Setta, Ahmed Mohamed Hassanin (2011) Immune response of the chicken in determination of

244 Pages·2017·9.96 MB·English

by  Christine Middleton

School of Veterinary Medicine and Science
Immune Response of the Chicken in
Determination of Virulence Profiles of
Salmonella enterica
Ahmed Mohamed Hassanin Setta
BVSc (Hons), MVSc
Thesis submitted to the University of Nottingham for
the degree of Doctor of Philosophy
October 2011
Abstract
Abstract
Salmonella enterica subspecies enterica (S. enterica) infection remains a
global problem in a wide range of animals and in man. Poultry-derived food is
a common source of human infection with the non-host-adapted Salmonella
strains while fowl typhoid and pullorum disease are serious diseases in poultry.
Development of novel immune-based control strategies against Salmonella
infection necessitates a better understanding of the host-pathogen interactions
at the cellular level. This study characterizes, in vitro and in vivo, the immune
responses that develop following infection of avian species with typhoid and
non-typhoid Salmonella serotypes. Salmonella serovars Typhimurium,
Enteritidis, Hadar and Infantis showed a greater level of invasion and/or uptake
characters to both chicken macrophages (HD11) and chicken kidney epithelial
cells (CKC), when compared with S. Pullorum or S. Gallinarum. Nitrate and
reactive oxygen species were greater in Salmonella-infected HD11 cells
compared with the non-infected controls. HD11 cells revealed higher mRNA
gene expression for CXCLi2 (IL-8), IL-6 and iNOS genes in response to S.
Enteritidis infection when compared to S. Pullorum-infected cells. S.
Typhimurium- and S. Hadar-infected HD11 showed higher gene expression for
CXCLi2 versus S. Pullorum-infected cells. Higher mRNA gene expression
levels of pro-inflammatory cytokine IL-6, chemokines CXCLi1 (K60) and
CXCLi2 and iNOS genes were detected in S. Typhimurium- and S. Enteritidis-
infected CKC followed by S. Hadar and S. Infantis while no significant
changes were observed in S. Pullorum or S. Gallinarum-infected CKC.
Epithelial cell response and production of pro-inflammatory cytokines and
chemokines were greatly influenced by Salmonella virulence markers,
i
Abstract
including Salmonella pathogenicity island type-1 (SPI-1), SPI-2 and bacterial
flagella. In chicken infections, S. Enteritidis and S. Infantis colonized the caeca
more efficiently than S. Gallinarum and S. Pullorum. High numbers of B-
lymphocytes and macrophages were observed in the caecal tonsils of infected
birds. S. Enteritidis infection in newly hatched birds elicited the expression of
CXCLi1 and CXCLi2 chemokines in the caecal tonsils, while S. Gallinarum
up-regulated the expression of LITAF. In older chickens, S. Enteritidis
infection resulted in a significantly higher expression of CXCLi2, iNOS,
LITAF and IL-10 while S. Pullorum appeared to down-regulate CXCLi1
expression in the caecal tonsils. Data from spleens showed either no expression
or down-regulation of the tested genes. In conclusion, data from the present
study provide further insights on the interaction of Salmonella with poultry,
and while both S. Typhimurium and S. Enteritidis are strong inflammatory
serotypes, S.Pullorum and S.Gallinarum arenot.
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Declaration
Declaration
Unless otherwise acknowledged, the work presented in this thesis is my own.
No part has been submitted for another degree at the University of Nottingham
orelsewhere.
Ahmed Setta
October2011
ii
Conferences
Conferences (oral and poster presentations)
I. A.M. Setta, M.A. Jones & P.A. Barrow, 2008. Salmonella invasion,
persistence and immune responses in chicken macrophages. Society for
General Microbiology (SGM) meeting, Trinity College, Dublin, Ireland.
pp.58
II. A.M. Setta, M.A. Jones & P.A. Barrow, 2009. Transcriptional analysis of
Salmonella enterica in avian cells. Society for General Microbiology
(SGM)meeting,Heriot-Watt University,Edinburgh,UK.pp.41
III. Setta A.M, Jones M.A and Barrow P.A, 2009. Cytokines and chemokines
expression in avian cells infected with Salmonella enterica. Tri-Society
Annual Conference, Lisbon,Portugal.Cytokine48(1-2):65-66
IV. Setta A.M., Jones M.A. and Barrow P. A., 2009. Immune responses of
avian cells infected with Salmonella enterica. World Veterinary Poultry
Association(WVPA)Congress,Marrakesh, Morocco.pp.138
V. British Veterinary Poultry Association (BVPA) meeting, 2009.
Harrogate,UK.
VI. Ahmed M Setta, Michael A Jones, Paul A Barrow, 2010. In-vitro and In-
vivo Differential Expression of Cytokines and Histological Changes
FollowingSalmonella Infections inPoultry.The American Associationof
Avian Pathologists (AAAP)Conference,Atlanta, USA.pp.9
VII. A. Setta, M. Jones, P. Barrow, 2010. Cytokine Gene Expression in
Chickens Following Infection with Salmonella Enterica Serovars.
EuropeanPoultryConference(EPC),Tours, France.pp.157
VIII. A. Setta, M. Jones, P. Kaiser and P. Barrow, 2010. Immune responses of
chicken macrophages to Salmonella enterica. British Society for
Immunology(BSI)Congress, Liverpool,UK. Immunology131(184).
IX. British Veterinary Poultry Association (BVPA) meeting, 2010. East
Midlands, UK.
X. O.C. Freitas Neto, A. Setta, A. Imre, M. Jones, A. Berchieri, P. Barrow,
2011. Working towards smart Salmonella vaccines. Congress of
EuropeanMicrobiologists, FEMS,Geneva,Switzerland,pp229.
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Acknowledgments
Acknowledgements
I would like to thank my supervisors Prof. Paul Barrow and Dr. Michael Jones
for their unwavering support and advice. I’m immensely grateful for the
opportunities you have given me while I’m studying under your kind
supervision over the last few years, including your encouragement to
communicate my research with others world-wide and broaden my research
network.Manythanks for yoursupervisionandinput.
I’m also very grateful to Prof. Pete Kaiser, Infection and Immunity Research,
Roslin Institute,forhis involvement inthis project andprovidingHD11cells.
I would like to acknowledge the other members of enteric pathogens group
within the Nottingham Veterinary School for help and support. I’m also
grateful tothestaff,technicianandpostgraduate communities withintheschool
fortheirhelp,support,interactions and advice.
I’m very grateful to the Egyptian Ministry of Higher Education for the
generous funding. I would also like to thank the University of Nottingham for
hostingthis project.
Many thanks should also go to Salmonella, the smallest member in our group.
Salmonella; you were always ready and fantastic and any obstacles have risen
were from otherparticipants (tissueculture,chickens ormyself).
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Acknowledgments
“To my wife, Sahar, and my sons, Mohamed and Yousuf, who are supporting
me and following me around the world. To my father, my mother and my
extendedfamilyinEgypt”
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Contents
Contents
Abstract................................................................................................................i
Declaration..........................................................................................................ii
Acknowledgements............................................................................................iv
Contents.............................................................................................................vi
List ofTables......................................................................................................x
List of Figures....................................................................................................xi
List ofabbreviations........................................................................................xiv
1 Introduction.................................................................................................1
1.1 General introduction...........................................................................1
1.2 Taxonomy...........................................................................................2
1.3 Salmonella serotypes..........................................................................3
1.4 Zoonoticinfections.............................................................................3
1.5 Epidemiology......................................................................................6
1.6 Salmonella infections inpoultry.......................................................10
1.6.1 Fowl typhoid andpullorum disease..........................................11
1.6.2 Paratyphoidinfections..............................................................12
1.6.3 Arizonosis.................................................................................14
1.7 Pathogenesis andvirulencefactors...................................................14
1.7.1 Attachment andcolonization....................................................15
1.7.2 Salmonella invasion..................................................................17
1.7.3 TTSS and Salmonella-inducedenteritis...................................19
1.7.4 PersistanceofSalmonella infection..........................................19
1.8 Avianimmunesystem......................................................................21
1.8.1 Primarylymphoidorgans.........................................................24
1.8.2 Secondarylymphoidorgans.....................................................24
1.8.2.1 Gut-associatedlymphoid tissues..........................................25
1.8.3 Chickens versus mammals........................................................30
1.8.3.1 Toll-likereceptors (TLRs)....................................................30
1.8.3.2 Heterophils............................................................................34
1.8.3.3 Antimicrobial peptides..........................................................34
1.8.3.4 Aviancytokines....................................................................35
1.9 Immune responses to Salmonella.....................................................37
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Contents
1.9.1 Mammalianimmuneresponses................................................38
1.9.2 Avianimmuneresponses..........................................................42
1.9.2.1 Cell culturestudies...............................................................43
1.9.2.1.1 Epithelial cells................................................................43
1.9.2.1.2 Macrophages...................................................................44
1.9.2.1.3 Heterophils......................................................................47
1.9.2.2 Animal studies......................................................................48
1.10 Reductionofthecaecal carriage.......................................................52
1.11 Vaccination.......................................................................................53
1.12 Aims andobjectives oftheproject:..................................................55
2 General Materials andMethods................................................................58
2.1 Bacteriology......................................................................................58
2.1.1 Media........................................................................................58
2.1.2 Antibiotics.................................................................................58
2.1.3 Bacterial strains........................................................................59
2.1.4 Nalidixicacidmutation.............................................................62
2.1.5 Growthpatterns ofdifferent serotypes.....................................62
2.1.6 Confirmationofmutations bypolymerasechainreaction(PCR)
63
2.1.6.1 Extractionofbacterial DNA.................................................63
2.1.6.2 PreparationofPCR MasterMix...........................................64
2.1.6.3 Agarose gel electrophoresis anddetectionofPCR products66
2.2 Salmonella infectionofaviancells (invitro)...................................68
2.2.1 Tissueculturecell lines............................................................68
2.2.1.1 Chickenmacrophages (HD11).............................................68
2.2.1.2 Chickenkidneycells (CKC).................................................69
2.2.2 Invasionassay...........................................................................71
2.2.3 Griess assay..............................................................................72
2.2.4 Oxidativeburst assay................................................................74
2.2.5 Formalized Salmonella.............................................................74
2.2.6 Isolationofbloodlymphocytes................................................75
2.2.7 Macrophage-lymphocyte co-culture and Salmonella infection75
2.3 Salmonella infectionofpoultry........................................................76
2.3.1 Chickens andexperimental design...........................................76
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Contents
2.3.2 Vancomycinsusceptibilitytest.................................................77
2.3.3 Vancomycinadministration......................................................77
2.3.4 Sampling...................................................................................78
2.3.5 Bacteriology..............................................................................78
2.3.6 Histopathology..........................................................................78
2.3.6.1 Tissuefixation......................................................................79
2.3.6.2 Paraffinembedding...............................................................79
2.3.6.3 Sectioning.............................................................................79
2.3.6.4 H&Estaining........................................................................80
2.3.7 Immunohistochemistry.............................................................80
2.3.8 Isolationoflymphocytes forflowcytometry...........................81
2.4 QuantificationofmRNA genetranscripts........................................82
2.4.1 RNAextractionandcDNAsynthesis.......................................82
2.4.2 Quantitativereal-timeRT-PCR (qRT-PCR)............................84
2.5 Statistical analysis.............................................................................90
3 Immunedynamics followingSalmonella infectionofculturedaviancells;
invasionandpersistence, nitricoxideandoxygen productionanddifferential
host gene expression.........................................................................................91
3.1 Introduction.......................................................................................91
3.2 Results..............................................................................................93
3.2.1 Invasionandintracellular survival ofSalmonella inaviancells
93
3.2.2 NitricoxideproductionbyHD11 cells inresponse to
Salmonella infection.................................................................................96
3.2.3 OxygenproductionfollowinginfectionofHD11 cells with
Salmonella................................................................................................96
3.2.4 Quantificationof geneexpressionofimmunemediators
followingSalmonella infectionofcultured aviancells............................99
3.3 Discussion.......................................................................................110
4 Immune responses of aviancells toinfectionwith Salmonella
pathogenicityisland-and flagellar assemblysystem-mutants ofSalmonella
enterica...........................................................................................................118
4.1 Introduction.....................................................................................118
4.2 Results............................................................................................119
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