investigation of campylobacter jejuni and campylobacter
TRANSCRIPT
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
Appendix 2.2.2 A: Clustering of Campylobacter jejuni isolates from
BD–B ....................................................................... 286
Appendix 2.2.2 B: Clustering of Campylobacter coli isolates from
BD–B ....................................................................... 288
Appendix 2.2.3 A: Clustering of Campylobacter jejuni isolates from
BD–C ....................................................................... 290
Appendix 2.2.3 B: Clustering of Campylobacter coli isolates from
BD–C ....................................................................... 292
Appendix 2.2.4 A: Clustering of Campylobacter jejuni isolates from
BD–F ....................................................................... 294
Appendix 2.2.4 B: Clustering of Campylobacter coli isolates from
BD–F ....................................................................... 297
Appendix 2.2.5 A: Clustering of Campylobacter jejuni isolates from
BD–G ....................................................................... 299
Appendix 2.2.5 B: Clustering of Campylobacter coli isolates from
BD–G ....................................................................... 302
Appendix 2.3: Summary of clustering Campylobacter jejuni and
Campylobacter coli isolates from all broiler farms in experiments 1 and 2
based on MALDI-TOF, PCR, flaA-HRM analysis and flaA sequencing303
VII
Appendix 2.3.1 A: Clustering of Campylobacter jejuni isolates from
free-range broiler farm 1 (FB1) in experiment 1 (Exp.1) .................. 303
Appendix 2.3.1 B: Clustering of Campylobacter jejuni isolates of
free-range broiler farm 1 (FB1) in experiment 2 (Exp.2) .................. 308
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 A: Clustering of Campylobacter jejuni isolates from
free-range broiler farm 2 (FB2) in experiment 1 (Exp.1) .................. 315
Appendix 2.3.2 B: Clustering of Campylobacter coli isolates from
free-range broiler farm 2 (FB2) in experiment 1 (Exp.1) .................. 319
Appendix 2.3.2 C: Clustering of Campylobacter jejuni isolates from
free-range broiler farm 2 (FB2) in experiment 2 (Exp.2) .................. 322
Appendix 2.3.3 A: Clustering of Campylobacter jejuni isolates from
free-range broiler farm 3 (FB3) in experiment 1 (Exp.1) .................. 327
Appendix 2.3.3 B: Clustering of Campylobacter coli isolates from
free-range broiler farm 3 (FB3) in experiment 1 (Exp.1) .................. 328
Appendix 2.3.3 C: Clustering of Campylobacter jejuni isolates from
free-range broiler farm 3 (FB3) in experiment 2 (Exp.2) .................. 332
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
Reaction targeting flaA gene (flaA-HRM PCR) analysis and flaA sequencing
................................................................................................................. 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
XII
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
Table 4.3: Oligonucleotide primer pairs used for DNA sequencing of the
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
clusters identified on free-range broiler farm 2 (FB2) in the experiment
2 ............................................................................................................. 100
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 ............................................................................................................. 103
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 ............................................................................................................. 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
Figure 3.2: Example of alignment analyses of the nucleotide sequences
and subsequent amino acid sequences generated from the cadF
amplicon of the selected C. jejuni and C. coli clusters. ........................ 144
XIV
Figure 3.3: Example of alignment analyses of the nucleotide sequences
and subsequent amino acid sequences generated from the peb1A
amplicon of the selected C. jejuni and C. coli clusters. ........................ 146
Figure 3.4 Example of alignment analyses of the nucleotide sequences
and subsequent amino acid sequences generated from the cjaA
amplicon of the selected C. jejuni and C. coli clusters. ........................ 148
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
cells from the transformed One Shot® Mach1™-T1 competent E. coli
colonies as DNA template in PCR reactions. ....................................... 150
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
cells from the transformed One Shot® Mach1™-T1 competent E. coli
colonies as DNA template in PCR reactions. ....................................... 151
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
Figure 4.4: Agarose gel electrophoresis of the PCR product containing
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
PCR products using whole cells from transformed One Shot® TOP10
chemically competent E. coli colonies as the DNA template. .............. 187
Figure 4.7: Example of agarose gel electrophoresis of the peb1A ORF
PCR products using whole cells from transformed One Shot® TOP10
chemically competent E. coli colonies as the DNA template. .............. 188
Figure 4.8: Example of agarose gel electrophoresis of the cjaA ORF
PCR products using whole cells from transformed One Shot® TOP10
chemically competent E. coli colonies as the DNA template. .............. 188
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
compared with the original PCR amplicon and the TOPO vector alone.
............................................................................................................... 191
XVI
Figure 4.13: Example of sequence alignment of the pcDNA3T-peb1A-1
compared with the original PCR amplicon and the TOPO vector alone.
............................................................................................................... 192
Figure 4.14: Example of sequence alignment of the pcDNA3T-cjaA-1
compared with the original PCR amplicon and the TOPO vector alone.
............................................................................................................... 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
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. ............................... 198
Figure 4.19: Example of agarose gel electrophoresis of PCR products
for the peb1A ORF fragment using whole cells from the transformed
One Shot® TOP10 E. coli colonies as a DNA template. ....................... 200
Figure 4.20: Example of agarose gel electrophoresis of PCR products
for the cjaA ORF fragment using whole cells from the transformed One
Shot® TOP10 E. coli colonies as a DNA template. ............................... 202
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
ORF after HindIII and BamHI-HF digestion of the recombinant
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
ORF after HindIII and BamHI-HF digestion of the recombinant
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
Figure 4.31 : Microscopic analysis of infected CEF cells with different
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.
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 & Gala´n, 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 & Gala´n, 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
Intramuscular Challenge
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
Intramuscular Challenge
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
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
FliD
C.
jejuni
M1
GST 1 TiterMax
Gold
Specific-
pathogen
free White
Leghorn
Chicken
Subcutaneous Challenge
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
Subcutaneous Challenge
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
Subcutaneous Challenge
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
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
SodB
C.
jejuni
M1
GST 1 TiterMax
Gold
SPF
White
Leghorn
Chickens
Subcutaneous Challenge
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
Subcutaneous Challenge
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)
Note: 1 GST - N-terminal glutathione S-transferase
37
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
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)
Note: 1 GST - N-terminal glutathione S-transferase
38
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
Adjuva
nt
Immune
responses
Vaccine
efficacy
Capsular
polysacchar
ide (CPS)
C.
jejuni
81-176
CRM197 3 N/A BALB/c
Mice
Subcutaneous Challenge
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
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
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
Subcutaneous Challenge
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
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
FlaA
C.
jejuni
81-176
–
CpG and
Montainde
ISA70
Ross
PM3
chickens
Intramuscular Challenge
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
Intramuscular Challenge
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
Subcutaneous Challenge
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
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
rCjaA
C.
jejuni
M1
His-
tagged TiterMax®
SPF
Light
Sussex
Chicken
s
Subcutaneous Challenge
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
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
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
Intramuscular Challenge
study
C. jejuni
81-176
Induced
specific
IgY
4 to 7
log10
CFU/g of
reduction
in caecal
contents
Nothaft et
al. (2016)
43
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
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
& Guzma´n, 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
Reference Strain Vector
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
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
Reference Strain Vector
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
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
Reference Strain Vector
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
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
Reference Strain Vector
Immune
responses
Vaccine
efficacy
CjaA C. jejuni
Attenuated
∆aroA S.
Typhimurium
Chickens Oral Challenge
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
Chickens Oral Challenge
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
Chickens Oral Challenge
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
Chickens Oral Challenge
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
Table 1.5: Summary of studies of anti-Campylobacter jejuni vaccines (live vector vaccine) evaluated in animal models (cont’)
Note: N/A; Non-applicable
Antigen(s)
Vaccine Animal
model
Route of
administration
Experiment
type
Challenge
strain
Outcomes
Reference Strain Vector
Immune
responses
Vaccine
efficacy
CjaA-cadF-
ciaB-cj1496
C.
jejuni
Attenuated
∆aroA S.
Typhimuri
um
Chickens Oral Challenge
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
campylobacteriosis by at least 76% (Romero-Barrios et al., 2013; Rosenquist
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
Adjacent1 12.1 × 69.4 17 × 50 11,980 FB3–A1–Exp.1 BD–C (47)
Target 12.1 × 69.4 17 × 50 11,980 FB3–T–Exp.1 BD–B (61) and BD–C (47)
Adjacent2 12.1 × 73.2 17 × 65 15,030 FB3–A2–Exp.1 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
Reaction targeting flaA gene (flaA-HRM PCR) analysis and flaA sequencing
flaA-HRM profile
(Cluster)
flaA allele
(Peptide, Nucleotide)
Breeder farm(s)
(number of isolates)
Free-range broiler shed(s)
(number of isolates)
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
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 (cont’)
flaA-HRM profile
(Cluster)
flaA allele
(Peptide, Nucleotide)
Breeder farm(s)
(number of isolates)
Free-range broiler shed(s)
(number of isolates)
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
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 (cont’)
flaA-HRM profile
(Cluster)
flaA allele
(Peptide, Nucleotide)
Breeder farm(s)
(number of isolates)
Free-range broiler shed(s)
(number of isolates)
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
Reaction targeting flaA gene (flaA-HRM PCR) analysis and flaA sequencing
flaA-HRM profile
(Cluster)
flaA allele
(Peptide, Nucleotide)
Breeder farm(s)
(number of isolates)
Free-range broiler shed(s)
(number of isolates)
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
Reaction targeting flaA gene (flaA-HRM PCR) analysis and flaA sequencing (cont’)
flaA-HRM profile
(Cluster)
flaA allele
(Peptide, Nucleotide)
Breeder farm(s)
(number of isolates)
Free-range broiler shed(s)
(number of isolates)
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.
92
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 26 1 105 2 21 5 3 2 1 5 46 ST-206 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. jejuni 29 27 2 4 73 10 4 1 7 1 128 ST-45 complex
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
94
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–
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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).
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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)
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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
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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
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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
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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
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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)
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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
campylobacteriosis by at least 76% (Romero-Barrios et al., 2013; Rosenquist
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;
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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
121
(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;
123
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 3´end 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
Forward/XhoI 58 Modified from
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
Forward/XhoI 55 Modified from
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
amplicons of the selected C. jejuni and C. coli clusters.
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
amplicons of the selected C. jejuni and C. coli clusters.
A) Example alignment of the peb1A 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 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
amplicons of the selected C. jejuni and C. coli clusters.
A) Example alignment of the cjaA 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 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
using whole cells from the transformed One Shot® Mach1™-T1
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 whole cells from the transformed One Shot® Mach1™-T1
competent E. coli colonies as DNA template in PCR reactions.
Using cadF primer set, the estimated size of PCR product is
approximately 910 bp (arrow). Colonies showing evidence of
transformed plasmids ligated with the inserted katA are detected in
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.
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.
Using peb1A primer set, the estimated size of PCR product is
approximately 770 bp (arrow). Colonies showing evidence of
transformed plasmids ligated with the inserted katA are detected in
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 whole cells from the transformed One Shot® Mach1™-T1
competent E. coli colonies as DNA template in PCR reactions.
Using cjaA primer set, the estimated size of PCR product is
approximately 840 bp (arrow). Colonies showing evidence of
transformed plasmids ligated with the inserted katA are detected in
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).
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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).
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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
1270 bp in length (Appendix 3.5.2), encoding a polypeptide with 517 amino
acid residues with an estimated molecular weight of 46.6 kDa. The Western
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
1124 bp in length (Appendix 3.5.3), encoding a polypeptide with 374 amino
acid residues with an estimated molecular weight of 41.2 kDa. The Western
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).
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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.
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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.
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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.
169
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
Reagent Volume (μL)
Fresh PCR amplicon X*
Salt solution 1
Sterile water to a total volume of 5
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).
Table 4.3: Oligonucleotide primer pairs used for DNA sequencing of the
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,
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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.
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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
Reagent Volume (μL)
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
Sterile water to a total volume of 10
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)
Figure 4.3: Agarose gel electrophoresis of the PCR product containing
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.
186
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
PCR products using whole cells from transformed One Shot® TOP10
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.
188
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
Figure 4.7: Example of agarose gel electrophoresis of the peb1A
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
PCR products using whole cells from transformed One Shot® TOP10
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.
190
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
compared with the original PCR amplicon and the TOPO vector alone.
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
sequences of the TOPO vector are indicated in green colour. The
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
sequences of the TOPO vector are indicated in green colour. The
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.
B) The estimated size of PCR product was approximately 1170 bp using the pEGFP-C1 primers (arrow). Colonies showing the evidence of
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
One Shot® TOP10 E. coli colonies as a DNA template.
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
One Shot® TOP10 E. coli colonies as a DNA template.
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.
B) The estimated size of PCR product was approximately 1095 bp using the pEGFP-C1 primers (arrow). Colonies showing the evidence of
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)
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.
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
ORF after HindIII and BamHI-HF digestion of the recombinant
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)
Figure 4.24: Example of agarose gel electrophoresis of the inserted
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).
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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).
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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.
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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
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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
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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).
Figure 4.30 : Microscopic analysis of infected CEF cells with different
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.
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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
Figure 4.31 : Microscopic analysis of infected CEF cells with different
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
223
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
224
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.
225
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.
226
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
227
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%.
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
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 170 1 Faecal sample C. jejuni C. jejuni 79.6 ± 0.02 – 22 1, 36b 25.95
BD–A 189 1 Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 14 1, 34a 21.01
BD–A 199 2 Faecal sample C. jejuni C. jejuni 79.7 ± 0.04 – 23 1, 467a 18.30
BD–A 205 2 Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 – 25 33, 222 20.44
BD–A 211 2 Faecal sample C. jejuni C. jejuni 79.8 ± 0.04 – 22 1, 36b 25.84
BD–A 224 3 Faecal sample C. coli C. jejuni 79.6 ± 0.01 – 14 1, 34a 24.20
BD–A 226 3 Faecal sample C. jejuni C. jejuni 79.8 ± 0.05 – 23 1, 467a 18.40
BD–A 231 3 Faecal sample C. jejuni C. jejuni 79.9 ± 0.01 – 23 1, 467a 26.91
BD–A 241 3 Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 – 8 125, 419 19.69
BD–A 246 4 Faecal sample C. jejuni C. jejuni 78.7 ± 0.01 79.5 ± 0.01 26 1, 105 19.35
BD–A 252 4 Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 25 33, 222 21.12
BD–A 256 4 Faecal sample C. jejuni C. jejuni 79.8 ± 0.02 – 22 1, 36b 26.63
BD–A 261 4 Faecal sample C. jejuni C. jejuni 79.4 ± 0.00 – 25 33, 222 21.28
BD–A 266 4 Faecal sample C. jejuni C. jejuni 79.7 ± 0.05 – 22 1, 36b 25.74
BD–A 276 5 Faecal sample C. jejuni C. jejuni 79.7 ± 0.12 – 22 1, 36b 25.85
BD–A 282 5 Faecal sample C. jejuni C. jejuni 79.8 ± 0.05 – 22 1, 36b 26.92
283
Identification and clustering of Campylobacter jejuni isolated from breeder farm A (BD–A) con’t
Farm
Isolate
no. Shed Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
BD–A 487 5 Faecal sample C. jejuni C. jejuni 79.7 ± 0.02 – 22 1, 36b 25.38
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
value MALDI-TOF PCR Peak 1 Peak 2
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 230 3 Faecal sample C. coli C. coli 79.9 ± 0.04 – 2 17 1, 467c 18.67
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 249 4 Faecal sample C. coli C. coli 79.8 ± 0.01 – 2 17 1, 467c 18.91
BD–A 272 5 Faecal sample C. coli C. coli 79.5 ± 0.01 – 5 6 21, 13 22.60
BD–A 277 2 Faecal sample C. coli C. coli 79.5 ± 0.02 – 1 5 1, 36d 18.19
BD–A 292 5 Faecal sample C. coli C. coli 79.5 ± 0.01 – 5 6 21, 13 22.89
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
value MALDI-TOF PCR Peak 1 Peak 2
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 1246 4 Faecal sample C. jejuni C. jejuni 79.2 ± 0.09 79.7 ± 0.07 10 8b 28.38
BD–B 1251 4 Faecal sample C. jejuni C. jejuni 79.6 ± 0.04 – 8 125, 419 30.89
BD–B 1256 4 Faecal sample C. jejuni C. jejuni 79.7 ± 0.06 – 11 1a 27.38
BD–B 1261 4 Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 79.6 ± 0.04 10 8b 27.20
BD–B 1271 5 Faecal sample C. jejuni C. jejuni 79.5 ± 0.5 – 8 125, 419 30.95
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 1291 6 Faecal sample C. jejuni C. jejuni 79.1 ± 0.23 79.6 ± 0.03 10 8b 26.79
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 1262 4 Faecal sample C. coli C. coli 79.6 ± 0.01 – 9 11d 21.15
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 1285 5 Faecal sample C. coli C. coli 79.5 ± 0.02 – 15 8d 21.18
BD–B 1289 6 Faecal sample C. coli C. coli 79.5 ± 0.03 – 10 11e 22.88
BD–B 1301 6 Faecal sample C. coli C. coli 79.5 ± 0.02 – 15 8d 21.16
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 1163 2 Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 – 8 125, 419 26.54
BD–C 1166 2 Faecal sample C. jejuni C. jejuni 79.1 ± 0.03 – 5 20, 18b 25.09
BD–C 1191 3 Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 5 20, 18b 26.54
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
BD–C 1211 4 Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 – 20 3, 106 26.70
BD–C 1216 4 Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 5 20, 18b 26.80
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 1146 2 Faecal sample C. coli C. coli 79.4 ± 0.02 – 6 21, 13 27.62
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 1172 3 Faecal sample C. coli C. coli 79.3 ± 0.03 – 6 21, 13 33.29
BD–C 1176 3 Faecal sample C. coli C. coli 79.5 ± 0.02 – 6 21, 13 30.25
BD–C 1183 3 Faecal sample C. coli C. coli 78.7 ± 0.00 79.2 ± 0.01 14 8c 25.08
BD–C 1189 3 Faecal sample C. coli C. coli 79.4 ± 0.03 – 6 21, 13 30.22
BD–C 1196 4 Faecal sample C. coli C. coli 79.5 ± 0.05 – 6 21, 13 29.38
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 2003 2 Faecal sample C. jejuni C. jejuni 79.6 ± 0.05 – 38 10, 28a 24.59
BD–F 2011 2 Faecal sample C. jejuni C. jejuni – 79.5 ± 0.02 10 8b 22.47
BD–F 2012 3 Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 6 9, 239a 23.66
BD–F 2027 3 Faecal sample C. jejuni C. jejuni 79.6 ± 0.05 – 24 1, 467b 22.25
BD–F 2032 3 Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 6 9, 239a 23.02
BD–F 2038 4 Faecal sample C. jejuni C. jejuni – 79.6 ± 0.04 36 1, 8a 23.03
BD–F 2042 4 Faecal sample C. jejuni C. jejuni 79.1 ± 0.03 – 7 9, 239b 20.64
BD–F 2047 4 Faecal sample C. jejuni C. jejuni – 79.5 ± 0.03 10 8b 22.9
BD–F 2061 4 Faecal sample C. jejuni C. jejuni 79.2 ± 0.00 – 7 9, 239b 23.38
BD–F 2072 5 Faecal sample C. jejuni C. jejuni 79.4 ± 0.00 – 39 2, 54 22.15
BD–F 2077 5 Faecal sample C. jejuni C. jejuni – 79.5 ± 0.03 10 8b 22.96
296
Identification and clustering of Campylobacter jejuni isolated from breeder farm F (BD–F) con’t
Farm
Isolate
no. Shed Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
BD–F 2085 5 Faecal sample C. jejuni C. jejuni 79.3 ± 0.00 – 5 20, 18b 22.75
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 1985 1 Faecal sample C. coli C. coli 78.8 ± 0.01 79.6 ± 0.03 22 1, 8b 17.17
BD–F 1999 2 Faecal sample C. coli C. coli 79.5 ± 0.03 – 19 10, 28b 19.87
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 2022 3 Faecal sample C. coli C. coli 79.5 ± 0.04 – 19 1, 467e 19.77
BD–F 2036 3 Faecal sample C. coli C. coli 79.5 ± 0.03 – 19 1, 467e 19.16
BD–F 2040 4 Faecal sample C. coli C. coli 79.2 ± 0.03 – 23 20, 18c 19.93
BD–F 2052 4 Faecal sample C. coli C. coli 79.5 ± 0.04 – 19 1, 467e 19.35
BD–F 2058 4 Faecal sample C. coli C. coli 79.6 ± 0.02 – 19 10, 28b 24.02
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
BD–F 2097 6 Faecal sample C. coli C. coli 79.2 ± 0.02 80.5 ± 0.00 13 12, 16b 17.53
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 1864 4 Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 30 2, 612 20.73
BD–G 1868 4 Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 30 2, 612 20.4
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 1880 5 Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 21 1, 36a 20.43
BD–G 1886 5 Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 21 1, 36a 20.16
BD–G 1891 5 Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 – 9 8a 20.79
BD–G 1896 5 Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 21 1, 36a 20.77
BD–G 1901 6 Faecal sample C. jejuni C. jejuni 79.5 ± 0.03 – 17 11a 26.89
BD–G 1906 6 Faecal sample C. jejuni C. jejuni 78.6 ± 0.02 79.4 ± 0.02 31 1, 32a 20.16
BD–G 1910 6 Faecal sample C. jejuni C. jejuni 79.9 ± 0.01 – 33 11, 30a 22.42
BD–G 1916 6 Faecal sample C. jejuni C. jejuni 79.6 ± 0.02 – 17 11a 26.34
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 1924 7 Faecal sample C. jejuni C. jejuni 79.5 ± 0.03 – 21 1, 36a 19.38
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
Farm Isolate no. Shed Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 1864 4 Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 30 2, 612 20.73
BD–G 1868 4 Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 30 2, 612 20.4
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
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)
Farm Isolate no. Shed Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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
Appendix 2.3: Summary of clustering Campylobacter jejuni and
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
Species isolated HRM flaA-HRM
Cluster
flaA
allele
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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–A1–Exp.1 792G Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 1 4, 57 32.03
FB1–A1–Exp.1 797G Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.02 – 1 4, 57 29.29
FB1–A1–Exp.1 802C Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 1 4, 57 28.77
FB1–A1–Exp.1 807C Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 1 4, 57 25.91
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
Species isolated HRM flaA-HRM
Cluster
flaA
allele
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB1–T–Exp.1 509 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 2 11, 14 23.13
FB1–T–Exp.1 514 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 2 11, 14 23.41
FB1–T–Exp.1 519 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.04 – 2 11, 14 23.09
FB1–T–Exp.1 524 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.00 – 2 11, 14 22.05
FB1–T–Exp.1 529 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.03 – 2 11, 14 23.19
FB1–T–Exp.1 534 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.04 – 2 11, 14 22.72
FB1–T–Exp.1 539 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.04 – 2 11, 14 23.12
FB1–T–Exp.1 544 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.05 – 2 11, 14 23.15
FB1–T–Exp.1 549 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.02 – 1 4, 57 26.64
FB1–T–Exp.1 554 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.04 – 1 4, 57 27.22
FB1–T–Exp.1 559 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 2 11, 14 23.64
FB1–T–Exp.1 564 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 26.35
FB1–T–Exp.1 569 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 1 4, 57 27.52
FB1–T–Exp.1 574 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.04 – 1 4, 57 28.36
FB1–T–Exp.1 579 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 – 1 4, 57 28.48
FB1–T–Exp.1 584 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.08 – 1 4, 57 30.51
FB1–T–Exp.1 589 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.09 – 1 4, 57 29.96
FB1–T–Exp.1 594 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.03 – 2 11, 14 24.91
FB1–T–Exp.1 599 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 2 11, 14 24.68
306
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.1) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
Cluster
flaA
allele
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB1–T–Exp.1 604 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.04 – 2 11, 14 25.45
FB1–T–Exp.1 609 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.02 – 2 11, 14 26.30
FB1–T–Exp.1 614 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.03 – 2 11, 14 25.22
FB1–T–Exp.1 619 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.00 – 2 11, 14 24.30
FB1–T–Exp.1 624 Day 22- Faecal sample C. jejuni C. jejuni 79.8 ± 0.07 – 2 11, 14 29.30
FB1–T–Exp.1 634 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 25.85
FB1–T–Exp.1 639 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 1 4, 57 26.09
FB1–T–Exp.1 644 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.04 – 1 4, 57 28.85
FB1–T–Exp.1 649 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.04 – 1 4, 57 29.09
FB1–T–Exp.1 652 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.04 – 2 11, 14 24.43
FB1–T–Exp.1 657 Day 22- Faecal sample C. jejuni C. jejuni 79.8 ± 0.00 – 2 11, 14 32.34
FB1–T–Exp.1 662 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.04 – 2 11, 14 26.88
FB1–T–Exp.1 667 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.05 – 1 4, 57 29.66
FB1–T–Exp.1 672 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 2 11, 14 24.30
FB1–T–Exp.1 677 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 32.54
FB1–A2–Exp.1 112 Day 15- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 2 11, 14 21.54
FB1–A2–Exp.1 118 Day 15- Faecal sample C. jejuni C. jejuni 79.6 ± 0.00 – 2 11, 14 22.20
FB1–A2–Exp.1 120 Day 15- Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 2 11, 14 23.06
FB1–A2–Exp.1 125 Day 15- Faecal sample C. jejuni C. jejuni 79.6 ± 0.03 – 2 11, 14 22.38
307
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.1) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
Cluster
flaA
allele
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB1–A2–Exp.1 131 Day 15- Faecal sample C. jejuni C. jejuni 79.5 ± 0.04 – 2 11, 14 22.02
FB1–A2–Exp.1 133 Day 15- Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 2 11, 14 22.34
FB1–A2–Exp.1 139 Day 15- Faecal sample C. jejuni C. jejuni 79.6 ± 0.03 – 2 11, 14 22.30
FB1–A2–Exp.1 146 Day 15- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 2 11, 14 21.78
FB1–A2–Exp.1 151 Day 15- Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 2 11, 14 20.85
FB1–A2–Exp.1 157 Day 15- Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 2 11, 14 21.30
FB1–A2–Exp.1 707 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.02 – 2 11, 14 23.32
FB1–A2–Exp.1 712 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.04 – 2 11, 14 24.34
FB1–A2–Exp.1 717 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.04 – 2 11, 14 24.22
FB1–A2–Exp.1 725 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.03 – 2 11, 14 23.53
FB1–A2–Exp.1 727 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 2 11, 14 22.58
FB1–A2–Exp.1 732 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.05 – 2 11, 14 23.42
FB1–A2–Exp.1 737 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.02 – 2 11, 14 23.37
FB1–A2–Exp.1 742 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.02 – 2 11, 14 24.51
FB1–A2–Exp.1 747 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.01 – 2 11, 14 24.82
FB1–A2–Exp.1 752 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.00 – 2 11, 14 21.75
308
Appendix 2.3.1 B: Clustering of Campylobacter jejuni isolates of free-
range broiler farm 1 (FB1) in experiment 2 (Exp.2)
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
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.2)
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 2150 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 80.5 ± 0.02 27 12, 16a 23.32
FB1–A1–Exp.2 2160 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 80.5 ± 0.01 27 12, 16a 23.97
FB1–A1–Exp.2 2166 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 – 6 9, 239a 27.75
FB1–A1–Exp.2 2170 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.00 80.5 ± 0.02 27 12, 16a 23.97
FB1–A1–Exp.2 2175 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 80.5 ± 0.02 27 12, 16a 24.94
FB1–A1–Exp.2 2180 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 80.5 ± 0.02 27 12, 16a 24.22
FB1–A1–Exp.2 2180 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 80.5 ± 0.01 27 12, 16a 23.28
FB1–A1–Exp.2 2190 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 80.6 ± 0.03 27 12, 16a 23.96
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
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.2) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB1–T–Exp.2 2282 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.03 27 12, 16a 21.11
FB1–T–Exp.2 2287 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.04 27 12, 16a 20.78
FB1–T–Exp.2 2292 Day 22- Faecal sample C. jejuni C. jejuni 79.1 ± 0.04 80.3 ± 0.04 27 12, 16a 19.69
FB1–T–Exp.2 2297 Day 22- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 80.4 ± 0.03 27 12, 16a 20.54
FB1–T–Exp.2 2302 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 80.4 ± 0.03 27 12, 16a 20.28
FB1–T–Exp.2 2307 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.04 27 12, 16a 21.64
FB1–T–Exp.2 2312 Day 22- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 80.4 ± 0.00 27 12, 16a 19.68
FB1–T–Exp.2 2317 Day 22- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 80.4 ± 0.02 27 12, 16a 19.62
FB1–T–Exp.2 2322 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.01 80.4 ± 0.02 27 12, 16a 20.69
FB1–T–Exp.2 2327 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 80.4 ± 0.03 27 12, 16a 22.37
FB1–T–Exp.2 2332 Day 22- Faecal sample C. jejuni C. jejuni 79.1 ± 0.03 80.4 ± 0.02 27 12, 16a 19.98
FB1–T–Exp.2 2337 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.03 27 12, 16a 22.24
FB1–T–Exp.2 2342 Day 22- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 80.4 ± 0.00 27 12, 16a 20.54
FB1–T–Exp.2 2347 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 80.4 ± 0.01 27 12, 16a 22.12
FB1–T–Exp.2 2352 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 80.3 ± 0.02 27 12, 16a 20.77
FB1–T–Exp.2 2357 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 80.4 ± 0.03 27 12, 16a 21.36
FB1–T–Exp.2 2362 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.03 80.6 ± 0.04 27 12, 16a 27.8
FB1–T–Exp.2 2367 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.07 80.4 ± 0.07 27 12, 16a 23.12
311
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.2) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB1–T–Exp.2 2372 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.06 80.5 ± 0.05 27 12, 16a 23.69
FB1–T–Exp.2 2377 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.5 ± 0.01 27 12, 16a 21.18
FB1–T–Exp.2 2382 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.08 80.5 ± 0.09 27 12, 16a 22.56
FB1–T–Exp.2 2387 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 80.5 ± 0.05 27 12, 16a 22.46
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
FB1–T–Exp.2 2397 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 80.4 ± 0.04 27 12, 16a 22.25
FB1–T–Exp.2 2402 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 80.5 ± 0.06 27 12, 16a 22.76
FB1–T–Exp.2 2407 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 80.4 ± 0.02 27 12, 16a 22.8
FB1–T–Exp.2 2412 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 80.4 ± 0.04 27 12, 16a 22.51
FB1–T–Exp.2 2417 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.04 27 12, 16a 23.04
FB1–T–Exp.2 2422 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 80.4 ± 0.03 27 12, 16a 22.54
FB1–T–Exp.2 2427 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.08 80.4 ± 0.07 27 12, 16a 22.65
FB1–T–Exp.2 2432 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.05 80.4 ± 0.04 27 12, 16a 21.79
FB1–T–Exp.2 2337 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 80.4 ± 0.03 27 12, 16a 22.76
FB1–T–Exp.2 2442 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 80.4 ± 0.01 27 12, 16a 22.1
FB1–T–Exp.2 2448 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.14 80.4 ± 0.10 27 12, 16a 21.69
FB1–A2–Exp.2 1803 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 80.4 ± 0.02 27 12, 16a 20.04
FB1–A2–Exp.2 1808 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.03 27 12, 16a 20.4
312
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 1 (FB1), experiment 1 (Exp.2) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB1–A2–Exp.2 1812 Day 15- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 80.5 ± 0.03 27 12, 16a 26.34
FB1–A2–Exp.2 1818 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 80.4 ± 0.03 27 12, 16a 20.67
FB1–A2–Exp.2 1822 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 80.4 ± 0.05 27 12, 16a 20.97
FB1–A2–Exp.2 1828 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.03 27 12, 16a 19.88
FB1–A2–Exp.2 1833 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.04 27 12, 16a 19.93
FB1–A2–Exp.2 1838 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.05 80.4 ± 0.04 27 12, 16a 20.14
FB1–A2–Exp.2 1843 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 80.4 ± 0.03 27 12, 16a 19.58
FB1–A2–Exp.2 1848 Day 15- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 80.4 ± 0.06 27 12, 16a 19.47
FB1–A2–Exp.2 2195 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 80.4 ± 0.06 27 12, 16a 21.05
FB1–A2–Exp.2 2200 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.05 80.4 ± 0.05 27 12, 16a 19.78
FB1–A2–Exp.2 2205 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.01 80.4 ± 0.01 27 12, 16a 20.52
FB1–A2–Exp.2 2210 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 80.4 ± 0.01 27 12, 16a 19.79
FB1–A2–Exp.2 2215 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.05 80.4 ± 0.05 27 12, 16a 19.67
FB1–A2–Exp.2 2220 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 80.5 ± 0.02 27 12, 16a 25.03
FB1–A2–Exp.2 2225 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.01 80.5 ± 0.02 27 12, 16a 23.37
FB1–A2–Exp.2 2230 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.09 80.4 ± 0.08 27 12, 16a 22.66
FB1–A2–Exp.2 2235 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 80.5 ± 0.02 27 12, 16a 24.25
FB1–A2–Exp.2 2240 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.06 80.5 ± 0.07 27 12, 16a 24.36
313
Appendix 2.3.1 C: Clustering of Campylobacter coli isolates of free-
range broiler farm 1 (FB1) in experiment 2 (Exp.2)
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 1792 Day 15- Faecal sample C. coli C. coli 79.9 ± 0.03 – 3 11, 30b 20.89
FB1–A1–Exp.2 1797 Day 15- Faecal sample C. coli C. coli 79.9 ± 0.05 – 3 11, 30b 20.18
FB1–A1–Exp.2 2119 Day 15- Faecal sample C. coli C. coli 80.0 ± 0.04 – 3 11, 30b 21.81
FB1–A1–Exp.2 2134 Day 15- Faecal sample C. coli C. coli 80.0 ± 0.03 – 3 11, 30b 20.89
FB1–A1–Exp.2 2159 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.1 – 3 11, 30b 20.09
FB1–A1–Exp.2 2161 Day 22- Faecal sample C. coli C. coli 80.0 ± 0.09 – 3 11, 30b 22.92
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-
range broiler farm 2 (FB2) in experiment 1 (Exp.1)
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
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 2 (FB2), experiment 1 (Exp.1)
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB2–A1–Exp.1 813 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 – 3 20, 208 25.99
FB2–A1–Exp.1 817 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 3 20, 208 26.86
FB2–A1–Exp.1 822 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 3 20, 208 26.18
FB2–A1–Exp.1 827 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 3 20, 208 26.36
FB2–A1–Exp.1 832 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 3 20, 208 24.70
FB2–A1–Exp.1 837 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 3 20, 208 25.57
FB2–A1–Exp.1 842 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 3 20, 208 25.13
FB2–A1–Exp.1 847 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 3 20, 208 24.97
FB2–A1–Exp.1 852 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 – 3 20, 208 25.95
FB2–A1–Exp.1 857 Day 22- Faecal sample C. jejuni C. jejuni 80.0 ± 0.02 – 2 11, 14 34.01
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
FB2–T–Exp.1 977 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 29.07
317
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 2 (FB2), experiment 1 (Exp.1) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB2–T–Exp.1 1002 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 3 20, 208 21.96
FB2–T–Exp.1 1037 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 28.06
FB2–T–Exp.1 1077 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 3 20, 208 23.18
FB2–T–Exp.1 942 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.00 – 3 20, 208 21.03
FB2–T–Exp.1 947 Day 22- Faecal sample C. jejuni C. jejuni 79.8 ± 0.03 – 2 11, 14 24.85
FB2–T–Exp.1 952 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.01 – 2 11, 14 24.30
FB2–T–Exp.1 962 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.05 – 3 20, 208 20.51
FB2–T–Exp.1 967 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.00 – 3 20, 208 23.52
FB2–T–Exp.1 972 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 – 3 20, 208 19.34
FB2–T–Exp.1 982 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.00 – 2 11, 14 24.96
FB2–T–Exp.1 987 Day 22- Faecal sample C. jejuni C. jejuni 79.7 ± 0.03 – 2 11, 14 25.42
FB2–T–Exp.1 992 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.00 – 2 11, 14 24.99
FB2–T–Exp.1 997 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 3 20, 208 19.63
FB2–T–Exp.1 1007 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.03 – 3 20, 208 19.54
FB2–T–Exp.1 1012 Day 22- Faecal sample C. jejuni C. jejuni 79.8 ± 0.06 – 2 11, 14 24.82
FB2–T–Exp.1 1017 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 3 20, 208 19.89
318
Identification and clustering of Campylobacter jejuni isolated from free-range broiler farm 2 (FB2), experiment 1 (Exp.1) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB2–T–Exp.1 1022 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.03 – 3 20, 208 19.66
FB2–T–Exp.1 1027 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.00 – 3 20, 208 20.05
FB2–T–Exp.1 1031 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.04 – 3 20, 208 20.29
FB2–T–Exp.1 1042 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 3 20, 208 20.69
FB2–T–Exp.1 1047 Day 22- Faecal sample C. jejuni C. jejuni 79.8 ± 0.02 – 2 11, 14 25.50
FB2–T–Exp.1 1052 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 – 3 20, 208 20.49
FB2–T–Exp.1 1058 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.00 – 3 20, 208 20.58
FB2–T–Exp.1 1062 Day 22- Faecal sample C. jejuni C. jejuni 79.8 ± 0.02 – 2 11, 14 25.47
FB2–T–Exp.1 1067 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 3 20, 208 23.53
FB2–T–Exp.1 1082 Day 22- Faecal sample C. jejuni C. jejuni 79.8 ± 0.01 – 2 11, 14 25.33
FB2–T–Exp.1 1087 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.02 – 3 20, 208 19.62
FB2–T–Exp.1 1092 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.01 – 3 20, 208 19.67
FB2–T–Exp.1 1097 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.03 – 3 20, 208 19.69
FB2–T–Exp.1 1107 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.02 – 3 20, 208 19.91
319
Appendix 2.3.2 B: Clustering of Campylobacter coli isolates from free-
range broiler farm 2 (FB2) in experiment 1 (Exp.1)
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 34 Day 8- Rodents faeces C. coli C. coli 79.7 ± 0.04 – 2 97, 256 22.65
FB2–T–Exp.1 295 Day 15- Rodents faeces C. coli C. coli 79.7 ± 0.02 – 2 97, 256 22.08
FB2–T–Exp.1 932 Day 22- Rodents faeces C. coli C. coli 79.7 ± 0.04 – 2 97, 256 22.84
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–T–Exp.1 957 Day 22- Faecal sample C. coli C. coli 80.0 ± 0.04 – 3 11, 30b 26.97
FB2–T–Exp.1 1072 Day 22- Faecal sample C. coli C. coli 80.0 ± 0.03 – 3 11, 30b 26.71
FB2–T–Exp.1 1102 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.02 – 3 11, 30b 26.94
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 1112 Day 15- Faecal sample C. coli C. coli 80.0 ± 0.01 – 3 11, 30b 25.97
FB2–A2–Exp.1 862 Day 22- Faecal sample C. coli C. coli 79.7 ± 0.01 – 2 97, 256 22.65
FB2–A2–Exp.1 867 Day 22- Faecal sample C. coli C. coli 80.0 ± 0.00 – 3 11, 30b 28.06
FB2–A2–Exp.1 873 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.00 – 3 11, 30b 26.87
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB2–A2–Exp.1 877 Day 22- Faecal sample C. coli C. coli 79.6 ± 0.03 – 2 97, 256 22.86
FB2–A2–Exp.1 882 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.01 – 3 11, 30b 27.94
FB2–A2–Exp.1 887 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.04 – 3 11, 30b 27.25
FB2–A2–Exp.1 892 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.05 – 3 11, 30b 27.06
FB2–A2–Exp.1 897 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.01 – 3 11, 30b 26.50
FB2–A2–Exp.1 903 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.02 – 3 11, 30b 26.65
FB2–A2–Exp.1 907 Day 22- Faecal sample C. coli C. coli 79.9 ± 0.04 – 3 11, 30b 27.74
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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–A1–Exp.2 2454 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 – 6 9, 239a 17.17
FB2–A1–Exp.2 2458 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 – 6 9, 239a 15.8
FB2–A1–Exp.2 2463 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 17.53
FB2–A1–Exp.2 2473 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 80.5 ± 0.03 27 12, 16a 16.17
FB2–A1–Exp.2 2478 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 80.5 ± 0.02 27 12, 16a 18.98
FB2–A1–Exp.2 2483 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.06 80.5 ± 0.03 27 12, 16a 18.95
FB2–A1–Exp.2 2468 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 6 9, 239a 19.1
FB2–A1–Exp.2 2488 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 80.5 ± 0.05 27 12, 16a 19.1
FB2–A1–Exp.2 2493 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 80.5 ± 0.04 27 12, 16a 18.49
FB2–A1–Exp.2 2498 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 80.5 ± 0.05 27 12, 16a 18.81
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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
FB2–T–Exp.2 2581 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 80.5 ± 0.03 27 12, 16a 23.13
FB2–T–Exp.2 2586 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.09 80.7 ± 0.07 27 12, 16a 27.47
FB2–T–Exp.2 2591 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.10 80.6 ± 0.12 27 12, 16a 25.39
FB2–T–Exp.2 2596 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 80.5 ± 0.04 27 12, 16a 22.03
FB2–T–Exp.2 2601 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 80.5 ± 0.05 27 12, 16a 22.25
FB2–T–Exp.2 2606 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 80.5 ± 0.04 27 12, 16a 23.6
FB2–T–Exp.2 2611 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 80.4 ± 0.01 27 12, 16a 19.32
FB2–T–Exp.2 2616 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 80.5 ± 0.03 27 12, 16a 22.76
FB2–T–Exp.2 2621 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 80.4 ± 0.03 27 12, 16a 19.93
FB2–T–Exp.2 2626 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.00 80.5 ± 0.00 27 12, 16a 23.09
FB2–T–Exp.2 2631 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 80.5 ± 0.03 27 12, 16a 22.5
FB2–T–Exp.2 2636 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.00 80.5 ± 0.01 27 12, 16a 24.3
FB2–T–Exp.2 2641 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.04 80.6 ± 0.02 27 12, 16a 25.72
325
Identification and clustering of Campylobacter jejuni isolated from breeder farm 2 (FB2), experiment 2 (exp.2) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB2–T–Exp.2 2646 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.05 80.7 ± 0.01 27 12, 16a 26.41
FB2–T–Exp.2 2651 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 80.6 ± 0.03 27 12, 16a 23.46
FB2–T–Exp.2 2656 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.07 80.6 ± 0.06 27 12, 16a 24.21
FB2–T–Exp.2 2661 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 80.6 ± 0.02 27 12, 16a 23.88
FB2–T–Exp.2 2666 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 80.6 ± 0.03 27 12, 16a 25.26
FB2–T–Exp.2 2671 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.05 80.6 ± 0.03 27 12, 16a 24.26
FB2–T–Exp.2 2676 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 80.6 ± 0.02 27 12, 16a 23.24
FB2–T–Exp.2 2681 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.07 80.6 ± 0.04 27 12, 16a 24.81
FB2–T–Exp.2 2686 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.06 80.8 ± 0.04 27 12, 16a 29.63
FB2–T–Exp.2 2691 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.09 80.6 ± 0.05 27 12, 16a 23.21
FB2–T–Exp.2 2696 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.05 80.7 ± 0.03 27 12, 16a 26.97
FB2–T–Exp.2 2701 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 80.6 ± 0.03 27 12, 16a 22.71
FB2–T–Exp.2 2706 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.10 80.6 ± 0.07 27 12, 16a 22.54
FB2–T–Exp.2 2711 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.05 80.7 ± 0.04 27 12, 16a 28.2
FB2–T–Exp.2 2716 Day 22- Faecal sample C. jejuni C. jejuni 79.5 ± 0.05 80.7 ± 0.04 27 12, 16a 27.18
FB2–T–Exp.2 2721 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 80.6 ± 0.01 27 12, 16a 22.18
FB2–T–Exp.2 2726 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 80.6 ± 0.01 27 12, 16a 24.19
326
Identification and clustering of Campylobacter jejuni isolated from breeder farm 2 (FB2), experiment 2 (exp.2) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB2–T–Exp.2 2731 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.05 80.6 ± 0.03 27 12, 16a 22.71
FB2–T–Exp.2 2736 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.01 80.6 ± 0.01 27 12, 16a 24.02
FB2–T–Exp.2 2741 Day 22- Faecal sample C. jejuni C. jejuni 79.4 ± 0.03 80.6 ± 0.02 27 12, 16a 22.71
FB2–T–Exp.2 2746 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.05 80.7 ± 0.05 27 12, 16a 27.82
FB2–T–Exp.2 2751 Day 22- Faecal sample C. jejuni C. jejuni 79.6 ± 0.02 80.8 ± 0.01 27 12, 16a 29.24
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
FB2–A2–Exp.2 2504 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 6 9, 239a 15.88
FB2–A2–Exp.2 2509 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 6 9, 239a 16.75
FB2–A2–Exp.2 2514 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.04 – 6 9, 239a 16.55
FB2–A2–Exp.2 2519 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.03 – 6 9, 239a 16.01
FB2–A2–Exp.2 2524 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.01 – 6 9, 239a 16.4
FB2–A2–Exp.2 2529 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 – 6 9, 239a 16.86
FB2–A2–Exp.2 2534 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.01 – 6 9, 239a 16.49
FB2–A2–Exp.2 2539 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 – 6 9, 239a 16.16
FB2–A2–Exp.2 2544 Day 22- Faecal sample C. jejuni C. jejuni 79.3 ± 0.02 – 6 9, 239a 16.36
FB2–A2–Exp.2 2549 Day 22- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 16.25
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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-
range broiler farm 3 (FB3) in experiment 1 (Exp.1)
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB3–A1–Exp.1 90 Day 10- Outside the shed C. coli C. coli 79.6 ± 0.01 – 5 1, 36d 20.92
FB3–A1–Exp.1 302 Day 17- Faecal sample C. coli C. coli 80.0 ± 0.01 – 3 11, 30b 25.65
FB3–A1–Exp.1 305 Day 17- Faecal sample C. coli C. coli 80.0 ± 0.03 – 3 11, 30b 25.74
FB3–A1–Exp.1 313 Day 17- Faecal sample C. coli C. coli 80.0 ± 0.04 – 3 11, 30b 25.83
FB3–A1–Exp.1 317 Day 17- Faecal sample C. coli C. coli 80.0 ± 0.03 – 3 11, 30b 25.74
FB3–A1–Exp.1 320 Day 17- Faecal sample C. coli C. coli 80.0 ± 0.06 – 3 11, 30b 25.51
FB3–A1–Exp.1 489 Day 17- Faecal sample C. coli C. coli 80.0 ± 0.04 – 3 11, 30b 25.25
FB3–A1–Exp.1 494 Day 17- Faecal sample C. coli C. coli 80.0 ± 0.06 – 3 11, 30b 24.97
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 77 Day 10- Faecal sample C. coli C. coli 79.5 ± 0.03 – 5 1, 36d 16.51
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.1 106 Day 10- Faecal sample C. coli C. coli 79.5 ± 0.02 – 5 1, 36d 16.78
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.1 340 Day 17- Farm boots C. coli C. coli 79.5 ± 0.03 – 5 1, 36d 16.60
FB3–T–Exp.1 499 Day 17- Water pan C. coli C. coli 79.5 ± 0.06 – 5 1, 36d 16.68
FB3–T–Exp.1 345 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.03 – 5 1, 36d 16.35
FB3–T–Exp.1 347 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.03 – 5 1, 36d 16.60
330
Identification and clustering of Campylobacter coli isolated from breeder farm 3 (FB3), experiment 1 (exp.1) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB3–T–Exp.1 358 Day 17- Faecal sample C. coli C. coli 79.6 ± 0.03 – 5 1, 36d 17.15
FB3–T–Exp.1 367 Day 17- Faecal sample C. coli C. coli 79.6 ± 0.02 – 5 1, 36d 16.94
FB3–T–Exp.1 379 Day 17- Faecal sample C. coli C. coli 79.6 ± 0.02 – 5 1, 36d 16.78
FB3–T–Exp.1 387 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.05 – 5 1, 36d 16.58
FB3–T–Exp.1 393 Day 17- Faecal sample C. coli C. coli 79.6 ± 0.03 – 5 1, 36d 17.50
FB3–T–Exp.1 397 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.04 – 5 1, 36d 16.50
FB3–T–Exp.1 407 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.01 – 5 1, 36d 17.07
FB3–T–Exp.1 412 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.02 – 5 1, 36d 16.49
FB3–T–Exp.1 417 Day 17- Faecal sample C. coli C. coli 79.6 ± 0.01 – 5 1, 36d 19.85
FB3–T–Exp.1 426 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.02 – 5 1, 36d 17.07
FB3–T–Exp.1 436 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.00 – 5 1, 36d 16.98
FB3–T–Exp.1 444 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.04 – 5 1, 36d 15.95
FB3–T–Exp.1 452 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.06 – 5 1, 36d 15.80
FB3–T–Exp.1 455 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.02 – 5 1, 36d 16.73
FB3–T–Exp.1 464 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.01 – 5 1, 36d 16.88
FB3–T–Exp.1 467 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.01 – 5 1, 36d 19.32
FB3–T–Exp.1 476 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.02 – 5 1, 36d 16.83
FB3–T–Exp.1 478 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.02 – 5 1, 36d 19.30
FB3–T–Exp.1 348 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.02 – 5 1, 36d 16.34
FB3–T–Exp.1 353 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.02 – 5 1, 36d 19.68
331
Identification and clustering of Campylobacter coli isolated from breeder farm 3 (FB3), experiment 1 (exp.1) con’t
Shed
Isolate
no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB3–T–Exp.1 362 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.0 – 5 1, 36d 19.85
FB3–T–Exp.1 371 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.06 – 5 1, 36d 20.07
FB3–T–Exp.1 374 Day 17- Faecal sample C. coli C. coli 79.3 ± 0.05 – 5 1, 36d 19.69
FB3–T–Exp.1 383 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.08 – 5 1, 36d 19.53
FB3–T–Exp.1 395 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.07 – 5 1, 36d 20.02
FB3–T–Exp.1 402 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.05 – 5 1, 36d 20.67
FB3–T–Exp.1 418 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.08 – 5 1, 36d 20.61
FB3–T–Exp.1 423 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.07 – 5 1, 36d 20.21
FB3–T–Exp.1 430 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.04 – 5 1, 36d 20.75
FB3–T–Exp.1 440 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.05 – 5 1, 36d 20.75
FB3–T–Exp.1 449 Day 17- Faecal sample C. coli C. coli 79.5 ± 0.06 – 5 1, 36d 20.78
FB3–T–Exp.1 459 Day 17- Faecal sample C. coli C. coli 79.4 ± 0.07 – 5 1, 36d 19.83
FB3–T–Exp.1 471 Day 17- Faecal sample C. coli C. coli 79.3 ± 0.01 – 5 1, 36d 19.82
332
Appendix 2.3.3 C: Clustering of Campylobacter jejuni isolates from free-
range broiler farm 3 (FB3) in experiment 2 (Exp.2)
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB3–A1–Exp.2 2756 Day 24- Outside the shed C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 16.68
FB3–A1–Exp.2 2759 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.04 – 6 9, 239a 16.5
FB3–A1–Exp.2 2764 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 – 6 9, 239a 19.63
FB3–A1–Exp.2 2772 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 – 6 9, 239a 15.7
FB3–A1–Exp.2 2774 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 17.25
FB3–A1–Exp.2 2779 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 18.18
FB3–A1–Exp.2 2784 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 18.73
FB3–A1–Exp.2 2789 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.00 – 6 9, 239a 16.82
FB3–A1–Exp.2 2794 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 17.56
FB3–A1–Exp.2 2799 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.03 – 6 9, 239a 16.33
FB3–A1–Exp.2 2804 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 6 9, 239a 17.73
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
FB3–T–Exp.2 2889 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.05 – 1 4, 57 21.88
334
Identification and clustering of Campylobacter jejuni isolated breeder farm 3 (FB3), experiment 2 (exp.2) con’t
Shed
Isolat
e no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB3–T–Exp.2 2894 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.00 – 1 4, 57 20.74
FB3–T–Exp.2 2899 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.04 – 1 4, 57 19.67
FB3–T–Exp.2 2904 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 – 1 4, 57 20.27
FB3–T–Exp.2 2909 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 – 1 4, 57 17.9
FB3–T–Exp.2 2914 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.04 – 1 4, 57 19.15
FB3–T–Exp.2 2919 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 19.57
FB3–T–Exp.2 2924 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 – 1 4, 57 22.32
FB3–T–Exp.2 2929 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.02 – 1 4, 57 22.41
FB3–T–Exp.2 2934 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 – 1 4, 57 20.66
FB3–T–Exp.2 2939 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 19.97
FB3–T–Exp.2 2944 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 – 1 4, 57 18.95
FB3–T–Exp.2 2949 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 20.23
FB3–T–Exp.2 2954 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.05 80.5 ± 0.03 27 12, 16a 19.55
FB3–T–Exp.2 2959 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 19.3
FB3–T–Exp.2 2964 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 18.11
FB3–T–Exp.2 2969 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 19.58
FB3–T–Exp.2 2974 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 17.37
335
Identification and clustering of Campylobacter jejuni isolated breeder farm 3 (FB3), experiment 2 (exp.2) con’t
Shed
Isolat
e no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
FB3–T–Exp.2 2979 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 – 1 4, 57 18.29
FB3–T–Exp.2 2984 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.04 – 1 4, 57 20.3
FB3–T–Exp.2 2989 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.02 – 1 4, 57 19.04
FB3–T–Exp.2 2994 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.01 – 1 4, 57 16.3
FB3–T–Exp.2 2999 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 18.34
FB3–T–Exp.2 3004 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.01 – 1 4, 57 16.58
FB3–T–Exp.2 3009 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.02 – 1 4, 57 16.75
FB3–T–Exp.2 3014 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.01 – 1 4, 57 16.77
FB3–T–Exp.2 3019 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.02 – 1 4, 57 15.76
FB3–T–Exp.2 3024 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.00 – 1 4, 57 16.07
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 3034 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.04 – 1 4, 57 17.16
FB3–T–Exp.2 3039 Day 24- Faecal sample C. jejuni C. jejuni 79.1 ± 0.01 – 1 4, 57 20.68
FB3–T–Exp.2 3044 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.02 – 1 4, 57 16.07
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
FB3–T–Exp.2 3074 Day 24- Faecal sample C. jejuni C. jejuni 79.2 ± 0.03 – 1 4, 57 21.42
336
Identification and clustering of Campylobacter jejuni isolated breeder farm 3 (FB3), experiment 2 (exp.2) con’t
Shed
Isolat
e no. Sample
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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 2812 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.03 – 6 9, 239a 18.33
FB3–A2–Exp.2 2817 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.01 – 6 9, 239a 15.24
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
FB3–A2–Exp.2 2827 Day 24- Faecal sample C. jejuni C. jejuni 79.0 ± 0.03 – 6 9, 239a 14.19
FB3–A2–Exp.2 2832 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.03 – 6 9, 239a 14.1
FB3–A2–Exp.2 2837 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.03 – 6 9, 239a 13.99
FB3–A2–Exp.2 2842 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.04 – 6 9, 239a 14.01
FB3–A2–Exp.2 2847 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.01 – 6 9, 239a 13.85
FB3–A2–Exp.2 2852 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.03 – 6 9, 239a 14.08
FB3–A2–Exp.2 2857 Day 24- Faecal sample C. jejuni C. jejuni 78.9 ± 0.01 – 6 9, 239a 14.06
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
Species isolated HRM flaA-HRM
cluster
flaA
Sequence
Ct
value MALDI-TOF PCR Peak 1 Peak 2
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
FB3–T–Exp.2 3061 Day 17- Farm boots C. coli C. coli 79.6 ± 0.01 – 5 1, 36d 19.29
FB3–A2–Exp.2 2825 Day 24- Faecal sample C. coli C. coli 79.6 ± 0.01 – 5 1, 36d 20.05
FB3–A2–Exp.2 2836 Day 24- Faecal sample C. coli C. coli 79.6 ± 0.01 – 5 1, 36d 19.94
FB3–A2–Exp.2 3064 Day 17- Faecal sample C. coli C. coli 79.6 ± 0.02 – 5 1, 36d 18.92
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).
Agarose gel electrophoresis of the fliD amplicon generated from
gradient temperature PCR reactions ranging 50-60°C using the new
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
Agarose gel electrophoresis of the fliD amplicon generated from
gradient temperature PCR reactions ranging 40-50°C using the new
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
2 2 C. jejuni Broiler 11 14 11, 14 Pos Pos Pos Pos Pos Pos Pos
3 3 C. jejuni Broiler 20 208 20, 208 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
6 6 C. jejuni Broiler and
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
flaA allele flaA type PCR analysis of antigenic genes
Peptide Nucleotide cadF katA peb cjaA-C.
coli
cjaA-C.
jejuni
omp18 flp
11 11 C. jejuni Breeder 1 1a Pos Pos Pos Pos Pos Pos Pos
12 12 C. jejuni Breeder 1 1b Pos Pos Pos Pos Pos Pos Pos
13 13 C. jejuni Breeder 1 56 1, 56 Pos Pos Pos Pos Pos Pos Pos
14 14 C. jejuni Breeder 1 34 1, 34a Pos Pos Pos Pos Pos Pos Pos
15 15 C. jejuni Breeder 1 34 1, 34b Pos Pos Pos Pos Pos Pos Pos
16 16 C. jejuni Breeder 1 34 1, 34c Pos Pos Pos Pos Pos Pos Pos
17 17 C. jejuni Breeder 11 11a 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
20 20 C. jejuni Breeder 3 106 3, 106 Pos Pos Pos Pos Pos Pos Pos
21 21 C. jejuni Breeder 1 36 1, 36a Pos Pos Pos Pos Pos Pos Pos
22 22 C. jejuni Breeder 1 36 1, 36b Pos Pos Pos Pos Pos Pos Pos
23 23 C. jejuni Breeder 1 467 1, 467a Pos Pos Pos Pos Pos Pos Pos
24 24 C. jejuni Breeder 1 467 1, 467b Pos Pos Pos Pos Pos Pos Pos
25 25 C. jejuni Breeder 33 222 33, 222 Pos Pos Pos Pos Pos Pos Pos
26 26 C. jejuni Broiler and
Breeder
1 105 1, 105 Pos Pos Pos Pos Pos Pos Pos
Note: Pos, Positive and Neg, Negative
344
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
flaA allele flaA type PCR analysis of antigenic genes
Peptide Nucleotide cadF katA peb cjaA-C.
coli
cjaA-C.
jejuni
omp18 flp
27 27 C. jejuni Broiler 12 16 12, 16a Pos Pos Pos Pos Pos Pos Pos
28 28 C. jejuni Broiler 257 1033 257, 1033 Pos Pos Pos Pos Pos Pos Pos
29 29 C. jejuni Broiler 72 2 72, 2 Pos Pos Pos Pos Pos Pos Pos
30 30 C. jejuni Breeder 2 612 2, 612 Pos Pos Pos Pos Pos Pos Pos
31 31 C. jejuni Breeder 1 32 1, 32a Pos Pos Pos Pos Pos Pos Pos
32 32 C. jejuni Breeder 1 32 1, 32b Pos Pos Pos Pos Pos Pos Pos
33 33 C. jejuni Breeder 11 30 11, 30a Pos Pos Pos Pos Pos Pos Pos
34 34 C. jejuni Breeder 8 67 8, 67 Pos Pos Pos Pos Pos Pos Pos
35 35 C. jejuni Breeder 5 5 Pos Pos Pos Pos Pos Pos Pos
36 36 C. jejuni Breeder 1 8 1, 8a Pos Pos Pos Pos Pos Pos Pos
37 37 C. jejuni Breeder 1 1c Pos Pos Pos Pos Pos Pos Pos
38 38 C. jejuni Breeder 10 28 10, 28a Pos Pos Pos Pos Pos Pos Pos
39 39 C. jejuni Breeder 2 54 2, 54 Pos Pos Pos Pos Pos Pos Pos
40 40 C. jejuni Breeder 5 5 5, 5a 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
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
flaA allele flaA type PCR analysis of antigenic genes
Peptide Nucleotide cadF katA peb cjaA-C.
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
46 5 C. coli Broiler and
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
50 9 C. coli Breeder 11 11d 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
54 13 C. coli Broiler and
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
56 15 C. coli Breeder 8 8d 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
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
flaA allele flaA type PCR analysis of antigenic genes
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 3050 ---------- ---------- -----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 2038 ---------- ---------- --------AT CAAGTAACCA TTCTTATGAG CGATAGAGGA
KatA C jejuni 2072 ---------- ---------- -----TACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA
KatA C jejuni 2114 ---------- ---------- -----TACAT CAAGTAACCA TTCTTATGAG CGATAGAGGA
KatA C jejuni 2170 ---------- ---------- --------AT CAAGTAACCA TTCTTATGAG CGATAGAGGA
KatA C jejuni 813 ---------- ---------- --------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 jejuni 3050 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 30 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 62 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 1162 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 2038 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 2072 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 2114 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 2170 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 813 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 1768 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 683 ATTCCTGCAA GTTATCGTCA TATGCATGGA TTTGGAAGCC ATACTTATAG TTTTATTAAT
KatA C jejuni 687 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
KatA C coli 1980 ATTCCAGCAA GTTATCGTCA TATGCACGGT TTTGGAAGCC ATACTTATAG CTTTATCAAT
KatA C coli 2119 ATTCCAGCAA GTTATCGTCA TATGCACGGT TTTGGAAGCC ATACTTATAG CTTTATCAAT
KatA C coli 2165 ATTCCAGCAA GTTATCGTCA TATGCACGGT TTTGGAAGCC ATACTTATAG CTTTATCAAT
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 3050 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 30 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 62 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 1162 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 2038 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 2072 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 2114 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 2170 GATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 813 CATAAAAATG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCAACAAGG GATTAAAAAT
KatA C jejuni 1768 GATAAAAATG 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
KatA C coli 175 GACAAAAACG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCTACAAGG TATTAAAAAT
KatA C coli 1980 GACAAAAACG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCTACAAGG TATTAAAAAT
KatA C coli 2119 GACAAAAACG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCTACAAGG TATTAAAAAT
KatA C coli 2165 GACAAAAACG AAAGATTTTG GGTGAAATTC CATTTTAAAA CCCTACAAGG TATTAAAAAT
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 3050 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 30 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 62 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 1162 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 2038 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 2072 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 2114 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 2170 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 813 CTTACCAACC AAGAAGCTGC CGAGCTTATA GCAAAAGATA GAGAAAGTCA TCAAAGAGAT
KatA C jejuni 1768 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
KatA C coli 1980 CTTAGCAATA AAGAAGCTGC TGAGCTTATC GCCAAAGATA GAGAAAGCCA CCAAAGAGAT
KatA C coli 2119 CTTAGCAATA AAGAAGCTGC TGAGCTTATC GCCAAAGATA GAGAAAGCCA CCAAAGAGAT
KatA C coli 2165 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 3050 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 30 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 62 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 1162 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 2038 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 2072 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 2114 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 2170 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 813 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 1768 CTCTATAATG CTATAGAAAA CAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 683 CTCTATAATG CTATAGAAAA TAAAGATTTT CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C jejuni 687 CTCTATAATG CTATAGAAAA TAAAGATTTT 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
KatA C coli 2887 CTTTACAATG CTATAGAAAA TAAAGATTTC CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C coli 175 CTTTACAATG CTATAGAAAA TAAAGATTTC CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C coli 1980 CTTTACAATG CTATAGAAAA TAAAGATTTC CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C coli 2119 CTTTACAATG CTATAGAAAA TAAAGATTTC CCAAAATGGA AAGTTCAAGT TCAAATTCTT
KatA C coli 2165 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 3050 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 30 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 62 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 1162 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 2038 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 2072 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 2114 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 2170 GCTGAAAAAG ATATAGAAAA ACTTGGATTT AATCCTTTTG ATTTAACAAA AATTTGGCCT
KatA C jejuni 813 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
KatA C coli 2887 GCTGAAAAAG ATGCTGACAA ACTAGGCTTT AATCCTTTTG ATTTAACTAA AATTTGGCCA
KatA C coli 175 GCTGAAAAAG ATGCTGACAA ACTAGGCTTT AATCCTTTTG ATTTAACTAA AATTTGGCCA
KatA C coli 1980 GCTGAAAAAG ATGCTGACAA ACTAGGCTTT AATCCTTTTG ATTTAACTAA AATTTGGCCA
KatA C coli 2119 GCTGAAAAAG ATGCTGACAA ACTAGGCTTT AATCCTTTTG ATTTAACTAA AATTTGGCCA
KatA C coli 2165 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 62 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT
KatA C jejuni 1162 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT
KatA C jejuni 2038 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT
KatA C jejuni 2072 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT
KatA C jejuni 2114 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT
KatA C jejuni 2170 CATAGTCTTG TGCCTTTGAT GGATATAGGC GAAATGATTC TAAACAAAAA TCCTCAAAAT
KatA C jejuni 813 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
KatA C coli 2887 CATAGCGTAG TGCCTTTAAT GGATATAGGC GAAATGATCT TAAATCAAAA TCCACAAAAT
KatA C coli 175 CATAGCGTAG TGCCTTTAAT GGATATAGGC GAAATGATCT TAAATCAAAA TCCACAAAAT
KatA C coli 1980 CATAGCGTAG TGCCTTTAAT GGATATAGGC GAAATGATCT TAAATCAAAA TCCACAAAAT
KatA C coli 2119 CATAGCGTAG TGCCTTTAAT GGATATAGGC GAAATGATCT TAAATCAAAA TCCACAAAAT
KatA C coli 2165 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 3050 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 30 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 62 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 1162 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 2038 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 2072 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 2114 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 2170 TATTTTAATG AAGTTGAACA AGCTGCCTTT AGTCCAAGCA ATATCGTTCC TGGAATTGGC
KatA C jejuni 813 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
KatA C coli 2887 TATTTTAATG AAGTAGAACA AGCAGCTTTT AGCCCAAGCA ATATAGTACC TGGTATAGGT
KatA C coli 175 TATTTTAATG AAGTAGAACA AGCAGCTTTT AGCCCAAGCA ATATAGTACC TGGTATAGGT
KatA C coli 1980 TATTTTAATG AAGTAGAACA AGCAGCTTTT AGCCCAAGCA ATATAGTACC TGGTATAGGT
KatA C coli 2119 TATTTTAATG AAGTAGAACA AGCAGCTTTT AGCCCAAGCA ATATAGTACC TGGTATAGGT
KatA C coli 2165 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
KatA C jejuni 3050 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 30 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 62 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 1162 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 2038 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 2072 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 2114 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 2170 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 813 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 1768 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
KatA C jejuni 683 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
365
KatA C jejuni 687 TTTAGCCCTG ATAAAATGTT GCAAGCTAGA ATTTTTTCAT ATCCTGATGC ACAAAGATAT
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
KatA C coli 2887 TTTAGTCCTG ATAAAATGCT ACAAGCTAGA ATTTTCTCAT ATCCTGATGC ACAAAGATAT
KatA C coli 175 TTTAGTCCTG ATAAAATGCT ACAAGCTAGA ATTTTCTCAT ATCCTGATGC ACAAAGATAT
KatA C coli 1980 TTTAGTCCTG ATAAAATGCT ACAAGCTAGA ATTTTCTCAT ATCCTGATGC ACAAAGATAT
KatA C coli 2119 TTTAGTCCTG ATAAAATGCT ACAAGCTAGA ATTTTCTCAT ATCCTGATGC ACAAAGATAT
KatA C coli 2165 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 3050 AGAATAGGAA CTAATTATCA TCTTTTACCA GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 30 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 62 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 1162 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 2038 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 2072 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 2114 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 2170 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 813 AGAATAGGAA CTAATTATCA TCTTTTACCA GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 1768 AGAATAGGAA CTAATTATCA TCTTTTGCCC GTAAATCGTG CAAAAAGCGA AGTGAATACT
366
KatA C jejuni 683 AGAATAGGAA CTAATTATCA TCTTTTACCA GTAAATCGTG CAAAAAGCGA AGTGAATACT
KatA C jejuni 687 AGAATAGGAA CTAATTATCA TCTTTTACCA GTAAATCGTG CAAAAAGCGA AGTGAATACT
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
KatA C coli 175 AGAATAGGAA CTAATTATCA TCTTTTACCT GTAAATCGTG CTAGAAGTGA AGTAAATACT
KatA C coli 1980 AGAATAGGAA CTAATTATCA TCTTTTACCT GTAAATCGTG CTAGAAGTGA AGTAAATACT
KatA C coli 2119 AGAATAGGAA CTAATTATCA TCTTTTACCT GTAAATCGTG CTAGAAGTGA AGTAAATACT
KatA C coli 2165 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 3050 TACAATGTC- ---------- ---------- ---------- ---------- ----------
KatA C jejuni 30 TACAATGTCG CTGGTGCTAT G--------- ---------- ---------- ----------
KatA C jejuni 62 TACAATGTC- ---------- ---------- ---------- ---------- ----------
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 683 TACAATGTC- ---------- ---------- ---------- ---------- ----------
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 1768 ---------- -----ACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG
CadF C jejuni 2072 ---------- -----ACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG
CadF C jejuni 813 ---------- -----ACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG
CadF C jejuni 683 GTAAAATTTG AAATCACTCC AACTTTAAAC TATAATTACT TTGAAGGTAA TTTAGATATG
CadF C jejuni 687 GTAAAATTTG AAATCACTCC 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 62 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 1206 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATTATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 30 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 1162 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 2038 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 2119 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 1768 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 2072 GATAATCGTT ATGCACCAGG GATTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 813 GATAATCGTT ATGCACCAGG TGTTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 683 GATAATCGTT ATGCACCAGG TGTTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 687 GATAATCGTT ATGCACCAGG TGTTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C jejuni 3050 GATAATCGTT ATGCACCAGG TGTTAGACTT GGTTATCATT TTGACGATTT TTGGCTTGAT
CadF C colistrainBG2108GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
CadF C coli 56 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
CadF C coli 175 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
CadF C coli 1980 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
CadF C coli 2119 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
376
CadF C coli 2165 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
CadF C coli 2887 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
CadF C coli 3064 GATAATCGCT ATGCACCAGG GATTAGACTA GGGTATCATT TTGATGATTT TTGGCTTGAT
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 62 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 1206 CAATTAGAAT TTGGGTTAGA GTATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 30 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 1162 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 2038 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 2119 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 1768 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 2072 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 813 CAATTAGAAT TTGGCTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 683 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 687 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C jejuni 3050 CAATTAGAAT TTGGGTTAGA GCATTATTCT GATGTTAAAT ATACAAATAC AAATAAAACT
CadF C colistrainBG2108CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
CadF C coli 56 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
CadF C coli 175 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
377
CadF C coli 1980 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
CadF C coli 2119 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
CadF C coli 2165 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
CadF C coli 2887 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
CadF C coli 3064 CAATTAGAAC TAGGTTTAGA ACATTACTCG GATGTAAAAT ATACAAATTC TACTCTTACC
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 62 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT
CadF C jejuni 1206 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT
CadF C jejuni 30 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT
CadF C jejuni 1162 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT
CadF C jejuni 2038 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT
CadF C jejuni 2119 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT
CadF C jejuni 1768 ACAGATATTA CAAGAACTTA TTTGAATGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT
CadF C jejuni 2072 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT
CadF C jejuni 813 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTGGG TGAGAAATTT
CadF C jejuni 683 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT
CadF C jejuni 687 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT
CadF C jejuni 3050 ACAGATATTA CAAGAACTTA TTTGAGTGCT ATTAAAGGTA TTGATGTAGG TGAGAAATTT
CadF C colistrainBG2108ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT
378
CadF C coli 56 ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT
CadF C coli 175 ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT
CadF C coli 1980 ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT
CadF C coli 2119 ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT
CadF C coli 2165 ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT
CadF C coli 2887 ACCGATATTA CTAGAACTTA TTTGAGTGCT ATTAAAGGCA TTGATTTAGG TGAGAAATTT
CadF C coli 3064 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 62 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 1206 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 30 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAAGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 1162 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAAGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 2038 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAAGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 2119 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAAGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 1768 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAAGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 2072 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 813 TATTTTTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 683 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT
CadF C jejuni 687 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT
379
CadF C jejuni 3050 TATTTCTATG GTTTAGCAGG TGGAGGATAT GAGGATTTTT CAAATGCTGC TTATGATAAT
CadF C colistrainBG2108TATTTTTATG GTTTAGCTGG TGGGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT
CadF C coli 56 TATTTTTATG GTTTAGCTGG TGTGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT
CadF C coli 175 TATTTTTATG GTTTAGCTGG TGTGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT
CadF C coli 1980 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 coli 2887 TATTTTTATG GTTTAGCTGG TGTGGGATAT GAGGATTTTT CTAAAGGCGC TTTTGATAAT
CadF C coli 3064 TATTTTTATG GTTTAGCTGG TGTGGGATAT 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 62 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 1206 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 30 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 1162 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 2038 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 2119 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 1768 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 2072 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGCCTTAG TGATTCTTTG
CadF C jejuni 813 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
380
CadF C jejuni 683 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 687 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C jejuni 3050 AAAAGCGGTG GATTTGGACA TTATGGCGCG GGTGTAAAAT TCCGTCTTAG TGATTCTTTG
CadF C colistrainBG2108AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA
CadF C coli 56 AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA
CadF C coli 175 AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA
CadF C coli 1980 AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA
CadF C coli 2119 AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA
CadF C coli 2165 AAAAGTGGAG GATTTGGCCA TTATGGAGCA GGTTTAAAAT TTCGCCTTAG TGATTCTTTA
CadF C coli 2887 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 62 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTCAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 1206 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTCAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 30 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 1162 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 2038 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 2119 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 1768 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
381
CadF C jejuni 2072 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 813 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTCAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 683 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 687 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C jejuni 3050 GCTTTAAGAC TTGAAACTAG AGATCAAATT AATTTTAATC ATGCAAACCA TAATTGGGTT
CadF C colistrainBG2108GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT
CadF C coli 56 GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT
CadF C coli 175 GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT
CadF C coli 1980 GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT
CadF C coli 2119 GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT
CadF C coli 2165 GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT
CadF C coli 2887 GCTTTAAGAC TTGAAACAAG AGATCAAATT TCTTTCCATG ATGCAGATCA TAGTTGGGTT
CadF C coli 3064 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--------
CadF C jejuni 62 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 1206 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 30 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 1162 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 2038 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
382
CadF C jejuni 2119 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 1768 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 2072 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 813 TCAACTTTAG GTATTAGTTT TGGTTTTGGT AGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 683 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 687 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG AAAAAGCTGT AG--------
CadF C jejuni 3050 TCAACTTTAG GTATTAGTTT TGGTTTTGGT GGCAAAAAGG 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 coli 2119 TCAACTTTGG GCATTAGTTT TGGTTTTGGC GCTAAGCAAG AAAAAGTTGT AGTGGAGCAA
CadF C coli 2165 TCAACTTTGG GCATTAGTTT TGGTTTTGGC GCTAAGCAAG AAAAAGTTGT AGTGGAGCAA
CadF C coli 2887 TCAACTTTGG GCATTAGTTT TGGTTTTGGC GCTAAGCAAG AAAAAGTTGT AGTGGAGCAA
CadF C coli 3064 TCAACTTTGG 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 62 ----AAGAAG TTGCTGATA- ---------- -------CTC GTGCAACTCC ---------A
CadF C jejuni 1206 ----AAGAAG TTGCTGATA- ---------- -------CTC GTGCAACTCC ---------A
CadF C jejuni 30 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
383
CadF C jejuni 1162 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 2038 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 2119 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 1768 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 2072 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 813 ----AAGAAG TTGGTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 683 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 687 ----AAGAAG TTGCTGATA- ---------- -------CTC GTCCAGCTCC ---------A
CadF C jejuni 3050 ----AAGAAG TTGCTGATA- ---------- -------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 coli 2119 ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG CTCCAGCTCC TGTAGTCTCA
CadF C coli 2165 ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG CTCCAGCTCC TGTAGTCTCA
CadF C coli 2887 ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG CTCCAGCTCC TGTAGTCTCA
CadF C coli 3064 ACAAAAGAAG TAGTTAATAA ACCTCAAGTT GTAACCCCTG CTCCAGCTCC 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 1206 CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C jejuni 30 CAAACAAAAT GTCCTGTAGA GCCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C jejuni 1162 CAAACAAAAT GTCCTGTAGA GCCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C jejuni 2038 CAAACAAAAT GTCCTGTAGA GCCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C jejuni 2119 CAAACAAAAT GTCCTGTAGA GCCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C jejuni 1768 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 jejuni 683 CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C jejuni 687 CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C jejuni 3050 CAAGCAAAAT GTCCTGTTGA ACCAAGAGAA GGTGCTTTGT TAGATGAAAA TGGTTGCGAA
CadF C colistrainBG2108CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA
CadF C coli 56 CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA
CadF C coli 175 CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA
CadF C coli 1980 CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA
CadF C coli 2119 CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA
CadF C coli 2165 CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA
CadF C coli 2887 CAATCAAAAT GTCCTGAAGA ACCAAGAGAG GGTGCTTTGT TGGATGAGAA TGGTTGCGAA
CadF C coli 3064 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 jejuni 62 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 1206 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 30 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 1162 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 2038 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 2119 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 1768 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 2072 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 813 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 683 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 687 AAAACTATTT CTTTGGAAGG TCATTTTGGT TTTGATAAAA CTACTATAAA TCCAACTTTT
CadF C jejuni 3050 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 coli 175 AAAACAATTT ATTTAGAGGG ACATTTTGAT TTTGATAAAG TAAATATCAA CCCAGCCTTT
CadF C coli 1980 AAAACAATTT ATTTAGAGGG ACATTTTGAT TTTGATAAAG TAAATATCAA CCCAGCCTTT
CadF C coli 2119 AAAACAATTT ATTTAGAGGG ACATTTTGAT TTTGATAAAG TAAATATCAA CCCAGCCTTT
CadF C coli 2165 AAAACAATTT ATTTAGAGGG ACATTTTGAT TTTGATAAAG TAAATATCAA CCCAGCCTTT
CadF C coli 2887 AAAACAATTT ATTTAGAGGG ACATTTTGAT TTTGATAAAG TAAATATCAA CCCAGCCTTT
CadF C coli 3064 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 jejuni 62 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 1206 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 30 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 1162 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 2038 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 2119 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 1768 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 2072 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 813 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 683 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 687 CAAGAAAAAA TCAAAGAAAT TGCAAAAGTT TTAGATGAAA ATGAAAGATA TGATACTATT
CadF C jejuni 3050 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 coli 175 GAAGAACAAA TCAAAGAAAT TGCTCAAATT TTAGATGAAA ATGTAAGATA TGATACTATT
CadF C coli 1980 GAAGAACAAA TCAAAGAAAT TGCTCAAATT TTAGATGAAA ATGTAAGATA TGATACTATT
CadF C coli 2119 GAAGAACAAA TCAAAGAAAT TGCTCAAATT TTAGATGAAA ATGTAAGATA TGATACTATT
CadF C coli 2165 GAAGAACAAA TCAAAGAAAT TGCTCAAATT TTAGATGAAA ATGTAAGATA TGATACTATT
CadF C coli 2887 GAAGAACAAA TCAAAGAAAT TGCTCAAATT TTAGATGAAA ATGTAAGATA TGATACTATT
CadF C coli 3064 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 62 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 1206 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 30 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 1162 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 2038 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 2119 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 1768 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 2072 CTTGAAGGAC ATACAGATAA TATAGGTTCA AGAGCTTATA ATCAAAAGCT TTCAGAAAGA
CadF C jejuni 813 CTTGAAGGAC ATACAGATAA TATCGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 683 CTTGAAGGAC ATACAGATAA TATTGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 687 CTTGAAGGAC ATACAGATAA TATTGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C jejuni 3050 CTTGAAGGAC ATACAGATAA TATTGGTTCA AGAGCTTATA ATCAAAAGCT TTCTGAAAGA
CadF C colistrainBG2108TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA
CadF C coli 56 TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA
CadF C coli 175 TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA
CadF C coli 1980 TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA
CadF C coli 2119 TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA
CadF C coli 2165 TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA
CadF C coli 2887 TTAGAGGGTC ATACTGATAA TATAGGTTCT AGATCATACA ATCAAAAACT TTCAGAAAGA
CadF C coli 3064 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 jejuni 62 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 1206 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 30 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 1162 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 2038 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 2119 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 1768 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 2072 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 813 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 683 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 687 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C jejuni 3050 CGTGCTAAAA GTGTTGCTAA TGAACTTGAA AAATATGGTG TAGAAAAAAG TCGCATCAAA
CadF C colistrainBG2108CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
CadF C coli 56 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
CadF C coli 175 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
CadF C coli 1980 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
CadF C coli 2119 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
CadF C coli 2165 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
389
CadF C coli 2887 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
CadF C coli 3064 CGCGCTAACA GCGTTGCAAA AGAGCTTGAA AAATTCGGTG TAGATAAAAG TCGTATCCAG
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 62 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 1206 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 30 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 1162 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 2038 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 2119 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 jejuni 813 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 683 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 687 ACAGTAGGTT ATGGTCAAGA TAATCCTCGC TCAAGCAATG ACACTAAAGA AGGTAGAGCG
CadF C jejuni 3050 ACAGTAGGTT ATGGTCAAGA 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 175 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG ACACTAAAGA GGGTAGAGCA
CadF C coli 1980 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG ACACTAAAGA GGGTAGAGCG
390
CadF C coli 2119 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG ACACTAAAGA GGGTAGAGCA
CadF C coli 2165 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG ACACTAAAGA GGGTAGAGCA
CadF C coli 2887 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCGAGCAATG ACACTAAAGA GGGTAGAGCA
CadF C coli 3064 ACAGTTGGTT ATGGTCAAGA TAAGCCACGC TCAAGCAATG 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
2072 C jejuni Peb ---GTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA
2114 C jejuni Peb --GGTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA
2170 C jejuni Peb ---GTTTTTA GAAAATCTTT GTTAAAGTTG GCAGTTTTTG CTCTAGGTGC TTGTGTTGCA
3050 C jejuni Peb --GGTTTTTA GAAAATCTTT 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
683 C jejuni Peb TTTAGTAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
687 C jejuni Peb TTTAGTAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
813 C jejuni Peb TTTAGTAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
393
1162 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
1206 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
1768 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
2038 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
2072 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
2114 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
2170 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAG CTTGAGTCTA TTAAATCTAA AGGACAATTA
3050 C jejuni Peb TTTAGCAATG CTAATGCAGC AGAAGGTAAA CTTGAGTCTA TTAAATCTAA AGGACAATTA
C coli strain YH502 Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
56 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
175 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
1980 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
2040 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
2119 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
2165 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
2887 C coli Peb TTTACTAGTG CAAATGCAGC TGAAGGAAAA CTTGAAGCTA TCAAGGCTAA AGGAGAGTTG
3064 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
687 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCACAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
394
813 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCACAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
1162 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
1206 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
1768 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
2038 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
2072 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGT AACAGGTGAA
2114 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
2170 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
3050 C jejuni Peb ATAGTTGGTG TTAAAAATGA TGTTCCGCAT TATGCTTTAC TTGATCAAGC AACAGGTGAA
C coli strain YH502 Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
56 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
175 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
1980 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
2040 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
2119 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
2165 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
2887 C coli Peb GTTATAGGTG TAAAAAATGA TGTACCACAC TATGCTTTAC TTGATCAAGC TACAGGCGAA
3064 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
687 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT AGGTGATGAT
813 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT AGGTGATGAT
1162 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCTAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT
1206 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT
1768 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCTAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT
2038 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT
2072 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT
2114 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCTAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT
2170 C jejuni Peb ATTAAAGGTT TCGAAGTAGA TGTTGCCAAA TTGCTAGCTA AAAGTATATT GGGTGATGAT
3050 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
175 C coli Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA
1980 C coli Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA
2040 C coli Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA
2119 C coli Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA
2165 C coli Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA
2887 C coli Peb ATTAAAGGCT TTGAAGTTGA TGTTGCTAAA ATGCTTGCTA AGAGTATTTT AGGAGATGAA
3064 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
62 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
396
683 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
687 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
813 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
1162 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
1206 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
1768 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
2038 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
2072 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
2114 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
2170 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
3050 C jejuni Peb AAAAAAATAA AACTAGTTGC AGTTAATGCT AAAACAAGAG GCCCTTTGCT TGATAATGGT
C coli strain YH502 Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
56 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
175 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
1980 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
2040 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
2119 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
2165 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
2887 C coli Peb AATAAAGTTA AACTTATAGC AGTAAATGCT AAAACAAGAG GTCCATTACT TGATAATGGT
3064 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
62 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
683 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
687 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
813 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
1162 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
1206 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCGG AGAGAAAAAG AATTTATAAT
1768 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
2038 C jejuni Peb AGTGTAGATG CGGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
2072 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
2114 C jejuni Peb AGTGTAGATG CAGTGATAGC AACTTTTACT ATTACTCCAG AGAGAAAAAG AATTTATAAT
2170 C jejuni Peb AGTGTAGATG CAGTGATAGC 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
175 C coli Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT
1980 C coli Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT
2040 C coli Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT
2119 C coli Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT
2165 C coli Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT
2887 C coli Peb AGCGTTGATG CGGTTATAGC AACTTTTACT ATCACTCCAG AGAGAAAAAG AGTGTATAAT
3064 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
683 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAATTAT
687 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAATTAT
813 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAATTAT
1162 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTAG TCTTAAAAGA AAAAAATTAT
1206 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAATTAT
1768 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTAG TCTTAAAAGA AAAAAATTAT
2038 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAAATAT
2072 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTGG TTTTAAAAGA AAAAAATTAT
2114 C jejuni Peb TTCTCAGAGC CTTATTATCA AGATGCTATA GGGCTTTTAG TCTTAAAAGA AAAAAATTAT
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
175 C coli Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT
1980 C coli Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT
2040 C coli Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT
2119 C coli Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT
2165 C coli Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT
2887 C coli Peb TTTTCAGAGC CGTATTATCA AGATGCTGTA GGGCTTTTAG TTTTAAAAGA GAAAAATTAT
3064 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
683 C jejuni Peb AAATCTCTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA
687 C jejuni Peb AAATCTCTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA
813 C jejuni Peb AAATCTCTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA
1162 C jejuni Peb AAATCTTTAG CTGATATGAA AGGTGCAAAC ATTGGAGTGG CTCAAGCTGC AACTACAAAA
1206 C jejuni Peb AAATCTCTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA
1768 C jejuni Peb AAATCTTTAG CTGATATGAA AGGTGCAAAC ATTGGAGTGG CTCAAGCTGC AACTACAAAA
2038 C jejuni Peb AAATCTTTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA
2072 C jejuni Peb AAATCTCTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA
2114 C jejuni Peb AAATCTTTAG CTGATATGAA AGGTGCAAAC ATTGGAGTGG CTCAAGCTGC AACTACAAAA
2170 C jejuni Peb AAATCTTTAG CTGATATGAA AGGTGCAAAT ATTGGAGTGG CTCAAGCTGC AACTACAAAA
3050 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
175 C coli Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA
1980 C coli Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA
2040 C coli Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA
2119 C coli Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA
2165 C coli Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA
2887 C coli Peb AAATCTTTAG CAGATATGAA TGGTGCTACT ATAGGGGTAG CTCAAGCAGC AACTACTAAA
3064 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
683 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT
687 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT
813 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT
1162 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGTATTGATG TTAAATTTAG TGAATTTCCT
1206 C jejuni Peb AAAGCTATAG GTAAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT
1768 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGTATTGATG TTAAATTTAG TGAATTTCCT
2038 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT
2072 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT
2114 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGTATTGATG TTAAATTTAG TGAATTTCCT
2170 C jejuni Peb AAAGCTATAG GTGAAGCTGC TAAAAAAATT GGCATTGATG TTAAATTTAG TGAATTTCCT
3050 C jejuni Peb AAAGCTATAG GTGAAGCTGC 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
175 C coli Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT
1980 C coli Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT
2040 C coli Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT
2119 C coli Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT
2165 C coli Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT
2887 C coli Peb AAAGTTATCA ATACTGCGGC TAAAAAAATA GGTGTTAAAG TAAAATTCAG CGAATTTCCT
3064 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
683 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
687 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
813 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
1162 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
1206 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
1768 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
2038 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
2072 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
2114 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
2170 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
3050 C jejuni Peb GATTATCCAA GTATAAAAGC TGCTTTAGAT GCTAAAAGAG TTGATGCGTT TTCTGTAGAC
C coli strain YH502 Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
56 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
175 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
1980 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
2040 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
2119 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
2165 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
2887 C coli Peb GATTATCCTA GCATAAAAGC AGCTTTAGAT GCAAAAAGAA TTGATGCGTT TTCAGTTGAT
3064 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
683 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
687 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
813 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
1162 C jejuni Peb AAATCAATAT TGTTAGGCTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
1206 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
1768 C jejuni Peb AAATCAATAT TGTTAGGCTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
2038 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
2072 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
2114 C jejuni Peb AAATCAATAT TGTTAGGCTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
2170 C jejuni Peb AAATCAATAT TGTTAGGTTA TGTGGATGAT AAAAGTGAAA TTTTGCCAGA TAGTTTTGAA
3050 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
175 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT
1980 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT
2040 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT
2119 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT
2165 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT
2887 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT
403
3064 C coli Peb AAATCTATTT TACTAGGTTA TAAAGATGAG AATAATGAAA TTTTACCTGA TAGTTTCGAT
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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
62 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
683 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
687 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
813 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
1162 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
1206 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
1768 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
2038 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
2072 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
2114 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
2170 C jejuni Peb CCACAAAGTT ATGGTATTGT AACCAAAAAA GATGATCCAG CTTTTGCAAA ATATGTTGAT
3050 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
175 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT
1980 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT
2040 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT
2119 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT
2165 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT
404
2887 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT
3064 C coli Peb CCTCAAAGTT ATGGCATAGT TACAAAAAAA GATGATGCAA ATTTTTCAAA TTATGTCAAT
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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
62 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTATAA
683 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 ----------
1206 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTATAA
1768 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTA---
2038 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTATAA
2072 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTA---
2114 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTATAA
2170 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTA---
3050 C jejuni Peb GATTTTGTAA AAGAACATAA AAATGAAATT GATGCTTTAG CGAAAAAATG GGGTTTATAA
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- ---------- ----------
1980 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTA- ---------- ----------
2040 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTA- ---------- ----------
2119 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTAG CGAAAAAATG GGGTTTATAA
405
2165 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTAG CGAAAAAATG GGGTTTATAA
2887 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTAG CGAAAAAATG GGGTTTATAA
3064 C coli Peb GATTTTGTAA AACAAAACAA AACTGAAATC GACGCTTTAG CGAAAAAATG GGGTTTATAA
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 1768 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA
CjaA-Cc C jejuni 2072 ---------C TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA
CjaA-Cc C jejuni 2114 ATTCAGATTC TGGTGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA
CjaA-Cc C jejuni 3050 ATTCAGATTC 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 175 ---------- ---TGCTTCA AATTCTCTTG AAAGAATCAA GCAAGATGGA GTAGTAAGAA
CjaA-Cc C coli 1980 ---------- ---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
CjaA-Cc C coli 3064 ---------- ---TGCTTCA 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 2170 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG
CjaA-Cc C jejuni 30 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG
CjaA-Cc C jejuni 687 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG
CjaA-Cc C jejuni 1162 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG
CjaA-Cc C jejuni 1768 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG
CjaA-Cc C jejuni 2072 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG
CjaA-Cc C jejuni 2114 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG
CjaA-Cc C jejuni 3050 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG
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
CjaA-Cc C coli 1980 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG
CjaA-Cc C coli 2040 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC ATAAATCAAG
CjaA-Cc C coli 2119 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG
CjaA-Cc C coli 2165 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG
CjaA-Cc C coli 2887 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG
CjaA-Cc C coli 3064 TAGGAGTTTT TGGAGATAAA CCGCCTTTTG GTTATGTAGA TGAAAAAGGC GTAAATCAAG
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-Cc C jejuni 2170 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C jejuni 30 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C jejuni 687 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C jejuni 1162 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C jejuni 1768 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C jejuni 2072 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C jejuni 2114 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C jejuni 3050 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
CjaA-Cc C coli 175 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C coli 1980 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C coli 2040 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C coli 2119 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C coli 2165 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C coli 2887 GTTATGATAT AGTCTTGGCG AAACGTATAG CAAAAGAACT CTTAGGAGAT GAAAATAAGG
CjaA-Cc C coli 3064 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-Cc C jejuni 2170 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C jejuni 30 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C jejuni 687 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C jejuni 1162 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C jejuni 1768 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C jejuni 2072 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C jejuni 2114 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C jejuni 3050 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
CjaA-Cc C coli 175 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C coli 1980 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C coli 2040 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C coli 2119 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C coli 2165 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
CjaA-Cc C coli 2887 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
422
CjaA-Cc C coli 3064 TGCAGTTTGT ATTAGTTGAA GCTGCAAATA GGGTGGAATT TTTAAAATCA AATAAAGTTG
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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-Cc C jejuni 2170 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C jejuni 30 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C jejuni 687 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C jejuni 1162 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C jejuni 1768 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C jejuni 2072 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C jejuni 2114 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C jejuni 3050 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 175 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C coli 1980 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C coli 2040 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
CjaA-Cc C coli 2887 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
CjaA-Cc C coli 3064 ATATTATTTT AGCTAATTTT ACTCAAACAC CTGAAAGAGC AGAGCAAGTG GATTTTTGCT
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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-Cc C jejuni 2170 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C jejuni 30 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C jejuni 687 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C jejuni 1162 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C jejuni 1768 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C jejuni 2072 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C jejuni 2114 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C jejuni 3050 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
CjaA-Cc C coli 175 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C coli 1980 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C coli 2040 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C coli 2119 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
424
CjaA-Cc C coli 2165 TACCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C coli 2887 TGCCTTATAT GAAGGTAGCT TTAGGTGTGG CTGTGCCTCA AGATAGCAAT ATCAGTAGCA
CjaA-Cc C coli 3064 TACCTTATAT 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-Cc C jejuni 2170 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C jejuni 30 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C jejuni 687 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C jejuni 1162 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C jejuni 1768 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C jejuni 2072 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C jejuni 2114 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C jejuni 3050 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
CjaA-Cc C coli 175 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C coli 1980 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C coli 2040 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
CjaA-Cc C coli 2887 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT GATGCGTATT
CjaA-Cc C coli 3064 TAGAAGATTT AAAAGATAAA ACTTTACTTT TAAACAAAGG AACTACTGCT 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-Cc C jejuni 2170 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C jejuni 30 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C jejuni 687 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C jejuni 1162 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C jejuni 1768 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C jejuni 2072 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C jejuni 2114 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C jejuni 3050 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
CjaA-Cc C coli 175 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C coli 1980 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
426
CjaA-Cc C coli 2040 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C coli 2119 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C coli 2165 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C coli 2887 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
CjaA-Cc C coli 3064 TTACAAAAGA ATATCCTGAT ATTAAAACAT TAAAATACGA TCAAAATACC GAAACTTTTG
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 2170 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C jejuni 30 CCGCTTTAAT AGATCAAAGA GGTGATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C jejuni 687 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C jejuni 1162 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C jejuni 1768 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C jejuni 2072 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C jejuni 2114 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C jejuni 3050 CCGCTTTAAT AGATCAAAGA GGTGATGCTT 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 1980 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
CjaA-Cc C coli 2040 CCGCTTTAAT AGATCAAAGA GGTAATGCTT TAAGTCATGA CAATACTTTG CTTTTTGCGT
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-Cc C jejuni 2170 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C jejuni 30 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C jejuni 687 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C jejuni 1162 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C jejuni 1768 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C jejuni 2072 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C jejuni 2114 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C jejuni 3050 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
CjaA-Cc C coli 175 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C coli 1980 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C coli 2040 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C coli 2119 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C coli 2165 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C coli 2887 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
CjaA-Cc C coli 3064 GGGTAAAAGA ACATCCTGAA TTTAAAATGG CCATTAAAGA ATTGGGCAAT AAAGATGTAA
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 2170 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C jejuni 30 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTGAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C jejuni 687 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C jejuni 1162 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C jejuni 1768 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C jejuni 2072 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C jejuni 2114 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C jejuni 3050 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA 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 175 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C coli 1980 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C coli 2040 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C coli 2119 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C coli 2165 TTGCTCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C coli 2887 TTGCCCCTGC TGTTAAAAAA GGTGATAAAG AGCTTAAAGA ATTTATTGAT AATCTAATCA
CjaA-Cc C coli 3064 TTGCTCCTGC 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
CjaA-Cc C jejuni 2170 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C jejuni 30 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C jejuni 687 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C jejuni 1162 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C jejuni 1768 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C jejuni 2072 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C jejuni 2114 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C jejuni 3050 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
CjaA-Cc C coli 175 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C coli 1980 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C coli 2040 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C coli 2119 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C coli 2165 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C coli 2887 CAAAATTAGG AGAAGAACAA TTCTTCCATA AAGCTTATGA TGAAACTTTA AAAAGTCATT
CjaA-Cc C coli 3064 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 1162 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTA------- ---------- ----------
CjaA-Cc C jejuni 1768 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTATTGAAGG CGGTAA---- ----------
CjaA-Cc C jejuni 2072 TTGGAGATGA TGTAAAAGCC GATGATGTAG TTA------- ---------- ----------
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|....| ....|....| ....|....| ....|....|
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 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 ---------- ---------- ---------- ---------- ---------- ----------
....|....| ....|....| ....|...
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
2119CcolikatA --------------------------------------------CPESLHQVTILMSDRG 16
2165CcolikatA --------------------------------------------CPESLHQVTILMSDRG 16
2887CcolikatA --------------------------------------------CPESLHQVTILMSDRG 16
1768CjejunikatA --------------------------------------------------QVTILMSDRG 16
813CjejunikatA --------------------------------------------------QVTILMSDRG 16
683CjejunikatA -------------------------------------------------HQVTILMSDRG 16
687CjejunikatA --------------------------------------------------QVTILMSDRG 16
CjejuniNCTC11186katA LVGNNTPTFFIRDAYKFPDFIHTQKRDPRTHLRSNNAAWDFWSLCPESLHQVTILMSDRG 180
1206CjejunikatA -------------------------------------------------HQVTILMSDRG 16
3050CjejunikatA -------------------------------------------------HQVTILMSDRG 16
30CjejunikatA -----------------------------------------------SLHQVTILMSDRG 16
62CjejunikatA ------------------------------------------------LHQVTILMSDRG 16
1162CjejunikatA --------------------------------------------------QVTILMSDRG 16
2038CjejunikatA --------------------------------------------------QVTILMSDRG 16
2072CjejunikatA -------------------------------------------------HQVTILMSDRG 16
2114CjejunikatA ------------------------------------------------LHQVTILMSDRG 16
2170CjejunikatA --------------------------------------------------QVTILMSDRG 16
CcolistrainRM4661katA LVGNNTPTFFIRDAYKFPDFIHTQKRDPRTHLRSNNAAWDFWSLCPESLHQVTILMSDRG 180
2040CcolikatA --------------------------------------------------QVTILMSDRG 16
3064CcolikatA -------------------------------------------------HQVTILMSDRG 16
**********
442
56CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTLQGIKNLSNKEAAELIAKDRESHQRD 76
175CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTLQGIKNLSNKEAAELIAKDRESHQRD 76
1980CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTLQGIKNLSNKEAAELIAKDRESHQRD 76
2119CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTLQGIKNLSNKEAAELIAKDRESHQRD 76
2165CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTLQGIKNLSNKEAAELIAKDRESHQRD 76
2887CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTLQGIKNLSNKEAAELIAKDRESHQRD 76
1768CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
813CjejunikatA IPASYRHMHGFGSHTYSFINHKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
683CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
687CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
CjejuniNCTC11186katA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 240
1206CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
3050CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
30CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
62CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
1162CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
2038CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
2072CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
2114CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
2170CjejunikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
CcolistrainRM4661katA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 240
2040CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
3064CcolikatA IPASYRHMHGFGSHTYSFINDKNERFWVKFHFKTQQGIKNLTNQEAAELIAKDRESHQRD 76
********************.************* ******:*:****************
443
56CcolikatA LYNAIENKDFPKWKVQVQILAEKDADKLGFNPFDLTKIWPHSVVPLMDIGEMILNQNPQN 136
175CcolikatA LYNAIENKDFPKWKVQVQILAEKDADKLGFNPFDLTKIWPHSVVPLMDIGEMILNQNPQN 136
1980CcolikatA LYNAIENKDFPKWKVQVQILAEKDADKLGFNPFDLTKIWPHSVVPLMDIGEMILNQNPQN 136
2119CcolikatA LYNAIENKDFPKWKVQVQILAEKDADKLGFNPFDLTKIWPHSVVPLMDIGEMILNQNPQN 136
2165CcolikatA LYNAIENKDFPKWKVQVQILAEKDADKLGFNPFDLTKIWPHSVVPLMDIGEMILNQNPQN 136
2887CcolikatA LYNAIENKDFPKWKVQVQILAEKDADKLGFNPFDLTKIWPHSVVPLMDIGEMILNQNPQN 136
1768CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLEFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
813CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
683CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
687CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
CjejuniNCTC11186katA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSFVPLMDIGEMILNKNPQN 300
1206CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSFVPLMDIGEMILNKNPQN 136
3050CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSFVPLMDIGEMILNKNPQN 136
30CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
62CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
1162CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
2038CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
2072CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
2114CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
2170CjejunikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
CcolistrainRM4661katA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 300
2040CcolikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
3064CcolikatA LYNAIENKDFPKWKVQVQILAEKDIEKLGFNPFDLTKIWPHSLVPLMDIGEMILNKNPQN 136
************************ :** *************.************:****
444
56CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRARSEVNT 196
175CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRARSEVNT 196
1980CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRARSEVNT 196
2119CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRARSEVNT 196
2165CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRARSEVNT 196
2887CcolikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRARSEVNT 196
1768CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
813CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
683CjejunikatA YFNEVEQAAFSPSNIIPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
687CjejunikatA YFNEVEQAAFSPSNIIPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
CjejuniNCTC11186katA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 360
1206CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
3050CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
30CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
62CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
1162CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
2038CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
2072CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
2114CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
2170CjejunikatA YFNEVEQAAFSPSNIVPGIGFSPDKMLQARIFSYPDAQRYRIGTNYHLLPVNRAKSEVNT 196
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
2114CjejunicadF -------------------------TPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42
2072CjejunicadF -------------------------TPTLNYNYFEGNLDMDNRYAPGIRLGYHFDDFWLD 42
****************:****:*******
450
CcolistrainBG2108cadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGGGYEDFSKGAFDN 120
3064CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGVGYEDFSKGAFDN 102
2119CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGGGYEDFSKGAFDN 102
2165CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGGGYEDFSKGAFDN 102
175CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGVGYEDFSKGAFDN 102
1980CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGVGYEDFSKGAFDN 102
56CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGVGYEDFSKGAFDN 102
2887CcolicadF QLELGLEHYSDVKYTNSTLTTDITRTYLSAIKGIDLGEKFYFYGLAGVGYEDFSKGAFDN 102
1206CjejunicadF QLEFGLEYYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
CjejuniNCTC11168cadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 120
2170CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
62CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
2040CcolicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
1768CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLNAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
813CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
683CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 101
687CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
3050CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
CcolistrainBP3183cadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 120
30CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
1162CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
2038CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
2114CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
2072CjejunicadF QLEFGLEHYSDVKYTNTNKTTDITRTYLSAIKGIDVGEKFYFYGLAGGGYEDFSNAAYDN 102
451
***:***:********:. *********.******:*********** ******:.*:**
CcolistrainBG2108cadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 180
3064CcolicadF KSGGFGHYGAGLKFRLNDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162
2119CcolicadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162
2165CcolicadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162
175CcolicadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162
1980CcolicadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162
56CcolicadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162
2887CcolicadF KSGGFGHYGAGLKFRLSDSLALRLETRDQISFHDADHSWVSTLGISFGFGAKQEKVVVEQ 162
1206CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
CjejuniNCTC11168cadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 180
2170CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
62CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
2040CcolicadF KSGGFGHYGTGVKFCLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
1768CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
813CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGSKKEKAVEEV 162
683CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 161
687CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
3050CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
CcolistrainBP3183cadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 180
30CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
1162CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
2038CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
2114CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
452
2072CjejunicadF KSGGFGHYGAGVKFRLSDSLALRLETRDQINFNHANHNWVSTLGISFGFGGKKEKAVEEV 162
*********:*:** *.*************.*:.*:*.************.*:**.* *
CcolistrainBG2108cadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 240
3064CcolicadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222
2119CcolicadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222
2165CcolicadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222
175CcolicadF TKEVVNKPQVVTPVPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222
1980CcolicadF TKEVVNKPQVVTPVPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222
56CcolicadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 222
2887CcolicadF TKEVVNKPQVVTPAPAPVVSQSKCPEEPREGALLDENGCEKTIYLEGHFDFDKVNINPAF 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
687CjejunicadF A-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209
3050CjejunicadF A-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209
CcolistrainBP3183cadF A-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 227
30CjejunicadF A-------------DTRPAPQTKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209
1162CjejunicadF A-------------DTRPAPQTKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209
2038CjejunicadF A-------------DTRPAPQTKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209
453
2114CjejunicadF A-------------DTRPAPQTKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209
2072CjejunicadF A-------------DTRPAPQAKCPVEPREGALLDENGCEKTISLEGHFGFDKTTINPTF 209
: . * *** ***************** *****.***..***:*
CcolistrainBG2108cadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 300
3064CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282
2119CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282
2165CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282
175CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282
1980CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282
56CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282
2887CcolicadF EEQIKEIAQILDENVRYDTILEGHTDNIGSRSYNQKLSERRANSVAKELEKFGVDKSRIQ 282
1206CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
CjejuniNCTC11168cadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 287
2170CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
62CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
2040CcolicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
1768CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
813CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
683CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 268
687CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
3050CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
CcolistrainBP3183cadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 287
30CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
1162CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
454
2038CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
2114CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
2072CjejunicadF QEKIKEIAKVLDENERYDTILEGHTDNIGSRAYNQKLSERRAKSVANELEKYGVEKSRIK 269
:*:*****::**** ****************:**********:***:****:**:****:
CcolistrainBG2108cadF TVGYGQDKPRSSNDTKEGRADNRRVEAKFILN 332
3064CcolicadF TVGYGQDKPRSSNDTKEGRAD----------- 314
2119CcolicadF TVGYGQDKPRSSNDTKEGRADNRR-------- 314
2165CcolicadF TVGYGQDKPRSSNDTKEGRADNRR-------- 314
175CcolicadF TVGYGQDKPRSSNDTKEGRADNRR-------- 314
1980CcolicadF TVGYGQDKPRSSNDTKEGRADN---------- 314
56CcolicadF TVGYGQDKPRSSNDTKEGRADNRR-------- 314
2887CcolicadF TVGYGQDKPRSSNDTKEGRADNRR-------- 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
687CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFILR 301
3050CjejunicadF TVGYGQDNPRSSNDTKEGRA------------ 301
CcolistrainBP3183cadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFILR 319
30CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFIL- 301
455
1162CjejunicadF TVGYGQDNPRSSNDTKEGRADNRRVDAKFILR 301
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
2119Ccolipeb -----SLLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60
2887Ccolipeb -----SLLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60
2165Ccolipeb -----SLLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60
CcoliYH501peb -----SLLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60
56Ccolipeb ------LLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60
175Ccolipeb ------LLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60
3064Ccolipeb -----SLLKLAALALGACMAFTSANAAEGKLEAIKAKGELVIGVKNDVPHYALLDQATGE 60
1206Cjejunipeb -----SLLKLAVFALGVCVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
62Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPYYALLDQATGE 60
2072Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQVTGE 60
CjejuniYH002peb MVFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
2038Cjejunipeb -----SLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
2170Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
3050Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
30Cjejunipeb -----SLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
683Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
687Cjejunipeb MVFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
813Cjejunipeb -----SLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
1162Cjejunipeb ------LLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
1768Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
2114Cjejunipeb -VFRKSLLKLAVFALGACVAFSNANAAEGKLESIKSKGQLIVGVKNDVPHYALLDQATGE 60
*****.:***.*:**:.*********:**:**:*::*******:******.***
458
1980Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
2040Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
2119Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
2887Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
2165Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
CcoliYH501peb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
56Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
175Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
3064Ccolipeb IKGFEVDVAKMLAKSILGDENKVKLIAVNAKTRGPLLDNGSVDAVIATFTITPERKRVYN 120
1206Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
62Cjejunipeb IKGFEIDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
2072Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
CjejuniYH002peb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
2038Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
2170Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
3050Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
30Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
683Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
687Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
813Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
1162Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
1768Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
2114Cjejunipeb IKGFEVDVAKLLAKSILGDDKKIKLVAVNAKTRGPLLDNGSVDAVIATFTITPERKRIYN 120
*****:****:********::*:**:*******************************:**
459
1980Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
2040Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
2119Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
2887Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
2165Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
CcoliYH501peb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
56Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
175Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
3064Ccolipeb FSEPYYQDAVGLLVLKEKNYKSLADMNGATIGVAQAATTKKVINTAAKKIGVKVKFSEFP 180
1206Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGKAAKKIGIDVKFSEFP 180
62Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
2072Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
CjejuniYH002peb FSEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
2038Cjejunipeb FSEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
2170Cjejunipeb FSEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
3050Cjejunipeb FSEPYYQDAIGLLVLKEKKYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
30Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
683Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
687Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
813Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
1162Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
1768Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
2114Cjejunipeb FSEPYYQDAIGLLVLKEKNYKSLADMKGANIGVAQAATTKKAIGEAAKKIGIDVKFSEFP 180
*********:********:*******:**.***********.*. ******:.*******
460
1980Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
2040Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
2119Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
2887Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
2165Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
CcoliYH501peb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
56Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
175Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
3064Ccolipeb DYPSIKAALDAKRIDAFSVDKSILLGYKDENNEILPDSFDPQSYGIVTKKDDANFSNYVN 240
1206Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
62Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
2072Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
CjejuniYH002peb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
2038Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
2170Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
3050Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
30Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
683Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
687Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
813Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
1162Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
1768Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
2114Cjejunipeb DYPSIKAALDAKRVDAFSVDKSILLGYVDDKSEILPDSFEPQSYGIVTKKDDPAFAKYVD 240
*************:************* *::.*******:************ *::**:
461
1980Ccolipeb DFVKQNKTEIDAL------ 259
2040Ccolipeb DFVKQNKTEIDALA----- 259
2119Ccolipeb DFVKQNKTEIDALAKKWGL 259
2887Ccolipeb DFVKQNKTEIDALAKKWGL 259
2165Ccolipeb DFVKQNKTEIDALAKKWGL 259
CcoliYH501peb DFVKQNKTEIDALAKKWGL 259
56Ccolipeb DFVKQNKTEIDALAKKW-- 259
175Ccolipeb DFVKQNKTEIDAL------ 259
3064Ccolipeb DFVKQNKTEIDALAKKWGL 259
1206Cjejunipeb DFVKEHKNEIDALAKKWGL 259
62Cjejunipeb DFVKEHKNEIDALAKKWGL 259
2072Cjejunipeb DFVKEHKNEIDALAKKWG- 259
CjejuniYH002peb DFVKEHKNEIDALAKKWGL 259
2038Cjejunipeb DFVKEHKNEIDALAKKWGL 259
2170Cjejunipeb DFVKEHKNEIDALAKKWG- 259
3050Cjejunipeb DFVKEHKNEIDALAKKWGL 259
30Cjejunipeb DFVKEHKNEIDALAKKWGL 259
683Cjejunipeb DFVKEHKNEIDALAKKWGL 259
687Cjejunipeb DFVKEHKNEIDALAKKWG- 259
813Cjejunipeb DFVKEHKNEIDALAKKW-- 259
1162Cjejunipeb DFVKEHKNEIDALAKK--- 259
1768Cjejunipeb DFVKEHKNEIDALAKKWG- 259
2114Cjejunipeb DFVKEHKNEIDALAKKWGL 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
2114CjejunicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30
175CcolicjaA -----------------------------------ASNSLERIKQDGVVRIGVFGDKPPF 30
1980CcolicjaA -----------------------------------ASNSLERIKQDGVVRIGVFGDKPPF 30
2040CcolicjaA ------------------------------------SNSLERIKQDGVVRIGVFGDKPPF 30
56CcolicjaA -----------------------------------ASNSLERIKQDGVVRIGVFGDKPPF 30
30CjejunicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30
CcolistrainYH502cjaA --------MKKMLLSIFTTFVAVFLAACGGNSDSGASNSLERIKQDGVVRIGVFGDKPPF 52
C.jejunicjaAgene,GenBank:Y10872.1 --------MKKMLLSIFTTFVAVFLAACGGNSDSGASNSLERIKQDGVVRIGVFGDKPPF 52
62CjejunicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30
683CjejunicjaA ---------------------------------SGASNSLERIKQDGVVRIGVFGDKPPF 30
813CjejuniajaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30
1206CjejunicjaA ---------------------------------SGASNSLERIKQDGVVRIGVFGDKPPF 30
2038CjejunicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30
2170CjejunicjaA ------------------------------------------IKQDGVVRIGVFGDKPPF 30
3050CjejunicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30
2119CcolicjaA ------------------------------------------IKQDGVVRIGVFGDKPPF 30
2165CcolicjaA ----------------------------------GASNSLERIKQDGVVRIGVFGDKPPF 30
2887CcolicjaA ------------------------------NSDSGASNSLERIKQDGVVRIGVFGDKPPF 30
3064CcolicjaA -----------------------------------ASNSLERIKQDGVVRIGVFGDKPPF 30
******************
464
687CjejunicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
1162CjejunicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
1768CjejunicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2072CjejunicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2114CjejunicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
175CcolicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
1980CcolicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2040CcolicjaA GYVDEKGINQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
56CcolicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
30CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
CcolistrainYH502cjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 112
C.jejunicjaAgene,GenBank:Y10872.1 GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 112
62CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
683CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
813CjejuniajaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
1206CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2038CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2170CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
3050CjejunicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2119CcolicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2165CcolicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
2887CcolicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
3064CcolicjaA GYVDEKGVNQGYDIVLAKRIAKELLGDENKVQFVLVEAANRVEFLKSNKVDIILANFTQT 90
*******:****************************************************
465
687CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
1162CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
1768CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2072CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2114CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
175CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
1980CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2040CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
56CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
30CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
CcolistrainYH502cjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 172
C.jejunicjaAgene,GenBank:Y10872.1 PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 172
62CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
683CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
813CjejuniajaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
1206CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2038CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2170CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
3050CjejunicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2119CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2165CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
2887CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
3064CcolicjaA PERAEQVDFCLPYMKVALGVAVPQDSNISSIEDLKDKTLLLNKGTTADAYFTKEYPDIKT 150
************************************************************
466
687CjejunicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
1162CjejunicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
1768CjejunicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2072CjejunicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2114CjejunicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
175CcolicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
1980CcolicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2040CcolicjaA LKYDQNTETFAALIDQRGNALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
56CcolicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFSWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
30CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
CcolistrainYH502cjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 232
C.jejunicjaAgene,GenBank:Y10872.1 LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 232
62CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
683CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
813CjejuniajaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
1206CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2038CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2170CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
3050CjejunicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2119CcolicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2165CcolicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
2887CcolicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
3064CcolicjaA LKYDQNTETFAALIDQRGDALSHDNTLLFAWVKEHPEFKMAIKELGNKDVIAPAVKKGDK 210
******************:**********:******************************
467
687CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGG-- 255
1162CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVV------ 255
1768CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGK- 256
2072CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVV------ 255
2114CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGK- 256
175CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDV------- 255
1980CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDV------- 255
2040CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDV------- 255
56CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDV------- 255
30CjejunicjaA ELEEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVV------ 255
CcolistrainYH502cjaA ELKEFIDNLITKLGEEQFFHKVYDETLKSHFGDDVKADDVVIEGGKI 279
C.jejunicjaAgene,GenBank:Y10872.1 ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGKI 279
62CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGK- 256
683CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGK- 256
813CjejuniajaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIE---- 255
1206CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVV------ 255
2038CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGK- 256
2170CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVK----------- 255
3050CjejunicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVIEGGK- 256
2119CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDV------- 255
2165CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADD-------- 255
2887CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDVVI----- 255
3064CcolicjaA ELKEFIDNLITKLGEEQFFHKAYDETLKSHFGDDVKADDV------- 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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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
pETSUMO-peb1A fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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 fusion protein ---------- ---------- ---------- ---------- ---------- ---------- ----------
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