni isolates (n=46) from Exp.1 were

assigned to flaA-HRM clusters 2, 3, and 5 (Table 2.12 and Appendix 2.3.2

A). The C. jejuni flaA-HRM clusters 2 and 3 were isolated from FB2–A1–

Exp.1 and FB2–T–Exp.1 on Day 22 for the first time. The C. jejuni flaA-

HRM cluster 2 was isolated from the faecal samples from FB2–A1–Exp.1

(n=1) and FB2–T–Exp.1 (n=11) and the environment of FB2–T–Exp.1 (walls

and the free-range area). The C. jejuni flaA-HRM cluster 3 was isolated from

faecal samples (FB2–A1–Exp.1, n=9; and FB2–T–Exp.1, n=20) and the

rodent faeces from FB2–T–Exp.1. In contrast, the C. jejuni flaA-HRM cluster

5 was isolated from the rodent faeces from FB2–T–Exp.1 on Day 8.

Moreover, all C. coli isolates (n=24) were arranged to flaA-HRM clusters 1,

2, and 3 (Table 2.12 and Appendix 2.3.2 B). The C. coli flaA-HRM cluster 1

was isolated from the free-range area of FB2–A1–Exp.1 on Day 8. The C.

coli flaA-HRM cluster 2 was first isolated from the rodent faeces from FB2–

T–Exp.1 and the free-range area of FB2–A2–Exp.1 on Day 1. Then, this

cluster was isolated from other samples from FB2–T–Exp.1 at different time

points, such as shed boots (Day 8) and rodent faeces (Days 8, 15 and 22) as

well as two faecal samples of FB2–A2–Exp.1 (Day 22). The C. coli flaA-

HRM cluster 3 was isolated from a faecal sample of FB2–A2–Exp.1 on Day

15 for the first time. One week later, Day 22, this cluster was found in faecal

samples of different sheds (FB2–A2–Exp.1, n=8; and FB2–T–Exp.1, n=4)

and the floors of FB2–T–Exp.1.

In Exp.2, only C. jejuni was identified and the dynamics of C. jejuni

colonisation are shown in Figure 2.3B. All C. jejuni isolates (n=67) were

assigned to flaA-HRM clusters 6, 26, 27, 28, and 29 (Table 2.12 and Appendix

2.3.2 C). The C. jejuni clusters 28, 26, 6, and 29, isolated from the rodent

faeces from FB2–T–Exp.2, were found on Days 0, 1, 8, and 15, respectively.

While the anteroom floor of the same shed was contaminated with C. jejuni

flaA-HRM cluster 29 on Day 15. Furthermore, the C. jejuni flaA-HRM cluster

27 was found among the sheds and the environment on Day 22, such as faecal

samples (FB2–A1–Exp.2, n=6; and FB2–T–Exp.2, n=35), the free-range area

98

of all three sheds (FB2–A1–Exp.2, FB2–T–Exp.2 and FB2–A2–Exp.2), the

environment of FB2–T–Exp.2 (floors and shed boots), and a sample of farm

boots. At the same time, the C. jejuni flaA nucleotide allele 239 was isolated

from faecal samples of FB2-A1-Exp.2 (n=4) and FB2-A2-Exp.2 (n=10).

99

Figure 2.3A: Schematic diagram of the dynamics of C. jejuni and C. coli clusters identified on free-range broiler farm 2 (FB2) in the

experiment 1

100

Figure 2.3B: Schematic diagram of the dynamics of C. jejuni and C. coli clusters identified on free-range broiler farm 2 (FB2) in the

experiment 2

101

Free-range Broiler Farm 3 (FB3): The dynamics of C. jejuni and C. coli

colonisation in Exp.1 are shown in Figure 2.4A. The C. jejuni flaA-HRM

cluster 6 was isolated from a sample of rodent faeces from FB3–T–Exp.1 on

Day 3 (Table 2.13 and Appendix 2.3.3 A). All C. coli isolates (n=53) from

Exp.1 were assigned to flaA-HRM clusters 3 and 5 (Table 2.13 and Appendix

2.3.3 B). The C. coli flaA-HRM cluster 5 was identified on Day 10 from the

samples of FB3–T–Exp.1 such as faeces (n=3) and shed boots (n=1). At the

same time, Day 10, it was found in the external environment, including farm

boots (n=1) and the free-range area of FB3–A1–Exp.1 (n=1) as well. After

that (Day 17), this cluster persisted in farm boots (n=1) and the samples of

FB3–T–Exp.1 such as the faecal samples (n=35), the free-range area of the

shed, and the internal environment (floor, wall, water pans, shed boots). The

C. coli flaA-HRM cluster 3 was isolated from seven faecal samples of FB3–

A1–Exp.1 on Day 17.

In Exp.2, C. jejuni and C. coli were identified on this farm. The dynamics of

C. jejuni and C. coli colonisation are shown in Figure 2.4B. All C. jejuni

isolates (n=62) from Exp.2 were identified on Day 24 for the first time and

they were assigned to flaA-HRM clusters 1, 6, 26, and 27 (Table 2.13 and

Appendix 2.2.3 C). The C. jejuni flaA-HRM cluster 1 was isolated from the

samples from FB3–T–Exp.2, including the free-range area (n=1), faecal

samples (n=32), and the environment (floors, and shed boots). The C. jejuni

flaA-HRM cluster 6, previously isolated from Exp.1, was also isolated from

farm boots, the free-range areas of FB3–A1–Exp.2 and FB3–A2–Exp.2 as

well as faecal samples of FB3–A1–Exp.2 (n=10) and FB3–A2–Exp.2 (n=9).

The C. jejuni flaA-HRM cluster 26 was isolated from two faecal samples of

FB3–T–Exp.2 and a faecal sample of FB3–A2–Exp.2. Furthermore, the C.

jejuni flaA-HRM cluster 27 was isolated from a faecal sample of FB3–T–

Exp.2. On the other hand, All C. coli isolates (n=8) from Exp.2 were assigned

to flaA-HRM clusters 2 and 5 (Table 2.13 and Appendix 2.2.3 D). The C. coli

cluster 5, previously isolated in Exp.1, was also found in Exp.2 as well. This

cluster was first isolated from the free-range area of FB3–A1–Exp.2 before

chick placement. Two weeks later, Day 17, this cluster was isolated from the

rodent faeces from FB3–T–Exp.2, a faecal sample of FB3–A2–Exp.2 and

farm boots. After that, Day 24, this cluster was isolated from a faecal sample

102

of FB3–A1–Exp.2, and two faecal samples of FB3–A2–Exp.2. Moreover, the

C. coli flaA-HRM cluster 2 was only isolated from the rodent faeces of FB3–

T–Exp.2 on Day 24.

103

Figure 2.4A: Schematic diagram of the dynamics of C. jejuni and C. coli clusters identified on free-range broiler farm 3 (FB3) in the

experiment 1

104

Figure 2.4B: Schematic diagram of the dynamics of C. jejuni and C. coli clusters identified on free-range broiler farm 3 (FB3) in the

experiment 2

105

2.3.5 Similarity of Campylobacter jejuni and Campylobacter coli isolates

from breeders and their progeny (broilers)

For the purposes of this study, vertical transmission was defined as the

identification of the same flaA-HRM cluster(s) of C. jejuni and/or C. coli

being isolated from faecal samples of breeders and their linked broilers. All

C. jejuni and C. coli flaA-HRM clusters obtained from the five breeder farms

and their progeny were assessed for the possibility of vertical transmission of

C. jejuni and C. coli by comparing the flaA-HRM clusters assigned on the

basis of the HRM analyses as summarised in Tables 2.6 and 2.7.

The results showed that the majority of the C. jejuni and C. coli flaA-HRM

clusters collected from the breeder and free-range broiler farms were

genetically distinct. However, three flaA-HRM clusters of C. jejuni (Table

2.7) and C. coli (Table 2.8) shared between some breeders and their linked

broilers.

One of three C. jejuni sharing flaA-HRM clusters, cluster 6, was isolated from

faecal samples of a breeder farm and in faecal samples from its broiler

offspring, despite being located in geographically distant areas (Figure 2.5B).

The C. jejuni flaA-HRM cluster 6 (ST583) was found in two faecal samples

of BD–F, located in QLD (Appendix 2.2.5 A) and one faecal sample of FB1–

A1–Exp.2, located in NSW, on Day 22 (Figure 2.5B).This cluster was also

identified in BF 2 which was supplied chicks from BD–F within the same

experiment (Exp.2) as well. This farm was located in NSW. However, this

cluster was first isolated from one sample of rodent faeces on Day 8 in FB2–

T–Exp.2 for the first time. (Figure 2.5B). Then, it was isolated from faecal

samples of FB2–A1–Exp.2 (n=4) and FB2–A2–Exp.2 (n=10) on Day 22

(Figure 2.5B).

Two of three C. coli sharing flaA-HRM clusters, clusters 3 and 13, were

isolated from the breeder farms were genetically similar to the isolates from

the broiler progeny (Table 2.8). The C. coli flaA-HRM cluster 3 was found in

the breeders and linked broiler, located in both the same and different regions

(Figure 2.5A). This cluster was isolated from two faecal samples of BD–A,

located in QLD (Appendix 2.2.1 B), and samples from FB2 (located in NSW)

in Exp.1 such as faecal samples of FB2–T–Exp.1(n=4, Day 22) and FB2–A2–

106

Exp.1 (n=1, Day 17; n=8, Day 22), and two floor samples of FB2–T–Exp.1

(Figure 2.5A and Appendix 2.3.2 B). Isolates with this cluster were also

isolated from a faecal sample from BD–C (located in NSW) and seven faecal

samples of linked broiler FB3–A1–Exp.1 (located in NSW) on Day 17

(Figure 2.5A and Appendix 2.3.3 B). In addition, the C. coli flaA-HRM

cluster 13 was isolated from the breeder and their progeny located in different

states (Figure 2.5B). This cluster was found in two faecal samples of BD–F

(located in QLD) and one faecal sample of FB1–A1–Exp.2 (located in NSW).

107

Figure 2.5: Schematic diagram of similarity of C. jejuni and C. coli clusters between breeder farms and their progeny in the

experiments 1 (A) and 2 (B)

108

2.4 Discussion

This study revealed that C. jejuni and C. coli were identified in samples from

both breeder and free-range broiler farms, in agreement with previous studies

conducted by O'Mahony et al. (2011) and Vandeplas et al. (2010) who

reported that these two species were found in breeder and free-range broiler

farms, respectively. C. jejuni was the most frequently isolated species in this

study, and this is similar to the results of previous studies of Ingresa-

Capaccioni et al. (2016) and Vandeplas et al. (2010) who reported that C.

jejuni was a predominant species in breeder farms and free-range broiler

flock, respectively. C. jejuni and C. coli were isolated from chicken faeces in

most free-range broiler sheds by 3 weeks of rearing, consistent with several

studies conducted in intensive chicken farming system (Ingresa-Capaccioni

et al., 2015; Ingresa-Capaccioni et al., 2016; Kalupahana et al., 2013;

Messens et al., 2009; Miflin et al., 2001; Thomrongsuwannakij et al., 2017).

By contrast, this study demonstrated that C. coli was the first species found

in chicken faeces of a free-range broiler shed as early as 10 days after chick

placement. Although this finding is in accordance with a study in the UK in

terms of the onset of Campylobacter isolation on a free-range broiler flock,

C. jejuni was the first isolated species at 8 days of rearing (El-Shibiny et al.,

2005). This suggests that the free-range broiler farming system may induce

earlier colonisation, compared with the intensive system, even if there was no

difference in colonisation between the two farming systems in general. Thus,

the further investigation of C. jejuni and C. coli colonisation between the two

different farming systems would improve knowledge of the factors involves

in colonisation of this microorganism.

Some relevant factors involved in the delay of Campylobacter colonisation

have been studied. For example, the persistence of preventive maternal

immunity from parent breeders which generally remains in commercial

chicks until 2–3 weeks of age is associated with the delay of Campylobacter

colonisation (Cawthraw & Newell, 2010; Laniewski et al., 2012; Rice et al.,

1997; Sahin, Luo, et al., 2003; Wyszynska et al., 2004). Biosecurity including

boot dip disinfection and sanitation of water can control Campylobacter

colonisation in chicken more than 50% at farm-level (Gibbens et al., 2001).

The antibiotics used at the farm can be another possible factor affecting the

109

C. jejuni and C. coli colonisation (Allain et al., 2014). However, antibiotics

have not been used on these farms for at least two years in this study prior to

the current study, according to the farm records of antibiotics use. This

suggests that antibiotics have not affected the colonisation of C. jejuni and C.

coli in the free-range broiler farm sampled during study.

The current study also found that once a few colonised chickens in the flock

were detected, most chickens in the same flock and the environment were

later found to be positive for Campylobacter within one week. This suggests

that Campylobacter can rapidly spread within flocks and the environment and

this has been reported previously (van Gerwe et al., 2009).

This study suggests that flaA-HRM PCR is a rapid, reliable and cost-effective

method to differentiate C. jejuni and C. coli isolated from various sources of

commercial free-range broiler farms. This method has been developed by

Merchant-Patel et al. (2010) who reported the flaA-HRM PCR provided a

high discriminatory power for genotyping C. jejuni and C. coli. The flaA gene

is a highly variable gene which is subject to rapid intra-and inter-genomic

recombination between Campylobacter populations but less different in a

clonal structure (Harrington et al., 1997; Meinersmann et al., 2005). The

current data showed that C. jejuni and C. coli flaA-HRM clusters identified

from commercial free-range broiler farms using flaA-HRM PCR were

correlated to flaA sequencing and MLST analysis. This suggests that this

method can be used to differentiate C. jejuni and C. coli genotypes in the

epidemiological studies.

The current data showed that C. jejuni and C. coli flaA-HRM clusters isolated

from colonised chickens were diverse and this was consistent with previous

studies (Bull et al., 2006; Colles et al., 2011; Prachantasena et al., 2016;

Ridley, Allen, et al., 2008; Vidal et al., 2016; Zbrun et al., 2017). In relation

to breeder farms, multiple Campylobacter genotypes were found to be

colonized (Colles et al., 2011). We too showed a wide range of C. jejuni

(thirty-five) and C. coli (twenty-three) flaA-HRM clusters colonising

chickens in the breeder farms. These findings suggest that Campylobacter

colonisation in breeder chickens is a dynamic and accumulative process, as

supported by the notion of repeat exposure in longer-lived breeders (Colles et

al., 2011). By contrast, C. jejuni (nine) and C. coli (five) flaA-HRM clusters

110

from both experiments were less genetic diversity in free-range broiler farms,

compared with those of breeder farms, suggesting that free-range chickens

were initially colonised with a low number of C. jejuni and C. coli genotypes

with some dominant genotypes. A reason for this could be the relatively short

period in this study, regarding our aims of this study. A recent study by

Templeton (2014) showed similar genotype numbers of C. jejuni (seven)

were identified in free-range broiler farm from NSW, based on MLST-HRM

analysis; however, no single C. jejuni genotype dominated. This suggests that

Campylobacter colonisation is a dynamic process from chick placement until

the end of rearing within free-rage farms. Thus, a further study with a full

period of free-range broiler farm production cycle (before chick placement

until slaughter) would provide more knowledge about the dynamics of C.

jejuni and C. coli colonisation and the genetic diversity on the free-range

broiler farming system. However, the relevant factors influencing the genetic

diversity of Campylobacter spp. remain unclear. Several studies suggest that

multiple Campylobacter genotypes from various sources could accumulate

and persist simultaneously within chicken flocks (Colles et al., 2011;

Prachantasena et al., 2016; Ridley, Allen, et al., 2008). By contrast, some

studies have suggested that genetic rearrangements within Campylobacter

populations occur due to their genetic instability and competitive

environmental pressure within the chicken gut, and thus, this could have led

to diverse genotypes (Alter et al., 2011; Ge et al., 2006; Hook et al., 2005;

Ridley, Toszeghy, et al., 2008; Wilson et al., 2009). Based on the results of

this study, the current data suggest that multiple C. jejuni and C. coli

genotypes isolated from free-range broiler faeces are most likely from various

environmental sources, whereas, that of breeder farms are still unclear due to

inadequate numbers of samples and sample types. So, the investigation on the

relevant mechanism involving genetic diversity of C. jejuni and C. coli is

required.

The dynamics of C. jejuni and C. coli colonisation among the free-range

broiler farms generally followed a similar pattern in this study. Most broilers

were colonised by multiple C. jejuni and C. coli clusters which were isolated

from the environment and faecal samples. Some C. jejuni and C. coli flaA-

HRM clusters identified in free-range farms were common among chicken

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faeces from different farms and their environments. This suggests these free-

range broiler farms, located in the same area, may be exposed to a common

environmental source leading to sharing the same genotypes. Some C. jejuni

and C. coli flaA-HRM clusters not only coexisted within a single free-range

broiler shed and its environment but were also found in chicken faeces of the

adjacent sheds and the farm environment, suggesting the spread of the

microorganisms between the broiler chickens and the surrounding

environment on the same farms. These findings indicate that free-range

broiler flocks are exposed to multiple Campylobacter sources contaminating

their environment, as per previous studies (Alter et al., 2011; Anderson et al.,

2012; Conlan et al., 2007; Rivoal et al., 2005; Zweifel et al., 2008). In

addition, Vidal et al. (2016) suggested that new genotypes could be

introduced into broiler flocks during rearing via some other routes.

The current study showed that the dominant C. jejuni and C. coli clusters

varied within each free-range broiler flock depending on the time of sample

collection; this agrees with El-Shibiny et al. (2005) who reported that various

Campylobacter spp. (C. jejuni and C. coli) identified and their genotypes (C.

jejuni; n=3 and C. coli; n=6) were found at different sampling time points

within a single broiler flock during the rearing cycle. The current data

revealed that the pre-existing dominant C. coli was replaced with a new

upcoming C. jejuni in some free-range broiler sheds (FB2–A1–Exp.2 and

FB3–A2–Exp.2; Tables 2.11 and 2.12). This implies that some newly

acquired species could potentially colonise the chickens. By contrast, we

found that when a novel C. coli flaA-HRM cluster isolated from the

environment was introduced to a broiler shed (FB2–A2–Exp.1) and it was

unable to replace the pre-existing C. coli flaA-HRM cluster. This implied that

the new genotype could be less competitive than the pre-existing genotype in

chickens. The inability to displace an existing genotype may be due to it being

highly adapted to its unknown source. It would be interesting to determine if

genotypes like this one would be able to colonise naïve chickens. Competitive

exclusion among multiple Campylobacter genotypes in chickens during

colonisation is suggested to lead to one genotype replicating rapidly and

becoming dominant (Colles et al., 2019; Hook et al., 2005; Pope, Wilson, et

al., 2007; Ridley, Toszeghy, et al., 2008). The current study revealed that a

112

single cluster of Campylobacter persisted in the same sheds (FB1–A2–Exp.1

and FB3–A2–Exp.2) throughout the sample collection, suggesting a single

contact with a particular genotype could persist within a free-range broiler

shed and the environment and, eventually, become a dominant genotype

(Zweifel et al., 2008).

Whether the genotype(s) were able to achieve uniform colonisation through

the competitive exclusion of other genotypes or were due to an introductory

single source is unknown. Cases such as these are of interest for two reasons.

Firstly, if the genotype is highly adept at chicken colonisation, to the point

where it can exclude all other genotypes, it could potentially be used as a

control method. Rather than try to exclude all genotypes from the production

environment, which this study has demonstrated is not possible with current

control methods, a dominant genotype could be introduced to prevent

colonisation by other genotypes. A single and controlled point of introduction

could provide the optimal solution. Subsequent controls to prevent product

contamination could be tailored to this genotype. Alternatively, in cases such

as this where a single genotype dominates the production environment, it may

suggest a single point of introduction or single reservoir in the production

system. If this source could be identified, then targeted control may be

possible. Of course, the risk in specifically targeting the dominant genotype

may result in the emergence of other genotypes. The further study of what

appears to be dominant genotypes are warranted as it should provide insight

into the underlying genetics of Campylobacter spp. fitness for chicken

colonisation.

In the present study, C. jejuni was a common species colonising free-range

broilers and C. jejuni ST 257 was the most frequently isolated genotype, with

the widest distribution. This MLST genotype has been previously identified

in humans and chickens in Australia (Djordjevic et al., 2007; Habib et al.,

2009; Wieczorek et al., 2017) Moreover, Mickan et al. (2007) investigated C.

jejuni isolated from patients in the Hunter region, NSW and reported that C.

jejuni ST 257 was one of the endemic genotypes in human. This suggests that

C. jejuni ST 257 is not only zoonotic but also a pathogenic agent. To identify

the correlation of C. jejuni infections between chickens and humans, a further

investigation on genetic diversity of C. jejuni between humans and chickens

113

from the same region and duration period would confirm this correlation and

the outcome could be useful for epidemiological studies to develop an

effective intervention of Campylobacter infection control in aspects of public

health concern.

In order to reduce or prevent Campylobacter colonisation in chicken farms, it

is important to understand how Campylobacter spp. establish in chicken

farms and which potential sources would affect colonisation of

Campylobacter spp. in chickens. Numerous potential sources affecting

Campylobacter colonisation and transmission have been investigated.

However, most studies have been conducted in conventional intensive poultry

production systems. As results from these studies may not be effectively

applied in free-range production systems such as those investigated in the

current study. The reason for this is that in free-range systems, chickens are

continuously exposed to the external environment which could increase risks

of Campylobacter transmission within flocks and between flocks (Nather et

al., 2009).

This study demonstrated that horizontal transmission plays an important role

in C. jejuni and C. coli colonisation in free-range broiler farms. The

environment such as shed walls, floors (bedding), water pans, and shed boots,

the free-range areas (soil), anteroom, and farm boots were found to be

potential sources of C. jejuni and C. coli colonisation within the free-range

broiler sheds and farms, consistent with previous results (Agunos et al., 2014;

Battersby et al., 2016; Bull et al., 2006; Ellis-Iversen et al., 2012; Newell et

al., 2011; O'Mahony et al., 2011; Patriarchi et al., 2009; Smith et al., 2016).

Other relevant sources related to Campylobacter colonisation were also found

in this study. For example, the current study found fresh rodent faeces in the

free-range broiler sheds to carry multiple Campylobacter strains and, thus, it

may be able to transmit them to the free-range broiler chickens, consistent

with studies from (Meerburg & Kijlstra, 2007); Messens et al. (2009) who

suggested that rodents can serve as a reservoir of Campylobacter and can

spread this microorganism to the broiler flocks. Similarly, the current data

indicate that drinking water in the shed was another potential source of

Campylobacter involved in the spread of Campylobacter within chicken

sheds. This finding agrees with a previous report by Perez-Boto et al. (2010)

114

indicating that shed drinking water was a potential source of Campylobacter

transmission within grandparent breeder farms.

Importantly, the current data reveal that the same C. coli cluster isolated from

the previous experiment (Exp.1) was found in the environment (before the

placement of chicks) and the chicken faeces in the associated flock for the

next experiment (Exp.2) on the same farm. This demonstrates the potential

for carryover or reintroduction of Campylobacter via the common

environment between consecutive free-range broiler sheds. By contrast, the

majority of C. jejuni and C. coli clusters identified in free-range broiler farms

were distinct between Exp.1 and Exp.2, suggesting that the all-in-all-out

system and farm practices (cleaning and disinfection) during the empty period

can eliminate C. jejuni and C. coli genotypes between cycles of free-range

farm productions. Thus, based upon the findings of the present study,

improved hygiene practices and appropriate biosecurity measures could

potentially reduce Campylobacter transmission in broiler farms (de Castro

Burbarelli et al., 2017; Smith et al., 2016).

As breeders supply the fertilised eggs for multiple generations of broiler

chickens (Australian Chicken Meat Federation-ACMF, 2018a), the

possibility of vertical transmission of Campylobacter transferring from

breeders to broilers is of interest. If vertical transmission was an important

source of broiler colonisation, Campylobacter spp. control in the breeder

birds could be an effective intervention point. Even though previous studies

have reported that vertical transmission of Campylobacter did not occur on

broiler farms (Battersby et al., 2016; Callicott et al., 2006; O'Mahony et al.,

2011; Prachantasena et al., 2016). However, Cox, Stern, et al. (2002a)

suggested that Campylobacter could be transmitted from the breeder flock to

the fertile eggs through the hatchery and then on to the broiler farms. Few

studies revealed that the same C. jejuni or C. coli strains were found in broiler

breeder flocks and their progeny (Cox, Stern, et al., 2002a; Idris et al., 2006).

These suggested that the layer hens can be a potential source of

Campylobacter spp. for the broiler chickens. Hence, the identification of

Campylobacter strains with shared genotypes between linked breeder and

broiler flocks is suggestive of vertical transmission.

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The current study identified C. jejuni and C. coli isolates from breeder farms

(n=3) with the same genotypes as those isolates from their progeny in broiler

sheds (n=4) from the same region (approximately 500 km apart) and different

regions (approximately 1000 km apart). These suggest the possibility of

vertical transmission. However, faecal samples from some breeder farms

could only be collected after their corresponding chicks were placed at broiler

farms or not at all in this study. Consequently, it was not possible to determine

what the specific genotypes, if any, of Campylobacter spp. were on the

breeder farm at the time of egg-laying. Therefore, there could alternatively be

a geographical connection between breeder and broiler farms and carry over

through other routes such as flies (Hald et al., 2008) or wild birds (Craven et

al., 2000; Waldenstrom et al., 2002). Another possibility of Campylobacter

spp. transmission in young chicks is that hatching birds could take up

Campylobacter spp. from contamination in eggshells (Messelhausser et al.,

2011) or tray liners in the hatchery (Byrd et al., 2007).

In the current study, fresh faecal samples from the breeder farms, collected

by the industry partner to maintain biosecurity in their enterprise, were

obtained after their broilers were placed at the farms and resulted in no faecal

samples from some farms. As a result, it was difficult to determine what the

specific genotypes, if any, of Campylobacter spp. were on the breeder farm

at the time of egg laying. Moreover, sampling at the hatchery was not possible

in this study for commercial reasons. Because of these factors, directly tracing

specific genotypes of Campylobacter spp. through the complete broiler

production system was not possible. Consequently, the linking of strain

genotypes between breeders and broilers in this study may not strictly be due

to vertical transmission. Further research is required to fully investigate this

aspect of colonisation. Ideally, the breeders would have been sampled, during

the laying period, when it was known which broiler sheds of the progeny were

to be placed. The samples from the same egg batches at hatchery would have

been collected before broiler chick placement at farms. However, this optimal

sampling strategy was not possible in this study due to constraints,

particularly biosecurity, associated with working in a commercial production

system. Nevertheless, given the paucity of data associated with free-range

broiler systems, this study still reports useful information. As common

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genotypes were identified between linked breeder and broiler flocks using a

suboptimal sampling approach this suggests further research is warranted to

clearly address this issue. Particularly, as if vertical transmission plays an

important role in broiler colonisation, the breeder flock would be an ideal

intervention point. An expanded longitudinal study of the entire chicken

production chain is required to gain a comprehensive understanding of

Campylobacter colonisation and transmission in commercial poultry farms.

Such a study would include sampling at all of the production sites, at various

stages of the chicken production cycle (e.g. breeder farms, hatchery,

transportation, and broiler farms).

Some other limitations were included in this study. First, we found that some

isolates were misidentified by MALDI-TOF. This method is a robust, rapid,

reliable and cost-effective tool which is commonly used to identify many

pathogens at the genus and species level (Calderaro et al., 2014; Deng et al.,

2014; Penny et al., 2016). Previous studies have reported that MALDI-TOF

correctly identified a number of Campylobacter spp. (Alispahic et al., 2010;

Bester et al., 2016; Mandrell et al., 2005). Consistent with this, the current

data too revealed that MALDI-TOF is an effective and rapid method for

screening Campylobacter spp. as the results were consistent with PCR

reactions. This study showed that 545 of 551 isolates (98.9%) were correctly

identified using the MALDI-TOF, consistent with a previous report which

showed the accuracy of MALDI-TOF was 99.4% for C. jejuni (Bessede,

Solecki, et al., 2011). A reason for the misidentification of the two species of

interest by the MALDI-TOF method could be caused by higher similarity at

the polypeptide level compared to the genetic level between these species

(Lee et al., 2015). Bessede, Solecki, et al. (2011) suggested that C. jejuni and

C. coli are in the same genus and genetically related to each other.

Consequently, proteins/polypeptides translated by them could generate

similar spectra and be detected by the MALDI-TOF, and thus, these may lead

to an inability to distinguish between them at the protein level. Alternatively,

the limitation of the reference database in the MALDI-TOF can affect the

ability to discriminate between bacterial species (Porte et al., 2017). To

overcome this problem, updating the reference database library with multiple

spectra of characterised Campylobacter strains can solve this since new

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Campylobacter genotypes are frequently identified (Bester et al., 2016).

Additional methods such as PCR and biochemical tests could be used for

further confirmation at species level.

Second, there were a few issues that may have affected the efficiency of

genotyping using flaA HRM-PCR in this study, since the criteria used in the

determination of the same HRM profile in this study were based on the HRM

profile and Tm. We found that variations of Tm and Ct value were identified

among samples of the same Campylobacter genotype in this study, this could

result in the assignment of a different HRM profile when we tried to merge

all data. These variations of Tm and Ct value are possibly due to the quality

and/or quantity of the genomic DNA template affecting amplicon yield,

which in turn affects the HRM profile assigned (Slomka et al., 2017). This

study was unable to measure DNA concentration and its purity because no

equipment was available at the commercial laboratory where the study was

undertaken. The measurement of genomic DNA purity and concentration

would have enabled standardisation of the amount of genomic DNA, and

thus, the HRM profile may be more informative. Moreover, evaluating the

quality of the genomic DNA would have identified any samples of low quality

and facilitated re-extraction. We found flaA-HRM PCR showed a high

discriminatory power with 98.5% (65/66 correct clusters, based on flaA-HRM

clusters and flaA sequences) for screening a large number of Campylobacter

isolates in this study. The majority of C. jejuni (n=41) and C. coli (n=24) flaA-

HRM genetic clusters identified in the 406 and 137 isolates in this study were

supported by nucleotide sequencing of the flaA amplicons used. However,

one flaA-HRM cluster of C. coli had different flaA allele numbers. This

cluster containing 8 C. coli isolates from the same breeder farm (BD-F) was

assigned to cluster 19 due to a similar HRM shape and Tm (Appendix 2.2.5

B) but two different flaA amplicon sequences. Therefore, flaA-HRM PCR can

be used as a screening method to differentiate C. jejuni and C. coli genotypes

among a large number of samples and other molecular methods such as flaA

sequencing, MLST, and PFGE are alternatively used to confirm the

genotypes.

The results of this study support that flaA-HRM PCR is a rapid, robust and

cost-effective method to differentiate genotypes of C. jejuni and C. coli from

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various sources in commercial free-range broiler farms. The horizontal

transmission was identified as the most frequent mode of colonization of free-

range broiler chickens. While dominant genotypes were identified, all free-

range broiler flocks studied were exposed to and/or colonized by multiple

genotypes earlier in the production cycle. Also, of interest was the detection

of diverse genotypes in the longer-lived layer birds, where it might be

expected that the colonizing genotype may stabilize over time. Collectively,

these data indicate that the colonization of chickens with Campylobacter is a

complex and dynamic process. Not surprisingly, these results suggest that

effective ongoing control of Campylobacter in the broiler production system

will require a multifaceted approach to reduce the impact of this important

foodborne pathogen. Further studies such as a larger longitudinal study of the

whole chicken production chain with the effective time of sample collection

and sample size based on the current prevalence of Campylobacter in

Australia are required to further elucidate a better understanding of

Campylobacter colonisation and transmission of chickens in poultry farms.

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Chapter 3 Identification and characterisation of Campylobacter genes

3.1 Introduction

Campylobacter jejuni and C. coli are important Campylobacter species which

are strongly associated with human gastrointestinal disease (Gurtler et al.,

2005; Taylor et al., 2013; Weinberger et al., 2013). Chickens are a reservoir

of Campylobacter and are the main source of human Campylobacter

infections (Hermans et al., 2011; Wingstrand et al., 2006). It has been

estimated that a reduction of the Campylobacter burden by 2–3 log10 CFU/g

of chicken caecal contents could lead to a decline of human


et al., 2003). Thus, control of Campylobacter colonisation in chickens is one

of the most potent strategies for reducing the prevalence of human

Campylobacter infections.

Multiple approaches aiming to control Campylobacter colonisation are used

on chicken farms such as biosecurity, feed additives, chicken genetic

selection, competitive exclusion (probiotics used), bacteriocin, and

bacteriophages have been evaluated. However, the interventions tested to date

have not been effective in preventing Campylobacter colonisation (Bailey et

al., 2018; Ghareeb et al., 2012; Gibbens et al., 2001; Kittler et al., 2013;

Ridley et al., 2011; Romero-Barrios et al., 2013; Smith et al., 2016; Solis de

los Santos et al., 2009; Stern et al., 2008).

Vaccine development against Campylobacter colonisation is one potential

intervention to control Campylobacter at the farm level. Over the past

decades, many researchers have developed various prototype vaccine

candidates containing identified antigens and evaluated the vaccine efficacies

against Campylobacter colonisation in chickens. For example, two decades

ago, the whole-killed cell vaccine of C. jejuni was developed and examined

as a vaccine candidate (Rice et al., 1997). Since then, subunit vaccines

containing Campylobacter antigenic proteins (Annamalai et al., 2013;

Chintoan-Uta et al., 2016; Godlewska et al., 2016; Hodgins et al., 2015;

Huang et al., 2010; Neal-McKinney et al., 2014; Zeng et al., 2010) and live-

attenuated microorganism vectors expressing Campylobacter antigens

(Buckley et al., 2010; Clark et al., 2012; Kobierecka, Olech, et al., 2016;

120

Laniewski et al., 2014; Laniewski et al., 2012; Layton et al., 2011; Nothaft et

al., 2016; Saxena, John, et al., 2013) have been evaluated in more recent

studies. However, the outcomes in terms of reduction of C. jejuni colonisation

following experimental challenge, were inconsistent, even amongst the

approaches that successfully induced significant immune responses. At

present, there is no commercially available vaccine to prevent Campylobacter

infection in poultry, and thus, identification of new antigens or improving the

delivery of known antigens for new vaccine formulations remains as an area

of considerable interest.

The selection of a suitable antigen which could potentially elicit a strong

immune response is crucial prior to vaccine construction. To date, various C.

jejuni genes encoding proteins (antigens) such as outer membrane vesicles,

CiaB, CadF, Peb1A, ChuA, GlnH, FliD, PorA, SodB, FspA, FlaA, FlpA, and

CmeC have been investigated for their immunogenicity and used in vaccine

development (Buckley et al., 2010; Chintoan-Uta et al., 2015; Chintoan-Uta

et al., 2016; Islam et al., 2010; Monteiro et al., 2009; Neal-McKinney et al.,

2014; Saxena, John, et al., 2013; Zeng et al., 2010). While many researchers

have investigated these genes obtained from one C. jejuni pathogenic strain

by testing them in vaccine efficacy studies, and none of them prevented the

colonisation of C. jejuni in chickens after challenge with a single strain

(Annamalai et al., 2013; Buckley et al., 2010; Chintoan-Uta et al., 2016;

Laniewski et al., 2014; Layton et al., 2011; Neal-McKinney et al., 2014;

Saxena, John, et al., 2013; Wyszynska et al., 2004). The genomic instability

of Campylobacter results in genetic diversity (Cody et al., 2009; Wassenaar

et al., 1998; Wilson et al., 2010) and this may lead to inadequate vaccine

protection (Ridley, Toszeghy, et al., 2008). The results of Chapter 2

demonstrated that C. jejuni and C. coli isolated from chicken farms are

genetically diverse, based on flaA sequencing. If conserved genes encoding

potentially protective antigens from various genotypes could be identified,

these antigens could potentially be used in the development of an efficacious

vaccine which prevents colonisation by both species.

This chapter reports the evaluation of seven Campylobacter genes identified

in previous studies, katA, cadF, peb1A, flpA, omp18, cjaA, and fliD, that have

been identified as encoding Campylobacter colonisation or virulence factors

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(Table 3.1). These genes encode polypeptides which have been previously

been investigated for their potential to be used as vaccine candidates with

varying levels of success in preventing Campylobacter colonisation

(Annamalai et al., 2013; Buckley et al., 2010; Chintoan-Uta et al., 2015;

Chintoan-Uta et al., 2016; Kobierecka, Olech, et al., 2016; Laniewski et al.,

2012; Layton et al., 2011; Neal-McKinney et al., 2014; Rickaby et al., 2015).

Table 3.1: Information of Campylobacter genes used in Chapter 3

The catalase-encoding katA gene, commonly found in both C. jejuni and C.

coli, is involved in oxidative stress defence, which is induced by free-radical

oxygen exposure, and converts hydrogen peroxide to water and dioxygen

(Garenaux et al., 2008; Palyada et al., 2009). Day et al. (2000) suggested that

catalase is essential for the persistence and growth of C. jejuni in

macrophages. Palyada et al. (2009) reported that the KatA protein is essential

for Campylobacter colonisation in vivo as the katA-deficient mutant C. jejuni

failed to colonise the caecum of chicks. A study reported that KatA from C.

jejuni was immunogenic in mice after intramuscular injection and the

Gene Functional

area

Predicted/identified

protein function

References

katA Oxidative

stress

response

Catalase KatA (Day et al., 2000);

Garenaux et al. (2008);

(Palyada et al., 2009)

cadF Adhesion Campylobacter adhesin

to fibronectin-like

protein (CadF)

(Konkel et al., 1997);

Konkel, Gray, et al.

(1999); (Monteville et

al., 2003)

flpA Adhesion Fibronectin-like protein

A (FlpA)

(Flanagan et al., 2009);

Konkel et al. (2010)

peb1A Adhesion Periplasmic-binding

protein (Peb1),

periplasmic ABC

transporter of amino

acids

(Pei & Blaser, 1993);

Pei et al. (1998); (Pei

et al., 1991)

omp18 Maintenance

cell wall

Peptidoglycan-

associated protein

Godlewska et al.

(2009)

cjaA The uptake of

amino acid

Campylobacter solute-

binding protein (CjaA),

a component of the

ABC transport system

Muller et al. (2005);

Pawelec et al. (1997);

Wyszynska et al.

(2008)

fliD Flagella

prevention

Flagellar cap protein or

flagella-hook associated

protein2

Freitag et al. (2017);

Maki et al. (1998); Yeh

et al. (2014)

122

antibody against KatA reduced adhesion and invasion in vitro, suggesting that

it can be used as a potential vaccine candidate (Rickaby et al., 2015).

Campylobacter adhesins, such as CadF, Peb1A, and fibronectin-like protein

A (FlpA), are important factors in Campylobacter colonisation as they

promote pathogen interaction with host cells (Flanagan et al., 2009; Konkel

et al., 2010; Monteville et al., 2003). The cadF gene, which encodes a 37-kDa

outer membrane protein (OMP), is conserved in C. jejuni and C. coli and

plays a vital role in their binding to fibronectin (Fn) in the host intestinal

epithelial cells (Konkel et al., 1997; Konkel, Gray, et al., 1999). Studies have

reported that prototype vaccines containing the CadF protein elicited high

immune responses and reduced by 2.26 log10 CFU/g of C. jejuni colonisation

in chicken models (Neal-McKinney et al., 2014; Saxena, John, et al., 2013).

The flpA gene encodes for FlpA, which is another putative adhesin protein

involved in C. jejuni colonisation, by binding to the Fn receptor of host cells

(Konkel et al., 2010). A subunit vaccine candidate based on the FlpA protein

elicited a significant immune response and reduced C. jejuni colonisation by

3.65 log10 CFU/g in chicken models (Neal-McKinney et al., 2014).

The peb1A gene is conserved in C. jejuni and C. coli and encodes a

periplasmic-binding protein (PEB1), which aids Campylobacter colonisation

through adherence to and invasion of host cells (Ó Croinin & Backert, 2012;

Oh et al., 2017; Pei & Blaser, 1993; Pei et al., 1998; Pei et al., 1991). PEB1,

a periplasmic protein mediating the interaction between C. jejuni and

epithelial cells, is similar to glutamine- and histidine-binding proteins from

ABC transporter systems (Leon-Kempis Mdel et al., 2006). A live-attenuated

Salmonella vector vaccine based on the peb1A gene reduced C. jejuni

colonisation by 1.64 log10 CFU/g in chicken models (Buckley et al., 2010).

The omp18 gene, which encodes the 18-kDa OMP, is associated with the

maintenance of the bacterial cell wall (Godlewska et al., 2009). The omp18

(Cj0113) gene has been named as Campylobacter jejuni antigen D (cjaD) and

peptidoglycan-associated lipoprotein (Pal) by (Laniewski et al., 2012; Layton

et al., 2011; Pawelec et al., 2000). This OMP has been investigated as a

candidate in many vaccine trials since it is conserved between C. jejuni and

C. coli and induces high immune responses in humans (Blaser et al., 1984;

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Konkel et al., 1996; Laniewski et al., 2012; Layton et al., 2011; Pawelec et

al., 2000). However, experiments with vaccine candidates based on omp18

have shown inconsistent efficacies in chickens. Laniewski et al. (2012)

reported that attenuated Salmonella vector vaccine expressing Omp18 did not

significantly reduce C. jejuni colonisation, whereas Layton et al. (2011) found

that live Salmonella vectors expressing Omp18 reduced by 4.8 log10 CFU/g

of C. jejuni colonisation in chickens.

The cjaA gene encodes the solute-binding protein or Glutamine-binding

protein (CjaA), which is a component of the ABC transport system and is

conserved in C. jejuni and C. coli (Muller et al., 2005; Wyszynska et al.,

2008). Shoaf-Sweeney et al. (2008) reported that the antibodies to the CjaA

protein were detected in chicken maternal antibodies, suggesting that it may

be a good candidate antigen in vaccine trials. Studies have reported that

chickens orally immunised with live-attenuated vaccines expressing CjaA

conferred IgG and IgA responses with various reductions between 1 and 6

log10 CFU/g in caecal colonisation after challenge with either homologous or

heterologous C. jejuni strains (Buckley et al., 2010; Clark et al., 2012; Layton

et al., 2011; Saxena, John, et al., 2013; Wyszynska et al., 2004). In contrast,

other studies have reported that immunisation of chickens with a purified

CjaA subunit vaccine or a live-attenuated Lactobacillus lactis expressing

CjaA did not protect against Campylobacter colonisation (Chintoan-Uta et

al., 2015; Kobierecka, Olech, et al., 2016). The fliD gene encodes a flagella

cap protein (FliD), an essential element in the assembly of the functional

flagella and a crucial factor for colonisation by binding to host epithelial cells

(Freitag et al., 2017). Yeh et al. (2014) and Yeh et al. (2016) found that

immunisation of chickens with FliD elicited strong immune reactions,

evaluated by Western blotting, suggesting that it can be used as an antigen in

vaccine development. Indeed, a subunit vaccine containing FliD elicited

immune responses and reduced by 2 log10 CFU/g of C. jejuni colonisation in

the chicken model (Chintoan-Uta et al., 2016).

3.2 Materials and Methods

124

3.2.1 Campylobacter strains and culture conditions

The C. jejuni and C. coli strains used in this study and the culture methods

used have been described in Chapter 2 (Section 2.2.5).

3.2.2 Genomic DNA extraction

Genomic DNA extraction from C. jejuni and C. coli has been described in

Chapter 2 (Section 2.2.7).

3.2.3 Campylobacter gene detection

To evaluate the conservation (presence/absence) of the genes of interest

(Table 3.1) among the C. jejuni and C. coli isolates representing flaA-HRM

clusters (clusters) identified in this study, specific oligonucleotide pairs were

used to amplify the genes of interest from all C. jejuni and C. coli strains from

the chicken farms. Based on Chapter 2, 408 C. jejuni and 145 C. coli from

526 culturable samples were grouped into 41 and 25 flaA-HRM clusters,

respectively. In this chapter, except for the C. coli cluster 19, a representative

isolate from each flaA-HRM cluster was selected for the detection of the

Campylobacter antigens encoding genes. The C. coli cluster 19 had two flaA

genotypes and consequently both were included in this analysis. Therefore,

all genomic DNA samples of 41 C. jejuni and 26 C. coli isolates were

evaluated using conventional PCR assays to amplify Campylobacter genes of

interest (Table 3.1).

3.2.3.1 Primers of gene amplification

Most of the oligonucleotides used in this study have been described

previously and shown in Table 3.2 (Buckley et al., 2010; Chintoan-Uta et al.,

2016; Gundogdu et al., 2015; Laniewski et al., 2012; Neal-McKinney et al.,

2014; Rickaby et al., 2015; Wyszynska et al., 2008). The exceptions were two

primer sets for the omp18 and cjaA genes of C. jejuni which were modified

in the present study. The oligonucleotides for omp18 amplification were

modified from that of Laniewski et al. (2012) by removing the restriction sites

of forward and reverse primers. Then the reverse primer of omp18 was

125

extended by six nucleotides at 3énd to ensure that the Tm (°C) of both

forward reverse primers were close to each other. For cjaA, C. jejuni

amplification, the forward and reverse primers used in this study were from

that of Buckley et al. (2010) and Chintoan-Uta et al. (2016), respectively with

some modifications. The oligonucleotides of restriction sites were removed

from both primers. Then five nucleotides were extended at 3' end of the

forward primer from that of Buckley et al. (2010). An extra six extra

nucleotides were extended at 5' of the reverse primer from that of Chintoan-

Uta et al. (2016). All modified primers with extended nucleotide sequences

are shown in Table 3.2.

All oligonucleotide primer sets (Table 3.2) were subjected to a BLAST search

in the NCBI nucleotide database to determine that each primer set was

specific for the particular gene of interest in C. jejuni and C. coli

(https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 02/03/2017)). Then,

each primer set was confirmed with the expected size of each PCR product,

and the suitable Tm (°C) of each gene amplification was determined using a

temperature gradient PCR. The estimated sizes of the PCR products are

summarised in Table 3.2.

3.2.3.2 Temperature gradient PCR

Each primer set was examined in gradient PCR reactions (annealing

temperature range of 50 to 60°C) to determine the optimal annealing

temperature using C. jejuni (NCTC 11168) and C. coli (ATCC 33559) as

DNA templates. All PCR reactions were performed in a Bio-Rad S1000TM

Thermal Cycler (Bio-Rad, Australia) and in a 25-µL reaction mixture

containing 2 U Platinum Taq polymerase (Invitrogen, Australia), 1 × PCR

Rxn Buffer-MgCl2 (Invitrogen, Australia) or 1 × Green PCR Rxn Buffer-

MgCl2 (Invitrogen, Australia), 1.5 mM MgCl2 (Invitrogen, Australia), 0.2

mM dNTPs Mix (Invitrogen, Australia), and 0.2 mM each of relevant forward

and reverse primers (Integrated DNA Technologies, Singapore), as described

in Table 3.2, as well as 10–30 ng of DNA template (Section 3.2.2) and RNAse

free water.

126

Table 3.2: Oligonucleotide primers used for the detection of genes in Campylobacter jejuni and Campylobacter coli and summary of the estimated sizes

of the PCR product

Gene Oligo

Name

Sequence 5' to 3' (include

modification codes if applicable) Reference

C. jejuni C. coli

GenBank

access

number

Estimated

PCR

amplicon (bp)

GenBank

access

number

Estimated

PCR

amplicon (bp)

C. coli

cjaA cjaAcoli-F AAT TCA GAT TCT GGT GCT TC Wyszynska et

al. (2008)

Y10872.1 767 CP018900.1 767

cjaAcoli-R TTA CCG CCT TCA ATA ACT AC

C.

jejuni

cjaA

cjaAjejuni-

F

ATG AAA AAA ATA CTT CTA

AGT GTT TTT A a Modified from

Buckley et al.

(2010)

NC_002163.1

840

CP018900.1

840

cjaAjejuni-

R

TAG TGA TTG AAG GTG GAA

AAA TTT AA a

Omp18 omp18-F

ACA AAA AGC ACT AGC GTA

AGC G Modified from

Laniewski et

al. (2012)

CP020766.1

485

CP007181.1

485

omp18-R CTT CTT GGA GCT ACT TTA CTT

TA a

cadF cadF-F

ACA ATG TAA AAT TTG AAA

TCA CTC C Neal-

McKinney et

al. (2014)

CP006688.1

902

CP017878.1 941

cadF-R GAA GAG TGG ATG CTA AAT

TTA TTT TAA GA

CP017871.1 902

katA katA-F TGT CCT GAA AGT TTA CAT C Gundogdu et

al. (2015)

NC_002163.1

609

CP007181.1

609 katA-R CAT AGC ACC AGC GAC ATT G Note: a Bold typed letter indicates the modified (extended) oligonucleotides of the primer

127

Table 3.2: Oligonucleotide primers used for the detection of genes in Campylobacter jejuni and Campylobacter coli and summary of the estimated sizes

of the PCR product (cont’)

Gene Oligo

Name

Sequence 5' to 3' (include modification

codes if applicable) Reference

C. jejuni C. coli

GenBank

access

number

Estimated

PCR

amplicon (bp)

GenBank

access

number

Estimated

PCR

amplicon (bp)

peb1A peb1A-F ATG GTT TTT AGA AAA TCT TT Buckley et al.

(2010)

CP020776.1 780 CP018900.1 780

peb1A-R CGA AAA AAT GGG GTT TAT AA

fliD fliD-F ATG GCA TTT GGT AGT CTA TC Chintoan-Uta

et al. (2016)

CP020766.1 1936 CP018900.1 1917

fliD-R TTA ATT ATT AGA ATT GTT TG

flpA flpA-F TCG CTA GCT TCA AGT AAA GAG C Neal-

McKinney et

al. (2014)

CP020766.1

1152

CP025281.1

1152 flpA-R GCA AAG TTA AGG CGG CTC A

fliD fliD-F set

1

ATG GCA TTT GGT AGT CTA TCT

AGT TTA GGA TTT This study

CP020766.1

1049

CP018900.1

1046

fliD-R set

1

GGC ATC AGT GAA GTA AAT TCA

ATA CGC TC

fliD fliD-F set

2

CAA AAG CCA TGC AAG ATT TGG

TGG ATG C This study

CP020766.1

1009

CP018900.1

994

fliD-R set

2

ACT GTG ACT AAT ATG ATT AAT

GCG GCA AAC AAT TC Note: a Bold typed letter indicates the modified (extended) oligonucleotides of the primer

128

3.2.3.3 Detection of katA, cadF, peb1A, cjaA, omp18, flpA, and fliD genes

Conventional PCR assays were used to detect katA, cadF, peb1A, cjaA,

omp18, flpA, and fliD genes using the most suitable annealing temperature

determined from the gradient PCR reactions from section 3.2.3.2. For each

PCR reaction, Campylobacter reference strains (C. jejuni NCTC 11168 and

C. coli ATCC 33559) and RNAse-free water served as positive control and

negative control, respectively. Based on the results from Section 3.2.3.2, the

cycling conditions were as follows: 94°C for 4 min (one cycle), 40 cycles of

denaturation at 94°C for 10 sec, annealing at 55oC (katA, cadF, and flpA) or

58°C (omp18) or 51°C (peb1A, C. jejuni-cjaA- and C. coli-cjaA) for 20 sec,

and extension at 72°C for 30 sec.

3.2.3.4 PCR amplicon analysis

The PCR products were analysed using agarose gel electrophoresis at 80 V

for 40 min in 1.5% (w/v) agarose gel stained with Midori Green Advanced

DNA stain (Nippon Genetics Europe GmbH, Germany) in 1× Tris-acetate-

EDTA (TAE) buffer (40 mM Tris-HCl pH 7.6, 20 mM acetic acid, 1 mM

EDTA). The PCR products were visualised using a Gel DocTM XR+ imaging

system (Bio-Rad, Australia) with Gel Green software (Bio-Rad, Australia),

and product sizes were determined using a standard molecular weight markers

(75–20000 bp from GeneRuler™ 1 Kb Plus DNA ladder, Thermo Scientific,

USA, or 100–15000 bp from 1 Kb Plus DNA ladder, Invitrogen, USA).

Representative PCR amplicons of each gene amplified from all C. jejuni and

C. coli clusters were further confirmed using DNA sequencing.

3.2.3.5 PCR product sequence analysis

3.2.3.5.1 PCR product preparation for sequencing

Before sequencing, the PCR products (Section 3.2.4.1) were purified using

ExoSAP-IT™ Express PCR Product Cleanup Reagent (Affymetrix, USA). A

total of 2 µL of ExoSAP-IT™ Express reagent was added to 5 µL of fresh

PCR amplicons, mixed by gentle vortexing and quick spin, and incubated at

37°C for 4 min. The reagent was subsequently inactivated by heating at 80°C

for 1 min in a thermocycler.

129

3.2.3.5.2 BigDye® Terminator sequencing reaction

All purified PCR products from Section 3.2.3.5.1 were subjected to DNA

sequencing using the BigDye® Terminator (BDT) v3.1 Cycle Sequencing Kit

according to the manufacturer’s protocol (Biosystems, 2010). Each reaction

was performed in a 20-µL reaction of BDT contained 3.5 µL of 5 × sequence

buffer (Applied Biosystems®, Foster City, Ca, USA), 10–40 ng of DNA

(Section 3.2.3.5.1), 1 µL of BDT v.3.1 Ready Reaction Mix (Applied

Biosystems®), 1 µL of 3.2 µM relevant primer (forward or reverse primer),

and nuclease-free water (to a final volume of 20 µL). Forward and reverse

primers specific for each gene (Table 3.2) were used for the forward and

reverse sequencing reactions, respectively.

The cycling conditions for sequencing were as follows: initial denaturation at

94°C for 1 min, followed by 25 cycles of denaturation at 96°C for 10 sec,

annealing at 50°C for 5 sec, polymerisation at 60°C for 4 min, and then

maintained at 4°C. Tubes were briefly pulsed spin, transferred to a plastic

rack, covered with aluminium foil, and then delivered to the Australian

Equine Genetics Research Centre (AEGRC) of the University of Queensland,

Brisbane, Australia for sequencing.

3.2.3.5.3 Nucleotide sequence alignment

The individual nucleotide sequence obtained for each PCR amplicon was

opened and analysed for alignment using the BioEdit Sequence Alignment

Editor (version 7.2.5). The nucleotide sequence alignment of each

Campylobacter gene was compared with the NCBI database. The nucleotide

sequences were aligned along with the data retrieved from BioEdit using the

multiple sequence alignment program Clustal Omega via

https://www.ebi.ac.uk/Tools/msa/clustalo/.

3.2.4 Cloning, sequencing, and expression of Campylobacter jejuni

genes

Champion™ pET SUMO® Expression System (Invitrogen, USA) was used

to express the protein of the construct (individually inserted katA or cadF or

peb1A or cjaA) in bacterial cells (E. coli). In this study, C. jejuni cluster 27

(ST-257 complex) served as the most frequently identified flaA-HRM cluster

130

in the broiler flocks (Chapter 2) and was selected for recombinant gene

construct and expression analyses. The construction of recombinant pET

SUMO expressing the Campylobacter gene was carried out as per the

manufacturer’s instructions (Invitrogen, 2010a).

3.2.4.1 Amplicon generation of Campylobacter jejuni genes

The genes detected in all C. jejuni and C. coli clusters from Section 3.2.3 were

further individually amplified using a conventional PCR method. Internal

oligonucleotide primers of each gene carrying unique restriction sites (Table

3.3) were used in each PCR reaction to generate unidirectional cloning into

pET SUMO vectors. All oligonucleotide primers used in this part were

subjected to a BLAST search in the NCBI nucleotide database to determine

that each primer set was specific for the particular gene of interest in C. jejuni

(Figures 3.1 and 3.2). The PCR reactions, cycling conditions and amplicon

analysis are previously described as above (sections 3.2.3.3 and 3.2.4). The

relevant primers and annealing temperatures are described in Table 3.3.

131

Table 3.3: Summary of oligonucleotides of the gene primers used for bacterial antigen expression

Oligo Name Sequence 5' to 3'α Product

size (bp)

Accession

number

Orientation/restriction

site

Tm

(°C)

Reference

katAFClone GAA GCT TCT ATG GAA AGT TTA CAT

CAA GTA ACC ATT CTT ATG AGC

686

NC_002163.1

Forward/HindIII 55 Modified from

Gundogdu et al.

(2015) katARClone CCA AAC AGC TAT GAT AAT AGC CCA

GGA TCC AC

Reverse/BamHI-HF

cadFFClone GCT CGA GCT GGT GCT GAT AAC AAT

GTA AAA TTT GAA

913

CP006688.1

Forward/XhoI 58 Modified from

Gundogdu et al.

(2015); Neal-

McKinney et al.

(2014)

cadFRClone GCG GAT AAT AGA AGA GTG GAT GCT

GGA TCC AC

Reverse/BamHI-HF

cjaAcoliFClone GCT CGA GCT ATG CTC TTA AGT ATT

TTT ACA ACC

839

Y10872.1


Wyszynska et al.

(2008) cjaAcoliRClone GAT GTA GTT ATT GAA GGC GGT GGA

TCC AC

Reverse/BamHI-HF

peb1AFClone GCT CGA GCT TCT TTG TTA AAG TTG

GCA GTT

767

CP020776.1


Buckley et al.

(2010) peb1ARClone GAA ATT GAT GCT TTA GCG AAA GGA

TCC AC

Reverse/BamHI-HF

Note: α Restriction recognition sites added for cloning purposes are underlined.

132

3.2.4.2 Plasmid ligation

The freshly amplified amplicons (Section 3.2.4.1) were ligated to the

linearised pET SUMO plasmid vector. Each ligation reaction was performed

using the vector: insert (PCR amplicon) molar ratio of 1:1. The ligation

reaction was performed in a total of 10 μL ligation reaction (Table 3.4) and

incubated at 15°C for overnight (16 ± 2 h) in a thermocycler.

Table 3.4: The ligation reaction for pET SUMO vector and PCR amplicons

Reagent Volume (μL)

Fresh PCR amplicon X*

10 × Ligation Buffer 1

pET SUMO vector (25 ng/μL) 2

Sterile water to a total volume of 9

T4 DNA Ligase (4.0 Weiss units) 2

Total 10 Note: * Each ligation reaction was performed using the vector: insert (PCR amplicon) molar ratio of

1:1.

3.2.4.3 Plasmid transformation

A 2 μL volume of the ligation reaction (Section 3.2.4.2) was transformed into

a vial of One Shot® Mach1™-T1R chemically competent E. coli cells

(Invitrogen, USA). The ligation reaction and the One Shot® Mach1™-T1R

chemically competent E. coli cells were incubated on ice for 30 min and then

heat-shocked in a water bath at 42°C for 30 sec. The cells were then

immediately transferred to ice, and 250 μL super-optimal catabolite

repression (SOC) medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl,

2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose) at 22 ± 2

°C was added. The cells were incubated at 37°C with shaking at 200 rpm for

60 min. An aliquot of each transformant (60 µL) was spread onto a pre-

warmed Luria–Bertani agar plate containing 50 μg/mL kanamycin sulfate

(LB-Kan50 plate) and incubated at 37°C for 16 ± 2 h. Non-ligated pET

SUMO plasmid was used as negative control.

133

3.2.4.4 Screening of transformed colonies

A conventional PCR method was used to assess the constructed plasmid in

transformed bacterial colonies (whole cells). A single bacterial colony on

each LB-Kan50 plate was collected using a sterile micropipette tip,

resuspended in 20 μL of nuclease-free water to generate a cell suspension,

and added as a template for the PCR reaction. All PCR reactions were

performed in a Bio-Rad S1000TM Thermal Cycler (Bio-Rad, Australia). Each

PCR reaction was performed in a total volume of 25 μL containing 2 μL of

the cell suspension and other reagents as described in Section 3.2.4.1.1. The

PCR cycling program and the PCR product assessment were performed as

described in Sections 3.2.4.1.2, 3.2.3.4, and 3.2.3.5. The remaining cell

suspensions of every colony yielding an amplicon of the expected size in the

PCR reaction were used to inoculate LB broth (5 mL) containing 50 μg/mL

kanamycin (LB-Kan50 broth) and incubated at 37°C for 16 ± 2 h with

vigorous shaking.

3.2.4.5 Plasmid isolation

The recombinant plasmids containing each gene were isolated from the

transformed E. coli cells using the QIAprep® Spin Miniprep Kit (Qiagen,

Germany) following the manufacturer’s instructions (Qiagen, 2015a). A 4 mL

aliquot of the incubation culture (Section 3.2.4.4) was pelleted by

centrifugation at 6800 g for 3 min at 22 ± 2 °C. The pelleted E. coli cells were

resuspended in 250 μL of Buffer P1 (Qiagen) and transferred to a

microcentrifuge tube. A volume of 250 μL of Buffer P2 (Qiagen) was then

added and mixed thoroughly by inverting the tube 4–6 times until the solution

became clear. Subsequently, a volume of 350 μL of Buffer N3 (Qiagen) was

added, mixed immediately and thoroughly by inverting the tube 4–6 times.

Then it was centrifuged for 10 min at 17,900 g. An 800-μL aliquot of the

supernatant was collected and transferred to a QIAprep 2.0 spin column by

pipetting. The spin column was centrifuged for 60 s, and the flow-through

was discarded. Then, the spin column was washed by adding 750 μL of Buffer

PE (Qiagen) and centrifuged for 60 sec, and the flow-through was discarded.

The residual wash buffer in the spin column was removed by centrifugation

134

for an additional 60 sec. Next, the spin column was placed in a clean 1.5-mL

microcentrifuge tube, and the DNA was eluted by adding 50 μL of MilliQ

water to the centre of the spin column, leaving for 1 min, and followed by

centrifugation for 1 min. The eluted plasmid DNA was collected, and DNA

concentration and purity were determined using a spectrophotometer

(Nanodrop® ND-1000, Wilmington, DE, USA). The eluted plasmid samples

were labelled and stored at -20°C until required.

3.2.4.6 Analysis of plasmid construct

The purified recombinant plasmids containing each gene from Section 3.2.4.5

were further analysed using restriction enzyme analysis and DNA

sequencing.

3.2.4.6.1 Double digestion with two restriction enzymes

The purified plasmids from Section 3.2.4.5 were used as the DNA template.

Double digestions with specific restriction enzymes were used to determine

the presence of the inserted gene. Each double digestion reaction was made

up to a volume of 50 μL containing 1 × restriction enzyme buffer, 10-20 U of

each enzyme, and an appropriate amount of DNA as described in Table 3.5.

The digest condition was at 37°C for 1 h using a Bio-Rad S1000TM Thermal

Cycler (BIO-RAD, Australia). The digestion reactions were analysed by

electrophoresis on a 1.5% agarose gel stained with Midori Green Advanced

DNA stain (Nippon Genetics Europe GmbH, Germany) as described in

Section 3.2.3.4. The inserted DNA sizes were estimated using a molecular

weight marker (1kb ladder; New England Biolabs, Australia).

135

Table 3.5: Information of restriction enzymes and buffer used

Reagent Gene

katA peb1A cjaA cadF

10 x CutSmart (New

England Biolabs, USA)

5 μL 5 μL 5 μL 5 μL

HindIII (Roche,

Mannheim, Germany)

10 U – – –

XhoI (New England

Biolabs)

– 20 U 20 U 20 U

BamHI-HF (New

England Biolabs)

20 U 20 U 20 U 20 U

Plasmid DNA 1 µg 1 µg 1 µg 1 µg

Sterile water (μL) Up to 50 Up to 50 Up to 50 Up to 50

Total (μL) 50 50 50 50

3.2.4.6.2 Sequence analysis

All purified plasmid DNA samples with the gene inserted (Section 3.2.4.5)

were selected for further DNA sequencing to confirm the nucleotide

orientation of each gene using the same protocol as described in Sections

3.2.3.5.2 and 3.2.5.3. The SUMO forward and T7 reverse primers were used

for forward and reverse sequencing reactions, respectively (Table 3.6).

Table 3.6: Oligonucleotide primer pairs used for DNA sequencing of the pET

SUMO plasmid containing Campylobacter genes

Primer Name Nucleotide sequence

SUMO Forward 5´-AGA TTC TTG TAC GAC GGT ATT AG-3

T7 Reverse 5´-TAG TTA TTG CTC AGC GGT GG-3´

3.2.4.7 Transformation of BL21 (DE3) One Shot® E. coli cells

All plasmids (5–10 ng) containing each open reading frame (ORF) from the

gene of interest in the correct nucleotide orientation (Section 3.2.4.5) were

transformed into the competent BL21 (DE3) E. coli cells and induced for

protein expression as follows. The constructed plasmid was transformed into

BL21 (DE3) One Shot® E. coli cells according to the manufacturer’s

instruction (Invitrogen, 2010a). A vial of BL21 (DE3) One Shot® chemically

136

competent E. coli cells (Invitrogen, USA) was thawed on ice. A 2 µL aliquot

of the plasmid DNA (5–10 ng) from Section 3.2.4.5 was added into the

thawed vial of BL21 (DE3) competent E. coli cells (Invitrogen, USA), stirred

gently with a pipette tip, and then incubated on ice for 30 min. The mixture

of plasmid DNA and the BL21 (DE3) cells were incubated in a water bath

(heat shock) at 42°C for 30 s, immediately transferred to ice, and then 250 μL

of SOC medium (22 ± 2 °C) was added. The transformed BL21 (DE3) cells

were incubated at 37°C for 60 min with shaking at 200 rpm; subsequently, 10

mL of LB-Kan50 broth was added and incubated at 37°C for 16 ± 2 h with

shaking at 200 rpm.

3.2.4.8 Induction of protein expression

A 500 µL aliquot of each incubated culture was inoculated in 10 mL of pre-

warmed LB-Kan50 broth and then incubated at 37°C with shaking for 2 h

until the culture reached an optical density (OD600) of 0.4–0.6. Isopropyl-D-

thiogalactoside (IPTG) was added to a 5-mL aliquot of the culture to make a

final concentration of 1 mM; IPTG was added to overexpress the protein of

interest at 37°C as follows. A 5 mL aliquot collected from the culture was

kept IPTG free and served as the negative control (uninduced cells). A 500

µL sample of each aliquot was collected and transferred into a new tube as a

zero-time point sample (T0). Then, both aliquots were incubated at 37°C with

shaking at 200 rpm for 6 h. A 500-µL sample of the aliquots was collected

and transferred into a new tube every hour for 6 h (time points T1–T6). All

samples collected from each time point (T0–T6) were initially centrifuged at

maximum speed for 30 sec, and the supernatant was discarded. The cell

pellets were stored at -20°C until required for protein extraction. In this study,

E. coli BL21 (DE3) transformed by the pET SUMO/CAT vector, which

expresses an N-terminally tagged chloramphenicol acetyltransferase (CAT)

fusion protein, was used as the positive control.

3.2.4.9 Protein extraction

The frozen bacterial cell pellets from Section 3.2.4.8 were thawed on ice and

subjected to cell lysis following the manufacturer’s instructions (Novex®’Life

Technologies, Carlsbad, CA, USA). The thawed cells were reconstituted in

137

40 µL of the lysis solution, which was made from 1× NuPAGE® LDS sample

buffer (Life Technologies), 1× sample-reducing agents (Invitrogen), and

deionised water (to a final volume of 40 µL). Then, a small number of glass

beads was added to the mixture. Five cycles of vortexing (30 sec) and

transferring to ice (30 sec) were further performed to ensure complete cell

lysis; then, the lysed cells were centrifuged at maximum speed for 5 min. The

supernatant containing the soluble antigenic protein fraction was carefully

transferred to a clean tube, heated at 95°C for 10 min, and stored at -20°C for

SDS-PAGE analysis.

3.2.4.10 SDS-PAGE analysis

The supernatants from Section 3.2.4.9 were separated using a Bolt 4–12%

Bris-Tris plus SDS polyacrylamide gel electrophoresis apparatus (SDS-

PAGE) (Invitrogen, USA) and the Mini-PROTEAN® Tetra Cell

Electrophoresis Module (Bio-Rad, Hercules, CA, USA) at 140 V for 45 min

in 1 × Run buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). The Precision

Plus Protein™ Kaleidoscope™ Standard (Bio-Rad, USA) was used as a

protein molecular weight marker. The separated proteins were transferred

onto polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences,

Piscataway, NJ, USA) using a semidry blotting apparatus (Amersham

Biosciences). The electrotransfer time was 1 hr, with 100 V in 1× transfer

buffer containing 20% methanol [25 mM Tris, 192 mM glycine, 20% (v/v)

methanol]. After transfer, the membranes were washed three times with 1×

Tris-buffered saline, 0.1% Tween 20 (TBST), for 5 min per wash; then, the

membranes were blocked with 5% w/v milk in TBST for 1 h with agitation

at 22 ± 2 °C.

3.2.4.11 Western blotting

The transferred membranes were incubated for 16 ± 2 h with shaking in

1:3000 of the anti-His-tag mouse (Cell Signaling Technology, USA) diluted

in 5% w/v milk in TBST at 4°C. After that, the membranes were subsequently

washed three times with 1× TBST. The membranes were further incubated at

22 ± 2 °C for 45 min with shaking in 1:4000 of rabbit anti-mouse IgG HRP

138

(Cell Signaling Technology) diluted in 5% w/v milk in TBST. Following this,

the membrane was washed three times, for 5 min per wash, using 1× TBST

and then incubated with a chemiluminescent Pierce™ ECL Western Blotting

Substrate (Pierce Biotechnology, USA) for visualisation according to the

manufacturer’s instructions (Thermo Scientific, 2013). The blots were

exposed to Fuji Super RX-N medical X-ray film (Fuji Corporation, Japan) in

a dark room for 1–10 sec. An image of the radiograph was digitised using a

Gel DocTM XR+ imaging system (Bio-Rad) with Image LabTM software

version 6.0.1 (Bio-Rad). The size of the recombinant protein was analysed

using Precision Plus Protein™ Dual Colour Standards (Bio-Rad, USA).

3.3 Results

3.3.1 Gradient PCR analysis

Genomic DNA samples of C. jejuni NCTC11168 and C. coli ATCC33559

reference strains were used for gradient PCR. All primer sets successfully

amplified the Campylobacter genes of interest (katA, cadF, C. jejuni cjaA, C.

coli cjaA, peb1A, omp18, and flpA), except for the primers of fliD which failed

to amplify the target gene (Table 3.7). The optimum annealing temperature

and the size of the PCR amplicon for each gene are summarised in Table 3.7.

Therefore, two primer sets of fliD were re-designed (Sets 1 and 2) (Appendix

3.1). The expected sizes of PCR products are summarised in Table 3.2. These

primer sets were then used to amplify fliD from C. jejuni and C. coli reference

strains using the gradient PCR reactions with the annealing temperatures of

40–70 °C (Appendix 3.1). The results indicated that these primer sets failed

to amplify fliD (Table 3.7), so fliD was not investigated further.

139

Table 3.7: Summary of gradient PCR results using Campylobacter jejuni and

Campylobacter coli reference strains

Gene

C. jejuni NCTC 11168 C. coli ATCC 33559 Optimal

annealing

temperature

(°C)

PCR

product

(bp)

Annealing

temperatures

(°C)

PCR

product

(bp)

Annealing

temperatures

(°C)

katA 600 50-60 600 50-60 55

cadF 900 50-60 900 50-60 55

peb1A 780 50.9-54 780 50-54 51

C.

jejuni

cjaA

850 50-60 850 50-56.3 51

C. coli

cjaA 770 50.9-54 770 50-60 51

flpA 1150 50-60 1150 50-60 55

omp18 490 50-60 490 56.3-59 58

fliD NS Failed NS Failed NIS

fliD

set 1 NS Failed NS Failed NIS

fliD

set 2 NS Failed NS Failed NIS

Note: NS indicates Non-specific PCR products and NIS indicates Not included in this study

3.3.2 Detection of katA, cadF, peb1A, cjaA, omp18, and flpA genes in C.

jejuni and C. coli isolates representing flaA-HRM clusters

All C. jejuni (n=41) and C. coli (n=26) clusters reported in Chapter 2 were

assessed to identify the presence and absence of these genes using a

conventional PCR method. The PCR results revealed that amplicons of four

(katA, cadF, peb1A and cjaA-C. coli) of the seven genes were detected in all

C. jejuni and C. coli clusters (100%) (Table 3.8 and Appendix 3.2). Some

PCR samples of the four genes (katA, cadF, peb1A and cjaA-C. coli) that

showed 100% detection of both C. jejuni and C. coli were further

characterised using nucleotide sequence analysis.

140

Table 3.8: PCR analysis of Campylobacter gene detections, using all

Campylobacter jejuni and Campylobacter coli isolates that represents the

flaA-HRM clusters identified from the breeder and broiler farms

Campylobacter

gene

Number of Campylobacter isolates representing

flaA-HRM clusters detected (%)

C. jejuni C. coli Total

katA 41/41 (100) 26/26 (100) 67/67 (100)

cadF 41/41 (100) 26/26 (100) 67/67 (100)

peb1A 41/41 (100) 26/26 (100) 67/67 (100)

C. coli cjaA 41/41 (100) 26/26 (100) 67/67 (100)

C. jejuni cjaA 41/41 (100) 8/26 (30.77) 49/67 (73.13)

omp18 41/41 (100) 6/26 (23.08) 47/67 (70.15)

flpA 41/41 (100) 24/26 (92.31) 65/67 (97.02)

3.3.3 Nucleotide sequence and amino acid sequence analysis

Thirteen C. jejuni clusters (clusters 1, 2, 3, 5, 6, 8, 12, 26, 27, 28, 29, 36, and

39) and eight C. coli clusters (clusters 1, 2, 3, 5, 6, 13, 21, and 23) were

selected as the representative clusters for sequencing analysis. Appendix 3.3

summarises the information on the selected isolate number representing each

cluster. The nucleotide sequences and the subsequent amino acids from the

selected amplicons were aligned with the corresponding genes and

polypeptides of the C. jejuni and C. coli reference strains from the NCBI

database (Appendices 3.3 and 3.4).

3.3.3.1 Sequence analysis of the katA PCR amplicon

The NCBI nucleotide sequences of C. jejuni NCTC 11168 and C. coli

RM4661 were used as references for aligning to the sequences of nucleotides

and subsequent amino acids identified from the katA PCR amplicons. The

alignment analysis confirmed that the katA amplicons amplified from the

selected clusters belonged to the katA gene, with a 609-bp length (Appendix

3.3.1), and eight sequence groups were identified (Figure 3.1A).

These katA amplicons were translated into 203 subsequent amino acids

(Appendix 3.4.1), which showed 97.9% and 95.8% alignment similarity to

the selected C. jejuni and C. coli clusters, respectively, and 94.2% alignment

similarity between the two species. Six different groups of the subsequent

amino acids were identified among the selected clusters. Figure 3.1B displays

141

an example of subsequent amino acid variants. Only one group consisted of

both selected C. jejuni (n=7) and C. coli (n=2) clusters and shared 100%

similarity of the KatA amino acids with C. coli RM4661 (Appendix 3.4.1).

The other five groups had 13 different amino acid positions, and each group

had either C. jejuni or C. coli. Of the 13 different positions, eight were found

in the same group including C. coli clusters 1, 2, 3, 6, 13 and 21. In the eight

positions above, six had conservations between amino acid groups (five with

strongly and one with weakly similar physicochemical properties) and two

had non-conserved amino acids. Three groups (C. jejuni clusters 1, 2, 3,

12and 26) had a different position of conserved amino acid showing strongly

or weakly similar physicochemical properties. The remaining group (C. jejuni

cluster 28) had a different position that was not conserved.

142

Figure 3.1: Example of alignment analyses of the nucleotide sequences and subsequent amino acid sequences generated from the katA

amplicons of the selected C. jejuni and C. coli clusters.

A) Example alignment of the katA amplicons revealed the variations of nucleotide sequences among selected C. jejuni and C. coli clusters.

Identical nucleotides for each sequence compared to the reference are shown as dots (.).

B) Example alignment of the subsequent amino acid sequences coding for KatA amino acid showed differences between C. jejuni and C. coli

clusters. Identical amino acid residues for each sequence compared to the reference are shown as dots (.).

A)

A

A

A

B)

A

A

A

143

3.3.3.2 Sequence analysis of the cadF PCR amplicon

Based on the NCBI database, there were differences in the length of cadF

ORFs between Campylobacter spp. and the following strains: C. jejuni NCTC

11168 (n=960 bp), C. coli BP3183 (n=960 bp) and C. coli BG2108 (n=999

bp) (Appendix 3.3.2). Therefore, the cadF nucleotide sequences of C. jejuni

NCTC 11168, C. coli BP3183 and C. coli BG2108 from the NCBI database

were used as references for alignment analysis. The nucleotide alignment

analysis confirmed that the cadF amplicons from the selected C. jejuni and

C. coli clusters had high nucleotide identity to the cadF gene of the reference

strains (Appendix 3.3.2). Sequence analysis identified 14 groups of the cadF

amplicon in the selected clusters (Figure 3.2A). Of these, eight and six groups

were found in the selected C. jejuni and C. coli clusters, respectively. All C.

coli clusters tested, except for C. coli cluster 23, showed 39 extra nucleotides,

compared with the selected C. jejuni clusters (Figure 3.2A and Appendix

3.3.2).

The cadF ORF amplicon from the selected C. jejuni clusters and one C. coli

cluster were translated into 259 amino acids (Appendix 3.4.2). By contrast,

the subsequent amino acid generated from C. coli clusters 1, 2, 3, 5, 6, 13 and

21 showed 13 extra amino acids in length (Figure 3.2B). Overall, 13 groups

of the subsequent amino acids were identified in either C. jejuni (eight

groups) or C. coli (five groups). Of the C. jejuni groups, nine different amino

acid positions were identified: six conserved with strongly similar

physicochemical properties and three not conserved. Of the five C. coli

groups, one group (clusters 3 and 13) shared 100% similarity of CadF amino

acid with C. coli BG2108 and the other four had six different amino acid

positions. Of the four groups, one group (clusters 1 and 2) had a different

amino position (position 108) that was not conserved between groups

(Appendix 3.4.2). The same position was also found in other two groups

(clusters 5, 6, and 12). Both groups had other different amino acids with

different positions, which were conserved between amino acid groups with

weakly similar physicochemical properties. By contrast, the remaining group

(cluster 23) did not have the extra 13 amino acids although three different

amino acid positions were identified: two conserved with strongly similar

physicochemical properties and one non-conserved (Appendix 3.4.2).

144

Figure 3.2: Example of alignment analyses of the nucleotide sequences and subsequent amino acid sequences generated from the cadF


A) Example alignment of the cadF amplicons revealed variations of nucleotide sequences among C. jejuni and C. coli clusters. Identical

nucleotides for each sequence compared to the reference are shown as dots (.). The inserted nucleotides are indicated with red triangles.

B) Example alignment of the subsequent amino acid sequences coding for CadF protein showed differences between C. jejuni and C. coli

clusters. Identical amino acid residues for each sequence compared to the reference are shown as dots (.). The inserted amino acid residues are

shown as dashes (-).

A) B)

145

3.3.3.3 Sequence analysis of the peb1A PCR amplicon

The NCBI nucleotide sequences of C. jejuni YH002 and C. coli YH501 were

used as references for aligning to the peb1A amplicons from the selected

clusters. The alignment analysis of the nucleotide sequences confirmed that

the peb1A amplicons obtained were correct, with a 780-bp amplicon size

(Appendix 3.3.3). The two species had different peb1A nucleotide sequences

(Appendix 3.3.3), with seven and one different groups in the selected C. jejuni

and C. coli clusters, respectively (Figure 3.3A).

The peb1A amplicons generated from the selected clusters were translated

into 259 subsequent amino acids (Appendix 3.4.3). Their alignments were

97.9% and 100% similar in the selected C. jejuni and C. coli clusters,

respectively, but 79.1% similar between the two species. Five and one

different groups of the subsequent amino acids were found in the selected C.

jejuni and C. coli clusters, respectively. Of the five groups, one group shared

100% similarity of the Peb1A amino acids with C. jejuni YH002, whereas the

other four groups had six differences in the subsequent amino acids. Of these,

one group (clusters 1, 2, 3, 6, 8, 28, and 29) had a conserved amino acid

substitution with strongly similar physicochemical properties. Two groups

(clusters 12 and 39) had a conserve amino acid substitution with weakly

similar physicochemical properties at different positions (Appendix 3.4.3).

One group (cluster 5) had two different amino acids, which were conserved

between amino acid groups, with strongly similar physicochemical properties

(Appendix 3.4.3). As for C. coli clusters, the subsequent amino acids shared

100% similarity in the selected C. coli clusters (n=8) (Figure 3.3B). Thirty-

four out of the 38 different subsequent amino acid positions identified in the

C. coli clusters were conserved between the amino groups, compared with the

C. jejuni YH002 (Appendix 3.4.3). These included 26 and 8 positions

showing strongly and weakly similar physicochemical properties,

respectively. The remaining four positions were not conserved between the

amino acid groups.

146

Figure 3.3: Example of alignment analyses of the nucleotide sequences and subsequent amino acid sequences generated from the peb1A


A) Example alignment of the peb1A amplicons revealed the variations of nucleotide sequences among selected C. jejuni and C. coli clusters.


B) Example alignment of the subsequent amino acid sequences coding for Peb1A amino acids showed differences between selected C. jejuni and

C. coli clusters. Identical amino acid residues for each sequence compared to the reference are shown as dots (.).

A)

A

A

A

B)

A

A

A

147

3.3.3.4 Sequence analysis of the cjaA PCR amplicon

The nucleotide sequence of C. jejuni (Accession number Y10872.1) and C.

coli strain YH502 from the NCBI database was used as a reference for

aligning with the selected clusters (Appendix 3.3.4). The alignment analysis

revealed that the cjaA amplicon size was 767 bp in the selected C. jejuni and

C. coli clusters, and the nucleotide sequences were different in the selected

clusters (Appendix 3.3.4). Five and six different groups of the cjaA nucleotide

sequences were found among the selected C. jejuni and C. coli clusters,

respectively (Figure 3.4A).

The cjaA ORF amplicons from the selected clusters were translated into 255

subsequent amino acids (Appendix 3.4.4). The alignment analysis identified

four different groups. Of these, one group had seven C. jejuni clusters

(clusters 1, 3, 5, 12, 26, 27 and 36) and four C. coli clusters (clusters 2, 3, 5

and 13), which shared 100% of CjaA amino acids with the C. jejuni reference.

The remaining three groups had four different amino acid positions, which

were conserved between amino groups with strongly similar physicochemical

properties. Two positions were found in one group consisting of five C. jejuni

(clusters 12, 8, 28, 29 and 39) and three C. coli (clusters 6, 21 and 23) (Figure

3.4B); and one was found in the other two groups.

148

Figure 3.4: Example of alignment analyses of the nucleotide sequences and subsequent amino acid sequences generated from the cjaA


A) Example alignment of the cjaA amplicons revealed the variations of nucleotide sequences among selected C. jejuni and C. coli clusters.


B) Example alignment of the subsequent amino acid sequences coding for CjaA amino acid showed differences between selected C. jejuni and

C. coli clusters. Identical amino acid residues for each sequence compared to the reference are shown as dots (.).

A)

A

A

A

B)

A

A

A

149

3.3.4 Screening of transformed E. coli cells containing the ligated pET

SUMO plasmid

The C. jejuni flaA-HRM cluster 27 was the most frequently detected genotype

in Chapter 2, consequently, it was selected as the representative genotype for

cloning and expressing of the genes of interest. All conserved genes- katA,

cadF, peb1A and cjaA were re-amplified and ligated in the bacterial

expression vector. All single colonies from the transformed One Shot®

Mach1™-T1 competent E. coli cells were screened using conventional PCR

and the gene-specific oligonucleotides of each gene (Table 3.3) for the

presence of the inserted Campylobacter gene.

The results showed that katA, cadF, peb1A, and cjaA genes were successfully

cloned into the pET SUMO expression vector. The katA ORF was amplified

from the transformed E. coli cells in three of the eight colonies tested and was

approximately 680 bp in length at (Figure 3.5; Lanes 2, 7 and 8). The plasmids

yielding a fragment of the expected size were designated pET-SUMO-KatA1,

pET-SUMO-KatA7, and pET-SUMO-KatA8.

Figure 3.5: Example of agarose gel electrophoresis of the katA

amplicon generated from the pET SUMO plasmid contained katA


competent E. coli colonies as DNA template in PCR reactions.

Using katA primer set, the estimated size of PCR product is

approximately 680 bp (arrow). Colonies showing evidence of

transformed plasmids ligated with the inserted katA are detected in

Lanes 2, 7 and 8. Lane 1,1 Kb Plus DNA ladder; Lane 2, colony no.1,

Lane 3, colony no.2, Lane 4, colony no.3, Lane 5, colony no.4, Lane 6,

colony no.5, Lane 7, colony no.6, Lane 8, colony no.7, Lane 9, colony

no.8 and, Lane 10, RNase water (Negative control).

150

The cadF ORF was amplified from the transformed cells in three of eight

colonies and was approximately 910 bp in length (Figure 3.6; Lanes 4, 5, and

8). The plasmids yielding a fragment of the expected size were designated

pET-SUMO-CadF4, pET-SUMO-CadF5, and pET-SUMO-CadF8.

The peb1A ORF was amplified from the transformed cells in two of eight

colonies and was approximately 770 bp in length (Figure 3.7; Lanes 8 and 9).

The plasmids yielding a fragment of the expected size were designated pET-

SUMO-Peb1A8 and pET-SUMO-Peb1A9.

Figure 3.6: Example of agarose gel electrophoresis of the cadF

amplicon generated from the pET SUMO plasmid contained cadF



Using cadF primer set, the estimated size of PCR product is



Lanes 4, 5 and 8. Lane 1,1 Kb Plus DNA ladder; Lane 2, colony no.1,

Lane 3, colony no.2, Lane 4, colony no.3, Lane 5, colony no.4, Lane 6,

colony no.5, Lane 7, colony no.6, Lane 8, colony no.7, Lane 9, colony

no.8 and, Lane 10, RNase water (Negative control).

151

The cjaA ORF was amplified from two of the eight colonies tested and was

approximately 840 bp in length (Figure 3.8; Lanes 5 and 9). The plasmids

yielding a fragment of the expected size were designated pET-SUMO-CjaA5

and pET-SUMO-CjaA9.


amplicon generated from the pET SUMO plasmid contained peb1A



Using peb1A primer set, the estimated size of PCR product is



Lane 8 and 9. Lane 1,1 Kb Plus DNA ladder; Lane 2, colony no.1,

Lane 3, colony no.2, Lane 4, colony no.3, Lane 5, colony no.4, Lane

6, colony no.5, Lane 7, colony no.6, Lane 8, colony no.7, Lane 9,

colony no.8 and, Lane 10, RNase water (Negative control).

Figure 3.8: Example of agarose gel electrophoresis of the cjaA

amplicon generated from the pET SUMO plasmid contained cjaA



Using cjaA primer set, the estimated size of PCR product is



Lane 5. Lane 1,1 Kb Plus DNA ladder; Lane 2, colony no.1, Lane 3,

colony no.2, Lane 4, colony no.3, Lane 5, colony no.4, Lane 6,

colony no.5, Lane 7, colony no.6, Lane 8, colony no.7, Lane 9,

colony no.8 and, Lane 10, RNase water (Negative control).

152

3.3.5 Confirmation of the ligated pET SUMO plasmids

The purified plasmid DNA samples encoding the ORFs for the katA, cadF,

peb1A, and cjaA genes of C. jejuni cluster 27 (Section 3.3.4) were selected

for further analyses using double digestion with restriction endonucleases and

DNA sequencing to confirm the presence of the ORFs and in-frame cloning

with the SUMO ORF respectively, before protein expression.

The results from double digestion using the restriction endonucleases

revealed the excision of DNA fragments of sizes consistent with the cloned

PCR amplicons. The excised fragments obtained from katA, cadF, peb1A,

and cjaA gene ORFs were approximately 680, 910, 770, and 840 bp in size,

respectively (Figure 3.9).

The nucleotide sequence analysis of the pET SUMO clones showed the

successful insertion of the katA, cadF, Peb1A, and cjaA ORFs. This was

evident as the ORFs were in the correct orientation and in the same reading

frame as the SUMO ORF of the pET SUMO vector (Appendices 3.5.1, 3.5.2,

3.5.3, and 3.5.4).

Figure 3.9: Agarose gel electrophoresis of the digestion of pET

SUMO clones after digestion with HindIII and BamHI-HF (for the

katA ORF) or XhoI and BamHI-HF (for the cadF, peb1A and cjaA

ORFs).

The expected product sizes from the inserted katA, peb1A, cadF and

cjaA genes are approximately 680, 770, 910 and 840 bp, respectively.

Lane 1,1 Kb Plus DNA ladder; Lane 2, pET SUMO plasmid

containing katA gene; Lane 3, pET SUMO plasmid containing peb1A

gene; Lane 4, pET SUMO plasmid containing cadF; and Lane 5, pET

SUMO plasmid containing cjaA.

153

The nucleotide sequence of katA obtained from the pET SUMO system was

identical to the original nucleotide sequences of katA PCR amplicon from the

C. jejuni cluster 27 (Appendix 3.5.1). The 686 bp ORF translated a

polypeptide with 338 amino acid residues, that was identical to the amino acid

sequences of the original C. jejuni cluster 27.

For the pET SUMO-cadF clone, one nucleotide mismatch was identified

compared with the original C. jejuni cluster 27 sequence (Appendix 3.5.2).

The 913-bp cadF ORF, translated into a 304 amino acid polypeptide, one

conserved amino acid substitution (histidine; H for asparagine; N at residue

position of 288), compared to the parental C. jejuni cluster 27 (Appendix

3.6.1).

Sequencing of the peb1A ORF ligated into the pET SUMO vector identified

two nucleotide mismatches in the nucleotide sequence compared with the

original (Appendix 3.5.3). The inserted peb1A amplicon was 767 bp long and

translated a polypeptide with 255 amino acid residues. The nucleotide

changes resulted in one conserved amino acid substitution (leucine; L for

phenylalanine; F at residue position of 214) between the pET SUMO

amplicon and the C. jejuni cluster 27 it was derived from (Appendix 3.6.2).

Three mismatches were identified in the nucleotide sequence of the cjaA ORF

ligated into the pET SUMO vector, compared with the original cjaA amplicon

from the C. jejuni cluster 27 (Appendix 3.5.4). The cloned cjaA ORF was 839

bp long and could be translated into a 279 amino acid polypeptide, which was

identical to the parent C. jejuni cluster 27.

3.3.6 Protein expression of pET SUMO carrying katA, peb1A, cjaA, and

cadF

To determine if the Campylobacter ORFs fused to the SUMO ORF would

express fusion polypeptides, the characterised pET SUMO clones were used

to transform the E. coli One Shot® BL21 (DE3) expression cells. The pET

SUMO/CAT plasmid was used as a positive control for the expression of a

SUMO-CAT fusion protein with the estimated size of 39 kDa (Figures 3.10A

and 3.11A).

154

The expression of the Campylobacter polypeptides of interest, as soluble

SUMO fusions, was assessed in both IPTG-induced and uninduced E. coli

cell cultures at hourly intervals for 6 h using SDS-PAGE and Western blot

analyses.

The ORF encoding the SUMO fusion polypeptide of pET SUMO was 357 bp

in length and could be translated into a 119 amino acid polypeptide an

estimated molecular weight of 13.09 kDa. The pET SUMO fused katA ORF

was 1043 bp in length (Appendix 3.5.1), which encodes 347 amino acid

polypeptides with a calculated molecular weight of 38.17 kDa. The Western

blotting analysis showed that four polypeptides with the molecular sizes of

28, 30, 38, and 80 kDa were reactive with the 6x-His Tag monoclonal

antibody. Among these reactive species, the 38 kDa protein had the strongest

intensity and as it was consistent with the expected size it was deemed to be

the SUMO-KatA fusion polypeptide (Figure 3.10B).

The ORF encoding the SUMO fusion polypeptide of pET SUMO-CjaA was

1196 bp in length (Appendix 3.5.4), encoding a polypeptide with 398 amino

acid residues with an estimated molecular weight of 43.8 kDa. The Western

blotting analysis identified a single reactive polypeptide with a molecular

weight of approximately 44 kDa, as the estimated was consistent with the

expected molecular weight it was deemed to be the SUMO-CjaA polypeptide

(Figure 3.10C).

155

Figure 3.10: Western blot analysis of the soluble protein fraction of BL21 (DE3) E. coli cells containing pET SUMO/CAT (control), pET

SUMO-katA, and pET SUMO-cjaA plasmids at 0 h (T0) and 6 h (T6) with and without after IPTG induction.

The exposure time of immunoblotting was 10 sec. A) pET SUMO/CAT (control), B) pET SUMO-KatA, C) pET SUMO-CjaA. All panels:

Lane 1: protein molecular weight markers; Lane 2: BL21 (DE3) soluble fraction without IPTG at T0; Lane 3: BL21 (DE3) soluble fraction

with 1 mM IPTG T0; Lane 4: soluble fraction of BL21 (DE3) without IPTG at T6; Lane 5: soluble fraction of BL21 (DE3) with IPTG at T6.

Arrows indicate the reactive fusion polypeptides of intertest with the estimated molecular weights in parenthesis.

156

The ORF encoding the SUMO fusion polypeptide of pET SUMO-CadF was



blot analysis showed two reactive polypeptides with molecular weights of

approximately 47 and 40 kDa with similar intensity (Figure 3.11B).

The ORF encoding the SUMO fusion polypeptide of pET SUMO-Peb1A was



blotting analysis identified a single reactive polypeptide with a molecular

weight of approximately 40 kDa, as this estimate was consistent with the

predicted weight it was deemed to be the SUMO-Peb1A fusion polypeptide

(Figure 3.11C). The band was not detected when the exposure time was less

than 10 sec, whereas, it was detected at the exposure time of 10 sec. The

expression level of the SUMO-Peb1A fusion polypeptide was lower than the

SUMO-CAT control (Figure 3.11C).

The results showed that all Campylobacter SUMO polypeptides proteins

increased with time in the IPTG-induced E. coli cells. The amount of the

SUMO-KatA polypeptide detected was the highest (Figure 3.11B), whereas

the SUMO-Peb1A polypeptide exhibited the lowest level of expression

(Figure 3.10C).

157

Figure 3.11: Western blot analysis of the soluble protein fraction of BL21 (DE3) E. coli cells containing the pET SUMO/CAT (control), pET

SUMO-cadF, and pET SUMO-peb1A plasmids at 0 h (T0) and 6 h (T6) with and without after IPTG induction.

Exposure time of immunoblotting was 10 sec. A) pET SUMO/CAT (control), B) pET SUMO-CadF, C) pET SUMO-Peb1A. All panels: Lane

1: protein molecular weight markers; Lane 2: BL21 (DE3) soluble fraction without IPTG at T0; Lane 3: BL21 (DE3) soluble fraction with 1

mM IPTG T0; Lane 4: soluble fraction of BL21 (DE3) without IPTG at T6; Lane 5: soluble fraction of BL21 (DE3)with IPTG at T6. Arrows

indicate the reactive fusion polypeptides of intertest with the estimated molecular weights in parenthesis.

158

3.4 Discussion

Campylobacter jejuni and C. coli are the two most common species

associated with human food-borne illness from poultry products. One way to

reduce the disease burden in the human population would be to

reduce/eliminate colonisation of chickens. As C. jejuni and C. coli are closely

related species, this study has examined the level of conservation between

genes encoding putative protective antigens.

The current data showed that four genes (katA, cadF, peb1A, and C. coli-

cjaA) were detected in all C. jejuni and C. coli genotypes examined, thus

suggesting these genes are conserved between these two species. This finding

was consistent with previous studies (Day et al., 2000; Grant & Park, 1995;

Konkel, Gray, et al., 1999; Muller et al., 2005; Oh et al., 2017; Park, 1999;

Pei & Blaser, 1993; Pei et al., 1991; Richardson & Park, 1997; Shang et al.,

2016; Wyszynska et al., 2008). In contrast, three genes including omp18, C.

jejuni-cjaA and flp were not detected in all C. jejuni and C. coli genotypes.

This suggests that these genes may not be conserved between C. jejuni and

C. coli. Alternatively, the primers used in this study may be highly specific

for gene amplification.

The current data showed that the PCR reactions showed very good detection

of both C. jejuni and C. coli reference strains using all primer sets except for

the fliD primers. Some oligonucleotide primers were not fully aligned to the

conserved sequences of the Campylobacter gene especially C. coli. For

example, based on the alignment with the cjaA-C. jejuni primers, four

oligonucleotides from the forward and three oligonucleotides from the

reverse primers were mismatched compared with the cjaA gene of C. coli

reference strain (Appendix 3.5). Eight and six mismatches of 22 nucleotides

were found in the forward and the reverse flp primers, respectively compared

with the corresponding C. coli reference strain (Appendix 3.7). These

findings suggest that the mismatch oligonucleotide primers may influence the

consistent amplification of the target gene; indeed, Green et al. (2015)

suggested that the mismatches between primers and the DNA template could

be inefficient in target DNA amplification.

159

Our data showed that these genes contained low guanine and cytosine (G+C)

content (Appendix 3.3.1), which is consistent with the literature on

Campylobacter genome (Liu et al., 2018; Mohan & Stevenson, 2013; Pearson

et al., 2013; Poly et al., 2004; Takamiya et al., 2011; Taylor et al., 1992). This

could affect the specificity of primers in a PCR reaction and could be

challenging for primer design. Consequently, specific primers derived from

conserved sequences of each gene from more C. jejuni and C. coli strains

having less adenine (A) and thymine (T) content would enhance the PCR

efficiency. For further study, the design of highly specific primers and the

alignment with more C. jejuni and C. coli strains are indicated. Nevertheless,

the current study revealed the failure of the amplification of fliD during PCR

optimisation. Therefore, designing more specific primers (more specific

regions) and optimisation of PCR reactions for this gene would enhance the

specific fliD amplicon in further study.

Due to limited information on the comparison of the nucleotide sequences in

katA, peb1A, and cjaA genes between C. jejuni and C. coli, the current data

first showed that the nucleotide sequences of each conserved gene were

different between C. jejuni and C. coli, resulting in variations in the

subsequent amino acid sequences. Although the nucleotide sequences of katA

varied among Campylobacter genotypes, the subsequent amino acid had high

similarities— 97.3% and 95.8% for C. jejuni and C. coli, respectively. The

identity of the subsequent amino acids between C. jejuni and C. coli was

94.2%. These findings imply that katA is a good antigen candidate. However,

further investigation is required, such as identification of the epitope and

protein expression in mammalian cells.

For peb1A, the nucleotide sequences identified between C. jejuni and C. coli

genotypes were distinct, with the amino acid sequences encoded by these

genes being 79.1% similar. However, the subsequent amino sequences among

C. jejuni isolates were 97.9% similarity, whereas those of C. coli genotypes

were identical (100% similarity). Pei and Blaser (1993) reported that peb1A,

especially in the Open Reading Frame D (ORF-D) part, was a mature native

Peb1A protein, which is involved both in binding to intestinal cells and in

amino acid transport. Based on the subsequent amino acid sequences of this

study, peb1A amplified from most C. jejuni genotypes was identical to C.

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jejuni strain 81-176, thus suggesting these genotypes can represent similar

epitopes. The nucleotide sequence of peb1A amplified from C. jejuni clusters

26, 27, and 36 was identical to that of C. jejuni strain 81-176 (ORF-D) which

reported Pei and Blaser (1993). Even though most C. jejuni genotypes

(clusters 1, 2, 3, 6, 8, 28, and 29) from this study had different nucleotide

sequences of peb1A, the subsequent amino acids were similar to the C. jejuni

strain 81-176 as well (Pei & Blaser, 1993). Du (2008) reported that the

recombinant Peb1A vaccine induced immune responses and reduced sickness

and mortality in mice challenged with C. jejuni. These findings indicate that

the Peb1A protein is a good candidate for vaccine development against C.

jejuni colonisation. Although translated amino acid polypeptide of Peb1A

from C. jejuni differed from those of C. coli, we do not know these would

show similar epitopes or not. Therefore, more research focusing on the

investigation of epitope expression among Campylobacter spp. is required. If

this gene could represent the similar epitopes, it would be a good antigen

candidate for vaccine development with cross-protection purposes.

The cjaA gene amplified from C. jejuni and C. coli was highly conserved in

this study using the cjaA-C. coli primers. The subsequent amino acid

translated from these two species had 98.29% similarity, suggesting this gene

could present similar epitopes and could be a good candidate for vaccine

development; therefore, further investigation of this gene such as gene

expression in the mammalian cells is necessary.

The data showed that the cadF gene was highly conserved between C. jejuni

and C. coli genotypes of this study, but the alignment of its nucleotide

sequence obtained from these two species was different, consistent with a

previous study (Konkel, Gray, et al., 1999) who reported that this gene was

found in both C. jejuni and C. coli but different nucleotide sequences. A total

of 39 bp insertion nucleotide sequences was found in most C. coli genotypes,

which resulted in the addition of 13 extra amino acids compared with the

CadF protein produced by the C. jejuni genotypes in this study. This finding

agrees with that by Krause-Gruszczynska et al. (2007) who reported that the

CadF protein generated from C. coli strains was slightly larger than C. jejuni

homologue, by approximately 13 amino acids due to its additional

nucleotides. By contrast, the present study also showed that one C. coli

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genotype did not have the extra amino acids and resulted in a predicted length

of 259 amino acids, similar to that of C. jejuni. The present study found

variations of these genes between C. jejuni and C. coli, possibly indicating

differences in epitopes and immunogenicity. These differences suggest that

CadF would be unsuitable for use as a cross-protective antigen for C. jejuni

and C. coli.

The genes from C. jejuni cluster 27, as the most frequent genotype in broiler

flocks, was used for evaluating protein expressions in this study. These

Campylobacter genes (katA, cadF, peb1A, and cjaA) were successfully

cloned into pET SUMO plasmids. These genes were verified using nucleotide

sequence alignment and the subsequent amino acid sequences. Nucleotide

sequence alignment showed that the inserted pET SUMO-katA ORF was

identical to that of C. jejuni cluster 27 (Appendix 3.5.1). The remaining ORFs

(cadF, peb1A, and cjaA) were at least 99% similarity (Appendices 3.5.2,

3.5.3, and 3.5.4). However, these differences did not affect the amino acid

properties. The subsequent amino acid sequences encoded by the pET SUMO

fusion ORFs were identical to the corresponding polypeptides of C. jejuni

cluster 27. While the pET SUMO ORF encoding the cadF and peb1A ORFs,

had one amino acid substitution compared with those of C. jejuni cluster 27,

the polypeptides were still conserved as the substitution was between amino

acid groups of strongly similar properties (Appendices 3.6.1 and 3.6.2). To

prove this, three recombinant pET SUMO plasmids containing each OFRS

were sent for DNA sequences and resulted in the different nucleotides were

randomly dispersed among plasmid samples. These findings suggest that

conservative substitution may have resided, in agreement with a study of

Potapov and Ong (2017), which suggested that lack of proof-reading by Taq

polymerase (during PCR reactions) and DNA damage (during temperature

cycling) could introduce mutations in PCR products. Another possible reason

is a low concentration of the DNA template used in PCR reactions, leading to

mutations (Akbari et al., 2005). Further studies need to check PCR products

from replicate PCR reactions using the same DNA template and measure the

DNA template prior to cloning in order to ensure that all PCR products are

identical to the original template.

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Western blotting revealed that the transformed E. coli cells harbouring the

cloned pET SUMO plasmids contained each gene and successfully expressed

the protein of interest. The Western blot of total cell lysates results showed

that two molecular weights of the CadF protein were detected with different

sizes (Figure 3.14B), consistent with a previous study (Krause-Gruszczynska

et al., 2007) who reported two different sizes of CadF were identified on

immunoblotting. The heat-modifiable CadF protein is a component of OMP

(Mamelli et al., 2007). Incomplete denaturation during cell lysis may have led

to a partially folded form of the cadF protein and resulted in a smaller size;

this agreed with a study by Krause-Gruszczynska et al. (2007), which

suggested that two different sizes of the CadF protein may be influenced by

their heat-modifiable conformational state.

The immunoblot of KatA expression showed the KatA protein expression

with multiple molecular weights; of these, the one with 38.2 kDa molecular

weight showed the strongest intensity. The higher molecular weight could be

a multimeric form of KatA, whereas, the lower band(s) could be broken down

products, suggesting it is unstable. However, the lack of information on

Campylobacter KatA protein, the investigation of KatA protein property is

required for further study.

In cjaA expression, only one protein with a molecular weight of 43.8 kDa,

belonging to the CjaA protein, was found; however, some background from

cell lysates was also observed with very low intensity. The E. coli cells

containing pET SUMO-peb1A expressed the 41.1 kDa Peb1A protein on

immunoblotting, but the expression was low. This finding suggests that pET

SUMO may not be a good expression vector for this gene in transformed E.

coli. Nielsen et al. (2012) too found that Peb1A was not detected in the

Western blot analysis and suggested that some C. jejuni extracellular protein

may be poorly expressed by E. coli and could be undetectable on Western

blot analysis. Alternatively, a protein produced during induction may have

been toxic to E. coli cells due to unrestricted regulation of the IPTG-induced

expression of T7 RNA polymerase in the BL21(DE3) E. coli cells (Hoppe et

al., 2012; Saïda et al., 2006; Studier, 1991). Addition of 1% glucose in the

medium during growth or incubation at 22 ± 2 °C for 1–2 days could

overcome this problem (Hoppe et al., 2012; Invitrogen, 2010a).

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However, some limitations may have affected the Western blotting results.

First, a high background on immunoblotting could be the result of long

exposure time or the use of excessive antibody or old buffer (Mahmood &

Yang, 2012). Second, a quaternary protein structure may have caused a too

large molecular weight of protein, which can be solved by reheating the

samples (Mahmood & Yang, 2012), whereas lower-molecular-weight bands

could result from the degradation of the protein of interest by endogenous

protease contamination (Ghosh et al., 2014; Mahmood & Yang, 2012).

Therefore, a rapid process between frozen cells and lysed cells can be

effective in avoiding protein degradation.

The current data revealed that ORFs for the katA, cadF, peb1A, and cjaA

genes were conserved in both C. jejuni and C. coli isolated from the chicken

farms. However, their nucleotide and subsequent amino acid sequences

varied between them. The subsequent amino acid sequences of CadF

polypeptides from C. jejuni and C. coli were distinct, suggesting it is not a

suitable candidate for use in a cross-protective vaccine. The subsequent

amino acid sequences of KatA and CjaA between C. jejuni and C. coli were

more than 94% similarity, whereas, that of Peb1A was 79.1% identical. On

the other hand, the different amino acid sequences of each gene between C.

jejuni and C. coli may present similar epitope, and thus further analysis is

required.

The C. jejuni flaA-HRM cluster 27 as the representative strain in the broiler

flocks was selected for gene identification and characterisation in this study.

These four conserved genes were successfully cloned into pET SUMO

plasmids and expressed the protein of interest in bacterial cell cultures.

Although some conservative substitutions were detected, the amino acids

(antigen) properties were strongly similar. As for the gene expression, three

proteins, KatA, CadF, and CjaA, were strongly expressed using the E. coli

cells. In contrast, Peb1A was poorly expressed, based on the Western blot

analysis. Further study on gene expression in mammalian cells is required to

determine if these conserved genes are expressed in a eukaryotic environment

(mammalian cells) before constructing a viral vector vaccine in Chapter 4.

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In conclusion, based on the cross-species conservation and level of expression

of the SUMO fusion polypeptides, KatA, CadF, and CjaA, were identified as

the most promising candidate for inclusion in a Campylobacter vaccine to

reduce/prevent the colonisation of chickens. These genes could be well suited

for use in either subunit vaccines generated using antigen expressed and

purified from bacterial cells or prokaryotic vectored vaccines. The SUMO

fusion Peb1A polypeptide exhibited the lowest expression in E. coli cells in

this study. However, a previous study found Peb1A to be a potential antigen

that could be used in a vaccine to reduce C. jejuni colonisation of chickens

(Buckley et al., 2010). Consequently, this gene was still included for further

evaluation of protein expression in a eukaryotic system (Chapter 4) to be

evaluated for its potential use in the development of a viral vector-based

vaccine.

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Chapter 4 Expression of Campylobacter genes and HVT vector

vaccine preparation

4.1 Introduction

Commercial chickens are commonly slaughtered by 6-8 weeks of age, based

upon market weight (Animal Liberation NSW, 2019). The immune system of

chickens including the development of humoral immunity and the generation

of functional antibodies is still developing until approximately 6 weeks of age

(Lacharme-Lora et al., 2017). Thus, antibodies may not effectively modulate

Campylobacter spp. present in the intestines before the slaughter of the

commercial broiler chicken. Moreover, the persistence of protective maternal

immunity which generally remains in commercial chicks until 2–3 weeks of

age is associated with the delay of Campylobacter spp. colonisation

(Laniewski et al., 2012; Rice et al., 1997; Sahin, Luo, et al., 2003; Wyszynska

et al., 2004). Thus, the main arm of the immune response that has the potential

to modulate Campylobacter spp. colonisation is cell-mediated immunity.

These factors underlie the challenges for vaccine development against

Campylobacter spp. colonisation in commercial chickens.

To provide a practical and effective solution to the commercial poultry

industry, a vaccine that rapidly induces a strong immune response at an early

age (especially a cell-mediated immune response) and significantly reduces

Campylobacter colonisation within commercial broiler chickens is of interest.

Accordingly, a viral vector vaccine could be an effective alternative solution

to reduce Campylobacter colonisation, since it has high infectivity and elicits

both humoral and cellular immune responses without being affected by pre-

existing immunity (Baron et al., 2018; Dey et al., 2017; Gerdts et al., 2006;

Ingrao et al., 2017; Santra et al., 2005).

Herpesvirus of turkeys (HVT) is one of the most potent delivery vectors for

vaccines and has been used to induce antigens of various chicken infections

such as Chlamydia psittaci (Liu et al., 2015), infectious bursal disease (Roh

et al., 2016), Newcastle disease (El Khantour et al., 2017), and infectious

laryngotracheitis (Vagnozzi et al., 2012). The recombinant HVT vector

vaccine is known to be safe and less sensitive to maternal immunity

interference (Baron et al., 2018; Dey et al., 2017; Ingrao et al., 2017). Li et al.

(2011) reported that HVT-based vector vaccines expressing antigens using

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the bacterial artificial chromosome provided very effective results in both in

vitro and in vivo. For these reasons, the construction of the HVT vector

harbouring conserved Campylobacter genes is of interest in this study.

In the previous chapter, four Campylobacter genes (katA, cadF, peb1A, and

cjaA) were shown to be conserved between C. jejuni and C. coli genotypes

and these were successfully cloned and expressed in transformed E. coli cells,

as described in Chapter 3. Before vaccine construction, recombinant gene

expression in eukaryotic cells is a crucial step to understand the biological

properties by producing the protein of interest (Kaufman, 2000; Khan, 2013).

Two vectors, pcDNA™ 3.1 D/V5-His-TOPO and pEGFP-C1, have been

commonly used as recombinant protein expression vectors for high-level

expression of protein in mammalian cells (Abis et al., 2019; Ding et al., 2012;

Jomary & Jones, 2008; Joseph et al., 2002; Shetty et al., 2004; Song et al.,

2015; Wang et al., 2012).

The pcDNA™ 3.1 D/V5-His-TOPO vector is a fast-recombinant cloning

vector. It can be expressed immediately and directly in mammalian cell lines

and provides highly efficient cloning (Udo, 2015). For cloning with this

vector, primer design is crucial since it has a GTGG overhang at the 5′ end of

the cloning site (Invitrogen, 2010b). As a result, the 5´ end of the forward

primer requires the inclusion of four bases, CACC, and corresponding to a

portion of the Kozak sequence to ensure correct cloning direction with the

inserted gene and proper translation (Invitrogen, 2010b; Kozak, 1984).

Furthermore, pcDNA3.1 allows the expression of the cloned gene of interest

in mammalian cells driven by a cytomegalovirus (CMV) promoter (Foecking

& Hofstetter, 1986).

pEGFP-C1, a eukaryotic expression vector, has been extensively used to

express animal and human genes in mammalian cells (Buelow et al., 2011; Li

et al., 2014; Wang et al., 2012; Xu et al., 2008). The pEGFP-C1 vector

encodes the enhanced green fluorescent protein (EGFP) gene, which

expresses the enhanced green fluorescent protein (EGFP) driven by the CMV

promoter and expresses EGFP fused with the protein of interest without

creating toxic effects on cells (Collares et al., 2011). pEGFP-C1 has been used

to investigate the intracellular activities of genes of interest by visualising

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EGFP fluorescence histologically (Broadway et al., 2003; Tamura et al.,

2011; Wang et al., 2012).

This chapter aimed to characterise the expression of the conserved

Campylobacter genes – katA, cadF, peb1A, and cjaA – in eukaryotic

expression systems to evaluate their suitability for delivery using a HVT viral

vector. Following that, the HVT and chicken embryonic fibroblast (CEF)

cells were initially prepared. Prior to the construction of the vector, TCID50

infectivity was determined using different multiplicities of infection (MOIs).

4.2 Materials and Methods

The pcDNA™ 3.1 Directional TOPO® Expression Kit (Invitrogen, 2010b)

and the pEGFP-C1 expression vectors were used to evaluate the expression

of the KatA, CadF, Peb1A, and CjaA proteins in eukaryotic cell culture.

4.2.1 Gene expression using the pcDNA™ 3.1 D/V5-His-TOPO® vector

The conserved katA, cadF, peb1A, and cjaA genes as reported in Chapter 3

were re-amplified from the genomic DNA of a C. jejuni cluster 27 isolate

using specific primers for cloning (Table 4.1). The pcDNA™ 3.1 D/V5-His-

TOPO® expression vector from the pcDNA TM 3.1 Directional TOPO®

Expression Kit (Invitrogen, USA) was used for cloning according to the

manufacturer’s instructions.

4.2.1.1 Amplification of katA, cadF, peb1A, and cjaA genes

The oligonucleotide primers used for amplifying the Campylobacter genes of

interest included, a four-nucleotide motif, CACC, at the 5′ end of the forward

amplification primers to facilitate directional cloning into the plasmid vector.

An in-frame ATG start codon was also added to the forward primer to allow

initiation of translation of the gene of interest. The estimated size of each PCR

product is summarised in Table 4.1.

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Table 4.1: Oligonucleotide primers used for gene amplification and

expression vector cloning

Gene Sequence 5’ to 3’ Estimated

PCR (bp)

katA Forward: C ACC ATG GAA AGT TTA CAT CAA

GTA ACC ATT CTT ATG AGC

Reverse: CCA AAC AGC TAT GAT AAT AGC CCA

670

cadF Forward: C ACC ATG GGT GCT GAT AAC AAT

GTA AAA TTT GAA ATC ACT CCA

Reverse: CCA CGC TCA AGC AAT GAC ACT AAA

870

peb1A Forward: C ACC ATG GCT CTA GGT GCT TGT

GTT GCA

Reverse: TTT GCA AAA TAT GTT GAT GAT TTT

GTA AAA

700

cjaA Forward: C ACC ATG GTC AAG CAA AAT GGA

GTT GTA

Reverse: ACT TTA AAA AGT CAT TTT GGA GAT

700

Note: The underlined letters indicate additional nucleotides (CACCATG) added to the 5′ end of the forward primer for directional cloning and translation initiation.

All PCR assays with gene-specific primers were performed in a BIO-RAD

S1000TM thermal cycler (BIO-RAD, Australia). The reaction volume was 25

µL and comprised 2 U of Platinum™ Pfx DNA Polymerase (Invitrogen,

Australia), 1× Pfx Amplification Buffer (Invitrogen), 0.3 mM of dNTP

mixture (Invitrogen), 1 mM MgSO4, 10 µM of primer mixture (Integrated

DNA Technologies, Singapore), 10–30 ng of DNA template, and RNase-free

water (Ambion®) to a final volume of up to 25 µL.

The cycling conditions were as follows: 94°C for 4 min (one cycle), 40 cycles

of denaturation at 94°C for 10 sec, annealing at 55°C (katA and cadF) or 51°C

(peb1A and cjaA-C. coli) for 20 sec, and extension at 72°C for 30 sec, as

described in Section 3.2.3.3. All PCR products were analysed using 1.5% gel

electrophoresis as described in Section 3.2.3.4.

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4.2.1.2 TOPO® cloning reaction and transformation

All freshly amplified PCR products of each gene (Section 4.2.1.1) were

cloned directly into the pcDNA™ 3.1 D/V5-His-TOPO® expression vector

from the pcDNA™3.1 Directional TOPO® Expression Kit according to the

manufacturer’s instructions (Invitrogen, 2010b). Each reaction was

performed in a 6 μL volume (Table 4.2) at 22 ± 2 °C for 5 min and placed on

ice.

Table 4.2: Cloning reaction for the TOPO® vector and gene amplicons


Fresh PCR amplicon X*

Salt solution 1


pcDNA™3.1D/V5-His-TOPO® vector 1

Total 6

Note: * Each ligation reaction was performed using the vector: insert (PCR amplicon) molar ratios of 1:1 in this study.

A 2-μL volume of the TOPO cloning reaction mixture (pcDNA™3.1D/V5-

His-TOPO® vector containing inserts of interest) was gently mixed into a vial

of One Shot® TOP10 chemically competent E. coli cells and incubated on ice

for 30 min. The mixture was subjected to heat-shock at 42°C for 30 sec in a

water bath and immediately transferred to ice. Then, the cells were mixed

with 250 μL of room-temperature SOC medium (Section 3.2.4.3) and then

horizontally incubated at 37°C with shaking at 200 rpm for 60 min. An aliquot

of each transformation (60 µL) was subsequently spread onto a pre-warmed

Luria–Bertani agar plate containing 100 μg/mL ampicillin (LB-Am100) and

incubated at 37°C for 16 ± 2 h. The pUC19 control plasmid DNA (10 pg),

provided from the kit, was used as a positive control transformation and

plating control. A pcDNA™3.1D/V5-His-TOPO® vector (without PCR

product), provided from the kit, was used as a negative control.

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4.2.1.3 Screening of transformed colonies

The presence of plasmids containing the inserts of interest in the transformed

ampicillin-resistant bacterial colonies (whole cells) was conducted using a

conventional PCR assay.

A single bacterial colony on each LB-Am100 plate was collected using a 40

μL sterile micropipette tip and gently resuspended in 20 μL of nuclease-free

water to generate a cell suspension. All PCR assays were performed in a BIO-

RAD S1000TM thermal cycler (BIO-RAD, Australia). Each PCR assay,

prepared in a total volume of 25 μL containing 2 μL of the cell suspension as

DNA template, and cycling parameters were as previously described in

Sections 4.2.1.1 and 3.2.3.4, respectively. The forward and reverse primers

used in each PCR assay are described in Table 4.1.

Single E. coli colonies yielding amplicons of the expected length were

cultured in LB broth containing 100 µg of Ampicillin (LB-Am 100 broth) and

incubated for 16 ± 2 h at 37°C. The incubated cultures were processed for

plasmid isolation using the QIAprep® Spin Miniprep Kit (Qiagen, Germany)

following the manufacturer’s instructions (Qiagen, 2015a).

4.2.1.4 Confirmation of recombinant TOPO plasmids

To confirm the plasmids contained an insert consistent with the amplicon of

interest, they were digested with XhoI and BamHI-HF restriction enzymes.

The reaction solution and the conditions of double digestion are described in

Section 3.2.4.6.1. The digested reactions were analysed by electrophoresis on

a 1.5% agarose gel as previously described (Section 3.2.3.4). The sizes of the

digestion fragments were estimated by comparison to a molecular weight

marker (1kb ladder, New England Biolabs, Australia). The estimated

restriction enzyme fragments relative to the relevant PCR amplicons are

summarised in Figure 4.1.

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Purified plasmid DNA with restriction enzyme fragments consistent with the

PCR amplicons of interest were selected for sequence analyses, to confirm

the nucleotide sequence and orientation of each ORF as previously described

(Sections 3.2.3.5.2 and 3.2.5.3). The sequencing primers provided from the

pcDNA™3.1 Directional TOPO® Expression Kit (Invitrogen) were used for

the forward and reverse sequencing reactions (Table 4.3).


plasmid containing Campylobacter genes and the recombinant pEGFP-C1

plasmids

Primer Name Nucleotide sequence

T7 5´-TAATACGACTCACTATAGGG-3´

BGH Reverse 5´-TAGAAGGCACAGTCGAGG-3´

EGFP-C Forward 5´- CATGGTCCTGCTGGAGTTCGTG -3´

SV40pA-R Reverse 5´- GAAATTTGTGATGCTATTGC -3´

4.2.1.5 Gene expression in eukaryotic cells

All recombinant TOPO plasmids carrying the required ORF were transfected

into the rabbit kidney-13 (RK-13) cells to analyse for recombinant gene

expression.

Figure 4.1: Schematic representation of the BamHI-HF and XhoI

restriction sites located on the recombinant TOPO vector containing

each inserted PCR amplicon from the gene of interest (green colour).

The estimated product of each gene cloned into each TOPO vector after

cutting with restriction enzymes (BamHI-HF and XhoI) is indicated with

arrow. The sizes of katA, cadF, peb1A and cjaA products were

approximately 737, 935, 764, and 764 bp, respectively.

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4.2.1.5.1 Cell culture preparation

RK-13 cells were grown in a 75-cm2 flask with a vented cap (Nunclon™

Delta Surface; Thermo Fisher Scientific, Denmark) containing 15 mL of

Dulbecco’s modification of Eagle medium (DMEM; Gibco, Life

Technologies, Carlsbad, CA, USA) supplemented with 5% foetal bovine

serum (FBS; Gibco), as described in Appendix 4.2 at 37°C for 16 ± 2 h in a

humidifying atmosphere containing 5% CO2. Cell growth was monitored

daily using an inverted microscope (Nikon Eclipse Ti-s; Tokyo, Japan) until

80% confluence was reached. At this point, the cells were subculture and cell

count.

4.2.1.5.2 Transfection into mammalian cells

Prior to transfection, RK-13 cells were seeded in a 6-well tissue culture plate

(Nunclon™ Delta Surface, Thermo Fisher Scientific, Denmark) at a density

of 5 × 105 cells per well and grown in 2 mL of DMEM with 5% FCS under

the same conditions as described above until cultures reached 70–80%

confluence.

The recombinant plasmids containing the ORF of interest were transfected

into RK-13 cells in the presence of Lipofectamine® and Plus™ reagents

(Invitrogen) according to the manufacturer’s instructions (Invitrogen).

Briefly, for each transfection, the recombinant TOPO plasmid DNA (1 µg)

was diluted to 100 µL of Opti-MEM® (Gibco); then 4 µL of Plus™ reagent

was added, and the mixture was incubated at 22 ± 2 °C for 15 min.

Lipofectamine® (5 µL) was diluted to 125 µL with Opti-MEM® (Gibco),

transferred to the mixture of the diluted plasmid DNA-Plus™ reagent, and

incubated at 22 ± 2 °C for 15 min to facilitate the formation of DNA-Plus™-

Lipofectamine® reagent complexes.

The RK-13 cell were washed three times with 1 mL of 1× phosphate-buffered

saline (PBS) and the medium was then replaced with 800 µL of Opti-MEM®

(Gibco); and the mixture of DNA-Plus™-Lipofectamine® Reagent (229 µL)

was added to monolayers in each well and incubated at 37°C with 5% CO2

for 3 h. The culture volume was increased with 2 mL of DMEM with 5% FBS

(Gibco) and incubated at 37°C with 5% CO2 for 48 h. The pcDNA™3.1D/V5-

His/lacZ vector plasmid containing the gene for β-galactosidase gene,

173

supplied in the pcDNA™3.1 Directional TOPO® Expression Kit (Invitrogen),

was used as a positive control for transfection and recombinant protein

expression. This control plasmid expresses β-galactosidase fusion protein

with an estimated molecular weight of 120 kDa that can be detected by

Western blotting (Invitrogen, 2010b). RK-13 cells without transfection were

used as a negative control for transfection and expression.

4.2.1.5.3 Cell lysis

Following 48 h incubation, the transfected cells were harvested by

trypsinisation for protein extraction. Old media from the cultures was

removed using Nunc™ serological pipettes (Thermo Fisher Scientific,

Waltham, MA, USA), and the transfected cells were washed three times with

1 mL of PBS. The cells were trypsinised using 600 µL of 1× trypsin and then

incubated at 37°C for 5 min to dissociate the transfected cells from the wells.

Cells were resuspended in maintenance media (DMEM with 5% FBS) and

transferred to new 15-mL high-clarity polypropylene conical centrifuge tubes

(Falcon®, Corning, Mexico). The resuspension tube was centrifuged at 800 g

for 10 min at 4°C and the supernatant was discarded. The cell pellets were

washed three times with 1 mL of PBS, centrifuged at 1000 g for 10 min each

time, and the supernatant was discarded. The cell pellets from each well were

resuspended in the 700 µL cell lysis solution, containing 400 µL of

BugBuster® Protein Extraction Reagent (EMD Millipore Corp., Billerica,

MA, USA), 200 µL of 4× Novex® LDS sample buffer (Life Technologies),

and 100 µL of 10× Nupage® sample reducing agent (Invitrogen). Then, a few

glass beads were added to the mixture. Five cycles of vortexing (30 sec) and

transferring to ice (30 sec) were performed to ensure complete cell lysis. The

cell lysates were heated at 95°C for 10 min and clarified by centrifuged at

10,000 g for 5 min. The supernatant containing the soluble protein fraction

was carefully transferred to a clean tube and stored at -20°C until required.

4.2.1.5.4 SDS-PAGE and Western blotting analysis

The cell supernatants were resolved using a Bolt 4-12% Bris-Tris plus SDS

polyacrylamide gel electrophoresis apparatus (SDS-PAGE) (Invitrogen,

USA) as described in Section 3.2.4.10.

174

Western blotting was performed as described in Section 3.2.4.11. The blots

were exposed to a Fuji Super RX-N medical X-ray film (Fuji Corporation,

Japan) in the darkroom for 1–3 min, and the radiograph was digitised using a

Gel DocTM XR+ imaging system (Bio-Rad) with Image LabTM software (Bio-

Rad). The size of the recombinant protein was analysed using Precision Plus

Protein™ Dual Colour Standards (Bio-Rad, USA).

4.2.2 Construction of recombinant pEGFP-C1 harbouring katA,

peb1A, cjaA, and cadF

The ORFs cloned into the pET SUMO plasmids described in Chapter 3 were

subcloned into the eukaryotic expression pEGFP-C1 vector for expression in

Vero cell cultures.

4.2.2.1 Double digestion with two restriction enzymes

The respective pET SUMO plasmids from Chapter 3 and the pEFPC1 plasmid

were digested with two restriction enzymes to excise the ORFs of interest as

described in Section 3.2.4.1.

For subcloning the katA ORF into pEGFP-C1 and pET SUMO-katA plasmids

were subjected to double digestion using HindIII (New England Biolabs,

USA) and BamHI-HF (New England Biolabs, USA), for the construction of

pEGFP-C1-katA.

For subcloning of cadF, peb1A, cjaA ORFs into pEGFP-C1 from the

respective pET SUMO plasmids double digestion with using XhoI (New

England Biolabs, USA) and BamHI-HF (New England Biolabs, USA) was

used to for the construction of pEGFP-C1-cadF, pEGFP-C1-peb1A, and

pEGFP-C1-cjaA. The restriction enzyme digestion solution was performed in

a BIO-RAD S1000TM Thermal Cycler (BIO-RAD, Australia) and conditions

are described in Section 3.2.4.6.1.

After completion of the restriction enzyme digestion, the products were

visualised by agarose gel electrophoresis (Section 3.2.3.4). The fragments of

interest (excised ORFs and double digested pEGFP-C1 vector) were excised

from the agarose gel and extracted using a QIAquick Gel Extraction Kit

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(Qiagen, 2015b). The concentrations and purities of all purified ORF inserts

and digested pEGFP-C1 vector were estimated using a Nanodrop® ND-1000

(Wilmington, DE, USA) using the manufacturer’s instructions.

4.2.2.2 Insert gene construction

The purified fragment of each ORF was ligated into the pEGFP-C1 in a 10

μL ligation reaction (Table 4.4) and then incubated at 4°C for 16 ± 2 h.

Table 4.4: Cloning reaction for the pEGFP-C1 vector and Campylobacter

ORF fragments


Freshly purified insert gene X*

Fresh purified pEGFP-C1 vector (50 ng) 1

T4 DNA ligase (Promega, Madison, WI, USA) 1

T4 DNA ligase 10× buffer (Promega, Madison,

WI, USA)

1


Total 10

Note: * Each ligation reaction was performed using the vector: insert (PCR amplicon) molar ratios of 1:3 in this study

A 2 μL aliquot of the ligation reaction was used to transform one vial of One

Shot® TOP10 chemically competent E. coli cells (Invitrogen, USA). The

ligation reaction and the competent E. coli cells were incubated on ice for 30

min and then heat-shocked at 42°C for 30 sec using a water bath. The cells

were immediately transferred to ice, and 250 μL of room-temperature SOC

medium was added. The cells were then incubated at 37°C with shaking at

200 rpm for 60 min. An aliquot of each transformant (60 µL) was

subsequently spread onto a pre-warmed LB-Kan50 plate and incubated at

37°C for 16 ± 2 h.

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4.2.2.3 Screening of transformed E. coli colonies

All single transformed E. coli colonies from each transformation were

resuspended in 10 µL of sterile MilliQ water. A conventional PCR was used

to assess the presence or absence of an insert consistent with the expected size

of the ORF of interest in the resuspended bacterial colonies (whole cells)

using two different primer sets. The first set was the cloning primers used to

detect the gene of interest in the transformed colonies. The PCR assays and

conditions for the cloning primers (Table 3.2) are described in Section 3.2.4.4.

The pEGFP-C1 sequencing primers (Table 4.3) were also used to ensure the

presence of recombinant pEGFP-C1 with the gene of interest. The PCR

mixture was prepared as described in Section 3.2.4.4. The PCR cycling

programs for the pEGFP-C1 primers were as follows: 94°C for 4 min (one

cycle), 40 cycles of denaturation at 94°C for 10 sec, annealing at 58°C for 20

sec and extension at 72°C for 30 sec. The PCR amplicons were analysed as

described in sections 3.2.4.1.2, 3.2.3.4, and 3.2.3.5. Any colony yielding an

amplicon of the expected size was grown in 5 mL of LB-Kan50 broth at 37°C

for 16 ± 2 h. Plasmid DNA was recovered from the cultures as previously

described (Section 3.2.4.5).

4.2.2.4 Identification of the recombinant pEGFP-C1 plasmids

All purified recombinant plasmids were analysed for the presence of the

inserted gene using specific restriction enzymes (with the same restriction

enzymes used for cloning) and were confirmed using DNA sequencing with

vector primers EGFP-C and SV40pA-R (Table 4.3) as previously described

in Section 4.2.1.4.

4.2.2.5 Transfection in eukaryotic cells

All the recombinant pEFGP-C1 plasmids confirmed that the correct

orientation of each gene was transfected into Vero cells to define protein

expression as follows.

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4.2.2.5.1 Cell culture preparation

Vero cells representing eukaryotic cells were grown to a confluent monolayer

with the same conditions as described in Section 4.2.1.5.1.

4.2.2.5.2 Transfection

One day before transfection, Vero cells were seeded in 6-well tissue culture

plates (Nunclon™ Delta Surface, Thermo Fisher Scientific, Denmark) at a

density of 5 × 105 cells per well, and 2 mL of DMEM with 5% FBS (Gibco)

was added. Then, the cell cultures were grown under the same conditions as

described in Section 4.2.1.5 as monolayers to 80% confluent.

The recombinant pEGFP-C1 plasmids containing the gene of interest were

transfected into Vero cell cultures (monolayers) using the Lipofectamine®

and Plus™ reagents (Invitrogen) according to the manufacturer’s instructions

(Invitrogen) and as described in Section 4.2.1.5. Non-transfected Vero cells

(mock) and transfected Vero cells with pEGFP-C1 alone were used as

negative and positive controls, respectively.

4.2.2.6 Evaluation of transfection efficiency

After transfection, monolayers of Vero cells transfected with pEGFP-C1-

katA, pEGFP-C1-cadF, pEGFP-C1-peb1A, pEGFP-C1-cjaA, and pEGFP-C1

alone were observed daily for EGFP expression using fluorescent

microscopy. Briefly, transfected cells and non-transfected cells were

identified as green-stained cells and non-stained cells, respectively, using BF

light path under an Olympus CKX41 inverted microscope equipped with an

Olympus DP 70 camera (Olympus, Japan). The cells were visualised using a

10× lens of the microscope and DP manager software version 2.2.1.195. The

images of GFP fluorescent transfected cells and non-transfected cells were

captured at equivalent exposure times with a DP70 camera using an Olympus

DP Controller software version 2.2.1.227 (Olympus, Japan).

4.2.2.7 Analysis of EGFP Campylobacter fusion protein expression

All transfected cells and non-transfected cells were subsequently determined

by the antigen (protein) expressions using immunoblot and mRNA analyses.

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All transfected cells and non-transfected cells were processed for cell lysis

and SDS-PAGE for resolving fusion proteins of interest as described in

Sections 4.2.1.5.3 and 4.2.1.4.

4.2.2.7.1 Western blot

Western blotting was modified from the protocol described in Section

3.2.4.11. Anti-GFP rabbit (Cell Signaling Technology, USA) diluted in 5%

w/v milk in TBST (1:3000) was the primary antibody. Goat anti-rabbit IgG

HRP (Cell Signaling Technology) diluted in 5% w/v milk in TBST (1:4000)

was the secondary antibody. The blots were exposed to a Fuji Super RX-N

medical X-ray film (Fuji Corporation, Japan) in the darkroom for 1–3 min.

An image of the radiograph was digitised using a Gel DocTM XR+ imaging

system (Bio-Rad) with Image LabTM software (Bio-Rad). The size of the

recombinant protein was analysed using Precision Plus Protein™ Dual

Colour Standards (Bio-Rad).

4.2.2.7.2 mRNA analysis

mRNA extraction on all cell cultures, including transfected and non-

transfected cells, was conducted using the RNeasy mini kit (Qiagen,

Germany) according to the manufacturer’s instructions (Qiagen, 2012) and

purified using Turbo DNA-free kit (Ambion®, Life Technologies). Following

this, first-strand cDNA synthesis was performed using SuperScript™ III

First-Strand Synthesis (Invitrogen) under SuperScript™ III reaction

conditions, and the cDNA samples were subjected to conventional PCR to

detect the cDNA of the gene of interest.

4.2.2.7.2.1 mRNA extraction

The transfected and non-transfected monolayers consisting of Vero cells

transfected with pEGFP-C1, pEGFP-C1-KatA, pEGFP-C1-Peb1A, pEGFP-

C1-CadF, or pEGFP-C1-CjaA were subjected to mRNA extraction using the

RNeasy mini kit (Qiagen, Germany) according to the manufacturer’s

instructions (Qiagen, 2012). The medium was removed from each well using

a 2-mL Nunc™ serological pipette (Thermo Fisher Scientific), and the cells

were washed twice with 1 mL of 1× PBS (Medicago AB). The cells were

disrupted by adding 350 µL of RLT (Qiagen) and dissociated using a cell

scraper (Sarstedt, Newton, NC, USA), and 1 v/v of 70% ethanol was added.

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Then, 750 µL of the cell lysates were applied to RNeasy Mini spin columns

(Qiagen), centrifuged at maximum speed for 15 sec, and the flow-through

was discarded. Subsequently, 700 µL of RW1 buffer (Qiagen) was added to

the spin column, centrifuged at 8000 g for 15 sec, and the flow-through was

discarded. The spin column was washed using 500 µL of RPE (Qiagen),

centrifuged at 8000 g for 15 sec, and the flow-through was discarded. The

washing step was repeated using 500 µL of RPE (Qiagen), centrifuged at

8000 g for 2 min, and the flow-through was discarded. The spin column was

placed in a new collection tube and centrifuged at 8000 g for 1 min. Then, the

spin column was placed in a clean 1.5-mL microcentrifuge tube (Sartedt), and

the mRNA was eluted with 30 μL of RNase-free water (Ambion®), left to

stand for 1 min and centrifuged for 1 min. The eluted mRNA samples were

digested with Turbo DNA-free kit (Ambion®, Life Technologies) to eliminate

DNA contamination as follows. Each 50-μL reaction mixture containing 30

μL of mRNA, 1 µL of DNase, and 1× buffer Turbo DNase buffer was

incubated at 37°C for 30 min. Following this, 1 × DNase inactivation reagent

(Ambion®) was added to stop the reaction, incubated at 22 ± 2 °C for 5 min,

and centrifuged at 10,000 g for 2 min. The supernatant was transferred to a

new 200-µL microcentrifuge tube (Sartedt). The purified mRNA

concentration and purity were determined using a spectrophotometer

(Nanodrop® ND-1000). The purified mRNA samples were stored at -80°C

until required.

4.2.2.7.2.2 cDNA synthesis

All mRNA samples were subsequently reverse transcribed to cDNA using the

SuperScript® III First-Strand Synthesis System (Invitrogen) according to the

manufacturer’s instructions (Invitrogen, 2013).

Each mRNA sample was mixed with 50 ng of random hexamers (Invitrogen)

and 1 mM of each dNTP (Invitrogen) in a 10-μL reaction with the addition of

DEPC-treated water (Invitrogen). The mixture was incubated at 65°C for 5

min using a thermocycler and placed on ice for at least 1 min. Following this,

the mixture was combined with 10 μL of cDNA synthesis reagents containing

1× RT buffer (Invitrogen), 10 mM MgCl2 (Invitrogen), 20 mM DTT

(Invitrogen), 40 U RNaseOUT™ (Invitrogen), and 200 U of SuperScript™

III reverse transcriptase (Invitrogen). Each reaction was incubated for 50 min

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at 50°C for dNTP primed, 10 min at 25°C, 50 min at 50°C for random

hexamer-primed, and then terminated at 85°C for 5 min and held on ice.

Following this, 1 μL of RNase H (Invitrogen) was added and incubated at

37°C for 20 min. The cDNA products were stored at -20°C. For each reaction,

a control reaction without reverse transcriptase was performed in parallel to

determine RNA template purity from DNA.

4.2.2.7.2.3 PCR assays

All cDNA samples from Section 4.2.2.8.2.1 were used to detect the presence

of genes using conventional PCR. All PCR assays were performed in a BIO-

RAD S1000TM thermal cycler (BIO-RAD) and made up in a 25-µL reaction

mixture containing 2 U Platinum Taq polymerase (Invitrogen), 1 × PCR Rxn

Buffer- MgCl2 (Invitrogen) or 1 × Green PCR Rxn Buffer- MgCl2

(Invitrogen), 1.5 mM MgCl2 (Invitrogen), 0.2 mM of dNTPs mixed

(Invitrogen), 0.2 mM of forward and reverse primers (Integrated DNA

Technologies) as described in Table 4.4, 10–30 ng of cDNA template, and

RNAse water (to a final volume of 25 µL). The PCR cycling conditions were

as follows: 94°C for 4 min (one cycle), 40 cycles of 94°C for 10 sec, 58°C for

20 sec, and 72°C for 30 sec. For each PCR, RNase water mixed with the PCR

solution served as the negative control. All PCR amplicons were analysed

using 1.5% gel electrophoresis as described in Section 3.2.3.4.

4.2.3 Preparations of HVT virus and CEF

The HVT wild-type strain CF126 kindly obtained from Professor Tim

Mahony’s laboratory was used for the construction of the HVT-peb1A.

4.2.3.1 Cell cultures

CEF cells were grown in CEF media, which consisted of medium 199 (M199;

Invitrogen), 20% FBS (Gibco), and 1% PSF. A vial of frozen CEF cells

containing 10% dimethyl sulfoxide (DMSO) from liquid nitrogen storage was

rapidly thawed at 37°C for 30 sec using a water bath, and 2 mL of CEF media

was added. The mixture was transferred to a new 15 mL high-clarity

polypropylene conical centrifuge tubes (Falcon®), and 10 mL of CEF media

was added. The solution was centrifuged at 1500 rpm for 4 min to form a cell

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pellet, and the supernatant was discarded. The cell pellets were resuspended

with 10 mL of CEF media. The resuspended cells were transferred to a new

T75 flask (Nunclon™ Delta Surface, Thermo Scientific, Roskilde, Denmark),

mixed with 10 mL of CEF media, and incubated at 37°C with 5% CO2. Cell

proliferation was monitored daily using an inverted microscope (Japan

Aviation Electronics Industries Ltd.). Cell culture conditions, cell monitoring,

and cell passaging were performed.

4.2.3.2 HVT preparation and passage

A vial of frozen HVT-associated cells from storage in liquid nitrogen was

rapidly thawed at 37°C for 30 sec using a water bath. The thawed HVT-

associated cells were transferred to a new centrifuge tube, and 1 mL of CEF

media was added. The solution was transferred to a new Falcon™ 15-mL

high-clarity polypropylene conical centrifuge tubes (Falcon®), mixed with 10

mL of CEF media, and centrifuged at 400 g for 4 min. The supernatant was

discarded, and cell pellets were resuspended in 500 μL of CEF medium. The

HVT- associated cell suspension was passaged on to a fresh CEF monolayer

at 70–80% confluency in a T75 flask (Thermo Scientific). The infection

efficiency of HVT was checked daily for cytopathic effect (CPE) using an

Axiovert 200 M microscope (Carl Zeiss, Germany). Images of infected and

non-infected cells were captured using Axio Vision LE Release 4.7 software

(Carl Zeiss, Germany).

The CEF cells infected with HVT showing 70% CPE were passaged as

follows. The CEF medium was removed, and the cells were washed with 1×

PBS (Medicago AB). The cells were detached from the culture vessel by

adding 2 mL of 0.25 × trypsin solution (Gibco) for 5 min at 37°C. Following

this, 10 mL of CEF media was added to inactivate the trypsin, and the cells

were transferred to 15-mL high-clarity polypropylene conical centrifuge tube

(Falcon®). The cells were pelleted by centrifugation at 400 g for 4 min at 22

± 2 °C (Sigma 3K18 rotor 12154-H, Germany), and the supernatant was

discarded. The cell pellets were resuspended in 900 µL and passaged on to a

fresh CEF monolayer at 70–80% confluency.

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4.2.3.3 Determination of TCID50 of HVT

TCID50 was performed to determine the infectious titre of HVT which can

cause CPE in CEF cell culture over 5–7 days, while cells in culture remain

viable.

Before the TCID50 test, CEF cells were seeded in 24-well plates at a density

of 1 × 105 cells per well and incubated for 16 ± 2 h to generate monolayers as

described above. On the following day, a vial of frozen HVT-CEF cells was

rapidly thawed at 37°C for 30 sec using a water bath, and the cells were then

diluted 10-fold, starting from 10-1 to 10-6. Each dilution (10-2–10-6) was

inoculated into the monolayers with 100 μL of a 10-fold dilution series of the

HVT in four replicates. The last column of CEF monolayers in a 24-well plate

were used as non-infection (negative control). The infected monolayers with

HVT were incubated for 1 week and daily monitored for the appearance of

CPE lesions at each dilution. A dilution showing CPE lesions at 50% of all

replicates was determined as TCID50. TCID50 was used to calculate the

average viral particles infecting each cell, called MOI.

The TCID50 titre was calculated using the Spearman-Karber method as

described by Hierholzer and Killington (1996) who used the following

formula: L – [d (∑p – 0.5)], where

L = the last dilution showing CPE in all replicates

d = the dilution factor

∑p = summation of CPE dilutions from the last dilution with positive CPE in

all replicates until the last dilution showing CPE

The volume of the virus was calculated using the following formula as

described by Sloutskin and Goldstein (2014):

Volume virus = MOI× seed cells

pfu

pfu = 0.7 × TCID50 as described by Anonymous (2012)

The entire HVT-CEF dilution was performed to detect and quantify HVT

using a duplex real-time quantitative PCR assay (qPCR) as previously

described by (Islam et al., 2004). Chicken α2 (VI) collagen gene was used as

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an internal control for qPCR. All multiplex qPCR reactions and specific

primers and probes (Table 4.5) were performed in a Rotor-Gene Q thermal

cycler (Qiagen, Hilden, Germany). For each qPCR assay, HVT and HVT

mixed with CEF (HVT-CEF) were used as positive controls. CEF and no

template control (NTC) were used as negative controls.

Table 4.5: Oligonucleotide primers and probes used for a duplex qPCR

Gene

(Target species)

Sequence 5’ to 3’

SORF1 (HVT) Forward: GGC AGA CAC CGC GTT GTA T

Reverse: TGT CCA CGC TCG AGA CTA TCC

Probe a: AAC CCG GGC TTG TGG ACG TCT TC

α2 (VI)

Collagen

(chicken)

Forward: GGG AAC TGG AGA ACC CAA TTT T

Reverse: CGT GCC GCT GTC TCT ACC AT

Probe b: CCC TTA ACT GAG TTC CCC AGC

TAC TGC AG Note: qPCR required channels detected on a ROX (Orange) and b JOE (Yellow)

The real-time PCR conditions were the following: 50°C for 2 min, 95°C for

15 min, followed by 40 cycles of 94°C for 45 sec, and 60°C for 75 sec. Each

reaction volume was 20 µL and comprised 1× Quantitect Multiplex RT-PCR

Master Mix (Qiagen), 0.3 M of each primer (Sigma, Australia), 0.2 M of the

corresponding probe (Sigma, Australia), 2 µL of DNA template, and RNase-

free water (Ambion®) to a final volume of up to 20 µL.

Results of qPCR were analysed using Rotor-Gene Q Software (version

2.0.2.4; Qiagen). A threshold value of 0.05 was used as the baseline for raw

data analysis. A standard curve of each primer set was used to acquire the

amount of HVT in each dilution. The channel of ROX (orange) was used to

analyse the HVT amplification.

4.2.3.4 Evaluation of HVT infection with different MOIs

Different MOIs were used to identify the most suitable virus for infection. A

suitable MOI generating CPE within an appropriate time was of interest. The

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CEF cells were seeded in 6-well plates at a density of 5 × 105 cells per well

and incubated for 16 ± 2 h to generate monolayers as described in Section

4.2.3.1. The monolayers with 70% confluency were infected by HVT with

MOIs of 0.02, 0.01, and 0.0035 in duplicate and incubated at the same

conditions as described above. CEF monolayers without infection were used

as negative controls. The infected cells were monitored daily for the presence

of CPE.

4.2.3.5 HVT stock

Virus stocks were typically prepared from T75 infected CEF monolayers. At

approximately 70–80% grossly observable CPE, the monolayers were

trypsinised as described above. After pelleting, the cells were resuspended in

1.8-mL FCS (Gibco) and 200-μL DMSO. Virus stocks were stored in 500-μL

aliquots and frozen at -80°C in a Mr Frosty freezing container (Nalgene)

before being stored for longer durations in liquid nitrogen.

4.2.3.6 CEF stock

A fresh CEF culture in a T75 flask at 70% confluence was harvested for cell

collection using the same cell passage protocol as described above. After

resuspending in CEF media, the cells were counted and visualised using a

haemocytometer under a Nikon inverted microscope (Japan Aviation

Electronics Industries Ltd.). Then, 4 × 107 CEF cells mixed with 10% DMSO

were seeded in each cryopreservation vial and stored at -80°C in a Mr Frosty

freezing container (Nalgene) before being stored for longer durations in liquid

nitrogen.

4.3 Results

4.3.1 5´-CACCATG-overhanging insert gene amplicons for directional

cloning

C. jejuni cluster 27 was used as a DNA template to amplify all conserved

genes (katA, cadF, peb1A, and cjaA) using a specific 5´-CACCATG-

overhang forward primer in each conventional PCR (Table 4.1). Agarose gel

electrophoresis revealed that the katA, cadF, peb1A, and cjaA amplicons were

approximately 670 (Figure 4.2), 870 (Figure 4.2), 700 (Figure 4.3), and 700

185

bp (Figure 4.4) in size, respectively. These sizes were consistent with the size

estimations shown in Figure 4.1. All these PCR products were directionally

cloned into TOPO plasmid vectors and then transformed into One Shot®

TOP10 chemically competent E. coli cells.

Figure 4.2: Agarose gel electrophoresis of the PCR products

containing the katA and cadF ORFs used for cloning into the TOPO

plasmid vector.

Each PCR amplicon was generated from the C. jejuni cluster 27 and

used as plasmid vector gene insert. The estimated sizes of katA and

cadF amplicons were approximately 670 and 870 bp, respectively

(arrows). Lane 1,1 Kb Plus DNA Ladder; Lane 2, katA amplicon; Lane

3, cadF amplicon; and Lane 4, MilliQ water (negative control).

850 bp 650 bp

1 2 3 4

katA amplicon (670 bp) cadF amplicon (870 bp)

1 2 3

850 bp 650 bp cjaA amplicon (700 bp)


the cjaA ORF used for cloning into the TOPO plasmid vector.

The PCR amplicon was generated from the C. jejuni cluster 27 and used

as plasmid vector gene insert. The estimated size of the cjaA amplicon

was approximately 700 bp (arrow). Lane 1,1 Kb Plus DNA Ladder;

Lane 2, MilliQ water (negative control); and Lane 3, cjaA amplicon.

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4.3.2 Screening of transformed E. coli cells harbouring the

recombinant TOPO plasmids

All ampicillin-resistant single colonies from the transformed E. coli cells

were screened for the gene of interest using conventional PCR. Clones of the

katA, cadF, peb1A, and cjaA ORFs yielding amplicons of the expected sizes

were identified from each E. coli transformation (Figures 4.5-4.8). Based on

the electrophoretic analyses, two of four katA colonies (Figure 4.5; Lanes 4

and 5;), one of four cadF colonies (Figure 4.6; Lane 5;), four of four peb1A

colonies (Figure 4.7; Lanes 3, 4, 5, and 6), and one of four cjaA colonies

(Figure 4.8; Lane 5) yielded amplicons of the expected sizes from the

transformed cells of approximately 650, 850, 700, and 700 bp in size,

respectively. Of these, the amplification of peb1A showed a very faint

amplicon (Figure 4.7). All colonies yielding amplicons consistent with the

expected sizes were individually cultured into a 5 mL of LB-Am100 broth

and plasmid DNA isolated. The confirmation of the inserted ORFs for each

gene in the isolated plasmids was further assessed using conventional PCR,

double restriction enzyme digestion, and DNA sequencing.

1 2 3 4

850 bp 650 bp

peb1A amplicon (700 bp)

Figure 4.4: Agarose gel electrophoresis of the PCR product

containing the peb1A ORF used for cloning into the TOPO plasmid

vector.

Each PCR amplicon was generated from the C. jejuni cluster 27 and

used as plasmid vector gene insert. The estimated size of the PCR

product was approximately 700 bp (arrow). Lane 1,1 Kb Plus DNA

Ladder; Lanes 2 and 3, MilliQ water (negative control); and Lane 4,

peb1A amplicons.

187

650 bp

1 2 3 4 5 6

katA amplicon (670 bp)

Figure 4.5: Example of agarose gel electrophoresis of the katA ORF

PCR products using whole cells from transformed One Shot® TOP10 chemically competent E. coli colonies as the DNA template.

Colonies showing the evidence of katA in transformed plasmids with

approximately 650 bp in size on the gel are indicated by the arrow.

Lane 1, 1 Kb Plus DNA Ladder; Lane 2, E. coli TOPO-katA colony

no. 1; Lane 3, E. coli TOPO-katA colony no. 2; Lane 4, E. coli TOPO-

katA colony no. 3; Lane 5, E. coli TOPO-katA colony no. 4, and Lane

6, RNase water (negative control).

cadF amplicon (870 bp)

Figure 4.6: Example of agarose gel electrophoresis of the cadF ORF


chemically competent E. coli colonies as the DNA template.

A colony showing the evidence of cadF in transformed plasmids with

approximately 870 bp in size on the gel is indicated by the arrow.

Lane 1, 1 Kb Plus DNA Ladder; Lane 2, RNase water; Lane 3, E.

coli TOPO-cadF colony no. 1; Lane 4, E. coli TOPO-cadF colony no.

2; Lane 5, E. coli TOPO-cadF colony no. 3, and Lane 6, E. coli

TOPO-cadF colony no. 4.

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4.3.3 Restriction enzyme analysis of recombinant TOPO plasmids

The putatively recombinant TOPO plasmids yielding PCR amplicons

consistent with the insertion of the katA, cadF, peb1A and cjaA ORFs were

further analysed using BamHI-HF and XhoI restriction enzymes. These two

enzymes flank the PCR amplicon insertion site within the TOPO vector and

850 bp peb1A amplicon (700 bp)

1 2 3 4 5 6


ORF PCR products using whole cells from transformed One Shot®

TOP10 chemically competent E. coli colonies as the DNA template.

Colonies showing evidence of peb1A in transformed plasmids with

approximately 700 bp in size on the gel are indicated by the arrow.

Lane 1, 1 Kb Plus DNA Ladder; Lane 2, RNase water; Lane 3, E.

coli TOPO-peb1A colony no. 1; Lane 4, E. coli TOPO-peb1A

colony no. 2; Lane 5, E. coli TOPO-peb1A colony no. 3, and Lane

6, E. coli TOPO-peb1A colony no. 4.

1 2 3 4 5 6

850 bp cjaA amplicon (700 bp)

Figure 4.8: Example of agarose gel electrophoresis of the cjaA ORF


chemically competent E. coli colonies as the DNA template.

Colonies showing evidence of cjaA in transformed plasmids are

indicated by the arrow. Lane 1, 1 Kb+ DNA Ladder; Lane 2, RNase

water; Lane 3, E. coli TOPO-cjaA colony no. 1; Lane 4, E. coli TOPO-

cjaA colony no. 2; Lane 5, E. coli TOPO-cjaA colony no. 3, and Lane 6,

E. coli TOPO-cjaA colony no. 4.

1 2 3 4 5 6

189

were expected to generate the estimated fragment sizes of each ORF as

described in Figure 4.1. Based on the double digestion, the inserted fragments

obtained from katA, cadF, peb1A, and cjaA genes were slightly larger than

the PCR product (Figures 4.1 and 4.4). The fragments of katA, cadF, peb1A,

and cjaA ORF fragments were approximately 730, 930, 790, and 790 bp in

size, respectively.

A digestion fragment of recombinant TOPO plasmid containing katA was

slightly larger than the expected size for katA and was designated as

pcDNA3T-katA-1 (Figure 4.9; Lane 3). A digestion fragment of recombinant

TOPO plasmid containing peb1A was slightly larger the expected size for

peb1A and was designated as pcDNA3T-peb1A-1 (Figure 4.9; Lane 6).

The plasmids containing cjaA with digestion fragments were slightly larger

than the expected size for cjaA, were designated as pcDNA3T-cjaA-1 and

pcDNA3T-cjaA-2 (Figure 4.9; Lanes 9 and 10). The plasmid with a digestion

fragment consistent with the expected size for cadF was designated as

pcDNA3T-cadF-4 (Figure 4.10).

1 2 3 4 5 6 7 8 9 10

850 bp

Figure 4.9: Agarose gel electrophoresis analysis of the TOPO plasmids

after double digestion with BamHI-HF and XhoI and the original PCR

used in the cloning process.

The amplicon size of each gene using restriction enzymes is slightly

larger than the original PCR amplicons. The expected product sizes from

the inserted katA, peb1A, and cjaA genes were approximately 690 (Lane

3), 790 (Lane 6), and 790 bp (Lanes 9 and 10), respectively. Lane 1, 1 Kb

Plus DNA Ladder; Lane 2, katA PCR amplicon; Lane 3, TOPO-katA

plasmid no. 1; Lane 4, No sampled; Lane 5, peb1A PCR amplicon; Lane

6, TOPO-peb1A plasmid no. 1: Lane7, No sampled; Lane 8, cjaA PCR

amplicon; Lane 9, TOPO-cjaA plasmid no. 1; and Lane 10, TOPO-cjaA

plasmid no. 2.

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4.3.4 Sequence analysis of recombinant TOPO plasmids

The nucleotide sequencing of the TOPO plasmids showed that the amplicons

for katA, cadF, and cjaA ORFs were successfully cloned into the vector. The

katA, cadF, and cjaA ORFs were cloned into the TOPO vector in the correct

orientation, as shown in Figures 4.11, 4.12, and 4.14. However, an additional

25 nucleotides were identified between the cloning region of the vector and

PCR amplicons for pcDNA3T-katA-1, pcDNA3T-cadF-4, and pcDNA3T-

cjaA-1 plasmids. The reverse oligonucleotide primer used to amplify the

clone cadF amplicon had one mismatch compared with the original isolate

(Figure 4.12), whereas two mismatches were found in the forward

oligonucleotide primer of the cjaA gene (Figure 4.14). The nucleotide

sequence of pcDNA3T-katA-1 showed that the restriction site for BamHI and

XhoI were located at different locations compared with the TOPO vector and

the start codon (ATG) was in the correct frame (Figure 4.13). Besides the

recombinant region, different nucleotides were also found at positions 0–112

and 836–871.

cadF amplicon (930 bp)

Figure 4.10: Agarose gel electrophoresis of insert cadF ORF of

cloned TOPO plasmids after double digestion using BamHI-HF

and XhoI and the cadF PCR amplicon used in the cloning process.

The amplicon size of each gene using restriction enzymes is larger

than the original PCR amplicons. The expected product size of

cadF was 930 bp (arrow). Lane 1, 1 Kb Plus DNA Ladder; Lane, 2

the original PCR of cadF; Lane 3, TOPO- cadF plasmid no. 1;

Lane 4, TOPO- cadF plasmid no. 2; Lane 5, TOPO- cadF plasmid

no. 3; Lane 6, TOPO- cadF plasmid no. 4; and Lane 7, TOPO-

cadF plasmid no. 5.

191

Figure 4.11: Example of sequence alignment of the pcDNA3T-katA-1


The katA gene was cloned into the TOPO vector. The nucleotide

sequences of the TOPO vector are indicated in green colour. The

restriction sites for BamHI-HF and XhoI located on the TOPO are

indicated in red and yellow colours, respectively. Letters in red font

indicate insertion of the nucleotides in the pcDNA3T-katA-1. Underlined

letters indicate the forward and reverse (5’ and 3’ ends) primers used.

Figure 4.12: Example of sequence alignment of the pcDNA3T-cadF-

4 compared with the original PCR amplicon and the TOPO vector

alone.

The cadF gene was cloned into the TOPO vector. The nucleotide


restriction sites for BamHI-HF and XhoI located on the TOPO

vectors indicated in red and yellow colours, respectively. Letters in

red font indicate the extra nucleotides in the pcDNA3T-cadF-4.

Underlined letters indicated the forward and reverse (5’ and 3’

ends) primers used. One mismatch nucleotide was found in the

reverse primer (blue letter).

192

Figure 4.13: Example of sequence alignment of the pcDNA3T-peb1A-

1 compared with the original PCR amplicon and the TOPO vector

alone.

The peb1A gene was cloned into the TOPO vector. The nucleotide


restriction sites for BamHI-HF and XhoI located on the TOPO

vectors are indicated in red and yellow colours, respectively. Letters

in brown font indicate different nucleotides of the pcDNA3T-peb1A-

1 and the TOPO plasmid vector. Underlined letters indicate the

forward and reverse (5’ and 3’ ends) primers used.

Figure 4.14: Example of sequence alignment of the pcDNA3T-cjaA-1

compared with the original PCR amplicon and the TOPO vector

alone.

The cjaA gene was cloned into the TOPO vector. The nucleotide

sequences of TOPO vector indicated in green colour. The restriction

sites for BamHI-HF and XhoI are indicated in red and yellow

colours, respectively. Letters in red font indicate the extra

nucleotides in the pcDNA3T-cjaA-1. Underlined letters indicate the

forward and reverse (5’ and 3’ ends) primers used. Two mismatches

of the nucleotide were found in the forward primer (blue letters).

193

4.3.5 Eukaryotic expression of Campylobacter polypeptides

To determine if the recombinant TOPO plasmids could express the

Campylobacter polypeptides of interest, the plasmids were transfected into

RK-13 cells. After 48 hr, the total protein content of cell lysates was resolved

using SDA-PAGE and subsequently transferred to a nitrocellulose membrane

by Western blotting. Staining of the nitrocellulose membrane with Ponceau S

did not identify any differentially expressed proteins compared to the

untransfected RK-13 cells (Figure 4.15). The membrane was subsequently

probed with an anti-6His antibody to detect any Campylobacter polypeptides.

Analysis of potential expression of the recombinant polypeptides of interest

by Western blotting did not identify any differentially expressed polypeptides

between any of the cell lysates (Figure 4.16). Several polypeptides were

detected; however, these were present in all extracts. These included a

reactive polypeptide in cells transfected with plasmid pcDNA™ 3.1D/V5-

His/lacZ, the expression positive control, where the evidence of a 120 kDa of

β-galactosidase fusion polypeptide was supposed to be visualised (Figure

4.16; Lane 6), using the specific anti-His tag mouse monoclonal antibody

(Section 3.2.4.11). All bands detected from all recombinant TOPO vector

Figure 4.15: SDS-PAGE analysis of total proteins from the RK-13

cells and the recombinant TOPO plasmids containing katA, cjaA,

peb1A, or cadF.

Lane 1, RK-13 cells alone (negative control); Lane 2, transfected RK-

13 cells with TOPO-katA; Lane 3, transfected RK-13 cells with

TOPO-cjaA; Lane 4 transfected RK-13 cells with TOPO-peb1A;

Lane 5, transfected RK-13 cells with TOPO-cadF; Lane 6,

pcDNA™3.1D/V5-His/lacZ (positive control); Lanes 7 and 8, blank;

Lanes 9–11, molecular weight markers.

194

containing genes and positive control were visualised and similar to the

negative controls at the exposure time of 3 min (Figure 4.16).

4.3.6 Screening of the transformed E. coli containing the recombinant

pEGFP-C1 plasmids

As no expression of the Campylobacter polypeptides of interest could be

detected in RK-13 cells transfected with the pcDNA3-TOPO plasmids, an

alternative expression strategy was devised whereby the ORF would be fused

to the 5′ end of the eGFP ORF of pEGFP-C1.

Analyses of the pET SUMO plasmids containing katA, cadF, peb1A, and cjaA

ORFs described in Chapter 3 identified suitable restriction enzyme sites

flanking these OFRs that would facilitate subcloning into the cut pEGFP-C1

vector.

The pET SUMO-katA and the pEGFP-C1 plasmids were digested using

HindIII and BamHI-HF, and after gel purification, the katA ORF fragment

was cloned into the pEGFP-C1 vector. Screening of kanamycin-resistant

50 kDa

37 kDa

25 kDa

100 kDa

1 2 3 4 5 6 7 8 9 10 11

Figure 4.16: The Western blot analysis of total cell protein extracts

from RK-13 cells transfected with plasmids encoding ORFS for katA,

cjaA, peb1A, and cadF.

Lane 1, RK-13 cells alone (negative control); Lane 2, transfected RK-

13 cells with TOPO-katA; Lane 3, transfected RK-13 cells with TOPO-

cjaA; Lane 4 transfected RK-13 cells with TOPO-peb1A; Lane 5,

transfected RK-13 cells with TOPO-cadF; Lane 6, pcDNA™3.1D/V5-

His/lacZ (positive control; 120 kDa); Lanes 7 and 8, blank; and Lanes

9–11, molecular weight markers.

195

colonies by PCR and agarose gel electrophoresis revealed that the katA gene

fragment was detected in the transformed E. coli cells using both katA cloning

and pEGFP-C1 primer pairs (Figure 4.17). However, while the four colonies

tested yielded an amplicon of the expected size (680 bp) with the katA cloning

primers (Figure 4.17A), only one colony (colony no.5) yielded an amplicon

of the expected size (950 bp) with the katA cloning and pEGFP-C1 primer

(Figure 4.17B). This colony designated pEGFP-C1-katA-5 was selected for

further analyses.

196

1 2 3 4 5 6 7

650 bp katA amplicon

(670 bp)

850 bp GFP-katA amplicon

(950 bp)

A) B)

300 bp

1 2 3 4 5 6 7

Figure 4.17: Example of agarose gel electrophoresis of PCR products for the katA ORF fragment using whole cells from the transformed One

Shot® TOP10 E. coli colonies as a DNA template.

A) The estimated size of PCR product was approximately 680 bp using the cloning katA primers (arrow). Colonies showing the evidence of

insert katA in transformed E. coli were on Lanes 3–6. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no 1; Lane 3, colony no 2; Lane 4,

colony no 3; Lane 5, colony no 4; Lane 6, colony no 5; and Lane 7, RNase water.

B) The estimated size of PCR product was approximately 950 bp using the pEGFP-C1 primers (arrow). Colonies showing the evidence of

insert pEGFP-C1-katA were on Lane 6. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no 1; Lane 3, colony no 2; Lane 4, colony no 3; Lane

5, colony no 4; Lane 6, colony no 5; and Lane 7, RNase water (Negative control).

197

The pET SUMO plasmids containing cadF, peb1A, or cjaA ORFs were

digested with XhoI and BamHI, and then these fragments were subsequently

ligated into the pEGFP-C1 vector digested with the same enzymes. Following

transformation into One Shot® TOP10 competent E. coli, kanamycin-resistant

colonies from each ligation/transformation were screened using two PCR

assays. The PCR products of the cadF gene ORF amplified from the E. coli

cells were expected to be approximately 910 bp and 1170 bp using the cloning

(Table 3.2) and the pEGFP-C1 primers on agarose gel electrophoresis (Table

4.3), respectively. Of the five colonies analysed, two were positive with the

cadF gene ORF amplicon with the cloning primers (Figure 4.18A; Lanes 4

and 5). In contrast, three of the five colonies were positive using the vector

primers (Figure 4.18B; Lanes 4-6). As the vector primers annealing sites are

located away from the cloning termini of the vector, the three colonies

(colony no 3, 4, and 5) positive with the cadF gene ORF amplicon with the

cloning primers, designated pEGFP-C1-cadF-3, pEGFP-C1-cadF-4, and

pEGFP-C1-cadF-5, were selected for further analyses.

198

cadF amplicon

(910 bp)

1 2 3 4 5 6 7

850 bp

1 2 3 4 5 6 7

GFP-cadF amplicon

(1170 bp) 1000 bp

300 bp

A) B)

Figure 4.18: Example of agarose gel electrophoresis of PCR products for the cadF ORF fragment using whole cells from the transformed

One Shot® TOP10 E. coli colonies as a DNA template.

A) The estimated PCR size of cadF was approximately 910 bp using the cloning cadF primers (arrow). Colonies showing the evidence of

insert cadF in transformed E. coli cells are on Lanes 4 and 5. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no 1; Lane 3, colony no 2; Lane

4, colony no 3; Lane 5, colony no 4; Lane 6, colony no 5; and Lane 7, RNase water.


insert pEGFP-C1-cadF were on Lanes 4–6. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no. 1; Lane 3, colony no. 2; Lane 4, colony no. 3;

Lane 5, colony no. 4; Lane 6, colony no. 8; and Lane 7, RNase water (Negative control).

199

The PCR products for the peb1A ORF amplified from the E. coli cells were

expected to be approximately 760 bp and 1020 bp on the agarose gel

electrophoresis using the cloning (Table 3.2) and the pEGFP-C1 primers

(Table 4.3), respectively. Of the five colonies analysed, two were positive

with the peb1A ORF amplicon with the cloning primers (Figure 4.19A; Lanes

5 and 6). Two of the five colonies were positive using the vector primers

(Figure 4.19B; Lanes 4 and 5). As the vector primers anneal sites are located

away from the cloning termini of the vector, the two colonies (colony no 4

and 5) positive with the peb1A ORF amplicon with the cloning primers,

designated pEGFP-C1-peb1A-4 and pEGFP-C1-peb1A-5, were selected for

further analyses.

200

peb1A amplicon

(760 bp)

1 2 3 4 5 6 7

700 bp

1000 bp

A) B)

1000 bp GFP-peb1A amplicon

(1023 bp)

1 2 3 4 5 6 7

300 bp

Figure 4.19: Example of agarose gel electrophoresis of PCR products for the peb1A ORF fragment using whole cells from the transformed


A) The estimated PCR size of peb1A was approximately 760 bp using the cloning primers. Colonies showing the evidence of insert peb1A in

transformed E. coli cells are on Lanes 5 and 6. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no 1; Lane 3, colony no 2; Lane 4, colony no 3;

Lane 5, colony no 4; Lane 6, colony no 5; and Lane 7, RNase water.

B) The estimated size of the PCR product was approximately 1020 bp using the pEGFP-C1 primers. Colonies showing the evidence of insert

pEGFP-C1-peb1A are on Lanes 4 and 5. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no 1; Lane 3, colony no 4; Lane 4, colony no 5; Lane

5, colony no 5; Lane 6, colony no 2; and Lane 7, RNase water (Negative control).

201

The PCR products for the cjaA ORF amplified from the E. coli cells were

expected to be approximately 840 and 1100 bp on the agarose gel

electrophoresis using the cloning (Table 3.2) and the pEGFP-C1 primers

(Table 4.3), respectively. Four of five colonies analysed were positive with

the cjaA ORF amplicon with the cloning primers (Figure 4.20A; Lanes 3-6).

While three of the five colonies were positive using the vector primers (Figure

4.20B; Lanes 4-6). Of these three, two bands were found in one colony

(Figure 4.20B; Lane 4). As the vector primers annealing sites are located

away from the cloning termini of the vector, the two colonies (colony no 5

and 6) positive with the cjaA ORF amplicon with the cloning primers (Figure

4.20B; Lanes 5 and 6), designated pEGFP-C-cjaA-5 and pEGFP-C1-cjaA-6,

were selected for further analyses.

202

1 2 3 4 5 6 7

cjaA amplicon

(840 bp)

850 bp

1 2 3 4 5 6 7

1000 bp GFP-cjaA amplicon

(1095 bp)

A) B)

250 bp

Figure 4.20: Example of agarose gel electrophoresis of PCR products for the cjaA ORF fragment using whole cells from the transformed


A) The estimated PCR size of cjaA was approximately 840 bp using the cloning primers (arrow). Colonies showing the evidence of insert

cjaA in transformed E. coli cells were on Lanes 3–6. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no 1; Lane 3, colony no 4; Lane 4,

colony no 5; Lane 5, colony no 2; Lane 6, colony no 5; and Lane 7, RNase water.


insert pEGFP-C1-cjaA were on Lanes 4–6. Lane 1,1 Kb Plus DNA Ladder; Lane 2, colony no 1; Lane 3, colony no 4; Lane 4, colony no 5;

Lane 5, colony no 2; Lane 6, colony no 5; and Lane 7, RNase water (Negative control).

203

4.3.7 Analysis of the recombinant pEGFP-C1 containing the genes

All colonies yielding PCR amplicons of the expected sizes were subjected to

plasmid isolation and further analysed using restriction enzymes and DNA

sequencing.

All purified recombinant pEGFP-C1 plasmids containing the katA, cadF,

peb1A, or cjaA ORFs were subjected to digestion with the restriction enzymes

used for cloning to determine if a fragment of the expected size was released

prior to DNA sequencing. Based on double digestion, the inserted fragments

obtained from the katA, cadF, peb1A, and cjaA ORFs were approximately

680 (Figure 4.21), 910 (Figure 4.22), 760 (Figure 4.23), and 840 (Figure 4.24)

bp in size, respectively. These sizes are consistent with the expected sizes of

the DNA fragments used for cloning the ORFs of interest. The nucleotide

sequence analysis confirmed that pEGFP-C1-katA-5, pEGFP-C1-cadF-4,

pEGFP-C1-peb1A-4, and pEGFP-C1-cjaA-5 were in the correct orientation

and in frame with the eGFP ORF (Appendices 4.1.1, 4.1.2, 4.1.3, and 4.1.4).

700 bp katA amplicon (680 bp)


katA ORF after HindIII and BamHI-HF digestion of the recombinant

pEGFP-C1 plasmids.

The expected product size of katA in the recombinant pEGFP-C1

plasmids was approximately 680 bp (arrow). The evidence of katA

detection was on Lanes 3–5. Lane 1,1 Kb Plus DNA Ladder; Lane 2,

the recombinant pEGFP-C1-katA plasmid no 1; Lane 3, the

recombinant pEGFP-C1-katA plasmid no 2; Lane 4, the recombinant

pEGFP-C1-katA plasmid no 3; and Lane 5, the recombinant pEGFP-

C1-katA plasmid no 4.

204

1000 bp cadF amplicon (910 bp)

Figure 4.22: Example of agarose electrophoresis of the inserted cadF


pEGFP-C1 plasmids.

The expected product size of cadF in the recombinant pEGFP-C1

plasmids was approximately 910 bp (arrow). The evidence of cadF

cloned in pEGFP-C1 plasmids was detected on Lanes 2–5. Lane 1,1 Kb

Plus DNA Ladder; Lane 2, the recombinant pEGFP-C1-cadF plasmid

no 1; Lane 3, the recombinant pEGFP-C1-cadF plasmid no 2; Lane 4,

the recombinant pEGFP-C1-cadF plasmid no 3; and Lane 5, the

recombinant pEGFP-C1-cadF plasmid no 4.

850 bp peb1A amplicon (760 bp)

Figure 4.23: Example of agarose gel electrophoresis of the inserted peb1A

ORF after HindIII and BamHI-HF digestion of the recombinant pEGFP-

C1 plasmids.

The expected product size of peb1A was 760 bp (arrow). The recombinant

pEGFP-C1-peb1A plasmids showing the evidence of peb1A was on Lanes

3–5. Lane 1,1 Kb Plus DNA Ladder; Lane 2, the recombinant pEGFP-C1-

peb1A plasmid no 6; Lane 3, the recombinant pEGFP-C1-peb1A plasmid

no 1; Lane 4, the recombinant pEGFP-C1-peb1A plasmid no 2; and Lane

5, the recombinant pEGFP-C1-peb1A plasmid no 3.

205

4.3.8 Evaluation of Campylobacter polypeptide expression as EGFP

fusions

The recombinant pEGFP-C1 plasmids containing katA, cadF, peb1A, or cjaA

ORFs in the correct orientation were transfected into Vero cells. Vero cells

transfected with pEGFP-C1 alone and untransfected cells were used as

positive and negative controls, respectively. At 48 h post-transfection, a large

number of the Vero cells transfected with the recombinant pEGFP-C1

containing the Campylobacter ORFs of interest and pEGFP-C1 alone were

observed to be expressing high levels of EGFP expression by fluorescent

microscopy (Figure 4.25). No expressing of EGFP expression was observed

in the untransfected Vero cells (Figure 4.25). On the basis of visual

fluorescence, there did not appear to be any qualitative differences in the

levels of expression of the Campylobacter fusion proteins (Figure 4.25).

850 bp cjaA amplicon

(840 bp)


cjaA ORF after HindIII and BamHI-HF digestion of the recombinant

pEGFP-C1 plasmids.

The expected product size of cjaA in the recombinant pEGFP-C1

plasmids was approximately 840 bp (arrow). The evidence of cjaA

detection was on Lanes 2–5. Lane 1,1 Kb Plus DNA Ladder; Lane 2,

the recombinant pEGFP-C1-cjaA plasmid no 1; Lane 3, the

recombinant pEGFP-C1-cjaA plasmid no 2; Lane 4, the recombinant

pEGFP-C1-cjaA plasmid no 3; and Lane 5, the recombinant pEGFP-

C1-cjaA plasmid no 4.

206

Figure 4.25: Transfection analysis of the recombinant pEGFP-C1 containing katA, cadF, peb1A, or cjaA ORFs in Vero cells visualised under

a fluorescent microscope with the 10 X objectives of at 48 h after transfection.

The transfected Vero cells with pEGFP-C1 alone (positive control) and the recombinant pEGFP-C1 plasmids containing katA, cadF, peb1A,

or cjaA showed uniform cytoplasmic distribution of eGFP in Vero cells. Vero cells without transfection showed negative result.

207

4.3.9 Western blot analyses

All proteins extracted from transfected and untransfected cells were analysed

using SDS-PAGE and Western blotting. Based on the pEGFP-C1 vector map,

start codon (ATG) and stop codon (TAA) were at positions 613–615 and

1408–1410, and thus, the eGFP protein was 795 bp in length, translating 265

amino acids, with an estimated molecular weight of 29.15 kDa. The pEGFP-

C1-katA consisted of 1435 nucleotides, translating 478 amino acids, and thus,

the estimated molecular weight of EGFP-KatA was 52.58 kDa. The pEGFP-

C1-cadF was 1652 bp long, translating 550 amino acids, with an estimated

molecular weight of 60.5 kDa. The pEGFP-C1-peb1A was 1502 bp in length,

translating 500 amino acids, with an estimated molecular weight of 55 kDa.

The pEGFP-C1-cjaA was 1577 bp long, translating 525 amino acids, with an

estimated molecular weight of 57.75 kDa.

The Western blot analysis showed that all recombinant pEGFP-C1 containing

either pEGFP-C1 alone or with one of the four genes were robustly expressed

in Vero cells (Figure 4.26). The untransfected Vero cells showed many bands

of protein (background) from the cell lysates. The EGFP and EGFP fusion

proteins were detected at approximately 29 kDa; EGFP-KatA, approximately

52 kDa (faint); EGFP-CadF, approximately 60 kDa; and EGFP-CjaA,

approximately 57 kDa. The EGFP-Peb1A showed a very strong intensity

band at approximately 55 kDa (Figure 4.26).

208

4.3.10 mRNA analysis

All transfected and non-transfected cells were subjected to mRNA extraction

and cDNA synthesis. cDNA from each sample was detected using

conventional PCR, as described in Section 4.2.2.7.2.2. Agarose

electrophoresis showed no DNA contamination in any cDNA sample. The

PCR products generated from cDNA of pEGFP-C1, pEGFP-C1-katA-5,

pEGFP-C1-cadF-4, pEGFP-C1-peb1A-4, and pEGFP-C1-cjaA-5 were

approximately 300, 950, 1100, 1200, and 1000 bp in size, respectively (Figure

4.27). No PCR amplicon resulted from the PCR reaction using cDNA from

untransfected Vero cells (Figure 4.27).

1 2 3 4 5 6 7

37 kDa

25 kDa

50 kDa

75 kDa

Figure 4.26: Western blot analyses of VERO cell extracts from cells

transfected with pEGFPC1, pEGFPC1-KatA, pEGFPC1-CjaA,

pEGFPC1-Peb1A, and pEGFPC1-CadF expression with the exposure

time of 10 sec.

The EGFP fusion protein expressed by cells transfected with pEGFPC1,

pEGFPC1-KatA, pEGFPC1-CjaA, pEGFPC1-Peb1A, and pEGFPC1-

CadF were detected (brown boxes). Lane 1: protein molecular weight

markers; Lane 2: pEGFPC1 (positive control); Lane 3: pEGFPC1-KatA;

Lane 4: pEGFPC1-CjaA; Lane 5: pEGFPC1-Peb1A; Lane 6: pEGFPC1-

CadF; and Lane 7: Vero cells (negative control).

209

4.3.11 TCID50 analysis

Each HVT-CEF dilution was assessed for the amount of HVT using a duplex

qPCR assay, which showed that each diluted HVT in CEF was amplified at a

different cycle corresponding to the degree of dilution and Ct values (Figure

4.28).

Figure 4.27: Agarose gel electrophoresis of the PCR amplicons

generated by PCR using from Vero cells transfected with pEGFP-C1,

pEGFP-C1-KatA, pEGFP-C1-CadF, pEGFP-C1-Peb1A, or pEGFP-

C1-CjaA.

The estimated PCR product sizes of EGFP, EGFP-katA, EGPF-cjaA,

EGFP-cadF, and EGFP-peb1A amplicons were approximately 300, 950,

1100, 1200, and 1000 bp, respectively. Lane 1: 1 Kb Plus DNA

molecular weight marker; Lane 2: pEGFPC1 without RT; Lane 3:

pEGFPC1 with RT (300 bp); Lane 4, pEGFPC1-KatA without RT;

Lane 5, pEGFPC1-KatA with RT (950 bp); Lane 6, pEGFPC1-CjaA

without RT; Lane 7, pEGFPC1-CjaA with RT (1100 bp); Lane 8,

pEGFPC1-CadF without RT; Lane 9, pEGFPC1-CadF with RT (1200

bp); Lane 10, pEGFPC1-Peb1A without RT; Lane 11, pEGFPC1-

Peb1A with RT (1000 bp); Lane 12, Vero cells without RT; Lane 13,

Vero cells with RT; and Lane 14, RNase-free water (Negative control).

Figure 4.28: Quantification data for Cycling A. Orange for HVT dilutions.

All positive controls showed amplifications from 10th to 15th cycles. The

amplifications of HVT-CEF samples with 10-1, 10-2, 10-3, 10-4, 10-5, and 10-6

dilutions occurred at the 15th, 18th, 20th, 25th, and 33rd cycles, respectively.

Negative controls (CEF and NTC) showed no amplification.

210

All negative controls (CEF and NTC) showed curves below the threshold and

no Ct value. The positive controls (HVT and HVT-CEF) were amplified

between the 10th and 15th cycles and the Ct values were between 13.46 and

16.96. The lowest dilution (10-1) and the highest dilution (10-6) showed

amplifications at the 15th and 33rd cycles, respectively, and the Ct values were

between 16.18 and 37.51 (Table 4.6).

Table 4.6: Analysis of Ct values of each HVT dilution from a duplex qPCR

No. Colour Name Ct value

A1

HVT 13.46

A2

HVT 13.45

A3

HVT-CEF 16.96

A4

HVT+CEF 16.92

A5

CEF –

A6

CEF –

A7

NTC –

A8

NTC –

B1

-1 16.18

B2

-1 16.18

B3

-2 19.34

B4

-2 19.47

B5

-3 21.85

B6

-3 21.87

B7

-4 26.17

B8

-4 26.08

C1

-5 29.51

C2

-5 29.70

C3

-6 37.51

C4

-6 36.84

211

At 7 days after infection, all transfected cells with 10-2, 10-3, and 10-4 dilutions

of HVT-CEF showed CFE lesions (Figure 4.29A), whereas CPE lesions were

not detected in the transfected cells with 10-5 and 10-6 dilutions and non-

infected CEF cells (Figure 4.29B). The CPE lesions were visualised in all four

replicates from the 10-2 and 10-3 dilutions, whereas two of four replicates were

evaluated from the 10-4 dilution (Table 4.7). The TCID50 titre was 1 × 105

(per mL).

Table 4.7: Appearance of CPE on the replicates of each dilution of HVT-

CEF

Replicate

10-fold serial dilution of HVT-CEF

10-2 10-3 10-4 10-5 10-6 Uninfected

1 + + + - - -

2 + + - - - -

3 + + + - - -

4 + + - - - - Note: + indicates CPE lesions observed and – indicates no CPE lesion observed.

4.3.12 Evaluation of HVT infections

Based on a result of TCID50 from Section 4.3.10, the MOIs 0.02, 0.01, and

0.0035 used to evaluate HVT infections were prepared from the HVT

volumes of 150, 75, and 25 µL, respectively. The percentage of CPE lesions

was estimated by visualising the proportion between CPE lesions and healthy

CEF monolayers under a microscope in all areas of each well. The infected

Figure 4.29: Samples of CPE lesions in CEF cells infected with HVT

and non-infected CEF cells were evaluated using an inverted

microscope at 7 days post-infection.

A) CPE lesion (red arrow) and cell death were found in the infected

CEF cells.

B) CEF cells (elongated and needle-like cells; black arrow) and death

cells without CPE (red arrow) were found in uninfected cell control

212

cells with virus demonstrate the changes of cell morphology such as rounding

of the infected cell, cell lysis (dissolution), polykaryocytes, nuclear or

cytoplasmic inclusion bodies (Albrecht et al., 1996). At 24 h after infection,

CPE lesions were found approximately 20% and 10% in the infected CEF

cells with MOIs 0.02 and 0.01, respectively (Figure 4.30). The infected cells

with MOI 0.0035 of HVT and non-infected cells were negative for CPE

(Figure 4.30).

At 2 days post-infection, infected cells with all MOIs of HVT showed the

appearance of CPE lesions (Figure 4.31). CPE lesions were approximately

80%, 70%, and 50% in the infected cells with MOIs 0.02, 0.01, and 0.0035,

respectively, whereas, non-infected cells remained negative for CPE (Figure

4.31).


MOIs of HVT using an inverted microscope at 1 day after infection.

The infected CEF cells with MOIs 0.02 and 0.01 of HVT showed CPE

lesions (red arrows). No CPE lesion was found in infected CEF cells

with HVT (MOI 0.0035) and in non-infected CEF cells.

213

At 3 days post-infection, CPE lesions were approximately 100%, 90%, and

70% in the infected cells with MOIs 0.02, 0.001, and 0.0035, respectively

(Figure 4.32).

4.4 Discussion

As the first step in the development of a viral vector system to deliver genes

encoding putative Campylobacter immunogenic antigens, this chapter has

examined the expression of these genes using eukaryotic expression vectors.

Eukaryotic expression systems, especially in mammalian cells, are commonly

used to explore gene functions and the biological functions of the proteins


MOIs of HVT using an inverted microscope at 2 days post-infection.

The infected CEF cells with all MOIs (0.02, 0.01, and 0.0035) of HVT

showed CPE lesions with high (red arrows). Non-infected CEF cells

showed no CPE lesion.

Figure 4.32: Microscopic analysis of infected CEF cells with different

MOIs of HVT using an inverted microscope at 3 days post-infection.

The infected CEF cells with all different MOIs (0.02, 0.01, and 0.0035)

of HVT showed more CPE lesions (red arrows). Non-infected CEF cells

showed no CPE lesion.

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they encode in in vitro systems (Kim & Eberwine, 2010; Lai et al., 2013;

Wurm, 2004). Initially, ORFs encoding the conserved Campylobacter genes

– katA, cadF, peb1A, and cjaA (as described in Chapter 3) – were cloned into

the pcDNA3.1 V5/HIS-TOPO vector for expression of the proteins of interest

in the mammalian cells. Udo (2015) suggested that this pcDNA3.1 directional

cloning vector is convenient and can be immediately used after cloning for

protein expression; thus, these genes cloned in this way could be directly used

in the construction of the HVT-based vector without testing in bacterial cells

for expression. However, the data from this study revealed that none of these

genes was expressed in the mammalian cells (RK-13 cells).

According to the manufacturer’s instructions, CACC motif is required for the

efficient and directional TOPO forward primers, followed by Kozak’s

sequences (ATG) as a start codon, and then the rest of gene-specific sequence

(Invitrogen, 2010b). As a result, the GTGG 5′ overhang of the vector

displaces GTGG in the PCR product and ATG is required to initiate

translation, resulting in a directional reaction and a correct initiation of

translation during the TOPO reaction.

All the genes of interest (katA, cadF, peb1A, and cjaA) were successfully

cloned into the pcDNA™ 3.1 D/V5-His-TOPO vector, based on the results

of PCR, double digestion, and DNA sequencing analyses. However, the

nucleotide sequence analyses showed either nucleotide insertions or

nucleotide substitutions in the regions upstream of the cloning/recombinant

site of the TOPO vector in the pcDNA3T-katA-1, pcDNA3T-peb1A-1,

pcDNA3T-cjaA-1, and pcDNA3T-cadF-4. This was consistent with the

results of double digestion using restriction enzymes, which revealed all gene

fragments to be larger than the estimated sizes. An additional 25 nucleotide

bases were found before the katA, cadF, and cjaA coding regions (Figure 4.5,

4.6 and 4.8). This insertion was initially considered unlikely to interrupt the

translation of the proteins of interest since it was located upstream of the start

codon (ATG) of the ORFs. In addition, the nucleotide sequences of the cloned

TOPO vector with peb1A showed the different nucleotide bases occurring

outside the regions between the insertions of peb1A region (Figure 4.7).

Although the start codon was still in the correct frame, the inserted peb1A

gene would likely disrupt the reading frame of the C-terminal tag.

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The Western blot analysis showed that neither the recombinant TOPO

plasmids nor the pcDNA™3.1D/V5-His/lacZ vector plasmid (positive

control), which provided from the kit, expressed the proteins of interest,

compared with the negative control, after being transfected into RK-13 cells,

thus indicating lack of TOPO vector transfection into RK-13 cells. There

could have been a technical problem during transfection since it appeared that

in the positive control, expression of the protein of interest was not detected

using Western blotting. Klupp et al. (2017) used the cloned pcDNA3.1

V5/HIS-TOPO with herpesvirus genes transfected into RK-13 cell and

showed successful transfection and protein expression, suggesting that the

TOPO vector and RK-13 cells are compatible for transfection studies. In

addition, cDNA and qPCR were not conducted in this current study due to

time constraint. Thus, repeating the transfection reaction more than twice with

positive controls in each step and cDNA analysis may be required. Even

though the vectors contained additional bases from DNA sequences in the

current study, these nucleotides did not disrupt the ORF as long as they were

outside of the coding regions of each construct. However, these types of

nucleotide modifications should not occur during the cloning processes. It is

possible that the extra bases may have disrupted the optimal spacing between

the promoter and the ORF, adversely affecting the translation. Alternatively,

the inserted nucleotides may have affected the translation of the proteins of

interest by affecting the stability or structure of the respective mRNAs. On

the basis of these results, all recombinant plasmids must be confirmed using

DNA sequencing in this system to ensure that all plasmid clones do not

contain any nucleotide insertions and/or rearrangements before further

analyses are attempted. Whereas, these experiments were conducted

simultaneously in the current study due to time constraints.

It is also possible that the cells used for transfection may have impacted on

the likelihood of successfully detecting the proteins of interest. However, RK-

13 cells have been used extensively and successfully in transfection with

plasmids containing viral genes and infection with viruses or yeasts (Duncan

et al., 2000; Flores Rodríguez et al., 2018; Kojima et al., 2003; Maruri-Avidal

et al., 2013). Moreover, in the present study, the pEGFP-C1 vector was used

as a transfection control for the RK-13 cells, and EGFP fluorescence was

216

clearly observed in the cytoplasm of the transfected cells. This indicates that

transfection of the pEGFP-C1 vector into RK-13 cells was successful,

suggesting the lack of detectable expression was due to the recombinant

TOPO vectors containing katA, cjaA, peb1A, and cadF ORFs and the

pcDNA™3.1D/V5-His/lacZ vector (positive control) plasmids rather than

transfection failure in this study. For further study, screening a larger number

of recombinant clones to identify those containing only the target sequences

(without additional bases) may provide a satisfactory outcome. Inclusion of

another vector such as pEGFP simultaneously used as a positive control in

the same cell would provide a further control to compare transfection

efficiency.

Due to unsuccessful protein expression using the recombinant pcDNA3.1

V5/HIS-TOPO vector, the pEGFP vector was selected for further

investigations into the expression of the proteins of interest in this study. The

pEGFP-C1 vector, encoding EGFP under the control of CMV promoter, has

been used as the model system to express the protein of interest in eukaryotic

cells to facilitate rapid confirmation of expression by visualising EGFP

(Broadway et al., 2003; Karagöz et al., 2018; Tamura et al., 2011; Wang et

al., 2012). The present study demonstrated that all recombinant pEGFP-C1

vectors containing the katA, cadF, peb1A, and cjaA ORFs were successfully

constructed, transfected and expressed the Campylobacter proteins of interest

as EGFP fusion proteins in Vero cells. The EGFP fusion proteins and EGFP

detected in the transfected Vero cells were uniformly distributed throughout

the cytoplasm. The fluorescence pattern observed in this study agreed with

that observed by Buelow et al. (2011) who reported that the Hela cells

transfected with pEGFP-C1 showed diffuse fluorescence with no specific

cellular localisation. The Western blot and mRNA analyses showed that all

recombinant pEGFP-C1 vectors containing the Campylobacter ORFs

expressed the KatA, CadF, Peb1A, and CJAA as EGFP fusion proteins.

However, the Western blot results showed that the EGFP-Peb1A polypeptide

from pEGFP-C1-peb1A-4 had the highest level of expression. In contrast, the

EGFP-KatA from pEGFP-C1-katA-5 had the lowest level of expression.

Analyses of the cDNA from cells transfected with the plasmids encoding the

fusion proteins did not suggest any differences in the levels of mRNA for

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these proteins. However, the PCR assay used was a conventional end-point

PCR. For further study, qPCR would be suggested to quantify the level of the

mRNA transcription.

These findings suggest that the pEGFP-C1 vector is useful for Campylobacter

gene expression and that the pEGFP-C1 with peb1A is a promising candidate

for vaccine construction. A pEGFP-C1 vector is a valuable vector which has

been widely used for protein expression in mammalian cells since it is readily

visualised the protein expression and transfection efficiency (Khezri et al.,

2018; Soboleski et al., 2005). However, it may interfere with the immune

recognition of the protective antigen of interest. This phenomenon has

previously been reported, where strong T cell responses were detected for

GFP, but not the heterologous antigen (Koelsch et al., 2013). In addition, we

do not know how the pEGFP-C1 vector synthesises the structure of the

protein of interest. Consequently, it may show either similar epitope as the

native antigen or different epitope and may affect the immune responses.

Thus, these are essential to account for the vaccine efficacy in vivo.

Limited resources were available for construction of a HVT-based vector.

Preparation of CEF cells, TCID50 analysis, and evaluation of HVT infection

efficacy were completed. The current data show that TCID50 of HVT was 1

× 105 units/mL at 7 days post-infection. After that, the confluent monolayers

of CEF cells were infected with HVT at different MOIs (0.02, 0.01, and

0.0035) to determine the suitable MOI, which can be used to construct the

HVT-based vaccine in an appropriate time. The MOI of 0.01 has been used

to evaluate the efficacy of the recombinant HVT vaccine (Dey et al., 2017;

Tarpey, 2007; Zhao et al., 2014). The present study showed that the CPE

lesions appeared in the infected CEF cells at 1 day after infection using the

MOIs 0.02 and 0.01, whereas the MOI of 0.0035 showed the CPE lesion at 2

days after infection. These findings suggest that HVT is a highly infectious

virus using CEF cells. However, Li et al. (2011) and Tang et al. (2018)

reported that the construction of recombinant HVT candidates with MOIs of

1 and 0.01 infected the CEF cells, resulted in the appearance of CPE by 3–4

days after infection. Hence, the approach using an MOI of 0.01 to infect the

CEF cells requires re-evaluation as CPE lesions were found 1 day after

infection. In the present study, CEF cells seeded in 24-well plates at a density

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of 1 × 105 cells/well resulted in cell aggregation. Dilnessa and Zeleke (2017)

and Leland and Ginocchio (2007) have suggested that infected monolayers

with viruses provide evidence of viral infection such as CPE. Hence, the

aggregation of the CEF cells in this study could interfere with CPE detection

under microscopic examination, resulting in lower TCID50 titres. Therefore,

repeating the TCID50 experiment with seeding a lower density of the cells,

and immediately shaking, may improve the protocol to promote monolayer

development of CEF cultures prior to infection with diluted HVT.

In the current study, the expression of target proteins following pcDNA3.1d

TOPO plasmid transfection was not achieved. Thus, refinement of the

approach and repeated experiments are required. The pEGFP-C1 plasmid was

found to be a good vector for cloning Campylobacter genes. Among

recombinant pEGFP-C1 vector containing four genes, the recombinant

pEGFP-C1-peb1A showed the highest level of expression in mammalian

cells. The peb1A gene encodes a periplasmic-binding protein (Peb1A)

involved in Campylobacter colonisation through adherence to and invasion

of host cells (Ó Croinin & Backert, 2012; Oh et al., 2017). The immunisation

with the Peb1A protein resulted in a significant reduction in caecal content

after C. jejuni challenge in a previous study (Buckley et al., 2010). Therefore,

Peb1A would be a good antigen candidate for a viral vector vaccine

development in the future. For future study, the peb1A gene showing the

strongest expression will be genetically cloned into the HVT vector to form a

recombinant HVT- peb1A vector vaccine by insertion of the peb1A gene into

the region of HVT-characterised peb1A using CRISPR/Cas9 gene-editing

system, as described by Tang et al. (2018).

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Chapter 5 General discussion

5.1 General aims and experimental chapter summaries

The purposes of this thesis were to improve the understanding of the

dynamics of C. jejuni and C. coli colonisation in commercial free-range

broiler farms and identify conserved antigen encoding genes that might

prevent the colonisation of chickens of the two species of interest following

delivery with a live viral vectored vaccine. Therefore, two major studies were

conducted.

The first study (Chapter 2) involved in the investigation of C. jejuni and C.

coli colonisation, the potential sources, and the genetic diversity in

commercial free-range broiler chickens. Subsequently, the C. jejuni (n=412)

and C. coli isolates (n=151) from various sources (fresh faeces and the

surrounding production environment) of the commercial free-range boiler

farms at different time points were differentiated into genotypes using flaA-

HRM-PCR. The flaA-HRM genotyping was validated by flaA amplicon

sequencing of selected isolates (n=229; C. jejuni and n=123; C. coli). While

a further subset of isolates (n=9; C. jejuni and n=5; C. coli) were also

subjected to MLST analyses to further support the flaA-HRM clusters C.

jejuni and C. coli isolates were assigned to. During this study, the fresh faecal

samples from the broilers’ parent breeders were included to evaluate the

possibility of vertical transmission. The same flaA-HRM clusters of C. jejuni

and C. coli were isolated from fresh faeces and the surrounding environment

on the same free-range broiler farms, suggesting horizontal transmission was

the main mode of colonisation in this study. Moreover, far less frequently, the

same flaA-HRM clusters of C. jejuni and C. coli were isolated from fresh

faeces from breeders and their progeny broilers, but not from any common

environmental sources. This suggests that vertical transmission cannot be

excluded as a potential source of transmission in this study.

The second study (Chapters 3 and 4) aimed to identify and characterise the

conserved genes, and encoded antigens (katA, cadF, cjaA, peb1A, omp18,

flpA, and fliD) shown to affect Campylobacter spp. colonisation efficiency,

which could be used for delivery using HVT viral vector vaccine. To achieve

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this, representative C. jejuni and C. coli isolates for the flaA-HRM clusters

isolated from the broiler farms (Chapter 2) were examined for the presence

of seven genes (katA, cadF, peb1A, cjaA, omp18, flp, and fliD) encoding

antigens using conventional PCR assays (Chapter 3). Of these, katA, cadF,

peb1A, and cjaA were detected in all of the C. jejuni and C. coli isolates

examined. As the first stage in the development of these genes as vaccine

candidates, the corresponding ORFs from an isolate of the most common C.

jejuni flaA-HRM cluster (cluster 27) from broiler farms (Chapter 2) were used

as a representative Campylobacter spp. genes to characterise the expression

capacities in bacterial (Chapter 3) and mammalian cells (Chapter 4). The

expression of all putative antigens was detected by Western blot and mRNA

analyses. The conserved genes showing strong expression in mammalian cells

(Chapter 4), suggesting they will be useful as antigen candidates for the future

construction of recombinant HVT viral vector vaccines.

5.2 Major findings and limitations

The current thesis showed that C. jejuni and C. coli were isolated from the

breeder and free-range broiler farms, with C. jejuni being the most frequently

isolated species (Chapter 2). The number of isolates (colonies) from each

positive sample on a culture plate varied from 1 to 415 in this study. With the

large number of isolates observed, it was not feasible to examine all colonies

from each plate. To resolve this issue, the international standard (ISO, 2006)

was adopted and this resulted in a maximum of 5 isolates per sample being

selected for speciation. The standardised ISO methodology has also been

applied to the investigation of Campylobacter genetic diversity in previous

studies (Ahmed et al., 2016; Greige et al., 2019; Marotta et al., 2015; Peyrat

et al., 2008). The ratio of one presumptive Campylobacter colony per sample

for genotyping has been commonly adopted to identify and differentiate

genotypes in other epidemiological studies (Ahmed et al., 2016; Broman et

al., 2002; Guyard-Nicodeme et al., 2015; Peyrat et al., 2008; Wieczorek et al.,

2019), while one isolate per sample has been suggested as a minimum

requirement for genotyping (Devane, 2006). To the best of my knowledge,

no studies have reported any variation in the genotypes between the

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Campylobacter colonies analysed from the same sample. Therefore, the ISO

standard method was considered appropriate for this study.

One of major findings of this study was that C. coli was the first species

isolated from chicken faeces in a commercial free-range broiler target flock

by 10 days of rearing, whereas, C. jejuni was first isolated from chickens after

15 days of rearing (Chapter 2). This finding is contrary to a previous study

from the UK conducted by El-Shibiny et al. (2005) who reported that C. jejuni

was the first species detected in a free-range broiler flock (Day 8). This

suggests that Campylobacter spp. can colonise free-range chicks at a very

young age, several studies; however, have demonstrated that C. jejuni and C.

coli could be first isolated from chickens by 14 to 21 days of rearing from in

commercial intensive broiler farms (Friis et al., 2010; Ingresa-Capaccioni et

al., 2015; Messens et al., 2009; Prachantasena et al., 2016). The commercial

intensive broilers are generally reared in the closed sheds throughout the

rearing period until slaughter, and this could delay the C. jejuni and C. coli

colonisation at farms (Huat et al., 2010). In contrast, the commercial free-

range broilers exposed to the external environment after 14-21 days of rearing

onwards depending on seasonality. However, research on the effect of

different farming systems (commercial intensive and free-range systems) on

C. jejuni and C. coli colonisation are further required. Once a few colonised

chickens were positive for Campylobacter spp. in a broiler flock, most

chickens within the same flock and the environment were found to be positive

within one week. This suggests that Campylobacter spp can rapidly spread

within the flocks and the environment and this has been reported previously

(van Gerwe et al., 2009).

Multiple genotypes of C. jejuni and C. coli were isolated from the breeder and

free-range broiler farms in this study and this agreed with previous studies

(Bull et al., 2006; Colles et al., 2011; Ridley, Allen, et al., 2008; Vidal et al.,

2016). The data showed a wide range of C. jejuni (n=35) and C. coli (n=25)

flaA-HRM clusters were isolated from colonised breeder chickens (Chapter

2). These findings suggest that Campylobacter spp. colonisation in breeder

chickens is a complex and dynamic process, supported by the notion of repeat

exposure in longer-lived breeders (compared with broilers) (Colles et al.,

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2011) and genetic rearrangement among Campylobacter genotypes in

colonised chickens (Ridley, Toszeghy, et al., 2008). However, the exact

mechanisms underpinning the genetic diversification of C. jejuni and C. coli

remain unclear. These data also suggest that controlling Campylobacter spp.

through a host immune system-based approach, such as vaccination, could be

challenging unless highly conserved antigens can be identified.

In contrast, fewer numbers of C. jejuni (n=9) and C. coli (n=5) flaA-HRM

clusters were isolated from free-range broiler farms and the majority of these

were distinct between Exp.1 and Exp.2 (Chapter 2). These findings suggest

that the all-in-all-out farming system and farm practices (cleaning and

disinfection) during the empty period are effective strategies which eliminate

most C. jejuni and C. coli genotypes between free-range broiler farming

production cycles. Another reason for the lower diversity could be the

relatively short period of sampling in this study. However, in a recent study

Templeton (2014) reported seven different genotypes of C. jejuni were

isolated from caecal contents of commercial free-range broiler chickens at a

slaughterhouse but did not examine the dynamics of C. jejuni colonisation on

the farms. Therefore, a future study designed to include sampling across the

full period of free-range broiler farm production cycle would provide more

knowledge about the dynamics of C. jejuni and C. coli colonisation and their

genetic diversity in the free-range broiler farming system.

The data obtained in this study suggest that multiple C. jejuni and C. coli flaA-

HRM clusters isolated from free-range broiler faeces are most likely from

various environmental sources (Chapter 2). These data indicate that

horizontal transmission was the major pathway of C. jejuni and C. coli

colonisation on free-range broiler farms sampled in this study. The

environment including drinking water, rodent faeces, shed walls, floors

(bedding), water pans, shed boots, the free-range areas (soil), anteroom floor,

and farm boots were identified as potential sources of Campylobacter

transmission within the free-range broiler flocks in this study (Chapter 2).

Some C. jejuni and C. coli flaA-HRM clusters were common between the

chicken faeces from different farms and the environments (in the same area)

in this study, suggesting these free-range chickens may have had contact with

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a common external environmental source of Campylobacter such as rodents

(Meerburg et al., 2006), flies (Hald et al., 2008), and wild birds (Craven et al.,

2000; Waldenstrom et al., 2002), resulting in the isolation of similar

genotypes. However, in this study it was only possible to collect the optimal

number of samples from one shed, the target shed, of three sheds sampled on

the free-range broiler farms for focusing on the C. jejuni and C. coli

colonisation and transmission. As fewer samples were collected from the

adjacent sheds, the results of the current study may not provide a full profile

of the genetic diversity present in the study farms. Therefore, a larger

longitudinal study looking at the dynamics of C. jejuni and C. coli

colonisation and identification of more potential sources of the transmission

on the free-range chicken farms in different regions is warranted. Importantly,

despite this limitation, the carryover transmission of Campylobacter between

consecutive free-range broiler flocks in one farm was identified in this study.

Therefore, improved hygiene practices (cleaning and disinfection) and

appropriate biosecurity measures could potentially reduce Campylobacter

transmission in broiler farms (de Castro Burbarelli et al., 2017; Newell et al.,

2011; Smith et al., 2016). One way to support improved on-farm hygiene

practices would be to conduct structured sampling prior to chick placement

to determine how effective the elimination of Campylobacter was.

The possibility of vertical transmission of Campylobacter from breeders to

broiler progeny was of interest in this study. If vertical transmission were an

important mode of transmission for broiler chickens, Campylobacter control

on the breeder farms could be a highly effective intervention point. In this

study, there was some evidence of vertical transmission with the same C.

jejuni and C. coli flaA-HRM clusters being isolated from faecal samples from

breeder farms and their corresponding broiler flocks (Chapter 2). However,

this only occurred in four of the 17 free-range broiler flocks sampled in this

study, suggesting that if the vertical transmission does occur it is not the major

mode of transmission.

One limitation of this current study was that we obtained fresh faecal samples

from the breeder farms after the broiler chicks had been placed on the broiler

farms for seven days. This sampling constraint was imposed by the operators

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of the enterprise involved the study. Consequently, the identification of

similar genomes of C. jejuni and C. coli in the linked breeders and broilers

may not be truly representative of vertical transmission events. Ideally, the

sample collection should be conducted on the breeder farms on the date of

laying the eggs which hatched the broilers included in this study. Thus, this

sampling should be conducted 21 days before the broiler chick placement at

farms. Therefore, further experimental studies focusing solely on vertical

transmission under commercial conditions are required to resolve this

question.

Given the diversity of genotypes identified on breeder farms, the common

genotypes identified possibly emerged in the 28 days prior to sampling in this

study, resulting in an over-estimation of the frequency of vertical

transmission. Paradoxically, this study may have also underestimated the

frequency of vertical transmission. The diversity of genotypes identified in

the sampled breeder farms in this study suggested that colonisation of these

birds is a highly dynamic process due to age, an observation which has also

been reported elsewhere (Colles et al., 2019). Consequently, it is possible that

the genotypes of C. jejuni and C. coli present in the laying flocks at the time

of laying were different to those present when the samples were collected. To

answer this important question, it would be necessary to sample the breeder

farms multiple times, before, during and after lay to understand the dynamics

of Campylobacter colonisation on these farms as well. This was not possible

in the current study as sampling processes were constrained for commercial

reasons imposed by the industry participants. For similar reasons sampling at

the hatchery was also not conducted in this study.

Due to the sampling constraints in this study, directly tracing specific

genotypes of Campylobacter through the complete broiler production system

was not possible. An expanded longitudinal study of the whole chicken

production chain, with the effective time of sample collection and sample size

based on the current prevalence of Campylobacter in Australia, is required to

further elucidate a better understanding of Campylobacter colonisation and

transmission of chickens in free-range poultry farms.

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For aspects of vaccine development, an important step is to ensure that the

antigens used in vaccine construction were conserved between C. jejuni and

C. coli isolates. If any conserved antigens with similar epitopes from various

C. jejuni and C. coli genotypes are identified, these antigens could be used to

generate broad protection as vaccine candidates. Currently, limited

information on Campylobacter conserved antigens is available. Therefore,

identification and characterisation of C. jejuni and C. coli conserved genes

encoding antigens among chicken farms were of interest in this thesis.

Selected C. jejuni and C. coli isolates representing flaA-HRM clusters from

chicken farms (Chapter 2) were assessed for the presence or absence of

Campylobacter conserved genes encoding antigens (Chapter 3). The

Campylobacter genes used in this thesis have been previously evaluated for

vaccine efficacies in published studies as described in Chapter 3.

C. jejuni and C. coli isolated from chicken farms were genetically diverse in

this study (Chapter 2). Due to time and resource constraints, not all C. jejuni

(n=412) and C. coli (n=151) isolates were tested for the presence of

Campylobacter spp. conserved genes encoding antigens. Therefore, selected

C. jejuni (n=41) and C. coli (n=26) isolates representing the flaA-HRM

clusters were used for this purpose. The data has shown that four

Campylobacter genes, katA, cadF, peb1A, and cjaA, were highly conserved

between C. jejuni and C. coli by conventional PCR assays in this study

(Chapter 3). Selected PCR amplicons of each conserved gene were sent for

sequencing, with the results showing some variations of the translated amino

acid sequences for each conserved gene were identified between C. jejuni and

C. coli. For example, the translated amino acid polypeptide of CjaA ORFs

was highly conserved between selected C. jejuni and C. coli isolates

representing flaA-HRM clusters in this study with 98.3% similarity, following

by KatA polypeptides with 94.2% similarity. In contrast, that of the Peb1A

polypeptides showed a lower similarity with 79.1%, compared with those two

CjaA and KatA polypeptides. However, it had a high similarity within the

same species. For example, the similarities of the amino acid polypeptide of

Peb1A were 98.0% and 100% in selected C. jejuni and C. coli isolates

representing flaA-HRM clusters, respectively.

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Moreover, a total of 13 amino acid insertions were identified in the CadF

polypeptide in most of the selected C. coli clusters, compared with that of

selected C. jejuni clusters. This finding is consistent with previous studies

(Konkel, Gray, et al., 1999; Krause-Gruszczynska et al., 2007). Therefore,

these findings suggest that the amino acid polypeptides encoded by the katA,

peb1A, and cjaA ORFs were highly conserved between selected C. jejuni and

C. coli isolates representing flaA-HRM clusters (more than 94% similarity),

whereas, that of cadF ORFs were distinct. However, it is unknown if these

variations would affect any of the epitopes of these polypeptides or not. If

these variations were present in different epitopes, these could affect the

efficacy of any vaccine they were used in. In addition, this study only

characterised the antigens of selected C. jejuni (n=13) and C. coli (n=8)

isolates representing flaA-HRM clusters, and thus, it may limit the

information about the variations of the translated amino acid polypeptide

from these ORFs encoding antigens in other flaA-HRM genotypes. Therefore,

characterisation of additional C. jejuni and C. coli isolates representing flaA-

HRM clusters of each ORF of interest would provide a better understanding

of the variations of nucleotide sequences and translated amino acid

polypeptide which may assist in the evaluation of how these may affect the

antigenic epitopes. Research on the investigation of structures of antigen

epitopes from more C. jejuni and C. coli isolates and identification of

additional conserved genes encoding antigens would reveal more information

on conserved genes and the precise epitope, and these would assist in

determining the most effective gene(s) for vaccine development.

As part of the pipeline for future vaccine construction activities, all these

conserved ORFs encoding antigens were evaluated for protein expression in

bacterial and mammalian cells. In this thesis, an isolate from the most

frequently C. jejuni flaA-HRM cluster (cluster 27) from broiler farms was

used as a representative genotype for these proof-of-concept studies. The pET

SUMO plasmid was used as a protein expression vector in bacterial cells. The

data showed that these four Campylobacter conserved ORFs (katA, cadF,

peb1A, and cjaA) were successfully cloned into pET SUMO plasmids and

expressed detectable polypeptides on the Western blots (Chapter 3). We

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found that the conservative substitution of translated amino acid was found

in the recombinant pET SUMO plasmids containing cadF, cjaA and peb1A,

based on DNA sequencing; however, these differences did not affect the

amino acid properties. Further studies are required to check PCR products

from repeat PCR reactions using the same DNA template and measure the

DNA template prior to cloning in order to ensure that all PCR products are

identical to the original template.

Western blot analysis demonstrated that all recombinant pET SUMO

plasmids containing these four Campylobacter conserved ORFs successfully

expressed the proteins (KatA, CadF, CjaA, and Peb1A) with correct

molecular weights (Chapter 3). Of these, two molecular weights of the CadF

protein were detected with different sizes due to incomplete denaturation

during cell lysis, consistent with a previous study (Krause-Gruszczynska et

al., 2007). The KatA protein had the strongest expression on the Western

blotting compared with other ORFs, whereas, Peb1A protein had the lowest

expression. This suggests that pET SUMO vectors effectively expressed

KatA, CadF, CjaA ORFs in bacterial cells, whereas, this vector may not be a

good expression promotor for peb1A ORF. While not the main goal of the

current study, these expressed polypeptides could be purified and formulated

into a conventional multi-dose vaccine for immunisation studies in chickens.

Such a formulation could be used to estimate the level of protection these

antigens may provide either in in vitro or in vivo studies. The expressed

polypeptides could also be used in various combinations to determine if there

is a cumulative effect on their capacities to inhibit Campylobacter

colonisation of chickens.

In Chapter 4, ORFs from katA, cadF, peb1A, and cjaA (Chapter 3) were

cloned into the pcDNA3.1 V5/HIS-TOPO and pEGFP-C1 vectors for

expression of the polypeptides of interest in the mammalian cells. The

purpose of this was to characterise the expression of these conserved ORFs

and the respective antigens in eukaryotic cells which is an essential step prior

to the development of a viral vector system. The data of this thesis showed

that the proteins of interest from the recombinant pcDNA3.1 V5/HIS-TOPO

plasmids containing these ORFs and the positive control were not detected on

228

the Western blotting assay (Chapter 4). In contrast, the pEGFP-C1 plasmid

was used as second control of the transfection, resulting in the GFP protein

detected on the Western blotting analysis. These findings suggest that either

the recombinant TOPO vector did not produce any polypeptides or expressed

them at levels below the detection limits of the Western blotting assay used.

The exact reason(s) for this lack of apparent expression was not determined.

However, a 25 bp insertion in the region upstream of the cloning/recombinant

site of the TOPO vector from the Campylobacter ORFs was identified in the

DNA sequencing results. It was initially considered unlikely to affect or

interrupt the translation of the polypeptides of interest since it was located

upstream of the start codon (ATG) of the ORFs. Therefore, a refinement of

this approach would have been to sequence plasmids from more colonies to

identify clones which lack the inserted DNA fragment. However, as all four

of the ORFs contained this insertion and as they were generated using

independent ligation reactions, the likelihood of identifying unaffected

plasmids was considered low. Consequently, an alternative eukaryotic

expression strategy was devised.

All recombinant pEGFP-C1 vectors containing the katA, cadF, peb1A, and

cjaA ORFs were successfully constructed, transfected and expressed the

Campylobacter polypeptides of interest as EGFP fusion polypeptides in Vero

cells (Chapter 4). These findings suggest that the pEGFP-C1 vector is useful

for Campylobacter conserved ORFs expression. The mRNA and Western

blot analyses showed that all recombinant pEGFP-C1 vectors containing the

Campylobacter ORFs expressed the KatA, CadF, Peb1A, and CjaA as EGFP

fusion proteins, albeit with different efficiencies. The EGFP-Peb1A

polypeptide showed the highest level of expression on the Western blot

analysis, whereas, the EGFP-KatA showed the lowest level of expression

(Chapter 4). A conventional reverse transcriptase PCR was used to detect

mRNA from cells transfected with the plasmids encoding the fusion proteins.

While the assay confirms the presence of transcripts, as this is not a

quantitative method, the relative levels of mRNA expression for the proteins

of interested could not be compared. Future studies could utilise a RT-qPCR

assay to quantify the levels of the mRNA (transcription) for each protein, in

229

parallel with the Western blotting (translation process). This assessment could

include the pEGFP-C1/eGFP controls to evaluate if the inability to detect the

proteins of interest was due to insufficient transcription or inefficient

translation from the pcDNA3.1 V5/HIS-TOPO vector. Furthermore, the

protein expressions of all recombinant pEGFP-C1 containing the katA, cadF,

peb1A, and cjaA ORFs in Chicken Embryonic Fibroblast (CEF) cells is also

required to determine if these ORFs can express the polypeptides of interest

in avian cells via pEGFP-C1 vectors before construction of a viral vector.

Subsequently, qPCR and Western blotting could be used to quantify mRNA

synthesis and detect the polypeptides of interest, respectively.

The results presented in this thesis showed that the pEGFP-C1 plasmid is an

excellent expression vector for C. jejuni conserved ORFs from the katA,

cadF, cjaA, and peb1A genes in mammalian cells. However, it is unknown if

the presence of the eGFP polypeptide will allow the Campylobacter

polypeptides form their native conformations, which could be particularly

important if they have non-linear epitope(s) which are important for

protective immune responses. Similarly, it is unknown if important epitopes

will be presented in polypeptides expressed by HVT or other viral vector

vaccines carrying these Campylobacter conserved putative antigens.

Therefore, the investigation of polypeptide structures of the EGFP fusion

proteins is required to determine if important epitopes present in the native

Campylobacter spp. cells during host colonisation.

5.3 Future directions

These studies have enhanced understanding C. jejuni and C. coli colonisation

and transmission in broiler chickens in free-range farms. Data from this thesis

have provided information to underpin future epidemiological studies and

further identification of suitable genes encoding antigens for vaccine

development. For example, C. coli was isolated from faeces of 10-day-old

chickens, suggesting chicken can uptake C. coli prior to this point. This

finding is useful in the design and selection of the best vaccine strategy in

future studies. This thesis has shown that horizontal transmission (from

230

various environmental sources to birds) is a major pathway of C. jejuni and

C. coli colonisation at farms, whereas, the evidence of vertical transmission

was minimal. These findings suggest more effective biosecurity measures

could be useful for Campylobacter transmission control at farms. However,

further research, focusing on more farms (a national study) and more potential

sources of C. jejuni and C. coli transmission, are required in order to provide

more knowledge of C. jejuni and C. coli epidemiology on Australian

commercial free-range broiler farms. This more expansive study is important

to investigate potential interventions to control/prevent C. jejuni and C. coli

colonisation of chickens in commercial farm production systems.

This thesis has contributed to the identification of the most suitable antigen

for vaccine development since no commercial vaccine against

Campylobacter is currently available in the poultry industry. The sequences

of 13 C. jejuni and eight C. coli isolates representing the major genotypes

identified were further selected for sequencing and in silico analysis. This

approach not only confirmed the correct gene amplicon of interest but also

provided the opportunity to study some variants of some genes. The analyses

confirmed the four conserved genes and identified some variations in the

polypeptides between C. jejuni and C. coli isolated from Australian chicken

farms. The outcome has identified some variations in the polypeptides of the

four Campylobacter homologous genes (katA, cadF, peb1A, and cjaA) which

encode potential antigens. As an example, the peb1A gene was conserved

between C. jejuni and C. coli genotypes but the polypeptides encoded by the

peb1A ORFs were different. Within the C. coli genotypes (n=8), all Peb1A

polypeptides were identical. In contrast, 38 amino acid variants (14.6%) were

identified in the 13 C. jejuni genotypes, compared with the C. coli genotypes.

Of these, 34 variants (13.1%) were conserved substitutions of amino acids

and four variants (1.5%) were not conserved amino acid substitutions. Of the

34 conserved variants, twenty-six and eight substitutions showed strong and

weak physicochemical similarities, respectively. Furthermore, among the 13

C. jejuni genotypes, only three C. jejuni genotypes shared identical amino

acids with the C. jejuni strain YH002. While six variations of Peb1A amino

acid sequence were identified in 10 C. jejuni genotypes, in the same

231

alignment. Of these 10 C. jejuni genotypes, only one genotype had a different

amino acid which was conserved between amino acid groups, with weak

physicochemical similarities. These findings suggest that the more highly

variable conserved genes between species are unlikely to be suitable for

inclusion in a vaccine with the aspect of the heterologous strain prevention.

Therefore, in silico analysis with additional ORFs encoding antigens of all C.

jejuni and C. coli isolates/genotypes identified from chicken farms and the

epitope predictions should be further investigated. This would provide more

information on the variation of ORF encoding antigens from Australian free-

range chicken farms and assist in the selection of the most suitable ORF for

future vaccine development. However, the only way to test the potential

influence of these variations on vaccine efficacy would be to conduct

homologous and heterologous challenge studies. In terms of antigenic

polypeptide expression, further studies looking at the expression of

polypeptides in CEF cells, investigation of recombinant antigens from

expressing other vaccine vectors, multivalent vaccine approaches, the

evaluation of vaccine efficacy in chickens, and identifying other conserved

ORF encoding antigen are required to warrant more information of effective

antigen candidates for vaccine development.

In conclusion, the findings from the current research have enhanced the

understanding of the potential sources, timing and genetic diversity of

Campylobacter colonisation in free-range broiler farms. This research thesis

also has provided the information of potential genes which could be used as

antigen candidates for the construction of recombinant HVT vector vaccine

or another vaccine delivery system. Hence, these can be useful for further

studies for developing appropriate measures to prevent Campylobacter

colonisation in the free-range broiler production system.

232

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Appendices

Appendix 1: Raw data of the notification rate of human gastroenteritis in Australia from 2002 and 2018

Gastrointestinal

diseases

Year

2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018

Botulism 0 0 0 0 <0.1 <0.1 0 <0.1 0 <0.1 0 <0.1 <0.1 0 0 0 0

Campylobacteriosis 113.1 116.4 116.1 121 111.1 119.9 107.4 110 114.1 117.2 101.6 93.5 124.9 94.7 100.2 116.6 130.5

Cryptosporidiosis 16.7 6.2 8.3 15.8 15.5 13.3 9.3 21.3 6.7 8.1 13.8 16.6 10.2 17.1 22.4 19.1 12.2

Haemolytic

uraemic syndrome 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 <0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Hepatitis A 2 2.2 1.8 1.6 1.4 0.8 1.3 2.6 1.2 0.6 0.7 0.8 1.0 0.8 0.6 0.9 1.8

Hepatitis E 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.1 0.1 0.2 0.2 0.2 0.2 0.2

Listeriosis 0.3 0.3 0.3 0.3 0.3 0.2 0.3 0.4 0.3 0.3 0.4 0.3 0.3 0.3 0.4 0.3 0.3

Salmonellosis 40.1 35.2 39 41.3 39.7 44.9 38.6 43.8 54.1 54.9 49.5 55.3 69.7 71.2 74.5 66.6 57.6

Shigellosis 2.6 2.2 2.6 3.6 2.6 2.8 3.9 2.8 2.5 2.2 2.4 2.3 4.5 4.4 5.8 7.2 10.6

Shiga toxin-

producing

Escherichia coli 0.3 0.3 0.2 0.4 0.3 0.5 0.5 0.6 0.4 0.4 0.5 0.8 0.5 0.6 1.4 2 2.3

Typhoid fever 0.4 0.3 0.4 0.3 0.4 0.4 0.5 0.5 0.4 0.6 0.5 0.7 0.5 0.5 0.4 0.6 0.7 Note: The raw data is modified from Australia's notifiable diseases status, NNDSS annual report 1991-2018 (NNDSS, 2015; OzFoodNet, 2010, 2015) and http://www9.health.gov.au/cda/source/rpt_2.cfm

280

Appendix 2.1: MALDI-TOF protocol

Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)

(VITEK® MS) (BioMérieux, France)

1. The edge of a presumptive single Campylobacter colony was directly

collected from the selective plate for Campylobacter isolation using a

sterile toothpick. Each sample was duplicated.

2. Then, the selected colony was smeared on a well of a micro-titre 64

targe plate ground steel (FlexiMass™, BioMérieux) containing three

acquisition groups of 16 spots each.

3. E. coli ATCC 8739 strain was used as a calibrator and internal control

for each acquisition group by adding in the middle well of each group.

4. After that, one microlitre of matrix solution (saturated solution of a

cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5%

trifluoroacetic acid; CHCA) prove was immediately added to the

sample.

5. The mixed sample was crystallised by air-drying at 22 ± 2 °C until the

sample becomes CHCA crystals. Then, the plate was load onto the

VITEK MS mass spectrometer for target interrogation.

6. The plate was analysed by the VITEK® MS machine to obtain the

identification of C. jejuni and C. coli.

7. The outcomes were shown on the screen monitor as C. jejuni and C.

coli with the best identification match(es) and confidence value(s)

between 0 and 99%.


Campylobacter coli isolates on breeder farms based on MALDI-TOF, PCR,

flaA-HRM analysis and flaA amplicon sequencing

Appendix 2.2.1 A: Clustering of Campylobacter jejuni isolates from BD–

A

Only one isolate was initially identified as C. coli by MALDI-TOF, but it was

later confirmed as C. jejuni by PCR as indicated with yellow colour in the

table below. All 19 C. jejuni isolates were grouped into 6 clusters: cluster 8

281

(flaA allele 125, 419), cluster 14 (flaA allele 1, 34a), cluster 22 (flaA allele 1,

36b), cluster 23 (flaA allele 1, 467a), cluster 25 (flaA allele 33, 222), and

cluster 26 (flaA allele 1, 105).

The results of HRM analysis of C. jejuni from breeder farm A (BD–A).

Six different HRM profiles were identified and were assigned to cluster

8, 14, 22, 23, 25, and 26.

282

Identification and clustering of Campylobacter jejuni isolated from breeder farm A (BD–A)

Farm

Isolate

no. Shed Sample

Species isolated HRM flaA-HRM

cluster

flaA

Sequence

Ct

value MALDI-TOF PCR Peak 1 Peak 2

BD–A 163 1 Faecal sample C. jejuni C. jejuni 79.7 ± 0.04 – 22 1, 36b 26.55


BD–A 189 1 Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 14 1, 34a 21.01


BD–A 205 2 Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 – 25 33, 222 20.44


BD–A 224 3 Faecal sample C. coli C. jejuni 79.6 ± 0.01 – 14 1, 34a 24.20




BD–A 246 4 Faecal sample C. jejuni C. jejuni 78.7 ± 0.01 79.5 ± 0.01 26 1, 105 19.35







283

Identification and clustering of Campylobacter jejuni isolated from breeder farm A (BD–A) con’t

Farm

Isolate

no. Shed Sample


cluster

flaA

Sequence

Ct



D–A 290 5 Faecal sample C. jejuni C. jejuni 79.7 ± 0.02 – 22 1, 36b 25.72

284

Appendix 2.2.1 B: Clustering of Campylobacter coli isolates from BD–A

All C. coli isolates were grouped into 5 clusters: cluster 3 (flaA allele 11, 30b),

cluster 4 (flaA allele 1, 36c), cluster 5 (flaA allele 1, 36d), cluster 6 (flaA allele

21, 13), and cluster 17 (flaA allele 1, 467).

The results of HRM analysis of C. coli from breeder farm A (BD–A).

Five different HRM profiles were identified and were assigned to

clusters 3, 4, 5, 6, and 17.

285

Identification and clustering of Campylobacter coli isolated from breeder farm A (BD–A)

Farm

Isolate

no. Shed Sample

Species isolated HRM HRM

group

Cluster

no.

flaA

Sequence

Ct


BD–A 169 1 Faecal sample C. coli C. coli 79.5 ± 0.01 – 1 5 1, 36d 18.85

BD–A 175 1 Faecal sample C. coli C. coli 79.4 ± .0.20 – 5 6 21, 13 22.85

BD–A 181 1 Faecal sample C. coli C. coli 80.0 ± 0.05 – 4 3 11, 30b 22.37

BD–A 217 2 Faecal sample C. coli C. coli 79.3 ± 0.01 – 3 4 1, 36c 18.30


BD–A 233 3 Faecal sample C. coli C. coli 80.1 ± 0.01 – 4 3 11, 30b 22.90

BD–A 236 3 Faecal sample C. coli C. coli 79.6 ± 0.02 – 5 6 21, 13 23.53



BD–A 277 2 Faecal sample C. coli C. coli 79.5 ± 0.02 – 1 5 1, 36d 18.19


286

Appendix 2.2.2 A: Clustering of Campylobacter jejuni isolates from BD–

B

Ten C. jejuni isolates were classified into 5 clusters: cluster 8 (flaA allele 125,

419), cluster 10 (flaA allele 8b), cluster 11 (flaA allele 1a), cluster 13 (flaA

allele 1, 56) and cluster 16 (flaA allele 1, 34c).

HRM analysis of C. jejuni isolated from breeder farm B (BD–B). Five

different HRM profiles were identified and were assigned to clusters

8, 10, 11, 13, and 16.

287

Identification and clustering of Campylobacter jejuni isolated from breeder farm B (BD–B)

Farm

Isolate

no. Shed Sample

Species isolated HRM flaA- HRM

Cluster

flaA

Sequence

Ct


BD–B 1221 3 Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 8 125, 419 30.25

BD–B 1241 3 Faecal sample C. jejuni C. jejuni 79.1 ± 0.03 79.6 ± 0.03 10 8b 26.49



BD–B 1256 4 Faecal sample C. jejuni C. jejuni 79.7 ± 0.06 – 11 1a 27.38



BD–B 1286 6 Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 80.1 ± 0.02 13 1, 56 27.03


BD–B 1296 6 Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 16 1, 34c 24.67

288

Appendix 2.2.2 B: Clustering of Campylobacter coli isolates from BD–B

MALDI-TOF showed two isolates were initially identified as C. jejuni but it was confirmed as C. coli with PCR as indicated with yellow colour in the

table below. Eleven isolates were grouped into 10 clusters: cluster 3 (flaA allele 11, 30b), 6 (flaA allele 21, 13), 7 (flaA allele 1d), 9 (flaA allele 11d), 10

(flaA allele 11e), 11 (flaA allele 1, 34d), 12 (flaA allele 1, 22), 13 (flaA allele 12, 16b), 15 (flaA allele 8d), and 16 (flaA allele 9, 239c).

HRM analysis of C. coli isolated from breeder farm B (BD–B). Ten different HRM profiles were identified and were assigned to clusters 3, 6,

7, 9, 10, 11, 12, 13, 15 and 16.

289

Identification and clustering of Campylobacter coli isolated from breeder farm B (BD–B)

Farm

Isolate

no. Shed Sample


cluster

flaA

Sequence

Ct


BD–B 1226 3 Faecal sample C. coli C. coli 80.1 ± 0.03 – 3 11, 30b 24.52

BD–B 1232 3 Faecal sample C. jejuni C. coli 79.7 ± 0.03 – 6 21, 13 32.24

BD–B 1240 3 Faecal sample C. coli C. coli 79.4 ± 0.01 – 7 1d 22.29


BD–B 1266 4 Faecal sample C. coli C. coli 79.7 ± 0.02 – 11 1, 34d 22.59

BD–B 1311 5 Faecal sample C. jejuni C. coli 79.5 ± 0.09 – 12 1, 22 27.20

BD–B 1276 5 Faecal sample C. coli C. coli 79.4 ± 0.00 80.0 ± 0.00 13 12, 16b 21.36


BD–B 1289 6 Faecal sample C. coli C. coli 79.5 ± 0.03 – 10 11e 22.88


BD–B 1306 6 Faecal sample C. coli C. coli 79.2 ± 0.04 – 16 9, 239c 17.32

290

Appendix 2.2.3 A: Clustering of Campylobacter jejuni isolates from BD–C

Twelve C. jejuni isolates were grouped into 7 clusters: cluster 5 (flaA allele 20, 18b), cluster 8 (flaA allele 125, 419), cluster 12 (flaA allele 1b), cluster

15 (flaA allele 1, 34b), cluster 16 (flaA allele 1, 34c), cluster 19 (flaA allele 11c), and cluster 20 (flaA allele 3, 106).

HRM analysis of C. jejuni in breeder farm C (BD–C). Twelve isolates were distinguished into 7 different HRM curve patterns. These 7 HRM

profiles were assigned to clusters 5, 8, 12, 15, 16, 19, and 20.

291

Identification and clustering of Campylobacter jejuni isolated from breeder farm C (BD–C)

Farm

Isolate

no. Shed Sample


cluster

flaA

Sequence

Ct


BD–C 1126 1 Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 16 1, 34c 23.89

BD–C 1131 1 Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 16 1, 34c 24.17

BD–C 1136 1 Faecal sample C. jejuni C. jejuni 79.4 ± 0.02 – 19 11c 23.89

BD–C 1154 2 Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 15 1, 34b 25.37

BD–C 1157 2 Faecal sample C. jejuni C. jejuni 79.2 ± 0.01 – 8 125, 419 25.24




BD–C 1205 4 Faecal sample C. jejuni C. jejuni 79.6 ± 0.05 – 12 1b 26.09

BD–C 1206 4 Faecal sample C. jejuni C. jejuni 79.7 ± 0.02 – 12 1b 27.76



292

Appendix 2.2.3 B: Clustering of Campylobacter coli isolates from BD–C

Two of ten isolates were initially identified as C. jejuni from MALDI-TOF

but they were confirmed as C. coli by PCR as indicated with yellow colour in

the table below. The ten C. coli isolates were assigned to in 4 clusters: cluster

3 (flaA allele 11, 30b), cluster 6 (flaA allele 21, 13), cluster 8 (flaA allele 1e),

and cluster 14 (flaA allele 8c).

HRM analysis of C. coli from breeder farm C (BD–C). The results of

HRM analysis revealed that all C. coli isolates had 4 different HRM

profiles. These HRM profiles were assigned to clusters 3, 6, 8, and 14.

293

Identification and clustering of Campylobacter coli isolated from breeder farm C (BD–C)

Farm

Isolate

no. Shed Sample


cluster

flaA

Sequence

Ct


BD–C 1121 1 Faecal sample C. coli C. coli 79.4 ± 0.04 – 6 21, 13 27.57

BD–C 1145 1 Faecal sample C. jejuni C. coli 79.5 ± 0.04 – 6 21, 13 29.63


BD–C 1159 2 Faecal sample C. coli C. coli 79.9 ± 0.04 – 3 11, 30b 28.52

BD–C 1168 2 Faecal sample C. coli C. coli 79.5 ± 0.04 – 8 1e 23.43



BD–C 1183 3 Faecal sample C. coli C. coli 78.7 ± 0.00 79.2 ± 0.01 14 8c 25.08



294

Appendix 2.2.4 A: Clustering of Campylobacter jejuni isolates from BD–F

One of 21 isolates was identified as C. coli from MALDI-TOF, but it was confirmed as cluster by PCR as indicated with yellow colour in the table below.

These 21 isolates generated 13 clusters: cluster 4 (flaA allele 20, 18a), cluster 5 (flaA allele 20, 18b), cluster 6 (flaA allele 9, 239a), cluster 7 (flaA allele

9, 239b), cluster 10 (flaA allele 8b), cluster 24 (flaA allele 1, 467b), cluster 26 (flaA allele 1, 105), cluster 36 (flaA allele 1, 8a), cluster 37 (flaA allele 1c),

cluster 38 (flaA allele 10, 28a), cluster 39 (flaA allele 2, 54), cluster 40 (flaA allele 5,5a), and cluster 41 (flaA allele 5).

HRM analysis of C. jejuni from breeder farm F (BD–F). The results of HRM analysis revealed that all C. jejuni isolates had 13 different

HRM profiles. These HRM profiles were assigned to cluster 4, 5, 6, 7, 10, 24, 26, 36, 37, 38, 39, 40, and 41.

295

Identification and clustering of Campylobacter jejuni isolated from breeder farm F (BD–F)

Farm

Isolate

no. Shed Sample


cluster

flaA

Sequence

Ct


BD–F 1972 1 Faecal sample C. coli C. jejuni – 79.5 ± 0.03 10 8b 18.26

BD–F 1977 1 Faecal sample C. jejuni C. jejuni 79.4 ± 0.04 – 4 20, 18a 23.55

BD–F 1983 1 Faecal sample C. jejuni C. jejuni – 79.5 ± 0.00 10 8b 23.8

BD–F 1988 2 Faecal sample C. jejuni C. jejuni 78.7 ± 0.01 79.6 ± 0.03 36 1, 8a 26.34

BD–F 1993 2 Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 5 20, 18b 23.26

BD–F 2001 2 Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 – 37 1c 22.24






BD–F 2038 4 Faecal sample C. jejuni C. jejuni – 79.6 ± 0.04 36 1, 8a 23.03




BD–F 2072 5 Faecal sample C. jejuni C. jejuni 79.4 ± 0.00 – 39 2, 54 22.15


296

Identification and clustering of Campylobacter jejuni isolated from breeder farm F (BD–F) con’t

Farm

Isolate

no. Shed Sample


cluster

flaA

Sequence

Ct



BD–F 2099 6 Faecal sample C. jejuni C. jejuni 78.6 ± 0.05 79.9 ± 0.06 40 5, 5a 26.84

BD–F 2102 6 Faecal sample C. jejuni C. jejuni 79.8 ± 0.02 – 41 15 22.97

BD–F 2107 6 Faecal sample C. jejuni C. jejuni 78.8 ± 0.03 79.7 ± 0.00 26 1, 105 30.96

297

Appendix 2.2.4 B: Clustering of Campylobacter coli isolates from BD–F

One isolate was initially identified as C. jejuni from MALDI-TOF, but it was confirmed as with PCR as indicated with yellow colour in the table below.

The 17 C. coli isolates were grouped into 10 clusters: cluster 19 (flaA allele 1, 467e and flaA allele 10, 28), cluster 18 (flaA allele 1, 467d), cluster 21

(unassigned flaA allele), cluster 22 (flaA allele 1, 8b), cluster 23 (flaA allele 201, 18c), cluster 24 (flaA allele 4), cluster 25 (flaA allele 5, 5b), and cluster

26 (flaA allele 33).

HRM analysis of C. coli isolated from breeder farm F (BD–F). The results of HRM analysis revealed that all C. coli isolates had 10 different

HRM profiles. These HRM profiles were assigned to cluster 13, 18, 19, 20, 21, 22, 23, 24, and 25.

298

Identification and clustering of Campylobacter coli isolated from breeder farm F (BD–F)

Farm

Isolate

no. Shed Sample


cluster

flaA

Sequence

Ct


BD–F 1962 1 Faecal sample C. coli C. coli 79.2 ± 0.04 80.4 ± 0.04 13 12, 16b 19.86

BD–F 1967 1 Faecal sample C. coli C. coli 79.5 ± 0.02 – 19 10, 28b 18.72

BD–F 1980 1 Faecal sample C. coli C. coli 79.4 ± 0.03 – 21 New 18.5



BD–F 2004 2 Faecal sample C. coli C. coli 79.5 ± 0.01 – 19 1, 467e 19.92

BD–F 2017 3 Faecal sample C. coli C. coli 79.5 ± 0.03 – 18 1, 467d 19.81



BD–F 2040 4 Faecal sample C. coli C. coli 79.2 ± 0.03 – 23 20, 18c 19.93



BD–F 2062 5 Faecal sample C. coli C. coli 79.1 ± 0.03 – 24 4 19.8

BD–F 2067 5 Faecal sample C. jejuni C. coli 78.5 ± 0.03 79.9 ± 0.05 25 5, 5b 20.06

BD– 2087 6 Faecal sample C. coli C. coli 79.6 ± 0.03 – 19 10, 28b 19.54

BD–F 2093 6 Faecal sample C. jejuni C. coli 79.4 ± 0.00 – 26 33 22.36


299

Appendix 2.2.5 A: Clustering of Campylobacter jejuni isolates from BD–G

Twenty-three C. jejuni isolates were grouped into 10 clsuters: cluster 9 (flaA allele 8a), cluster 17 (flaA allele 11a), cluster 18 (flaA allele 11b), cluster

21 (flaA allele 1, 36a), cluster 30 (flaA allele 2, 612), cluster 31 (flaA allele 1, 32a), cluster 32 (flaA allele 1, 32b), cluster 33 (flaA allele 11, 30a), cluster

34 (flaA allele 8, 67), and cluster 35 (flaA allele 5).

HRM analysis of C. jejuni from breeder farm G (BD–G). The results of HRM analysis revealed that all C. jejuni isolates had 10 different

HRM profiles. These HRM profiles were assigned to clusters 9, 17, 18, 21, 30, 31, 32, 33, 34, and 35.

300

Identification and clustering of Campylobacter jejuni isolated from breeder farm G (BD–G)

Farm Isolate no. Shed Sample


cluster

flaA

Sequence

Ct


BD–G 1854 4 Faecal sample C. jejuni C. jejuni 79.1 ± 0.00 – 30 2, 612 20.54

BD–G 1858 4 Faecal sample C. jejuni C. jejuni – 79.4 ± 0.01 31 1, 32a 19.63



BD–G 1873 4 Faecal sample C. jejuni C. jejuni 78.6 ± 0.02 79.4 ± 0.02 31 1, 32a 19.19

BD–G 1876 5 Faecal sample C. jejuni C. jejuni 79.5 ± 0.04 – 21 1, 36a 19.55



BD–G 1891 5 Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 – 9 8a 20.79






BD–G 1921 6 Faecal sample C. jejuni C. jejuni 78.4 ± 0.01 79.4 ± 0.01 34 8, 67 19.04


BD–G 1928 7 Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 – 18 11b 24.73

301

Identification and clustering of Campylobacter jejuni isolated from breeder farm G (BD–G) con’t



cluster

flaA

Sequence

Ct



BD–G 1858 4 Faecal sample C. jejuni C. jejuni – 79.4 ± 0.01 31 1, 32a 19.63





302

Appendix 2.2.5 B: Clustering of Campylobacter coli isolates from BD–G

Two C. coli isolates were grouped into cluster 3.

Identification and clustering of Campylobacter coli isolates from breeder farm G (BD–G)



cluster

flaA

Sequence

Ct


BD–G 1910 6 Faecal sample C. coli C. coli 80.0 ± 0.01 – 3 11, 30b 24.41

BD–G 1922 7 Faecal sample C. coli C. coli 80.0 ± 0.04 – 3 11, 30b 24.52

HRM analysis of C. coli from breeder farm G (BD–G). The result from HRM analysis showed that one HRM profile was seen and assigned to

cluster 3.

303


Campylobacter coli isolates from all broiler farms in experiments 1 and 2

based on MALDI-TOF, PCR, flaA-HRM analysis and flaA sequencing

Appendix 2.3.1 A: Clustering of Campylobacter jejuni isolates from free-

range broiler farm 1 (FB1) in experiment 1 (Exp.1)

Seventy-three C. jejuni isolates were grouped into 2 clusters: cluster 1 (flaA

allele 4, 57) and cluster 2 (flaA allele 11, 14).

HRM analysis of C. jejuni isolated from free-range farm 1 (FB1),

experiment 1 (Exp.1). The results of HRM analysis revealed that all

C. jejuni isolates were classified into 2 HRM profiles. These HRM

profiles were assigned to cluster 1 and 2.

304

Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.1)

Shed

Isolate

no. Sample


Cluster

flaA

allele

Ct


FB1–A1–Exp.1 758C Day 22- outside shed C. jejuni C. jejuni 79.4 ± 0.02 – 1 4, 57 28.99

FB1–A1–Exp.1 764D Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.04 – 1 4, 57 29.86

FB1–A1–Exp.1 767S Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.02 – 1 4, 57 29.78

FB1–A1–Exp.1 772B Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 30.72

FB1–A1–Exp.1 778G Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 1 4, 57 29.88

FB1–A1–Exp.1 782C Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.06 – 1 4, 57 30.13

FB1–A1–Exp.1 788D Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.04 – 1 4, 57 28.71





FB1–T–Exp.1 682 Day 22- Shed boots C. jejuni C. jejuni 79.4 ± 0.00 – 1 4, 57 26.80

FB1–T–Exp.1 687 Day 22- Farm boots C. jejuni C. jejuni 79.5 ± 0.09 – 2 11, 14 29.56

FB1–T–Exp.1 692 Day 22- Left wall C. jejuni C. jejuni 79.5 ± 0.08 – 1 4, 57 22.99

FB1–T–Exp.1 697 Day 22- outside shed C. jejuni C. jejuni 79.6 ± 0.06 – 2 11, 14 30.38

FB1–T–Exp.1 698 Day 22- Back floor C. jejuni C. jejuni 79.5 ± 0.08 – 1 4, 57 27.63

FB1–T–Exp.1 703 Day 22- Front floor C. jejuni C. jejuni 79.5 ± 0.05 – 1 4, 57 29.86

FB1–T–Exp.1 1116 Day 22- Drinking water C. jejuni C. jejuni 79.5 ± 0.04 – 2 11, 14 29.52

FB1–T–Exp.1 504 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 – 1 4, 57 28.29

305

Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.1) con’t

Shed

Isolate

no. Sample


Cluster

flaA

allele

Ct





















306


Shed

Isolate

no. Sample


Cluster

flaA

allele

Ct

















FB1–A2–Exp.1 112 Day 15- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 2 11, 14 21.54




307


Shed

Isolate

no. Sample


Cluster

flaA

allele

Ct


















308

Appendix 2.3.1 B: Clustering of Campylobacter jejuni isolates of free-


Seventy-two C. jejuni isolates were grouped into two clusters: cluster 6 (flaA

allele 9,239a) and cluster 27 (flaA allele 12, 16a).

HRM analysis of C. jejuni isolated from free-range farm 1 (FB1),

experiment 2 (Exp.2). The results of HRM analysis revealed that all C.

jejuni isolates were classified into 2 HRM profiles. These HRM profiles

were assigned to cluster 1 and 2.

309


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB1–A1–Exp.2 2140 Day 22- Outside shed C. jejuni C. jejuni 79.3 ± 0.02 80.4 ± 0.02 27 12, 16a 24.75

FB1–A1–Exp.2 2145 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 80.4 ± 0.01 27 12, 16a 23.01



FB1–A1–Exp.2 2166 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 – 6 9, 239a 27.75






FB1–T–Exp.2 2245 Day 22- Front floor C. jejuni C. jejuni 79.2 ± 0.04 80.5 ± 0.04 27 12, 16a 23.52

FB1–T–Exp.2 2250 Day 22- Back floor C. jejuni C. jejuni 79.2 ± 0.01 80.4 ± 0.02 27 12, 16a 22.98

FB1–T–Exp.2 2255 Day 22- Anteroom C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.03 27 12, 16a 22.73

FB1–T–Exp.2 2259 Day 22- Outside shed C. jejuni C. jejuni 79.2 ± 0.01 80.4 ± 0.02 27 12, 16a 23.14

FB1–T–Exp.2 2262 Day 22- Left wall C. jejuni C. jejuni 79.2 ± 0.01 80.4 ± 0.01 27 12, 16a 23.63

FB1–T–Exp.2 2267 Day 22- Shed boots C. jejuni C. jejuni 79.2 ± 0.01 80.4 ± 0.03 27 12, 16a 21.09

FB1–T–Exp.2 2272 Day 22- Farm boots C. jejuni C. jejuni 79.2 ± 0.02 80.5 ± 0.01 27 12, 16a 24.58

FB1–T–Exp.2 2277 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.03 27 12, 16a 21.77

310


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct




















311


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct






FB1–T–Exp.2 2392 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.05 80.4 ± 0.04 27 12, 16a 23














312


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct




















313

Appendix 2.3.1 C: Clustering of Campylobacter coli isolates of free-


Nine C. coli isolates were grouped into 3 clusters: cluster 3 (flaA allele

11,30b), cluster 5 (flaA allele 11,16b), and cluster 13 (flaA allele 1,36d).

HRM analysis of C. coli from breeder farm 1 (FB1), experiment 2

(exp.2). The results of HRM analysis revealed that all C. coli isolates

were classified into 3 HRM profiles. These HRM profiles were assigned

to clusters 3, 5, and 13.

314

Identification and clustering of Campylobacter coli isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.2)

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB1–A1–Exp.2 1787 Day 15- Faecal sample C. coli C. coli 80.0 ± 0.03 – 3 11, 30b 23.41







FB1–A1–Exp.2 2165 Day 22- Faecal sample C. coli C. coli 79.3 ± 0.04 80.5 ± 0.03 13 12, 16b 19.98

FB1–A2–Exp.2 2112 Day 15- Outside shed C. coli C. coli 79.6 ± 0.04 – 5 1, 36d 18.58

315

Appendix 2.3.2 A: Clustering of Campylobacter jejuni isolates from free-


Forty-six C. jejuni isolates were grouped into 3 clusters: cluster 2 (flaA allele

11, 14), cluster 3 (flaA allele 20, 208), and cluster 5 (flaA allele 20, 18b).

HRM analysis of C. jejuni from breeder farm 2 (FB2), experiment 1

(exp.1). The results of HRM analysis revealed that all C. jejuni isolates

were classified into 3 HRM profiles. These HRM profiles were assigned

to cluster 2, 3, and 5.

316


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct












FB2–T–Exp.1 35 Day 8- Rodents faeces C. jejuni C. jejuni 79.2 ± 0.04 – 5 20, 18b 24.50

FB2–T–Exp.1 917 Day 22- Outside the shed C. jejuni C. jejuni 79.7 ± 0.08 – 2 11, 14 29.55

FB2–T–Exp.1 922 Day 22- Left wall C. jejuni C. jejuni 80.0 ± 0.00 – 2 11, 14 32.41

FB2–T–Exp.1 927 Day 22- Right wall C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 29.63

FB2–T–Exp.1 934 Day 22 -Rodents faeces C. jejuni C. jejuni 79.3 ± 0.03 – 3 20, 208 25.20


317


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


















318


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct
















319

Appendix 2.3.2 B: Clustering of Campylobacter coli isolates from free-


Twenty-four C. coli isolates were grouped into 3 clusters: cluster 1 (flaA allele

1, 769), cluster 2 (flaA allele 97, 256), and cluster 3 (flaA allele 11, 30b).

HRM analysis of C. coli from breeder farm 2 (FB2), experiment 1 (exp.1).

The results of HRM analysis revealed that all C. coli isolates were

classified into 3 HRM profiles. These HRM profiles were assigned to

cluster 1, 2, and 3.

320

Identification and clustering of Campylobacter coli isolated from free-range broiler farm 2 (FB2), experiment 1 (Exp.1)

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB2–A1–Exp.1 42 Day 8- Outside shed C. coli C. coli 79.5 ± 0.01 – 1 1, 769 21.55

FB2–T–Exp.1 2 Day 1- Rodents faeces C. coli C. coli 79.7 ± 0.01 – 2 97, 256 21.50

FB2–T–Exp.1 57 Day 8- Shed boots C. coli C. coli 79.4 ± 0.02 – 2 97, 256 22.79




FB2–T–Exp.1 913 Day 22- Back floor C. coli C. coli 80.0 ± 0.07 – 3 11, 30b 27.77

FB2–T–Exp.1 632 Day 22- Front floor C. coli C. coli 80.0 ± 0.02 – 3 11, 30b 26.95

FB2–T–Exp.1 937 Day 22- Faecal sample C. coli C. coli 80.0 ± 0.04 – 3 11, 30b 26.72




FB2–A2–Exp.1 15 Day 1- Outside Shed C. coli C. coli 79.7 ± 0.02 – 2 97, 256 21.93


FB2–A2–Exp.1 862 Day 22- Faecal sample C. coli C. coli 79.7 ± 0.01 – 2 97, 256 22.65



321

Identification and clustering of Campylobacter coli isolated from free-range broiler farm 2 (FB2), experiment 1 (Exp.1) con’t

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB2–A2–Exp.1 877 Day 22- Faecal sample C. coli C. coli 79.6 ± 0.03 – 2 97, 256 22.86







322

Appendix 2.3.2 C: Clustering of Campylobacter jejuni isolates from free-range broiler farm 2 (FB2) in experiment 2 (Exp.2)

Sixty-seven C. jejuni isolates were grouped into 5 clusters: cluster 6 (flaA allele 9,239a), cluster 26 (flaA allele 1,105), cluster 27 (flaA allele 12, 16a),

cluster 28 (flaA allele 257, 1033), and cluster 29 (flaA allele 27, 2).

HRM analysis of C. jejuni from breeder farm 2 (FB2), experiment 2 (exp.2). The HRM analysis revealed that all C. jejuni isolates were

classified into 5 HRM profiles and they were assigned to clusters 6, 26, 27, 28 and 29.

323

Identification and clustering of Campylobacter jejuni isolated from breeder farm 2 (FB2), experiment 2 (exp.2)

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB2–A1–Exp.2 2452 Day 22- Outside the shed C. jejuni C. jejuni 79.3 ± 0.02 80.6 ± 0.04 27 12, 16a 22.81











FB2–T–Exp.2 1769 Day 0 - Outside the shed C. jejuni C. jejuni 79.1 ± 0.05 80.0 ± 0.04 28 257, 1033 13.95

FB2–T–Exp.2 1773 Day 1- Rodent faeces C. jejuni C. jejuni 78.6 ± 0.01 79.4 ± 0.01 26 1, 105 19.67

FB2–T–Exp.2 1783 Day 8- Rodent faeces C. jejuni C. jejuni 79.3 ± 0.03 – 6 9, 239a 21.64

FB2–T–Exp.2 2114 Day 15- Anteroom C. jejuni C. jejuni 78.9 ± 0.04 80.5 ± 0.01 29 27, 2 18.45

FB2–T–Exp.2 2124 Day 15- Rodent faeces C. jejuni C. jejuni 78.9 ± 0.03 80.5 ± 0.02 29 27, 2 18.53

FB2–T–Exp.2 2554 Day 22-Front floor C. jejuni C. jejuni 79.2 ± 0.04 80.4 ± 0.03 27 12, 16a 19.07

324

Identification and clustering of Campylobacter jejuni isolated from breeder farm 2 (FB2), experiment 2 (exp.2) con’t

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB2–T–Exp.2 2559 Day 22- Back floor C. jejuni C. jejuni 79.3 ± 0.04 80.5 ± 0.03 27 12, 16a 22.64

FB2–T–Exp.2 2569 Day 22- Outside the shed C. jejuni C. jejuni 79.3 ± 0.02 80.4 ± 0.01 27 12, 16a 19.3

FB2–T–Exp.2 2574 Day 22- Shed boots C. jejuni C. jejuni 79.3 ± 0.02 80.5 ± 0.03 27 12, 16a 24.27

FB2–T–Exp.2 2579 Day 22- Farm boots C. jejuni C. jejuni 79.3 ± 0.03 80.5 ± 0.03 27 12, 16a 22.62














325


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct



















326


Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct







FB2–A2–Exp.2 2503 Day 22- Outside the shed C. jejuni C. jejuni 79.4 ± 0.07 80.6 ± 0.08 27 12, 16a 18.9











327

Appendix 2.3.3 A: Clustering of Campylobacter jejuni isolates from free-range broiler farm 3 (FB3) in experiment 1 (Exp.1)

Only C. jejuni was found and it was assigned to cluster 6 (flaA allele 9,239a).

Identification and clustering of Campylobacter jejuni isolated from breeder farm 3 (FB3), experiment 1 (exp.1)

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB3–T–Exp.1 30 Day 3- Rodents faeces C. jejuni C. jejuni 79.3 ± 0.01 – 6 9, 239a 17.45

HRM analysis of C. jejuni from breeder farm 3 (FB3), experiment 1 (exp.1). The result of HRM analysis revealed that this C. jejuni isolate

was distinguished and was assigned to cluster 6.

328

Appendix 2.3.3 B: Clustering of Campylobacter coli isolates from free-


Fifty-three C. coli isolates were grouped into 2 clusters: cluster 3 (flaA allele

11,30b) and cluster 5 (flaA allele 1,36d).

HRM analysis of C. coli from breeder farm 3 (FB3), experiment 1 (exp.1).

The HRM analysis revealed that all C. coli isolates were classified into 2

HRM profiles and they were assigned to clusters 3 and 5.

329

Identification and clustering of Campylobacter coli isolated from breeder farm 3 (FB3), experiment 1 (exp.1)

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB3–A1–Exp.1 90 Day 10- Outside the shed C. coli C. coli 79.6 ± 0.01 – 5 1, 36d 20.92








FB3–T–Exp.1 67 Day 10- Faecal sample C. coli C. coli 79.6 ± 0.03 – 5 1, 36d 16.77


FB3–T–Exp.1 95 Day 10- Shed boots C. coli C. coli 79.5 ± 0.05 – 5 1, 36d 16.87

FB3–T–Exp.1 100 Day 10- Farm boots C. coli C. coli 79.5 ± 0.03 – 5 1, 36d 17.55


FB3–T–Exp.1 328 Day 17- Back floor C. coli C. coli 79.4 ± 0.01 – 5 1, 36d 16.24

FB3–T–Exp.1 329 Day 17- Right wall C. coli C. coli 79.4 ± 0.01 – 5 1, 36d 16.50

FB3–T–Exp.1 335 Day 17- Shed boots C. coli C. coli 79.5 ± 0.02 – 5 1, 36d 16.71


FB3–T–Exp.1 499 Day 17- Water pan C. coli C. coli 79.5 ± 0.06 – 5 1, 36d 16.68



330

Identification and clustering of Campylobacter coli isolated from breeder farm 3 (FB3), experiment 1 (exp.1) con’t

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct






















331

Identification and clustering of Campylobacter coli isolated from breeder farm 3 (FB3), experiment 1 (exp.1) con’t

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct















332

Appendix 2.3.3 C: Clustering of Campylobacter jejuni isolates from free-


Sixty-two C. jejuni isolates were grouped into 6 clusters: cluster 1 (flaA allele

4, 57), cluster 6 (flaA allele 9,239a), cluster 26 (flaA allele 1,105), and cluster

27 (flaA allele 12,16a).

HRM analysis of C. jejuni from breeder farm 3 (FB3), experiment 2

(exp.2). The HRM analysis revealed that all C. jejuni isolates were

classified into 4 HRM profiles and they were assigned to clusters 1, 6, 26

and 27.

333

Identification and clustering of Campylobacter jejuni isolated breeder farm 3 (FB3), experiment 2 (exp.2)

Shed

Isolat

e no. Sample


cluster

flaA

Sequence

Ct


FB3–A1–Exp.2 2756 Day 24- Outside the shed C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 16.68











FB3–T–Exp.2 2862 Day 24- Front floor C. jejuni C. jejuni 79.1 ± 0.04 – 1 4, 57 21.37

FB3–T–Exp.2 2867 Day 24- Back floor C. jejuni C. jejuni 79.1 ± 0.05 – 1 4, 57 20.1

FB3–T–Exp.2 2873 Day 24- Outside the shed C. jejuni C. jejuni 79.1 ± 0.06 – 1 4, 57 19.01

FB3–T–Exp.2 2877 Day 24- Shed boots C. jejuni C. jejuni 79.2 ± 0.05 – 1 4, 57 23.55

FB3–T–Exp.2 2882 Day 24- Farm boots C. jejuni C. jejuni 79.1 ± 0.03 – 6 9, 239a 15.93


334

Identification and clustering of Campylobacter jejuni isolated breeder farm 3 (FB3), experiment 2 (exp.2) con’t

Shed

Isolat

e no. Sample


cluster

flaA

Sequence

Ct



















335


Shed

Isolat

e no. Sample


cluster

flaA

Sequence

Ct












FB3–T–Exp.2 3029 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.02 – 1 4, 57 17




FB3–T–Exp.2 3050 Day 24- Faecal sample C. jejuni C. jejuni 78.5 ± 0.00 79.3 ± 0.00 26 1, 105 14.82

FB3–T–Exp.2 3055 Day 24- Faecal sample C. jejuni C. jejuni 78.5 ± 0.01 79.3 ± 0.02 26 1, 105 14.77


336


Shed

Isolat

e no. Sample


cluster

flaA

Sequence

Ct


FB3–A2–Exp.2 2809 Day 24- Outside the shed C. jejuni C. jejuni 79.0 ± 0.00 – 6 9, 239a 17.03



FB3–A2–Exp.2 2822 Day 24- Faecal sample C. jejuni C. jejuni 78.4 ± 0.04 79.3 ± 0.05 26 1, 105 15.65








337

Appendix 2.3.3 D: Clustering of Campylobacter coli isolates from free-range broiler farm 3 (FB3) in experiment 2 (Exp.2)

Eight C. coli isolates were grouped into 2 clusters: cluster 2 (flaA allele 97, 256) and cluster 5 (flaA allele 1, 36d).

Identification and clustering of Campylobacter coli isolated breeder farm 3 (FB3), experiment 2 (exp.2)

Shed

Isolate

no. Sample


cluster

flaA

Sequence

Ct


FB3–A1–Exp.2 1778 Day 0- Outside the shed C. coli C. coli 79.6 ± 0.02 – 5 1, 36d 20.02

FB3–A1–Exp.2 2806 Day 24- Faecal sample C. coli C. coli 79.6 ± 0.09 – 5 1, 36d 19.65

FB3–T–Exp.2 2135 Day 17- Rodent faeces C. coli C. coli 79.6 ± 0.01 – 5 1, 36d 18.05

FB3–T–Exp.2 2887 Day 24- Rodent faeces C. coli C. coli 79.7 ± 0.01 – 2 97, 256 20.18





HRM analysis of C. coli from breeder farm 3 (FB3), experiment 2 (exp.2). The HRM analysis revealed that all C. coli isolates were classified into

2 HRM profiles and they were assigned to clusters 2 and 5.

338

Appendix 3.1: Analysis of fliD primers and gradient temperature PCR

Gradient PCR showed no amplification of the fliD amplicon from C. jejuni

NCTC 11168 at the annealing temperatures ranging from 50°C to 60°C as

below (A). Non-specific PCR products were found at the annealing

temperatures ranging from 50°C to 52.3°C generated approximately 1750 and

1900 bp in size using C. coli ATCC 33559 as the DNA template as below

(B).

Therefore, the fliD oligonucleotide primers were redesigned and named as the

fliD set 1 and set 2. The fliD set 1 conserved within the fliD gene of C. jejuni

strain YH002 and C. coli strain YH502 by blasting in the NCBI database and

resulted in 1046 bp as below (A). The fliD oligonucleotide primer set 2

conserved within the fliD gene of C. jejuni strain YH002 and C. coli strain

YH502 by BLAST search in the NCBI database. Using the fliD

oligonucleotide primer set 2, the estimated size of fliD amplicon in C. jejuni

strain YH002 and C. coli strain YH502 were 1009 and 994 bp, respectively

as below (B).

Agarose gel electrophoresis of the fliD amplicon generated from gradient

temperature PCR reactions using genomic DNA from C. jejuni NCTC

11168 (A) and C. coli ATCC 33559 (B)

The annealing temperatures used in PCR reactions ranged from 50°C to

60°C. Lane 1: 1 Kb+ DNA molecular weight marker; Lane 2: 60°C; Lane

3: 59.3°C; Lane 4: 58.1°C; Lane 5: 56.3°C; Lane 6: 54.0°C; Lane 7:

52.3°C; Lane 8: 50.9°C; and Lane 9: 50.0°C.

A) PCR reaction products from C. jejuni NCTC11168 using the

oligonucleotide primer pair of the fliD gene.

B) PCR reaction products from C. coli ATCC 33559 using the

oligonucleotide primer pair of fliD gene.

339

Gradient PCR showed unsuccessful fliD amplification from C. jejuni (NCTC

11168) and C. coli ATCC 33559 using new fliD primer sets at the annealing

temperatures ranging from 40°C to 70°C as below.

Schematic representation of fliD-F and fliD-R oligonucleotide primer

set 1 and 2 locations on the fliD gene of C. jejuni strain YH002

(Accession number: CP020776.1) and C. coli strain YH502 fliD gene

(Accession number: CP018900.1).

A) The alignment of the fliD-F and fliD-R oligonucleotide primer set 1

in from both C. jejuni and C. coli reference strains.

B) The alignment of the fliD-F and fliD-R oligonucleotide primer set 2

in the C. jejuni and C. coli reference strains.

340

Agarose gel electrophoresis of the fliD amplicon generated from

gradient temperature PCR reactions ranging 60-70°C using the new

fliD primers and genomic DNA from C. jejuni and C. coli reference

strains

A) Use of the fliD primer set 1

B) Use of the fliD primer set 2

PCR reaction products from C. jejuni (Lanes 2-9) and C. coli (Lanes

11-18). Lane 1: 1 Kb+ DNA molecular weight marker; Lanes 2 and

11: 70°C; Lanes 3 and 12: 69.3°C; Lanes 4 and 13: 68.1°C; Lanes 5

and 14: 66.3°C; Lanes 6 and 15: 64.0°C; Lanes 7 and 16: 62.3°C;

Lanes 8 and 17: 61°C; Lanes 9 and 18: 60°C, Lanes 10 and 19; RNase

water (Negative control).



fliD primers and genomic DNA from C. jejuni and C. coli reference

strains

C) Use of the fliD primer set 1

D) Use of the fliD primer set 2

PCR reaction products from C. jejuni (Lanes 2-9) and C. coli (Lanes

11-18). Lane 1: 1 Kb+ DNA molecular weight marker; Lane 2: 60°C;

Lane 3: 59.3°C; Lane 4: 58.1°C; Lane 5: 56.3°C; Lane 6: 54°C; Lane

7: 52.3°C; Lane 8: at 50.9°C; Lane 9: 50°C, Lane 10: Blank, Lane 11:

60°C; Lane 12: 59.3°C; Lane 13: 58.1°C; Lane 14: 56.3°C; Lane 15:

54°C; Lane 16: 52.3°C; Lane 17: 50.9°C, and Lane 18: 50°C.

341



fliD primers1 and genomic DNA from C. jejuni and C. coli reference

strains

E) Use of of the fliD primer set 1 (E; Lane2-9, 11-18)

F) Use of of the fliD primer set 1 and set 2 (F; Lane 2-15 and E;

Lane19-20)

PCR reaction products from C. jejuni (E; Lane 2-8 and 18) and C. coli

(E; Lane 9,11-17,19-20 and F;10-15). Lane 1: 1 Kb+ DNA molecular

weight marker; Lane 2: 50°C; Lane 3: 49.2°C; Lane 4: 48.1°C; Lane 5:

46.3°C; Lane 6: 43.9°C; Lane 7: 42.3°C; Lane 8: at 40.9°C; Lane 9: 50

°C (E) and 40°C (F), Lane 10: Blank (E) and 50 °C (F), Lane 11:49.2°C

(E and F); Lane 12: 48.1°C (E and F); Lane 13: 46.3°C (E and F); Lane

14: 43.9°C (E and F); Lane 15: 42.3°C (E and F); Lane 16: 40.9°C (E);

Lane 17: at 40°C (E), Lane 18; 40°C (E), Lane 19, 40.9°C (E), and Lane

20; 40°C (E).

342

Appendix 3.2: PCR analysis of Campylobacter antigenic gene detection

The summary of the detection of katA, cadF, peb1A, and C. coli-cjaA genes in all C. jejuni and C. coli representing genotypes (Tables), followed by

examples of agarose gel electrophoresis of katA, cadF, peb1A, and C. coli-cjaA gene (two figures), as below.

PCR analysis of all Campylobacter jejuni and Campylobacter coli clusters isolated from breeder and broiler farms

ID Cluster

number

Species

isolated

Chicken

type

flaA allele flaA type PCR analysis of antigenic genes

Peptide Nucleotide cadF katA peb cjaA-C.

coli

cjaA-C.

jejuni

omp18 flp

1 1 C. jejuni Broiler 4 57 4, 57 Pos Pos Pos Pos Pos Pos Pos



4 4 C. jejuni Breeder 20 18 20, 18a Pos Pos Pos Pos Pos Pos Pos

5 5 C. jejuni Broiler and

Breeder

20 18 20, 18b Pos Pos Pos Pos Pos Pos Pos


Breeder

9 239 9, 239a Pos Pos Pos Pos Pos Pos Pos

7 7 C. jejuni Breeder 9 239 9, 239b Pos Pos Pos Pos Pos Pos Pos

8 8 C. jejuni Breeder 125 419 125, 419 Pos Pos Pos Pos Pos Pos Pos

9 9 C. jejuni Breeder 8 8a Pos Pos Pos Pos Pos Pos Pos

10 10 C. jejuni Breeder 8 8b Pos Pos Pos Pos Pos Pos Pos Note: Pos, Positive and Neg, Negative

343

PCR analysis of all Campylobacter jejuni and Campylobacter coli clusters isolated from breeder and broiler farms con’t

ID Cluster

number

Species

isolated

Chicken

type



coli

cjaA-C.

jejuni

omp18 flp


12 12 C. jejuni Breeder 1 1b Pos Pos Pos Pos Pos Pos Pos




16 16 C. jejuni Breeder 1 34 1, 34c Pos Pos Pos Pos Pos Pos Pos


18 18 C. jejuni Breeder 11 11b Pos Pos Pos Pos Pos Pos Pos

19 19 C. jejuni Breeder 11 11c Pos Pos Pos Pos Pos Pos Pos








Breeder

1 105 1, 105 Pos Pos Pos Pos Pos Pos Pos

Note: Pos, Positive and Neg, Negative

344


ID Cluster

number

Species

isolated

Chicken

type



coli

cjaA-C.

jejuni

omp18 flp

27 27 C. jejuni Broiler 12 16 12, 16a Pos Pos Pos Pos Pos Pos Pos








35 35 C. jejuni Breeder 5 5 Pos Pos Pos Pos Pos Pos Pos


37 37 C. jejuni Breeder 1 1c Pos Pos Pos Pos Pos Pos Pos




41 41 C. jejuni Breeder 15 15 Pos Pos Pos Pos Pos Pos Pos

42 1 C. coli Broiler 1 769 1, 769 Pos Pos Pos Pos Neg Neg Pos

43 2 C. coli Broiler 97 256 97, 256 Pos Pos Pos Pos Neg Neg Pos Note: Pos, Positive and Neg, Negative

345


ID Cluster

number

Species

isolated

Chicken

type



coli

cjaA-C.

jejuni

omp18 flp

44 3 C. coli Broiler and

Breeder

11 30 11, 30b Pos Pos Pos Pos Neg Neg Pos

45 4 C. coli Breeder 1 36 1, 36c Pos Pos Pos Pos Neg Neg Pos


Breeder

1 36 1, 36d Pos Pos Pos Pos Pos Neg Pos

47 6 C. coli Breeder 21 13 21, 13 Pos Pos Pos Pos Pos Pos Pos

48 7 C. coli Breeder 1 1d Pos Pos Pos Pos Neg Neg Pos

49 8 C. coli Breeder 1 1e Pos Pos Pos Pos Neg Neg Pos


51 10 C. coli Breeder 11 11e Pos Pos Pos Pos Neg Neg Pos

52 11 C. coli Breeder 1 34 1, 34d Pos Pos Pos Pos Neg Neg Pos

53 12 C. coli Breeder 1 22 1, 22 Pos Pos Pos Pos Pos Pos Pos


Breeder

12 16 12, 16b Pos Pos Pos Pos Pos Pos Pos

55 14 C. coli Breeder 8 8c Pos Pos Pos Pos Neg Neg Pos


57 16 C. coli Breeder 9 239 9, 239c Pos Pos Pos Pos Neg Neg Neg

58 17 C. coli Breeder 1 467 1, 467c Pos Pos Pos Pos Neg Neg Pos Note: Pos, Positive and Neg, Negative

346


ID Cluster

number

Species

isolated

Chicken

type


Peptide Nucleotide cadF katA peb cjaA-C. coli cjaA-C. jejuni omp18 flp

59 18 C. coli Breeder 1 467 1, 467d Pos Pos Pos Pos Pos Neg Pos

60 19 C. coli Breeder 1 467 1, 467e Pos Pos Pos Pos Neg Neg Neg

61 19 C. coli Breeder 10 28 10, 28b Pos Pos Pos Pos Neg Neg Pos

62 20 C. coli Breeder New New Pos Pos Pos Pos Neg Neg Pos

63 21 C. coli Breeder 1 8 1, 8b Pos Pos Pos Pos Neg Neg Pos

64 22 C. coli Breeder 20 18 20, 18c Pos Pos Pos Pos Pos Pos Pos

65 23 C. coli Breeder 4 4 Pos Pos Pos Pos Neg Neg Pos

66 24 C. coli Breeder 5 5 5, 5b Pos Pos Pos Pos Pos Pos Pos

67 25 C. coli Breeder 33 33 Pos Pos Pos Pos Pos Pos Pos Note: Pos, Positive and Neg, Negative

347

Appendix 3.3: Nucleotide sequence analysis

Thirteen C. jejuni and eight C. coli clusters were selected for sequencing analysis in this study. The selected 13 C. jejuni clusters were cluster 1 (Isolate

no. 683), cluster 2 (Isolate no. 687), cluster 3 (Isolate no. 813), cluster 5 (Isolate no. 62), cluster 6 (Isolate no. 30), cluster 8 (Isolate no. 1162), cluster 12

(Isolate no. 1206), cluster 26 (Isolate no. 3050), cluster 27 (Isolate no. 2170), cluster 28 (Isolate no. 1768 or 1769), cluster 29 (Isolate no. 2114), cluster

36 (Isolate no. 2038), and cluster 39 (Isolate no.2072). The selected 8 C. coli clusters were cluster 1 (Isolate no. 56), cluster 2 (Isolate no. 2887), cluster

3 (Isolate no. 2119), cluster 5 (Isolate no. 3064), cluster 6 (Isolate no. 175), cluster 13 (Isolate no. 2165), cluster 21 (Isolate no. 1980), and cluster 23

(Isolate no. 2040). The green and yellow colours indicate the forward and reverse primers used, respectively. The red font indicates the mismatches of

the oligonucleotide (Appendices 3.3.1-3.3.4).

Appendix 3.3.1: Nucleotide sequence of katA amplicons

The nucleotide sequences of the katA amplicon obtained from the NCBI database (C. jejuni NCTC 11168 and C. coli strain RM4661) used as references

for aligning with the selected C. jejuni and C. coli clusters are shown below.

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60

KatA C jejuniNCTC11168 ATGAAAAAAT TGACTAACGA TTTTGGAAAC ATTATAGCTG ATAACCAAAA TTCATTAAGC

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

348

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 ATGAAAAAAT TAACTAACGA CTTCGGAAAC ATTATAGCCG ATAATCAAAA CTCTTTAAGC

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

70 80 90 100 110 120

KatA C jejuniNCTC11168 GCAGGCGCAA AAGGACCTTT ACTTATGCAA GATTATCTTT TGCTTGAAAA ACTTGCTCAT

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

349

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 GCAGGTGCAA AAGGCCCTTT ACTTATGCAA GATTATCTTT TACTTGAAAA ACTTGCTCAT

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

130 140 150 160 170 180

KatA C jejuniNCTC11168 CAAAATAGAG AAAGAATTCC AGAAAGAACC GTTCATGCTA AGGGAAGTGG AGCTTATGGC

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

350

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 CAAAATAGAG AAAGAATTCC AGAAAGAACA GTGCATGCCA AGGGAAGTGG GGCTTATGGA

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

190 200 210 220 230 240

KatA C jejuniNCTC11168 GAAATAAAAA TTACAGCAGA CTTAAGTGCT TATACCAAAG CTAAAATTTT TCAAAAAGGC

351

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 GAAATAAAAA TCACCGCTGA TTTATCTGCT TATACTAAGG CAAAAATATT TCAAAAAGGA

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

250 260 270 280 290 300

352

KatA C jejuniNCTC11168 GAAGTTACTC CATTATTTTT ACGCTTTTCA ACAGTAGCAG GTGAAGCAGG TGCAGCAGAT

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 GAAATAACTC CTCTTTTCCT ACGCTTTTCT ACTGTTGCAG GTGAAGCAGG TGCAGCAGAT

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

353

310 320 330 340 350 360

KatA C jejuniNCTC11168 GCTGAACGCG ATGTGAGAGG TTTTGCTATT AAATTTTACA CTAAAGAAGG AAACTGGGAC

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 GCTGAGCGTG ATGTACGTGG ATTTGCCATT AAATTTTACA CCAAAGAAGG AAACTGGGAT

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

354

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

370 380 390 400 410 420

KatA C jejuniNCTC11168 TTGGTAGGAA ATAACACTCC GACATTCTTC ATCCGCGATG CTTATAAATT CCCTGATTTC

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 TTAGTAGGAA ATAATACTCC AACTTTTTTT ATTCGTGATG CGTATAAATT TCCTGATTTC

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

355

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480

KatA C jejuniNCTC11168 ATCCATACTC AAAAAAGAGA TCCAAGAACT CATCTAAGAA GTAATAATGC TGCTTGGGAT

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 ATCCATACTC AAAAAAGAGA TCCAAGAACT CACCTAAGAA GTAATAATGC TGCTTGGGAT

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

356

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

490 500 510 520 530 540

KatA C jejuniNCTC11168 TTTTGGAGTT TATGTCCTGA AAGTTTACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA

KatA C jejuni 1206 ---------- ---------- -----TACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA


KatA C jejuni 30 ---------- ---------A AAGTTTACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA

KatA C jejuni 62 ---------- ---------- ----TTACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA

KatA C jejuni 1162 ---------- ---------- --------AT CAAGTAACCA TTCTTATGAG CGATAGAGGA






KatA C jejuni 1768 ---------- ---------- --------AT CAAGTAACCA TTCTTATGAG TGATAGAGGA

KatA C jejuni 683 ---------- ---------- -----TACAT CAAGTAACCA TTCTTATGAG TGATAGAGGA

KatA C jejuni 687 ---------- ---------- --------AT CAAGTAACCA TTCTTATGAG TGATAGAGGA

KatA C coliRM4661 TTTTGGAGTT TATGTCCTGA AAGTTTACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA

KatA C coli 2040 ---------- ---------- --------AT CAAGTAACCA TTCTTATGAG CGATAGAGGA

KatA C coli 3064 ---------- ---------- -----TACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA

KatA C coli 56 ---------- --TGTCCTGA AAGTTTACAT CAAGTAACTA TTCTTATGAG CGATAGAGGA

KatA C coli 2887 ---------- --TGTCCTGA AAGTTTACAT CAAGTAACTA TTCTTATGAG CGATAGAGGA

KatA C coli 175 ---------- ---------- ------ACAT CAAGTAACTA TTCTTATGAG TGATAGAGGA

KatA C coli 1980 ---------- ---------- --------AT CAAGTAACTA TTCTTATGAG TGATAGAGGA

357

KatA C coli 2119 ---------- --TGTCCTGA AAGTTTACAT CAAGTAACTA TTCTTATGAG TGATAGAGGA

KatA C coli 2165 ---------- --TGTCCTGA AAGTTTACAT CAAGTAACTA TTCTTATGAG TGATAGAGGA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

550 560 570 580 590 600

KatA C jejuniNCTC11168 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT

KatA C jejuni 1206 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT













KatA C coliRM4661 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT

KatA C coli 2040 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT

KatA C coli 3064 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT

KatA C coli 56 ATTCCGGCAA GTTATCGCCA TATGCATGGT TTTGGAAGCC ATACTTATAG CTTTATCAAT

KatA C coli 2887 ATTCCGGCAA GTTATCGCCA TATGCATGGT TTTGGAAGCC ATACTTATAG CTTTATCAAT

KatA C coli 175 ATTCCAGCAA GTTATCGTCA TATGCACGGT TTTGGAAGCC ATACTTATAG CTTTATCAAT

358




....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

610 620 630 640 650 660

KatA C jejuniNCTC11168 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT

KatA C jejuni 1206 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT









KatA C jejuni 813 CATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT


KatA C jejuni 683 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG AATTAAAAAT

KatA C jejuni 687 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG AATTAAAAAT

KatA C coliRM4661 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT

KatA C coli 2040 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT

KatA C coli 3064 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT

KatA C coli 56 GACAAAAACG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCTACAAGG TATTAAAAAT

KatA C coli 2887 GACAAAAACG AGAGATTTTG GGTGAAATTC CATTTTAAAA CCCTACAAGG TATTAAAAAT

359





....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

670 680 690 700 710 720

KatA C jejuniNCTC11168 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT

KatA C jejuni 1206 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT











KatA C jejuni 683 CTTACCAACC AAGAAGCTGC AGAGCTTATA GCAAAGGATA GGGAAAGTCA TCAAAGAGAT

KatA C jejuni 687 CTTACCAACC AAGAAGCTGC AGAGCTTATA GCAAAGGATA GGGAAAGTCA TCAAAGAGAT

KatA C coliRM4661 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT

KatA C coli 2040 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT

KatA C coli 3064 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT

KatA C coli 56 CTTAGCAATA AAGAAGCTGC TGAACTTATC GCCAAAGATA GAGAAAGCCA CCAAAGAGAT

360

KatA C coli 2887 CTTAGCAATA AAGAAGCTGC TGAACTTATC GCCAAAGATA GAGAAAGCCA CCAAAGAGAT

KatA C coli 175 CTTAGCAATA AAGAAGCTGC TGAGCTTATC GCCAAAGATA GAGAAAGCCA CCAAAGAGAT




....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

730 740 750 760 770 780

KatA C jejuniNCTC11168 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT

KatA C jejuni 1206 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT










KatA C jejuni 1768 CTCTATAATG CTATAGAAAA CAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT



KatA C coliRM4661 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT

KatA C coli 2040 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT

KatA C coli 3064 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT

361

KatA C coli 56 CTTTACAATG CTATAGAAAA TAAAGATTTC CCAAAATGGA AAGTTCAAGT TCAAATTCTT






....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

790 800 810 820 830 840

KatA C jejuniNCTC11168 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT

KatA C jejuni 1206 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT










KatA C jejuni 1768 GCTGAAAAAG ATATAGAAAA ACTTGAATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT

KatA C jejuni 683 GCTGAAAAAG ATATAGAAAA GCTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT

KatA C jejuni 687 GCTGAAAAAG ATATAGAAAA GCTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT

KatA C coliRM4661 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT

KatA C coli 2040 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT

362

KatA C coli 3064 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT

KatA C coli 56 GCTGAAAAAG ATGCTGACAA ACTAGGCTTT AATCCTTTTG ATTTAACTAA AATTTGGCCA






....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

850 860 870 880 890 900

KatA C jejuniNCTC11168 CATAGTTTTG TACCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

KatA C jejuni 1206 CATAGTTTTG TACCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

KatA C jejuni 3050 CATAGTTTTG TACCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

KatA C jejuni 30 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT








KatA C jejuni 1768 CATAGTCTTG TACCTTTGAT GGATATAGGC GAAATGATTT TAAACAAAAA TCCTCAAAAT

KatA C jejuni 683 CATAGTCTTG TACCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

KatA C jejuni 687 CATAGTCTTG TACCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

KatA C coliRM4661 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

363

KatA C coli 2040 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

KatA C coli 3064 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT

KatA C coli 56 CATAGCGTAG TGCCTTTAAT GGATATAGGC GAAATGATCT TAAATCAAAA TCCACAAAAT






....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

910 920 930 940 950 960

KatA C jejuniNCTC11168 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC

KatA C jejuni 1206 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC










KatA C jejuni 1768 TATTTCAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC

KatA C jejuni 683 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCATTCC TGGAATTGGC

KatA C jejuni 687 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCATTCC TGGAATTGGC

364

KatA C coliRM4661 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATAGTACC TGGTATAGGT

KatA C coli 2040 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC

KatA C coli 3064 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC

KatA C coli 56 TATTTTAATG AAGTAGAACA AGCAGCTTTT AGCCCAAGCA ATATAGTACC TGGTATAGGT






....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

970 980 990 1000 1010 1020

KatA C jejuniNCTC11168 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT

KatA C jejuni 1206 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT












365


KatA C coliRM4661 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT

KatA C coli 2040 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT

KatA C coli 3064 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT

KatA C coli 56 TTTAGTCCTG ATAAAATGCT ACAAGCTAGA ATTTTCTCAT ATCCTGATGC ACAAAGATAT






....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1030 1040 1050 1060 1070 1080

KatA C jejuniNCTC11168 AGAATAGGAA CTAATTATCA TCTTTTACCA GTAAATCGTG CAAAAAGCGA AGTGAATACT

KatA C jejuni 1206 AGAATAGGAA CTAATTATCA TCTTTTACCA GTAAATCGTG CAAAAAGCGA AGTGAATACT


KatA C jejuni 30 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT









366



KatA C coliRM4661 AGAATAGGAA CTAATTATCA TCTTTTACCA GTAAATCGTG CAAAAAGCGA AGTGAATACT

KatA C coli 2040 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT

KatA C coli 3064 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT

KatA C coli 56 AGAATAGGAA CTAATTATCA TCTTTTACCT GTAAATCGTG CTAGAAGTGA AGTAAACACT

KatA C coli 2887 AGAATAGGAA CTAATTATCA TCTTTTACCT GTAAATCGTG CTAGAAGTGA AGTAAATACT





....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1090 1100 1110 1120 1130 1140

KatA C jejuniNCTC11168 TACAATGTCG CTGGTGCTAT GAATTTTGAT AGTTATAAAA ATGATGCTGC TTATTATGAA

KatA C jejuni 1206 TACAATGTC- ---------- ---------- ---------- ---------- ----------


KatA C jejuni 30 TACAATGTCG CTGGTGCTAT G--------- ---------- ---------- ----------


KatA C jejuni 1162 TA-------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 TACAATGTCG CTGGTG---- ---------- ---------- ---------- ----------

KatA C jejuni 2072 TACAATGTCG CTGG------ ---------- ---------- ---------- ----------

KatA C jejuni 2114 TACAATG--- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 TACAAT---- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 TACAATGT-- ---------- ---------- ---------- ---------- ----------

367

KatA C jejuni 1768 TACAAT---- ---------- ---------- ---------- ---------- ----------


KatA C jejuni 687 TACAATG--- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 TACAATGTCG CTGGTGCTAT GAATTTTGAT AGTTATAAAA ATGATGCAGC TTATTATGAA

KatA C coli 2040 TACAA----- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 TACAATGTC- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 TACAATGTCG CTGGTG---- ---------- ---------- ---------- ----------

KatA C coli 2887 TACAATGTCG CTGGTGCTAT G--------- ---------- ---------- ----------

KatA C coli 175 TACAAT---- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 TACAAT---- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 TACAATGTCG CTGGTG---- ---------- ---------- ---------- ----------

KatA C coli 2165 TACAATGTC- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1150 1160 1170 1180 1190 1200

KatA C jejuniNCTC11168 CCAAACAGCT ATGATAATAG CCCAAAAGAA GACAAAAGCT ATCTTGAACC TGATTTAGTC

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

368

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 CCAAACAGCT ATGATAACAG CCCAAAAGAA GACAAAAGCT ATCTTGAACC TGATTTAGTC

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1210 1220 1230 1240 1250 1260

KatA C jejuniNCTC11168 TTAGAAGGCG TAGCACAAAG ATATGCTCCA CTAGATAATG ACTTTTATAC TCAACCAAGA

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

369

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 TTAGAAGGCG TAGCACAAAG ATATACTCCA CTAGATAATG ACTTTTATAC TCAACCAAGA

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1270 1280 1290 1300 1310 1320

KatA C jejuniNCTC11168 GCTTTATTTA ATCTTATGAA TGATGATCAA AAAACTCAAC TTTTTCATAA TATCGCCGCT

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

370

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 GCTTTATTTA ATCTTATGAA TGATGATCAA AAAACTCAAC TTTTTCATAA TATCGCCGCT

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1330 1340 1350 1360 1370 1380

KatA C jejuniNCTC11168 TCTATGGAAG GAGTTGATGA AAAAATTATC ACTAGAGCTT TAAAACATTT TGAAAAAATT

KatA C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

371

KatA C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coliRM4661 TCTATGGAGG GAGTTGATGA AAAAATTATC ACTAGAGCTT TAGAACATTT TGAAAAAATT

KatA C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

KatA C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|

1390 1400 1410 1420

KatA C jejuniNCTC11168 TCACCTGATT ATGCAAAAGG AATTAAAAAA GCTTTAGAAA AATAA

KatA C jejuni 1206 ---------- ---------- ---------- ---------- -----

KatA C jejuni 3050 ---------- ---------- ---------- ---------- -----

KatA C jejuni 30 ---------- ---------- ---------- ---------- -----

KatA C jejuni 62 ---------- ---------- ---------- ---------- -----

KatA C jejuni 1162 ---------- ---------- ---------- ---------- -----

372

KatA C jejuni 2038 ---------- ---------- ---------- ---------- -----

KatA C jejuni 2072 ---------- ---------- ---------- ---------- -----

KatA C jejuni 2114 ---------- ---------- ---------- ---------- -----

KatA C jejuni 2170 ---------- ---------- ---------- ---------- -----

KatA C jejuni 813 ---------- ---------- ---------- ---------- -----

KatA C jejuni 1768 ---------- ---------- ---------- ---------- -----

KatA C jejuni 683 ---------- ---------- ---------- ---------- -----

KatA C jejuni 687 ---------- ---------- ---------- ---------- -----

KatA C coliRM4661 TCACCTGATT ATGCAAAAGG AATTAAAAAA GCTTTAGAAA AATAA

KatA C coli 2040 ---------- ---------- ---------- ---------- -----

KatA C coli 3064 ---------- ---------- ---------- ---------- -----

KatA C coli 56 ---------- ---------- ---------- ---------- -----

KatA C coli 2887 ---------- ---------- ---------- ---------- -----

KatA C coli 175 ---------- ---------- ---------- ---------- -----

KatA C coli 1980 ---------- ---------- ---------- ---------- -----

KatA C coli 2119 ---------- ---------- ---------- ---------- -----

KatA C coli 2165 ---------- ---------- ---------- ---------- -----

Appendix 3.3.2: Nucleotide sequence of cadF amplicons

The cadF nucleotide sequences of C. jejuni NCTC 11168, C. coli strain BP3183, and C. coli strain BG2108 obtained from the NCBI database were used

as references for aligning with the selected C. jejuni and C. coli clusters.

373

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60

CadF C jejuniNCTC11168 ATGAAAAAAA TATTCTTATG TTTAGGTTTG GCAAGTGTTT TATTTGGTGC TGATAACAAT

CadF C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 62 ---------- ---------- ---------- ---------- ---------- ---TAACAAT

CadF C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 2119 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 813 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CadF C jejuni 687 ---------- ---------- ---------- ---------- ---------- ---------T

CadF C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

CadF C colistrainBG2108ATGAGAAAGT TATTGCTATG TTTAGGGTTG TCAAGCGTTT TATTTGGTGC AGATAACAAT

CadF C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CadF C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CadF C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CadF C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CadF C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CadF C coli 2887 ---------- ---------- ---------- ---------- ---------- ---TAACAAT

CadF C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

CadF C colistrainBP3183ATGAAAAAAA TATTCTTATG TTTAGGTTTG GCAAGTGTTT TATTTGGTGC TGATAACAAT

374

CadF C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

70 80 90 100 110 120

CadF C jejuniNCTC11168 GTAAAATTTG AAATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C jejuni 2170 --------TG AAATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C jejuni 62 GTAAAATTTG AAATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C jejuni 1206 ---------- ---------- AACTTTAAAC TATAATTATT TTGAAGGTAA TTTAGATATG

CadF C jejuni 30 ---------- ---------- -ACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C jejuni 1162 --------TG AAATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C jejuni 2038 ---------- -AATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C jejuni 2119 ---------- -----ACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG






CadF C jejuni 3050 ---------- ---------- ---TTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C colistrainBG2108GTAAAATTTG AAATCACTCC TACTTTGAAT TACAATTATT TTGAAGGTAA TTTAGATATG

CadF C coli 56 ---------- ---------- ---------- ---AATTATT TTGAAGGTAA TTTAGATATG

CadF C coli 175 ---------G AAATCACTCC TACTTTGAAT TACAATTATT TTGAAGGTAA TTTAGATATG

CadF C coli 1980 ---------- ---------- ---------- ---AATTATT TTGAAGGTAA TTTAGATATG

CadF C coli 2119 ---------- ---------- --CTTTGAAT TACAATTATT TTGAAGGTAA TTTAGATATG

CadF C coli 2165 ---------- ---------- TACTTTGAAT TACAATTATT TTGAAGGTAA TTTAGATATG

CadF C coli 2887 GTAAAATTTG AAATCACTCC TACTTTGAAT TACAATTATT TTGAAGGTAA TTTAGATATG

375

CadF C coli 3064 --------TG AAATCACTCC TACTTTGAAT TACAATTATT TTGAAGGTAA TTTAGATATG

CadF C colistrainBP3183GTAAAATTTG AAATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

CadF C coli 2040 --------TG AAATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

130 140 150 160 170 180

CadF C jejuniNCTC11168 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT

CadF C jejuni 2170 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT


CadF C jejuni 1206 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATTATT TTGACGATTT TTGGCTTGAT







CadF C jejuni 813 GATAATCGTT ATGCACCAGG TGTTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT




CadF C colistrainBG2108GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT

CadF C coli 56 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT




376




CadF C colistrainBP3183GATAATCGTT ATGCACCAGG TGTTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT

CadF C coli 2040 GATAATCGTT ATGCACCAGG TGTTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

190 200 210 220 230 240

CadF C jejuniNCTC11168 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT

CadF C jejuni 2170 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT


CadF C jejuni 1206 CAATTAGAAT TTGGGTTAGA GTATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT







CadF C jejuni 813 CAATTAGAAT TTGGCTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT




CadF C colistrainBG2108CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC

CadF C coli 56 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC


377






CadF C colistrainBP3183CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT

CadF C coli 2040 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

250 260 270 280 290 300

CadF C jejuniNCTC11168 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT

CadF C jejuni 2170 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT



CadF C jejuni 30 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT




CadF C jejuni 1768 ACAGATATTA CAAGAACTTA TTTGAATGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT






CadF C colistrainBG2108ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT

378

CadF C coli 56 ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT







CadF C colistrainBP3183ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT

CadF C coli 2040 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

310 320 330 340 350 360

CadF C jejuniNCTC11168 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT

CadF C jejuni 2170 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT



CadF C jejuni 30 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAAGATTTTT CAAATGCTGC TTATGATAAT






CadF C jejuni 813 TATTTTTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT



379


CadF C colistrainBG2108TATTTTTATG GTTTAGCTGG TGGGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT

CadF C coli 56 TATTTTTATG GTTTAGCTGG TGTGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT



CadF C coli 2119 TATTTTTATG GTTTAGCTGG TGGGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT

CadF C coli 2165 TATTTTTATG GTTTAGCTGG TGGGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT



CadF C colistrainBP3183TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT

CadF C coli 2040 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

370 380 390 400 410 420

CadF C jejuniNCTC11168 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG

CadF C jejuni 2170 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG








CadF C jejuni 2072 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGCCTTAG TGATTCTTTG


380




CadF C colistrainBG2108AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA

CadF C coli 56 AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA






CadF C coli 3064 AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAA TGATTCTTTA

CadF C colistrainBP3183AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG

CadF C coli 2040 AAAAGCGGTG GATTTGGACA TTATGGCACG GGTGTAAAAT TCTGTCTTAG TGATTCTTTG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480

CadF C jejuniNCTC11168 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTCAATC ATGCAAACCA TAATTGGGTT

CadF C jejuni 2170 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTCAATC ATGCAAACCA TAATTGGGTT



CadF C jejuni 30 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT





381






CadF C colistrainBG2108GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT

CadF C coli 56 GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT







CadF C colistrainBP3183GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT

CadF C coli 2040 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

490 500 510 520 530 540

CadF C jejuniNCTC11168 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------

CadF C jejuni 2170 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------






382




CadF C jejuni 813 TCAACTTTAG GTATTAGTTT TGGTTTTGGT AGCAAAAAGG AAAAAGCTGT AG--------




CadF C colistrainBG2108TCAACTTTGG GCATTAGTTT TGGTTTTGGC GCTAAGCAAG AAAAAGTTGT AGTGGAGCAA

CadF C coli 56 TCAACTTTGG GCATTAGTTT TGGTTTTGGC GCTAAGCAAG AAAAAGTTGT AGTGGAGCAA

CadF C coli 175 TCGACTTTGG GCATTAGTTT TGGTTTTGGC GCTAAGCAAG AAAAAGTTGT AGTGGAGCAA

CadF C coli 1980 TCGACTTTGG GCATTAGTTT TGGTTTTGGC GCTAAGCAAG AAAAAGTTGT AGTGGAGCAA





CadF C colistrainBP3183TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------

CadF C coli 2040 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

550 560 570 580 590 600

CadF C jejuniNCTC11168 ----AAGAAG TTGCTGATA- ---------- -------CTC GTGCAACTCC ---------A

CadF C jejuni 2170 ----AAGAAG TTGCTGATA- ---------- -------CTC GTGCAACTCC ---------A



CadF C jejuni 30 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A

383






CadF C jejuni 813 ----AAGAAG TTGGTGATA- ---------- -------CTC GTCCAGCTCC ---------A




CadF C colistrainBG2108ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG CTCCAGCTCC TGTAGTCTCA

CadF C coli 56 ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG CTCCAGCTCC TGTAGTCTCA

CadF C coli 175 ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG TTCCAGCTCC TGTAGTCTCA

CadF C coli 1980 ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG TTCCAGCTCC TGTAGTCTCA





CadF C colistrainBP3183----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A

CadF C coli 2040 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

610 620 630 640 650 660

CadF C jejuniNCTC11168 CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA

CadF C jejuni 2170 CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA

CadF C jejuni 62 CAAGTAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA

384


CadF C jejuni 30 CAAACAAAAT GTCCTGTAGA GCCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA





CadF C jejuni 2072 CAAGCAAAAT GTCCTGTAGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA

CadF C jejuni 813 CAAGCAAAAT GTCCTGTAGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA




CadF C colistrainBG2108CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA

CadF C coli 56 CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA







CadF C colistrainBP3183CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA

CadF C coli 2040 CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

670 680 690 700 710 720

CadF C jejuniNCTC11168 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT

385

CadF C jejuni 2170 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT













CadF C colistrainBG2108AAAACAATTT ATTTAGAGGG ACATTTTGAT TTTGATAAAG TAAATATCAA CCCAGCCTTT

CadF C coli 56 AAAACAATTT ATTTAGAGGG ACATTTTGAT TTTGATAAAG TAAATATCAA CCCAGCCTTT







CadF C colistrainBP3183AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT

CadF C coli 2040 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

386

730 740 750 760 770 780

CadF C jejuniNCTC11168 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT

CadF C jejuni 2170 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT













CadF C colistrainBG2108GAAGAACAAA TCAAAGAAAT TGCTCAAATT TTAGATGAAA ATGTAAGATA TGATACTATT

CadF C coli 56 GAAGAACAAA TCAAAGAAAT TGCTCAAATT TTAGATGAAA ATGTAAGATA TGATACTATT







CadF C colistrainBP3183CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT

CadF C coli 2040 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT

387

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

790 800 810 820 830 840

CadF C jejuniNCTC11168 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA

CadF C jejuni 2170 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA








CadF C jejuni 2072 CTTGAAGGAC ATACAGATAA TATAGGTTCA AGAGCTTATA ATCAAAAGCT TTCAGAAAGA


CadF C jejuni 683 CTTGAAGGAC ATACAGATAA TATTGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA



CadF C colistrainBG2108TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA

CadF C coli 56 TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA







388

CadF C colistrainBP3183CTTGAAGGAC ATACAGATAA TATTGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA

CadF C coli 2040 CTTGAAGGAC ATACAGATAA TATTGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

850 860 870 880 890 900

CadF C jejuniNCTC11168 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA

CadF C jejuni 2170 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA













CadF C colistrainBG2108CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG

CadF C coli 56 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG





389



CadF C colistrainBP3183CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA

CadF C coli 2040 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

910 920 930 940 950 960

CadF C jejuniNCTC11168 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG

CadF C jejuni 2170 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG







CadF C jejuni 1768 ACAGTAGGTT GTGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG

CadF C jejuni 2072 ACAGTAGGTT ATGGACAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG





CadF C colistrainBG2108ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG ACACTAAAGA GGGTAGAGCA

CadF C coli 56 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG ACACTAAAGA GGGTAGAGCA


CadF C coli 1980 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG ACACTAAAGA GGGTAGAGCG

390



CadF C coli 2887 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCGAGCAATG ACACTAAAGA GGGTAGAGCA


CadF C colistrainBP3183ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG

CadF C coli 2040 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG

....|....| ....|....| ....|....| ....|....

970 980 990

CadF C jejuniNCTC11168 GATAATAGAA GAGTGGATGC TAAATTTATT TTAAGATAA

CadF C jejuni 2170 GATAATAGAA GAGTGGA--- ---------- ---------

CadF C jejuni 62 GATAATAGAA GAGTGGATGC TAAATTTATT TTAAGA---

CadF C jejuni 1206 GATAATAGAA GAGTGGATG- ---------- ---------

CadF C jejuni 30 GATAATAGAA GAGTGGATGC TAAATTTATT TTAA-----

CadF C jejuni 1162 GATAATAGAA GAGTGGAT-- ---------- ---------

CadF C jejuni 2038 GATAATAGAA ---------- ---------- ---------

CadF C jejuni 2119 GATAATAGAA GA-------- ---------- ---------

CadF C jejuni 1768 GATAATAGAA GAGT------ ---------- ---------

CadF C jejuni 2072 GATAATAGAA GAGTGGATGC TAAATT---- ---------

CadF C jejuni 813 GATAATAGAA GAGTGGAT-- ---------- ---------

CadF C jejuni 683 GATAATAGAA GAGTGGATGC TAAATTTATT TT-------

CadF C jejuni 687 GATAATAGAA GAGTGGATGC TAAATTTATT TTAAGA---

CadF C jejuni 3050 GA-------- ---------- ---------- ---------

CadF C colistrainBG2108GATAATAGAA GAGTAGAGGC TAAATTTATT TTAAATTAA

CadF C coli 56 GATAATAGAA GA-------- ---------- ---------

391

CadF C coli 175 GATAATAGAA GA-------- ---------- ---------

CadF C coli 1980 GATAATAG-- ---------- ---------- ---------

CadF C coli 2119 GATAATAGAA GAG------- ---------- ---------

CadF C coli 2165 GATAATAGAA GAGTA----- ---------- ---------

CadF C coli 2887 GATAATAGAA GAGTGGATGC TAAATTTATT TTAA-----

CadF C coli 3064 GATA------ ---------- ---------- ---------

CadF C colistrainBP3183GATAATAGAA GAGTGGATGC TAAATTTATT TTAAGATAA

CadF C coli 2040 GATAATAGAA GAG------- ---------- ---

Appendix 3.3.3: Nucleotide sequence of peb1A amplicons

The nucleotide sequences of the peb1A gene obtained from the NCBI database (C. jejuni strain YH0002 and C. coli strain YH502) were used as references

for aligning with the selected C. jejuni and C. coli clusters.

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60

C jejuni strain YH002 Peb ATGGTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

30 C jejuni Peb ---------- ----ATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

62 C jejuni Peb -TGGTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

683 C jejuni Peb --GGTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

687 C jejuni Peb ATGGTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

813 C jejuni Peb ---------- ----ATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

1162 C jejuni Peb ---------- ------CTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

392

1206 C jejuni Peb ---------- ----ATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGT TTGTGTTGCA

1768 C jejuni Peb ---GTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA

2038 C jejuni Peb ---------- -----TCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA





C coli strain YH502 Peb ATGGTTTTTA GAAATTCTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

56 C coli Peb ---------- GAAATTCTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

175 C coli Peb ---------- ------CTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

1980 C coli Peb ---------- ------CTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

2040 C coli Peb ---GTTTTTA GAAATTCTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

2119 C coli Peb --GGTTTTTA GAAATTCTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

2165 C coli Peb --GGTTTTTA GAAATTCTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

2887 C coli Peb -TGGTTTTTA GAAATTCTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

3064 C coli Peb ATGGTTTTTA GAAATTCTTT ATTAAAATTA GCAGCACTTG CTTTAGGAGC TTGTATGGCT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

70 80 90 100 110 120

C jejuni strain YH002 Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA

30 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA

62 C jejuni Peb TTTAGTAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA




393







2170 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAG CTTGAGTCTA TTAAATCTAA AGGACAATTA


C coli strain YH502 Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG

56 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

130 140 150 160 170 180

C jejuni strain YH002 Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA

30 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA

62 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCATAT TATGCTTTAC TTGATCAAGC AACAGGTGAA

683 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCACAT TATGCTTTAC TTGATCAAGC AACAGGTGAA


394






2072 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGT AACAGGTGAA




C coli strain YH502 Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA

56 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

190 200 210 220 230 240

C jejuni strain YH002 Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT

30 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCTAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT

62 C jejuni Peb ATTAAAGGTT TCGAAATAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT AGGTGATGAT

683 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT AGGTGATGAT

395




1206 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT







C coli strain YH502 Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA

56 C coli Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

250 260 270 280 290 300

C jejuni strain YH002 Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT

30 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT


396












C coli strain YH502 Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT

56 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

310 320 330 340 350 360

C jejuni strain YH002 Peb AGTGTAGATG CGGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT

30 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT

397






1206 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCGG AGAGAAAAAG AATTTATAAT


2038 C jejuni Peb AGTGTAGATG CGGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT




3050 C jejuni Peb AGTGTAGATG CGGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT

C coli strain YH502 Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT

56 C coli Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

370 380 390 400 410 420

C jejuni strain YH002 Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAAATAT

398

30 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTAG TCTTAAAAGA AAAAAATTAT

62 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAATTAT







2038 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAAATAT



2170 C jejuni Peb TTCTCAGAAC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAAATAT

3050 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAAATAT

C coli strain YH502 Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT

56 C coli Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480

399

C jejuni strain YH002 Peb AAATCTTTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA

30 C jejuni Peb AAATCTTTAG CTGATATGAA AGGTGCAAAC ATTGGAGTGG CTCAAGCTGC AACTACAAAA

62 C jejuni Peb AAATCTCTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA







2038 C jejuni Peb AAATCTTTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA





C coli strain YH502 Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA

56 C coli Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

400

490 500 510 520 530 540

C jejuni strain YH002 Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT

30 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGTATTGATG TTAAATTTAG TGAATTTCCT

62 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT





1206 C jejuni Peb AAAGCTATAG GTAAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT







C coli strain YH502 Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT

56 C coli Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT








401

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

550 560 570 580 590 600

C jejuni strain YH002 Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC

30 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC

62 C jejuni Peb GATTATCCAA GTATAAAAGC TGCGTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC












C coli strain YH502 Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT

56 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT








402

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

610 620 630 640 650 660

C jejuni strain YH002 Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA

30 C jejuni Peb AAATCAATAT TGTTAGGCTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA

62 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA












C coli strain YH502 Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT

56 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT







403


....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

670 680 690 700 710 720

C jejuni strain YH002 Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT

30 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT













C coli strain YH502 Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT

56 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT






404



....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

730 740 750 760 770 780

C jejuni strain YH002 Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTATAA

30 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTATAA



687 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTT-----

813 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG G---------

1162 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG ----------


1768 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTA---






C coli strain YH502 Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTAG CTAAAAAATG GGGTTTATAA

56 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTAG CGAAAAAATG GGGTT-----

175 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTA- ---------- ----------



2119 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTAG CGAAAAAATG GGGTTTATAA

405




Appendix 3.3.4: Nucleotide sequence of cjaA amplicons

The nucleotide sequences of the cjaA gene obtained from C. jejuni cjaA gene (GenBank: Y10872.1) and C. coli strain YH502 in the NCBI database used

as references for aligning with the selected C. jejuni and C. coli clusters as shown below.

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60

C.jejuni cjaA gene GTCGACGGTA TCGATAAGCT TGATATCGCT GATTACTTTT TCTCCAAGTT TAAATCCTTC

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

406

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

CjaA C colistrainYH502 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

70 80 90 100 110 120

C.jejuni cjaA gene TAGGGTAAAA CTTGTTTTTA AATTTTTATT TTTACTCAAT AGATTATCGT AATAATCTTT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

407

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

130 140 150 160 170 180

C.jejuni cjaA gene TAAAGAGATT AAGCCTTGCT CCTCTCCTGT GTAAATTTCA TTAGAATAAA TTTCTAAATT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

408

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

190 200 210 220 230 240

C.jejuni cjaA gene TTTTGCTTCG ATGAGTTTTT GCTCTGTTTG GGTATTGTCT TTTGCAAAAA CATTAAAATT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

409

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

250 260 270 280 290 300

C.jejuni cjaA gene AGGGCTTGAA CATCTAAGAA ACATTCCATC GCCTTTACAA GAAAAATTAG AAATTTCTAT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

410

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

310 320 330 340 350 360

C.jejuni cjaA gene TCTTACACCC ATATCTTGTT GAAAATCCTT AAGGGTTTCA TTTATTTGAG TGTTAAGACG

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

411

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

370 380 390 400 410 420

C.jejuni cjaA gene CTCCTCATAT TTTGCAACTG TTGTTTTATC AAGCGAGTTG CTGCACGCTG AAAAGAAAAA

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

412

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480

C.jejuni cjaA gene TATAGTACCT AGGGTTAGAG CAATACTTTT TTTCATAAAA TCTTCTCCTT AATATAGTTT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

413

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

490 500 510 520 530 540

C.jejuni cjaA gene TATAAATTAT AACAATCTTA TACTTAAATT TTTATGCTTT GATATTGATA TTACTTTTTT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

414

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

550 560 570 580 590 600

C.jejuni cjaA gene ACACAAGAAG AAATAATTTT ATCTTAAAAA ATTGAAATAT GCTTTTTTTT AAAGTATAAT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

415

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

610 620 630 640 650 660

C.jejuni cjaA gene GCTTTTGCAT TTTTTGCAAA ATAAATTAGG GTTTATACCA AGAAAGGAAA AGTATGAAAA

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

416

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------

CjaA C colistrainYH502 ---------- ---------- ---------- ---------- ---------- ---ATGAAAA

CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

670 680 690 700 710 720

C.jejuni cjaA gene AAATGCTCTT AAGTATTTTT ACAACCTTTG TTGCAGTATT TTTGGCTGCT TGTGGAGGAA

417

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA C colistrainYH502 AAATGCTCTT AAGTATTTTT ACAACCTTTG TTGCAGTATT TTTGGCTGCT TGTGGAGGAA

CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ---------A

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

730 740 750 760 770 780

418

C.jejuni cjaA gene ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-CC C jejuni 62 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA_Cc C jejuni 683 -------TTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C jejun 813 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C jejuni 1206 -------TTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-CC C jejuni 2038 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---GAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C jejuni 30 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C jejuni 687 ---------C TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C jejuni 1162 ---------- -----CTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA


CjaA-Cc C jejuni 2072 ---------C TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA



CjaA C colistrainYH502 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C coli 56 ---------- ---TGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA



CjaA-Cc C coli 2040 ---------- ------TTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---GAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C coli 2165 ---------- -GGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA

CjaA-Cc C coli 2887 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA


....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

419

790 800 810 820 830 840

C.jejuni cjaA gene TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG

CjaA-CC C jejuni 62 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG

CjaA_Cc C jejuni 683 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG

CjaA-Cc C jejun 813 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG

CjaA-Cc C jejuni 1206 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG

CjaA-CC C jejuni 2038 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG



CjaA-Cc C jejuni 687 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG






CjaA C colistrainYH502 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG

CjaA-Cc C coli 56 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG

CjaA-Cc C coli 175 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG







420

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

850 860 870 880 890 900

C.jejuni cjaA gene GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG

CjaA-CC C jejuni 62 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG

CjaA_Cc C jejuni 683 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG

CjaA-Cc C jejun 813 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG

CjaA-Cc C jejuni 1206 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG

CjaA-CC C jejuni 2038 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG









CjaA C colistrainYH502 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG

CjaA-Cc C coli 56 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG








421

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

910 920 930 940 950 960

C.jejuni cjaA gene TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG

CjaA-CC C jejuni 62 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG

CjaA_Cc C jejuni 683 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG

CjaA-Cc C jejun 813 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG

CjaA-Cc C jejuni 1206 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG

CjaA-CC C jejuni 2038 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG









CjaA C colistrainYH502 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG

CjaA-Cc C coli 56 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG







422


....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

970 980 990 1000 1010 1020

C.jejuni cjaA gene ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT

CjaA-CC C jejuni 62 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT

CjaA_Cc C jejuni 683 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT

CjaA-Cc C jejun 813 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT

CjaA-Cc C jejuni 1206 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT

CjaA-CC C jejuni 2038 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT









CjaA C colistrainYH502 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT

CjaA-Cc C coli 56 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT




CjaA-Cc C coli 2119 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAACAAGTG GATTTTTGCT

CjaA-Cc C coli 2165 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAACAAGTG GATTTTTGCT

423



....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1030 1040 1050 1060 1070 1080

C.jejuni cjaA gene TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA

CjaA-CC C jejuni 62 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA

CjaA_Cc C jejuni 683 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA

CjaA-Cc C jejun 813 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA

CjaA-Cc C jejuni 1206 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA

CjaA-CC C jejuni 2038 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA









CjaA C colistrainYH502 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA

CjaA-Cc C coli 56 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA





424


CjaA-Cc C coli 2887 TGCCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA


....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1090 1100 1110 1120 1130 1140

C.jejuni cjaA gene TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT

CjaA-CC C jejuni 62 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT

CjaA_Cc C jejuni 683 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT

CjaA-Cc C jejun 813 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT

CjaA-Cc C jejuni 1206 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT

CjaA-CC C jejuni 2038 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT









CjaA C colistrainYH502 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT

CjaA-Cc C coli 56 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT




425

CjaA-Cc C coli 2119 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACCGCT GATGCGTATT

CjaA-Cc C coli 2165 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACCGCT GATGCGTATT



....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1150 1160 1170 1180 1190 1200

C.jejuni cjaA gene TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG

CjaA-CC C jejuni 62 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG

CjaA_Cc C jejuni 683 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG

CjaA-Cc C jejun 813 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG

CjaA-Cc C jejuni 1206 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG

CjaA-CC C jejuni 2038 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG









CjaA C colistrainYH502 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG

CjaA-Cc C coli 56 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG



426






....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1210 1220 1230 1240 1250 1260

C.jejuni cjaA gene CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-CC C jejuni 62 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA_Cc C jejuni 683 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-Cc C jejun 813 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-Cc C jejuni 1206 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-CC C jejuni 2038 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT



CjaA-Cc C jejuni 687 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT






CjaA C colistrainYH502 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-Cc C coli 56 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTTCGT

CjaA-Cc C coli 175 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

427



CjaA-Cc C coli 2119 CCGCTTTAAT AGATCAAAGA GGGGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-Cc C coli 2165 CCGCTTTAAT AGATCAAAGA GGGGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-Cc C coli 2887 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

CjaA-Cc C coli 3064 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1270 1280 1290 1300 1310 1320

C.jejuni cjaA gene GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA

CjaA-CC C jejuni 62 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA

CjaA_Cc C jejuni 683 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA

CjaA-Cc C jejun 813 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA

CjaA-Cc C jejuni 1206 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA

CjaA-CC C jejuni 2038 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA









CjaA C colistrainYH502 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA

CjaA-Cc C coli 56 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA

428








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1330 1340 1350 1360 1370 1380

C.jejuni cjaA gene TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA

CjaA-CC C jejuni 62 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA

CjaA_Cc C jejuni 683 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA

CjaA-Cc C jejun 813 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA

CjaA-Cc C jejuni 1206 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA

CjaA-CC C jejuni 2038 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA


CjaA-Cc C jejuni 30 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTGAAGA ATTTATTGAT AATCTAATCA







CjaA C colistrainYH502 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA

429

CjaA-Cc C coli 56 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA






CjaA-Cc C coli 2887 TTGCCCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA


....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1390 1400 1410 1420 1430 1440

C.jejuni cjaA gene CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT

CjaA-CC C jejuni 62 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT

CjaA_Cc C jejuni 683 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT

CjaA-Cc C jejun 813 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT

CjaA-Cc C jejuni 1206 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT

CjaA-CC C jejuni 2038 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT









430

CjaA C colistrainYH502 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGTTTATGA TGAAACTTTA AAAAGTCATT

CjaA-Cc C coli 56 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT








....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1450 1460 1470 1480 1490 1500

C.jejuni cjaA gene TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAAAATT TAACAAAAAA

CjaA-CC C jejuni 62 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAA---- ----------

CjaA_Cc C jejuni 683 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAA---- ----------

CjaA-Cc C jejun 813 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG ---------- ----------

CjaA-Cc C jejuni 1206 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTAT------ ---------- ----------

CjaA-CC C jejuni 2038 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAA---- ----------

CjaA-Cc C jejuni 2170 TTGGAGATGA TGTAAAAG-- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTAT------ ---------- ----------

CjaA-Cc C jejuni 687 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTA------- ---------- ----------


CjaA-Cc C jejuni 1768 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAA---- ----------


CjaA-Cc C jejuni 2114 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAA---- ----------

431

CjaA-Cc C jejuni 3050 TTGGAGATGA TGTAAAAGCT GATGATGTAG TTATTGAAGG CGGTAA---- ----------

CjaA C colistrainYH502 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAAAATT TAA-------

CjaA-Cc C coli 56 TTGGAGATGA TGTAAAAGCC GATGATGTA- ---------- ---------- ----------

CjaA-Cc C coli 175 TTGGAGATGA TGTAAAAGCC GATGATGTAG ---------- ---------- ----------

CjaA-Cc C coli 1980 TTGGAGATGA TGTAAAAGCC GATGATGTAG ---------- ---------- ----------

CjaA-Cc C coli 2040 TTGGAGATGA TGTAAAAGCC GATGATGTAG T--------- ---------- ----------

CjaA-Cc C coli 2119 TTGGAGATGA TGTAAAAGCT GATGATGTAG ---------- ---------- ----------

CjaA-Cc C coli 2165 TTGGAGATGA TGTAAAAGCC GATGATGT-- ---------- ---------- ----------

CjaA-Cc C coli 2887 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTG---- ---------- ----------

CjaA-Cc C coli 3064 TTGGAGATGA TGTAAAAGCT GATGATGTAG ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1510 1520 1530 1540 1550 1560

C.jejuni cjaA gene GGGCTTTTGC CCTTTAGTTG ATTTAGGATA AAATATGCAA AAAAAATACA AAAATATAAT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

432

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1570 1580 1590 1600 1610 1620

C.jejuni cjaA gene TTATGCTTCT TTGGGCGGAA TTTTAGAATT TTATGATTTT GTGCTCTTTG CCTTTTTTTT

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

433

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1630 1640 1650 1660 1670 1680 C.jejuni

cjaA gene GGATATTTTT GCTAAGGTTT TCTTTCCTCA AAATGATACT TTTTGGATGC AAATAAATGC

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

434

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1690 1700 1710 1720 1730 1740

C.jejuni cjaA gene TTATATAGCC TTTGGTGCTG CTTATTTGGC GCGTCCTTTT GGATCTATTG TTATGGCGCA

CjaA-CC C jejuni 62 ---------- ---------- ---------- ---------- ---------- ----------

CjaA_Cc C jejuni 683 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejun 813 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1206 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-CC C jejuni 2038 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 30 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 687 ---------- ---------- ---------- ---------- ---------- ----------

435

CjaA-Cc C jejuni 1162 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 1768 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2072 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 2114 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C jejuni 3050 ---------- ---------- ---------- ---------- ---------- ----------


CjaA-Cc C coli 56 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 175 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 1980 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2040 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2119 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2165 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 2887 ---------- ---------- ---------- ---------- ---------- ----------

CjaA-Cc C coli 3064 ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|...

1750 1760

C.jejuni cjaA gene TTTTGGCGAT AGATACGGGC GTAAAAAT

CjaA-CC C jejuni 62 ---------- ---------- --------

CjaA_Cc C jejuni 683 ---------- ---------- --------

CjaA-Cc C jejun 813 ---------- ---------- --------

CjaA-Cc C jejuni 1206 ---------- ---------- --------

CjaA-CC C jejuni 2038 ---------- ---------- --------

CjaA-Cc C jejuni 2170 ---------- ---------- --------

CjaA-Cc C jejuni 30 ---------- ---------- --------

436

CjaA-Cc C jejuni 687 ---------- ---------- --------

CjaA-Cc C jejuni 1162 ---------- ---------- --------

CjaA-Cc C jejuni 1768 ---------- ---------- --------

CjaA-Cc C jejuni 2072 ---------- ---------- --------

CjaA-Cc C jejuni 2114 ---------- ---------- --------

CjaA-Cc C jejuni 3050 ---------- ---------- --------

CjaA C colistrainYH502 ---------- ---------- --------

CjaA-Cc C coli 56 ---------- ---------- --------

CjaA-Cc C coli 175 ---------- ---------- --------

CjaA-Cc C coli 1980 ---------- ---------- --------

CjaA-Cc C coli 2040 ---------- ---------- --------

CjaA-Cc C coli 2119 ---------- ---------- --------

CjaA-Cc C coli 2165 ---------- ---------- --------

CjaA-Cc C coli 2887 ---------- ---------- --------

CjaA-Cc C coli 3064 ---------- ---------- --------

437

Appendix 3.4: The alignment of subsequence amino acids

The Clustal Omega program was used for multiple sequence alignment of

subsequent amino acids in this study. The results showed three levels of

conservative subsequent amino acid substitution for each sequence position

using the Gonnet’s Point Accepted Mutation (PAM) 250 scoring matrix

(Gonnet et al., 1992), as described below:

1. A position with a conservation between amino groups of strongly similar

(physicochemical) properties (the score was > 0.5 in the Gonnet PAM 250

matrix)

2. A position with a conservation between amino groups of weakly similar

(physicochemical) properties (the score was ≤ 0.5 in the Gonnet PAM 250

matrix)

3. A position with a fully conserved amino acid

Some C. jejuni (n=13; clusters 1, 2, 3, 5, 6, 8, 12, 26, 27, 28, 29, 36, and 39)

and C. coli (n=7; clusters 2, 3, 5, 6, 13, 21, and 23) genotypes were selected

for amino acid sequence alignment compared with C. jejuni and C. coli

reference strains obtained from the NCBI database using the Clustal Omega

program version 1.2.4. The alignment analysis of the subsequent amino acid

sequences was provided in Appendices 3.4.1-3.4.4. The fully conserved

amino acids are *. The amino acids conserved between groups of strongly

similar properties are indicated in green (:). The amino acids conserved

between groups of weakly similar properties are indicated in yellow (.). The

amino acids not conserved between groups are indicated in grey.

Appendix 3.4.1: KatA amino acid

The primer used in this study generated the predicted amino acids starting at

the positions 165–171 and ending at the positions 360–367, compared with

the reference strains.

A total of 203 amino acids identified among the C. jejuni and C. coli

genotypes resulted in six different sequence patterns of the subsequent amino

acids. Of the six groups, Group 1 (C. jejuni clusters 5, 6, 8, 27, 28, 29, and 36

and C. coli clusters 3 and 25) shared 100% similarity in the KatA amino acid

438

sequences with C. coli strain RM4661. Group 2 (C. coli clusters 1, 2, 3, 6, 13,

and 21) had eight different amino acid positions. Six positions were conserved

between the amino groups. These included five and one showing strong

(positions 222, 224, 266, and 355) and weak (positions 215) physicochemical

similarities, respectively. The remaining two positions, positions 265 and

269, were not conserved between amino acid groups. Group 3 (C. jejuni

clusters 12 and 26) had the same KatA amino acids as in C. jejuni NCTC

11168. One different position identified (F: the position 283) in this group

was conserved between amino groups with weak physicochemical

similarities to Group 1 (L) and Group 2 (V). The remaining four C. jejuni

clusters (clusters 1,2, 3 and 28) had one amino acid substitution but different

positions. The subsequent amino acids of C. jejuni clusters 1 and 2 were

identical but had a different amino acid (I; the positions 316) compared with

others (V), but it was conserved between groups of strongly similar

properties. A different position of C. jejuni cluster 3 (H: the positions

201position 201) had weak physicochemical similarities, whereas, that of C.

jejuni cluster 28 was not conserved amino acid (E) between groups at position

269 compared with others (G).

439

56CcolikatA ------------------------------------------------------------ 0

175CcolikatA ------------------------------------------------------------ 0

1980CcolikatA ------------------------------------------------------------ 0

2119CcolikatA ------------------------------------------------------------ 0

2165CcolikatA ------------------------------------------------------------ 0

2887CcolikatA ------------------------------------------------------------ 0

1768CjejunikatA ------------------------------------------------------------ 0

813CjejunikatA ------------------------------------------------------------ 0

683CjejunikatA ------------------------------------------------------------ 0

687CjejunikatA ------------------------------------------------------------ 0

CjejuniNCTC11186katA MKKLTNDFGNIIADNQNSLSAGAKGPLLMQDYLLLEKLAHQNRERIPERTVHAKGSGAYG 60

1206CjejunikatA ------------------------------------------------------------ 0

3050CjejunikatA ------------------------------------------------------------ 0

30CjejunikatA ------------------------------------------------------------ 0

62CjejunikatA ------------------------------------------------------------ 0

1162CjejunikatA ------------------------------------------------------------ 0

2038CjejunikatA ------------------------------------------------------------ 0

2072CjejunikatA ------------------------------------------------------------ 0

2114CjejunikatA ------------------------------------------------------------ 0

2170CjejunikatA ------------------------------------------------------------ 0

CcolistrainRM4661katA MKKLTNDFGNIIADNQNSLSAGAKGPLLMQDYLLLEKLAHQNRERIPERTVHAKGSGAYG 60

2040CcolikatA ------------------------------------------------------------ 0

3064CcolikatA ------------------------------------------------------------ 0

440

56CcolikatA ------------------------------------------------------------ 0

175CcolikatA ------------------------------------------------------------ 0

1980CcolikatA ------------------------------------------------------------ 0

2119CcolikatA ------------------------------------------------------------ 0

2165CcolikatA ------------------------------------------------------------ 0

2887CcolikatA ------------------------------------------------------------ 0

1768CjejunikatA ------------------------------------------------------------ 0

813CjejunikatA ------------------------------------------------------------ 0

683CjejunikatA ------------------------------------------------------------ 0

687CjejunikatA ------------------------------------------------------------ 0

CjejuniNCTC11186katA EIKITADLSAYTKAKIFQKGEVTPLFLRFSTVAGEAGAADAERDVRGFAIKFYTKEGNWD 120

1206CjejunikatA ------------------------------------------------------------ 0

3050CjejunikatA ------------------------------------------------------------ 0

30CjejunikatA ------------------------------------------------------------ 0

62CjejunikatA ------------------------------------------------------------ 0

1162CjejunikatA ------------------------------------------------------------ 0

2038CjejunikatA ------------------------------------------------------------ 0

2072CjejunikatA ------------------------------------------------------------ 0

2114CjejunikatA ------------------------------------------------------------ 0

2170CjejunikatA ------------------------------------------------------------ 0

CcolistrainRM4661katA EIKITADLSAYTKAKIFQKGEITPLFLRFSTVAGEAGAADAERDVRGFAIKFYTKEGNWD 120

2040CcolikatA ------------------------------------------------------------ 0

3064CcolikatA ------------------------------------------------------------ 0

441

56CcolikatA --------------------------------------------CPESLHQVTILMSDRG 16

175CcolikatA -------------------------------------------------HQVTILMSDRG 16

1980CcolikatA --------------------------------------------------QVTILMSDRG 16




1768CjejunikatA --------------------------------------------------QVTILMSDRG 16


683CjejunikatA -------------------------------------------------HQVTILMSDRG 16


CjejuniNCTC11186katA LVGNNTPTFFIRDAYKFPDFIHTQKRDPRTHLRSNNAAWDFWSLCPESLHQVTILMSDRG 180



30CjejunikatA -----------------------------------------------SLHQVTILMSDRG 16

62CjejunikatA ------------------------------------------------LHQVTILMSDRG 16




2114CjejunikatA ------------------------------------------------LHQVTILMSDRG 16


CcolistrainRM4661katA LVGNNTPTFFIRDAYKFPDFIHTQKRDPRTHLRSNNAAWDFWSLCPESLHQVTILMSDRG 180

2040CcolikatA --------------------------------------------------QVTILMSDRG 16

3064CcolikatA -------------------------------------------------HQVTILMSDRG 16

**********

442

56CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTLQGIKNLSNKEAAELIAKDRESHQRD 76






1768CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76

813CjejunikatA IPASYRHMHGFGSHTYSFINHKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76



CjejuniNCTC11186katA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 240










CcolistrainRM4661katA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 240

2040CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76

3064CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76

********************.************* ******:*:****************

443

56CcolikatA LYNAIENKDFPKWKVQVQILAEKDADKLGFNPFDLTKIWPHSVVPLMDIGEMILNQNPQN 136






1768CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLEFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136

813CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136



CjejuniNCTC11186katA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSFVPLMDIGEMILNKNPQN 300

1206CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSFVPLMDIGEMILNKNPQN 136

3050CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSFVPLMDIGEMILNKNPQN 136








CcolistrainRM4661katA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 300

2040CcolikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136

3064CcolikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136

************************ :** *************.************:****

444

56CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRARSEVNT 196






1768CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196


683CjejunikatA YFNEVEQAAFSPSNIIPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196

687CjejunikatA YFNEVEQAAFSPSNIIPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196

CjejuniNCTC11186katA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 360










CcolistrainRM4661katA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 360

2040CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196

3064CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196

***************:**************************************:*****

445

56CcolikatA YNVAG------------------------------------------------------- 203

175CcolikatA YN---------------------------------------------------------- 203

1980CcolikatA YN---------------------------------------------------------- 203

2119CcolikatA YNVAG------------------------------------------------------- 203

2165CcolikatA YNV--------------------------------------------------------- 203

2887CcolikatA YNVAGAM----------------------------------------------------- 203

1768CjejunikatA YN---------------------------------------------------------- 203

813CjejunikatA YN---------------------------------------------------------- 203

683CjejunikatA YNV--------------------------------------------------------- 203

687CjejunikatA YN---------------------------------------------------------- 203

CjejuniNCTC11186katA YNVAGAMNFDSYKNDAAYYEPNSYDNSPKEDKSYLEPDLVLEGVAQRYAPLDNDFYTQPR 420

1206CjejunikatA YNV--------------------------------------------------------- 203

3050CjejunikatA YNV--------------------------------------------------------- 203

30CjejunikatA YNVAGAM----------------------------------------------------- 203

62CjejunikatA YNV--------------------------------------------------------- 203

1162CjejunikatA ------------------------------------------------------------ 203

2038CjejunikatA YNVAG------------------------------------------------------- 203

2072CjejunikatA YNVA-------------------------------------------------------- 203

2114CjejunikatA YN---------------------------------------------------------- 203

2170CjejunikatA YN---------------------------------------------------------- 203

CcolistrainRM4661katA YNVAGAMNFDSYKNDAAYYEPNSYDNSPKEDKSYLEPDLVLEGVAQRYTPLDNDFYTQPR 420

2040CcolikatA Y----------------------------------------------------------- 203

3064CcolikatA YNV--------------------------------------------------------- 203

446

56CcolikatA ------------------------------------------------------ 203

175CcolikatA ------------------------------------------------------ 203

1980CcolikatA ------------------------------------------------------ 203

2119CcolikatA ------------------------------------------------------ 203

2165CcolikatA ------------------------------------------------------ 203

2887CcolikatA ------------------------------------------------------ 203

1768CjejunikatA ------------------------------------------------------ 203

813CjejunikatA ------------------------------------------------------ 203

683CjejunikatA ------------------------------------------------------ 203

687CjejunikatA ------------------------------------------------------ 203

CjejuniNCTC11186katA ALFNLMNDDQKTQLFHNIAASMEGVDEKIITRALKHFEKISPDYAKGIKKALEK 474

1206CjejunikatA ------------------------------------------------------ 203

3050CjejunikatA ------------------------------------------------------ 203

30CjejunikatA ------------------------------------------------------ 203

62CjejunikatA ------------------------------------------------------ 203

1162CjejunikatA ------------------------------------------------------ 203

2038CjejunikatA ------------------------------------------------------ 203

2072CjejunikatA ------------------------------------------------------ 203

2114CjejunikatA ------------------------------------------------------ 203

2170CjejunikatA ------------------------------------------------------ 203

CcolistrainRM4661katA ALFNLMNDDQKTQLFHNIAASMEGVDEKIITRALEHFEKISPDYAKGIKKALEK 474

2040CcolikatA ------------------------------------------------------ 203

3064CcolikatA ------------------------------------------------------ 203

447

Appendix 3.4.2: CadF amino acid

The primer used in this study generated the predicted amino acids starting at

the positions 20–32 and ending at the positions 321–324 for most C. coli

samples or ending at the positions 307–319 for all selected C. jejuni and some

C. coli clusters, compared with the reference strains.

The alignment analysis of subsequent amino acid sequences coding for CadF

proteins showed the differences between C. jejuni and C. coli clusters,

resulting in 13 groups. Eight and five groups were identified in the selected

C. jejuni and C. coli clusters, respectively. Of these eight groups of the C.

jejuni, Group 1 had 4 C. jejuni clusters (clusters 6, 8, 29, and 36). This group

showed one different amino acid identified at position 202 that was conserved

between amino acid groups with strong physicochemical similarities. Group

2 (C. jejuni cluster 28) had two different amino acid positions. One position

(position 202) was the same in Group 1. The other position was found in

position 96 that was non- conserved amino acid. Group 3 (C. jejuni clusters

1, 2, and 26) shared 100% similarity of the CadF amino acids with the C. coli

BP3183 and one conserved amino acid substitution with strong

physicochemical similarities was found at position 48. Group 4 (C. jejuni

cluster 3) had 2 conserved amino acid substitution positions with strong

physicochemical similarities. One position (position 48) was the same as in

Group 3 and the other position was identified at position 181. Group 5 (C.

jejuni cluster 27) had the same CadF amino acids as in the C. jejuni

NCTC11168 (Appendix 3.4.2). This group had two different amino acid

positions that were one non-conserved amino acid (position 198) and one

conserved amino acid substitution with strong physicochemical similarities

(position 199). Group 6 (C. jejuni cluster 5) had three different amino acid

positions. Two of them were the same as in Group 5. The other position was

found at 202 that was non-conserved amino acids. Group 7 (C. jejuni cluster

12) had 4 different positions/ There of them (positions 53, 68, and 199) were

conserved amino acid substitutions with strong physicochemical similarities

strong. One position was a non-conserved amino acid at position 198. Group

8 consisted of one C. jejuni cluster (cluster 12). As for the 5 groups in C. coli

clusters, Group 1 (C. coli clusters 3 and 13) shared 100% similarity of CadF

448

amino acids with C. coli BP3183. Three different amino acids were found in

three groups (Groups 2-4) at position 106, 137, and 194. At the position 106,

one non-conserved amino acid was identified in Group 2 (C. coli clusters 1

and 2), Group 3 (C. coli clusters 6and 21) and Group 4 (C. coli clusters 6).

Groups 3 and 4 had another one different amino acid at positions 194 and

137, respectively. These differences were conserved substitution with weak

physicochemical similarities. Group 5 (C. coli cluster 23) had no extra 13

amino acids (between positions 184-194) and three different amino acid

positions. Two positions (positions 48 and 130) was conserved amino acid

substitution with strong physicochemical similarities and one position was a

non-conserved amino acid. Also, the presence of extra 13 amino acids was

identified in all C. coli clusters tested (clusters 1, 2, 3, 5, 6, 13, and 21), except

for C. coli cluster 23) and the reference C. coli strain BP3183, compared to

that of C. jejuni. Based on the alignment analysis. the 13 extra amino acids in

the C. coli sequences are shaded in purple, as below.

449

CcolistrainBG2108cadF MRKLLLCLGLSSVLFGADNNVKFEITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 60

3064CcolicadF -----------------------EITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

2119CcolicadF ----------------------------LNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

2165CcolicadF ----------------------------LNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

175CcolicadF -----------------------EITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

1980CcolicadF -------------------------------NYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

56CcolicadF -------------------------------NYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

2887CcolicadF -------------------NVKFEITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

1206CjejunicadF ---------------------------TLNYNYFEGNLDMDNRYAPGIRLGYYFDDFWLD 42

CjejuniNCTC11168cadF MKKIFLCLGLASVLFGADNNVKFEITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 60

2170CjejunicadF -----------------------EITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

62CjejunicadF -------------------NVKFEITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

2040CcolicadF -----------------------EITPTLNYNYFEGNLDMDNRYAPGVRLGYHFDDFWLD 42

1768CjejunicadF -------------------------TPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

813CjejunicadF -------------------------TPTLNYNYFEGNLDMDNRYAPGVRLGYHFDDFWLD 42

683CjejunicadF --------------------VKFEITPTLNYNYFEGNLDMDNRYAPGVRLGYHFDDFWLD 41

687CjejunicadF --------------------VKFEITPTLNYNYFEGNLDMDNRYAPGVRLGYHFDDFWLD 42

3050CjejunicadF ----------------------------LNYNYFEGNLDMDNRYAPGVRLGYHFDDFWLD 42

CcolistrainBP3183cadF MKKIFLCLGLASVLFGADNNVKFEITPTLNYNYFEGNLDMDNRYAPGVRLGYHFDDFWLD 60

30CjejunicadF ---------------------------TLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

1162CjejunicadF -----------------------EITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42

2038CjejunicadF ------------------------ITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42



****************:****:*******

450

CcolistrainBG2108cadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGGGYEDFSKGAFDN 120

3064CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGVGYEDFSKGAFDN 102

2119CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGGGYEDFSKGAFDN 102

2165CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGGGYEDFSKGAFDN 102





1206CjejunicadF QLEFGLEYYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102

CjejuniNCTC11168cadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 120

2170CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102


2040CcolicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102

1768CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLNAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102





CcolistrainBP3183cadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 120






451

***:***:********:. *********.******:*********** ******:.*:**

CcolistrainBG2108cadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 180

3064CcolicadF KSGGFGHYGAGLKFRLNDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162

2119CcolicadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162






1206CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162

CjejuniNCTC11168cadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 180



2040CcolicadF KSGGFGHYGTGVKFCLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162


813CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGSKKEKAVEEV 162




CcolistrainBP3183cadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 180





452


*********:*:** *.*************.*:.*:*.************.*:**.* *

CcolistrainBG2108cadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 240

3064CcolicadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222



175CcolicadF TKEVVNKPQVVTPVPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222

1980CcolicadF TKEVVNKPQVVTPVPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222



1206CjejunicadF A-------------DTRATPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209

CjejuniNCTC11168cadF A-------------DTRATPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 227

2170CjejunicadF A-------------DTRATPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209

62CjejunicadF A-------------DTRATPQVKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209

2040CcolicadF A-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209

1768CjejunicadF A-------------DTRPAPQTKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209

813CjejunicadF G-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209

683CjejunicadF A-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 208



CcolistrainBP3183cadF A-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 227




453



: . * *** ***************** *****.***..***:*

CcolistrainBG2108cadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 300

3064CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282







1206CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269

CjejuniNCTC11168cadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 287



2040CcolicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269






CcolistrainBP3183cadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 287



454




:*:*****::**** ****************:**********:***:****:**:****:

CcolistrainBG2108cadF TVGYGQDKPRSSNDTKEGRADNRRVEAKFILN 332

3064CcolicadF TVGYGQDKPRSSNDTKEGRAD----------- 314

2119CcolicadF TVGYGQDKPRSSNDTKEGRADNRR-------- 314



1980CcolicadF TVGYGQDKPRSSNDTKEGRADN---------- 314



1206CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVD------ 301

CjejuniNCTC11168cadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFILR 319

2170CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRV------- 301

62CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFILR 301

2040CcolicadF TVGYGQDNPRSSNDTKEGRADNRR-------- 301

1768CjejunicadF TVGCGQDNPRSSNDTKEGRADNRR-------- 301

813CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVD------ 301

683CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFI-- 300


3050CjejunicadF TVGYGQDNPRSSNDTKEGRA------------ 301

CcolistrainBP3183cadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFILR 319

30CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFIL- 301

455


2038CjejunicadF TVGYGQDNPRSSNDTKEGRADNR--------- 301

2114CjejunicadF TVGYGQDNPRSSNDTKEGRADNRR-------- 301

2072CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVDAK---- 301

*** ***:************

456

Appendix 3.4.3: Peb1A amino acid

The peb1A primer used in this study demonstrated the subsequent amino acid

sequence starting at positions 1–7 and ending at positions 253–259. A total of

259 amino acids were generated from the selected C. jejuni and C. coli

clusters.

Five different groups were identified in the C. jejuni clusters, whereas, the C.

coli clusters had a unique group. Group 1 contained the majority of the

selected C. jejuni (clusters 1, 2, 3, 6, 8, 28, and 29). Group 2 had three C.

jejuni clusters (clusters 26, 27, and 36), sharing 100% similarity to Peb1A of

the C. jejuni YH002. This group had one different amino acid (K) at position

139, which was conserved between amino acid groups with strong

physicochemical similarities. Group 3 (C. jejuni cluster 5) had two different

amino acids at positions 50 (Y) and 66 (I) which were conserved between

amino acid groups with strong physicochemical similarities. Group 4 (C.

jejuni cluster 12) had two different amino acids (positions 17 and 165). The

position 17 (V) was conserved between amino acid groups with strongly

similar properties, compared among C. jejuni. In contrast, the amino acid at

position 165 from this group (K) and other C. jejuni clusters (E) was non-

conserved to that of all C. coli clusters (T). Group 5 (C. jejuni cluster 39) had

a different amino acid at the position 57 (V) which was conserved between

amino acid groups of weak physicochemical similarities. By contrast, all C.

coli clusters shared 100% similarity of Peb1A amino acids to each other and

C. coli YH501and. The alignment analysis of the selected C. coli and C. jejuni

clusters showed 38 different amino acid position identified. Twenty-six

positions were conserved between amino acid groups showing of strong

physicochemical similarities (positions 13, 19, 22, 33, 36, 39, 41, 42, 71, 80,

81, 83, 86, 118, 130, 147, 172, 194, 210, 211, 220, 236, 237, 240, 245, and

246). Seven amino acid positions were conserved between amino acids with

weak physicochemical similarities (positions 23, 150, 162, 164, 173, 212, and

248). Four amino acid positions were non-conserved at positions 12, 165,

208, 233, and 234.

457

1980Ccolipeb ------LLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60

2040Ccolipeb -----SLLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60




CcoliYH501peb -----SLLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60




1206Cjejunipeb -----SLLKLAVFALGVCVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60

62Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPYYALLDQATGE 60

2072Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQVTGE 60

CjejuniYH002peb MVFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60

2038Cjejunipeb -----SLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60

2170Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60




687Cjejunipeb MVFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60


1162Cjejunipeb ------LLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60



*****.:***.*:**:.*********:**:**:*::*******:******.***

458

1980Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120





CcoliYH501peb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120




1206Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120

62Cjejunipeb IKGFEIDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120


CjejuniYH002peb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120











*****:****:********::*:**:*******************************:**

459

1980Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180





CcoliYH501peb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180




1206Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGKAAKKIGIDVKFSEFP 180

62Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180


CjejuniYH002peb FSEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180

2038Cjejunipeb FSEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180










*********:********:*******:**.***********.*. ******:.*******

460

1980Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240





CcoliYH501peb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240




1206Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240



CjejuniYH002peb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240











*************:************* *::.*******:************ *::**:

461

1980Ccolipeb DFVKQNKTEIDAL------ 259

2040Ccolipeb DFVKQNKTEIDALA----- 259

2119Ccolipeb DFVKQNKTEIDALAKKWGL 259



CcoliYH501peb DFVKQNKTEIDALAKKWGL 259

56Ccolipeb DFVKQNKTEIDALAKKW-- 259

175Ccolipeb DFVKQNKTEIDAL------ 259


1206Cjejunipeb DFVKEHKNEIDALAKKWGL 259


2072Cjejunipeb DFVKEHKNEIDALAKKWG- 259

CjejuniYH002peb DFVKEHKNEIDALAKKWGL 259







813Cjejunipeb DFVKEHKNEIDALAKKW-- 259

1162Cjejunipeb DFVKEHKNEIDALAKK--- 259



****::*.*****

462

Appendix 3.4.4: CjaA amino acid

The cjaA-C. coli primer used in this study demonstrated the starting the

subsequent amino acid at positions 23–35 and ending at the positions 268–

278. A total of 255 amino acids was generated from the selected C. jejuni and

C. coli clusters using the cjaA-C. coli oligonucleotide primers. Four different

groups were identified among the selected C. jejuni and C. coli clusters.

Group 1 consisted of 7 C. jejuni (clusters 1, 3, 5, 12, 26, 27, and 36) and 4 C.

coli (clusters 2, 3, 5, and 13), which shared 100% similarity of CjaA amino

acids with the reference C. jejuni (Accession number Y10872.1). Group 2

consisted of C. jejuni clusters 2, 8, 28, 29, and 39, and C. coli clusters 6, 21,

and 23. This group had two different amino acids at the positions 60 (I) and

191 (N), compared with other genotypes. These amino acids were conserved

between amino acid groups with strong physicochemical similarities. Group

3 had one (C. jejuni cluster 6) and Group 4 (C. coli cluster 1) had a different

amino acid at positions 235 (E) and 202 (S), respectively. These two different

amino acids were conserved between amino acid groups with strong

physicochemical similarities. For C. coli strain YH502, an amino acid

conserved substitution with weakly similar properties was found at the

position 254 (V), compared with others (A).

463

687CjejunicjaA ---------------------------------SGASNSLERIKQDGVVRIGVFGDKPPF 30

1162CjejunicjaA ------------------------------------SNSLERIKQDGVVRIGVFGDKPPF 30

1768CjejunicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30

2072CjejunicjaA ----------------------------------GASNSLERIKQDGVVRIGVFGDKPPF 30


175CcolicjaA -----------------------------------ASNSLERIKQDGVVRIGVFGDKPPF 30


2040CcolicjaA ------------------------------------SNSLERIKQDGVVRIGVFGDKPPF 30



CcolistrainYH502cjaA --------MKKMLLSIFTTFVAVFLAACGGNSDSGASNSLERIKQDGVVRIGVFGDKPPF 52

C.jejunicjaAgene,GenBank:Y10872.1 --------MKKMLLSIFTTFVAVFLAACGGNSDSGASNSLERIKQDGVVRIGVFGDKPPF 52



813CjejuniajaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30



2170CjejunicjaA ------------------------------------------IKQDGVVRIGVFGDKPPF 30


2119CcolicjaA ------------------------------------------IKQDGVVRIGVFGDKPPF 30

2165CcolicjaA ----------------------------------GASNSLERIKQDGVVRIGVFGDKPPF 30

2887CcolicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30


******************

464

687CjejunicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90





175CcolicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90



56CcolicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90

30CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90

CcolistrainYH502cjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 112

C.jejunicjaAgene,GenBank:Y10872.1 GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 112



813CjejuniajaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90









*******:****************************************************

465

687CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150





175CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150





CcolistrainYH502cjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 172

C.jejunicjaAgene,GenBank:Y10872.1 PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 172



813CjejuniajaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150









************************************************************

466

687CjejunicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210





175CcolicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210



56CcolicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFSWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210

30CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210

CcolistrainYH502cjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 232

C.jejunicjaAgene,GenBank:Y10872.1 LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 232



813CjejuniajaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210





2119CcolicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210




******************:**********:******************************

467

687CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGG-- 255

1162CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVV------ 255

1768CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGK- 256



175CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDV------- 255




30CjejunicjaA ELEEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVV------ 255

CcolistrainYH502cjaA ELKEFIDNLITKLGEEQFFHKVYDETLKSHFGDDVKADDVVIEGGKI 279

C.jejunicjaAgene,GenBank:Y10872.1 ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGKI 279



813CjejuniajaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIE---- 255



2170CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVK----------- 255



2165CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADD-------- 255

2887CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVI----- 255


**:******************.**************

468

Appendix 3.5: Nucleotide sequence analysis from pET SUMO

C. jejuni cluster 27 was used as the original DNA template for cloning into pET SUMO. Consequently, the ligated pET SUMO plasmids carrying katA,

cadF, peb1A, or cjaA were analysed for DNA sequencing. In Appendices 3.13.1-3.13.4, the red colour indicates the nucleotide sequence of the pET

SUMO fusion protein. The green and yellow colours indicate the forward and reverse primers used, respectively. The red font indicates the mismatch of

the oligonucleotide in the cloned pET SUMO vector contained inserted gene and the original inserted gene of C. jejuni cluster 27.

Appendix 3.5.1: Nucleotide sequence analysis of pET SUMO-katA

The inserted katA gene was found at the SUMO cleavage site and that the nucleotide sequences obtained from the pET SUMO-katA and the original

katA gene were identical.

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60 70

pET SUMO-katA fusion protein ATGGGCAGCA GCCATCATCA TCATCATCAC GGCAGCGGCC TGGTGCCGCG CGGCAGCGCT AGCATGTCGG

pET SUMO-katA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

80 90 100 110 120 130 140

pET SUMO-katA fusion protein ACTCAGAAGT CAATCAAGAA GCTAAGCCAG AGGTCAAGCC AGAAGTCAAG CCTGAGACTC ACATCAATTT

pET SUMO-katA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

469

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

150 160 170 180 190 200 210

pET SUMO-katA fusion protein AAAGGTGTCC GATGGATCTT CAGAGATCTT CTTCAAGATC AAAAAGACCA CTCCTTTAAG AAGGCTGATG

pET SUMO-katA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

220 230 240 250 260 270 280

pET SUMO-katA fusion protein GAAGCGTTCG CTAAAAGACA GGGTAAGGAA ATGGACTCCT TAAGATTCTT GTACGACGGT ATTAGAATTC

pET SUMO-katA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

290 300 310 320 330 340 350

pET SUMO-katA fusion protein AAGCTGATCA GACCCCTGAA GATTTGGACA TGGAGGATAA CGATATTATT GAGGCTCACA GAGAACAGAT

pET SUMO-katA 2170 ---------- ---------- ---------- TGGAGGATAA CGATATTATT GAGGCTCACA GAGAACAGAT

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

360 370 380 390 400 410 420

pET SUMO-katA fusion protein TGGTGGT--- ---------- ---------- ---------- ---------- ---------- ----------

pET SUMO-katA 2170 TGGTGGTGAA GCTTCTATGG AAAGTTTACA TCAAGTAACC ATTCTTATGA GCGATAGAGG AATTCCTGCA

KatA C jejuni 2170 PCR ---------- ---------- ---------A TCAAGTAACC ATTCTTATGA GCGATAGAGG AATTCCTGCA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480 490

470

pET SUMO-katA fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------

pET SUMO-katA 2170 AGTTATCGTC ATATGCATGG ATTTGGAAGC CATACTTATA GTTTTATTAA TGATAAAAAT GAAAGATTTT

KatA C jejuni 2170 PCR AGTTATCGTC ATATGCATGG ATTTGGAAGC CATACTTATA GTTTTATTAA TGATAAAAAT GAAAGATTTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

500 510 520 530 540 550 560


pET SUMO-katA 2170 GGGTGAAATT CCATTTTAAA ACCCAACAAG GGATTAAAAA TCTTACCAAC CAAGAAGCTG CCGAGCTTAT

KatA C jejuni 2170 PCR GGGTGAAATT CCATTTTAAA ACCCAACAAG GGATTAAAAA TCTTACCAAC CAAGAAGCTG CCGAGCTTAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

570 580 590 600 610 620 630


pET SUMO-katA 2170 AGCAAAAGAT AGAGAAAGTC ATCAAAGAGA TCTCTATAAT GCTATAGAAA ATAAAGATTT TCCAAAATGG

KatA C jejuni 2170 PCR AGCAAAAGAT AGAGAAAGTC ATCAAAGAGA TCTCTATAAT GCTATAGAAA ATAAAGATTT TCCAAAATGG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

640 650 660 670 680 690 700


pET SUMO-katA 2170 AAAGTTCAAG TTCAAATTCT TGCTGAAAAA GATATAGAAA AACTTGGATT TAATCCTTTT GATTTAACAA

KatA C jejuni 2170 PCR AAAGTTCAAG TTCAAATTCT TGCTGAAAAA GATATAGAAA AACTTGGATT TAATCCTTTT GATTTAACAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

710 720 730 740 750 760 770


pET SUMO-katA 2170 AAATTTGGCC TCATAGTCTT GTGCCTTTGA TGGATATAGG CGAAATGATT CTAAACAAAA ATCCTCAAAA

471

KatA C jejuni 2170 PCR AAATTTGGCC TCATAGTCTT GTGCCTTTGA TGGATATAGG CGAAATGATT CTAAACAAAA ATCCTCAAAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

780 790 800 810 820 830 840


pET SUMO-katA 2170 TTATTTTAAT GAAGTTGAAC AAGCTGCCTT TAGTCCAAGC AATATCGTTC CTGGAATTGG CTTTAGCCCT

KatA C jejuni 2170 PCR TTATTTTAAT GAAGTTGAAC AAGCTGCCTT TAGTCCAAGC AATATCGTTC CTGGAATTGG CTTTAGCCCT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

850 860 870 880 890 900 910


pET SUMO-katA 2170 GATAAAATGT TGCAAGCTAG AATTTTTTCA TATCCTGATG CACAAAGATA TAGAATAGGA ACTAATTATC

KatA C jejuni 2170 PCR GATAAAATGT TGCAAGCTAG AATTTTTTCA TATCCTGATG CACAAAGATA TAGAATAGGA ACTAATTATC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

920 930 940 950 960 970 980


pET SUMO-katA 2170 ATCTTTTGCC CGTAAATCGT GCAAAAAGCG AAGTGAATAC TTACAATGTC GCTGGTGCTA TGAATTTTGA

KatA C jejuni 2170 PCR ATCTTTTGCC CGTAAATCGT GCAAAAAGCG AAGTGAATAC TTACAAT--- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

990 1000 1010 1020 1030 1040 1050

pET SUMO-katA fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ---AGACAAG

pET SUMO-katA 2170 TAGTTATAAA AATGATGCAG CTTATTATGA ACCAAACAGC TATGATAATA GCCCAGGATC CACAGACAAG

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

472

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1060 1070 1080 1090 1100 1110 1120

pET SUMO-katA fusion protein CTTAGGTATT TATTCGGCGC AAAGTGCGTC GGGTGATGCT GCCAACTTAG TCGAGCACCA CACCACCACA

pET SUMO-katA 2170 CTTAGGTATT TATTCGGCGC AAAGTGCGTC GGGTGATGCT GCCAACTTAG TC-------- ----------

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1130 1140 1150 1160 1170 1180 1190

pET SUMO-katA fusion protein CTGAGATCCG GCTGCTACCA ACCCCGAAAG GAGCTGAGTT GGTGCTGCCC CGCTGAGCAA AACTAGCTAA

pET SUMO-katA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

KatA C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....|

1200 1210 1220 1230

pET SUMO-katA fusion protein CCCCCTGGGG CCTCAAACGG GTCTGGGGGG TTTTTGCTGG

pET SUMO-katA 2170 ---------- ---------- ---------- ----------

KatA C jejuni 2170 PCR ---------- ---------- ---------- ----------

Appendix 3.5.2: Nucleotide sequence analysis of pET SUMO-cadF

The insertion of cadF gene occurred at the SUMO cleavage site, and one mismatch in the nucleotide sequences was found between the pET SUMO-

cadF and the original cadF gene. One mismatch nucleotide was found at the position of 1140 bp. The nucleotide base T was identified from the PCR

detection, but it was C from cloned plasmid at the same position.

473

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60 70

pET SUMO fusion protein ATGGGCAGCA GCCATCATCA TCATCATCAC GGCAGCGGCC TGGTGCCGCG CGGCAGCGCT AGCATGTCGG

pET SUMO-cadF 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

cadF C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

80 90 100 110 120 130 140

pET SUMO fusion protein ACTCAGAAGT CAATCAAGAA GCTAAGCCAG AGGTCAAGCC AGAAGTCAAG CCTGAGACTC ACATCAATTT

pET SUMO-cadF 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

cadF C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

150 160 170 180 190 200 210

pET SUMO fusion protein AAAGGTGTCC GATGGATCTT CAGAGATCTT CTTCAAGATC AAAAAGACCA CTCCTTTAAG AAGGCTGATG

pET SUMO-cadF 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

cadF C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

220 230 240 250 260 270 280

pET SUMO fusion protein GAAGCGTTCG CTAAAAGACA GGGTAAGGAA ATGGACTCCT TAAGATTCTT GTACGACGGT ATTAGAATTC

pET SUMO-cadF 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

cadF C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

290 300 310 320 330 340 350

pET SUMO fusion protein AAGCTGATCA GACCCCTGAA GATTTGGACA TGGAGGATAA CGATATTATT GAGGCTCACA GAGAACAGAT

pET SUMO-cadF 2170 ---------- ---------- ---------A TGGAGGATAA CGATATTATT GAGGCTCACA GAGAACAGAT

474

cadF C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

360 370 380 390 400 410 420

pET SUMO fusion protein TGGTGGTG-- ---------- ---------- ---------- ---------- ---------- ----------

pET SUMO-cadF 2170 TGGTGGTGCT CGAGCTGGTG CTGATAACAA TGTAAAATTT GAAATCACTC CAACTTTAAA CTATAATTAC

cadF C jejuni 2170 PCR ---------- ---------- ---------- ---------T GAAATCACTC CAACTTTAAA CTATAATTAC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480 490

pET SUMO fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------

pET SUMO-cadF 2170 TTTGAAGGTA ATTTAGATAT GGATAATCGT TATGCACCAG GGATTAGACT TGGTTATCAT TTTGACGATT

cadF C jejuni 2170 PCR TTTGAAGGTA ATTTAGATAT GGATAATCGT TATGCACCAG GGATTAGACT TGGTTATCAT TTTGACGATT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

500 510 520 530 540 550 560


pET SUMO-cadF 2170 TTTGGCTTGA TCAATTAGAA TTTGGGTTAG AGCATTATTC TGATGTTAAA TATACAAATA CAAATAAAAC

cadF C jejuni 2170 PCR TTTGGCTTGA TCAATTAGAA TTTGGGTTAG AGCATTATTC TGATGTTAAA TATACAAATA CAAATAAAAC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

570 580 590 600 610 620 630


pET SUMO-cadF 2170 TACAGATATT ACAAGAACTT ATTTGAGTGC TATTAAAGGT ATTGATGTAG GTGAGAAATT TTATTTCTAT

cadF C jejuni 2170 PCR TACAGATATT ACAAGAACTT ATTTGAGTGC TATTAAAGGT ATTGATGTAG GTGAGAAATT TTATTTCTAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

475

640 650 660 670 680 690 700


pET SUMO-cadF 2170 GGTTTAGCAG GTGGAGGATA TGAGGATTTT TCAAATGCTG CTTATGATAA TAAAAGCGGT GGATTTGGAC

cadF C jejuni 2170 PCR GGTTTAGCAG GTGGAGGATA TGAGGATTTT TCAAATGCTG CTTATGATAA TAAAAGCGGT GGATTTGGAC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

710 720 730 740 750 760 770


pET SUMO-cadF 2170 ATTATGGCGC GGGTGTAAAA TTCCGTCTTA GTGATTCTTT GGCTTTAAGA CTTGAAACTA GAGATCAAAT

cadF C jejuni 2170 PCR ATTATGGCGC GGGTGTAAAA TTCCGTCTTA GTGATTCTTT GGCTTTAAGA CTTGAAACTA GAGATCAAAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

780 790 800 810 820 830 840


pET SUMO-cadF 2170 TAATTTCAAT CATGCAAACC ATAATTGGGT TTCAACTTTA GGTATTAGTT TTGGTTTTGG TGGCAAAAAG

cadF C jejuni 2170 PCR TAATTTCAAT CATGCAAACC ATAATTGGGT TTCAACTTTA GGTATTAGTT TTGGTTTTGG TGGCAAAAAG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

850 860 870 880 890 900 910


pET SUMO-cadF 2170 GAAAAAGCTG TAGAAGAAGT TGCTGATACT CGTGCAACTC CACAAGCAAA ATGTCCTGTT GAACCAAGAG

cadF C jejuni 2170 PCR GAAAAAGCTG TAGAAGAAGT TGCTGATACT CGTGCAACTC CACAAGCAAA ATGTCCTGTT GAACCAAGAG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

920 930 940 950 960 970 980


pET SUMO-cadF 2170 AAGGTGCTTT GTTAGATGAA AATGGTTGCG AAAAAACTAT TTCTTTGGAA GGTCATTTTG GTTTTGATAA

cadF C jejuni 2170 PCR AAGGTGCTTT GTTAGATGAA AATGGTTGCG AAAAAACTAT TTCTTTGGAA GGTCATTTTG GTTTTGATAA

476

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

990 1000 1010 1020 1030 1040 1050


pET SUMO-cadF 2170 AACTACTATA AATCCAACTT TTCAAGAAAA AATCAAAGAA ATTGCAAAAG TTTTAGATGA AAATGAAAGA

cadF C jejuni 2170 PCR AACTACTATA AATCCAACTT TTCAAGAAAA AATCAAAGAA ATTGCAAAAG TTTTAGATGA AAATGAAAGA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1060 1070 1080 1090 1100 1110 1120


pET SUMO-cadF 2170 TATGATACTA TTCTTGAAGG ACATACAGAT AATATCGGTT CAAGAGCTTA TAATCAAAAG CTTTCTGAAA

cadF C jejuni 2170 PCR TATGATACTA TTCTTGAAGG ACATACAGAT AATATCGGTT CAAGAGCTTA TAATCAAAAG CTTTCTGAAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1130 1140 1150 1160 1170 1180 1190


pET SUMO-cadF 2170 GACGTGCTAA AAGTGTTGCC AATGAACTTG AAAAATATGG TGTAGAAAAA AGTCGCATCA AAACAGTAGG

cadF C jejuni 2170 PCR GACGTGCTAA AAGTGTTGCT AATGAACTTG AAAAATATGG TGTAGAAAAA AGTCGCATCA AAACAGTAGG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1200 1210 1220 1230 1240 1250 1260


pET SUMO-cadF 2170 TTATGGTCAA GATAATCCTC GCTCAAGCCA TGACACTAAA GAAGGTAGAG CGGATAATAG AAGAGTGGAT

cadF C jejuni 2170 PCR TTATGGTCAA GATAATCCTC GCTCAAGCAA TGACACTAAA GAAGGTAGAG CGGATAATAG AAGAGTGGA-

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1270 1280 1290 1300 1310 1320 1330

477

pET SUMO fusion protein ---------- -AGACAAGCT TAGGTATTTA TTCGGCGCAA AGTGCGTCGG GTGATGCTGC CAACTTAGTC

pET SUMO-cadF 2170 GCTGGATCCA CAGACAAGCT TAGGTATTTA TTCGGCGCAA AGTGCGTCGG GTGATGCTGC CAACTTAGTC

cadF C jejuni 2170 PCR ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....

1340 1350

pET SUMO fusion protein GAGCACCACA CCACCACACT GAGATCCGG

pET SUMO-cadF 2170 GAGCACCACA CCACCACACT GAGATCCGG

cadF C jejuni 2170 PCR .......... .......... .........

Appendix 3.5.3: Nucleotide sequence analysis of pET SUMO-peb1A

Two mismatch nucleotides were found at the position of 570 and 1006 bp (red font).

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60 70

pETSUMO-peb1A fusion protein ATGGGCAGCA GCCATCATCA TCATCATCAC GGCAGCGGCC TGGTGCCGCG CGGCAGCGCT AGCATGTCGG

pETSUMOpeb1A 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

PCR C jejuni 2170 peb1A ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

80 90 100 110 120 130 140

pETSUMO-peb1A fusion protein ACTCAGAAGT CAATCAAGAA GCTAAGCCAG AGGTCAAGCC AGAAGTCAAG CCTGAGACTC ACATCAATTT

pETSUMOpeb1A 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

478

PCR C jejuni 2170 peb1A ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

150 160 170 180 190 200 210

pETSUMO-peb1A fusion protein AAAGGTGTCC GATGGATCTT CAGAGATCTT CTTCAAGATC AAAAAGACCA CTCCTTTAAG AAGGCTGATG

pETSUMOpeb1A 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

PCR C jejuni 2170 peb1A ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

220 230 240 250 260 270 280

pETSUMO-peb1A fusion protein GAAGCGTTCG CTAAAAGACA GGGTAAGGAA ATGGACTCCT TAAGATTCTT GTACGACGGT ATTAGAATTC

pETSUMOpeb1A 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

PCR C jejuni 2170 peb1A ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

290 300 310 320 330 340 350

pETSUMO-peb1A fusion protein AAGCTGATCA GACCCCTGAA GATTTGGACA TGGAGGATAA CGATATTATT GAGGCTCACA GAGAACAGAT

pETSUMOpeb1A 2170 ---------- ---------- ---------- ---------- ---------- ---------A GAGAACAGAT

PCR C jejuni 2170 peb1A ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

360 370 380 390 400 410 420

pETSUMO-peb1A fusion protein TGGTGGT--- ---------- ---------- ---------- ---------- ---------- ----------

pETSUMOpeb1A 2170 TGGTGGTGCT CGAGCTTCTT TGTTAAAGTT GGCAGTTTTT GCTCTAGGTG CTTGTGTTGC ATTTAGCAAT

PCR C jejuni 2170 peb1A ----GTTTTT AGAAAATCTT TGTTAAAGTT GGCAGTTTTT GCTCTAGGTG CTTGTGTTGC ATTTAGCAAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480 490

479

pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------

pETSUMOpeb1A 2170 GCTAATGCAG CAGAAGGTAA GCTTGAGTCT ATTAAATCTA AAGGACAATT AATAGTTGGT GTTAAAAATG

PCR C jejuni 2170 peb1A GCTAATGCAG CAGAAGGTAA GCTTGAGTCT ATTAAATCTA AAGGACAATT AATAGTTGGT GTTAAAAATG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

500 510 520 530 540 550 560


pETSUMOpeb1A 2170 ATGTTCCGCA TTATGCTTTA CTTGATCAAG CAACAGGTGA AATTAAAGGT TTCGAAGTAG ATGTTGCCAA

PCR C jejuni 2170 peb1A ATGTTCCGCA TTATGCTTTA CTTGATCAAG CAACAGGTGA AATTAAAGGT TTCGAAGTAG ATGTTGCCAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

570 580 590 600 610 620 630


pETSUMOpeb1A 2170 ATTGCTAGCC AAAAGTATAT TGGGTGATGA TAAAAAAATA AAACTAGTTG CAGTTAATGC TAAAACAAGA

PCR C jejuni 2170 peb1A ATTGCTAGCT AAAAGTATAT TGGGTGATGA TAAAAAAATA AAACTAGTTG CAGTTAATGC TAAAACAAGA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

640 650 660 670 680 690 700


pETSUMOpeb1A 2170 GGCCCTTTGC TTGATAATGG TAGTGTAGAT GCAGTGATAG CAACTTTTAC TATTACTCCA GAGAGAAAAA

PCR C jejuni 2170 peb1A GGCCCTTTGC TTGATAATGG TAGTGTAGAT GCAGTGATAG CAACTTTTAC TATTACTCCA GAGAGAAAAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

710 720 730 740 750 760 770


pETSUMOpeb1A 2170 GAATTTATAA TTTCTCAGAA CCTTATTATC AAGATGCTAT AGGGCTTTTG GTTTTAAAAG AAAAAAAATA

PCR C jejuni 2170 peb1A GAATTTATAA TTTCTCAGAA CCTTATTATC AAGATGCTAT AGGGCTTTTG GTTTTAAAAG AAAAAAAATA

480

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

780 790 800 810 820 830 840


pETSUMOpeb1A 2170 TAAATCTTTA GCTGATATGA AAGGTGCAAA TATTGGAGTG GCTCAAGCTG CAACTACAAA AAAAGCTATA

PCR C jejuni 2170 peb1A TAAATCTTTA GCTGATATGA AAGGTGCAAA TATTGGAGTG GCTCAAGCTG CAACTACAAA AAAAGCTATA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

850 860 870 880 890 900 910


pETSUMOpeb1A 2170 GGTGAAGCTG CTAAAAAAAT TGGCATTGAT GTTAAATTTA GTGAATTTCC TGATTATCCA AGTATAAAAG

PCR C jejuni 2170 peb1A GGTGAAGCTG CTAAAAAAAT TGGCATTGAT GTTAAATTTA GTGAATTTCC TGATTATCCA AGTATAAAAG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

920 930 940 950 960 970 980


pETSUMOpeb1A 2170 CTGCTTTAGA TGCTAAAAGA GTTGATGCGT TTTCTGTAGA CAAATCAATA TTGTTAGGTT ATGTGGATGA

PCR C jejuni 2170 peb1A CTGCTTTAGA TGCTAAAAGA GTTGATGCGT TTTCTGTAGA CAAATCAATA TTGTTAGGTT ATGTGGATGA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

990 1000 1010 1020 1030 1040 1050


pETSUMOpeb1A 2170 TAAAAGTGAA ATTTTGCCAG ATAGTCTTGA ACCACAAAGT TATGGTATTG TAACCAAAAA AGATGATCCA

PCR C jejuni 2170 peb1A TAAAAGTGAA ATTTTGCCAG ATAGTTTTGA ACCACAAAGT TATGGTATTG TAACCAAAAA AGATGATCCA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1060 1070 1080 1090 1100 1110 1120

481


pETSUMOpeb1A 2170 GCTTTTGCAA AATATGTTGA TGATTTTGTA AAAGAACATA AAAATGAAAT TGATGCTTTA GCGAAAGGAT

PCR C jejuni 2170 peb1A GCTTTTGCAA AATATGTTGA TGATTTTGTA AAAGAACATA AAAATGAAAT TGATGCTTTA GCGAAAAAAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1130 1140 1150 1160 1170 1180 1190

pETSUMO-peb1A fusion protein ----AGACAA GCTTAGGTAT TTATTCGGCG CAAAGTGCGT CGGGTGATGC TGCCAACTTA GTCGAGCACC

pETSUMOpeb1A 2170 CCACAGACAA GCTTAGGTAT TTATTCGGCG CAAAGTGCGT CGGGTGATGC TGCCAACTTA GTCGAGCACC

PCR C jejuni 2170 peb1A GGGGTTTA-- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....|

1200 1210 1220

pETSUMO-peb1A fusion protein ACACCACCAC ACTGAGATCC GG--------

pETSUMOpeb1A 2170 ---------- ---------- ----------

PCR C jejuni 2170 peb1A ---------- ---------- ----------

Appendix 3.5.4: Nucleotide sequence analysis of pET SUMO-cjaA

Three mismatch nucleotides were found at the position of 708, 831, and 927 bp.

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

10 20 30 40 50 60 70

pETSUMO fusion protein ATGGGCAGCA GCCATCATCA TCATCATCAC GGCAGCGGCC TGGTGCCGCG CGGCAGCGCT AGCATGTCGG

pETSUMO-cjaA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

PCR cjaA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

482

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

80 90 100 110 120 130 140

pETSUMO fusion protein ACTCAGAAGT CAATCAAGAA GCTAAGCCAG AGGTCAAGCC AGAAGTCAAG CCTGAGACTC ACATCAATTT

pETSUMO-cjaA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

PCR cjaA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

150 160 170 180 190 200 210

pETSUMO fusion protein AAAGGTGTCC GATGGATCTT CAGAGATCTT CTTCAAGATC AAAAAGACCA CTCCTTTAAG AAGGCTGATG

pETSUMO-cjaA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

PCR cjaA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

220 230 240 250 260 270 280

pETSUMO fusion protein GAAGCGTTCG CTAAAAGACA GGGTAAGGAA ATGGACTCCT TAAGATTCTT GTACGACGGT ATTAGAATTC

pETSUMO-cjaA 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

PCR cjaA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

290 300 310 320 330 340 350

pETSUMO fusion protein AAGCTGATCA GACCCCTGAA GATTTGGACA TGGAGGATAA CGATATTATT GAGGCTCACA GAGAACAGAT

pETSUMO-cjaA 2170 ---------- ---------- ---------- TGGAGGATAA CGATATTATT GAGGCTCACA GAGAACAGAT

PCR cjaA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

360 370 380 390 400 410 420

pETSUMO fusion protein TGGTGGT--- ---------- ---------- ---------- ---------- ---------- ----------

483

pETSUMO-cjaA 2170 TGGTGGTGCT CGAGCTATGC TCTTAAGTAT TTTTACAACC TTTGTTGCAG TATTTTTGGC TGCTTGTGGA

PCR cjaA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

430 440 450 460 470 480 490

pETSUMO fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------

pETSUMO-cjaA 2170 GGAAATTCAG ATTCTGGTGC TTCAAATTCT CTTGAAAGAA TCAAGCAAGA TGGAGTAGTA AGAATAGGAG

PCR cjaA C jejuni 2170 ---------- ---------- ---------- -------GAA TCAAGCAAGA TGGAGTAGTA AGAATAGGAG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

500 510 520 530 540 550 560


pETSUMO-cjaA 2170 TTTTTGGAGA TAAACCGCCT TTTGGTTATG TAGATGAAAA AGGCGTAAAT CAAGGTTATG ATATAGTCTT

PCR cjaA C jejuni 2170 TTTTTGGAGA TAAACCGCCT TTTGGTTATG TAGATGAAAA AGGCGTAAAT CAAGGTTATG ATATAGTCTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

570 580 590 600 610 620 630


pETSUMO-cjaA 2170 GGCGAAACGT ATAGCAAAAG AACTCTTAGG AGATGAAAAT AAGGTGCAGT TTGTATTAGT TGAAGCTGCA

PCR cjaA C jejuni 2170 GGCGAAACGT ATAGCAAAAG AACTCTTAGG AGATGAAAAT AAGGTGCAGT TTGTATTAGT TGAAGCTGCA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

640 650 660 670 680 690 700


pETSUMO-cjaA 2170 AATAGGGTGG AATTTTTAAA ATCAAATAAA GTTGATATTA TTTTAGCTAA TTTTACTCAA ACACCTGAAA

PCR cjaA C jejuni 2170 AATAGGGTGG AATTTTTAAA ATCAAATAAA GTTGATATTA TTTTAGCTAA TTTTACTCAA ACACCTGAAA

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

484

710 720 730 740 750 760 770


pETSUMO-cjaA 2170 GAGCAGAACA AGTGGATTTT TGCTTACCTT ATATGAAGGT AGCTTTAGGT GTGGCTGTGC CTCAAGATAG

PCR cjaA C jejuni 2170 GAGCAGAGCA AGTGGATTTT TGCTTACCTT ATATGAAGGT AGCTTTAGGT GTGGCTGTGC CTCAAGATAG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

780 790 800 810 820 830 840


pETSUMO-cjaA 2170 CAATATCAGT AGCATAGAAG ATTTAAAAGA TAAAACTTTA CTTTTAAACA AAGGAACTAC CGCTGATGCG

PCR cjaA C jejuni 2170 CAATATCAGT AGCATAGAAG ATTTAAAAGA TAAAACTTTA CTTTTAAACA AAGGAACTAC TGCTGATGCG

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

850 860 870 880 890 900 910


pETSUMO-cjaA 2170 TATTTTACAA AAGAATATCC TGATATTAAA ACATTAAAAT ACGATCAAAA TACCGAAACT TTTGCCGCTT

PCR cjaA C jejuni 2170 TATTTTACAA AAGAATATCC TGATATTAAA ACATTAAAAT ACGATCAAAA TACCGAAACT TTTGCCGCTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

920 930 940 950 960 970 980


pETSUMO-cjaA 2170 TAATAGATCA AAGAGGGGAT GCTTTAAGTC ATGACAATAC TTTGCTTTTT GCGTGGGTAA AAGAACATCC

PCR cjaA C jejuni 2170 TAATAGATCA AAGAGGTGAT GCTTTAAGTC ATGACAATAC TTTGCTTTTT GCGTGGGTAA AAGAACATCC

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

990 1000 1010 1020 1030 1040 1050


pETSUMO-cjaA 2170 TGAATTTAAA ATGGCCATTA AAGAATTGGG CAATAAAGAT GTAATTGCTC CTGCTGTTAA AAAAGGTGAT

485

PCR cjaA C jejuni 2170 TGAATTTAAA ATGGCCATTA AAGAATTGGG CAATAAAGAT GTAATTGCTC CTGCTGTTAA AAAAGGTGAT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1060 1070 1080 1090 1100 1110 1120


pETSUMO-cjaA 2170 AAAGAGCTTA AAGAATTTAT TGATAATCTA ATCACAAAAT TAGGAGAAGA ACAATTCTTC CATAAAGCTT

PCR cjaA C jejuni 2170 AAAGAGCTTA AAGAATTTAT TGATAATCTA ATCACAAAAT TAGGAGAAGA ACAATTCTTC CATAAAGCTT

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|....|

1130 1140 1150 1160 1170 1180 1190


pETSUMO-cjaA 2170 ATGATGAAAC TTTAAAAAGT CATTTTGGAG ATGATGTAAA AGCTGATGAT GTAGTTATTG AAGGCGGTGG

PCR cjaA C jejuni 2170 ATGATGAAAC TTTAAAAAGT CATTTTGGAG ATGATGTAAA AG-------- ---------- ----------

....|....| ....|....| ....|....| ....|....| ....|....| ....|....| ....|.

1200 1210 1220 1230 1240 1250

pETSUMO fusion protein -------GAC AAGCTTAGGT ATTTATTCGG CGCAAAGTGC GTCGGGTGAT GCTGCCACTT AGTCGA

pETSUMO-cjaA 2170 ATCCACAGAC AAGCTTAGGT ATTTATTCGG CGCAAAGTGC GTCGGGTGAT GCTGCCACTT AGTCGA

PCR cjaA C jejuni 2170 ---------- ---------- ---------- ---------- ---------- ---------- ------

Appendix 3.6.: The alignment analysis of subsequent amino acids of the ligated pET SUMO contained cadF or peb1A

The subsequent amino acids of pET SUMO contained cadF or peb1A were separately aligned with the original subsequent amino acid residues from C.

jejuni cluster 27 used as the DNA template. The green and yellow colours indicate the restriction site and extra nucleotide bases added in forward and

486

reverse primers, respectively. The fully conserved amino acids are indicated as “*”. The amino acids conserved between groups of strongly similar

properties are indicated as “:”.

Appendix 3.6.1: The alignment analysis of subsequent amino acids between pET SUMO-cadF and the original cadF gene

A protein of 304 amino acid residues was generated from the pET SUMO-cadF. The alignment analysis showed a different amino acid which was

conserved between amino acid groups, with strong physicochemical similarities.

PCRcadF2170 -----------EITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLDQLEFGLEHYSDV 49

pETSUMOcadF2170 ARAGADNNVKFEITPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLDQLEFGLEHYSDV 60

*************************************************

PCRcadF2170 KYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDNKSGGFGHYGAGV 109

pETSUMOcadF2170 KYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDNKSGGFGHYGAGV 120

************************************************************

PCRcadF2170 KFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEVADTRATPQAKCP 169

pETSUMOcadF2170 KFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEVADTRATPQAKCP 180

************************************************************

PCRcadF2170 VEPREGALLDENGCEKTISLEGHFGFDKTTINPTFQEKIKEIAKVLDENERYDTILEGHT 229

pETSUMOcadF2170 VEPREGALLDENGCEKTISLEGHFGFDKTTINPTFQEKIKEIAKVLDENERYDTILEGHT 240

************************************************************

487

PCRcadF2170 DNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIKTVGYGQDNPRSSNDTKEGRADNRRV 289

pETSUMOcadF2170 DNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIKTVGYGQDNPRSSHDTKEGRADNRRV 300

***********************************************:************

PCRcadF2170 ----- 289

pETSUMOcadF2170 DADPQ 305

Appendix 3.6.2: The alignment analysis of subsequent amino acids between pET SUMO-peb1A and the original peb1A gene

A protein of 255 amino acid residues was generated from the peb1A gene ligated into pET SUMO. One conserved amino acid between groups was found

and it had strong physicochemical similarities.

PCRpeb1A2170 VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGEI 60

pETSUMOpeb1A2170 -ARASLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGEI 56

* ********************************************************

PCRpeb1A2170 KGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYNF 120

pETSUMOpeb1A2170 KGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYNF 116

************************************************************

PCRpeb1A2170 SEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFPD 180

pETSUMOpeb1A2170 SEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFPD 176

************************************************************

PCRpeb1A2170 YPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVDD 240

488

pETSUMOpeb1A2170 YPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSLEPQSYGIVTKKDDPAFAKYVDD 236

*************************************:**********************

PCRpeb1A2170 FVKEHKNEIDALAKKWGL 258

pETSUMOpeb1A2170 FVKEHKNEIDALAKGS-- 252

**************

489

Appendix 4.1: DNA sequencing analysis of the recombinant pEGFP-C1 plasmids

The sequence alignment of the ORF amplicon of interest from the recombinant pEGFP-C1plasmid was compared with the ORF of interest in recombinant

pET SUMO and the pEGFP-C1 vector alone. The restriction sites were indicated as underlined letters. The nucleotide sequences of pEGFP-C1vector

and pET SUMO are indicated in green and yellow colours, respectively. Similar nucleotide sequences are indicated as *.

Appendix 4.1.1: Nucleotide analysis of pEGFP-C1-katA plasmid

The katA gene was cloned into the pEGFP-C1 vector in the correct orientation. The restriction sites for HindIII and BamHI-HF were found at positions

of 32–37 and 709–714, respectively.

pETSUMO-katA ATTGAGGCTCACAGAGAACAGATTGGTGGTGAAGCTTCTATGGAAAGTTTACATCAAGTA 8

pEGFPC1katA CTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCTATGGAAAGTTTACATCAAGTA 60

*****************************

Original ACCATTCTTATGAGCGATAGAGGAATTCCTGCAAGTTATCGTCATATGCATGGATTTGGA 68

pEGFPC1katA ACCATTCTTATGAGCGATAGAGGAATTCCTGCAAGTTATCGTCATATGCATGGATTTGGA 120

************************************************************

pETSUMO-katA AGCCATACTTATAGTTTTATTAATGATAAAAATGAAAGATTTTGGGTGAAATTCCATTTT 128

pEGFPC1katA AGCCATACTTATAGTTTTATTAATGATAAAAATGAAAGATTTTGGGTGAAATTCCATTTT 180

************************************************************

490

pETSUMO-katA AAAACCCAACAAGGGATTAAAAATCTTACCAACCAAGAAGCTGCCGAGCTTATAGCAAAA 188

pEGFPC1katA AAAACCCAACAAGGGATTAAAAATCTTACCAACCAAGAAGCTGCCGAGCTTATAGCAAAA 240

************************************************************

pETSUMO-katA GATAGAGAAAGTCATCAAAGAGATCTCTATAATGCTATAGAAAATAAAGATTTTCCAAAA 248

pEGFPC1katA GATAGAGAAAGTCATCAAAGAGATCTCTATAATGCTATAGAAAATAAAGATTTTCCAAAA 300

************************************************************

pETSUMO-katA TGGAAAGTTCAAGTTCAAATTCTTGCTGAAAAAGATATAGAAAAACTTGGATTTAATCCT 308

pEGFPC1katA TGGAAAGTTCAAGTTCAAATTCTTGCTGAAAAAGATATAGAAAAACTTGGATTTAATCCT 360

************************************************************

pETSUMO-katA TTTGATTTAACAAAAATTTGGCCTCATAGTCTTGTGCCTTTGATGGATATAGGCGAAATG 368

pEGFPC1katA TTTGATTTAACAAAAATTTGGCCTCATAGTCTTGTGCCTTTGATGGATATAGGCGAAATG 420

************************************************************

pETSUMO-katA ATTCTAAACAAAAATCCTCAAAATTATTTTAATGAAGTTGAACAAGCTGCCTTTAGTCCA 428

pEGFPC1katA ATTCTAAACAAAAATCCTCAAAATTATTTTAATGAAGTTGAACAAGCTGCCTTTAGTCCA 480

************************************************************

pETSUMO-katA AGCAATATCGTTCCTGGAATTGGCTTTAGCCCTGATAAAATGTTGCAAGCTAGAATTTTT 488

pEGFPC1katA AGCAATATCGTTCCTGGAATTGGCTTTAGCCCTGATAAAATGTTGCAAGCTAGAATTTTT 540

************************************************************

491

pETSUMO-katA TCATATCCTGATGCACAAAGATATAGAATAGGAACTAATTATCATCTTTTGCCCGTAAAT 548

pEGFPC1katA TCATATCCTGATGCACAAAGATATAGAATAGGAACTAATTATCATCTTTTGCCCGTAAAT 600

************************************************************

pETSUMO-katA CGTGCAAAAAGCGAAGTGAATACTTACAATGTCGCTGGTGCTATGAATTTTGATAGTTAT 578

pEGFPC1katA CGTGCAAAAAGCGAAGTGAATACTTACAATGTCGCTGGTGCTATGAATTTTGATAGTTAT 660

************************************************************

pETSUMO-katA AAAAATGATGCAGCTTATTATGAACCAAACAGCTATGATAATAGCCCAGGATCCACAGAC 578

pEGFPC1katA AAAAATGATGCAGCTTATTATGAACCAAACAGCTATGATAATAGCCCAGGATCCACCGGA 720

******************************************************

pETSUMO-katA AAGCTTAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCCAACTTAGTCGAGCA 578

pEGFPC1katA TCTAGATAACTGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAA 780

Appendix 4.1.2: Nucleotide analysis of pEGFP-C1-cadF plasmid

The cadF gene was cloned into the pEGFP-C1 vector in the correct orientation. The restriction site for XhoI was found at positions 16–21.

pETSUMO-cadF AACAGATTGGTGGTGCTCGAGCTGGTGCTGATAACAATGTAAAATTTGAAATCACTCCAA 60

pEGFPC1cadF AGTCCGGACTCAGATCTCGAGCTGGTGCTGATAACAATGTAAAATTTGAAATCACTCCAA 60

*********************************************

492

pETSUMO-cadF CTTTAAACTATAATTACTTTGAAGGTAATTTAGATATGGATAATCGTTATGCACCAGGGA 120

pEGFPC1cadF CTTTAAACTATAATTACTTTGAAGGTAATTTAGATATGGATAATCGTTATGCACCAGGGA 120

************************************************************

pETSUMO-cadF TTAGACTTGGTTATCATTTTGACGATTTTTGGCTTGATCAATTAGAATTTGGGTTAGAGC 180

pEGFPC1cadF TTAGACTTGGTTATCATTTTGACGATTTTTGGCTTGATCAATTAGAATTTGGGTTAGAGC 180

************************************************************

pETSUMO-cadF ATTATTCTGATGTTAAATATACAAATACAAATAAAACTACAGATATTACAAGAACTTATT 240

pEGFPC1cadF ATTATTCTGATGTTAAATATACAAATACAAATAAAACTACAGATATTACAAGAACTTATT 240

************************************************************

pETSUMO-cadF TGAGTGCTATTAAAGGTATTGATGTAGGTGAGAAATTTTATTTCTATGGTTTAGCAGGTG 300

pEGFPC1cadF TGAGTGCTATTAAAGGTATTGATGTAGGTGAGAAATTTTATTTCTATGGTTTAGCAGGTG 300

************************************************************

pETSUMO-cadF GAGGATATGAGGATTTTTCAAATGCTGCTTATGATAATAAAAGCGGTGGATTTGGACATT 360

pEGFPC1cadF GAGGATATGAGGATTTTTCAAATGCTGCTTATGATAATAAAAGCGGTGGATTTGGACATT 360

************************************************************

pETSUMO-cadF ATGGCGCGGGTGTAAAATTCCGTCTTAGTGATTCTTTGGCTTTAAGACTTGAAACTAGAG 420

pEGFPC1cadF ATGGCGCGGGTGTAAAATTCCGTCTTAGTGATTCTTTGGCTTTAAGACTTGAAACTAGAG 420

************************************************************

pETSUMO-cadF ATCAAATTAATTTCAATCATGCAAACCATAATTGGGTTTCAACTTTAGGTATTAGTTTTG 480

493

pEGFPC1cadF ATCAAATTAATTTCAATCATGCAAACCATAATTGGGTTTCAACTTTAGGTATTAGTTTTG 480

************************************************************

pETSUMO-cadF GTTTTGGTGGCAAAAAGGAAAAAGCTGTAGAAGAAGTTGCTGATACTCGTGCAACTCCAC 540

pEGFPC1cadF GTTTTGGTGGCAAAAAGGAAAAAGCTGTAGAAGAAGTTGCTGATACTCGTGCAACTCCAC 540

************************************************************

pETSUMO-cadF AAGCAAAATGTCCTGTTGAACCAAGAGAAGGTGCTTTGTTAGATGAAAATGGTTGCGAAA 600

pEGFPC1cadF AAGCAAAATGTCCTGTTGAACCAAGAGAAGGTGCTTTGTTAGATGAAAATGGTTGCGAAA 600

************************************************************

pETSUMO-cadF AAACTATTTCTTTGGAAGGTCATTTTGGTTTTGATAAAACTACTATAAATCCAACTTTTC 660

pEGFPC1cadF AAACTATTTCTTTGGAAGGTCATTTTGGTTTTGATAAAACTACTATAAATCCAACTTTTC 660

************************************************************

pETSUMO-cadF AAGAAAAAATCAAAGAAATTGCAAAAGTTTTAGATGAAAATGAAAGATATGATACTATTC 720

pEGFPC1cadF AAGAAAAAATCAAAGAAATTGCAAAAGTTTTAGATGAAAATGAAAGATATGATACTATTC 720

************************************************************

pETSUMO-cadF TTGAAGGACATACAGATAATATCGGTTCAAGAGCTTATAATCAAAAGCTTTCTGAAAGAC 780

pEGFPC1cadF TTGAAGGACATACAGATAATATCGGTTCAAGAGCTTATAATCAAAA-------------- 766

**********************************************

pETSUMO-cadF GTGCTAAAAGTGTTGCCAATGAACTTGAAAAATATGGTGTAGAAAAAAGTCGCATCAAAA 840

pEGFPC1cadF ------------------------------------------------------------ 766

494

pETSUMO-cadF CAGTAGGTTATGGTCAAGATAATCCTCGCTCAAGCCATGACACTAAAGAAGGTAGAGCGG 900

pEGFPC1cadF ------------------------------------------------------------ 766

pETSUMO-cadF ATAATAGAAGAGTGGATGCTGGATCCACAGACAAGCTTAGGTATTTATTCGGCGCAAAGT 960

pEGFPC1cadF ------------------------------------------------------------ 766

pETSUMO-cadF GCGTCGGGTGATGCTGCCAACTTAGTCGAGCACCACACCACCACACTGAGATCCGG 1016

pEGFPC1cadF -------------------------------------------------------- 766

Appendix 4.1.3: The nucleotide analysis of pEGFP-C1-peb1A plasmid

The peb1A gene was cloned into the pEGFP-C1 vector in the correct orientation. The restriction sites for XhoI and BamHI-HF were found at positions

24–29 and 782–787, respectively.

pETSUMOpeb1A TCACAGAGAACAGATTGGTGGTGCTCGAGCTTCTTTGTTAAAGTTGGCAGTTTTTGCTCT 60

pEGFPC1peb1A GCTGTACAAGTCCGGACTCAGATCTCGAGCTTCTTTGTTAAAGTTGGCAGTTTTTGCTCT 60

*************************************

pETSUMOpeb1A AGGTGCTTGTGTTGCATTTAGCAATGCTAATGCAGCAGAAGGTAAGCTTGAGTCTATTAA 120

495

pEGFPC1peb1A AGGTGCTTGTGTTGCATTTAGCAATGCTAATGCAGCAGAAGGTAAGCTTGAGTCTATTAA 120

************************************************************

pETSUMOpeb1A ATCTAAAGGACAATTAATAGTTGGTGTTAAAAATGATGTTCCGCATTATGCTTTACTTGA 180

pEGFPC1peb1A ATCTAAAGGACAATTAATAGTTGGTGTTAAAAATGATGTTCCGCATTATGCTTTACTTGA 180

************************************************************

pETSUMOpeb1A TCAAGCAACAGGTGAAATTAAAGGTTTCGAAGTAGATGTTGCCAAATTGCTAGCCAAAAG 240

pEGFPC1peb1A TCAAGCAACAGGTGAAATTAAAGGTTTCGAAGTAGATGTTGCCAAATTGCTAGCCAAAAG 240

************************************************************

pETSUMOpeb1A TATATTGGGTGATGATAAAAAAATAAAACTAGTTGCAGTTAATGCTAAAACAAGAGGCCC 300

pEGFPC1peb1A TATATTGGGTGATGATAAAAAAATAAAACTAGTTGCAGTTAATGCTAAAACAAGAGGCCC 300

************************************************************

pETSUMOpeb1A TTTGCTTGATAATGGTAGTGTAGATGCAGTGATAGCAACTTTTACTATTACTCCAGAGAG 360

pEGFPC1peb1A TTTGCTTGATAATGGTAGTGTAGATGCAGTGATAGCAACTTTTACTATTACTCCAGAGAG 360

************************************************************

pETSUMOpeb1A AAAAAGAATTTATAATTTCTCAGAACCTTATTATCAAGATGCTATAGGGCTTTTGGTTTT 420

pEGFPC1peb1A AAAAAGAATTTATAATTTCTCAGAACCTTATTATCAAGATGCTATAGGGCTTTTGGTTTT 420

************************************************************

pETSUMOpeb1A AAAAGAAAAAAAATATAAATCTTTAGCTGATATGAAAGGTGCAAATATTGGAGTGGCTCA 480

pEGFPC1peb1A AAAAGAAAAAAAATATAAATCTTTAGCTGATATGAAAGGTGCAAATATTGGAGTGGCTCA 480

496

************************************************************

pETSUMOpeb1A AGCTGCAACTACAAAAAAAGCTATAGGTGAAGCTGCTAAAAAAATTGGCATTGATGTTAA 540

pEGFPC1peb1A AGCTGCAACTACAAAAAAAGCTATAGGTGAAGCTGCTAAAAAAATTGGCATTGATGTTAA 540

************************************************************

pETSUMOpeb1A ATTTAGTGAATTTCCTGATTATCCAAGTATAAAAGCTGCTTTAGATGCTAAAAGAGTTGA 600

pEGFPC1peb1A ATTTAGTGAATTTCCTGATTATCCAAGTATAAAAGCTGCTTTAGATGCTAAAAGAGTTGA 600

************************************************************

pETSUMOpeb1A TGCGTTTTCTGTAGACAAATCAATATTGTTAGGTTATGTGGATGATAAAAGTGAAATTTT 660

pEGFPC1peb1A TGCGTTTTCTGTAGACAAATCAATATTGTTAGGTTATGTGGATGATAAAAGTGAAATTTT 660

************************************************************

pETSUMOpeb1A GCCAGATAGTCTTGAACCACAAAGTTATGGTATTGTAACCAAAAAAGATGATCCAGCTTT 720

pEGFPC1peb1A GCCAGATAGTCTTGAACCACAAAGTTATGGTATTGTAACCAAAAAAGATGATCCAGCTTT 720

************************************************************

pETSUMOpeb1A TGCAAAATATGTTGATGATTTTGTAAAAGAACATAAAAATGAAATTGATGCTTTAGCGAA 780

pEGFPC1peb1A TGCAAAATATGTTGATGATTTTGTAAAAGAACATAAAAATGAAATTGATGCTTTAGCGAA 780

************************************************************

pETSUMOpeb1AA GGATCCACAGACAAGCTTAGGTATTTATTCGGCGCAAAGTGCGTCGGGTGATGCTGCC 839

pEGFPC1peb1AA GGATCCACCGGATCTAGATAACTGATCATAATCAGCCATACCACATTTGTAGAGGTTT 839

********

497

Appendix 4.1.4: The nucleotide analysis of pEGFP-C1-cjaA plasmid

The cjaA gene was cloned into the pEGFP-C1 vector in the correct orientation. The restriction sites for XhoI and BamHI-HF were found at positions 22–

27 and 852–857, respectively.

pETSUMO-cjaA ACAGAGAACAGATTGGTGGTGCTCGAGCTATGCTCTTAAGTATTTTTACAACCTTTGTTG 60

pEGFPC1cjaA CTGTACAGTCCGGACTCAGATCTCGAGCTATGCTCTTAAGTATTTTTACAACCTTTGTTG 60

***************************************

pETSUMO-cjaA CAGTATTTTTGGCTGCTTGTGGAGGAAATTCAGATTCTGGTGCTTCAAATTCTCTTGAAA 120

pEGFPC1cjaA CAGTATTTTTGGCTGCTTGTGGAGGAAATTCAGATTCTGGTGCTTCAAATTCTCTTGAAA 120

************************************************************

pETSUMO-cjaA GAATCAAGCAAGATGGAGTAGTAAGAATAGGAGTTTTTGGAGATAAACCGCCTTTTGGTT 180

pEGFPC1cjaA GAATCAAGCAAGATGGAGTAGTAAGAATAGGAGTTTTTGGAGATAAACCGCCTTTTGGTT 180

************************************************************

pETSUMO-cjaA ATGTAGATGAAAAAGGCGTAAATCAAGGTTATGATATAGTCTTGGCGAAACGTATAGCAA 240

pEGFPC1cjaA ATGTAGATGAAAAAGGCGTAAATCAAGGTTATGATATAGTCTTGGCGAAACGTATAGCAA 240

************************************************************

pETSUMO-cjaA AAGAACTCTTAGGAGATGAAAATAAGGTGCAGTTTGTATTAGTTGAAGCTGCAAATAGGG 300

pEGFPC1cjaA AAGAACTCTTAGGAGATGAAAATAAGGTGCAGTTTGTATTAGTTGAAGCTGCAAATAGGG 300

498

************************************************************

pETSUMO-cjaA TGGAATTTTTAAAATCAAATAAAGTTGATATTATTTTAGCTAATTTTACTCAAACACCTG 360

pEGFPC1cjaA TGGAATTTTTAAAATCAAATAAAGTTGATATTATTTTAGCTAATTTTACTCAAACACCTG 360

************************************************************

pETSUMO-cjaA AAAGAGCAGAACAAGTGGATTTTTGCTTACCTTATATGAAGGTAGCTTTAGGTGTGGCTG 420

pEGFPC1cjaA AAAGAGCAGAACAAGTGGATTTTTGCTTACCTTATATGAAGGTAGCTTTAGGTGTGGCTG 420

************************************************************

pETSUMO-cjaA TGCCTCAAGATAGCAATATCAGTAGCATAGAAGATTTAAAAGATAAAACTTTACTTTTAA 480

pEGFPC1cjaA TGCCTCAAGATAGCAATATCAGTAGCATAGAAGATTTAAAAGATAAAACTTTACTTTTAA 480

************************************************************

pETSUMO-cjaA ACAAAGGAACTACCGCTGATGCGTATTTTACAAAAGAATATCCTGATATTAAAACATTAA 540

pEGFPC1cjaA ACAAAGGAACTACCGCTGATGCGTATTTTACAAAAGAATATCCTGATATTAAAACATTAA 540

************************************************************

pETSUMO-cjaA AATACGATCAAAATACCGAAACTTTTGCCGCTTTAATAGATCAAAGAGGGGATGCTTTAA 600

pEGFPC1cjaA AATACGATCAAAATACCGAAACTTTTGCCGCTTTAATAGATCAAAGAGGGGATGCTTTAA 600

************************************************************

pETSUMO-cjaA GTCATGACAATACTTTGCTTTTTGCGTGGGTAAAAGAACATCCTGAATTTAAAATGGCCA 660

pEGFPC1cjaA GTCATGACAATACTTTGCTTTTTGCGTGGGTAAAAGAACATCCTGAATTTAAAATGGCCA 660

************************************************************

499

pETSUMO-cjaA TTAAAGAATTGGGCAATAAAGATGTAATTGCTCCTGCTGTTAAAAAAGGTGATAAAGAGC 720

pEGFPC1cjaA TTAAAGAATTGGGCAATAAAGATGTAATTGCTCCTGCTGTTAAAAAAGGTGATAAAGAGC 720

******************************************** ***************

pETSUMO-cjaA TTAAAGAATTTATTGATAATCTAATCACAAAATTAGGAGAAGAACAATTCTTCCATAAAG 780

pEGFPC1cjaA TTAAAGAATTTATTGATAATCTAATCACAAAATTAGGAGAAGAACAATTCTTCCATAAAG 780

************************************************************

pETSUMO-cjaA CTTATGATGAAACTTTAAAAAGTCATTTTGGAGATGATGTAAAAGCTGATGATGTAGTTA 840

pEGFPC1cjaA CTTATGATGAAACTTTAAAAAGTCATTTTGGAGATGATGTAAAAGCTGATGATGTAGTTA 840

************************************************************

pETSUMO-cjaA TTGAAGGCGGTGGATCCACAGACAAGCTTAGGTATTTATTCGGCGCAAAGTGCGTCGGGT 899

pEGFPC1cjaA TTGAAGGCGGTGGATCCACCGGATCTAG-------------------------------- 868

*******************

500

Appendix 4.2: Maintenance media used for Vero and RK-13 (rabbit

kidney-13) cells

Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% FCS

was prepared as follows.

Reagent Percentage (%) Volume (mL)

Heat Inactivated donor calf

serum

5 25

Non-essential amino acids

(NEAA)

1 5

GlutaMax 100x 1 5

HEPES (1M) 2.5 12.5

MEM 10x (Earle’s) 10 50

Sodium bicarbonate (7.5%) 3 15

Sodium pyruvate 100x 1 5

Sterile water 76.5 382.5

Documents

Investigation of Campylobacter jejuni and Campylobacter