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Salmonella and Salmonellosis

Abstract book 27 - 28 - 29 May 2013

Salmonella and Salmonellosis Abstract book 27 - 28 - 29 May 2013 Saint-Malo FRANCE

Saint-Malo

FRANCE

This symposium is arranged by:

French Agency for Food, Environmental and Occupational Health & Safety (ANSES), French Institute for Public Health Surveillance (InVS),

French National Institute for Agricultural Research (INRA),

Institut Pasteur, High Institute for Animal Production and Agro-Business (ISPAIA) and ZOOPOLE.

Chairman: Pierre Colin - UBO Brest - France Secretary-Treasurer: Geneviève Clément - ISPAIA-ZOOPOLE Développement - Ploufragan France

Scientific Committee

Jean-Christophe AUGUSTIN (France) Anne BRISABOIS (France) Marianne CHEMALY (France) Axel CLOECKAERT (France) Rob DAVIES ( United Kingdom) Rene HENDRIKSEN (Denmark) Michael HENSEL (Germany) Nathalie JOURDAN DA SILVA (France) Bob TAUXE (USA) Nicholas THOMSON (United Kingdom) Philippe VELGE (France) Pierre WATTIAU (Belgium) François Xavier WEILL (France) Marcel ZWIETERING (the Netherlands)

Organising Committee

Anne Brisabois ANSES Maisons Alfort Marianne Chemaly ANSES Ploufragan Axel Cloeckaert INRA Nouzilly Réjane Deluce ISPAIA -ZOOPOLE Dév. Ploufragan Nathalie Jourdan Da Silva InVS Renaud Lailler ANSES Maisons Alfort Simon Le Hello - Institut Pasteur Gilles Salvat - ANSES Ploufragan Véronique Vaillant - InVS Philippe Velge - INRA Nouzilly François-Xavier Weill Institut Pasteur

Proceedings edited by:

Pierre COLIN Geneviève CLEMENT © 2013 by ZOOPOLE développement - ISPAIA

SYMPOSIUM SECRETARIAT ISPAIA BP 7 22440 PLOUFRAGAN France Tel. +33 2 96 78 61 30 - Fax; +33 2 96 78 61 31 ispaia@zoopole.asso.fr - www.zoopole.asso.fr

TABLE OF CONTENTS

Session 1:

Interactions between hosts and bacteria

Chairpersons: Michael HENSEL (Germany) and Philippe VELGE (France)

Introduction

New insights on cell entry mechanisms and intracellular lifestyle of Salmonella Michael HENSEL – (Germany) ‐ Philippe VELGE (France)

2

Oral Communications

Toward a Novel, Transdermal Challenge Model that Adequately Explains the Route of Infection of Peripheral Lymph Nodes by Salmonella EDRINGTON Tom, United States of America

8

Salmonella associated taxonomic and functional changes in the pig digestive tract during application of feed associated mitigation option in FRAVALO Philippe, Canada (Qc)

12

Resistance to essential oils affects survival of Salmonella enterica serovars in growing and harvested basil KISLUK Guy, Israel

17

The effect of post-transcriptional regulators on the dynamics of Salmonella biofilm formation VAN PUYVELDE Sandra, Belgium

22

Characterization of chicken spleen transcriptome after infection with Salmonella enterica serovar Enteritidis RYCHLIK Ivan, Czech Republic

27

Deciphering why Salmonella Gallinarum is less invasive in vitro than Salmonella Enteritidis ROSSIGNOL Aurore, France

31

Posters

Invasiveness of monophasic and aphasic Salmonella strains in point-of-lay chickens MARTELLI Francesca, United Kingdom

35

SPI-1 encoded genes of Salmonella Typhimurium influence differential polarization of porcine alveolar macrophages in vitro KYROVA Kamila, Czech Republic

38

Flagella changed the pathogenicity of a Salmonella Gallinarum mutant strain FREITAS NETO Oliveiro, Brazil

41

Identification of Redundant Metabolic Pathways Essential for Virulence of Salmonella Typhimurium OLSEN John Elmerdahl, Denmark

44

Gene expression profile of enterocytes purified from two lines of chicken during the Salmonella carrier VELGE Philippe, France

45

The expression of cytokines in spleen of chickens infected with S. enterica SE 147 after administration of probiotic bacteria SPISAKOVA Viera, Slovakia

46

Cytokine signaling in splenic leukocytes of vaccinated and naive chickens after intravenous infection with Salmonella Enteritidis MATULOVA Marta, Czech Republic

50

Virulence Gene Profiling and Pathogenicity Characterization of Non-Typhoidal Salmonella Accounted for Invasive Disease in Humans GAL-MOR Ohad, Israel

53

Cellular immune response by CD4+ T cells in White Layer-hens vaccinated with live and killed Salmonella vaccines and infected with Salmonella Enteritidis BECHIERI Jr. Angelo, Brazil

54

Efficacy of different vaccine programs against Fowl Typhoid using a live attenuated strain of Salmonella Gallinarum as a vaccine candidate BECHIERI Jr. Angelo, Brazil

57

Evaluation of protection provided by an inactivated Salmonella vaccine in point-of- lay chickens challenged with monophasic Salmonella Typhimurium MARTELLI Francesca, United Kingdom

59

Session 2:

Genomic and applications

Chairpersons: Nicholas Thomson (United Kingdom) and François Xavier WEILL (France)

Oral communications

Genomic epidemiological studies of the global occurrence of S. Typhimurium DT104 LEEKITCHAROENPHON Pimlapas, Denmark

63

Multiplexed SNP-typing method for characterizing Salmonella isolates at the subserovar level BOLAND Cecile, Belgium

71

Massive parallel sequencing identify Salmonella single-nucleotide polymorphism (SNP) markers for host origin and antimicrobial resistance SCHIFFERLI Dieter, United States of America

76

Molecular subtyping of Salmonella Typhimurium combining different types of markers in a multiplex liquid bead suspension array WUYTS Véronique, Belgium

80

One-year surveillance of human and non-human Salmonella enterica serotype Typhimurium and its monophasic variant based on CRISPOL subtyping, France, 2011 LE HELLO Simon, France

85

Posters

Genetic basis of lack of expression of phase 2 flagellar antigen in Italian isolates of S.

4,[5],12:i:-

89

LONGO Alessandra, Italy

Validation of a Salmonella Geno-Serotyping Array (SGSA) in Three International Laboratories LINGOHR Erika, Canada

91

Genomic regions of difference between Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum and biovar Pullorum FREITAS NETO Oliveiro, Brazil

94

Session 3 :

Detection, identification, quantification

Chairpersons: Pierre WATTIAU (Belgium) and Anne BRISABOIS (France)

Introduction

Pierre WATTIAU (Belgium) ‐ Anne BRISABOIS (France)

 

97

Oral communications

 

The effect of pooling of poultry meat samples on the detection of Salmonella MOOIJMAN Kirsten, The Netherlands

 

101

Impact of routine use of two novel real-time polymerase chain reaction assays for identification of Salmonella enterica subspecies within a National Reference Laboratory PETERS Tansy, United Kingdom

105

Molecular categorization of Salmonella enterica isolated from bovine lymph nodes BUGAREL Marie, United States of America

108

Multiple

loci

VNTR

Analysis

(MLVA)

for

molecular

characterization

of

the

monophasic

variants

of

Salmonella enterica serotype Typhimurium outbreaks’

 

occurred in 2011 BRISABOIS Anne, France

 

113

Characterisation of a novel 18.4 kb genomic island in endemic monophasic Salmonella Typhimurium strains from Europe SIMON Sandra, Germany

118

Posters

Study on the effect of storage conditions on the recovery of Salmonella from boot swabs and hairnets RABIE Andre, United Kingdom

123

Sensitivity of different sampling methods in the detection of Salmonella in flocks of laying hens MARTELLI Francesca, United States of America

125

Improved Isolation of Salmonella from Poultry Environmental Samples using direct Tetrathionate Enrichment followed by MSRV WALTMAN Doug, United States of America

127

Validation of selective enrichment broths and chromogenic media for Salmonella detection in highly contaminated chicken carcass rinse SEO Kun-Ho, South Korea

130

A comparison between Rambach agar, Rapid Salmonella Complete agar and Brilliance Salmonella agar for detection of Salmonella RABIE Andre, United Kingdom

131

Performance of media plates for detection of Salmonella LE GALL Françoise, France

133

State of play of international standardisation of Salmonella methods MOOIJMAN Kirsten, The Netherlands

136

Performance of reference laboratories in interlaboratory comparison studies on serotyping of Salmonella JACOBS-REITSMA Wilma, The Netherlands

138

MicroVal/ISO-16140 validation of a PCR method for Salmonella detection in powdered infant formula, probiotic culture powders and ingredients, after a first screening for JACOBS-REITSMA Wilma, The Netherlands

140

AOAC-RI Validation of RAPID´Salmonella method, a Single 18 h Selective Enrichment and Chromogenic Media Detection of Salmonella spp. from Food BICHOT Yannick, France

142

VIDAS® UP SALMONELLA (VIDAS SPT) to detect Salmonella in Primary Production Samples: Certification according to the ISO16140 Standard AIGUILHON Christine, France

145

Evaluation of a Rapid Lateral Flow Method for the Detection of Salmonella AIGUILHON Christine, France

146

NF Validation Extension Study of an Immunoassay Method for The Detection of Salmonella in 375g food Samples PITTET Jean-Louis, France

148

Simultaneous detection of Salmonella enterica serovars Typhi, Enteritidis, Infantis and Typhimurium by multiplex PCR MAMMINA Caterina, Italia

150

Real-time PCR for detection of Salmonella in environmental samples from empty cleaned and disinfected poultry SODOLEVSKY Ekaterina, Israel

153

Two novel real-time PCR methods for rapid detection of Salmonella Enteritidis and

FRENKIEL-LEBOSSE Hélène, France

Molecular identification in monophasic and non-motile variants of Salmonella enterica serovar Typhimurium BRISABOIS Anne, France

155

158

Characterization of the emerging Salmonella 4,[5],12:i:- in Danish animal

ARGÜELLO RODRIGUEZ Héctor, Spain

Monitoring of monophasic and non-motile variants of serotype Typhimurium among non-human strains collected by the French Salmonella Network LAILLER Renaud, France

Stability study of the MLVA profiles BRISABOIS Anne, France

The Application of MultiLocus Variable-number tandem-repeat Analysis (MLVA) for the epidemiological subtyping of Salmonella enterica serovar Typhimurium in Scotland BROWN Derek, United Kingdom

Pulsenet MLVA protocol for Salmonella enterica serovar Enteritidis applied to Salmonella enterica serovar OCONNOR Jean, Ireland

Pulsenet Multi-locus VNTR Analysis MLVA protocol for Salmonella enterica subtype Enteritidis compliments phage typing OCONNOR Jean, Ireland

Multiple locus variable number of tandem repeats analysis of Salmonella enterica subsp. enterica serovar Dublin TORPDAL Mia, Denmark

A new immunoenzymatic strategy for the rapid and selective growth of Salmonella WATTIAU Pierre, Belgium

A Comparison of subtyping methods for Differentiating Salmonella enterica serovar Enteritidis Isolates Obtained from Food and Human Sources

SEO Kun-Ho, South Korea

161

163

166

168

170

172

174

175

178

Fast Salmonella Typhimurium sub-typing method based on CRISPR polymorphisms MARAULT Muriel, France

179

An Inter-laboratory proficiency trial for Salmonella enumeration ELLOUZE Mariem, France

182

Development of MPN-real time PCR for enumeration of Salmonella from pork meat DENIS Martine, France

185

Determination of Minimal Inhibitory Concentration (MIC) values for clinical isolates from the poultry practice using a microdilution antimicrobial susceptibility testing procedure WINDHORST Daniel, Germany

187

Establishment of a multiplex PCR-Lateral Flow Assay (mPCR-LFA) for the detection of DNA from S. Typhi and S. Paratyphi A AZIAH Ismail, Malaysia

188

Session 4:

Antimicrobial resistance

Chairpersons: Rene HENDRIKSEN (Denmark) and Axel CLOECKAERT (France)

Introduction

Antimicrobial resistance in a genomic era going from phenotype to genotype Rene HENDRIKSEN (Denmark)

190

Oral communications

Regulation of the AcrAB multidrug efflux pump in Salmonella enterica serovar Typhimurium NISHINO Kunihiko, Japan

199

Epidemiological and genetic features associated with the international spread of multi-drug resistant Salmonella Kentucky ST198 LE HELLO Simon, France

204

Dramatic increase of AmpC- and ESBL- producing d-Tartrate-positive Salmonella enterica serotype Paratyphi B from broilers in Belgium, 2008-2010 DOUBLET Benoit, France

209

Nationwide human outbreak of cephalosporin resistant Salmonella serotype Typhimurium in France associated with consumption of unpasteurized cheese, a link with a warm-blood horse reservoir? GRANIER Sophie A., France

210

Highly ciprofloxacin resistant Salmonella enterica serovar Kentucky isolates of human and animal origin in Germany BEUTLICH Janine, Germany

212

Antibiotic resistance trends in Salmonella, 2008-2012 WASYL Dariusz, Poland

216

Posters

Investigation of Salmonella in pig slaughterhouses in Southern Brazil and characterization of phenotypic and genotypic antimicrobial resistance of the isolates VOLZ LOPES Graciela, Germany

222

Phenotypic and genotypic profiles of antimicrobial resistance (AMR) in Salmonella spp. isolates from faeces of different canine populations of Northern and Central Italy PASOTTO Daniela, Italy

223

Salmonella spp. prevalence and antimicrobial resistance patterns (phenotypic profiles and presence of genetic AMR determinants) of serotypes isolated from pet reptiles in Northern Italy PASOTTO Daniela, Italy

226

Mechanisms of antimicrobial resistance in biofilm-associated Salmonella

 

228

SCHLISSELBERG Dov, Israel

Monitoring 3rd generation cephalosporin resistance in French non-human Salmonella : 2010-2012 overview GRANIER Sophie A., France

230

Emergence of the ASSuT Multidrug Resistant Profile in Salmonella enterica serotype 4,[5],12:i:- in Canada MULVEY Michael, Canada

232

The Emergence of Ciprofloxacin-Resistant Salmonella enterica serovar Kentucky in Canada MULVEY Michael, Canada

233

Effect of ramR mutations on efflux genes expression and on fluoroquinolone susceptibility in Salmonella enterica serotype Kentucky ST198 BAUCHERON Sylvie, France

234

Plasmid-mediated quinolone resistance in domestic and imported food and in food production animals in Finland 2003-2010 KURONEN Henry, Finland

235

Prevalence and characterisation of quinolone resistance mechanisms in Salmonella isolated in Poland WASYL Dariusz, Poland

237

Session 5 Ecology and animal epidemiology

Chairpersons: Rob DAVIES (United Kingdom) and Marianne CHEMALY (France)

Introduction

Salmonella control in food animals in the EU: successes, threats and future challenges Rob DAVIES (United Kingdom)

241

Oral communications

Salmonella in soybean meal, what is the source of infection? HÄGGBLOM Per, Sweden

249

Salmonella in wild birds in Israel: Surveillance studies in healthy and dead birds LUBLIN Avishai, Israel

253

Polyphasic characterization of Salmonella Enteritidis isolates on persistently contaminated layer farms during the implementation of a national control program with obligatory vaccination: a longitudinal study DE REU Koen, Belgium

258

Cattle, Beef, Consumers and Salmonella: A Novel Pathway of Consumer Exposure and a New Understanding of the Agent-Host Ecology LONERAGAN Guy, United States of America

261

Estimating the prevalence of Reptile acquired salmonellosis in England and Wales using a novel demographic based attribution model SINCLAIR Chantil, United Kingdom

265

Posters

A longitudinal study, from weaning to carcass, to determine the prevalence of Salmonella on pig farms in Northern Ireland MADDEN Bob, Northern Ireland

271

Salmonella in pig farms in Reunion Island: Prevalence assessment and identification of risk factors TESSIER Claire, France

272

Longitudinal investigation of Salmonella spp. from farm to fork in the pig industry of Reunion Island TESSIER Claire, France

273

Tracking in trucks, lairage, slaughter line and quartering as a useful tool to study Salmonella prevalence in a free-range pig processing plant ASTORGA Rafael J., Spain

275

Prevalence of Salmonella in Northern Ireland pigs at MADDEN Bob, Northern Ireland

278

Occurrence and genetic diversity of Salmonella in organic and conventional pig productions in France KEROUANTON Annaelle, France

279

Observations on the occurrence and persistence of monophasic Salmonella Typhimurium (S.4, [5],12:i:-) in poultry DAVIES Robert, United Kingdom

281

A

study of the dynamics of Salmonella infection in turkey breeding, rearing and

finishing houses with special reference to elimination, persistence and introduction

 

of

Salmonella

283

MUELLER-DOBLIES Doris, United Kingdom

Epidemiological investigation of Salmonella enterica subsp. enterica isolated from chicken carcasses and environment at slaughter in Reunion Island: prevalence, genetic characterization and antibiotic susceptibility HENRY Isabelle, France

286

Salmonella occurrence and antimicrobial resistance in nestlings of two seagull species, Larus michahellis and Larus audouinii in Ebro Delta (NE ANTILLES Noelia, Spain

289

Prevalence of Salmonella spp. on duck products at the retail level CHEMALY Marianne, France

291

Salmonella-contaminated eggs in flocks identified as positive by the National Control Programme for Salmonella in the UK MARTELLI Francesca, United Kingdom

293

Fate of Salmonella enterica serovar Infantis, the predominant serovar in Israel, in table eggs LUBLIN Avishai, Israel

295

The prevalence of Salmonella enterica subspecies diarizonae serovar 61:(k):1, 5, (7)

in

Swedish sheep herds

297

MELIN Lennart, Sweden

A longitudinal study on the persistence of non-avian Salmonella Enteritidis on cattle

farms in the UK GOSLING Rebecca, United Kingdom

299

Analysis of salmonellosis epidemic situation in the RF in 2008-2012 PRUNTOVA Olga, Russia

301

Genotypic diversity of Salmonella Kentucky isolated from reptiles, poultry food, feed and environment samples in Poland ZAJAC Magdalena, Poland

303

Prevalence of Salmonella isolates in exotic reptiles in Poland ZAJAC Magdalena, Poland

305

Prevalence and antimicrobial resistance of Salmonella spp. in healthy reptiles OVERESCH Gudrun, Switzerland

307

Are free-living turtles an important source of Campylobacter and Salmonella? MARIN Clara, Spain

309

GFP promoter fusion library for the study of Salmonella biofilm formation and the mode of action of biofilm inhibitors VAN PUYVELDE Sandra, Belgium

A

312

Monitoring of Salmonella in food, feed and veterinary samples by the French Salmonella network MOURY Frédérique, France

314

Set up of a National molecular typing database for Salmonella surveillance FEURER Carole, France

317

Session 6:

Risk assessment and control

Chairpersons: Marcel ZWIETERING (the Netherlands) and Jean-Christophe AUGUSTIN (France)

Introduction Marcel H. ZWIETERING (The Netherlands)

320

Oral communications

Changing the presentation of pigs feed: a cost effective solution to reduce Salmonella spp. LEBEL Philippe, Canada (Qc)

323

Decreasing prevalence in Dutch dairy herds under salmonellosis WEBER Maarten F., The Netherlands

328

Evaluation of the impact of the refrigerated transport of pig carcasses loaded above 7°C on their microbial and safety ELLOUZE Mariem, France

329

A comparison of Salmonella test results from microbiological testing undertaken by Food Business Operators and that Officialy undertaken EGAN John, Ireland

334

Application of forensic evaluation of evidence to the tracing of Salmonella ANDERSSON Gunnar, Sweden

338

Posters

Investigation of low prevalence Salmonella contamination in poultry DAVIES Robert, United Kingdom

344

Organic acids for control of Salmonella serotypes in different feed materials KOYUNCU Sevinc, Sweden

347

Assessment of anti-Salmonella activity of boot dip samples RABIE Andre, United Kingdom

349

Investigation of laboratory testing issues in the context of the Salmonella National Control Programme in GB GOSLING Rebecca, United Kingdom

351

Escherichia coli as indicator of the human Salmonella risk caused by consumption of pork BOLLERSLEV Anne Mette, Denmark

354

Assessment of biosecurity and uptake of advice in chicken layer farms in England and Wales MARTELLI Francesca, United Kingdom

357

Assessment of the social science aspect of biosecurity and uptake of advice on chicken layer farms in England and Wales GOSLING Rebecca, United Kingdom

360

Effect of litter aeration in the spread of Salmonella in poultry INGRESA Sofía, Spain

362

Importance of cleaning and disinfection in Salmonella persistence in poultry farms MARIN Clara, Spain

365

The first bivalent Salmonella live vaccine for chicken and ducks WINDHORST Daniel, Germany

367

Testing of the immunogenicity of the Salmonella Enteritidis live vaccine SALMOVAC SE against non PT4- Salmonella Enteritidis strains and Salmonella enterica subsp. enterica 4,[5],12:i:- SPRINGER Sven, Germany

369

Change of interventions in Swedish dairy herds under restrictions due to infection with Salmonella ÅGREN Estelle, Sweden

371

Risk factors for Salmonella contamination in chicken carcasses at the slaughterhouse GONZALEZ BODI Sara, Spain

373

The logistic slaughter experience in a Spanish slaughterhouse. Lessons learned and

 

375

ARGÜELLO RODRIGUEZ Héctor, Spain

Inter- and intraserovar variation in in vitro pathogenicity of Salmonella spp KUIJPERS Angelina, The Netherlands

377

Revised Estimates of the Burden of Food-borne Illness in Canada THOMAS Kate, Canada

379

Source Attribution of Salmonella enterica in Ireland using the Microbial Subtyping method DE LAPPE Niall, Ireland

380

Session 7:

Epidemiology and Public Health

Chairpersons: Bob TAUXE (USA) and Nathalie JOURDAN DA SILVA (France)

Introduction Changes and Challenges: Salmonellosis in the 21st Century Robert V. Tauxe, USA

383

Oral communications

An outbreak of Salmonella Newport associated with mung bean sprouts, Germany, October/November 2011 ROSNER Bettina, Germany

392

Changing trends in antimicrobial resistance patterns in Salmonella enterica serovar Typhimurim DT193 in England and Wales, 1981-2011 ESAN Oluwaseun, United Kingdom

396

The benefit of Salmonella control in Sweden who benefits and who pays the cost? STERNBERG LEWERIN Susanna, Sweden

401

Multistate outbreak of Salmonella Stanley infection in the European Union, 2011- 2012: investigations from fork to GOSSNER Céline, Sweden

405

First identification of multi-resistant Salmonella Brandenburg in patients hospitalized in the same clinic, France, 2010-2012 MOULY Damien, France

411

Seventeen years of extra-intestinal nontyphoidal human Salmonella infections in the United States, 1995 to 2011 FULLERTON Katie, United States of America

416

A European outbreak of S. Newport associated with melons from South America FISHER Ian, United Kingdom

417

Evidence for “Reptile-Exotic-Pet-Associated-Salmonellosis” (REPAS) in Germany RABSCH Wolfgang, Germany

421

Posters

Phenotypic and Molecular Characterisation of multiresistant monophasic Salmonella Typhimurium (1,4,[5],12:i:-) in Greece, 2006-2011 POLEMIS Michalis, Greece

430

Molecular Typing of Monophasic Salmonella 4,[5],12:i:- Strains Isolated in Belgium,

433

BOLAND Cecile, Belgium

Diversity of human Salmonella Typhimurium and Salmonella enterica 4,[5],12:i- strains isolated in Emilia Romagna Region, Italy CORPUS Francesco, Italy

435

Salmonella serovars and phage types associated with specific animal reservoirs and their epidemiology situation in the United Kingdom MUELLER-DOBLIES Doris, United Kingdom

437

About three cases of intestinal carriage of Salmonella enterica serovar Toulon in NICU MAMMINA Caterina, Italia

439

A

Multi-Country Outbreak of Salmonella Newport Associated with Watermelon

442

DE PINNA Elizabeth, United Kingdom

Foodborne disease outbreaks associated with the consumption of raw milk cheese

in

France, August September 2012

445

MOULY Damien, France

Phage and MLVA typing of Salmonella Enteritidis isolated from layers and humans in Belgium from 2000 2010, a period in which vaccination of laying hens was introduced DE REU Koen, Belgium

448

Epidemiology and antimicrobial resistance of human and avian Salmonella isolated

in

N'Djamena, Tchad

450

MILLEMANN Yves, France

“Ad hoc” study on Salmonella Stanley outbreak strains WASYL Dariusz, Poland

453

perfect storm: Misdiagnosed typhoid fever in a laboratory worker in a context of possible vaccine failure

A

456

KEDDY Karen, South Africa

Salmonella and Salmonellosis 2013

Salmonella and Salmonellosis 2013 May 27-28-29, 2013 Saint-Malo France

Session 1

Interactions between hosts and bacteria

Chairpersons:

Michael HENSEL (Germany) and Philippe VELGE (France)

1

Table of contents >>

Salmonella and Salmonellosis 2013

New insights on cell entry mechanisms and intracellular lifestyle of Salmonella

Michael HENSEL – (Germany) Philippe VELGE (France)

INTRODUCTION

Salmonella is a facultative intracellular zoonotic pathogen causing a wide spectrum of diseases according to serotype and hostspecific factors. In humans, typhoid fever due to the hostrestricted serotype S. Typhi affects 22 million people in developing countries, while gastroenteritis due to the ubiquitous serotypes is a predominant cause of foodborne illness worldwide. Moreover, ubiquitous serotypes can induce a wide spectrum of diseases such as typhoidlike fever, gastroenteritis and asymptomatic infections, depending on the host. Although several genetic loci are associated with hostspecificity and disease, the stages at which host–pathogen interactions affect the host range or the disease outcome are not fully understood. The importance of Salmonella cell invasion on these phenotypes has been often neglected because of the belief that Salmonella invades nonphagocytic cells only in a Type Three Secretion System (T3SS1) dependent mechanism. However, this belief has recently been abandoned because it has been shown that: i) some strains exhibiting nonactive T3SS1 are able to infect humans and cause gastroenteritis, ii) Salmonella invade fibroblasts and polarized enterocytes by a

T3SS1independent mechanism, iii) PagN mediates a T3SS1independent entry. Moreover, we demonstrated that iv) Salmonella can enter cells through a Zipper mechanism mediated by Rck that is completely different from the Trigger mechanism induced by the T3SS1 and v) a mutant strain able to express neither T3SS1 nor Rck and PagN is still able to enter different cell types and cell lines by inducing discrete and intense membrane rearrangements, suggesting that Salmonella can enter cells by other Zipper and Triggerlike mechanisms that have not yet been identified. The multiplicity of the entry routes would modify our understanding of the mechanisms leading to the different Salmonella induced diseases and to Salmonella host specificity. After entry into host cells, Salmonella resides in a membranebound compartment termed

Salmonella containing vacuole’ or SCV. A key virulence determinant for intracellular life is the

SPI2encoded T3SS or T3SS2 that translocates a cocktail of more than 30 effector proteins across the SCV membrane. Intracellular Salmonella induce a massive remodeling of host cell vesicular trafficking and organization of the endosomal system. One remarkable phenotype is the induction of extensive tubular membrane compartments, referred to as Salmonella induced filaments or SIF. Recruitment of membrane vesicle is required for the extension of the SCV during intracellular proliferation, however, the physiological role of SIF is still unclear. Application of live cell imaging, ultrastructural analyses and correlative microscopy revealed a number of unexpected features of the membrane organization and luminal content of SIF and SCV. These findings indicate unique mechanisms of remodeling of the host cells endomembranes. A specific requirement for adaptation of life inside the SCV is the recruitment of nutrients. We investigated the nutritional basis of intracellular Salmonella and observed a remarkable flexibility in utilization of various nutrients upon availability. Using live cell microscopy, the exchange of endocytosed material with the SCV was analyzed, as well as the interchange of membrane material in SIF and the SCV. Intracellular Salmonella in SCV connected to SIF are in exchange with endocytosed material and nutrients, while bacteria in SCV without connection to SIF are without access to nutrient and growth restricted. The implications of our observations and recent findings by other groups for the multiplicity of cell

entry mechanisms and intracellular lifestyle of Salmonella will be discussed in the introduction to

2

Table of contents >>

new pathogenicity mechanisms.

this

session.

These

Salmonella and Salmonellosis 2013

paradigms

should

modify

our

understanding

of

the

Salmonella

According to serotypes and hosts, Salmonella enterica cause a wide range of foodand waterborne diseases ranging from selflimiting gastroenteritis to systemic typhoid fever. Moreover, no other bacterial pathogen belonging to a single species shows such a remarkable ability to infect different hosts and induce so many different diseases. The reasons why some Salmonella serotypes are confined to the intestine while others translocate to distal organs are unclear. An essential feature of the pathogenicity of Salmonella is its interaction with phagocytic and nonphagocytic cells and Salmonella entry into host cells is known to be critical for bacterial survival and establishment of disease in a host. In general, intracellular bacterial pathogens enter nonphagocytic eukaryotic cells via two mechanisms, which are initially differentiated according to morphological criteria based on membrane remodeling. The "Trigger" mechanism involves dramatic cytoskeletal rearrangements known as "membrane ruffles". In contrast, in the "Zipper" mechanism, or "receptor mediated entry", the invading bacteria are tightly bound to the host cell membrane, and only minor cytoskeletal protein rearrangements are initiated by specific contact between bacterial ligands (invasin) and host cell surface receptors (1). An important mechanistic difference between the Trigger and Zipper modes of entry is that, the former is triggered from "inside" via the action of bacterial effectors delivered by secretion systems whereas the latter is promoted from "outside" through activation of host cell receptors. However, in both cases bacteria hijack the cell's physiological processes through the modulation of existing cell signaling cascades. We recently reported that Salmonella is the first bacteria shown to be able to enter cells using both these mechanisms (2). Moreover, in contrast to the prevailing paradigm of Salmonella pathogenesis asserting that the Salmonella pathogenicity island1 Type Three Secretion System (SPI1 T3SS or T3SS1) is essential for bacterial invasion of host cells, an emerging idea is that Salmonella strains can enter nonphagocytic cells by multiple pathways (3). The study of host cell invasion by Salmonella has been initiated in 1967 by Takeuchi (4). For decades, it was described in the literature that Salmonella can enter cells only via a “Trigger” mechanism mediated by the T3SS1 (5). T3SSs are supramolecular complexes that cross both inner and outer membranes of bacteria and are able to create a pore in eukaryotic membrane upon contact with a host cell to inject bacterial protein effectors directly into host cells in an energydependent (ATP) manner (6). This effector translocation is precisely coordinated ensuring that bacterial proteins engage in a coherent order through a cytoplasmic sorting platform, which ensures secretion of the translocases before the effectors. The sequential loading on this platform may be facilitated by the different affinities of the T3SSchaperones, ensuring the hierarchy in type three effector secretion (7). The T3SS1 contribution to pathogenesis depends on the model used. In vivo studies with S. Dublin and S. Typhimurium serotypes have demonstrated that the T3SS1 is essential for intestinal colonization and is required to induce enterocolitis in bovine, rabbit and murine models (8). In contrast, recent studies demonstrate that different serotypes of Salmonella lacking T3SS1 still have the ability to infect several hosts. For example, a SPI1 mutant of S. Gallinarum is fully virulent in adult chicken (9). Moreover, in S. Enteritidis and S. Typhimurium, the T3SS1 is not essential during systemic infection of one weekold chicken or BalB/C mouse nor during the intestinal colonization of rabbit ileal loops (1012). Moreover, S. Seftenberg strains lacking SPI1 were isolated from human clinical cases, suggesting that the T3SS1 is dispensable by this serotype for the establishment of infection in humans (13). Taken together, these results indicate that T3SS1independent invasion mechanisms also play an important role in Salmonella infection and

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Salmonella and Salmonellosis 2013

pathogenesis. This observation could be related to the demonstration that a Salmonella mutant, unable to express its T3SS1 is still able to invade numerous cell lines and cell types (14). The role of outer membrane proteins (OMP) as possible adhesion molecules and virulence factors has been demonstrated in different pathogenic bacteria. In Salmonella, some Salmonella OMP, namely Rck and PagN, have been shown to be able to induce cell invasion (15) (2) (16).

Rck is a 17kDa OMP encoded by the large virulence plasmid of S. Enteritidis and S. Typhimurium. Rck is a member of a family which consists of five members (Rck, Ail, Lom, OmpX and PagC). Even

if these proteins present a similar conformation, they have different functions. Only, Rck and Ail

(encoded by a chromosomal gene of Yersinia Enterocolitica ) share the ability to promote serum resistance and epithelial cell invasion. The cell adhesion/invasion property is associated with the region from the amino acids 113 to 159 (G113V159) (2, 17). By using both an initially noninvasive E. coli strain overexpressing Rck and latex beads coated with this minimal region of Rck inducing invasion, it was demonstrated that Rck alone is able to induce entry by a receptormediated process. This mechanism promotes local actin remodelling and weak and closely adherent membrane extensions (2). This Zipper entry process requires protein tyrosine kinase and cSrc (18), which activates in cascade, class I PI3 kinase, Akt, the small GTPase Rac1 and Cdc42 (but not Rho), leading to activation of the Arp2/3 complex and actin polymerization (19). The cellular receptor of Rck required in this process, however, remains unkown. The role of Rck in Salmonella invasion is clearly demonstrated in vitro , but its role in Salmonella pathogenesis is poorly understood. The fact that Rck is regulated by SdiA, a quorum sensing regulator suggests an intestinal role of this invasin (20). However, the mechanisms leading to SdiA activation and rck expression are not well characterized. As rck is also regulated by an unidentified SdiAindependent system (21), it is conceivable that Rck invasion mechanism is not restricted to the gastrointestinal tract. Considering that Rck is also involved in the resistance to complement killing, a systemic function of Rck is possible.

In addition to the T3SS1 and Rck, the PagN outer membrane protein is also involved in Salmonella

invasion (16). pagN , is localized on the specific centisome 7 genomic island and is widely distributed among Salmonella enterica serotypes (22). PagN is required for survival in BALB/c mice and a pagN mutant is less competitive to colonize the spleen of mice compared to its parental strain (23). However, the role of PagN in Salmonella pathogenesis is still unclear as well as the PagNmediated cell entry process. At the cellular level, pagN deletion in S. Typhimurium leads to a 3fold decrease in invasion of enterocytes without altering cell adhesion (16). PagN is able to interact with extracellular heparin proteoglycans and the entry process require actin polymerization (24). However, proteoglycans cannot transduce a signaling cascade, so it is probable that they act as coreceptors for invasion and not as the receptor itself (3). Other studies have revealed that invasion mechanisms of S. Typhimurium and S. Enteritidis are not restricted to the T3SS1, Rck and PagN. Indeed, a strain which does not express Rck, PagN and the T3SS1 is still able to significantly invade fibroblasts, epithelial and endothelial cells (14). Results obtained by Aiastui et al. and van Sorge et al. have reinforced the idea that nonidentified invasion factors are involved during entry into these cell types (25, 26). Moreover, invasion analyses of a 3

D intestinal epithelium by S. Typhimurium have also highlighted the fact that Salmonella possess noncharacterized invasion factors (27).

We can thus speculate that these different entry processes may contribute to the invasion of specific host cells, and be involved in cell and tissue tropism as well as host specificity and could thus be involved in particular diseases induced by the different Salmonella serotypes. The coexistence of several invasion processes also raises the interesting possibility that synergy might exist between these different bacterial entry proteins. The new paradigm presented here stating

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that Salmonella strains are able to enter nonphagocytic cells by various routes should modify our view of the mechanisms that lead to the different Salmonella induced diseases and should encourage us to revisit the host specificity bases. Future studies will undoubtedly focus on whether cell entry mechanisms are different according to the host and the serotype, and whether there is a link between host specificity, cell tropism, cell entry mechanism, cell response, intracellular bacterial behavior and disease outcomes.

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subspecies enterica serovar Enteritidis, mediates Zipperlike internalization. Cell Res. 2010; 20(6): 64764.

3. Velge P, Wiedemann A, Rosselin M, Abed N, Boumart Z, Chausse AM, et al . Multiplicity of Salmonella

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5. Ibarra JA, SteeleMortimer O. Salmonella ‐‐the ultimate insider. Salmonella virulence factors that

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8. Wallis TS, Galyov EE. Molecular basis of Salmonellainduced enteritis. Mol Microbiol. 2000; 36(5): 997

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9. Jones MA, Wigley P, Page KL, Hulme SD, Barrow PA. Salmonella enterica serovar Gallinarum requires the

Salmonella Pathogenicity Island 2 Type III Secretion System but not the Salmo nella Pathogenicity Island 1 Type III Secretion System for virulence in chickens. Infect Immun. 2001; 69(9): 54716.

10. Coombes BK, Wickham ME, Lowden MJ, Brown NF, Finlay BB. Negative regulation of Salmonella

pathogenicity island 2 is required for contextual control of virulence during typhoid. Proc Natl Acad Sci U S

A. 2005; 102(48): 174605. Epub 2005/11/23.

11. Jones MA, Hulme SD, Barrow PA, Wigley P. The Salmonella pathogenicity island 1 and Salmonella

pathogenicity island 2 type III secretion systems play a major role in pathogenesis of systemic disease and gastrointestinal tract colonization of Salmonella enterica serovar Typhimurium in the chicken. Avian Pathol. 2007; 36(3): 199203.

12. Karasova D, Sebkova A, Havlickova H, Sisak F, Volf J, Faldyna M, et al . Influence of 5 major Salmonella

pathogenicity islands on NK cell depletion in mice infected with Salmonella enterica serovar Enteritidis. BMC Microbiol. 2010; 10:75.

13. Hu Q, Coburn B, Deng W, Li Y, Shi X, Lan Q, et al . Salmonella enterica serovar Senftenberg human

clinical isolates lacking SPI1. J Clin Microbiol. 2008;46(4):13306.

14. Rosselin M, Abed N, VirlogeuxPayant I, Bottreau E, Sizaret PY, Velge P, et al . Heterogeneity of type III

secretion system (T3SS)1independent entry mechanisms used by Salmonella Enteritidis to invade

different cell types. Microbiology. 2011;157(Pt 3):83947.

15. Heffernan EJ, Wu L, Louie J, Okamoto S, Fierer J, Guiney DG. Specificity of the complement resistance

and cell association phenotypes encoded by the outer membrane protein genes rck from Salmonella

Typhimurium and ail from Yersinia enterocolitica . Infect Immun. 1994; 62: 51836.

16. Lambert MA, Smith SG. The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and

invasin. BMC Microbiol. 2008; 8: 142.

17. Miller VL, Beer KB, Heusipp G, Young BM, Wachtel MR. Identification of regions of Ail required for the

invasion and serum resistance phenotypes. Mol Microbiol. 2001; 41(5): 105362.

18. Wiedemann A, Rosselin M, Mijouin L, Bottreau E, Velge P. Involvement of cSrc tyrosine kinase

upstream of class I phosphatidylinositol (PI) 3kinases in Salmonella Enteritidis Rck proteinmediated invasion. J Biol Chem. 2012; 287(37): 3114854.

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19. Mijouin L, Rosselin M, Bottreau E, PizarroCerda J, Cossart P, Velge P, et al . Salmonella Enteritidis Rckmediated invasion requires activation of Rac1, which is dependent on the class I PI 3kinasesAkt signaling pathway. Faseb J. 2012; 26(4): 156981.

20. Ahmer BM, van Reeuwijk J, Timmers CD, Valentine PJ, Heffron F. Salmonella Typhimurium encodes an

SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid.

J Bacteriol. 1998; 180(5): 118593.

21. Smith JN, Dyszel JL, Soares JA, Ellermeier CD, Altier C, Lawhon SD, et al. SdiA, an Nacylhomoserine

lactone receptor, becomes active during the transit of Salmonella enterica through the gastrointestinal tract of turtles. PLoS ONE. 2008; 3(7): e2826.

22. Folkesson A, Advani A, Sukupolvi S, Pfeifer JD, Normark S, Lofdahl S. Multiple insertions of fimbrial

operons correlate with the evolution of Salmonella serovars responsible for human disease. Mol Microbiol. 1999; 33(3): 61222.

23. Heithoff DM, Conner CP, Hentschel U, Govantes F, Hanna PC, Mahan MJ. Coordinate intracellular

expression of Salmonella genes induced during infection. J Bacteriol. 1999; 181(3): 799807.

24. Lambert MA, Smith SG. The PagN protein mediates invasion via interaction with proteoglycan. FEMS

Microbiol Lett. 2009; 297(2): 20916.

25. Aiastui A, Pucciarelli MG, Garciadel Portillo F. Salmonella enterica serovar Typhimurium invades

fibroblasts by multiple routes differing from the entry into epithelial cells. Infect Immun. 2010; 78(6): 2700

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van Sorge AJ, Termote JU, de Vries MJ, Boonstra FN, Stellingwerf C, SchalijDelfos NE. The incidence of

visual impairment due to retinopathy of prematurity (ROP) and concomitant disabilities in the Netherlands:

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27. Radtke AL, Wilson JW, Sarker S, Nickerson CA. Analysis of interactions of Salmonella type three

secretion mutants with 3D intestinal epithelial cells. PLoS ONE. 2010; 5(12): e15750.

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Salmonella and Salmonellosis 2013 May 27-28-29, 2013 Saint-Malo France

Session 2

Genomic and applications

Chairpersons:

Nicholas Thomson (United Kingdom) and François Xavier WEILL (France)

Communications

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Toward a Novel, Transdermal Challenge Model that Adequately Explains the Route of Infection of Peripheral Lymph Nodes by Salmonella

Tom EDRINGTON 1 , Guy LONERAGAN 2 , Joshua HILL 1 , Ken GENOVESE 1 , Haiqi HE 1 , Todd CALLAWAY 1 , Robin ANDERSON 1 , Dayna BRICHTA HARHAY 4 , and David NISBET 1 1 United States Department of Agriculture, Agricultural Research Service, Food and Feed Safety Research Unit, COLLEGE STATION, TX USA 2 Department of Animal and Food Sciences, Texas Tech University, LUBBOCK, TX USA 3 United States Department of Agriculture, Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center, CLAY CENTER, NB USA

ABSTRACT

Salmonella, contained within the peripheral lymph nodes (PLN), is protected from current post-harvest intervention strategies and, therefore, is a concern for the beef industry as a potential contaminate of ground beef. To assess if Salmonella could be recovered from PLN following intradermal administration, studies were conducted in which Salmonella was inoculated in cattle using an allergy skin testing device. Results demonstrated the successful uptake of multiple Salmonella serotypes by the PLN following intradermal administration that was detectable up to 8 days post- challenge. Further, Salmonella recovery was regionally specific to the lymph drainage for each specific lymph node. For example, popliteal lymph nodes were only infected by Salmonella administered to the rear legs and the superficial cervical nodes by that administered to the front legs. This model not only provides a method for the evaluation of potential pre-harvest interventions but allows for the examination of multiple serotypes and infection times within an individual animal. Saliently, our novel model will allow estimation of duration of infection and facilitate design of interventions that decrease duration of infection. Additionally, this research implicates biting flies as an important vector for transmission of Salmonella to the PLN.

INTRODUCTION Peripheral lymph nodes (PLN) in cattle have recently been reported to harbor Salmonella (1, 7) and implicated as a potential source of Salmonella in ground beef (9) . Recently completed and ongoing research has found that prevalence and concentration of Salmonella within the PLN varies among cattle type, region and season (personal communication with G. Loneragan and D. BrichtaHarhay). Seasonal and regional variations have been reported previously for fecal and hide Salmonella prevalence in cattle (4, 5) with the seasonal differences (higher in the summer months, lower in winter) tending to mirror fly populations. Flies have been documented as potential vectors of Salmonella transmission, carrying Salmonella both externally and internally (6, 8, 10) . Biting flies could potentially transmit Salmonella either from their own saliva to the animal, and/or as cattle hides have been shown to be frequently contaminated by Salmonella (24) , inject Salmonella present on the hide during the biting process. Therefore, as transdermal inoculation and subsequent uptake of Salmonella by the PLN has not been reported, the objective of the current research is to determine if intradermal inoculation, similar to that of a fly bite, will result in Salmonella positive PLN in cattle.

MATERIALS AND METHODS All steers were individually penned outdoors in covered, concrete floored pens with feed (hay + grain) and water provided. Steers were euthanized (Euthasol®, euthanasia solution; Delmarva Laboratories, Inc., Midlothian VA) and the right and left subiliac, popliteal, and superficial cervical lymph nodes were collected, weighed and cultured for the challenge strains of Salmonella as described below. Experiment I: Five mature Holstein steers Salmonella (n = 3) or Control (n = 2) treatments. Treatments were injected intradermally above the metacarpus and metatarsus utilizing a 1.0 ml tuberculin syringe fitted with a 22 gauge 1.5 inch needle. One ml of tryptic soy broth (TSB)

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containing the Salmonella or corn oil (Control) was administered in a series of five injections [0.2 ml/injection (10 8 cfu Salmonella /ml)] in each of the four legs. For the Salmonella treatment, four different serovars were utilized: pan susceptible S. Montevideo right front leg, multidrug resistant (MDR) S. Newport left front leg, MDR S. Typhimurium right rear leg, and pansusceptible S. Senftenberg left rear leg. Steers were necropsied 2, 3 and 4 days following treatment administration (one treated steer on day 1; one control and one treated steer on each of days 3 and 4).

Experiment II: One Holstein steer (approx. BW 150 kg) was utilized to evaluate a new method for intradermal administration of Salmonella . The ComforTen® Multiple Skin Test Device (HollisterSteir Allergy, Spokane, WA) was utilized to administer S. Typhimurium (MDR; left legs; 4.5 x 10 8 /ml) and S. Senftenberg (pansusceptible; right legs; 3.8 x 10 8 /ml). Each device consists of 10 testing probes fitted at the end with a stainless steel lancet tip that protrudes from the probe providing intradermal, but