Proceddings I3S 2013

Salmonella and Salmonellosis
Abstract book
27 - 28 - 29 May 2013

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: Genevive Clment - ISPAIA-ZOOPOLE Dveloppement - Ploufragan France

Scientific Committee Organising 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)
Franois Xavier WEILL (France)
Marcel ZWIETERING (the Netherlands)

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

Proceedings edited by:
Pierre COLIN
Genevive CLEMENT
2013 by ZOOPOLE dveloppement - 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 ....................... 2
Michael HENSEL (Germany) Philippe VELGE (France)

Oral Communications
Toward a Novel, Transdermal Challenge Model that Adequately Explains the Route
of Infection of Peripheral Lymph Nodes by Salmonella ............................................................... 8
EDRINGTON Tom, United States of America
Salmonella associated taxonomic and functional changes in the pig digestive tract
during application of feed associated mitigation option in production. ..................................... 12
FRAVALO Philippe, Canada (Qc)
Resistance to essential oils affects survival of Salmonella enterica serovars in
growing and harvested basil ........................................................................................................ 17
KISLUK Guy, Israel
The effect of post-transcriptional regulators on the dynamics of Salmonella biofilm
formation ...................................................................................................................................... 22
VAN PUYVELDE Sandra, Belgium
Characterization of chicken spleen transcriptome after infection with Salmonella
enterica serovar Enteritidis .......................................................................................................... 27
RYCHLIK Ivan, Czech Republic
Deciphering why Salmonella Gallinarum is less invasive in vitro than Salmonella
Enteritidis ..................................................................................................................................... 31
ROSSIGNOL Aurore, France

Posters
Invasiveness of monophasic and aphasic Salmonella strains in point-of-lay chickens ............... 35
MARTELLI Francesca, United Kingdom
SPI-1 encoded genes of Salmonella Typhimurium influence differential polarization
of porcine alveolar macrophages in vitro .................................................................................... 38
KYROVA Kamila, Czech Republic
Flagella changed the pathogenicity of a Salmonella Gallinarum mutant strain .......................... 41
FREITAS NETO Oliveiro, Brazil
Identification of Redundant Metabolic Pathways Essential for Virulence of
Salmonella Typhimurium ............................................................................................................. 44
OLSEN John Elmerdahl, Denmark
Gene expression profile of enterocytes purified from two lines of chicken during the
Salmonella carrier state. .............................................................................................................. 45
VELGE Philippe, France
The expression of cytokines in spleen of chickens infected with S. enterica SE 147
after administration of probiotic bacteria ................................................................................... 46
SPISAKOVA Viera, Slovakia
Cytokine signaling in splenic leukocytes of vaccinated and naive chickens after
intravenous infection with Salmonella Enteritidis ....................................................................... 50
MATULOVA Marta, Czech Republic
Virulence Gene Profiling and Pathogenicity Characterization of Non-Typhoidal
Salmonella Accounted for Invasive Disease in Humans ............................................................... 53
GAL-MOR Ohad, Israel
Cellular immune response by CD4+ T cells in White Layer-hens vaccinated with live
and killed Salmonella vaccines and infected with Salmonella Enteritidis ................................... 54
BECHIERI Jr. Angelo, Brazil
Efficacy of different vaccine programs against Fowl Typhoid using a live attenuated
strain of Salmonella Gallinarum as a vaccine candidate .............................................................. 57
BECHIERI Jr. Angelo, Brazil
Evaluation of protection provided by an inactivated Salmonella vaccine in point-of-
lay chickens challenged with monophasic Salmonella Typhimurium .......................................... 59

Session 2:
Genomic and applications
Chairpersons: Nicholas Thomson (United Kingdom) and Franois Xavier WEILL (France)

Oral communications
Genomic epidemiological studies of the global occurrence of S. Typhimurium DT104 .............. 63
LEEKITCHAROENPHON Pimlapas, Denmark
Multiplexed SNP-typing method for characterizing Salmonella isolates at the
subserovar level ........................................................................................................................... 71
BOLAND Cecile, Belgium
Massive parallel sequencing identify Salmonella single-nucleotide polymorphism
(SNP) markers for host origin and antimicrobial resistance ........................................................ 76
SCHIFFERLI Dieter, United States of America
Molecular subtyping of Salmonella Typhimurium combining different types of
markers in a multiplex liquid bead suspension array .................................................................. 80
WUYTS Vronique, Belgium
One-year surveillance of human and non-human Salmonella enterica serotype
Typhimurium and its monophasic variant based on CRISPOL subtyping, France, 2011 ................... 85
LE HELLO Simon, France

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 ................................................................................................................................. 91
LINGOHR Erika, Canada
Genomic regions of difference between Salmonella enterica subsp. enterica serovar
Gallinarum biovar Gallinarum and biovar Pullorum .................................................................... 94
FREITAS NETO Oliveiro, Brazil

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 ......................... 101
MOOIJMAN Kirsten, The Netherlands
Impact of routine use of two novel real-time polymerase chain reaction assays for
identification of Salmonella enterica subspecies within a National Reference
Laboratory .................................................................................................................................... 105
PETERS Tansy, United Kingdom
Molecular categorization of Salmonella enterica isolated from bovine lymph nodes. ............... 108
BUGAREL Marie, United States of America
Multiple loci VNTR Analysis (MLVA) for molecular characterization of the
monophasic variants of Salmonella enterica serotype Typhimurium outbreaks
occurred in 2011........................................................................................................................... 113
BRISABOIS Anne, France
Characterisation of a novel 18.4 kb genomic island in endemic monophasic
Salmonella Typhimurium strains from Europe ............................................................................ 118
SIMON Sandra, Germany

Posters
Study on the effect of storage conditions on the recovery of Salmonella from boot
swabs and hairnets ....................................................................................................................... 123
RABIE Andre, United Kingdom
Sensitivity of different sampling methods in the detection of Salmonella in flocks of
laying hens .................................................................................................................................... 125
MARTELLI Francesca, United States of America

Improved Isolation of Salmonella from Poultry Environmental Samples using direct
Tetrathionate Enrichment followed by MSRV ............................................................................. 127
WALTMAN Doug, United States of America
Validation of selective enrichment broths and chromogenic media for Salmonella
detection in highly contaminated chicken carcass rinse ............................................................. 130
SEO Kun-Ho, South Korea
A comparison between Rambach agar, Rapid Salmonella Complete agar and
Brilliance Salmonella agar for detection of Salmonella ............................................................... 131
Performance of media plates for detection of Salmonella. ......................................................... 133
LE GALL Franoise, France
State of play of international standardisation of Salmonella methods ....................................... 136
MOOIJMAN Kirsten, The Netherlands
Performance of reference laboratories in interlaboratory comparison studies on
serotyping of Salmonella spp. ...................................................................................................... 138
JACOBS-REITSMA Wilma, The Netherlands
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 Enterobacteriaceae. ............................................................................................... 140
JACOBS-REITSMA Wilma, The Netherlands
AOAC-RI Validation of RAPIDSalmonella method, a Single 18 h Selective Enrichment
and Chromogenic Media Detection of Salmonella spp. from Food ............................................. 142
BICHOT Yannick, France
VIDAS UP SALMONELLA (VIDAS SPT) to detect Salmonella in Primary Production
Samples: Certification according to the ISO16140 Standard ....................................................... 145
AIGUILHON Christine, France
Evaluation of a Rapid Lateral Flow Method for the Detection of Salmonella ............................. 146
AIGUILHON Christine, France
NF Validation Extension Study of an Immunoassay Method for The Detection of
Salmonella in 375g food Samples ................................................................................................ 148
PITTET Jean-Louis, France
Simultaneous detection of Salmonella enterica serovars Typhi, Enteritidis, Infantis
and Typhimurium by multiplex PCR ............................................................................................. 150
MAMMINA Caterina, Italia
Real-time PCR for detection of Salmonella in environmental samples from empty
cleaned and disinfected poultry houses. ..................................................................................... 153
SODOLEVSKY Ekaterina, Israel
Two novel real-time PCR methods for rapid detection of Salmonella Enteritidis and
Typhimurium. ............................................................................................................................... 155
FRENKIEL-LEBOSSE Hlne, France
Molecular identification in monophasic and non-motile variants of Salmonella
enterica serovar Typhimurium. .................................................................................................... 158
Characterization of the emerging Salmonella 4,[5],12:i:- in Danish animal
production. ................................................................................................................................... 161
ARGELLO RODRIGUEZ Hctor, Spain
Monitoring of monophasic and non-motile variants of serotype Typhimurium
among non-human strains collected by the French Salmonella Network ................................... 163
LAILLER Renaud, France
Stability study of the MLVA profiles ............................................................................................. 166
The Application of MultiLocus Variable-number tandem-repeat Analysis (MLVA) for
the epidemiological subtyping of Salmonella enterica serovar Typhimurium in
Scotland. ....................................................................................................................................... 168
BROWN Derek, United Kingdom
Pulsenet MLVA protocol for Salmonella enterica serovar Enteritidis applied to
Salmonella enterica serovar Dublin. ............................................................................................ 170
OCONNOR Jean, Ireland
Pulsenet Multi-locus VNTR Analysis MLVA protocol for Salmonella enterica subtype
Enteritidis compliments phage typing ......................................................................................... 172
OCONNOR Jean, Ireland
Multiple locus variable number of tandem repeats analysis of Salmonella enterica
subsp. enterica serovar Dublin. .................................................................................................... 174
TORPDAL Mia, Denmark
A new immunoenzymatic strategy for the rapid and selective growth of Salmonella ............... 175
WATTIAU Pierre, Belgium
A Comparison of subtyping methods for Differentiating Salmonella enterica serovar
Enteritidis Isolates Obtained from Food and Human Sources ..................................................... 178
SEO Kun-Ho, South Korea
Fast Salmonella Typhimurium sub-typing method based on CRISPR polymorphisms ................ 179
MARAULT Muriel, France
An Inter-laboratory proficiency trial for Salmonella enumeration .............................................. 182
ELLOUZE Mariem, France
Development of MPN-real time PCR for enumeration of Salmonella from pork meat .............. 185
DENIS Martine, France
Determination of Minimal Inhibitory Concentration (MIC) values for clinical isolates
from the poultry practice using a microdilution antimicrobial susceptibility testing
procedure ..................................................................................................................................... 187
WINDHORST Daniel, Germany
Establishment of a multiplex PCR-Lateral Flow Assay (mPCR-LFA) for the detection
of DNA from S. Typhi and S. Paratyphi A ...................................................................................... 188
AZIAH Ismail, Malaysia

Session 4:
Antimicrobial resistance
Chairpersons: Rene HENDRIKSEN (Denmark) and Axel CLOECKAERT (France)

Introduction
Antimicrobial resistance in a genomic era going from phenotype to genotype ......................... 190
Rene HENDRIKSEN (Denmark)

Oral communications
Regulation of the AcrAB multidrug efflux pump in Salmonella enterica serovar
Typhimurium ................................................................................................................................ 199
NISHINO Kunihiko, Japan
Epidemiological and genetic features associated with the international spread of
multi-drug resistant Salmonella Kentucky ST198 ........................................................................ 204
LE HELLO Simon, France
Dramatic increase of AmpC- and ESBL- producing d-Tartrate-positive Salmonella
enterica serotype Paratyphi B from broilers in Belgium, 2008-2010 .......................................... 209
DOUBLET Benoit, France
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? ............................................................................................ 210
GRANIER Sophie A., France
Highly ciprofloxacin resistant Salmonella enterica serovar Kentucky isolates of
human and animal origin in Germany .......................................................................................... 212
BEUTLICH Janine, Germany
Antibiotic resistance trends in Salmonella, 2008-2012 ............................................................... 216
WASYL Dariusz, Poland

Posters
Investigation of Salmonella in pig slaughterhouses in Southern Brazil and
characterization of phenotypic and genotypic antimicrobial resistance of the
isolates .......................................................................................................................................... 222
VOLZ LOPES Graciela, Germany
Phenotypic and genotypic profiles of antimicrobial resistance (AMR) in Salmonella
spp. isolates from faeces of different canine populations of Northern and Central
Italy ............................................................................................................................................... 223
PASOTTO Daniela, Italy
Salmonella spp. prevalence and antimicrobial resistance patterns (phenotypic
profiles and presence of genetic AMR determinants) of serotypes isolated from pet
reptiles in Northern Italy .............................................................................................................. 226
PASOTTO Daniela, Italy
Mechanisms of antimicrobial resistance in biofilm-associated Salmonella
Typhimurium. ............................................................................................................................... 228
SCHLISSELBERG Dov, Israel
Monitoring 3rd generation cephalosporin resistance in French non-human
Salmonella : 2010-2012 overview ................................................................................................ 230
GRANIER Sophie A., France
Emergence of the ASSuT Multidrug Resistant Profile in Salmonella enterica serotype
4,[5],12:i:- in Canada .................................................................................................................... 232
MULVEY Michael, Canada
The Emergence of Ciprofloxacin-Resistant Salmonella enterica serovar Kentucky in
Canada .......................................................................................................................................... 233
MULVEY Michael, Canada
Effect of ramR mutations on efflux genes expression and on fluoroquinolone
susceptibility in Salmonella enterica serotype Kentucky ST198 .................................................. 234
BAUCHERON Sylvie, France
Plasmid-mediated quinolone resistance in domestic and imported food and in food
production animals in Finland 2003-2010 ................................................................................... 235
KURONEN Henry, Finland
Prevalence and characterisation of quinolone resistance mechanisms in Salmonella
isolated in 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 ........... 241
Rob DAVIES (United Kingdom)
Oral communications
Salmonella in soybean meal, what is the source of infection? .................................................... 249
HGGBLOM Per, Sweden
Salmonella in wild birds in Israel: Surveillance studies in healthy and dead birds ...................... 253
LUBLIN Avishai, Israel
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 ........................................................................ 258
DE REU Koen, Belgium
Cattle, Beef, Consumers and Salmonella: A Novel Pathway of Consumer Exposure
and a New Understanding of the Agent-Host Ecology ................................................................ 261
LONERAGAN Guy, United States of America
Estimating the prevalence of Reptile acquired salmonellosis in England and Wales
using a novel demographic based attribution model .................................................................. 265
SINCLAIR Chantil, United Kingdom
Posters
A longitudinal study, from weaning to carcass, to determine the prevalence of
Salmonella on pig farms in Northern Ireland ............................................................................... 271
MADDEN Bob, Northern Ireland
Salmonella in pig farms in Reunion Island: Prevalence assessment and identification
of risk factors ................................................................................................................................ 272
TESSIER Claire, France
Longitudinal investigation of Salmonella spp. from farm to fork in the pig industry of
Reunion Island .............................................................................................................................. 273
TESSIER Claire, France
Tracking in trucks, lairage, slaughter line and quartering as a useful tool to study
Salmonella prevalence in a free-range pig processing plant ....................................................... 275
ASTORGA Rafael J., Spain
Prevalence of Salmonella in Northern Ireland pigs at slaughter. ................................................ 278
MADDEN Bob, Northern Ireland
Occurrence and genetic diversity of Salmonella in organic and conventional pig
productions in France ................................................................................................................... 279
KEROUANTON Annaelle, France
Observations on the occurrence and persistence of monophasic Salmonella
Typhimurium (S.4, [5],12:i:-) in poultry farms. ............................................................................ 281
DAVIES Robert, United Kingdom
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 ................................................................. 286
HENRY Isabelle, France
Salmonella occurrence and antimicrobial resistance in nestlings of two seagull
species, Larus michahellis and Larus audouinii in Ebro Delta (NE Spain). ................................... 289
ANTILLES Noelia, Spain
Prevalence of Salmonella spp. on duck products at the retail level ............................................ 291
CHEMALY Marianne, France
Salmonella-contaminated eggs in flocks identified as positive by the National
Control Programme for Salmonella in the UK .............................................................................. 293
Fate of Salmonella enterica serovar Infantis, the predominant serovar in Israel, in
table eggs ..................................................................................................................................... 295
LUBLIN Avishai, Israel
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. ............................................................................................................................ 299
GOSLING Rebecca, United Kingdom
Analysis of salmonellosis epidemic situation in the RF in 2008-2012 ......................................... 301
PRUNTOVA Olga, Russia
Genotypic diversity of Salmonella Kentucky isolated from reptiles, poultry food, feed and
environment samples in Poland ................................................................................................... 303
ZAJAC Magdalena, Poland
Prevalence of Salmonella isolates in exotic reptiles in Poland.....................................................305
ZAJAC Magdalena, Poland
Prevalence and antimicrobial resistance of Salmonella spp. in healthy reptiles ........................ 307
OVERESCH Gudrun, Switzerland
Are free-living turtles an important source of Campylobacter and Salmonella? ........................ 309
MARIN Clara, Spain
A GFP promoter fusion library for the study of Salmonella biofilm formation and the
mode of action of biofilm inhibitors ............................................................................................ 312
VAN PUYVELDE Sandra, Belgium
Monitoring of Salmonella in food, feed and veterinary samples by the French
Salmonella network ...................................................................................................................... 314
MOURY Frdrique, France
Set up of a National molecular typing database for Salmonella surveillance ............................. 317
FEURER Carole, France

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. excretion. ........................................................................................................... 323
LEBEL Philippe, Canada (Qc)
Decreasing prevalence in Dutch dairy herds under salmonellosis surveillance. ......................... 328
WEBER Maarten F., The Netherlands
Evaluation of the impact of the refrigerated transport of pig carcasses loaded above
7C on their microbial and safety. ................................................................................................ 329
ELLOUZE Mariem, France
A comparison of Salmonella test results from microbiological testing undertaken by
Food Business Operators and that Officialy undertaken ............................................................. 334
EGAN John, Ireland
Application of forensic evaluation of evidence to the tracing of Salmonella .............................. 338
ANDERSSON Gunnar, Sweden

Posters
Investigation of low prevalence Salmonella contamination in poultry feedmills. ...................... 344
DAVIES Robert, United Kingdom
Organic acids for control of Salmonella serotypes in different feed materials ........................... 347
KOYUNCU Sevinc, Sweden
Assessment of anti-Salmonella activity of boot dip samples ....................................................... 349
Investigation of laboratory testing issues in the context of the Salmonella National
Control Programme in GB ............................................................................................................ 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 ..................................................................................................................................... 357
Assessment of the social science aspect of biosecurity and uptake of advice on
chicken layer farms in England and Wales. .................................................................................. 360
Effect of litter aeration in the spread of Salmonella in poultry ................................................... 362
INGRESA Sofa, Spain
Importance of cleaning and disinfection in Salmonella persistence in poultry farms ................ 365
MARIN Clara, Spain
The first bivalent Salmonella live vaccine for chicken and ducks ................................................ 367
WINDHORST Daniel, Germany
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:- ....................................................................................................................... 369
SPRINGER Sven, Germany
Change of interventions in Swedish dairy herds under restrictions due to infection
with Salmonella Dublin. ............................................................................................................... 371
GREN Estelle, Sweden
Risk factors for Salmonella contamination in chicken carcasses at the
slaughterhouse ............................................................................................................................. 373
GONZALEZ BODI Sara, Spain
The logistic slaughter experience in a Spanish slaughterhouse. Lessons learned and
implications. ................................................................................................................................. 375
ARGELLO RODRIGUEZ Hctor, Spain
Inter- and intraserovar variation in in vitro pathogenicity of Salmonella spp. ............................ 377
KUIJPERS Angelina, The Netherlands
Revised Estimates of the Burden of Food-borne Illness in Canada ............................................. 379
THOMAS Kate, Canada
Source Attribution of Salmonella enterica in Ireland using the Microbial Subtyping
method ......................................................................................................................................... 380
DE LAPPE Niall, Ireland

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 ..................................................... 383
Robert V. Tauxe, USA
Oral communications
An outbreak of Salmonella Newport associated with mung bean sprouts, Germany,
October/November 2011 ............................................................................................................. 392
ROSNER Bettina, Germany
Changing trends in antimicrobial resistance patterns in Salmonella enterica serovar
Typhimurim DT193 in England and Wales, 1981-2011 ................................................................ 396
ESAN Oluwaseun, United Kingdom
The benefit of Salmonella control in Sweden who benefits and who pays the cost? .............. 401
STERNBERG LEWERIN Susanna, Sweden
Multistate outbreak of Salmonella Stanley infection in the European Union, 2011-
2012: investigations from fork to farm. ....................................................................................... 405
GOSSNER Cline, Sweden
First identification of multi-resistant Salmonella Brandenburg in patients
hospitalized in the same clinic, France, 2010-2012 ..................................................................... 411
MOULY Damien, France
Seventeen years of extra-intestinal nontyphoidal human Salmonella infections in
the United States, 1995 to 2011 .................................................................................................. 416
FULLERTON Katie, United States of America
A European outbreak of S. Newport associated with melons from South America. ................... 417
FISHER Ian, United Kingdom
Evidence for Reptile-Exotic-Pet-Associated-Salmonellosis (REPAS) in Germany ..................... 421
RABSCH Wolfgang, Germany

Posters
DT7a: a very uncommon Salmonella phage type related to a human outbreak ......................... 428
LETTINI Antonia Anna, Italy
Phenotypic and Molecular Characterisation of multiresistant monophasic
Salmonella Typhimurium (1,4,[5],12:i:-) in Greece, 2006-2011 .................................................. 430
POLEMIS Michalis, Greece
Molecular Typing of Monophasic Salmonella 4,[5],12:i:- Strains Isolated in Belgium,
2008-2011. ................................................................................................................................... 433
BOLAND Cecile, Belgium
Diversity of human Salmonella Typhimurium and Salmonella enterica 4,[5],12:i-
strains isolated in Emilia Romagna Region, Italy.......................................................................... 435
CORPUS Francesco, Italy
Salmonella serovars and phage types associated with specific animal reservoirs and
their epidemiology situation in the United Kingdom ................................................................ 437
MUELLER-DOBLIES Doris, United Kingdom
About three cases of intestinal carriage of Salmonella enterica serovar Toulon in
NICU .............................................................................................................................................. 439
MAMMINA Caterina, Italia
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 .................................................................................................................................... 448
DE REU Koen, Belgium
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 ............................................................. 453
A perfect storm: Misdiagnosed typhoid fever in a laboratory worker in a context of
possible vaccine failure ................................................................................................................ 456
KEDDY Karen, South Africa

Salmonella and Salmonellosis 2013
May 27-28-29, 2013
Saint-Malo France
Session 1
Chairpersons:
Michael HENSEL (Germany) and Philippe VELGE (France)
Table of contents >>
1
NewinsightsoncellentrymechanismsandintracellularlifestyleofSalmonella
MichaelHENSEL(Germany)PhilippeVELGE(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 host
restricted serotype S. Typhi affects 22 million people in developing countries, while gastro
enteritisduetotheubiquitousserotypesisapredominantcauseoffoodborneillnessworldwide.
Moreover,ubiquitousserotypescaninduceawidespectrumofdiseasessuchastyphoidlikefever,
gastroenteritisandasymptomaticinfections,dependingonthehost.
Althoughseveralgeneticlociareassociatedwithhostspecificityanddisease,thestagesatwhich
hostpathogeninteractionsaffectthehostrangeorthediseaseoutcomearenotfullyunderstood.
The importance of Salmonella cell invasion on these phenotypes has been often neglected
becauseofthebeliefthatSalmonellainvadesnonphagocyticcellsonlyinaTypeThreeSecretion
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
humansandcausegastroenteritis,ii)Salmonellainvadefibroblastsandpolarizedenterocytesbya
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
strainabletoexpressneitherT3SS1norRckandPagNisstillabletoenterdifferentcelltypesand
celllinesbyinducingdiscreteandintensemembranerearrangements,suggestingthatSalmonella
canentercellsbyotherZipperandTriggerlikemechanismsthathavenotyetbeenidentified.
Themultiplicityoftheentryrouteswouldmodifyourunderstandingofthemechanismsleadingto
the
differentSalmonellainduceddiseasesandtoSalmonellahostspecificity.
After entry into host cells, Salmonella resides in a membranebound compartment termed
Salmonellacontaining vacuole or SCV. A key virulence determinant for intracellular life is the
SPI2encodedT3SSorT3SS2thattranslocatesacocktailofmorethan30effectorproteinsacross
theSCVmembrane.
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 Salmonellainduced filaments or SIF.
Recruitment of membrane vesicle is required for the extension of the SCV during intracellular
proliferation,however,thephysiologicalroleofSIFisstillunclear.
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.Thesefindingsindicateuniquemechanismsofremodelingofthehostcellsendomembranes.
A specific requirement for adaptation of life inside the SCV is the recruitment of nutrients. We
investigated the nutritional basis ofintracellular Salmonellaand 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
materialinSIFandtheSCV.IntracellularSalmonellainSCVconnectedtoSIFareinexchangewith
endocytosed material and nutrients, while bacteria in SCV without connection to SIF are without
accesstonutrientandgrowthrestricted.
Theimplicationsofourobservationsandrecentfindingsbyothergroupsforthemultiplicityofcell
entrymechanismsandintracellularlifestyleofSalmonellawillbediscussedintheintroductionto
2
this session. These new paradigms should modify our understanding of the Salmonella
pathogenicitymechanisms.
According to serotypes and hosts, Salmonella enterica cause a wide range of food and water
borne 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 non
phagocytic cells and Salmonella entry into host cells is known to be critical for bacterial survival
andestablishmentofdiseaseinahost.
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
physiologicalprocessesthroughthemodulationofexistingcellsignalingcascades.
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
Salmonellastrainscanenternonphagocyticcellsbymultiplepathways(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
ensuressecretionofthetranslocasesbeforetheeffectors.Thesequentialloadingonthisplatform
maybefacilitatedbythedifferentaffinitiesoftheT3SSchaperones,ensuringthehierarchyintype
threeeffectorsecretion(7).
TheT3SS1contributiontopathogenesisdependsonthemodelused.InvivostudieswithS.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
wereisolatedfromhumanclinicalcases,suggestingthattheT3SS1isdispensablebythisserotype
fortheestablishmentofinfectioninhumans(13).Takentogether,theseresultsindicatethatT3SS
1 independent invasion mechanisms also play an important role in Salmonella infection and
3
pathogenesis. This observation could be related to the demonstration that a Salmonella mutant,
unabletoexpressitsT3SS1isstillabletoinvadenumerouscelllinesandcelltypes(14).
Theroleofoutermembraneproteins(OMP)aspossibleadhesionmoleculesandvirulencefactors
has been demonstrated in different pathogenic bacteria. In Salmonella, some Salmonella OMP,
namelyRckandPagN,havebeenshowntobeabletoinducecellinvasion(15)(2)(16).
Rckisa17kDaOMPencodedbythelargevirulenceplasmidofS.EnteritidisandS.Typhimurium.
Rckisamemberofafamilywhichconsistsoffivemembers(Rck,Ail,Lom,OmpXandPagC).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
regionfromtheaminoacids113to159(G113V159)(2,17).Byusingbothaninitiallynoninvasive
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),whichactivatesincascade,classIPI3kinase,Akt,thesmallGTPaseRac1andCdc42(butnot
Rho), leading to activation of the Arp2/3 complex and actin polymerization (19). The cellular
receptorofRckrequiredinthisprocess,however,remainsunkown.
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
regulatorsuggestsanintestinalroleofthisinvasin(20).However,themechanismsleadingtoSdiA
activationandrckexpressionarenotwellcharacterized.Asrckisalsoregulatedbyanunidentified
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,asystemicfunctionofRckispossible.
InadditiontotheT3SS1andRck,thePagNoutermembraneproteinisalsoinvolvedinSalmonella
invasion (16). pagN, is localized on the specific centisome 7 genomic island and is widely
distributedamongSalmonellaentericaserotypes(22).PagNisrequiredforsurvivalinBALB/cmice
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
PagNmediatedcellentryprocess.Atthecellularlevel,pagNdeletioninS.Typhimuriumleadstoa
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
probablethattheyactascoreceptorsforinvasionandnotasthereceptoritself(3).
OtherstudieshaverevealedthatinvasionmechanismsofS.TyphimuriumandS.Enteritidisarenot
restrictedtotheT3SS1,RckandPagN.Indeed,astrainwhichdoesnotexpressRck,PagNandthe
T3SS1 is still able to significantly invade fibroblasts, epithelial and endothelial cells (14). Results
obtainedbyAiastuietal.andvanSorgeetal.havereinforcedtheideathatnonidentifiedinvasion
factorsareinvolvedduringentryintothesecelltypes(25,26).Moreover,invasionanalysesofa3
D intestinal epithelium by S. Typhimurium have also highlighted the fact that Salmonella possess
noncharacterizedinvasionfactors(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
coexistenceofseveralinvasionprocessesalsoraisestheinterestingpossibilitythatsynergymight
exist between these different bacterial entry proteins. The new paradigm presented here stating
4
thatSalmonellastrainsareabletoenternonphagocyticcellsbyvariousroutesshouldmodifyour
view of the mechanisms that lead to the
different Salmonellainduced diseases and should

encourage us to revisit the host specificity bases. Future studies will undoubtedly focus on
whethercellentrymechanismsaredifferentaccordingtothehostandtheserotype,andwhether
there is a link between host specificity, cell tropism, cell entry mechanism, cell response,
intracellularbacterialbehavioranddiseaseoutcomes.
REFERENCES:
1. Cossart P, Sansonetti PJ. Bacterial invasion: the paradigms of enteroinvasive pathogens. Science. 2004;
304(5668):2428.
2. RosselinM,VirlogeuxPayantI,RoyC,BottreauE,SizaretPY,MijouinL,etal.RckofSalmonellaenterica,
subspeciesentericaserovarEnteritidis,mediatesZipperlikeinternalization.CellRes.2010;20(6):64764.
3. Velge P, Wiedemann A, Rosselin M, Abed N, Boumart Z, Chausse AM, et al. Multiplicity of Salmonella
entrymechanisms,anewparadigmforSalmonellapathogenesis.MicrobiologyOpen.2012;1(3):24358.
4. Takeuchi A. Electron microscope studies of experimental Salmonella infection. I. Penetration into the
intestinalepitheliumbySalmonellaTyphimurium.AmJPathol.1967;50(1):10936.
5. Ibarra JA, SteeleMortimer O. Salmonellathe ultimate insider. Salmonella virulence factors that
modulateintracellularsurvival.CellMicrobiol.2009;11(11):157986.
6. Galan JE, WolfWatz H. Protein delivery into eukaryotic cells by type III secretion machines. Nature.
2006;444(7119):56773.
7. LaraTejero M, Kato J, Wagner S, Liu X, Galan JE. A sorting platform determines the order of protein
secretioninbacterialtypeIIIsystems.Science.2011;331(6021):118891.
8. Wallis TS, Galyov EE. Molecular basis of Salmonellainduced enteritis. Mol Microbiol. 2000; 36(5): 997
1005.
9. JonesMA,WigleyP,PageKL,HulmeSD,BarrowPA.SalmonellaentericaserovarGallinarumrequiresthe
Salmonella Pathogenicity Island 2 Type III Secretion System but not the Salmonella Pathogenicity Island 1
TypeIIISecretionSystemforvirulenceinchickens.InfectImmun.2001;69(9):54716.
10. Coombes BK, Wickham ME, Lowden MJ, Brown NF, Finlay BB. Negative regulation of Salmonella
pathogenicityisland2isrequiredforcontextualcontrolofvirulenceduringtyphoid.ProcNatlAcadSciUS
A.2005;102(48):174605.Epub2005/11/23.
11. Jones MA, Hulme SD, Barrow PA, Wigley P. The Salmonella pathogenicity island 1 and Salmonella
pathogenicityisland2typeIIIsecretionsystemsplayamajorroleinpathogenesisofsystemicdiseaseand
gastrointestinaltractcolonizationofSalmonellaentericaserovarTyphimuriuminthechicken.AvianPathol.
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.
BMCMicrobiol.2010;10:75.
13. Hu Q, Coburn B, Deng W, Li Y, Shi X, Lan Q, et al. Salmonella enterica serovar Senftenberg human
clinicalisolateslackingSPI1.JClinMicrobiol.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
differentcelltypes.Microbiology.2011;157(Pt3):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
TyphimuriumandailfromYersiniaenterocolitica.InfectImmun.1994;62:51836.
16. LambertMA,SmithSG.ThePagNproteinofSalmonellaentericaserovarTyphimuriumisanadhesinand
invasin.BMCMicrobiol.2008;8:142.
17. MillerVL,BeerKB,HeusippG,YoungBM,Wachtel MR.IdentificationofregionsofAilrequiredforthe
invasionandserumresistancephenotypes.MolMicrobiol.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.JBiolChem.2012;287(37):3114854.
5
19. MijouinL,RosselinM,BottreauE,PizarroCerdaJ,CossartP,VelgeP,etal.SalmonellaEnteritidisRck
mediatedinvasionrequiresactivationofRac1,whichisdependentontheclassIPI3kinasesAktsignaling
pathway.FasebJ.2012;26(4):156981.
20. Ahmer BM, van Reeuwijk J, Timmers CD, Valentine PJ, Heffron F. Salmonella Typhimurium encodes an
SdiAhomolog,aputativequorumsensoroftheLuxRfamily,thatregulatesgenesonthevirulenceplasmid.
JBacteriol.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
tractofturtles.PLoSONE.2008;3(7):e2826.
22. Folkesson A, Advani A, Sukupolvi S, Pfeifer JD, Normark S, Lofdahl S. Multiple insertions of fimbrial
operonscorrelatewiththeevolutionofSalmonellaserovarsresponsibleforhumandisease.MolMicrobiol.
1999;33(3):61222.
23. Heithoff DM, Conner CP, Hentschel U, Govantes F, Hanna PC, Mahan MJ. Coordinate intracellular
expressionofSalmonellagenesinducedduringinfection.JBacteriol.1999;181(3):799807.
24. Lambert MA, Smith SG. The PagN protein mediates invasion via interaction with proteoglycan. FEMS
MicrobiolLett.2009;297(2):20916.
25. Aiastui A, Pucciarelli MG, Garciadel Portillo F. Salmonella enterica serovar Typhimurium invades
fibroblastsbymultipleroutesdifferingfromtheentryintoepithelialcells.InfectImmun.2010;78(6):2700
13.
26. vanSorgeAJ,TermoteJU,deVriesMJ,BoonstraFN,StellingwerfC,SchalijDelfosNE.Theincidenceof
visualimpairmentduetoretinopathyofprematurity(ROP)andconcomitantdisabilitiesintheNetherlands:
a30yearoverview.BrJOphthalmol.2011;95(7):93741.
27. Radtke AL, Wilson JW, Sarker S, Nickerson CA. Analysis of interactions of Salmonella type three
secretionmutantswith3Dintestinalepithelialcells.PLoSONE.2010;5(12):e15750.

6
May 27-28-29, 2013
Saint-Malo France
Session 2
Chairpersons:
Nicholas Thomson (United Kingdom) and Franois Xavier WEILL (France)
Communications
7
TowardaNovel,TransdermalChallengeModel
thatAdequatelyExplainstheRouteofInfection
ofPeripheralLymphNodesbySalmonella
TomEDRINGTON
1
,GuyLONERAGAN
2
,JoshuaHILL
1
,KenGENOVESE
1
,HaiqiHE
1
,
ToddCALLAWAY
1
,RobinANDERSON
1
,DaynaBRICHTAHARHAY
4
,andDavidNISBET
1
1
UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,FoodandFeedSafety
ResearchUnit,COLLEGESTATION,TXUSA
2
DepartmentofAnimalandFoodSciences,TexasTechUniversity,LUBBOCK,TXUSA
3
UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,RomanL.HruskaU.S.
MeatAnimalResearchCenter,CLAYCENTER,NBUSA
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
ongoingresearchhasfoundthatprevalenceandconcentrationofSalmonellawithinthePLNvaries
amongcattletype,regionandseason(personalcommunicationwithG.LoneraganandD.Brichta
Harhay). Seasonal and regional variations have been reported previously for fecal and hide
Salmonellaprevalenceincattle(4,5)withtheseasonaldifferences(higherinthesummermonths,
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).
BitingfliescouldpotentiallytransmitSalmonellaeitherfromtheirownsalivatotheanimal,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
currentresearchistodetermineifintradermalinoculation,similartothatofaflybite,willresultin
SalmonellapositivePLNincattle.
MATERIALSANDMETHODS
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.,MidlothianVA)andtherightandleftsubiliac,popliteal,andsuperficialcervical
lymph nodes were collected, weighed and cultured for the challenge strains of Salmonella as
describedbelow.
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)
8
containingtheSalmonellaorcornoil(Control)wasadministeredinaseriesoffiveinjections[0.2
ml/injection(10
8
cfuSalmonella/ml)]ineachofthefourlegs.FortheSalmonellatreatment,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 pan
susceptibleS.Senftenbergleftrearleg.
Steerswerenecropsied2,3and4daysfollowingtreatmentadministration(onetreatedsteeron
day1;onecontrolandonetreatedsteeroneachofdays3and4).
ExperimentII:OneHolsteinsteer(approx.BW150kg)wasutilizedtoevaluateanewmethodfor
intradermal administration of Salmonella. The ComforTen Multiple Skin Test Device (Hollister
Steir 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 not subcutaneous, administration of the Salmonella. The device was
dipped into TSB containing the appropriate Salmonella strain and then applied medially and
laterally(twiceeach)abovethemetacarpusandmetatarsusofthesteer,providing40applications
ofSalmonella/leg.ThedevicewasdippedintotheSalmonellabrothpriortoeachadministration
and a new device used for each leg. Two days following Salmonella treatment, the steer was
euthanizedandnecropsied.
Experiment III. Two Holstein steers (approx. BW 180 kg) were utilized to further examine the
suitabilityoftheskintestdevicedescribedabovefortransdermalapplicationofSalmonella.Each
steerwastreatedwithMDRS.Newport(1.93.7x10
8
/ml)(approx.BW=635kg)wereassigned
toadministered to each leg (5 device applications or 50 Salmonella administrations/leg; one
anterior and two each on medial and lateral sides of metacarpus and metatarsus). Each leg was
inoculated at different times: the right front, right rear, left front and left rear 2, 4, 6 and 8 days
priortonecropsy,respectively.
Lymph Node Processing and Bacterial Culture: Lymph nodes were transferred to the laboratory
within 30 minutes of collection and processed as described previously (1). Salmonella isolates
wereserogrouped(5isolates/positivesample)usingslideagglutinationwithSalmonellaantiserum
(DifcoLaboratories,Detroit,MI,USA).
RESULTSANDDISCUSSION
Results of this research demonstrated the successful transdermal inoculation and subsequent
uptake of Salmonella by the PLN in cattle. In the first experiment, the majority of lymph nodes
examinedinallthreeSalmonellatreatedsteers,wereculturepositiveforthechallengestrainsand
most contained significant quantifiable concentrations [2.9 to 5.3 CFU (log
10
)/g lymph node
tissue].Theexceptionwasthesubiliaclymphnodeswhichwereallculturenegative.Salmonella
isolatesthatwereserogroupedmatchedperfectlywiththeserogroupsoftheinoculatedstrainsof
Salmonellaadministeredtoeachlegcontainingthatparticularlymphnode.Alllymphnodesfrom
thetwocontrolsteerswereculturenegativeforSalmonella.Resultsofthisfirstexperiment,while
encouraging,alsoindicatedaproblemwithourdeliverysystemasevidencedbymildtomoderate
swelling and lameness observed in the Salmonellainoculated, but not the control, steers. While
every effort was made to administer the Salmonella intradermally, controlling the depth of the
injection was a challenge which was exacerbated by the difficulty in restraining the steers. The
infectionproducedbythes.c.deliveryofsomeoftheSalmonellawouldlikelyexplainmost,ifnot
all,oftheSalmonellaacquiredmethodofapplicationwasexamined,theComforTenMultipleSkin
Test Device. Utilizing this device, we were able to inoculate intradermally (as indicated by an
absence of swelling or lameness in all of the treated steers), with minimal animal restraint and a
9
vast improvement in ease of application. Further examination of the PLN demonstrated the
uptake of the challenge strains of Salmonella. In the second experiment both the left popliteal
andleftsuperficialcervicallymphnodeswereSalmonellapositivewhereasthoseintherightlegs
and all of the subiliac nodes were culture negative. In contrast to Experiment I, none of the
lymphnodescontainedquantifiablepopulationsoftheinoculatedstrains.Isolatesculturedfrom
thelymphnodesoftheleftlegallbelongedtoserogroupB,thesameserogroupadministeredto
theleftlegs.Resultsfromthethirdexperimentweresimilartothesecond.Inonesteerbothright
and left superficial cervical and popliteal lymph nodes were positive while in the other steer the
leftsuperficialcervicallymphnodewasSalmonellapositive.Noneofthelymphnodescontained
quantifiableSalmonellapopulations.AlllymphnodeisolatesbelongedtoserogroupC
2
,thesame
serogrouputilizedtoinoculatealllegsinbothanimals.Salmonellawasrecoveredfromthelymph
nodes2,4,6and8dayspostinoculation.Allsubiliaclymphnodeswereculturenegative.
Somewhat surprising was the complete absence of Salmonella uptake by the subiliac lymph
nodesinthesecondandthirdpilotstudies,asweexpectedthattheymayaccountforsomeofthe
drainage of the hind legs, and Salmonella positive subiliac lymph nodes have been reported
previously (1). Subsequent research using this intradermal model has produced Salmonella
positivesubiliacsfollowingadministrationtothepaunchregion(Edrington,unpublishedresults).
Wecannotcompletelyruleoutthepossibilitythatsomeoftheisolateswerecoveredwerearesult
ofpreviousexposureandnotourinoculations.However,theconsistentandperfectmatchofthe
serogroupsoftherecoveredisolateswiththoseoftheinoculatedstrains,suggestthiswasnotthe
bythePLN.Basedontheseresults,another
caseanddemonstratesthesightspecificlymphdrainagesystemofeachnode.
Herein,wereportthetransdermaluptakeandtransmissionofSalmonellatothePLNincattleand
our progress towards developing an animal model for transdermal inoculation and subsequent
infection of the PLN. A distinct advantage of this model allows for the examination of multiple
serotypesandinfectiontimeswithinanindividualanimal,therebypotentiallyreducingtheanimal
numbers required for terminal experiments such as these and providing a method for the
evaluation of potential preharvest interventions to prevent and/or eliminate lymph node
acquisition of Salmonella. As PLN are immune to current postharvest intervention strategies,
successfulcontrolwillrequireapreharvestapproach.Priortoexpensiveliveanimalfieldstudies,
ourexperimentalmodelmaybeusedtoprescreenlikelyinterventionstrategies.
ACKNOWLEDGEMENTS
ThisprojectwasfundedinpartbytheUSDANationalInstitutesofFoodandAgriculture'sNational
Integrated Food Safety Initiative award number: 20115111031081, and by the beef and veal
producers and importers through their $1perhead checkoff and was produced for the
CattlemensBeefBoardandstatebeefcouncilsbytheNationalCattlemensBeefAssociation.
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9. Koohmaraie, M., J.A. Scanga, M.J. De La Zerda, B. Koohmaraie, L. Topay, V. Beskhlebnaya, T. Mai, K.
Greeson, and M. Samadpour. 2012. Tracking the sources of Salmonella in ground beef produced from
nonfedcattle.J.FoodProt.75:14641468.
10. Mian, L.S., H. Maag, ant J.V. Tacal. 2002. Isolation of Salmonella from muscoid flies at commercial
animalestablishmentsinSanBernardinoCounty,California.J.VectorEcol.27:8285.

11
Salmonella associated taxonomic and functional changes
in the pig digestive tract during application
of feed associated mitigation option in production
Philippe FRAVALO
1
, Philippe LEBEL
1
, Jessie LONGPR
1
, Benot LAPLANTE
2
,
Etienne YERGEAU
3
, Daniel MASS
4
and Ann LETELLIER
1

1
Chaire de Recherche industrielle du CRSNG en Salubrit des Viandes,
Facult de Mdecine Vtrinaire, Universit de Montral, ST-HYACINTHE,
Qubec, Canada
2
F. Mnard Inc, LANGE-GARDIEN, Qubec, Canada
3
Centre National de Recherche du Canada, avenue Royalmount, MONTREAL,
Qubec, Canada
4
Agriculture Canada SHERBROOKE, Qubec, Canada
ABSTRACT
Salmonella spp carriage and excretion by pigs during production has to be controlled as it increases the risk of
introduction of the pathogen in the food chain. Feed related options developed to mitigate excretion and carriage are
numerous. But whatever the effectiveness of the option, excretion still exists for some pigs. The question that raised is:
are there some individual characteristics that could explain why some pigs are still or became Salmonella shedders in
the context of the application of an efficient mitigation option? Coarse feed has been suspected (Mikkelsen et al 2004),
and recently demonstrated (Lebel et al, I3S2013), to decrease excretion of Salmonella in pigs. In the present study, a
group of 46 eight-weeks aged pigs, fed with 1250 m granulometry during 21 days, was followed. Among the 18 initially
shedders, 15 changed their status but 6 out of the 28 non-shedders presented Salmonella in their feces. To compare
function or composition of flora associated with excretion, short-chain fatty acids (SCFA) concentration in colon were
measured: individual Enterobacteriaceae, Lactobacillus and Bifidobacterium were quantified (Q-PCR) and phylogenetic
and functional metagenomic analyses were conducted. The group presented high level of SCFA: mean value: 4.6, 3.0
and 2.1g/L in colon content for acetic, propionic and butyric acids respectively. Salmonella new shedders showed higher
mean value of fecal Lactobacillus (0.6 Log gene copy more at D21) and lower Bifidobacterium (0.3 Log gene copy less)
when Enterobacteriaceae (5.2 Log gene copy /10ng of fecal DNA extract) didnt changed. Metagenomic analyses,
taxonomic after 16S PCR, were conducted on Illumina platform. Sequences were analyzed using publicly available
pipelines (MG-RAST). This permit to describe taxonomic flora characteristics in pigs at D0 and to follow individual
evolution, depending on they still shed (n=6) or clear (n=3) Salmonella and those, initially non shedders that became
shedders (n=3) in this context of favourable feeding strategy.
INTRODUCTION
Salmonella still represent a major concern for public health policy makers and industrials as the
zoonotic agent is frequently reported in foodborne grouped infections cases. In Quebec, 44% of
these cases are associated to Salmonella (1). The surveillance system specifies rarely the food
origin of the cases but, according to EFSA (2) the part of pig tends to be high (10 to 20%)
particularly in the situation where other implicated production have implemented a control
program as the Salmonella control program in egg production in Quebec. A Salmonella
surveillance program in pig production is present since 2004 in Qubec province. Control
measures at farm were proposed (3) mainly based on good hygiene practices during production.
Then when applicable, food derived mitigation options are recommended, including feed additives
or modification (pre-biotic probiotic and acidification) (4). These authors underlined that research
should document the benefits, and sometimes the absence of effect, using the recently open field
of digestive microbiota analyses. Working on a curative method for Salmonella colonisation
mitigation in pigs we recently demonstrated the previously suggested benefits of using coarse
feed to mitigate the Salmonella load during the growing phase (5). The object of the present study
is to compare fecal contents of pigs that eliminated Salmonella, from those which failed or
acquired Salmonella despite the favorable situation. To do so, compositions of the fecal
microbiota and concentrations of acids in the digestive tracts were compared.
12
MATERIAL AND METHODS
Samples :
10g faeces from individual pigs in a group of 46 naturally Salmonella challenged pigs (exposed
during post nursery phase) and submitted to a 1250 m coarse grinded feed from the growing
facilities period (D0) to day 21 of the trial (D21). Colon contents for 22 out of 46 pigs were
available when slaughter.
Salmonella spp. Detection :
Salmonella presence was assessed using a modified version of the ISO 6579 annexe D (BPW 18-
24h, MRSV 48h, isolation on BGS and XLD followed by biochemical and sero-agglutination
confirmations).
Real-time PCR :
DNA extraction (glass beads lysis followed by a phenol-chloroform extraction) was evaluated
(quality and quantity) by NanoDrop. Real-time PCR using the parameters from (5), have been
conducted using a Eco Illumina real-time PCR and EvaGreen qPCR mix. For the Lactobacillus,
Bifidobacterium genus and Enterobacteriaceae groups the standard curves were obtained by
dilution of the precipitate of a regular PCR product of L. acidophilus ATCC314, B. longum ATCC
15707 and E. coli 25922 respectively.
Short Chain Fatty Acid (SCFA) :
At the end of growing phase, at slaughter, some colon contents were available for acetate,
propionate and butyrate quantitation. Contents were sampled in a 15 ml tube and stored at -20C.
Ten grams of 1/1 diluted in distilled water sample were centrifuged at 41 000g, 30 min at 15 C.
One microliter of 0.5 M H
2
SO
4
was added to 5 mL of the supernatant and centrifuged at 21,800g,
15 min at 15 C. An internal standard (2-ethylbutyrate at 2 g L
1
) was added (0.5 mL) to a tube with
0.5 mL of the acidifiedcentrifuged supernatant and 0.1 g of DOWEX 50WX8 resin (The Dow
Chemical Company, Midland, MI) and vortexed. SCFA were measured with a Perkin Elmer gas
chromatograph model 8310 (Perkin Elmer, Waltham, MA), mounted with a DB-FFAP high
resolution column. Results were analyzed using TurboChrom version 6.2.1 software (Perkin Elmer).
Metagenomic analyses :
V2 16S rRNA amplicons were obtained after amplifications primed with adapters and MIDS
according to match Ion Torrent recommendations. Sequence data were analyzed by using the
Ribosomal Database Project Pyro-sequencing Pipeline (http://pyro.cme.msu.edu/, RDP release 10,
update 26). The sequences were deconvoluted and binned according to their MID tags, and the
MID and forward primers were trimmed by using the Pipeline Initial Process tool. Datasets
containing MID sequences associated with the 16S rRNA gene amplifications were individually
classified using the RDP Classifier tool with an 80% bootstrap cutoff (6).
Statistics Analyses were conducted on SPSS version 17.0 (Lic. UdeMontreal). Non parametric
Man&Wtihney test was used for date to date or type to type data comparisons.

RESULTS
Among the 46 pigs, 8 week aged, submitted to a 1250 m diet, 18 were initially (D0) shedders.
After 21 days in the mitigating conditions (D21), only 9 pigs out of the 46 shed Salmonella (Fig 1)
(Khi2 p<0.05), 3 of them were previously Salmonella shedders at D0 and 6 could be considered as
new shedders. This evolution allows defining 4 groups (types) depending on their Salmonella
status evolution (Table 1).
No significant difference in fecal content for Lactobacillus, Enterobacteriaceae and Bifidobacterium
appear at D0 (Table 1) between groups. At D21 it should be noticed that the mean value in fecal
Enterobacteriaceae was 0.6Log Unit (copy/10ng DNA extract) lower in the type +/+ compared to
other types. A significant increase of Lactobacillus occurs during the 3 weeks period. This increase
is equivalent whatever the type considered. But it should be underlined that, at D21 the type -/+
13
presented the highest value in Lactobacillus concentration (at least 0.6 Log Unit over compared
with the other types) and the lowest concentration in Bifidobacterium.

Table 1 Quantitative assessment of Lactobacillus (Lact), Enterobacteriaceae (Ent) and Bifidobacterium (Bif)
at D0 and D21, depending on Salmonella type.
Type : Salmonella Status at D0/D21, pos. +, neg. (n number of pig/group). Q-PCR mean value values in Log nb of
target copy /10ng DNA extract. SD :standard deviation
Type
D0/D21 Lacto D0
(SD)
Ent D0
(SD)
BifidoD0
(SD)
+/+ (n=3)
4.1
(0.8)
4.7
(0.6)
4.1
(0.3)

+/- (n=15)
3.9
(0.7)
5.4
(1.0)
3.8
(0.9)

-/+ (n=6)
4.1
(1.1)
5.6
(1.3)
3.8
(0.2)

-/- (n=22)
4.3
(0.9)
5.1
(1.1)
3.3
(0.5)

Type
D0/D21 Lacto D21
(SD)
EntD21
(SD)
Bifido D21
(SD)
+/+ (n=3)
5.3
(0.5)
5.6
(1.0)
4.3
(0.6)

+/- (n=15)
5.1
(1.4)
5.2
(0.8)
3.8
(0.8)

-/+ (n=6)
6.0
(0.6)
5.2
(0.8)
3.5
(0.4)

-/- (n=22)
5.3
(0.9)
5.3
(0.8)
3.7
(0.9)

The ability of these two last bacterial populations to contribute to the presence of SCFA in the
contents justified the analyses of SCFA in colon at slaughter (Table 2). The concentrations of the 3
SCFA in the colon are in the range documented for high particle size feed (7). No difference could
be registered for the concentration of SCFA in the type defined by the excretion status but for pig
fed with 1250mm particle size diet.

Table 2 SCFA concentrations in colon contents of pigs depending on their Salmonella shedding evolution
during 3 weeks
+ shedder, Salmonella not present in (n= number) colon samples. SD standard deviation.

Metagenomic analyses of fecal content were done and results corresponded to a mean value of
37.6 X1000 reads per sample (8.2-78.8). Most important changes in phylum proportion appeared
related to the age of the pigs in the groups. Firmicutes and Bacteroidetes represent 90% of the
flora, with increased part of Firmicutes with age (Fig 1). The greatest evolution of phylum
proportion between 0 and 21 was observed for Spirochetes (Fig 2). Their representation increased
less than10 fold for type -/+ and -/- and respectively more than 20 and 50 fold for the groups that
shed Salmonella at D0 and shed at D21 (+/+) or no more (+/-), respectively.
Fig 1) Metagenomic taxonomic classification of fecal contents of pigs (n=3) raised 21 days in coarse grid
feeding.
Type
D0/D21
Acetic
g/L
(SD)

Propionic
g/L
(SD)

Butyric
g/L
(SD)

+/+(n=2) 3.6
(0.6)
3.3
(0.4)
2.3
(0.1)

+/- (n=8) 4.9
(0.8)
2.9
(1.0)
2.0
(0.7)

-/+ (n=4) 4.7
(1.7)
2.7
(1.6)
2.0
(1.4)

-/- (n=8) 4.6
(0.7)
3.2
(1.1)
2.2
(0.8)

14
Salmonella Status at D0/D21, pos. +, neg.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
D0 D21 D0 D21 D0 D21 D0 D21
+/+ +/- -/+ -/-
P
r
o
p
o
r
t
i
o
n

o
f

t
h
e

p
h
y
l
u
m
Day in growing phase
D0/D21
Salmonella status
unclass._Bacteria
Spirochaetes
Firmicutes
Proteobacteria
Bacteroidetes
Actinobacteria

DISCUSSION
Whatever the mitigation option considered in a pig primary production it should be kept in mind
that the Salmonella shedding is associated to multiple factors. We analysed a group of pig that is
raised in conditions that were demonstrated as favourable to mitigate Salmonella excretion. Some
pigs were still shedders or some became shedders.

Fig 2 Evolution of the representation of the more frequent phyla in fecal contents of pig from D0 to D21.
Salmonella Status at D0/D21, pos. +, neg.

15
Different level of analyses is proposed here. At the bacteriological one, the three populations
quantified are often considered as key players in the context of orientation or analyse of the effect
on flora (7, 8).
No significant difference in quantitative measure was obtained depending on types, moreover the
highest concentration of Lactobacillus or Bifidobacterium (considered as protectives) were
observed in group + at D21. The establishment of Enterobacteria is remarkably homogeneous
among our types at D21. However among them a specific effect against Salmonella could be
mediated by SCFA concentration. Both butyrate (8) and propionate (9) were quite recently
described as specifically inhibiting Salmonella colonisation, by perturbation of some SPI-1 major
effectors. No Salmonella type specific increase or decrease could be associated with the
evolution of the Salmonella status at D21. This should be considered keeping in mind the delay
between D21 of growing phase and slaughter time (after 90 days growing), but other studies
confirmed elevated level of SCFA during the entire application of coarse grinded feeding (7).
Metagenomic analyses revealed a difference in Spirochetes representation in flora for group that
were shedders at D0 and moreover for the group that eliminate Salmonella at D21. At the
moment no association between this phylum and Salmonella behaviour was documented. This
constitute a research avenue for our further work.
Our work indicates that despite the application of an efficient mitigation option for the pig group,
individual shedding could occur. This could not be explained easily by conventional assessment
method. Subtle changes should be involved need adaptation of the investigation methodology to
be described.

ACKNOWLEDGEMENT
Thank to F. Mnard Inc. for the experimental design and help on the farm (special thank to Guy
Maynard and Martine Messier for their help).

REFERENCES
1. Bilan TIA 2010-2011 MAPAQ www.mapaq.gouv.qc.ca.
2. EFSA Journal 2010; 8(4):1547
3.http://www.mapaq.gouv.qc.ca/SiteCollectionDocuments/Santeanimale/Evenements/Journees%20scienti
fiques%202008/9_Surveillance_contr_salmon_Lettelier_A.pdf
4. Berge AC, Wierup M. Nutritional strategies to combat Salmonella in mono-gastric food animal
production. Animal. 2012 Apr;6(4):557-64.
5. Lebel Ph, Fravalo Ph; Longpr J.; Laplante; B. Letellier A. Changing the presentation of pigs feed: a cost
effective solution to reduce Salmonella excretion. I3S2013
6. Bell TH, Yergeau E, Martineau C, Juck D, Whyte LG, Greer CW. 2011. Identification of nitrogen-
incorporating bacteria in petroleum-contaminated Arctic soils by using [
15
N]DNA-based stable isotope
probing and pyro-sequencing. Appl Environ Microbiol 2011 77: 41634171
7. Mikkelsen LL, Naughton PJ, Hedemann MS, Jensen BB. Effects of physical properties of feed on microbial
ecology and survival of Salmonella enterica serovar Typhimurium in the pig gastrointestinal tract. Appl
Environ Microbiol. 2004 Jun;70(6):3485-92.
8. Gantois I, Ducatelle R, Pasmans F, Haesebrouck F, Hautefort I, Thompson A, Hinton JC, Van Immerseel F.
Butyrate specifically down-regulates Salmonella pathogenicity island 1 gene expression. Appl Environ
Microbiol. 2006 Jan;72(1):946-9.
9. Hung CC, Garner CD, Slauch JM, Dwyer ZW, Lawhon SD, Frye JG, McClelland M, Ahmer BM, Altier C. The
intestinal fatty acid propionate inhibits Salmonella invasion through the post-translational control of HilD.
Mol Microbiol. 2013 Mar;87(5):1045-60.
16
ResistancetoessentialoilsaffectssurvivalofSalmonellaentericaserovars
ingrowingandharvestedbasil
GuyKISLUK,EmmanuelKALILYandSimaYARON
TechnionIsraelInstituteofTechnology,FacultyofBiotechnologyandFoodEngineering,
HAIFA,Israel
ABSTRACT
The number of outbreaks of foodborne illness associated with consumption of fresh products has increased. A recent
and noteworthy outbreak occurred in 2007. Basil contaminated with Salmonella enterica serovar Senftenberg was the
source of this outbreak. Since basil produces high levels of antibacterial compounds the aim of this study was to
investigate if the emerging outbreak reflects ecological changes that occurred as a result of development of resistance
to ingredients of the basil oil. We irrigated basil plants with contaminated water containing two Salmonella serovars,
Typhimurium and Senftenberg, and showed that Salmonella can survive on the basil plants for at least 100 days. S.
Senftenberg counts in the phyllosphere were significantly higher than S. Typhimurium, moreover, S. Senftenberg was
able to grow on stored harvested basil leaves. Susceptibility experiments demonstrated that S. Senftenberg is more
resistant to basil oil and to its antimicrobial constituents: linalool, estragole and eugenol. This may indicate that S.
Senftenberg had adapted to the basil environment by developing resistance to the basil oil. The emergence of resistant
pathogens has a significant potential to change the ecology, and opens the way for pathogens to survive in new niches
in the environment such as basil and other plants.
INTRODUCTION
Todatemostoutbreaksofentericpathogensassociatedwithreadytoeatgreenshavebeenlinkedto
plantsthatdonotproducehighamountsofantimicrobialcompounds.Recently,outbreaksoriginated
from fresh greens that demonstrate natural antimicrobial activity have emerged. One of the most
recent and noteworthy outbreaks of such fresh produce occurred in 2007. Basil contaminated with
Salmonella enterica serovar Senftenberg was determined to be the source of this outbreak, which
causedmorethan50primarycasesinEuropeandtheUS(1).Theseemergingoutbreaksraiseconcern,
because basil produces various antimicrobial components which are well known for their wide
antibacterialactivity(24).Contaminatedirrigationwaterisconsideredtobeoneofthemajorsources
ofentericpathogensinthefield,buttheimpactofirrigationmethodsontheoccurrenceofSalmonella
onleafyvegetablesisnotwellunderstood(57).Furthermore,littleisknownaboutthepersistenceof
Salmonella on plants that produce high levels of antimicrobial compounds such as basil. We
hypothesize that the emerging outbreaks may reflect ecological changes that occurred as a result of
developmentofresistancetoessensialoils(EOs).Thustheobjectivesofthisstudyweretoinvestigate
transfer,persistenceandsurvivaloftwoSalmonellaserovars,S.TyphimuriumandS.Senftenberg,on
basil,andtodeterminepossiblecorrelationsbetweentheabilityofthebacteriatosurviveontheplant
anditssusceptibilitytoEOs(8).
MATERIALANDMETHODS
Bacterialstrains:
Salmonella enterica serovar Typhimurium (S. Typhimurium) ATCC 14028 and Salmonella enterica
serovarSenftenberg(S.Senftenberg)20070885wereusedinthisstudy.
Irrigationprocedure:
Sprayirrigation(applicationoftheinoculumdirectlyontothephyllospherewithahandsprayer)and
dripirrigation(theinoculumwasdrippeddirectlyintothesoil)methodswereemployed.
Samplingandrecoveryforplatecounting:
Sampling and recovery of GFPexpressing Salmonella from the basil were performed as recently
described(8,9).

17
SurvivalofSalmonellaserovarsoninoculatedbasilleavesduringstorage:
LeaveswerecontaminatedasdescribedpreviouslybyinoculumsofS.SenftenbergandS.Typhimurium
(10),andstoredat4
o
Cand25
o
Cpriortorecovery.
DeterminationofsusceptibilityofS.SenftenbergandS.TyphimuriumtoEos:
AntibacterialactivityagainstS.SenftenbergandS.Typhimuriumwastestedbythepaperdiskdiffusion
assayandinLBbroth.
S.Typhimuriumcountsfollowingsprayanddripirrigationsofbasil.
TodetermineifS.Typhimuriuminirrigationwaterisabletotransferandpersistonbasil,plantswere
irrigatedwithcontaminatedwatercontainingdifferentlevelsofthehumanpathogenS.Typhimurium.
Theirrigationmethodhadasignificanteffectonbacterialcountsinthephyllosphere,therhizosphere
and in the soil (P<0.05). Phyllosphere contamination by S. Typhimurium was significantly higher
following spray irrigations in comparison to drip irrigations. An inverse effect was observed in the
rhizosphere and soil (P<0.05). Following spray irrigation the pathogen was readily detected by plate
counting when heavily contaminated water was applied, but was not detected in any of the leaf
samples following irrigations with water harbouring 3.5 log CFU/ml of the pathogen or less. In roots
and stalks detectable levels of S. Typhimurium were observed following irrigation with water
containingatleast6.5logCFU/ml,andinsoilfollowingirrigationwithwatercontainingatleast5.5log
CFU/ml. Following application of contaminated water by drip irrigation, S. Typhimurium was not
detected in the phyllosphere, with the exception of plants irrigated with water harboring extremely
highconcentrations(i.e.8.5logCFU/ml).
Basil is commonly used fresh, or cooked for a very short time, thus, presence of pathogens on the
leaveshidespotentialhealthrisksfortheconsumers.Directcontactbetweenirrigationwaterandthe
basilphyllosphereresultedinrecoveryofcultivablecellsfromthephyllosphere,andthusturnedtobe
acontributingcauseforcontamination.Avoidanceofsuchcontact,asperformedinourstudybydrip
irrigation, significantly reduced the levels of Salmonella in the phyllosphere of basil and provided
scientific evidence to support the general agreement that such irrigation lowers the risk for
contaminationofgrowingplants(5).Overviewofirrigationmethodsinfruitandvegetableproduction
intheU.S.revealsthatsprinklerirrigationisthemainmethodology(6).Furthermore,raworpartially
treated wastewater is widely used for irrigation worldwide, increasing the risk for contamination (6,
11). Hence, in order to minimize the potential for contamination of growing produce, to promote
microbialsafetyoffreshreadytoeatleafygreensandtosafeguardthehealthoftheconsumers,edible
plantsshouldbeirrigatedbymethodsofdripirrigationratherthansprayirrigation.
ShortandlongtermpersistenceofS.Typhimurium.
S.Typhimuriumwasstilldetectableonbasilleaves100daysfollowingtheirrigationchallenge.Thebasil
phyllosphereharbouredthehighestinitiallevelsofS.Typhimurium(i.e.1hourfollowingtheirrigation
challenge),followedbythesoilandtherhizosphere.Anoteworthydieoffofthepathogen,ofabout1
log CFU/g, was observed in the phyllosphere within as little as the first 10 hours. The rapid decline
continued and after 48 hours the pathogen counts were about 3 log CFU/g lower. Pathogen dieoff
occurred throughout the sampling period but at a lower rate. Comparing the reduction in bacterial
counts over time, from 1h to 48h, reveals that a more significant bacterial dieoff occurred in the
phyllosphereincomparisontotherhizosphere(P<0.05).DieoffofS.Typhimuriummaybetheresultof
thehostileenvironmentsonthephyllospherewhichimposesvariousstressconditionsthatcanrestrict
bacterial survival (12, 13). It was already suggested that the interval between final irrigation and
harvest may influence the extent of contamination, however, lack of data prevents establishment of
effective guidelines for the production of leafy vegetables and herbs (5). Due to the rapid dieoff of
Salmonella in the initial hours post inoculation, as observed in this study, a time interval of 48 hours
18
betweenfinalirrigationandharvestmaybesufficienttoreducethepotentialforillness.Withregardto
field application, persistence of the pathogen in soil leads to a contaminated environment that can
serveasareservoirforfurthercontaminationwithSalmonelladuringorpostharvest.
SurvivalofS.Senftenbergvs.S.Typhimuriumonbasilplants.
Survival of both Salmonella serovars was compared in growing basil plants following application of
contaminated irrigation water (Figure 1). Susceptibility of basil phyllosphere and rhizosphere to both
Salmonellaserovarsdependedonthemethodofapplicationofcontaminatedirrigationwater(P<0.05).
The extent of phyllosphere contamination following spray irrigations depended on the Salmonella
serovarinquestion,withsignificantlyhighercountsofS.Senftenberginthephyllospherecomparedto
S. Typhimurium (P<0.05). Significant differences between the serovars were observed not only in
growing plants but in harvested basil leaves as well (Table 1). Higher bacterial counts on stored basil
leaves were recorded for S. Senftenberg in comparison to S. Typhimurium (P<0.05). The different
ability of S. Typhimurium and S. Senftenberg to contaminate growing and harvested basil implies on
serovardependenceandacquiredadaptationtotypesofplants.
AntibacterialactivityofbasiloilanditscompoundsagainstSalmonellaserovars.
TheabilityofS.SenftenbergtosurviveinhigherlevelscomparedtoS.Typhimurium,bothongrowing
plantsandonharvestedleaves,canbeexplainedbylessersusceptibilitytocomponentsofbasiloil.To
confirmthishypothesisweanalyzedthesensitivityofbothserovarstobasiloil,parsleyoilandselected
purifiedcomponentsofbasiloilusingthediskdiffusionassay(Figure2).Theinhibitionzoneofeachof
the tested material depended on the Salmonella serovar (P<0.05). Both Salmonella serovars are
resistanttoparsleyoilbutsusceptibletobasiloilanditscomponents,withS.Senftenbergbeingmore
resistanttobasiloilandallitstestedcompounds(i.e.linalool,estragoleandeugenol),comparedtoS.
Typhimurium (P<0.05). The difference in the effect of linalool on the two Salmonella serovars was
significantlygreatercomparedtotheeffectoftheotherpurifiedEOsandthebasiloil(P<0.05).Lesser
susceptibility of S. Senftenberg to antimicrobial compounds in basil may be the result of specific
adaptationandmayexplaintheabilityofthisserovartosurviveonbasilplantspreandpostharvest.
Persistence of Salmonella in the plant environment and variations in adaptation of serovars to plant
constituents are in line with the arguments that plants serve as secondary habitat for enteric
pathogens(12,14,15).Furthermore,itindicatesthatentericbacteriasuchasSalmonellaareableto
develop strategies to adapt to the stress conditions on the plant, including different antimicrobial
compounds.
CONCLUSIONS
We have shown that irrigation methodology had a significant effect on levels of plants and soil
contamination. Both serovars survived onthe plant, but theS. Senftenbergcounts were significantly
higher. S. Senftenberg survived on preharvested plants, and was able to grow on stored harvested
leaves.Sincethisserovarshowedincreasedtolerancetoatleast3compoundsandtoamixtureofthe
basilingredients,wesuggestthatS.Senftenberghadadaptedtothebasilenvironmentbydeveloping
resistance to the basil EO. We show here a first indication for adaptation of an enteric pathogen to
stress conditions on plants, and specifically, natural development of multiple resistances to different
EOconstituents.
ACKNOWLEDGMENTS
We thank Prof. Gad Frankel from the Imperial College, London as he helped us to obtain the
SalmonellaSenftenbergisolatefromthe2007outbreak.

19
TABLESANDFIGURES
Figure 1. Levels of S. Senftenberg and S. Typhimurium in basil following spray and drip irrigations. Salmonella
serovars were recovered 48 h following irrigations with water harboring 8.5 log CFU/ml. Following recovery of
the bacteria, GFPtagged Salmonella was enumerated by plating. Columns assigned with asterisks indicate
statisticalsignificancebetweenthetwoserovars(P<0.05).
Figure2.AntibacterialactivityofbasiloilanditscompoundsagainstSalmonellaserovars.A.S.Senftenbergand
B.S.Typhimurium.1.saline2.parsleyoil3.basiloil4.linalool5.estragole.
Table1.SurvivalofS.SenftenbergandS.Typhimuriumonleavesduringstorage.
logCFU/g
a
initial
level
3days
at4C
3days
at25C
S.Senftenberg 5.1
(0.4)
A
6.0
(0.4)
B
8.0
(0.4)
C
S.Typhimurium 5.2
(0.3)
A
5.0
(0.3)
A
7.2
(0.3)
D
a
Average bacterial counts and standard deviation. Numbers assigned with a different letter indicate
statisticalsignificancebetweenbacterialcounts(P<0.05).

20
REFERENCES
1. Pezzoli, L., R. Elson, C.L. Little, H. Yip, I. Fisher, R. Yishai, E. Anis, L. Valinsky, M. Biggerstaff,N. Patel, H.
Mather, D.J. Brown, J.E. Coia, W. van Pelt, E.M. Nielsen, S. Ethelberg, E. de Pinna, M.D. Hampton, T.
Peters,andJ.Threlfall,PackedwithSalmonellainvestigationofaninternationaloutbreakofSalmonella
Senftenberginfectionlinkedtocontaminationofprepackedbasilin2007.FoodbornePathogDis,2008.
5(5):p.661668.
2. Suppakul, P., J. Miltz, K. Sonneveld, and S.W. Bigger, Antimicrobial properties of basil and its possible
applicationinfoodpackaging.JAgricFoodChem,2003.51(11):p.31973207.
3. Kim,J.,Marshall,M.R.,andWei,C.,Antibacterialactivityofsomeessentialoilcomponentsagainstfive
foodbornepathogens.JAgricFoodChem,1995.43:p.28392845.
4. Elgayyar, M., F.A. Draughon, D.A. Golden, and J.R. Mount, Antimicrobial activity of essential oils from
plants against selected pathogenic and saprophytic microorganisms. J Food Prot, 2001. 64(7): p. 1019
1024.
5. FAO/WHO, Food and agriculture organization of the united nations/world health organization.
Microbiological hazards in fresh leafy vegetables and herbs: Meeting report. Microbiological risk
assessmentseriesno.14.Rome.151pp.2008b.
6. Suslow, T.V., Produce safety project issue brief: Standards for irrigation and foliar contact water
http://www.Producesafetyproject.Org/reports?Id=0007.2010.
7. Warriner, K., A. Huber, A. Namvar, W. Fan, and K. Dunfield, Recent advances in the microbial safety of
freshfruitsandvegetables.AdvFoodNutrRes,2009.57:p.155208.
8. Kisluk, G., E. Kalily, and S. Yaron, Resistance to essential oils affects survival of Salmonella enterica
serovarsingrowingandharvestedbasil.Submitted,2013.
9. Kisluk, G. and S. Yaron, Presence and persistence of Salmonella enterica serotype Typhimurium in the
phyllosphere and rhizosphere of sprayirrigated parsley. Appl Environ Microbiol, 2012. 78(11): p. 4030
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12.Brandl, M.T., Fitness of human enteric pathogens on plants and implications for food safety. Annu Rev
Phytopathol,2006.44:p.367392.
13.Warriner, K. and A. Namvar, The tricks learnt by human enteric pathogens from phytopathogens to
persistwithintheplantenvironment.CurrOpinBiotechnol,2010.21(2):p.131136.
14.Teplitski, M., J.D. Barak, and K.R. Schneider, Human enteric pathogens in produce: Unanswered
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21
Theeffectofposttranscriptionalregulatorsonthedynamics
ofSalmonellabiofilmformation
SandraVANPUYVELDE,NathalieMARIEN,HansSTEENACKERSandJosVANDERLEYDEN
CentreofMicrobialandPlantGenetics,KULeuven,KasteelparkArenberg20,
B3001Leuven,Belgium
ABSTRACT
Salmonella has the ability to form structured, multicellular communities, namely biofilms. The formation of a Salmonella
biofilm is triggered by stresses and offers a protected environment. The regulation of biofilm formation is complex and
much progress has been made to unravel the regulatory network. Recently it became clear that small non-coding RNAs
(sRNAs) play an important role in biofilm regulation. sRNAs are an important class of regulators that act post-
transcriptionally. These sRNAs are dependent on the chaperone Hfq for their activity and stability.
Based on this observation we performed a comparative phenotypic analysis of S. Typhimurium wild-type and hfq
knockout mutant under biofilm inducing conditions. The hfq mutant is severely reduced in its capacities to form biofilms.
In our study we could describe a switch at which the population of planktonic cells decreases, while the biofilm fraction
increases. This shows more insights in the dynamics during initial biofilm formation.
With a time lapse gene expression experiment, we could identify specific genes within the complex biofilm regulatory
cascade that are responsible for a disturbed biofilm phenotype caused by the hfq mutation, and are thus main
candidates to be controlled post-transcriptionally. Our data points towards the CsgD regulon, which plays a central role
in the control of biofilm formation.
INTRODUCTION
One of the mechanisms that the Salmonella bacterium uses to protect itself from harmful
environments is the formation of structured multicellular communities embedded within a self
produced matrix, namely biofilms. These biofilms offer a protection to unfavourable conditions,
suchaschemicalandphysicalstresses.Theimportanceofbacterialbiofilmsisreflectedbythefact
that about 80 % of bacterial infections can be linked to biofilms (3). Biofilms are formed on
different surfaces, where for Salmonella bacteria food processing and production environments
arethemostrelevantenvironments.
The regulatory network controlling the biofilm phenotype (described in recent reviews, see refs.
(17,19),ismanagedbyacomplexcascadeofregulators.Manyprocessesareaffectedduringthe
switch from a planktonic, freeliving to a multicellular, sessile state. Examples are the production
of extracellular matrix components, motility and global metabolism. The extracellular matrix of
Salmonella biofilms mainly consists of curli and cellulose, which are encoded by respectively the
csgBACcsgDEFG and bcsABZCbcsEFG operons (5, 18). The CsgD transcription factor, encodedon
thecsgDEFGoperon,hasbeendescribedasacentralregulatorinbiofilmformation(5).
Recently different cases were described where small noncoding RNA regulators (sRNAs) act on
core regulatory genes in the biofilm cascade (8, 9, 15, 20). Research on sRNAs in prokaryotes is
booming.PioneeringstudieshavedemonstratedthatsRNAsconstituteahighlyabundantclassof
regulatorsintheEnterobacteriaceae(1,23).ThebestdescribedclassoftranssRNAregulatorsact
by base pairing with their mRNA targets, which can affect the stability, processing and/or
translationefficiencyofthesemRNAs.TheirfunctionisfacilitatedbytheRNAchaperoneHfq(24).
Such sRNA regulators at the posttranscriptional level are especially important for bacteria in
dealing with stress conditions (13). Particularly for envelope stress, the impact of sRNAs on the
regulationoftheoutermembraneproteins(OMPs)iswelldocumented(6,22).
Previously, we reported that a Salmonella Typhimurium hfq mutant strain, in which the sRNA
regulation capacity is expected to be disturbed, cannot produce mature biofilms (10), strongly
suggestingthatsRNAsplayacrucialroleintheregulatorycascadesofbiofilmformation.Thiswas
confirmedrecentlybyanindependentstudy(16)andunderlinesacrucialroleforsRNAsasanovel
layerintheregulationofbiofilmformation.
22
MATERIALANDMETHODS
Bacterialstrains,plasmids
InthisstudythewildtypeSalmonellaTyphimuriumSL1344wasused(7).Thehfqknockoutstrain
(CMPG5628) was constructed with the method described by Datsenko and Wanner (2, 10).
Salmonella overnight cultures were grown at 37C with aeration in Luria Burtana (LB) broth. For
microscopy the pFPV25.1 plasmid was electroporated to Salmonella, which causes a constitutive
expressionofGFP(21).
Biofilmassay
Bacterialcellsofanovernightculture,washedin1/20trypticsoybroth(TSB)medium,wereused
forbiofilmformation.Theculturewasdiluted1:100infreshmedium.BiofilmsofSalmonellawere
grownin50mL1/20TSBinasquarepetridish(120x120mm,polystyrene)nonshaking,at25C
for24h.Biofilmformationoccursatthebottomandbordersofthepolystyreneplate.
Measurementofopticaldensity
The optical density at 595 nm (OD
595
) was determined of the liquid, planktonic phase of
Salmonellabiofilms.ThemeasurementwasperformedwithThermoSpectronicGenesys6.
Microscopy
Biofilm formation was visualised with light microscopy (Zeiss Imager Z1 Microscope). The liquid
phasewascarefullydiscardedandacoverglasswasplacedontheattachedbiofilmfraction.
RNApurificationandqRTPCR
Samples from planktonic cells during Salmonella biofilm formation were taken from the liquid
phase in the Petridish. 10 mL culture sample was added to 2.5 ml of an icecold phenol:ethanol
(5:95) solution, snapfrozen in liquid N
2
and stored at 80C. Samples were thawed, centrifuged
(4000 rpm, 15 min, 4C) and pellets were incubated for 4 min at room temperature with 5 mg
lysozyme (Sigma). Sequentially RNA was purified with the SV Total RNA Isolation System
(Promega) and DNA was removed with the TURBO DNAfree kit (Ambion). The presence of DNA
was checked with PCR, using primers PRO2041 (GCACCATAATCAACGCTAGACTGTTCT) and
PRO2042(CGCAGCGACAGCGGCAGAAA).RNAqualitywascontrolledwithNanoDropandcapillary
gel electrophoresis (Expirion system, BioRad). 1.5 g of RNA was reverse transcribed to cDNA
with the RevertAid H Minus First strand cDNA synthesis Kit (Fermentas). The qRTPCR assay was
performed on a StepOnePlus Real Time system (Life Technologies). Each PCR reaction was
performed in 20 l, containing 10 ng cDNA, 18 pmol per primer and 10 l Power SYBR Green
mastermix (Life Technologies). A list of the used primers is given in Table 1. All measurements
wereperformedwith3replicates.NormalisationwasperformedwiththeDataAssistsoftware(Life
Technologies),rpoD,rfaH,rpsM,gyrAandrecAwereusedasreferences.
RESULTS
Planktoniccellsshowaswitchduringinitialbiofilmformation
Duringthefirst24hoursofSalmonellabiofilmformation,westudiedthegrowthoftheplanktonic,
liquidphaseofabiofilmsetupinPetridishes.Thedynamicsoftheplanktoniccellsshowaspecific
pattern,whereafterexponentialgrowthatadefinedtimepointthecellsdisappearfromtheliquid
phase (Figure 1). We will describe this period between 6 and 12 h after biofilm inoculation as a
switch where biofilm formation is started. Images of light microscopy during biofilm formation
show that during this switch initial attachment and biofilm formation is started for wild type
Salmonellacells(Figure2).
23
ASalmonellahfqknockouthasadisturbedphenotypeduringthebiofilmswitch
MeasurementsoftheOD
595
oftheplanktonicphaseofaSalmonellahfqknockoutshowsthatthe
switch where freeliving cells disappear from the liquid medium is not present in this mutant. In
contrast to wild type Salmonella the planktonic cells increase and stay at a high level (Figure 1).
Imageswithlightmicroscopyconfirmthatthemutantcannotattachtoformabiofilm(Figure2).
Hfqiscrucialforawelltimedinductionofbiofilmgenes
To determine the genetic factors that can explain the effect of Hfq during the switch, we
measured RNA levels of different genes involved in biofilm formation. For wild type Salmonella,
RNA samples of the planktonic phase were taken during the switch: 6, 8, 10 and 12 hours after
inoculation. For the hfq mutant samples were taken at 8, 10, 12 and 20 hours after inoculation.
Theexpressionlevelof20genesthatarepartofthecomplexbiofilmregulatorynetwork(19)were
measured (data not shown). The most significant changes are found within the branch of the
master regulator CsgD. In the wild type, the levels of csgD and two direct targetscsgB and adrA,
are induced during the biofilm switch (Figure 3). The hfq mutant strain shows significant lower
inductions of expression levels in the planktonic cells (Figure 3), suggesting the need for an Hfq
dependentinductionofthesegenesduringthespecifictimepointofthebiofilmswitch.
DISCUSSION
The regulatory network controlling Salmonella biofilm formation is complex, and still not yet
completelyunderstood(19).RecentstudiesarepointingtowardsthefractionofsRNAregulators,
which form an additional layer in this regulatory network (4, 9, 15, 20). As described earlier, a
knockoutinthehfqgene,whichseverelydisturbssRNAaction,isunabletoformbiofilms(10,16).
Here, we could further specify the effects of this knockout. During initial biofilm formation, a
switch occurs whereby planktonic cells gradually start to organise themselves in a multicellular
biofilm structure. This switch is welltimed and is dependent on Hfq, and thus sRNAs. During this
conversion,wedescribeastrongHfqdependentinductionofthetranscriptionfactorCsgDandhis
targets. Very recently, different studies identified sRNAs with targets in the biofilm regulatory
cascade. Four studies described direct sRNA regulators of csgD, all acting negatively: OmrA/B,
RprA, GcvB and McaS (8, 9, 15, 20). Our study points towards the need to study the action of
sRNAs in a condition dependent way. Contrary to the many sRNAs acting directly on the csgD
transcript,itseemsthattheydonotplayanimportantroleduringinitialbiofilmformationasthey
allactnegatively.Inanhfqmutant,weresRNAactionisdisturbed,thereisnoinductionofCsgD.
Forthephenotypewedescribed,thereareprobablysRNAsresponsiblethatactmoreupstreamin
the cascade or that are not yet characterised as inducers of the CsgD cascade. csgD expression
itself is highly regulated: OmpR, HNS, IHF, MlrA, RpoS, RstA, Crl and CpxR were all described to
influence csgD transcription (19). One of these factors, RpoS, has been described to be highly
regulated by sRNAs as well, among which 3 act positively: RprA, ArcZ and DsrA (11, 12, 14). This
raises the hypothesis that an induction via these sRNAs is necessary during initial biofilm
formation.ThegroupofRomlingshowedthedependencyofSalmonellabiofilmformationonthe
sRNAArcZ.However,theydescribedthiseffecttobeindependentofRpoS(16).
Our study describes a welltimed Hfqdependent switch during Salmonella biofilm formation.
Although the biofilm regulatory network is complex, our data points towards one specific branch
inthecascadethatisbeinginducedduringthisswitch.
REFERENCES
1. Argaman,L.etal.,CurrBiol11,941(2001).
2. Datsenko,K.A.,Wanner,B.L.,ProcNatlAcadSciUSA97,6640(2000).
3. Davies,D.,NatRevDrugDiscov2,114(2003).
4. DeLay,N.,Gottesman,S.,MolMicrobiol,(2012).
24
5. Gerstel,U.,Romling,U.,ResMicrobiol154,659(2003).
6. Guillier,M.,Gottesman,S.,Storz,G.,GenesDev20,2338(2006).
7. Hoiseth,S.K.,Stocker,B.A.,Nature291,238(1981).
8. Holmqvist,E.etal.,EMBOJ29,1840(2010).
9. Jorgensen,M.G.etal.,MolMicrobiol,(2012).
10. Kint,G.etal.,BMCMicrobiol10,(2010).
11. Majdalani,N.etal.,MolMicrobiol39,1382(2001).
12. Majdalani,N.etal.,ProcNatlAcadSciUSA95,12462(1998).
13. Majdalani,N.,Vanderpool,C.K.,Gottesman,S.,CritRevBiochemMolBiol40,93(2005).
14. Mandin,P.,Gottesman,S.,EMBOJ29,3094(2010).
15. Mika,F.etal.,MolMicrobiol,(2012).
16. Monteiro,C.etal.,RNABiol9,(2012).
17. Povolotsky,T.L.,Hengge,R.,JBiotechnol,(2011).
18. Solano,C.etal.,MolMicrobiol43,793(2002).
19. Steenackers,H.etal.,FoodResInt45,502(2012).
20. Thomason,M.K.etal.,MolMicrobiol,(2012).
21. Valdivia,R.H.,Falkow,S.,MolMicrobiol22,367(1996).
22. Vogel,J.,Papenfort,K.,CurrOpinMicrobiol9,605(2006).
23. Wassarman,K.M.etal.,GeneDev15,1637(2001).
24. Waters,L.S.,Storz,G.,Cell136,615(2009).
TABLESANDFIGURES
Figure1:Opticaldensityat595nm(OD595)ofplanktonicphaseduring24hbiofilmformationofSalmonella
wildtypeandhfqmutant.
Figure2:ImagesofbiofilmformationofSalmonellawildtypeandhfqmutant(lightmicroscopy).
25
Figure 3: Gene expression levels of csgD, csgB and adrA during initial biofilm formation of Salmonella. All
samplesweretakenfromtheplanktonicphase(P).Shownaretherelativeexpressions(RQ)inregardtothe
levelsinwildtypeafter12h.
Table1:ListofusedprimersforqRTPCRanalysis
Name Sequence Target Forward/
Reverse
Referencegenes
PRO3532 AGGCTGAAGAGGCTCGTTTG rpoD F
PRO3533 TCCGCGTTCGGATCGA rpoD R
PRO4146 GTCAGGCAACTTACCGCTTGT rfaH F
PRO4147 AAACGCGGGCAACTTCAG rfaH R
PRO4148 GCGCCGTGAAATCAGCAT rpsM F
PRO4149 CAAACCGCGATAGCAACCA rpsM R
PRO4253 CGCACGGCCAACAATGA gyrA F
PRO4254 CAACATTGAGGAGGAGCTGAAGA gyrA R
PRO4261 ACGGCGCGGCGATT recA F
PRO4262 TGTCGTGGGTAGCGAAACG recA R
Biofilmtargets
PRO3650 TGACAGCTGCCCCCATAAA csgD F
PRO3657 CCCACAGCAGTGCAACATCT csgD R
PRO6496 TCAGGCGGCCATTATTGGT csgB F
PRO6497 TTGATCCTTCCTGGCGTACTCT csgB R
PRO6498 GAAGCTCGTCGCTGGAAGTC adrA F
PRO6499 CGAACAGAAGCGGATACAACATAAT adrA R
26
Characterizationofchickenspleentranscriptomeafterinfection
withSalmonellaentericaserovarEnteritidis
IvanRYCHLIK,MartaMATULOVA,JanaRAJOVA,LenkaVLASATIKOVA,JiriVOLF,
HanaSTEPANOVA,andFrantisekSISAK
VeterinaryResearchInstitute,BacteriologyDepartment,BRNO,CzechRepublic
ABSTRACT
In this study we were interested in identification of new markers of chicken response to Salmonella Enteritidis infection.
To reach this aim, gene expression in the spleens of naive chickens and those intravenously infected with S. Enteritidis
was determined by 454 pyrosequencing of splenic mRNA/cDNA. Forty genes with increased expression at the level of
transcription were identified. The most inducible genes encoded avidin (AVD), extracellular fatty acid binding protein
(EXFABP), immune responsive gene 1 (IRG1), chemokine ah221 (AH221), trappin-6-like protein (TRAP6) and serum
amyloid A (SAA). Using cDNA from sorted splenic B-lymphocytes, macrophages, CD4, CD8 and T-lymphocytes, we
found that the above mentioned genes were preferentially expressed in macrophages. AVD, EXFABP, IRG1, AH221,
TRAP6 and SAA were induced also in the cecum of chickens orally infected with S. Enteritidis on day 1 of life or day 42
of life. Unusual results were obtained for the immunoglobulin encoding transcripts. Prior to the infection, transcripts
coding for the constant parts of IgM, IgY, IgA and Ig light chain were detected in B-lymphocytes. However, after the
infection, immunoglobulin encoding transcripts were expressed also by T-lymphocytes and macrophages. Expression of
AVD, EXFABP, IRG1, AH221, TRAP6, SAA and all immunoglobulin genes can be therefore used for the characterization
of the course of S. Enteritidis infection in chickens.
INTRODUCTION
Except for the infection with S. enterica serovars Gallinarum or Pullorum, chicken infection
with the other remaining S. enterica serovars is usually not associated with any obvious
clinical signs. Despite the absence of gross clinical signs, chickens respond to oral infection
by an inflammatory response associated with heterophil and monocyte/macrophage
infiltrationintothececalmucosa.Inagreementwiththis,proinflammatorycytokinessuchas
IL1, IL6, IL17 and IL22, together with IFN and iNOS are induced in the cecum after
infection, either by epithelial cells, resident phagocytes, or infiltrating phagocytes or
lymphocytes [13]. A similar cytokine gene expression can be recorded also in the spleen,
although the induction rates in the spleen after oral infection are usually lower than those
observed in the cecum [4]. The low response of splenic leukocytes to S. enterica infection
can be overcome by intravenous infection. The chicken response to intravenous infection
with S. enterica is characterized by splenomegaly associated with macrophage and
heterophil infiltration and Th1 and Th17 cytokine signaling similar to the response in the
cecumafteroralinfection[3,4].
InthesearchformarkersofchickensresponsetoS.Enteritidisinfectionweusedthemodel
of intravenous infection. We hypothesized that if the spleen sizes differ between the
infected and noninfected chickens [4, 5], there must be significant differences in gene
expressions among these 2 groups of chickens. We therefore purified mRNA from the
spleensofintravenouslyinfectedchickensandsubjectedittotranscriptomecharacterization
by 454 pyrosequencing. This approach resulted in an unbiased identification of chicken
geneswhichareinducedinresponsetosystemicS.entericainfection.
27
MATERIALANDMETHODS
Experimentalanimals,samplecollectionandpyrosequencing
Two samples of spleen from ISA Brown chickens were used for RNA isolation. The first spleen
originated from a 46dayold, noninfected chicken and the second one from a chicken infected
intravenouslywithS.Enteritidisonday42oflifeandsacrificed4dayslater.Approximately30mg
of spleen was collected from each chicken and stored in RNALater (Qiagen) at 70 C for RNA
isolation.
Total RNA was isolated with RNeasy Mini Kit (Qiagen) followed by mRNA Isolation Kit (Roche) to
enrich the total RNA for mRNA species. cDNA libraries from the three spleen samples were
preparedwiththeGSRapidLibraryPreparationKit(Roche)andapprox.2moleculesofcDNAper
bead were used in emulsion PCR. All steps of the cDNA library preparation for sequencing were
performed with the GS Junior Titanium series kits according to the manufacturers instructions
(Roche).ThepyrosequencingwasperformedwiththeGSJunior454sequencer(Roche)andeach
ofthe2sampleswassequencedinaseparatesequencingrun.
Dataanalysis
In the first step we used the De Novo Assembler software provided with the GS Junior. This
software was used to assemble the chicken spleen transcriptome using the sff files generated as
an output of sequencing of each of the 2 samples. Out of this analysis we took the 454Isotig.fna
filewhichcontainedsequencesofalltranscriptsassembledandidentifiedaftermergingthedata
from both splenic samples. The data present in this file were used in two independent analyses.
Firstly, the 454Isotig.fna file was uploaded into Blast2GO software to associate each transcript
with a gene designation. In a second independent analysis we used the 454Isotig.fna file as a
reference file using GS Reference Mapper software provided with GS Junior and analyzed the sff
sequencing files of each of the 2 samples. Transcripts predicted as being downregulated after S.
Enteritidis infection included those for which we identified at least 10 independent reads in the
transcriptome of noninfected spleen and when the fold downregulation was 3fold or higher. A
slightly less stringent selection criteria were applied for the transcripts predicted as being
upregulated after S. Enteritidis infection since these were subjected to verification by realtime
PCR. The tentatively upregulated transcripts were chosen as those which were present in the
infected at 10 or more reads and the calculated induction was at least twofold. In addition, for
further analysis we also included the transcripts which were detected once or not at all in the
transcriptomeofthenoninfectedspleenbutwererecordedinthetranscriptomeoftheinfected
atleast8times.
RealtimePCRverificationofthepyrosequencingdata
Realtime PCR was used for the verification of the pyrosequencing data. Primers for the
quantification of expression realtime PCR were designed using Primer3 software. First we used
thesamecDNAsasinthepyrosequencingreactionsfollowedbyanadditional2spleensamplesfor
eachgroupofchickens.Aftersuchscreeningwereducedthenumberoftestedgenestothosein
whichtherealtimePCRconfirmedtheresultsfrompyrosequencing.Usingthereducedsetofreal
timePCRs,wefinallydeterminedthegeneexpressioninsortedsplenicleukocytesubpopulations.
RESULTS
Expressioninthespleen
Combining both samples in the de novo assembly resulted in the identification of 6,633
transcripts. After applying all the quality selective criteria, 23,663 reads from the spleen of the
noninfected chicken and 21,442 reads from the spleen of the infected chicken were finally
includedinthequantificationofexpression(themajorityoftheexcludedtranscriptscomprisedof
28
rRNA, polyA sequences or repeated sequences). For 99 and 78 genes we predicted that these
might be down or upregulated in the spleen after i.v. S. Enteritidis infection, respectively. In the
next step we designed real time PCRs to verify the expression of all 78 genes predicted as
upregulatedafteri.v.infectionwithS.Enteritidis.Significantupregulationwasconfirmedin40of
them.
Expressioninsortedsplenicleukocytes
Five different groups of genes in relation to their basal expression in particular leukocyte
subpopulationspriortoinfectionandtotheirexpressionprofileaftertheinfectionwereidentified
as follows: i) genes similarly expressed in all leukocyte subpopulations, ii) genes preferentially
expressedinBlymphocytes,iii)genesconstitutivelyexpressedinmacrophages,iv)genesinducible
inmacrophagesandv)genescodingforimmunoglobulins.
Genes that were equally expressed in macrophages, Blymphocytes, CD4, CD8 and T
lymphocytescomprisedofLMNB1,PRDX1,SLC35B1andTRAM1.
Prior to the infection, Blymphocytes were the major producers of PRDX4, SEC11C, ERLEC1 and
TXNDC5 and all immunoglobulins. The expression of PRDX4, SEC11C, ERLEC1 and TXNDC5
decreasedinexpressioninBlymphocytesafterS.Enteritidisinfectionsuggestingasuppressionof
common Blymphocyte function and specialization of Blymphocyte towards immunoglobulin
expressionaftertheinfection.
Genes transcribed constitutively in macrophages were the most numerous and included AH221,
ANG, ANXA2, ASAH, ASS, C1QA, C1QB, C1QG, CCDC86, CTSB, CTSC, CTSD, CTSS, EXFABP, FN1,
FTH1, GSTA, HMOX1, IRG1, MD1, MGST1, OTFB and VIM. Upregulation of these genes in the
spleen without their induction in their main producers indicates that upregulation in the spleen
wascausedmainlybyinfiltrationofmacrophageswiththeircharacteristicexpressionprofile.
Macrophageinducible genes included genes coding for avidin, serum amyloid A and trappin6
(AVD,SAA,TRAP6).Averageupregulationsofthesegenesinmacrophageswere46,23and61,
respectively.
The last group was formed by the transcripts coding for the constant part of immunoglobulins.
Prior to infection, these genes were transcribed the most in Blymphocytes. However, the
transcription of all 4 immunoglobulin genes did not increase after the infection in Blymphocytes
and, instead, there was a significant increase in the abundance of mRNA coding for the constant
part of immunoglobulins in both Tlymphocytes and macrophages in response to the infection.
Consequently, the transcription of the constant parts of immunoglobulins in Tlymphocytes and
macrophagesaftertheinfectionreachednearlythesamelevelasinBlymphocytes.
DISCUSSION
ThemajorityofthesegeneshaveneverbeenassociatedwithS.Enteritidisandchickensalthough
some of these transcripts were described as responsive to Salmonella in other experimental
animalsorwerecharacterizedasLPSinducibleorasbelongingamongacutephaseproteins.Thisis
true mainly for genes coding for serum amyloid A, avidin, immune responsive gene 1 or
extracellular fatty acid binding protein. The main motif of the immune response to the i.v.
infectionwithS.EnteritidiswasLPSinactivation.Inagreementwiththisobservation,threeclasses
of heavy immunoglobulin chains (IgM, IgG and IgA) together with a light chain were induced
afterS.Enteritidisinfection.TherapidincreaseinimmunoglobulinmRNAintheinfectedchickens
4dayspostinfectionindicatesthattheantibodyresponsemightbeaTcellindependentresponse
29
totheLPS.Furthermore,thespleenalsorespondedbyanincreasedtranscriptionofall3subunits
of the C1q complement complex which binds to the conserved domains of IgG and IgM in a
complex with antigen. Several types of cathepsin proteases were induced, as well as TRAP6, a
protease inhibitor, likely protecting host tissues against activity of its own proteases released
during inflammation. Six genes could be characterized as having a detoxification function
(glutathione Stransferase class, microsomal glutathione Stransferase 1, peroxiredoxin 1,
peroxiredoxin 4, thioredoxin domaincontaining protein 5, endoplasmic reticulum lectin 1), most
of them dismutating reactive oxygen species. Finally, the restoration of damaged tissues during
the initial immune response by angiogenesis was induced as early as 4 days post infection as
documentedbyanincreasedexpressionofangiogenin,annexina2,fibronectinandferritinheavy
chain.Foradditionaldetails,seereference[5].
REFERENCES
1. Berndt A, Wilhelm A, Jugert C, Pieper J, Sachse K, et al. (2007). Chicken cecum immune response to
Salmonellaentericaserovarsofdifferentlevelsofinvasiveness.InfectImmun75:59936007.
2. Crhanova M, Hradecka H, Faldynova M, Matulova M, Havlickova H et al. (2011). Immune response of
chicken gut to natural colonization by gut microflora and to Salmonella enterica serovar Enteritidis
infection.InfectImmun79:275563.
3. MatulovaM,StepanovaH,SisakF,HavlickovaH,FaldynovaMetal.(2012).Cytokinesignalinginsplenic
leukocytes from vaccinated and nonvaccinated chickens after intravenous infection with Salmonella
Enteritidis.PLoSOne7:e32346
4. Matulova M, Havlickova H, Sisak F, Rychlik I (2012). Vaccination of chickens with Salmonella
Pathogenicity Island (SPI) 1 and SPI2 defective mutants of Salmonella enterica serovar Enteritidis. Vaccine
30:20907.
5. Matulova M, Rajova J, Vlasatikova L, Volf J, Stepanova H, Havlickova H, Sisak F, Rychlik I (2012).
Characterization of chicken spleen transcriptome after infection with Salmonella enterica serovar
Enteritidis.PLoSOne7:e48101.
30
DecipheringwhySalmonellaGallinarumislessinvasiveinvitro
thanSalmonellaEnteritidis
AuroreROSSIGNOL
1
,SylvieROCHE
1
,IsabelleVIRLOGEUXPAYANT
1
,OlivierGREPINET
1
,
NadiaABED
2
,JrmeTROTEREAU
1
,LouiseBAUGE
3
,AgnsWIEDEMANN
1
,ValentinLOUX
4
,Hlne
CHIAPELLO
4
,JenniferFREDLUND
5
,JostENNINGA
5
andPhilippeVELGE
1
1
INRA,UMR1282InfectiologieetSantpublique,37380NOUZILLY,France;
2
CNRS,UMR8612,5rueJBClment,92290CHATENAYMALABRY,France;
3
UCOBretagneNord,CampusdelaTourdAuvergne,37rueduMarchalFoch,
22200GUINGAMP,France;
4
INRA,UnitMathmatique,InformatiqueetGnome,78352JOUYENJOSAS,France;
5
InstitutPasteur,GroupeDynamiquedesinteractionshtepathogne,
25rueduDrRoux,75724PARIS,France
INTRODUCTION
Salmonella Gallinarum and Salmonella Enteritidis are genetically highly related serotypes with
99.7%ofhomologybetweenorthologs(1).Inspiteofthis geneticsimilarity,theyareresponsible
for very different infections in poultry. S. Gallinarum causes a lethal systemic disease whereas S.
Enteritidis causes a transient systemic infection and an asymptomatic intestinal carriage.
Moreover, these two serotypes also present different behavior in vitro. More particularly,
epithelial cell invasion assays have shown that S.Gallinarum is poorly invasive compared to S.
Enteritidis(2,3).
Salmonellaharborsseveralvirulencefactorsallowingcellinvasion.Themaininvasionfactoristhe
Type Three Secretion System 1 (T3SS1), a secretion system encoded by the Salmonella
Pathogenicity Island 1 (SPI1). This secretion system is similar to a syringe, delivering bacterial
effectors encoded by SPI1 or not, directly into the host cell cytosol. Some effectors translocated
by the T3SS1 trigger the host cell invasion by manipulating the actin cytoskeleton while some
otherscontributetoavarietyofpostinvasionprocesses,suchasintracellulardevelopment(4)and
modulation of the inflammatory response (5, 6). Five effectors have been shown to be essential
fortheinvasionprocess:SipA,SipC,SopB,SopE,SopE2(7).SipCandSipAdirectlybindtoactinand
modulate cytoskeleton dynamics in different ways. SipA reduces the critical concentration of G
actin in the cytosol required for polymerization induced by SipC and stabilizes actin filaments by
displacing ADF/cofilin factor and interacting with TPlastin (8, 9). SopE and SopE2, which have
redundant function, act as eukaryotic Guanine nucleotide Exchange Factors (GEFs), straight
activating host cell RhoGTPases inducing membrane ruffling at the site of entry (10). SopB
contributestoSalmonellauptakebyfacilitatingmembranefissionattheentrysiteleadingtothe
formation of the Salmonellacontaining vacuole (SCV)(11). Among the effectors involved in post
invasion processes, some as SopA and SopD, have been shown to be also implicated in the
epithelialcellinvasionprocessonlyunderparticularconditions(5).
The implication in the entry process has been demonstrated for two others Salmonella invasion
factors,RckandPagN(12,13).Thesetwooutermembraneproteins,namedinvasins,interactwith
a host cell receptor resulting in the bacteria uptake. These entry mechanisms are responsible for
smallactinrufflescomparedtoT3SS1mechanism(14).
TheaimofthisstudyistounderstandwhyS.GallinarumstrainsarelessinvasivethanS.Enteritidis
strainsinvitro.
31
RESULTS
Gentamicinprotectionassayshavebeenperformedwithseveralavianandnonavianepithelial
cell lines, from different tissues (hepatic, pulmonary, intestinal), to determine if the
invasivenessofS.EnteritidisandS.Gallinarumvaryasafunctionoftheoriginofthehostcell.
Results have shown that invasiveness of S. Gallinarum strain SG287/91 varies from 0.0001 to
0.05% of the inoculum, according to the cell type. However, invasion rate of the SG287/91
strain is at least always 10 times inferior to S. Enteritidis strain SELA5, regardless of the host
cell origin and could be 1000 times inferior with some cell lines. These results suggested that
invasion abilities of the S. Gallinarum serotype are altered compared to the S.Enteritidis
serotypeindependentlyoftheavianoriginofthecells.
A preliminary fluorescence microscopy experiment, performed with actinGFP transfected
cells, has shown that during the same time lapse of infection, mCherrytagged SG287/91
causes about 20 times less of actin ruffles compared to a mCherrytagged SELA5. Since
important actin ruffles have been shown to be associated with a T3SS1 dependent entry
mechanism, these results suggested that S. Gallinarum invasion abilities are altered at the
T3SS1levelcomparedtoS.Enteritidis.
Whole genome comparison of two S. Enteritidis strains (SEP125109 and SELA5) and 4
S.Gallinarum strains (SG287/91, SG2210, SG9, SG9S) allowed us to confirm the high genetic
similarity between these serotypes and showed that S. Gallinarum genome encodes all genes
oftheT3SS1,presentinSPI1orinotherpathogenicityislands.
As S. Gallinarum has impaired invasion abilities compared to S. Enteritidis, whereas both
possess SPI1 genes, we have checked if S. Gallinarum has a functional T3SS1. In vitro
secretionassay,inamediuminducingT3SS1secretion,hasrevealedthatSG287/91isableto
secrete proteins in the culture medium in a T3SS1 dependent manner. In vitro translocation
assay, performed with avian and human cells, has shown that SG287/91 is able to translocate
SopD in both cell lines. Thus, low invasion abilities of S. Gallinarum compared to S. Enteritidis
werenotexplainedbyasecretionortranslocationdefectofT3SS1effectorsintothehostcell.
As S. Gallinarum is able to secrete and to translocate T3SS1 effectors in epithelial cells, we
have further investigated if the invasion difference between the two serotypes could be
explainedattheeffectorslevel.Sequencecomparison ofgenesencoding T3SS1effectorshas
shown that sipA and sopE harbor point mutations resulting in potentially important amino
acidssubstitutionsinthefourS.GallinarumstrainscomparedtoS.Enteritidisstrains.Another
differenceisthatsopAisapseudogeninS.Gallinarum.Thesedifferencescouldexplainthelow
invasionphenotypeofS.Gallinarum.
PERSPECTIVES
We report here that S. Gallinarum is less invasive than S. Enteritidis in vitro, is spite of their
genetic similarity and independently of the host cell origin. Results have shown that T3SS1,
the main invasion factor known in Salmonella, is functional. However, T3SS1 effectors gene
sequence analysis has revealed potentially important point mutations in essential effectors of
S.GallinarumcomparedtoS.Enteritidis.TofigureoutifthesemutationshaveanimpactonS.
Gallinarum invasion abilities, a complementation experiment will be performed soon to
determineifSopE,SipAandSopAfromS.Gallinarumareabletorestoreinvasionabilitiesofa
SELA5deletedstrain,atthesamelevelofSELA5wildtypestrain.Wewillalsostudyifthelow
invasionrateofS.GallinarumcouldbeexplainedbyalowSPI1expression,bytestinginvasion
abilities of SG287/91 strain complemented with hilA, a gene encoding a positive regulator of
SPI1expression.
32
REFERENCES
1. ThomsonNR,ClaytonDJ,WindhorstD,VernikosG,DavidsonS,ChurcherC,QuailMA,StevensM,Jones
MA,WatsonM,BarronA,LaytonA,PickardD,KingsleyRA,BignellA,etal.Comparativegenomeanalysisof
Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host
adaptationpathways.GenomeRes2008;18(10):16241637.
2. WilsonRL,ElthonJ,CleggS,JonesBD.SalmonellaentericaserovarsGallinarumandPullorumexpressing
Salmonella enterica serovar Typhimurium type 1 fimbriae exhibit increased invasiveness for mammalian
cells.InfectionandImmunity2000;68(8):47824785.
3. SettaA,BarrowPA,KaiserP,JonesMA.Immunedynamicsfollowinginfectionofavianmacrophagesand
epithelial cells with typhoidal and nontyphoidal Salmonella enterica serovars; bacterial invasion and
persistence,nitricoxideandoxygenproduction,differentialhostgeneexpression,NFkappaBsignallingand
cellcytotoxicity.VetImmunolImmunopathol2012;146(34):212224.
4. MalikKaleP,WinfreeS,SteeleMortimerO.ThebimodallifestyleofintracellularSalmonellainepithelial
cells:replicationinthecytosolobscuresdefectsinvacuolarreplication.PLoSOne2012;7(6):e38732.
5. Raffatellu M, Wilson RP, Chessa D, AndrewsPolymenis H, Tran QT, Lawhon S, Khare S, Adams LG,
Baumler AJ. SipA, SopA, SopB, SopD, and SopE2 contribute to Salmonella enterica serotype Typhimurium
invasionofepithelialcells.InfectImmun2005;73(1):146154.
6. Galyov EE, Wood MW, Rosqvist R, Mullan PB, Watson PR, Hedges S, Wallis TS. A secreted effector
protein of Salmonella Dublin is translocated into eukaryotic cells and mediates inflammation and fluid
secretionininfectedilealmucosa.MolMicrobiol1997;25(5):903912.
7. Patel JC, Galan JE. Manipulation of the host actin cytoskeleton by Salmonellaall in the name of entry.
CurrOpinMicrobiol2005;8(1):1015.
8. Zhou D, Mooseker MS, Galan JE. Role of the S. Typhimurium actinbinding protein SipA in bacterial
internalization.Science1999;283(5410):20922095.
9. Zhou D, Mooseker MS, Galan JE. An invasionassociated Salmonella protein modulates the actin
bundlingactivityofplastin.ProcNatlAcadSciUSA1999;96(18):1017610181.
10.OrchardRC,AltoNM.MimickingGEFs:acommonthemeforbacterialpathogens.Cellularmicrobiology
2012;14(1):1018.
11.Bakowski MA, Cirulis JT, Brown NF, Finlay BB, Brumell JH. SopD acts cooperatively with SopB during
SalmonellaentericaserovarTyphimuriuminvasion.CellMicrobiol2007;9(12):28392855.
12.Rosselin M, VirlogeuxPayant I, Roy C, Bottreau E, Sizaret PY, Mijouin L, Germon P, Caron E, Velge P,
Wiedemann A. Rck of Salmonella enterica, subspecies enterica serovar Enteritidis, mediates Zipperlike
internalization.CellRes2010;20(6):647664.
13.LambertMA,SmithSG.ThePagNproteinofSalmonellaentericaserovarTyphimuriumisanadhesinand
invasin.BMCMicrobiol2008;8:142.
14.VelgeP,WiedemannA,RosselinM,AbedN,BoumartZ,ChausseAM,GrepinetO,NamdariF,RocheSM,
Rossignol A, VirlogeuxPayant I. Multiplicity of Salmonella entry mechanisms, a new paradigm for
Salmonellapathogenesis.MicrobiologyOpen2012;1(3):243258.
33
May 27-28-29, 2013
Saint-Malo France

Session 1


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

Posters

34
InvasivenessofmonophasicandaphasicSalmonellastrains
inpointoflaychickens
FrancescaMARTELLI,RebeccaGOSLING,EmmaKENNEDY,AndrRABIE,HannahREEVES,
FelicityCLIFTONHADLEY,RobDAVIESandRobertoLARAGIONE
BacteriologyandFoodSafetyDepartment
AHVLAWEYBRIDGE,UnitedKingdom

INTRODUCTION
SalmonellaTyphimurium(ST)infectsasmallproportionoflayingflocksacrossEuropeandisable
tocontaminateeggs(6).EggassociatedSToutbreakshavebeenreported(1).Inothergeographic
regions,suchasIndiaandAustralia,SalmonellaEnteritidis(SE)israrelyisolatedfromlayingflocks
or eggs, and ST is responsible for most egg contamination and egg related outbreaks. In recent
yearsanincreaseofthenumberofhumanoutbreakscausedbymonophasicvariantsofST(mST)
has been reported throughout the European Union, the far East and USA. According to EU
zoonoseslegislation(Reg2160/2003)theisolationofmSTstrainsfromacommercialflockoflaying
henstriggersthesamerestrictionsasSTstrains.
TheobjectiveofthisstudywastoundertakeafirstassessmentofthepathogenicityofselectedST,
mSTandaphasicSTstrainsincommerciallayinghens.Toacquireinformationontheinvasiveness
of these strains in layer chickens, six groups of point of lay commercial hens were challenged
separatelywithoneST,4mSTandoneaphasicSTstrain.Aseventhgroupofhenswaschallenged
withanegginvasiveSEstrainforcomparison.
MATERIALANDMETHODS
Seven strains of Salmonella were selected for use in this study. Strain A was a ST DT 8, originally
isolatedintheUKfromaflockofducklayersthatproducedeggsforhumanconsumptionandwas
linkedtohumanoutbreaks(5).StrainsB,DandEweretetraresistantmST4,5:i:and4,5,12:i:DT
193strainsofUKorigin.StrainCwasanonresistantS.4,5,12:i:strain.Fwasanonmotileaphasic
STDT104bstrainoriginatingfromalayingflockinFrance,whereitwasresponsibleforeggrelated
humanoutbreaks(4).StrainGwasanegginvasiveSEphagetype(PT)14bstrainthatspreadfrom
mainlandEuropetotheUKthroughcontaminatedeggs(2).
One hundred and forty commercial Hyline layer chickens were sourced at day old. All remained
unvaccinatedagainstSalmonella.Thechallengestrainwasadministeredbyoralgavagewhenthe
birdswere20weeksofage(pointoflay).Eachbirdreceived1mlofapproximately1x10
9
cfu/ml
of the Salmonella challenge strain by oral gavage. After challenge, cloacal swabs were serially
collectedfromallbirdsineachgrouptoevaluatelevelsofexcretionofSalmonella.Theexperiment
lastedfor21daysafterchallenge(dpc)andpostmortemexaminations(PM)wereperformedon3,
7and17,20dpc.AtPM,liver,spleen,ovaryandcaecaofeachbirdwereasepticallysampled.
Cloacal swabs were plated directly into Rambach agar and incubated at 371
o
C for 24 3 hours.
All swabs were also incubated in 18 ml of Buffered Peptone Water (BPW) at 371
o
C for 1620
hours and subsequently inoculated into MuellerKauffmann tetrathionate (MKTTn) broth at
371
o
Cfor243hours,thenplatedontoRambachagar.InordertoenumerateSalmonellafrom
tissues, 1g subsamples were homogenised, and decimal dilutions of homogenates were made in
PBS (pH 7.4) and plated onto Rambach agar. The tissues were also enriched in BPW and any
negative tissues from the necropsy was enriched in MKTTn broth and replated onto Rambach
agar. A comparison between numbers of positive samples was carried out with a Fishers Exact
Test. The log 10 transformed cfu/g counts for the positive birds were compared at each PM for
eachorganbyaonewayanalysisofvarianceusingBonferroniadjustedttests.

35
RESULTS
Allthestrainscolonisedwellinthebirds,yieldingpositivesinallthetissuesharvestedatPMand
inthecloacalswabstakenseriallyduringtheexperiment.
Nostatisticallysignificantdifferences(p>0.05,FishersExacttest)couldbedetectedbetweenthe
recovery of the different strains after direct plating of cloacal swabs. After enrichment of the
cloacal swabs, there was significantly (p<0.001, Fishers Exact test) less recovery of strain A (ST
DT8)thantheotherstrains.
The birds were still shedding Salmonella in their faeces at 16 dpc in all challenge groups, apart
from the group challenged with strain A. Table 1 shows the number of positive sample and the
numberofsalmonellasdetectedateachPM.SalmonellawasrecoveredinovariesinthefirstPM
afterdirectplating,andafterenrichmentalsointhethirdandfourthPM.Inthespleen,10
1
to10
3
Salmonella cfu/g were recovered in the last postmortem for all strains except B (4,12:i:) and G
(SE). Statistically significant differences between strains, both in the cloacal swabs and in the PM
results, were only sporadic. Overall, all strains were readily recovered from tissue samples and
cloacal swabs at all sampling occasions. No significant difference could be consistently detected
betweentheST,mST,aphasicSTandSEstrainsinthefinalsamplingorcumulativeresults.
DISCUSSION
All the strains were recovered at high levels from cloacal swabs and tissue samples immediately
after challenge. The number of positive samples and the Salmonella counts decreased
progressivelyduringthestudy,butmostSalmonellastrainswererecoveredafterenrichmentinall
tissuesatthelastpostmortemexamination.ThissuggeststhatalthoughtheSalmonellashedding
might have progressively decreased over time, an active or latent Salmonella infection was still
present 20 days post challenge. Significant differences in the number of positive samples and in
the Salmonella counts between strains were variable, occurring on individual sampling occasions
only and not as an overall finding, indicating that the tissue invasion and shedding levels of
monophasic/aphasicstrainsiscomparablewiththoseofafullytypableSTstrainandanoutbreak
associated SE strain. These results are in agreement with those of previous in vivo challenge
studies,thathavehighlightednodifferenceintheinternalorgancolonizationandcloacalshedding
between chickens infected with SE and ST (3). These findings are however generated by a high
dose challenge and might therefore not be comparable with natural infections, where
contaminationusuallyinvolveslownumbersoforganisms,atleastinmostcases.STinfectionsare
likely to evoke a stronger immune response in the birds, and therefore can be cleared more
quickly when compared with SE infections (6), but longer term studies would be needed to
evaluatethepersistencecharacteristicsofthestrainsusedinthisstudy.
Inconclusion,ahighdosemSTandaphasicSTexperimentalchallengeinpointoflayhensresults
intissueinvasionandprolongedshedding.Fieldstudieswouldberequiredinordertoaccurately
assesstheleveloftheinvasivenessofmSTstrainsinnaturallyinfectedchickens,andtoelucidate
theimpactoftheinfectiononthecontaminationoftableeggs.
ACKNOWLEDGEMENTS
ThisworkwasfundedbyDefraprojectOZ0341.
REFERENCES
1. EFSA. 2010. Trends and Sources of Zoonoses and Zoonotic Agents and Foodborne Outbreaks in the
EuropeanUnionin2008.EFSAJournal8:1368.
2. Janmohamed, K., D. Zenner, C. Little, C. Lane, J. Wain, A. Charlett, B. Adak, and D. Morgan. 2011.
NationaloutbreakofSalmonellaEnteritidisphagetype14binEngland,SeptembertoDecember2009:case
controlstudy.EuroSurveill16.
36
3. Keller, L. H., D. M. Schifferli, C. E. Benson, S. Aslam, and R. J. Eckroade. 1997. Invasion of chicken
reproductivetissuesandformingeggsisnotuniquetoSalmonellaEnteritidis.AvianDis41:535539.
4. Le Hello, S., A. Brisabois, M. AccouDemartin, A. Josse, M. Marault, S. Francart, N. J. Da Silva, and F. X.
Weill. 2012. Foodborne outbreak and nonmotile Salmonella enterica variant, France. Emerg Infect Dis
18:132134.
5. Noble,D.J.,C.Lane,C.L.Little,R.Davies,E.DePinna,L.Larkin,andD.Morgan.2012.Revivalofanold
problem:anincreaseinSalmonellaentericaserovarTyphimuriumdefinitivephagetype8infectionsin2010
inEnglandandNorthernIrelandlinkedtoduckeggs.EpidemiolInfect140:146149.
6. Wales, A. D., and R. H. Davies. 2011. A critical review of Salmonella Typhimurium infection in laying
hens.AvianPathol40:429436.

A
STDT8
B
4,12:i:
C
4,5,12:i:
D
4,5,12:i:
E
4,5,12:i:
F
4,5,12::
G
SE
n Positive Positive Positive Positive Positive Positive Positive
PM1 Ovary
4
4
(4.6)
4
(3.5)
4
(4.1)
4
(3.2) 4
4
(3.0)
4
(2.9)
Liver 4
4
(3)
4
(3)
4
(3.4)
4
(3.3)
4
(3.3)
4
(3.3)
4
(3.3)
Spleen 4
4
(2.7)
4
(2.9)
4
(3.4)
4
(3.1)
4
(3.1)
4
(3.4)
4
(3.2)
Caeca 4
4
(7.6)
4
(7.7)
4
(7.7)
4
(7.9)
4
(7.8)
4
(7.6)
4
(8.4)
PM2 Ovary 4 2
4
(2.6)
4
(3) 4
4
(2.3)
4
(2.7) 4
Liver 4
4
(3)
4
(2.9)
4
(3.1)
4
(2.6)
4
(2.8)
4
(3.2)
4
(3)
Spleen 4
4
(2.9)
4
(3.1)
4
(3.2)
4
(2.7)
4
(3.5)
4
(3.4)
4
(4.1)
Caeca 4 4
4
(5) 4 4 4
4
(8.3)
4
(5.6)
PM3 Ovary 6 0 0 3 1 1 0 1
Liver 6 0 3 0 2 2 0 5
Spleen 6
4
(3.3)
6
(3.2)
6
(2.6)
6
(3.1)
6
(2.8)
6
(3.3)
4
(3)
Caeca 6 4 6 6 6 6 6 6
PM4 Ovary 6 0 0 0 1 0 4 2
Liver 6 0 1 1 1 1 1 1
Spleen 6
4
(2.7)
4
2
(2.6)
4
(2.6)
5
(2.6)
6
(3.8) 5
Caeca 6 0 6 6 6 5 4 6
Total 80 52.5% 65% 62.5% 66.2% 66.2% 68.7% 70)%
Table 1. Number of positive tissue samples (including post enrichment data) at post mortem examination
(PM) 1, 2, 3 and 4 (3, 7, 17 and 20 dpc, respectively). When Salmonella was isolated after direct plating
meansof thelog
10
transformedcfu/g (colonyformingunits/gram)countsforthepositivesamples arealso
showninbrackets.

37
SPI1encodedgenesofSalmonellaTyphimurium
influencedifferentialpolarization
ofporcinealveolarmacrophagesinvitro
KamilaKYROVA,HanaSTEPANOVA,IvanRYCHLIK,MartinFALDYNAandJiriVOLF
VeterinaryResearchInstitute,BRNO,CzechRepublic
INTRODUCTION
ThetypeIIIsecretionsystems(TTSS)representessentialvirulencefactorsofmanyGramnegative
bacteria. Salmonella enterica uses two functionally distinct TTSS encoded on the pathogenicity
islandsSPI1(TTSS1)andSPI2(TTSS2).TTSS1mediatestheinvasionofhostcellswhereasTTSS2
is required for intracellular survival and replication. Macrophages represent the key cells of host
immune defence against Salmonella. Recently, macrophages have been shown to have a plastic
phenotype, which is dependent on microenvironmental stimuli. Based on these stimuli,
macrophagescanbeactivated.Activationofmacrophagescanresultintotwodistinctsubsets,M1
(classically activated) with a high antimicrobial potential and M2 (alternatively activated) with
lower ability to kill bacteria. Some pathogens are capable of interfering with macrophage
polarizationinordertodownregulatetheantimicrobialactivityandenhancetheirsurvivalinthe
host. To test this ability of Salmonella enterica serovar Typhimurium DT104 (STM), we infected
porcinealveolarmacrophages,whichconstituteamodelofporcinetissuemacrophages,withwild
typeSTM(WT)anditsisogenicmutantslackingSPI1(SPI1)orSPI2(SPI2)respectively.
MATERIALANDMETHODS
The experimental design included infection of isolated primary porcine alveolar macrophages
(PAM) infected either with WT, SPI1 or SPI2. LPS (1 g/ml) was used as a control. In the first
partofexperimentthemultiplicityofinfection(MOI)10wasused.Theresponseofmacrophages
was measured at the transcriptional level by the quantitative realtime PCR 24 hours post
infection. The number of intracellular bacteria was determined bacteriologically by plating.
BecauseofthedeletionofSPI1ledtoasignificantlylowerinvasionratewecarriedoutthesecond
experiment.TheinfectiondoseofWTandSPI1waschangedtoMOI2.5,10and40toreachthe
samecountsofintracellularlylocatedSalmonella.OnewayANOVAfollowedbyTuckeysposthoc
testwasusedforstatisticalanalysis.Thestatisticalsignificancesaremarkedbydifferentlettersa
differsfrombc,butnotab,etc.,(P<0.05,)orasfollows0.05(*),0.01(**),and0.001(***).The
dataarepresentedasmeanandSD.
RESULTS
ThenumberofintracellularbacteriawasdependentonthepresenceofSPI1. TheSPI1invaded
significantly less efficiently than the WT or SPI2 (Fig. 1). When different MOI were used, the
numbers of intracellular bacteria increased with increasing MOI however intracellular counts of
theSPI1werealwayslowerthanthecountsofWT(Fig.1).
PAM responded by higher expression of proinflammatory genes related to M1 activation (IL1,
TNF, IL8, NFBI) when were infected with SPI1 than with WT or SPI2 (Fig. 2). All these
differences disappeared when heatkilled WT or SPI1 were used for stimulation. Except for
NFBI,heatkilledbacteriatriggeredhigherexpressionofthesegeneswhencomparedtoWTand
SPI2butlowerthanthoseinducedbySPI1.PurifiedLPSstimulatedproinflammatorysignalling
ofPAMtothesameextentasWTortheSPI2(Fig.2).
To assess the role of invasion various MOI (2.5, 10, 40) were used to reach the equal number of
intracellular bacteria 4 hours post infection. Since the behaviour of WT and SPI2 did not differ,
SPI2wasexcludedfromthisstudy.Nosignificantdifferenceswereobservedinproinflammatory
signalling of PAM after infection with WT at increasing MOI (Fig. 2) despite the fact that the
38
numbers of intracellular Salmonella correspondingly increased. On the other hand, the
transcription of the same genes showed a clear dosedependent profile in PAM infected with
SPI1.
ExceptforIL10,MMP9andarginase1,theantiinflammatorygenesrelatedtoM2activation(IL4
R,mannosereceptor,Tfr1,CD163,MMP12,TIMP1)showedlowerexpressionafterinfectionwith
SPI1 in comparison to WT and SPI2 (Fig. 3). As in the case of M1related signalling, the
differencesbetweenWTandSPI2weremostlyinsignificant.Unliketheproinflammatorygenes,
theM2relatedgeneswerenotinducedinPAMexposedtoheatkilledbacteriaofWTandSPI1
or LPS. The dosedependent antiinflammatory response to the infection with WT and SPI1 was
opposite to the proinflammatory response. The transcription of antiinflammatory genes
exhibitedacleardosedependentprofileafterinfectionwithWTbuttheincreasinginfectiondose
ofSPI1didnot(Fig.3).
CONCLUSION
In this study, we have shown that the presence of intact SPI1 genes enables Salmonella
polarization of PAM towards the less bactericidal M2related response. Moreover, there is no
difference in the response of PAM stimulated with LPS, heatkilled bacteria and WT at variable
MOI.TheproinflammatorysignallingappearedtobeamereresponsetoLPSafterinfectionwith
WT.ThereforeitseemsthatSPI1encodedgenesareprobablyrequirednotonlyforinvasionbut
alsoforthesuppressionofproinflammatorycytokineexpression.
ACKNOWLEDGMENT
This work has been supported by the projects MZE0002716202 of the Czech Ministry of
Agriculture, AdmireVet project CZ.1.05/2.1.00/01.0006 ED0006/01/01 from the Czech Ministry
ofEducationandprojectGA524/08/1606fromtheCzechScienceFoundation.
TABLESANDFIGURES
Figure 1: Invasion of Salmonella Typhimurium and isogenic SPI1 and SPI2 mutants into PAM 4 h post
infection.

39
Figure2:M1relatedgeneexpressions(DarkgreycolumnsMOI10,lightgreycolumnsMOI2.5and10).
Figure3:M2relatedgeneexpressions.(DarkgreycolumnsMOI10,lightgreycolumnsMOI2.5and10).

40
Flagellachangedthepathogenicity
ofaSalmonellaGallinarummutantstrain
OliveiroCaetanodeFREITASNETO,PriscilaDinizLOPES,DiegoAlvesBATISTA,
JanineDENADAI,AdrianaMariadeALMEIDA,AndreiSecundodeSOUZA,SilviaAcelasDIAZ
andAngeloBERCHIERIJUNIOR
UNESPUnivEstadualPaulista,DepartmentofVeterinaryPathology,JABOTICABAL,Brazil
INTRODUCTION
It has been suggested the absence of flagella helps Salmonella Gallinarum to invade the blood
stream from the alimentary tract without provoking a strong inflammatory response, favoring
systemic infection in chickens (Chappell et al., 2009; Kaiser et al., 2000). To investigate this
hypothesis,wepreviouslygeneratedaSGmutantstraincapableofproducingflagella(SGFla
+
).SG
Fla+ was less pathogenic to chickens and elicited higher levels of CXCLi2, IL6 and iNOS gene
expression than the wild type (SG) did (Freitas Neto, 2012). However, when successively
inoculated on solid culture medium this mutant stops flagella production and becomes non
flagellated (SG Fla
). The aim of this study was to compare the pathogenicity and systemic
infectionofSGFla
+
,SGFlaandSGinchickens.
MATERIALANDMETHODS
Twoexperimentswerecarriedout.Firstly,180brownlayerchicksweredividedinsixgroupsand
orallychallenged,atfivedaysold,withtwodoses(10
8
and10
6
CFU/mL)ofeachstrain.Birdswere
observedfor28dayspostinfection(dpi).Mortalitywasdailyrecorded.Inthesecondexperiment,
150 birds were divided in three groups and orally challenged, at five daysold, with 10
8
CFU of
eachstrain.Samplesofliverandspleenwerecollectedfromfivebirdsofeachgroupat0,6,12,24
e 48 hours postinfection and 5, 7, 14, 21, 28 dpi and bacterial counts were estimated by serial
dilution.
RESULTS
The mortality data of infected birds are described on Table 1. There were no differences in
mortality among the groups of birds challenged with the higher dose (P>0.05).The mortality
provoked by 10
6
CFU of SG Fla
+
was lower than that caused by SG Fla
and SG (P<0.05).The
recoveryofSalmonellastrainsfromliverandspleenofbirdsareshownaslinecharts(Figure1).SG
Fla
+
wasrecoveredinlowerlevelsinbothspleenandliver(P<0.05).
DISCUSSION
The role of flagella on the pathogenesis of salmonellosis has been extensivelly investigated using
Salmonella Typhimurium infecting mammalian cells as model (Gewirtz et al., 2001). However,
only fews studies have tryied to explore this subject during the avian salmonellosis (Iqbal et al.,
2005).IthasbeensuggestedthattheabsenceofflagellashouldassistSGininvadingfromthebird
alimentary tract without triggering a robust proinflammatory response, thus favouring the
developmentofsystemicinfection(Chappelletal.,2009).
In order to further investigate this subject we previously generated a flagellated and motile SG
mutant strain (SG Fla
+
) what has contributed significantly to a better understanding of the avian
salmonellosisthepathogenesis(FreitasNeto,2012).However,SGFla
+
tendedtoreverttoanon
flagellate variant (SG Fla
) after a few passages on solid medium but easily recovered flagellation

whenstimulated.TheflagellarsynthesisisahighlyenergydemandingprocessandSGisnaturally
deficientinenergyproduction(Thomsonetal.,2008);therefore,SGFla+,inordertosaveenergy,
41
may abrogate flagellar production when stimulus to produce flagella is not present as could be
noticedwhencultivatedonsolidmedium.
It was tempting to compare the pathogenicity of the SG mutant with different phenotypes (Fla
+
and Fla
) with the parental strain in vivo. Although SG Fla
is still able to produce flagella, the

results suggest that during the infection the production of this organelle is probably not
stimulated. The SG Fla
behaved similarly to the wild type strain; it was more invasive and more
pathogenictobirdsthantheSGFla
+
.
CONCLUSION
Theseresultssuggestthepresenceofflagellachangedthepathogenicityofthemutantstrain.
ACKNOWLEDGMENT
TheauthorswouldliketothanksFAPESP,CNPqandCAPESbyfinancialsupport.
REFERENCES
1. Chappell, L. et al. The immunobiology of avian systemic salmonellosis. Veterinary Immunology and
Immunopathology,2009.
2. Freitas Neto, O.C. Role of flagellum in the pathogenicity of Salmonella enterica subspecies enterica
serovar Gallinarum biovar Gallinarum. PhD. Thesis, UNESP Univ Estadual Paulista, Jaboticabal Brazil,
2012.
3. Gewirtz, A. T. et al. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce
epithelialproinflammatorygeneexpression.Journalofimmunology,2001.
4. Iqbal, M. et al. Identification and functional characterization of chicken tolllike receptor 5 reveals a
fundamental role in the biology of infection with Salmonella enterica serovar Typhimurium. Infection and
immunity,2005.
5. Kaiser, P. et al. Differential cytokine expression in avian cells in response to invasion by Salmonella
Typhimurium,SalmonellaEnteritidisandSalmonellaGallinarum.Microbiology,2000.
6. Thomson, N. R. et al. Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella
Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways. Genome research,
2008.

42
TABLESANDFIGURES
Table1.Accumulatedmortalityofbirdsafter28daysofchallengewithSG,SGFla
+
andSGFla
Strain
Grou
p
Totalof
dead/30
birds
Mortality
(%)
SG A
1
26a 87%
SGFla
+
B
1
27a 90%
SGFla
C
1
25a 83%
SG A
2
12bc 40%
SGFla
+
B
2
7b 23%
SGFla
C
2
18c 60%
BirdsfromgroupsA
1
,B
1
andC
1
received1x10
8
CFUofeachstrain.MeanwhilebirdsfromgroupsA
2
,B
2
and
C
2
received 1 x 10
6
CFU of the strains. Different letters after the total of dead birds indicate significant
differencesbyChisquaretestat5%.
Figure 1. Bacterial counts (Log10 CFU/g) in spleen and liver collected at different times from birds
experimentallychallengedwithSG,SGFla
+
and
SGFla

43
IdentificationofRedundantMetabolicPathwaysEssential
forVirulenceofSalmonellaTyphimurium
LotteJELSBAK
1
,HassanHARTMAN
2
,PeterRuhdalJENSEN
3
,MarkPOOLMAN
2
andJohnElmerdahlOLSEN
1
1
DepartmentofVeterinaryDiseaseBiology,UniversityofCopenhagen,Denmark;
2
SchoolofLifeSciences,OxfordBrookesUniversity,UK;
3
DepartmentofSystemsBiology,DanishTechnicalUniversity,Denmark
INTRODUCTION
Metabolicpathwaysthatarenonessentialbecausethebacteriumhasmorethanonealternative
to perform the reaction are not well characterized. We hypothesised that such redundant
pathways might be useful in control of infection, provided one could identify all components of
theredundantpathwaysandblocktheminparallel.
MATERIALANDMETHODS
TotestthehypothesiswedevelopedagenomescalemodelofSalmonellaTyphimuriumfromthe
published genome of strain LT2. After having curated the model, we performed damage analysis
whereallpossiblepairsofnonessentialreactionwereremovedsequentiallyfromthemodel.
The validity of predictions was tested by constructing single and double mutants in genes
encoding the relevant enzymes, and further we constructed double mutants complemented in
trans.
AllstrainswerecharacterizedforgrowthinM9withaddedglucoseandaminoacids(M9+),which
wasthemediausedinthesimulationstudy,andwherepossiblealsoforvirulenceinC57/BL6black
miceinacompetitionassay.
RESULTS
The genome scale model contained 1165 reactions, 975 of which were automatically generated
from database information, 74 were artificially created exchange reactions, 5 partial electron
chainreactionsand111gabfillingreactionstoaccountforliteraturereportsonmetabolicactivity
ofSalmonella.
The damage analysis identified 102 synthetic lethal pairs. Sixty three of these involved fully
annotated pathways only, 32 of which involved only 2 or 3 genes. We selected 10 pairs from
these,constructedmutantsandtestedthepredictions.
Despitetheprediction,threepairsturnedouttocontainessentialgenes.Fourofthepairsshowed
theexpectedphenotypewhengrowninM9+,i.e.growthinsinglegenemutantsbutnogrowthin
doublemutants.Threepairsdidnothaveagrowthphenotype.
The seven pairs where all mutants could be constructed were tested for virulence in mice. Only
twopairs,encodedby1)asnA/asnB)and2)speB/speF+speC)showedtheexpectedoutcome,i.e.
doublebutnosinglemutantswereattenuated.
CONCLUSION
Inconclusion,thegenomescalemodelcouldbeusedtopredictsyntheticlethalpairsofpathways,
butaccuracyofpredictionwasbelowtheexpected.

44
Geneexpressionprofileofenterocytespurifiedfromtwolinesofchicken
duringtheSalmonellacarrierstate
AnneMarieCHAUSSE
1
,OlivierGREPINET
1
,ElisabethBOTTREAU
1
,
ChristelleHENNEQUETANTIER
2
,CatherineBEAUMONT
2
andPhilippeVELGE
1
1
INRA,UMR1282InfectiologieetSantPublique.Site311.37380NOUZILLY,France
2
INRA,UR0083RecherchesAvicoles,37380NOUZILLY,France

INTRODUCTION
In chicken infection with Salmonella may be followed by a carrierstate characterized by
persistenceofbacteriainthececaforlongperiodoftime.
MATERIALSANDMETHODS
By use of microarrays, we have compared gene expression profiles in two lines of chicken
which differ with regard to the susceptibility to carrierstate. At three weeks post
inoculation, infected chicks were compared to controls in lines resistant or susceptible to
carrierstate.
In total we identified 271 genes differentially expressed with an absolute change greater
than 1.5. In a global approach we have determined interaction networks between these
differentially expressed genes. Then in an apriori approach, we have analyzed
differentially expressed genes which were transcriptionally linked to cytokines playing a
major role in the fate of the immune response. Globally, our results suggested that the
responsetoSalmonelladuringtheacutephaseandcarrierstateisdifferent.Indeed,ifacute
phase is characterized by a stimulation of innate immune response, carrier state is
characterized by the inhibition of immune gene expression in both lines ; especially those
transcriptionally linked to type I interferon (IFN) and to TNF. Expression of genes
transcriptionallylinkedtoIFNandIL12whichcharacterizetheTh1axissuggestedthatthis
latterisinhibitedinbothlines.Incontrast,expressionofgeneslinkedtoIL4,IL5andIL13
indicatesthatsusceptibilitytocarrierstatecouldbeassociatedwithaTh2bias.

45
Theexpressionofcytokinesinspleenofchickensinfected
withS.entericaSE147afteradministrationofprobioticbacteria
VieraSPIKOV
1
,MartinaKOLESROV
2
,MriaLEVKUTOV
2
,RbertHERICH
1
,
VieraREVAJOV
1
,MikulLEVKUT
1
andEvaHUSKOV
1
DepartmentofPathologicalAnatomy,DepartmentofEpizootologyandPreventiveVeterinary
Medicine,UniversityofVeterinarymedicineandPharmacy,Komenskho73,04181,
KOICE,SlovakRepublic
INTRODUCTION
Salmonellaentericaisoneofthemajorcauseofhumanfoodbornegastroenteritisandpoultryis
consideredtobethemostimportantsourceofS.Enteritidisforhumans(5).Therefore,knowledge
of the host immune response against Salmonella, is essential for the understanding of its
pathophysiology, prophylaxis and treatment. Manipulation of the gut microbiota of chickens by
administration of probiotic bacteria may help to control enteric bacterial infections, including
Salmonella infection (12). The mechanisms of effects of probiotics are, however, poorly
understood, especially at the molecular level (7). These activities include the ability to induce
cytokineproduction,leadingtoregulationofinnateandacquiredimmuneresponses.
Therefore,thecurrentstudywasundertakentodeterminatetheinfluenceofprobioticbacteriaon
theproinflammatorycytokineandchemokinemRNAprofilesduringS.EnteritidisSE147infection
inyoungchicks.

MATERIALANDMETHODS
The experiment was selected 100 one dayold ISA Brown breed in Provsk Hje in the Slovak
republic. Chickens were randomly divided into 4 groups: control (C), E. faecium EF55 (EF),
combined E. faecium EF55+S. Enteritidis SE147 (EF+SE) and S. Enteritidis SE147 (SE), each group
contain 25 chicks. Probiotic strain Enterococcus faecium EF55 (provided by Laukov, IAP SAS,
Koice, Slovakia) was per orally administered to EF and EF+SE groups from 0 to 7 days (1.10
9
CFU/0.2 ml PBS). Experimental infection of SE and EF+SE groups was carried out per orally using
SalmonellaentericaserovarEnteritidisSE147(providedbyRychlk,VRI,Brno,CzechRepublic)ina
single dose (1.10
8
CFU/0.2 ml PBS) on day 4. Five chickens from each group were euthanized on
days1.,2.,3.,4and7.postinfection(p.i.)withsalmonella.Ateachtimepointsamplesofspleen,
weretakenfromeveryanimal.
Tissue samples taken during necropsies were cut into approx. 20mg pieces which were
immediately placed into RNALater (Qiagen, UK) and stored at 70 C prior to RNA purification.
After storage, a single tissue fragment was transferred into 1 ml of TRI Reagent (Molecular
Research Center, USA) and homogenized using zirconium silica beads (BioSpec Products, USA) in
vortex mixer (Labnet, USA). To separate the phases, 50 l of 4bromanisole (Molecular Research
Center, USA) was added. The whole content of the tube was centrifuged and the upper aqueous
phase was collected for RNA purification using the RNAeasy mini kit (Qiagen, UK) following the
manufacturer's instructions. The purity and concentration of RNA was determined
spectrophotometrically on NanoDrop 200c (Thermo Scientific, USA). The resulting cDNA was 10x
diluted in sterile distilled water and used as a template in realtime PCR or stored at 20 C until
use.
QUANTITATIVEREALTIMEPCR
AmplificationanddetectionofspecificproductswereperformedusingCFX96RTsystem(BioRad,
USA) and iQ SYBR GREEN SUPERMIX (BioRad). Each sample was subjected to realtime PCR in
duplicateandthemeanvaluesoftheduplicateswereusedforsubsequentanalysis.
46
TheCt(cyclethreshold)valuesofgenesofinterestwerenormalisedtoanaverageCtvalueofthe
housekeepinggenes(Ct)andtherelativeexpressionofeachrepresentativewascalculatedas2
Ct.AllprimersusedintherealtimePCRinthisstudyarelistedinTable1.
StatisticalanalysisoftheresultswasperformedusingonewayANOVAwithTukeyposttest).
Primer Sequence5'3' Reference

GAPDHFor CCTGCATCTGCCCATTT DeBoeveretal.,2008 (6)
GAPDHRev GGCACGCCATCACTATC
UBFor GGGATGCAGATCTTCGTGAAA DeBoeveretal.,2008 (6)
UBRev CTTGCCAGCAAAGATCAACCTT
IFN GCCGCACATCAAACACATATCT Berndtetal.,2007(1)
IFN TGAGACTGGCTCCTTTTCCTT
IL2For CATCTCGAGCTCTACACACCA Rychliketal.,2009 (14)
IL2Rev AGAGTAACCACTTCTCCCAGGT
IL6For GCTACAGCACAAAGCACCTG Rychliketal.,2009 (14)
IL6Rev GACTTCAGATTGGCGAGGAG
IL15For TGGAGCTGATCAAGACATCTG Kolesarovaet.al.,2011 (10)
IL15Rev CATTACAGGTTCCTGGCATTC
IL18For ACGTGGCAGCTTTTGAAGAT Rychliketal.,2009 (14)
IL18Rev GCGGTGGTTTTGTAACAGTG
TGF4For AGGATCTGCAGTGGAAGTGGAT Swaggertyetal.,2004 (15)
TGF4Rev CCCCGGGTTGTGTGTTGGT
LITAFFor AATTTGCAGGCTGTTTCTGC Kolesarovaetal.,2011 (10)
LITAFRev TATGAAGGTGGTGCAGATGG
LymphotactinFor CATAGTCTGGCTTGGCGTCTT Whithnageetal.,2004 (19)
LymphotactinRev GCGCATTGACTGACTTGCA
iNOSFor GAACAGCCAGCTCATCCGATA Berndtetal.,2007 (1)
iNOSRev CCCAAGCTCAATGCACAACTT
Table1.ListofprimersusedforchickencytokinemRNAquantification
RESULTS
We found that the first collection (on the first day after infection) is the biggest stimulus for the
expression of IL2 and iNOS in the spleen in all groups, but especially in the EF group, suggesting
theactionofEF55inthedirectionofTh1polarization.IL18showedthehighestexpression,butit
wasnotsignificantintheSEgroupfirstcollectioncomparedtoothergroups. Overall,thehighest
expression of IFN and LITAF in the spleen was observed in the control group at I. collection
comparedtoothergroups.WeobservednonsignificantincreaseinexpressionTGF4inC,SEand
combined SE+EF groups in IV. collection (the fourth day after infection) compared to other
collection. LyAct expression in the spleen peaked at C, EF and combined SE+EF groups in V.
collection and in SE group in IV. collection. However, the highest level of expression among all
evaluated cytokines in the spleen reached IL15 in I. sampling in SE, combined SE+ EF and EF
groupscomparedtothecontrol.
DISCUSSIONANDCONCLUSIONS
Cytokines are essential effector molecules which initiate and coordinate cellular and humoral
immune response against pathogens. Specifically, the increased mRNA expression and protein
secretion of chemokines, proinflammatory and Th1 cytokines such asIFN, IL1, IL6, IL8, IL12
and MIP1 are observed following infection with various Salmonella species (3). The current
results demonstrate the modulation of cytokines in chickens after administration of E.faecium
EF55 and challenged with S. Enteritidis SE147. IL18 is produced at high levels in the liver by
macrophages and Kupffer cells leading to induction of IFN and a Th1type response (18). This
cytokne is the most important growth factor for avian CD4+cells and enhances the cytotoxic
activity of NK cells (7). IL15 is indispensable for longterm survival of memory CD8 + T cells and
enhancestheproliferationofcytotoxicandhelperTcellsdirectedheterophilsandNKcellstothe
47
site of inflammation (11). Chickens infected with S. Enteritidis SE147 in current trial showed the
highestincreaseoftheexpressionofIL18andIL15cytokinesonthefirstdayafterinfection,that
confirming the activation of specific immune components. However, further sampling showed
declined level of expression of IL18. Modulation of these cytokines suggests the stimulatory
actionofE.faeciumEF55inthedirectionofTh1immuneresponses.Severalauthorsdemonstrate
in their work suppressive effect of some probiotic strains to produce LITAF host immune cells
(4,13,17).SimilareffectofE.faeciumEF55onproductionof LITAFinspleenwasfoundinthefirst
dayafterinfectioningroupSE+EF.Macrophagesandothereffectorscellsexpressinduciblenitric
oxidesynthase(iNOS).Thissynthaseisanintegralpartofthehostdefensemechanisms.Synthases
activate the nitric oxide (NO), which is typical of their strong bacteriostatic effect against
intracellular bacteria(16). In mice infected with S. Typhimurium increased level of NO was found
in serum (9), similar to in vitro condition chickens macrophages infected with Salmonella
accounted iNOS enzyme important role in the defense against Salmonella (19). In current
experiment,weobservedthehighestbutnonsignificantexpressionmRNAofiNOSintheEFgroup
inthespleen(firstdayp.i),suggestingthattheactionofE.faeciumEF55isinthedirectionofTh1
polarization.
The application of probiotic strain E. faecium EF55 showed immunomodulatory effect in the
selected organ (spleen) of chickens with salmonellosis. The beneficial effect of probiotic bacteria
was manifested by higher level of proinflammatory cytokines IL15, IL18, LITAF and chemokine
LyTact in spleen of S. Enteritidis infected group. Finally, the results suggest that preventive
administrationofE.faeciumEF55canmodulateimmuneresponseofcytokinesandchemokines.
ACKNOWLEDGMENTS
ThisworkwassupportedbytheSlovakResearchandDevelopmentAgencyunderthecontractNo.
LPP021909.
REFERENCES
1. Berndt, A., Wilhelm, A., Jugert, C., Pieper, J., Sachse, K., Methner, U. 2007. Chicken cecum immune
response to Salmonella enterica serovars of different levels of invasiveness. Infection and Immunity. 75,
59936007.
2. Blaschke S., Brandt P., Wessels J.T., Mller G.A. 2009 Expression and function of the Cclass chemokine
lymphotactin(XCL1)inWegenersgranulomatosis.JRheumatol.24912500.
3.CheesemanJ.H.,KaiserM.G.,CiraciC.,KaiserP.,LamontS.J.2007.BreedeffectonearlycytokinemRNA
expresioninspleenandcecumofchickenwithandwithoutSalmonellaEnteritidisinfection.Dev.andComp.
Immunology.31:5260.
4.ChristensenH.R,FrkirH.,PestkaJ.J.2002.Lactobacillidifferentiallymodulateexpressionofcytokines
andmaturationsurfacemarkersinmurinedendriticcells.J.Immunol.171178.
5.CrhanovaM.,HradeckaH.,FaldynovaM., MatulovaM.,HavlickovaH.,SisakF., Rychlik I.2011.Immune
Response of Chicken Gut to Natural Colonization by Gut Microflora and to Salmonella enterica serovar
EnteritidisInfectionInfect.Immun.7:27552763
6. De Boever S., Vangestel C., De Backer P., Croubels S.U. 2008. Identification and validation of
housekeeping genes as internal control for gene expression in an intravenous LPS inflammation model in
chickens.VeterinaryImmunologyandImmunopathology.122:312317.
7.Hamid,R.,Haghighi,R.,Darab,A.,James,R.,Chambersc,andSharifa,S.2008.Cytokinegeneexpression
in chicken cecal tonsils following treatment with probiotics and Salmonella infection. Veterinary
Microbiology.126,225233.
8.KinoshitaM.,MiyazakiH.,OnoS.,InatsuA.,NakashimaH.,TsujimotoH.,ShinomiyaN.,SaitohD.,Seki.S.
2011. Enhancement of neutrophil function by interleukin18 therapy protects burninjured mice from
methicillinresistantStaphylococcusaureus.InfectImmun.26702680.
48
9. Koebernick H., Grode L., David J.R., Rohde W., Rolph M. S.,. Mittrcker H.W, Kaufmann S. H. 2002.
Macrophage migration inhibitory factor (MIF) plays a pivotal role in immunity against Salmonella
Typhimurium.PNAS.1368113686.
10. Kolesarova M., Spisakova V., Matulova M., Crhanova M., Sisak F., Rychlik I. 2011. Characterisation of
basalexpressionofselectedcytokinesintheliver,spleen,andrespiratory,reproductiveandintestinaltract
ofhens.VeterinarniMedicina,7:325332.
11.LillehojH.S.,MinW.,ChoiK.D.,BabuU.S.,BurnsideJ.,MiyamotoT.,RosenthalB.M.,LillehojE.P.2001.
Molecular, cellular, and functional characterization of chicken cytokines homologous to mammalian IL15
andIL2.VetImmunolImmunopathol.22944.
12. Mead G.C. 2000. Prospects for competitive exclusion treatment to control salmonellas and other
foodbornepathogens.PoultryVet.Journal.159:111123.
13. Pea J.A., Rogers A.B., Ge Z., Ng V., Li S.Y., Fox J.G., Versalovic J. 2005. Probiotic Lactobacillus spp.
diminish Helicobacter paticus induced inflammatory bowel disease in interleukin10deficient mice. Infect
Immun.912920.
14.RychlikI.,KarasovaD.,SebkovaA.,VolfJ.,SisakF.,HavlickovaH.,KummerV.,ImreA.,SzmolkaA.,Nagy.
B. 2009. Virulence potential of five major pathogenicity islands (SPI1 to SPI5) of Salmonella enterica
serovarEnteritidisforchickens.BMCMicrobiology.9:268.
15. Swaggerty CL, Kogut MH, Ferro PJ, Rothwell L, Pevzner IY, Kaiser P (2004): Differential cytokine mRNA
expression in heterophils isolated from Salmonellaresistant and susceptible chickens. Immunology 113,
139148.
16.ThaxtonJ.P.,CutlerS.A.,GriffithR.,ScanesC.G.2006.Changesintissuenitriteconcentrationinthecrop
oftheturkeypoultinresponsetoSalmonellatyphimuriumchallenge.PoultSci.10151019.
17. Thomas C. M., Hong T., Van Pijkeren J. P., Hemarajata P., Trinh D. V, Hu W., Britton R. A., Kalkum M.,
VersalovicJ.2012.HistaminederivedfromprobioticLactobacillusreuterisuppressesTNFviamodulationof
PKAandERKsignaling.PLoSone.2.
18. Wigley P. and Kaiser P. 2003. Avian Cytokines in Health and Disease. Brazilian Journal of Poultry
Science.5:114.
19. Withanage G.S., Mastroeni P., Brooks H.J., Maskell D.J., McConnell I. 2005. Oxidative and nitrosative
responsesofthechickenmacrophagecelllineMQNCSUtoexperimentalSalmonellainfection.BrPoultSci.
261267.

49
Cytokinesignalinginsplenicleukocytesofvaccinatedandnaivechickens
afterintravenousinfectionwithSalmonellaEnteritidis
MartaMATULOVA,HanaSTEPANOVA,KamilaKYROVA,FrantisekSISAKandIvanRYCHLIK
VeterinaryResearchInstitute,BacteriologyDepartment,BRNO,CzechRepublic
INTRODUCTION
InordertodesignanewSalmonellavaccineforchickens,itisnecessarytounderstandhownaive
and vaccinated chickens respond to S.enterica infection. Salmonella infection of chicken results
into intestine colonisation which is associated with heterophil, macrophage and Tlymphocyte
infiltration[1,4]andproductionofcytokines,e.g.IL1,IL6,IL8,IL17,IL22andIFN[3].Alower
level of cellular infiltrate and cytokine expression were commonly observed in the tissues of
chickensthathadbeenvaccinatedpriortotheinfectionthaninthoseexposedtotheinfectionfor
thefirsttime[2,5].
Since cytokine signaling is usually determined by real time PCR using cDNA from whole tissue,
information about the contribution of individual cellular subpopulations in chickens is not
available.Inthisstudywethereforedeterminedtheimmuneresponseofleukocytessortedfrom
thespleenofnonvaccinatedandvaccinatedchickensafterintravenousinfectionwithSalmonella
entericaserovarEnteritidis(S.Enteritidis).
MATERIALANDMETHODS
ISA Brown chickens were vaccinated on day 1 and revaccinated on day 21 of their life with
S.Enteritidis 147 SPI1. Naive and vaccinated groups of chickens were intravenously challenged
with wild type S.Enteritidis 147 on day 42 and sacrificed 4 days later along with the non
vaccinated noninfected group. Cells from chicken spleens were sorted on FACSAria instrument
(BD Biosciences) using antiCD45:APC, antiCD4:FITC, antiCD8a:SPRD, antiTCR1:PE, anti
monocyte/macrophage:FITCandantiBu1:PEantibodies(allfromSouthernBiotech).
RNAfromthecellpopulationswasisolatedwithTRIReagent(MRC),purifiedwithRNeasyMiniKit
(Qiagen) and reverse transcribed into cDNA using MMLV reverse transcriptase (Invitrogen). Real
time PCR for IL1, Il6, IL8, IL17, IL18, IL22, LITAF, IFN and iNOS was performed with
LightCyclerII(Roche)usingQuantiTectSYBRGreenPCRMasterMix(Qiagen)andCtvaluesofthe
genes of interest were normalized to Ct values of glyceraldehyde 3phosphate dehydrogenase,
TATAbindingproteinandubiquitin.
RESULTS
Four days post infection (DPI), counts of CD4 and Blymphocytes did not change, CD8 and T
lymphocytes decreased and macrophages and heterophils increased in the spleen (Fig.1). When
vaccinatedandnonvaccinatedchickenswerecompared,onlymacrophagesandheterophilswere
found in significantly higher counts in the spleens of the nonvaccinated chickens. The non
vaccinated chickens also expressedhigher antiLPS antibodies than the vaccinated chickens (data
notshown).
TheexpressionofinterleukinIL1,IL6,IL8,IL18,LITAF,IFNandiNOSdidnotexhibitanyclear
pattern in the cells sorted from the spleens of vaccinated or nonvaccinated chickens. Only IL17
and IL22 showed a differential expression in the CD4 Tlymphocytes of the vaccinated and non
vaccinated chickens at 4 DPI, both being expressed at a higher level in the nonvaccinated but
infectedchickens(Fig.2).

50
DISCUSSION
Due to a similar IFN expression in the CD4 Tlymphocytes in both the vaccinated and non
vaccinatedchickens,andavariableIL17expressionoscillatingaroundIFNexpressionlevels,the
IL17:IFN ratio in CD4 Tlymphocytes was found to be central for the outcome of the immune
response(Fig.3).
When IL17 was expressed at higher levels than IFN in the nonvaccinated chickens, the Th17
immune response with a higher macrophage and heterophil infiltration in the spleen dominated.
However,whentheexpressionofIL17waslowerthanthatofIFNasinthevaccinatedchickens,
theTh1responsewithahigherresistancetoS.Enteritidisinfectiondominated.
REFERENCES
1. Berndt A, Methner U (2001) Gamma/delta T cell response of chickens after oral administration of
attenuatedandnonattenuatedSalmonellaTyphimuriumstrains.VetImmunolImmunopathol78:14361.
2. Carvajal BG, Methner U, Pieper J, Berndt A (2008) Effects of Salmonella enterica serovar Enteritidis on
cellularrecruitmentandcytokinegeneexpressionincaecumofvaccinatedchickens.Vaccine26:542333.
3. Santos RL, Raffatellu M, Bevins CL, Adams LG, Tukel C, et al. (2009) Life in the inflamed intestine,
Salmonellastyle.TrendsMicrobiol17:498506.
4.vanDijkA,TersteegZijderveldMH,TjeerdsmavanBokhovenJL,JansmanAJ,VeldhuizenEJ,etal.(2009)
Chicken heterophils are recruited to the site of Salmonella infection and release antibacterial mature
cathelicidin2uponstimulationwithLPS.MolImmunol46:151726.
5. Withanage GS, Wigley P, Kaiser P, Mastroeni P, Brooks H, et al. (2005) Cytokine and chemokine
responsesassociatedwithclearanceofaprimary
Salmonella enterica serovar Typhimurium infection in the chicken and in protective immunity to
rechallenge.InfectImmun73:517382.
TABLESANDFIGURES
Figure 1 Macrophage and heterophil increase in the spleen of nonvaccinated and vaccinated chickens
after infection with Salmonella Enteritidis. nonvac nonvaccinated, vac vaccinated, inf infected
(challenged),ninoninfected

51
Figure 2 Expression of IFN, IL17 and IL22 in CD4 Tlymphocytes after infection with Salmonella
Enteritidis.nonvacnonvaccinated,vacvaccinated,infinfected(challenged),ninoninfected
Figure 3 IL17/IFN expression ratio in CD4 Tlymphocytes from spleen of nonvaccinated and vaccinated
chickensafterinfectionwithSalmonellaEnteritidis.

52
VirulenceGeneProfilingandPathogenicityCharacterization
ofNonTyphoidalSalmonellaAccounted
forInvasiveDiseaseinHumans
JothamSUEZ
1,2
,SteffenPORWOLLIK
3
,AmirDAGAN
1
,AlexMARZEL
1,2
,YosefILANSCHORR
4
,
PrerakTDESAI
3
,VeredAGMON
4
,MichaelMCCLELLAND
3,5
,GaliaRAHAV
1,2
andOhadGALMOR
1
1
TheInfectiousDiseasesResearchLaboratory,ShebaMedicalCenter,
TELHASHOMER,Israel;
2
SacklerFacultyofMedicine,TELAVIVUniversity,Israel;
3
TheVaccineResearchInstituteofSanDiego,SANDIEGO,California,USA;
4
GovernmentCentralLaboratories,MinistryofHealth,JERUSALEM,Israel;5Department
ofPathologyandLaboratoryMedicine,UniversityofCaliforniaIrvine,IRVINE,California,USA
Human infection with nontyphoidal Salmonella serovars (NTS) infrequently causes invasive
systemic disease and bacteremia. To understand better the nature of invasive NTS (iNTS), we
studied the gene content and the pathogenicity of bacteremic strains from twelve serovars
(Typhimurium, Enteritidis, Choleraesuis, Dublin, Virchow, Newport, Bredeney, Heidelberg,
Montevideo, Schwarzengrund, 9,12:l,v: and Hadar). Comparative genomic hybridization using a
Salmonella enterica microarray revealed a core of 3233 genes present in all of the iNTS strains,
which include the Salmonella pathogenicity islands 15, 9, 13, 14; five fimbrial operons (bcf, csg,
stb,sth,sti);threecolonizationfactors(misL,bapA,sinH);andtheinvasiongene,pagN.IntheiNTS
variable genome, we identified 16 novel genomic islets; various NTS virulence factors; and six
typhoidassociated virulence genes (tcfA, cdtB, hlyE, taiA, STY1413, STY1360), displaying a wider
distribution among NTS than was previously known. Characterization ofthe bacteremic strains in
C3H/HeN mice showed clear differences in disease manifestation. Previously unreported
characterization of serovars Schwarzengrund, 9,12:l,v:, Bredeney and Virchow in the mouse
model showed low ability to elicit systemic disease, but a profound and elongated shedding of
serovarsSchwarzengrund,9,12:l,v:(aswellasEnteritidisandHeidelberg)duetochronicinfection
of the mouse. Phenotypic comparison in macrophages and epithelial cell lines demonstrated a
remarkableintraserovarvariation,butalso showedthatS. Typhimuriumbacteremicstrainstend
to present lower intracellular growth than gastroenteritis isolates. Collectively, our data
demonstrated a common core of virulence genes, which might be required for invasive
salmonellosis,butalsoanimpressivedegreeofgeneticandphenotypicheterogeneity,highlighting
that bacteremia is a complex phenotype, which cannot be attributed merely to an enhanced
invasionorintracellulargrowthofaparticularstrain.

53
CellularimmuneresponsebyCD4+TcellsinWhiteLayerhens
vaccinatedwithliveandkilledSalmonellavaccines
andinfectedwithSalmonellaEnteritidis
RafaelANTONIOCASARINPENHAFILHO,AdrianaMARIADEALMEIDA,BrunaSILVAMOURA,
andAngeloBERCHIERIJr.
UniversidadeEstadualPaulistaJliodeMesquitaFilho,DepartmentofVeterinaryPathology,
LaboratoryofAvianPathology,JaboticabalCampus,14884900,SP,Brazil
INTRODUCTION
Vaccination of chickens has become common in countries with industrial poultry production to
prevent the contamination of meat and eggs with Salmonella enterica subsp. enterica serovar
Enteritidis (SE) and others Salmonella enterica serovars (Barrow, 2007). CD4+ T Lymphocytes are
important cells for the control of the immune response, specially by the production of IFN and
ChemokineCLigand4(CCL4),cytokinesinvolvedintheactivationandattractionofmacrophages,
respectively(Mizunoetal.,2003).
MATHERIALANDMETHODS
Experimentalbirds
One hundred and twenty white layerhens, susceptible to SE infection, were obtained at day of
hatchandsubmittedtobacteriologicalandserologicaltestsinordertochecktheSalmonellafree
status. All birds used tested negative for Salmonella infection. Birds were reared in controlled
ambientconditions.
Bacterialstrainsandvaccines
A bacterin in oil emulsion containing SE phagotype (PT) 4, PT8 and PT13a antigens, was
administered subcutaneously in the nape (0,3mL/bird) as the killed vaccine (KV). An attenuated
mutantSGstrain,withdeletionongenescobSandcbiA,unabletosynthesizecianocobalaminand
immunogenic against SE, was used as the live vaccine strain (LV). An invasive SE PT4 strain was
used to challenge birds. Bacterial cultures were prepared in LuriaBertani (LB) broth (Invitrogen,
USA)at100rpmat37C/24h.TheLVandSEchallengeinoculaconsistedof108CFUinPhosphate
Buffer Saline (PBS) pH 7.4 (Merck, Germany), administered orally, into the crop. Vaccination was
carried
for IFN F5AGCTGACGGT GGACCTATTATT3 and R5GGCTTT GCGCTGGATTC3. The real time
PCRwascarriedoutaccordingtomethodsdescribedin(HONGetal.,2006).
RESULTS
ThehighernumbersofCD4+TcellsinliversampleswerenoticedingroupLV+KV,whileincaecal
tonsil samples this population was seen in higher numbers in group LV+LV at 6 dpi. An influx of
CD4+Tcellswasseenintheunvaccinatedgroupat1dpi,howeveritwasseverelydiminishedat6
and9dpi(Figure1).ProductionofIFNgammawassignificantlyhigheringroupsLV+KVandLV+LV
at1dbiincaecaltonsils.ExpressionofCCL4wassignificantlyhigherintheunvaccinatedgroupat9
dpi.
54
1
d
b
i
1
d
p
i
6
d
p
i
9
d
p
i
1
d
b
i
1
d
p
i
6
d
p
i
9
d
p
i
1
d
b
i
1
d
p
i
6
d
p
i
9
d
p
i
1
d
b
i
1
d
p
i
6
d
p
i
9
d
p
i
1
d
b
i
1
d
p
i
6
d
p
i
9
d
p
i
0
5
10
15
20
LIVER
b
b
a a
a
a a
a
a
a
a
a
a
a
a
a
a
a
%

R
E
A
1

d
b
i
1

d
p
i
6

d
p
i
9

d
p
i
1

d
b
i
1

d
p
i
6

d
p
i
9

d
p
i
1

d
b
i
1

d
p
i
6

d
p
i
9

d
p
i
1

d
b
i
1

d
p
i
6

d
p
i
9

d
p
i
1

d
b
i
1

d
p
i
6

d
p
i
9

d
p
i
0
10
20
30
40
50 unvaccinated (A)
LV (B)
LV +LV (C)
KV (D)
LV +KV (E)
CAECAL TONSIL
a
a
a
a
a
a
b
c
c
b
a
b
a
c
a
a
a
b
c
b
%

R
E
A

Figure1.ImmunohistochemistrystainingofCD4+Tcellsinliverandcaecaltonsilofunvaccinated(groupA)
andvaccinatedchickens(groupsB,C,DandE),before
(1dbi)andafterchallenge(1,6and9dpi).ColumnsrepresentthemeanpercentageofstainedareasSD.
*, p < 0.05; ns, not significative. Different letters on columns from the same moment indicate statistically
differentresults(p<0.05).
Table1.VaccineschemestestedagainstS.Enteritidis
Groups Number
ofbirds
1
st
dose
(5days)
2
nd
dose
(25days)
A 20 unvaccinated
B
C
D
20
20
20
20
LV
LV
LV
LV
KV
KV E
LV:livevaccine (0.5mLviaoral); KV:killedvaccine(0.3mLsubcutaneous).At45days ofage,allbirdsinall
groupswerechallengedwithSE.
LV:livevaccine (0.5mLviaoral); KV:killedvaccine(0.3mLsubcutaneous).At45days ofage,allbirdsinall

groupswerechallengedwithSE.
DISCUSSION
CD4+Tlymphocytesweredetectedinallgroupsinliversamplesandincaecaltonsils.CD4+Tcells
can be divided in three subsets T helper 1 (secreting mainly IFN) that stimulates the cellular
immuneresponse,accordingtotheschemesdescribedinTable1.
Immunohistochemistry
Immunohistochemistry was used to determine the influx of CD4+ T cells as described previously
Carvajaletal., 2008). Briefly, frozen tissue sections (8m) of liver andcaecal tonsil samples were
fixed in icecold acetone. Sections were incubated overnight at 4C with antichicken CD4+
antibody(5ng/mL;SouthernBiotech,USA).ReactionwasdevelopedwithEnvisionHRPKitand3,3
diaminobenzidine (DAB; Dako, USA). Tissue sections were randomly photographed in light
microscope (Eclipse Moticam, Nikon, Japan). The percentage of positively stained areas was
analysedusingImageProPlusSoftware(MediaCybernetics,USA).

55
QuantificationofmRNAbyRTqPCRinrealtime
For the quantification of mRNA for the expression of CCL4 (ortholog to MIP1 in mammals) the
primersusedwereF5GTGCCCTCATGCTGGTGT3andR5GGTTGGATGCGGATTATTTC3,and
T helper 2(secretes IL4 and IL13) that develops the humoral immune response and T helper 17
(Treg) that are not effector cells. The increased expression of IFN (Th1) in caecal tonsils from
groupsLV+LVandLV+KVindicatesthedevelopmentofastrongcellularimmuneresponseinthese
groups which could be associated with the good efficacy of these vaccine schemes to control SE
shownelsewhere(PenhaFilhoIetal.,2012).
CONCLUSION
This experiment showed that IFNgamma is stimulated by vaccination which could contribute to
the controlof SE, activating the CD4+ T cells. CCL4 could be associated with the innate immunity
onceitwasnotsignificantlyexpressedinvaccinatedbirds.
ACKNOWLEDGEMENTS
TheauthorsthankFAPESP,CAPESandCNPqfortheconstantsupportforourresearch.
REFERENCES
1. Carvajal,B.G.;Methner,U.;Pieper,J.; Berndt,A. EffectsofSalmonella entericaserovarEnteritidis
on cellular recruitment and cytokine gene expression in caecum of vaccinated chickens. Vaccine, v. 26, n.
42,p.542333,2008.
2. Hong, Y. H.; Lillehoj, H. S.; Lee, S. H.; Dalloul, R. A.; LillehojI, E. P. Analysis of chicken cytokine and
chemokine gene expression following Eimeria acervulina and Eimeria tenella infections. Veterinary
immunologyandimmunopathology,v.114,n.34,p.20923,2006
3. Mizuno,Y.;Takada,H.;Nomura,A.;Jin,C.H.;HattoriI,H.;Ihara,K.;Aoki,T.;Eguchi,K.;Hara,T.Th1
and Th1inducing cytokines in Salmonella infection. Clinical and experimental immunology, v. 131, n. 1, p.
1117,2003.
4. PenhaFilho,R.A.;Moura,B.S.;DeAlmeida,A.M.;Montassier,H.J.;Barrow,P.A.;BerchieriJr,A.
Humoral and cellular immune response generated by different vaccine programs. Vaccine, v. 30, n. 52, p.
763743,2012

56
EfficacyofdifferentvaccineprogramsagainstFowlTyphoid
usingaliveattenuatedstrainofSalmonellaGallinarum
asavaccinecandidate
FbioTAVARESZANCAN,PriscilaDINIZLOPES,AdrianaMARIADEALMEIDA,
RafaelANTONIOCASARINPENHAFILHOandAngeloBERCHIERIJr.
UniversidadeEstadualPaulistaJliodeMesquitaFilho,DepartmentofVeterinaryPathology,
LaboratoryofAvianPathology,JaboticabalCampus,14884900,SP,Brazil
INTRODUCTION
Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum (SG) causes Fowl
Typhoidincommercialchickens(Shivaprasad,2000).FowlTyphoidisadiseaseofconcernforthe
poultry industry because it causes high morbidity and mortality within infected flocks. In many
countries, SG isolation in layers and breeders demands the sanitary slaughter of the flock to
control the disease. Vaccination is an important control measure in a biosafety program. The
attenuated mutant strain of SG prepared in our laboratory (SGcobScbiA) has proven efficacy
against Fowl Typhoid. However, this study was done to evaluate the long term protection after
immunizationwiththeSGmutant.
MATERIALSANDMETHODS
Twohundredandtenfemalebrownlayerhens(semiheavyline)wereseparatedinto7groupsof
thirtybirdseach.ThevaccinationingroupsA,BandCwasdonewithtwodosesat4and8weeks
of age (w). In groups E and F three doses were administered at 4, 8 and 12w. The vaccine was
inoculated by the oral, subcutaneous (SC) or intramuscular (IM) routes. The vaccine programs
were the following: Group A (oral+oral), Group B (SC+oral), Group C (oral+IM), Group D
(unvaccinated), Group E (oral+oral+oral), Group F (SC+oral+oral), Group G (unvaccinated).
Chickens in groups A, B, C and D were challenged with wild SG strain at 16w and chickens in
groups E, F and G were challenged at 20w. The mortality was followed through 30 days post
infection (dpi) in all groups. The number of laid eggs was recorded in groups E, F and G thorugh
outtheexperiment.
RESULTS
GroupsA,BandC,showedmortalityrateof3,33%whileintheunvacinatedgroupD,themortality
ratereached33,3%(Table1).ThemortalityingroupsEandFthatreceivedthreevaccinedoses
reached 16,67% whilst in the unvaccinated group G 50% of the chickens died, additionally the
number of laid eggs was significantly superior in the vaccinated hens in comparison with
unvaccinatedhens(Table2).
Table1.Mortalityofvaccinated(twodoses)andunvaccinatedbrownlayerhenschallengedat16weeksof
agewith10
8
CFUofSG.
Groups mortality %
A 1/30 3,33
a
B 1/30 3,33
a
C 1/30 3,33
a
D 10/30 33.33
b
%=mortalityrates
Differentlettersindicatestatisticallydifferentresults(p<0.05)

57
Table2.Mortalityofvaccinated(threedoses)andunvaccinatedbrownlayerhenschallengedat20weeksof
agewith10
8
CFUofSG
Groups mortality Eggs
E 5/30 155
a
F 5/30 117
b
G 16/30 39
c
Differentlettersindicatestatisticallydifferentresults(p<0.05)
DISCUSSION
The use of vaccines against fowl typhoid is very important in countries with industrial poultry
production.SGcauseshighmortalityininfectedflocks.TheuseofSGvaccineshasbeenimplicated
in good protection against fowl typhoid in other studies (LEE et al., 2005; PENHA FILHO et al.,
2010).
As shown in our results, the SGcobScbiA was capable to protect layers when used in different
vaccine using both SC, IM and oral route, decreasing the mortality rates in all vaccinated groups.
Moreover, the live vaccine contributed to the higher egg production in vaccinated layers in
comparisonwiththeunvaccinatedchickens.
CONCLUSION
Vaccination with SGcobScbiA is safe for use in brown layer hens and it is capable to protect
chickensagainstFowlTyphoidduringcriticallifeperiodsastheonsetofthesexualmaturity.
ACKNOWLEDGEMENTS
TheauthorsthankFAPESP,CAPESandCNPqfortheconstantsupportforourresearch.
REFERENCES
1. Lee,Y.J.;Mo,I.P.;Kang,M.S.SafetyandefficacyofSalmonellaGallinarum9Rvaccineinyounglaying
chickens.AvianPathol,v.34,n.4,p.3626,2005.
2. Penha Filho, R. A.; De Paiva, J. B.; Da Silva, M. D.; De Almeida, A. M.; Berchieri, A., Jr. Control of
Salmonella Enteritidis and Salmonella Gallinarum in birds by using live vaccine candidate containing
attenuatedSalmonellaGallinarummutantstrain.Vaccine,v.28,n.16,p.28539,2010.
3. Shivaprasad,H.L.Fowltyphoidandpullorumdisease.RevSciTech,v.19,n.2,p.40524,2000.

58
EvaluationofprotectionprovidedbyaninactivatedSalmonellavaccine
inpointoflaychickenschallenged
withmonophasicSalmonellaTyphimuriumstrains
FrancescaMARTELLI,RebeccaGOSLING,EmmaKENNEDY,AndrRABIE,MarkBRESLIN,
RobDAVIESandRobertoLARAGIONE
BacteriologyandFoodSafetyDepartmentAHVLAWEYBRIDGE,UnitedKingdom
INTRODUCTION
In order to reduce the prevalence of Salmonella in poultry and thus the potential for human
infection, vaccination of poultry flocks against Salmonella Enteritidis (SE) has been used in the UK
sincethemid1990sandSalmonellaTyphimurium(ST)vaccinationwasaddedformanyflocksduring
the 2000s. This is likely to have contributed to the decline in SE infections in humans from 1998/9
whenSEvaccinationoflayingflocksbecamewidelyadopted.AsSEandSTareconsideredtobemost
important for public health in Europe, existing commercially available live and inactivated
Salmonella vaccines for poultry are intended for use against one or both of these serovars.
Vaccinationisusedtopreventsystemicinfection(andlocalizationinthereproductivetract)andto
reduce faecal shedding (and consequently carcass and/or egg contamination). Both live and killed
vaccines are available to vaccinate laying flocks (3). The Member States and the European
CommissionhaveagreedcriteriatocontrolSEandSTinfectionsinlayingflocks,toreducetheriskof
contaminated eggs entering the food chain. Monophasic (lacking the second phase flagellar
antigens) variants of ST (mST) are included in this legislation. Currently used vaccination
programmesarelicensedforuseagainstbiphasicvariantsofST,andtheirefficacyagainstmSThas
notbeenyetinvestigated.ItislikelythatSTvaccineswouldhaveasimilarprotectiveeffectformST
as for ST, however there are no data available concerning the efficacy of current vaccination
programmes. The aim of this study was to assess the protection provided by an inactivated SE/ST
vaccineinpointoflaychickenschallengedwithtwomSTstrains(one4,5,12:i:andone4,12:i:),one
STDT8strainandoneSEPT14bstrain.
MATERIALANDMETHODS
FourSalmonellastrainswereusedinthisstudy.StrainonewasSTDT8,originallyisolatedintheUK
from a flock of laying ducks that produced eggs for human consumption and was linked to human
outbreaks(5).StrainstwoandthreeweretetraresistantmST4,12:i:and4,5,12:i:DT193strainsof
UK origin. Strain four was a nalidixic acid resistant egg invasive SE phage type (PT) 14b strain that
spreadfrommainlandEuropetotheUKthroughcontaminatedeggs(4).
Two hundred and forty commercial Hyline layer chickens were sourced at day old. During the
rearingperiod,120birdsreceivedintramuscularlytwodosesofacommercialinactivatedSEPT4and
ST DT 104 vaccine. The other half of the birds remained unvaccinated against Salmonella. Before
challenge, the birds were randomly assigned to 8 groups of 30 birds, 4 vaccinated and 4
unvaccinated.Thechallengestrainwasadministeredbyoralgavagewhenthebirdswere20weeks
of age (point of lay). Each bird received 1 ml of approximately 5 x 10
8
cfu/ml of the Salmonella
challengestrain.Eachstrainwasadministeredtoagroupofvaccinatedandagroupofunvaccinated
birds.Afterchallenge,cloacalswabswereseriallycollectedfromallbirdsineachgrouptoevaluate
levels of excretion of Salmonella. Eggs were collected and tested individually, shells and contents
separately.Theshellswereswabbedandthendisinfectedwith100%ethanolbeforetestingtheegg
contents. The experiment lasted for 27 days after challenge (dpc) and postmortem examinations
(PM)wereperformedon3,7and24and27dpc.AtPM,liver,spleen,ovaryandcaecaofeachbird
were aseptically sampled. Cloacal swabs and tissues collected at PM were direct plated onto
Rambach agar for enumeration, and then subjected to pre enrichment in Buffered Peptone Water
59
(BPW).ThesamplesinBPWwereincubatedat37Cfor1620hoursand0.1mlofincubatedbroth
was inoculated onto modified semisolid Rapaport Vassiliadis medium (MRSV) and incubated at
41.5C.FromtheMSRVplatea0.1mlloopwastaken,andstreakedontoRambachagar.Inoculated
plates were incubated at 37C for 24 hours, and presumptive Salmonella colonies were confirmed
serologically.
RESULTS
AllSalmonellastrainswereabletosuccessfullycolonisethebirds,andallgroupsapartfromtheone
challenged with ST DT8 were still shedding Salmonella in their faeces at 21 dpc. Salmonella was
readilyisolatedfromtissuesatallPMexaminations,inbothvaccinatedandunvaccinatedgroups.In
the last two PMs, no positives could be detected by direct plating, but all the groups had positive
tissuesafterenrichment.Salmonellawasrecoveredfromeggshellsinallgroups,andfromoneegg
contentinthegroupchallengedwiththe4,12:i:strain;thereweresignificantdifferencesbetween
the strains (p=0.023) and the effect of the vaccine was nearly significant (p=0.070). The birds
challenged with ST DT8 produced a significantly lower number of contaminated eggshells.
Vaccinated birds tended to have a lower percentage of positive egg shells and lower Salmonella
countsatPM.Thetotalnumberofeggslaidwasreduced,butnotstatisticallysignificantlyaffected
bychallengestrainorpresenceorabsenceofvaccination,althoughtherewasatemporarydropin
eggproductioninmostnonvaccinatedgroupsafterchallenge.Table1summarizesthepercentage
of positive samples (PM and cloacal swabs combined and eggs) in each group. The unvaccinated
groupsalwayshadahigherproportionofpositivesampleswhencomparedtothevaccinatedgroup
challengedwiththesamestrain,butthisdifferencewasseldomstatisticallysignificant.ThetwomST
strains and the SE strain produced similar proportions of positive samples. The groups challenged
withSTDT8hadalowerproportionofpositivecloacalswabs,eggshellsandpositivebirdsatPM.
DISCUSSION
High dose challenge of laying hens with nonhost restricted Salmonella serovars causes systemic
infection, normally in absence of clinical signs. Salmonella is isolated from tissues and shed in the
faeces for variable periods of time, depending on serovar and strain. Whilst little information is
available on the invasiveness of mST strains, several studies have reported a high degree of
colonizationandsheddinginlayinghenscausedbySEandST(7).Vaccinationisnotabletoprevent
tissueinvasion,faecalsheddingoreggcontaminationinthepresenceofhighSalmonellachallenge,
butcanreducethelevelofshedding(1).Inthisstudy,contaminatedeggshellswereidentifiedboth
invaccinatedandunvaccinatedgroups,withthevaccinatedflocksproducingalowerproportionof
contaminatedeggshells.Thisconfirmspreviousobservationscarriedoutinfarmbasedstudies(2).In
this study, mST strains gave higher levels of tissue invasion, eggshell contamination and faecal
sheddingthananSTDT8strain.Thiscouldbeduetointrinsicdifferencesbetweenthestrainsused,
or to the fact that mST strains may be more effective in evading the hosts immune response, as
thereisaconsiderabledegreeofvariabilityinthelevelofinvasivenessbetweenSTstrains(6).TheST
DT8strainwaschosenbecauseoftheoccurrenceofDT8infectioninsomelayinghenflockswhere
therewerealsoducksonsiteandtheknownegginvasiveofthisstrainonthefarmoforigin,butitis
possiblethattheparticularstrainmayhavebeenlesswelladaptedtochickensthantoducks.
Some level of protection was provided in this study by a killed ST/SE vaccine in point of lay hens
challenged with high doses of mST strains. Vaccinal protection against mST was similar to that
provided against SE and ST strains. Further studies will be necessary to clarify the effectiveness of
vaccinationinpreventingtheintroductionofinfectionintocommerciallayingflocksinthepresence
ofthelowerdosesofmSTchallengethatwouldbeexpectedinafieldsituation.

60
ACKNOWLEDGEMENTS
REFERENCES
1. CliftonHadley, F. A., M. Breslin, L. M. Venables, K. A. Sprigings, S. W. Cooles, S. Houghton, and M. J.
Woodward.2002.AlaboratorystudyofaninactivatedbivalentironrestrictedSalmonellaentericaserovars
EnteritidisandTyphimuriumdualvaccineagainstTyphimuriumchallengeinchickens.VetMicrobiol89:167
179.
2. Davies, R., and M. Breslin. 2004. Observations on Salmonella contamination of eggs from infected
commerciallayingflockswherevaccinationforSalmonellaentericaserovarEnteritidishadbeenused.Avian
Pathol33:133144.
3. EFSA.2004.TheuseofvaccinesforthecontrolofSalmonellainpoultry.EFSAJournal114:174.
4. Janmohamed, K., D. Zenner, C. Little, C. Lane, J. Wain, A. Charlett, B. Adak, and D. Morgan. 2011.
NationaloutbreakofSalmonellaEnteritidisphagetype14binEngland,SeptembertoDecember2009:case
controlstudy.EuroSurveill16.
5. Noble,D.J.,C.Lane,C.L.Little,R.Davies,E.DePinna,L.Larkin,andD.Morgan.2012.Revivalofanold
problem:anincreaseinSalmonellaentericaserovarTyphimuriumdefinitivephagetype8infectionsin2010
inEnglandandNorthernIrelandlinkedtoduckeggs.EpidemiolInfect140:146149.
6. Rabsch, W., H. L. Andrews, R. A. Kingsley, R. Prager, H. Tschape, L. G. Adams, and A. J. Baumler. 2002.
SalmonellaentericaserotypeTyphimuriumanditshostadaptedvariants.InfectImmun70:22492255.
7. Suzuki,S.1994.PathogenicityofSalmonellaEnteritidisinpoultry.IntJFoodMicrobiol21:89105.
STDT8
Vacc
STDT8
Unvacc
4,12:i:
Vacc
4,12,:i:
Unvacc
4,5,12:i:
Vacc
4,5,12:i:
Unvacc SEVacc
SE
Unvacc
PMandcloacalswabsd/p 16.5% 19.2% 25.5% 27.5% 18.0% 29.8% 20.8% 23.9%
PMandcloacalswabsd/pandenrich 20.7% 21.9% 66.7% 67.4% 55.4% 62.1% 63.5% 67.4%
Eggshells 2.6% 4.5% 40.3% 53.1% 23.8% 57.8% 47.6% 56.4%
Eggcontents 0.0% 0.0% 0.0% 0.3% 0.0% 0.0% 0.0% 0.0%
Totaleggslaid 309 246 352 352 369 301 315 305
Table1:Percentageofpositivesamplesdetectedineachgroup(Vacc=vaccinated;Unvacc=Unvaccinated).
Results after direct plating only (d/p) and cumulative after direct plating and enrichment are presented
separately for cloacal swabs and post mortem samples. Percentages of positives between groups
challengedwiththesamestrainwerecompared,andthegroupwiththehighestpercentageishighlighted
inbold.

61

May 27-28-29, 2013
Saint-Malo France

Session 2


Chairpersons:

Communications
62
Genomicepidemiologicalstudiesoftheglobaloccurrence
ofS.TyphimuriumDT104
PimlapasLEEKITCHAROENPHON
1,2
,EvaM.NIELSEN
3
,andFrankM.AARESTRUP
1
1
NationalFoodInstitute,DivisionforEpidemiologyandMicrobialGenomics,Technical
UniversityofDenmark,KGS.LYNGBY,Denmark;
2
CenterforBiologicalSequenceAnalysis,DepartmentofSystemBiology,TechnicalUniversity
ofDenmark,KGS.LYNGBY,Denmark;
3
DepartmentofMicrobiologicalSurveillanceandResearch,StatensSerumInstitut,
COPENHAGEN,Denmark
ABSTRACT
Salmonella is one of the prevalent infectious pathogens. Salmonella outbreaks continue to emerge in various countries.
To achieve successful monitoring of infectious disease, the reliable and rapid sub-typing is essential. WGS promises to
be used as a routine clinical epidemiology. Here we presented the proof of concept of applying WGS in an outbreak
investigation.

A number of selected WGS typing techniques including pan-genome tree, k-mer tree, and SNP tree have been
evaluated on a collection of well defined thirty-four S. Typhimurium strains from 6 different outbreaks. This data set
consisted of 18 isolates from six well-defined outbreaks and 16 epidemiologically unrelated background strains. The aim
was to determine which sub-typing would be superior for routine clinical outbreak investigation. The results of the three
phylogenetic analysis were also compared to PFGE typing. Among the sub-typing approaches under study, SNP tree
has shown the best resolution as the outbreak isolates are clustered together and distinguish from the background
strains with 100 % concordance.

In conclusion, for S. Typhimurium, SNP analysis of WGS data seem to be the superior method for epidemiological typing
compared to other phylogenetic analytic approaches that may be used on WGS data. Our study also indicated that
without WGS techniques, traditional molecular typing alone is not able to capture very little variation occurring among
outbreak strains meanwhile, the results from WGS alone are not capable to be meaningful without epidemiological data.
Furthermore, The cost and time for WGS will soon be the same or less range as the traditional typing methods. Thus,
WGS is transforming microbiology research and will soon transform the clinical routine diagnostic and public health
microbiology.
INTRODUCTION
Salmonellaisacommoncauseofinfectiousdiseaseinhumanandanimals.Salmonellaisclassically
divided into species S.bongori and S.enterica. The latter can be further defined into more than
2,500differentserotypes[1,2].S.entericaoftencausesminorandmajorfoodborneoutbreaks,for
example, the emerging of multidrugresistant S. Typhimurium DT104 [3], the intracontinental
spread of invasive S. Typhimurium in subSaharan Africa [4] and the recent outbreak of S.
Montevideo associated with contaminated black and red pepper in United States [5]. In Europe,
themostprevalentS.entericaisolatedfromhumansareEnteritidisandTyphimurium,responsible
for over 75% of the human cases of salmonellosis [6]. In United States, there are approximately
1.4millioncasesofSalmonellainfectionsannually[7].
In order to achieve successful and rapid monitoring the epidemiology of infectious disease, the
reliableandrapidsubtypingofbacterialpathogensisanessentialkey[8,9].Traditionalmolecular
typing methods are commonly used as a central part of the detection and investigation of
Salmonella outbreaks, for instance, serotyping, phage typing, pulsefield gel electrophoresis
(PFGE) andmultilocus variable number of tandem repeat analysis (MLVA) [7]. PFGE has beenthe
most golden standard for epidemiological investigations of foodborne bacterial pathogens
including Salmonella [6]. A drawback of PFGE is that it is labor intensive and unable to separate
verycloselyrelatedstrainsbecausethelowrateofgeneticvariationdoesnotsignificantlyimpact
the electrophoretic mobility of a restriction fragment [7]. MLVA has major benefits in
63
epidemiologicalsurveillanceofSalmonella,butitistimeconsumingandtheresultsaredifficultto
standardize[10].
Recently, the advances in whole genome sequencing (WGS) have resulted in the reduction of
economiccostforbacterialgenomesequencing.Moreover,thespeedofsequencingisapproving
from several days or weeks to perhaps hours for a bacterial genome in the near future [11]. The
combinationoflowcostandhighspeedofWGS,itopensanopportunityforWGSbecomingvery
useful and practical in various bacterial infectious studies [4, 12, 13] including the routine use in
diagnostic and public health microbiology [11, 14]. Nevertheless, prior using WGS as a routine
surveillance, WGS typing needs to be evaluated to gain better understanding of analysis,
classificationandclusteringstrainsfromagivingcluster.
TheaimofthisstudywastoevaluateWGStypingtechniquesonacollectionofepidemiologically
related S. Typhimurium outbreak and nonrelated outbreak strains isolated from 6 different
outbreaks [6] in order to elucidate the ability of clustering related isolates and distinguish these
fromnonrelatedones.
MATERIALANDMETHODS
Bacterialisolatesandmoleculartyping
S. Typhimurium strains were derived from the Danish laboratorybased surveillance system of
humangastrointestinalinfectionsin20002010.Bacterialpathogenswereisolatedfromhuman
fecal samples at the diagnostic laboratory at Statens Serum Institut [6]. The procedures for
isolation, identification, serotyping, antimicrobial susceptibility testing, PFGE and MLVA of the
isolatesincludedinthisstudyhavebeendescribedpreviously[6,15].
Wholegenomesequencing
Thirtyfour S. Typhimurium genomes were selected for WGS by Illumina GAIIx genome analyzer
(Illumina,Inc.,SanDiego,CA).Denovoshortreadassemblywasperformedonthesetofrawreads
usingVelvet[16],whichisapartofthepipelineavailableontheCenterforGenomicEpidemiology
(www.genomicepidemiology.org)[17,18].
Pangenometree
Pangenometreewasconstructedfromthepangenomematrixinwhichcomposedofgenesand
genomes(denovoassembledgenomes)asrowsandcolumnsrespectively.Thematrixconsistsof
profileof0sand1srepresentedastheabsenceandpresenceofgenesacrossgenomes.Fromthe
profile(0sand1s),therelativeManhattandistancebetweengenomes(columns)wascalculated
and used for hierarchical clustering. The confidence of branches was represented by bootstrap
values[19].
Kmertree
Akmerisacontiguoussequenceofkbases.Kcanbeanypositiveinteger.Inprinciplesequences
with high similarity likely share kmers [20, 21]. Based on this idea, the de novo assembled
genomes were split into short sequences with the size of k (kmers). If the kmer size is tiny, the
alignmentspecificityofkmerswillbelow.Ifthekmersaretoolarge,theywillbeseldomaligned.
Kmers were aligned against all the genomes. The number of hits or the frequency of kmers
across genomes was constructed as a matrix. The matrix consists of kmers and genomes (rows
and columns respectively) with the frequency of kmers hits as a profile. The hierarchical
clusteringwasperformedinordertobuildthekmertree.

64
Identificationofcoregenes
The set of 2,882 Salmonella core genes was downloaded from supplementary data of a previous
publication [2]. This set of core genes was estimated based on 73 publicly available Salmonella
genomesusingapreviouslypublishedclusteringmethod,whichemployssinglelinkageclustering
ontopofBLASTPalignmentsinordertoclustertheconservedgenes(coregenes)[22,23].
SNPtree
Singlenucleotidepolymorphisms(SNPs)wereidentifiedusingagenoboxpipelineavailableonthe
Center for Genomic Epidemiology (www.genomicepidemiology.org) [24]. The pipeline consists of
various freely available programs. Basically, the raw reads were aligned against the reference
genomeusingBurrowsWheelerAligner(BWA)[25].WeusedthewellstudiedS.Typhimuriumstr.
LT2asareferencegenome(NationalCenterforBiotechnologyInformation,accession:AE006468).
SAMtools[26]andbedtools[27]wereusedtofilteranddetermineSNPs.ThequalifiedSNPswere
selected once they pass a minimum coverage of 20 and a minimum distance of 20 bps between
each SNP. The qualified SNPs found within Salmonella core genes were only used to make SNP
tree as SNPs in the noncore reflect the high proportion of mobile or extrachromosomal
elements,includingprophageandgenomicislands[13,28].
From each genome, indels were ignored and the qualified SNPs were concatenated to a single
alignment relatively to the position of the reference genome by a perl script. Subsequently,
multiple alignments were employed by MUSCLE from MEGA5 [29]. SNP tree was constructed by
MEGA5 using maximum parsimony method [29]. Bootstrap analysis with 1,000 replicates was
appliedtoevaluatethesupportofthenotes.
RESULTS
The collection of thirtyfour S. Typhimurium isolates was well defined into 18 outbreakrelated
strains from 6 different outbreak sources and 16 background strains according to MLVA profile
andepidemiologicalinformation[6].
TraditionalSalmonellatyping
Pulsedfield gel electrophoresis has been using as a standard procedure for an epidemiological
outbreak investigation of Salmonella [7]. Nonetheless, PFGE discrimination power decrease
dramatically when applying to closely outbreak related strains. Some strains from different
outbreaks were grouped together and some outbreak strains were mixed with background
isolates.
WholegenomeSalmonellatyping
Pangenometree
The pan genome tree is the phylogenetic tree based on the profile of presence and absence of
genes across the genomes [2, 19]. The tree failed to cluster the outbreak strains into the
corresponding groups of six different outbreak sources (Figure 1A). Additionally, some different
outbreak strains were mixed together. This method showed 65 % concordance that is relatively
low compared to the other approaches (Table 1). Alternatively, the pangenome tree revealed
highperformanceforclusteringstrainsaccordingtotheirphagetype(Figure2).
Kmertree
Thekmertreewasconstructedfromthefrequencyprofileofkmersacrossthegenomes.Figure
1B showed that kmer tree gave higher resolution and more discriminatory tree than the pan
65
genome tree. However, the kmer tree exhibited 74 % concordance. Furthermore, two different
outbreakrelated strains were mixed up and some were clustered together with the background
strains(Figure1B).
SNPtree
SNP tree was computed from concatenated qualified SNPs identified from mapping raw reads to
coregenesofthereferencegenome[13,28].Fromfigure1C,theSNPtreeexceptionallyclustered
the outbreakrelated strains into six clusters with 100 % concordance (Table1) and differentiated
them accurately from the background strains. This made the SNP tree having the best typing
abilitybeyondtheothermethodsunderstudy.
Figure 3 revealed that minimum and maximum number of SNP difference within the outbreak
strainswereobviouslylessthanthosenumbersbetweentheoutbreakandbackgroundstrains.
CONCLUSIONS
The objective of this study was to determine the strengths and drawbacks of different typing
methods for Salmonella in order to ultimately identify which approaches would be the most
superior for routine clinical outbreak investigation. A set of thirtyfour human isolated S.
Typhimuriumstrainsfromsixdifferentoutbreakstogetherwithbackgroundstrainswasusedasa
testset[6].Varioustypingapproacheshadbeenevaluatedincludingtraditionalmoleculartyping
likePFGEandWGStyping.RecentstudiesalreadyvalidatedWGStypingbuttheyevaluatedmainly
SNPanalysis.Inthisstudy,itisnotonlySNPbutalsoanumberofWGSbasedtypingbeingselected
andtested.
PFGE, a longestablished golden standard for epidemiological studies, was unsuccessful to

competewithWGSbasedtypingsuchasSNPtree.PFGEislaborintensiverequiringmultipledays
toobtaintheresults[30].Additionally,fortheoutbreakorclonallevel,ageneticvariationisvery
littleanddoesnotsignificantlyimpacttheelectrophoreticmobility,thenthebanchangemightnot
be identified as a separate pulsotype [7]. Another problem is that PFGE patterns can vary within
anoutbreak,forexample,duetoinsertionofprophages[6].
Ultimately, we have found that SNP tree is the most superior method for Salmonella outbreak
detection. Recent studies support SNP tree as an outbreak surveillance tool such as Salmonella
Montevedio outbreak in United States [5, 31] and other retrospective studies, for example,
SalmonellaTyphimuriuminvasivestrainsinsubSaharanAfrica[4].
This study showed that SNP tree is epidemiologically useful for Salmonella outbreak detection.
However, the SNP detection and validation need to be improved, and this method needs to be
furtherevaluatedinotherbacterialpathogenstoelucidatetheusefulnessofusingSNPtree.
The longterm evolution and transmission history are also important to study the origin of the
strains causing outbreaks. This should be somewhat extension of using SNPs. Recent studies also
proofofapplyingSNPsfortrackingtheoriginofSalmonellaprevalentinsubSaharanAfricawithin
40 years [4]. This opens the door for SNPs being applied to identify the ancient ancestor that
happenedinmillionyearsago.
ACKNOWLEDGEMENTS
This study was supported by the Center for Genomic Epidemiology (09 067103/DSF)
http://www.genomicepidemiology.org.
PimlapasandRolfwouldliketoacknowledgefundingfromtheTechnicalUniversityofDenmark.
66
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TABLESANDFIGURES
Table1Evaluationresults
WGStypingmethods Percentageof
concordance
Pangenometree 65
Kmertree 74
SNPtree 100

68
Figure 1 WGS typing results from (A) pangenome tree, (B) Kmer tree, and (C) SNP tree. The tested set
consists of outbreakrelated strains displayed with color label and nonrelated outbreak strains shown
withoutcoloring.Theoutbreakstrainswerelabeledaccordingtothesixdifferentoutbreaksources.

69
Figure2.Pangenometreewithphagetypinglabels.
Figure3MinimumandmaximumnumberofSNPdifferencewithinandbetweentheoutbreakstrains
.

70
MultiplexedSNPtypingmethodforcharacterizingSalmonellaisolates
atthesubserovarlevel
CcileBOLANDandPierreWATTIAU
VeterinaryandAgrochemicalResearchCentre,DepartmentofBacterialDiseases,
BRUSSELS,Belgium
ABSTRACT
A multiplexed method able to subtype Salmonella isolates belonging to the most prevalent serovars and aiming at
displaying discriminative power comparable to PFGE was developed. The prototype assay is based on the detection of
SNPs with discriminative capacity reaching the subserovar level. SNPs were computed from published nucleotide
sequences and derived into phosphorylated, padlock-shaped oligonucleotide probes that were combined in a robust and
highly multiplexable assay. This assay is based on the selective ligation and amplification of reactive probes which are
further identified on a DNA array platform. An initial version of the assay in which size-based differentiation of the
reactive probes was assessed by capillary electrophoresis, was capable to identify >20 SNPs and evaluated on 95
Belgian isolates distributed among S. Typhimurium, S. Enteritidis, S. Derby, S. Brandenburg, S. Infantis, S. Virchow, S.
Hadar, S. Paratyphi B, S. Kentucky, S. Ohio, S. Newport, S. Choleraesuis and S. Gallinarum. Preliminary results
demonstrated subtyping capacity of the developed prototype comparable to PFGE for some serovars and under PFGE
performance for others. In summary, the proof-of-concept of the developed assay was established. The system will be
updated in future versions as to reach adequate sensitivity for all shortlisted serovars and will be transposed to a bead-
array platform in order to reduce analysis time and laboratory work.
INTRODUCTION
During the last two decades, numerous molecular methods have been described for typing
Salmonella enterica subsp. enterica at the subserovar level. Many of these methods are either
designed for a specific Salmonella serovar, which has to be identified first, require substantial
handwork or are too costly for routine use. A universal method able to subtype quickly and
cheaply any Salmonella isolate belonging to the most prevalent serovars and displaying a
discriminative power comparable to PFGE, which is widely considered the gold standard, is still
awaited.
A prototype assay was developed based on the detection of Single Nucleotide Polymorphisms
(SNPs)withpotentialdiscriminatorypowerfor13serovarsmostcommonlyencounteredinEurope
or posing a threat to animal health (Enteritidis, Typhimurium, Derby, Brandenburg, Infantis,
Virchow, Hadar, Paratyphi B, Kentucky, Ohio, Newport, Choleraesuis and Gallinarum). A Ligase
ChainReaction(LCR)assayabletointerrogatetheseSNPswasfirstvalidatedonreferencestrains.
The discriminatory power of the assay was further evaluated on a total of 95 Salmonella field
strains distributed among the target serovars and its performance was compared to Pulsed Field
GelElectrophoresis(PFGE)andMultilocusvariablenumberoftandemrepeatsanalysis(MLVA).
MATERIALANDMETHODS
Multiplesequencealignments
The full genome sequence of 10 S. Typhimurium, 8 from GenBank (FN424405, AE006468,
CP001363, CP002487, FQ312003, CP002614, AP011957, AERV00000000) and 2 from WTSI
(WellcomeTrustSangerInstituteNCTC13348andSTmDT2),2S.Hadar(GenBankABFG00000000
and WTSI HADAR), 2 S. Kentucky (GenBank ABEI00000000 and ABAK02000001), 2 S. Newport
(GenBankCP001113andABEW00000000),2S.Infantis(GenBankCM001274andWTSISIN),2S.
Choleraesuis (GenBank NC_006905.1 and CM001062.1) and 3 S. Gallinarum (GenBank
NC_011274.1, CP003047, CM001153.1) were aligned by serovar category with the reference
genomeofS.TyphimuriumLT2usingtheBioNumericssoftware(version6.6).Individualsequence
types gathered from the Multiple Locus Sequence Type (MLST) database of the Environmental
Research Institute of the University College of Cork (http://mlst.ucc.ie/mlst/dbs/Senterica) were
multiple aligned with BioNumerics 6.6. All fliC, fljB and rfb nucleotide sequences matching the
71
shortlisted serovars were retrieved from GenBank and aligned separately with BioNumerics (168
entriesalltogether).
Bacterialstrains
NinetyfiveSalmonellafieldisolatesdistributedamongthe13targetserovarsweretestedbyLCR.
ThesestrainswereisolatedinBelgiumduringtheperiod20052011fromanimal,food,humanor
environmental sources and were chosen because of their diverse origin or year of isolation.
TwentyS.Typhimurium,15S.Enteritidis,6S.Derby,6S.Brandenburg,6S.Infantis,6S.Virchow,6
S. Hadar, 5 S. Paratyphi B, 6 S. Kentucky, 6 S. Ohio, 6 S. Newport, 3 S. Choleraesuis and 4 S.
Gallinarum were assayed. PFGE was conducted in parallel on most of these strains in order to
evaluate genomic diversity with a reference method. Eight S. Typhimurium and 11 S. Enteritidis
wereanalysedbyMLVA.
ReferencestrainsusedforLCRvalidationoriginatedfromtheSalmonellaGeneticStockCentre(S.
Enteritidis SARB17, S. Typhimurium LT2 and 14028S, S. Newport SL254, S. Paratyphi B SPB7 and
SARA62,S.PullorumRKS5078,S.BrandenburgSARB3,S.InfantisSARB27),fromWTSI(S.Enteritidis
P125109 and S. Gallinarum 287/91), from the ATCC collection (S. Enteritidis ATCC13076, E. coli
ATCC25922)orfrominhousecollections(S.KentuckyandS.Infantis).
DNAextraction
Genomic DNA used as template for LCR assays was extracted from bacteria grown on Brilliant
Green Agar plates with the DNeasy blood and tissue kit according to the manufacturers
instructionsforGramnegativebacteria(Qiagen,Valencia,CA).
LigaseChainReaction(LCR)
The22PadlockshapedProbes(PLPs)targeting21SNPsandtheSalmonellapositivecontrolwere
designed as described (4). LCR and capillary electrophoresis (CE) analysis were conducted as
described (4), using 200 to 800pM of each PLP. CE was conducted using a CEQ8000 instrument
(BeckmanCoulter,Fullerton,CA).
PulsedFieldGelElectrophoresis(PFGE)
PFGE analyses were performed on a Biorad instrument (Biorad, La Jolla, CA) in accordance with
thePulseNetprotocolusingXbaIrestrictionenzymeandS.BraenderupH9812asreferencestrain.
Multilocusvariablenumberoftandemrepeatsanalysis(MLVA)
MLVAanalysesofS.TyphimuriumandS.Enteritidisisolateswereperformedasdescribed(3)(1).
RESULTS
Salmonella nucleotide sequences were computed in different ways in order to identify SNPs
displaying subtyping capacity at the subserovar level. First, full genome sequences of 10 S.
Typhimurium, 2 S. Hadar, 2 S. Kentucky, 2 S. Newport, 2 S. Infantis, 2 S. Choleraesuis and 3 S.
GallinarumwereretrievedfromGenBankandWTSIwebsitesandalignedwiththegenomeofthe
referencestrainS.TyphimuriumLT2usingBioNumerics(version6.6).TheSNPlistsgeneratedfrom
this alignment were compared to the list of SNPs retrieved from the alignment of three S.
Enteritidis full genome sequences published on the NCBI website
(http://www.ncbi.nlm.nih.gov/genomes/static/Salmonella_SNPS.html). SNPs displaying discriminative power
within3or4serovarswereidentifiedbythisapproach.Second,theSalmonellaMLSTdatabaseof
theIrishEnvironmentalResearchInstitutewascomputed.Thisdatabasestoresthesequenceof7
housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, thrA) collected from > 4,500 Salmonella
strains. From these, 208 sequence types representing 1,895 strains belonging to any of the
shortlisted serovars were extracted from the MLST database. Onehundred eightysix SNPs
allowing to subtype at least 2 serovars were identified by this method. Third, sequences of the
72
flagellar genes fliC and fljB and of the rfb cluster found in GenBank (July 2011 release) for all the
shortlisted serovars but S. Choleraesuis and S. Gallinarum, were aligned with BioNumerics. Six
SNPsabletosubtypeatleast2serovarswereidentifiedinfliC.
TwentyoneSNPswereselectedamongtheSNPsidentifiedbybiocomputing(9fromthegenomic
analysis, 9 from the MLST analysis and 3 from fliC). The following criteria were chosen to select
theseSNPs:(i)lackofsignificanthomologyofthe~40nucleotidesflankingtheSNPwiththerestof
thegenome,(ii)highestdiscriminationpotentialatsubserovarlevel,(iii)discriminationpotential
for more than one serovar, (iv) genetic polymorphism limited to 2 possible alleles, preferentially,
(v)scatteredpositionovertheentiregenomeand(vi)nucleotidesequencecompatibilitywiththe
technicalrequirementsofPLPsasdescribed(4).
TheprincipleofthemethodisbasedontheconditionalligationofPLPsuponannealingtoafully
complementary template DNA sequence. Template sequences differing by a single nucleotide at
the critical position will therefore not cause ligation of the annealed probes. Twentyone PLPs
weredesignedinordertotargettheselectedSNPs.Oneadditionalprobewasaddedtotheprobe
set in order to ensure that the analyzed strain belongs to the Salmonella species. This positive
control is a 45nucleotide sequence derived from a published invA probe sequence (2). In its
current development, our prototype assay makes use of capillary electrophoresis to detect the
ligatedprobesafteranamplificationstep.Sizebasedprobedetectionbycapillaryelectrophoresis
enabled3differentmixestobesetup(Mix1:9probestargetingthesocalledgenomicSNPs;Mix
2: 9 probes targeting the SNPs in housekeeping genes; Mix 3: 3 probes targeting the SNPs in fliC
and 1 probe for the Salmonella control). All probes were validated with reference strains
displayingtheexpectedSNPprofile(positivecontrol)oranothernucleotideatthisplace(negative
control).Onlyprobesdisplayingoptimalsignaltonoiseratiosuponassayingpositiveandnegative
controlswerekeptforfurtherincorporationintheassay.
A first evaluation of the discriminative power of the 21 PLPs was obtained with a small set of
strains belonging to each of the 13 shortlisted serovars. All probes except the Salmonellacontrol
showedvariabilityinatleastoneserovar.Twentyoutof21probeswerediscriminativeinatleast
3 different serovars (Figure 1). When compared to PFGE profiles, SNP typing profiles showed
comparable discriminatory capacity for isolates belonging to the S. Hadar, S. Choleraesuis and S.
Gallinarum serovars. The discriminatory power observed for S. Virchow, S. Newport, S.
Brandenburg,S.ParatyphiB,S.KentuckyandS.Ohioisolateswassatisfactorybutcurrentlylower
than observed by PFGE. S. Infantis isolates displayed a unique profile with our assay and hence
couldnotbesubtyped.Theseareprobablycloselyrelatedastheydisplayhighlysimilarprofilesin
PFGE analysis. Poor discrimination was observed for S. Typhimurium, S. Derby and S. Enteritidis
isolates, as compared with either PFGE or MLVA. Regardless of the method used, comparable
genetic clustering was observed for both LCR and PFGE methods. The Salmonella control
(invA_1437A)waswelldetectedinallSalmonellastrains(Figure1).
Simplification of the assay to a singletube assay and detection on a beadarray platform

(Luminex200
TM
)arecurrentlyunderprocess.Forthislatterpurpose,allprobesweredesignedas
tocontainauniquesequencetagmatchingoneoftheMagPlexTAGmicrospherescommercialized
byLuminex.
DISCUSSION
This study allowed proofofconcept demonstration for the developed SNPtyping method. The
whole assay delivered results in 1.5 day and globally fulfilled the initial objectives. All selected
SNPs showed discriminatory power in at least one serovar. The capacity to subtype different
serovars was reached for 20 out of the 21 selected SNPs. Though not optimal, the experimental
73
approachconsistingintheuseof3differentprobemixesperassayandsizebaseddifferentiation
of the amplified probes was found efficient. The use of a beadarray platform should enable the
detectionofall22probesinasinglemix.Althoughthediscriminatorypoweroftheassayneedsto
be improved for a number of serovars, a first screening conducted on a few field isolates was
globallysuccessfulandreachedasensitivitylevelcomparabletoPFGEforanumberofserovars.If
thisturnsouttobeconfirmedonlargercollectionsofisolates,itwillreinforcethecapacityofthe
developed assay to compete with PFGE in discriminating Salmonella strains at the subserovar
level. Future developments will include additional markers able to better subtype isolates
belongingtoserovarsTyphimuriumandEnteritidis,whicharehighlyprevalentserovarsinEurope.
Since the LCR methodology is insensitive to interferences resulting from the use of multiple
oligonucleotides in a single mixture, the way is open for smooth future updates of the proposed
assay. Transposition of the assay to a bead array platform is expected to simplify the associated
laboratoryworkandreducecostswhileshorteningtheanalysistime.
ACKNOWLEDGEMENTS
TheauthorsareverygratefultoMarieThirionetfortechnicalsupport.
REFERENCES
1. Hopkins,K.L.,T.M.Peters,E.dePinna,andJ.Wain.2011.Standardisationofmultilocusvariablenumber
tandemrepeat analysis (MLVA) for subtyping of Salmonella enterica serovar Enteritidis. Euro Surveill
16:19942.
2. Lauri, A., B. Castiglioni, and P. Mariani. 2011. Comprehensive analysis of Salmonella sequence
polymorphisms and development of a LDRUA assay for the detection and characterization of selected
serotypes.ApplMicrobiolBiotechnol91:189210.
3. Lindstedt, B. A., T. Vardund, L. Aas, and G. Kapperud. 2004. Multiplelocus variablenumber tandem
repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and
multicolorcapillaryelectrophoresis.JMicrobiolMethods59:16372.
4. Wattiau, P., A. M. Whatmore, M. Van Hessche, J. Godfroid, and D. Fretin. 2011. Nucleotide
polymorphismbasedsingletubetestforrobustmolecularidentificationofallcurrentlydescribedBrucella
species.ApplEnvironMicrobiol77:66749.
74
strainsofagivenserovar.Incontrast,whitecellsindicatenopolymorphismatthecorrespondingposition.
invA_1437AisanonpolymorphicSalmonellacontrol.
E
n
t
e
r
i
t
i
d
i
s
(
1
5
)
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p
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m
(
2
0
)
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e
r
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y
(
6
)
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(
6
)
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i
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6
)
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(
6
)
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6
)
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(
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)
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a
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i
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a
r
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m
(
4
)
LT2_3466121C Y Y Y Y Y Y
LT2_1659055C Y Y Y Y
LT2_4846502A Y Y Y Y Y Y
LT2_1375257A Y Y Y Y Y Y Y
LT2_2376100C Y Y Y Y
LT2_2461419A Y Y Y Y Y Y Y
LT2_141192C Y Y Y
LT2_4168743C Y Y Y Y Y Y Y
LT2_4317766G Y Y Y Y Y Y
purE_444A Y Y Y Y Y Y
purE_447A Y Y Y
aroC_711A Y Y Y Y Y
hisD_798G Y Y Y Y Y Y
aroC_420A Y Y Y Y Y
hemD_189A Y Y Y Y Y
dnaN_528C Y Y Y Y Y Y Y
hisD_759C Y Y Y
dnaN_195C Y Y Y Y Y Y
fliC_165G Y
fliC_525T Y Y Y Y Y
fliC_342G Y Y Y
invA_1437A
FIGURE
Figure 1. Genetic variability of 21 polymorphic nucleotides assayed by LCR in 13 different Salmonella
serovars. Single nucleotide polymorphisms targeted in the assay are listed in the left column, target
serovars are listed on the upper row together with the number of field strains assayed for each serovar
(into brackets). Black cells point to nucleotides displaying polymorphism when interrogated in different
75
Massiveparallelsequencingidentify
Salmonellasinglenucleotidepolymorphism(SNP)markers
forhostoriginandantimicrobialresistance
MinYUE
1
,RobertSCHMIEDER
2
,JeffWASHELESKI
3
,ShaohuaZHAO
4
,
RobertA.EDWARDS
2,5
,GeorgeP.FRASER
3
,PatrickF.MCDERMOTT
4
,ShelleyC.RANKIN
1
andDieterM.SCHIFFERLI
1,*
1
DepartmentofPathobiology,SchoolofVeterinaryMedicine,UniversityofPennsylvania,
Philadelphia,PENNSYLVANIA,USA;
2
DepartmentofComputerScience,CollegeofSciences,SanDiegoStateUniversity,
SANDIEGO,California,USA;
3
PennsylvaniaDepartmentofHealth,BureauofLaboratories,Exton,PENNSYLVANIA,USA;
4
DivisionofAnimalandFoodMicrobiology,OfficeofResearch,CenterforVeterinaryMedicine,
U.S.FoodandDrugAdministration,LAUREL,Maryland,USA;
5
MathematicsandComputerScienceDivision,ArgonneNationalLaboratory,
ARGONNE,Illinois,USA
INTRODUCTION
Salmonella enterica serovarsforma group of pathogens that differ widely in their host range within
mammalsandotheranimals(4).Themorethan2,600differentserovarscanbesubdividedintothree
groups on the basis of host prevalence. The first group consists of hostrestricted serovars. These
typicallycausesystemicdiseaseinalimitednumberofphylogeneticallyrelatedspecies.Forexample,
S.TyphiandGallinarumarealmostexclusivelyassociatedwithsystemicdiseaseinhumansandfowl,
respectively.Thesecondgroupincludeshostadaptedstrains.Theseareprimarilyassociatedwithone
or two closely related host species but may also infrequently cause disease in other hosts. For
example, S. Dublin and Choleraesuis are generally associated with severe systemic disease in
ruminants and pigs respectively. The third group comprises broad range colonizers, such as S.
Typhimurium that usually induce gastroenteritis in a variety of unrelated host species, including
humans,livestock,wildmammalsandbirds.However,eventheseserovarsdemonstratespecificityby
behaving differently in distinct hosts, S. Typhimurium causing a systemic disease in rodents.
Moreover,somestrainsofS.Typhimuriumvariantshaveanarrowhostrange,whileothersareclearly
abletoinfectandpersistinmultiplespecies(5).MostintriguingaretheinvasiveS.Typhimuriumthat
areinvolvedinbloodstreaminfectioninAfricanadultsandchildren,withanassociatedcasefatalityof
2025%(1),whichsuggeststhatspecificstrainsofS.Typhimuriummighthavebecomehostadapted,
or even hostrestricted in Africa. Although evolution to hostrestriction involves a genomic
degradation,nonsynonymousSNPsmightparticipateinthisprocess.Atypicalexampleoftheimpact
ofallelicvariantsonhostspecificityistheFimHadhesinofS.Gallinarumtype1fimbriaethatmediates
mannoseresistant binding to chicken leucocytes (2) and efficient systemic bacterial dissemination
andcolonizationofinternalorgansinchicks(3).SwitchingthisFimHforthemannosesensitivetype1
fimbrial adhesin, which varies only by 5 amino acids, affected significantly all the latter phenotypes
(3).
The hypothesis of this study is that the genetic variation in certain colonization factors, including
fimbrialadhesins,regulateshostrangeandthedegreeofhostadaptation.Werecentlystartedtotest
this hypothesis by genomewide association studies with a focus on fimbrial adhesins. Eight adhesin
genesof48S.Newportgenomesweresequenced(6).ThenumberofSNPsvariedbetween5and85
foreachofthe8genes,whereaseachadhesinwasrepresentedby6to12differentalleles.Phylogenic
treesclearlyseparatedeachoffiveadhesinsintotwoseparategroups.Insupportofourhypothesis,
76
FimHofthetwogroupsdifferentiatedstrainsfrombovineandnonbovineorigin.Theallelicadhesin
groupscorrelatedalsosignificantlywithstrainsthatdidordidnotshowantimicrobialresistance(and
with or without mobile DNA elements carrying antibiotic resistance genes such as plasmids or
integrons).Thisfindingwasconsistentwithasecondhypothesiswhichsuggestedthatadhesinalleles
ofpersistentlycolonizingSalmonellaoptimizetheopportunityforbacterialencountersandhorizontal
gene transfer (HGT) in intestines, resulting in the accumulation of antimicrobial resistance genes in
suchstrains.
MATERIALSANDMETHODS
We used the previously developed targeted sequencing strategy with the Fluidigm Access Array
systemandtheRoche454sequencer(6).
RESULTS
Totestwhetherthepotentialassociationsofadhesinalleleswithhostorantibioticresistancecanbe
confirmed with another Salmonella serovar, the sequence of all the 15 predicted fimbrial adhesin
genes detectable in S. Typhimurium (5) and of three genes for outer membrane proteins with
potential adhesive properties were determined for 394 wellcharacterized S. Typhimurium isolates.
The lpfD and fimH genes had the highest numbers of allelic variants (Table 1). 174 lpfD genes had a
unique 7 base pair deletion, resulting in a pseudogene. The nucleotide diversity of the 220 lpfD
sequences lacking the deletion (*) was lower (= 0.0001050). 173 isolates lacked the pefA gene,
suggestingthatthesestrainsalsolackedtheplasmidthatnormallycarriesthepefgenecluster.There
wasnoassociationbetweenthelpfDdeletionandtheabsenceofpefA.
Phylogeneticgroupingofstrainswithindividualgenesshowedthattherewasastatisticallysignificant
association of the FimH2 allele with horses. Significant associations were also determined for the
FimH3orBcfD3allelesandantibioticresistance.ThelpfDgenesthatencodedthefullproteinwere
positivelyassociatedwithbovineandporcinehosts,whilethepresenceofpefAcorrelatednegatively
withbovinehosts.
Thestudywasfurtherexpandedbycollectingandanalyzingatotalof1442availableSalmonellafimH
sequences from 74 serovars (NCBI and Welcome Trust Sanger Institute). A total of 131 fimH and 90
FimH alleles were identified. The sequences were from strains collected over 20 years (Fig. 1) and
originatedmainlyfromNorthAmerica(Fig.2).
Although most serovars were carrying different fimH alleles, many of them were mainly associated
withonetotwoofthesealleles(Fig.3).Onetofiveadditionalallelesperserovarrepresentedtypically
less than 10% of all the alleles. Thus each serovar carried only a selected small group offimH allele,
someonlyone(e.g.S.Dublin).Conversely,severalfimHalleleswereserovarspecific,beingidentified
only or mainly in one or two serovars. The data also highlighted large numbers of negative
associationsbetweenspecificserovarsandfimHalleles.
Fig. 4 confirms wellknown serovar specificities for hosts (e.g. S. Enteritidis and the chickenegg
human link). Moreover, fimH SNPs were associated with original hosts, clinical relevance or
environments. Most noticeable was the preponderance of the fimH2 (which also serves as the
reference allele) for serovar Typhimurium in humans and all studied farm animals. This was in
contrastwithfimH1,whichwasnotfoundonequineisolates.Incontrasttothenonsynonymous(ns)
fimHSNPsforthehumanspecificserovars,severalnsSNPswereassociatedwithbovine,equineand
porcineisolatesorwithplants(notshown).

77
CONCLUSION
InadditiontopreviousfindingswiththefimHofS.Gallinarum,thepresentedresultsfurthersupport
ourhypothesisthatallelicvariationofSalmonellaadhesinsparticipateinspecificbindingphenotypes
thataredirectedtowardspreferentialhost species orenvironmental niches, potentiallyfavoringthe
accumulationofantibioticresistancegenesbyHGT.Identifiedallelehostassociationsshouldserveas
diagnosticandepidemiologicaltoolstoconfirmandcomplementserotyping.
ACKNOWLEDGMENT:
FundingfromNIHNIAID:AI098041(toD.M.S.),NSFDBI:0850356(toR.A.E.),andWellcomeTrust
(DECIPHERproject).
REFERENCES:
1. Feasey,N.A., G.Dougan,R.A.Kingsley,R.S.Heyderman, andM.A. Gordon.2012.Invasivenontyphoidal
salmonelladisease:anemergingandneglectedtropicaldiseaseinAfrica.Lancet379:248999.
2. Guo, A., S. Cao, L. Tu, P. Chen, C. Zhang, A. Jia, W. Yang, Z. Liu, H. Chen, and D. M. Schifferli. 2009. FimH
alleles direct preferential binding of Salmonella to distinct mammalian cells or to avian cells. Microbiology
155:162333.
3. KuzminskaBajor, M., M. Kuczkowski, K. Grzymajlo, L. Wojciech, M. Sabat, D. Kisiela, A. Wieliczko, and M.
Ugorski. 2012. Decreased colonization of chicks by Salmonella enterica serovar Gallinarum expressing
mannosesensitiveFimHadhesinfromSalmonellaentericaserovarEnteritidis.VetMicrobiol158:20510.
4. Rabsch, W., H. L. Andrews, R. A. Kingsley, R. Prager, H. Tschape, L. G. Adams, and A. J. Baumler. 2002.
SalmonellaentericaserotypeTyphimuriumanditshostadaptedvariants.InfectImmun70:224955.
5. Yue,M.,S.C.Rankin,R.T.Blanchet,J.D.Nulton,R.A.Edwards,andD.M.Schifferli.2012.Diversificationof
theSalmonellaFimbriae:AModelofMacroandMicroevolution.PLoSONE7:e38596.
6. Yue, M., R. Schmieder, R. A. Edwards, S. C. Rankin, and D. M. Schifferli. 2012. Microfluidic PCR Combined
with Pyrosequencing for Identification of Allelic Variants with Phenotypic Associations among Targeted
SalmonellaGenes.ApplEnvironMicrobiol78:74802.
Table 1. Sequence diversity for 15
genes of 394 S. Typhimurium
isolates. m, number of sequences;
A, number of predicted allelic
genes; S, number of segregating
sites(sumofpositionswithSNPs);
,nucleotidediversity.
Table2.FimHdominantalleles. Table3.BcfDdominant
alleles.
78
Fig.4.Geneticrelationofmajorallele,andtheirhostandreservoirdistribution.
Fig.1:NumbersofSalmonellastrains
sampledperyear.
Fig.2:PercentagesofSalmonellastrains
sampledpergeographicregion
Fig.3.MostfrequentfimHandFimHallelesversusspecificserovars.Thereference
allelefimH2isfromS.TyphimuriumstrainSL1344.
79
MolecularsubtypingofSalmonellaTyphimuriumcombiningdifferenttypes
ofmarkersinamultiplexliquidbeadsuspensionarray
VroniqueWUYTS
1,2
,SophieBERTRAND
3
,NancyROOSENS
2
,KathleenMARCHAL
1,4
andSigridDEKEERSMAECKER
2
1
DepartmentofMicrobialandMolecularSystems,KULeuven,Leuven,Belgium;
2
PlatformBiotechnologyandMolecularBiology,ScientificInstitute
ofPublicHealth(WIVISP),BRUSSELS,Belgium;
3
NationalReferenceCentreforSalmonellaandShigella,BacterialDiseasesDivision,
CommunicableandInfectiousDiseases,ScientificInstituteofPublicHealth(WIVISP),
BRUSSELS,Belgium;
4
DepartmentofPlantBiotechnologyandBioinformatics,GhentUniversity(VIB),
GENT,Belgium
ABSTRACT
Subtyping of Salmonella enterica subsp. enterica serovar Typhimurium, a frequent cause of food-borne diseases, is critical
for surveillance and identification, tracking and ultimately confinement of outbreaks. We present a preliminary proof of
concept of a molecular subtyping method for S. Typhimurium which combines different types of markers in a multiplex
assay with detection on a liquid bead suspension array in a high-throughput format. Selected markers include, amongst
others, markers based on AFLP fragments, prophage genomes, sequence repeats, antibiotic resistance genes and SNPs.
The optimal multiplex design was evaluated and a ligation dependent amplification (LDA) assay was found to be the most
efficient strategy to target the selected markers. The in vitro stability of the selected markers was verified. To determine the
discriminatory ability, S. Typhimurium isolates of most common phage types in Belgium were subjected to the proposed
subtyping method. The capability to identify outbreaks was tested with strains from two outbreaks in Belgium that occurred
in 2008 and 2011. The resulting profiles were examined for correlation with phage types, MLVA and antimicrobial resistance
profiles. The potential of the new method to provide a new subtyping scheme for S. Typhimurium will be discussed.
INTRODUCTION
Classically in surveillance, isolates of Salmonella enterica subsp. enterica serovar Typhimurium are
phagetypedand,ofteninoutbreaksituations,furthersubtypedbymoleculartechniquesaspulsed
fieldgelelectrophoresis(PFGE),multiplelocusvariablenumbertandemrepeatsanalysis(MLVA)or
multilocussequencetyping(MLST).Althoughproventohaveadditionalvalueforsubtyping,eachof
these techniques has its intrinsic disadvantages and scientists still search for an ideal subtyping
method,whichshouldbeinexpensive,rapid,highlydiscriminativeandrobust(2,4).
Untilnow,differentkindsofDNAmarkershavebeenstudied,aloneorcombined,fortheirsuitability
for a molecular subtyping scheme for S. Typhimurium. The number of markers included in the
resulting methods and the level of multiplexing still feasible, often determines the discriminatory
powerandrapidity(4).
In this study we deliver a preliminary proof of concept for a molecular subtyping method for S.
Typhimurium that combines different types of markers, such as amplified fragment length
polymorphisms (AFLP), prophage genomes, sequence repeats, antibiotic resistance markers and
SNPs.Toachieveasufficientlevelofmultiplexinginahighthroughputmanner,wemakeuseofthe
Luminextechnology.Thistechnology,whichemergedinthe1990s,allowsdistinguishingupto500
differenttargetsinonesamplethroughitsbeadbasedassays.
MATERIALSANDMETHODS
Bacterialisolates
S.TyphimuriumisolateswereselectedfromthecollectionoftheBelgianNationalReferenceCentre
for Salmonella and Shigella. The selection includes 100 isolates, of which 13 isolates from two
outbreaks, with different combinations of phage type, antimicrobial resistance pattern and MLVA
profile. Besides the most frequent phage types over the past years in Belgium, i.e. DT12, DT104,
DT120, DT193, DT195 and U302, also phage types DT1, DT35, DT138 and one reactionsdonot
80
conform (RDNC) isolate are represented. In total, these isolates cover 26 different antimicrobial
resistancepatternsand55distinctMLVAprofiles.
DNAisolation
DNA was isolated by dissolving a single colony from an overnight culture on LB agar into 300l
sterilewaterandincubatingat100Cfor10minutes.Aftercoolingto4Candcentrifugationfor10
minutesat10,000rpm,thesupernatantwasstoredat20Candusedforfurtheranalysis.
Stabilityexperiment
Stability of selected markers was evaluated in 31 S. Typhimurium isolates of the most frequent
phage types in Belgium. A series of 50 passages, starting from a single colony, was performed.
GlycerolstocksweremadebeforeeachfifthpassageandDNAwasisolatedfromculturesafterthe
finalpassage(3).
Markerselection
Markers were taken from literature. Markers that are informative through presence or absence
were screened by PCR and gel electrophoresis on 30 isolates of the most common phage types in
Belgium. Those markers which were not present or absent in all 30 isolates and thus have
discriminatorypower,wereselectedfortheliquidbeadsuspensionarraydevelopment.
Beadbasednucleicacidassayselection
The Luminex technology, which is implemented through a MAGPIX platform in this study, utilizes
polystyrene beads with a different colour code for each set. Each bead set can be coated with a
different capture probe. Multiplexing is then achieved by combining different bead sets to analyze
onesample.Duringanalysis,thesettowhichthebeadbelongsisdeterminedbymeasuringthered
fluorescencesignalfromthebead.Afterwards,thepresenceofahybridizedtargetoligonucleotideis
detectedbymeasuringthegreenfluorescencesignalonthattarget.Eachbeadbasedassayshould
thus incorporate a fluorophore in the target oligonucleotide. Hereto, different possibilities exist.
Threebeadbasedassayformatswerecomparedwithregardtotheusagepossibilities,multiplexing
capacity,workflow,optimizationandcost.
The direct hybridization assay starts with a multiplex PCR with labelled primers, after which the
products are hybridized to beads, to which capture probes were coupled. If primers labelled with
biotin are used, an incubation step with streptavidinphycoerythrin (SAPE) is necessary before
analysisonaLuminexplatform.
IncontrasttothexMAP
technologythatisused indirecthybridizationassayswhere selfdesigned

capture probes have to be attached to the beads, the xTAG
technology utilizes beads with pre

coupled 24 bp antiTAGsequences. Allele specific primer extension (ASPE) and ligation dependent
amplification (LDA) are built upon this technology and should integrate the complementary TAG
sequenceinthetargetoligonucleotide.
ASPE commences with a multiplex PCR, after which the products are treated with ExoSAPIT to
degrade remaining primers and dNTPs. Target specific probes with a TAGsequence at 5 are then
annealed to the PCR product and extended with inclusion of biotindCTP. The ASPE products are
analyzedonaLuminexplatformafterhybridizationtoxTAGbeadsandincubationwithSAPE.
The first step in a LDA assay (figure 1) is a multiplex ligation with a probe pair for each target. The
upstream probe has a universal primer site, TAGsequence and target specific part, the adjacent
downstreamprobehasatargetspecificpartanduniversalprimersite.Thesecondstepcomprisesa
singleplex PCR with universal primers, one of which is labelled. After hybridization to xTAG beads
and an optional incubation with SAPE, the amplified ligation products are read out on a Luminex
platform.

81
Multiplexoligonucleotidedesign
OligonucleotidesforthemultiplexliquidbeadsuspensionarrayweredesignedwiththeVisualOMP
software(DNASoftware).
Figure1:Ligationdependentamplification(LDA).Probeshaveatargetspecificpart(black),auniversal
primersite(striped)andaTAGsequence(dotted).Afluorescentlabel(star)isintroducedviaalabelled
universalprimer.
RESULTS
Markerselection
ThroughPCRscreeningon30isolatesofphagetypesDT12,DT104,DT120,DT193,DT195andU302,34
literaturebasedmarkers,detectingAFLPfragments,prophagegenomes,sequencerepeats,SGI1and
allantoinutilization,havebeenfoundinformative.
Also included in the current selection of markers, but not screened through PCR, are antibiotic
resistancegenes,SNPsandamarkerspecifictoSalmonellaentericasubsp.enterica.
Beadbasednucleicacidassayselection
Tocombinetheselectedmarkersinamultiplexliquidbeadsuspensionarray,threenucleicacidassay
formats were considered. Advantages and disadvantages of the different beadbased nucleic acid
assaysaresummarizedintable1.TheLDAassaywaschosenfordevelopmentofasubtypingmethod
for S. Typhimurium, since the multiplex step in this assay format is a ligation, which allows a higher
multiplexcapacityandanadditionofmarkerswithoutredesignoftheassay,incontrasttomultiplex
PCR.Additionally,theLDAdoesnotrequiretheuseofneurotoxicTMACbuffer,ofcouplingofcapture
probes and of optimization of the hybridization to Luminex beads. Also the costs and total time to
completetheassaywerefoundtobeacceptable.
Preliminaryproofofconcept
From the 34 markers found informative through PCR screening, 5 markers with high discriminatory
power, including prophages and SGI1, were selected for the first LDA tests. A marker detecting
SalmonellaentericawasincludedasacontrolfortheDNAtemplates.
The resulting 6plex LDA was applied to 100 isolates and the 31 isolates after 50 passages in the
stabilityexperiment.TheamplifiedligationproductswerereadoutonaMAGPIXplatform.Eachassay
included a negative control, containing all reagents but no DNA template, for background signal
measurement, and a positive control, containing all reagents and a mixture of DNA template
representingall6markers,toverifythereaction.Aclearseparationwasobservedbetweennegative
and positive signals, measured as mean fluorescence intensity (MFI), which is illustrated by the
boxplotsinfigure2.
Target DNA
Target specific
probe pair
Ligation
PCR with universal primers
Hybridization to xTAG bead
xTAG bead
82
Figure 2: Boxplots of MFIs of negative (0) and positive (1) signals of amplified LDA products analyzed on a
MAGPIXdevice
Presence or absence of markers, as detected by the LDA, resulted in 7 distinct profiles in the 131
testedisolates.ThePCRprofilesofthe30isolatesusedinthePCRscreeningofmarkers,allcorrespond
with the profile determined by the LDA. The isolates in the stability experiment gave an identical
profile before and after the 50 serial passages in LB medium, thereby demonstrating the stability of
the6markersincludedintheassay.
AllDT104isolateshadthesameprofile,differingfromtheoneDT104Aisolatetested.Someisolatesof
DT12 and U302 shared the same profile as the DT104 isolates, others were found with different
profiles.IsolatesofDT193with2,3or4repeatsatMLVAlocusSTTR9showeddistinctLDAprofiles;the
profileoftheisolatewith4repeatswasnotsharedwithotherisolates.
With the current 6 selected markers, correlation with antimicrobial resistance patterns could not be
observedandalsonodistinctprofilewasobtainedfortheoutbreakisolates.Thesefindingsnecessitate
extendingtheLDAassaywithmoreinformativemarkerstoahigherlevelofmultiplexing.
DISCUSSION
ManyoftheproposedmolecularmethodsforsubtypingofS.Typhimuriumarelimitedbythenumber
ofmolecularmarkersandbythecapacityofmultiplexingthosemarkersinasinglerun.TheLDAassay
can achieve a high level of multiplexing by separating detection and amplification. LDA is also cost
effectivesincethespecificprobepairsareunlabelled.Numeroustypesofmolecularmarkers,including
SNPs, can be combined and straightforwardly added or left out of the assay. New probe pairs are
assignedadifferentTAGfordetectionontheLuminexplatform,butstillexploittheuniversalprimer
pair for amplification of the signal, which makes the LDA a modular assay. In addition, LDA is very
specificduetosevereconstraintsontheligationreaction:theupstreamprobemustannealadjacent
to the downstream probe and a strict complementarity is necessary for the base pairs flanking the
ligationsite.Amplificationbias,asmightbeobservedinmultiplexPCR,isunlikelytoaffectLDA,since
ligatedprobepairshaveaboutthesamelength(1).
Processing of the results generated by the LDA assay is facilitated by the single file output of the
Luminexplatform,whichcaneasilybehandledwithouttheneedofexpensivesoftware.
CONCLUSION
We have presented a preliminary proof of concept for the use of a LDA assay with analysis on a
MAGPIXplatformforthesubtypingofS.Typhimurium.Moremarkerswillbeincludedtoincreasethe
83
discriminatorypowerandoptimizationoftheworkflowandoftheassaywillfurtherreducecostand
time.Thisshouldleadtoacosteffective,rapid,highlydiscriminative,knowntargetbased,androbust
method, more efficiently addressing the needs of S. Typhimurium subtyping than MLVA and phage
typing.
ACKNOWLEDGEMENTS
WewouldliketothankG.DeLaminnedeBexforexecutingthestabilityexperiment,C.Wildemauwe
forphagetyping,andthetechniciansoftheBelgianNRCforSalmonellaandShigellaforantimicrobial
susceptibilityandMLVAtesting.
REFERENCES
1. Deshpandeetal.(2010).J.MicrobiolMethods,80:155163.
2. Sabatetal.(2013).EuroSurveill,18:pii=20380.
3. Struelensetal.(1996).ClinMicrobiolInfect,2:2131.
4. Wattiauetal.(2011).ApplEnvironMicrobiol,77:78777885.
Table 1: Overview of advantages (+) and disadvantages () of direct hybridization, ASPE and LDA assays.
mPCRdenotesmultiplexPCR.
Feature Directhybridization ASPE LDA

Usage Unrelatedsequences,SNPs,multiplepolymorphisms
Multiplexstep mPCR mPCR +Ligation
Additionofmarker Redesign Redesign +Noredesign
Buffers TMAC +Tm +Tm
Couplingofcaptureprobe Yes +No +No
Hybridizationtobeads Optimize:4555C +Standard:37C +Standard:37C
PCRamplicon <300bp +allsizes +allsizes
Patent +No +No Yes
Cost ++
Totaltime +3.5hours 7hours 6hours

84
OneyearsurveillanceofhumanandnonhumanSalmonellaenterica
serotypeTyphimuriumanditsmonophasicvariantbased
onCRISPOLsubtyping,France,2011
SimonLEHELLO
1
,MurielMARAULT
2
,LaetitiaFABRE
1
,NathalieJOURDANDASILVA
3
SabrinaCADELSIX
2
,LucileSONTAG
1
,AnneBRISABOIS
2
,FranoisXavierWEILL
1
1
InstitutPasteur,UnitdesBactriesPathognesEntriques28rueDrRoux,
75724Pariscedex,FRANCE
2
ANSES,23avenuedugnraldegaulle,94703MaisonsAlfort,France
3
InVS,12rueduVald'Osne,94410SaintMaurice,France

ABSTRACT
A new high-throughput method based on the polymorphisms of the CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeats) regions, named CRISPOL, for subtyping serotype Typhimurium and its monophasic variant
1,4,[5],12:i:- have been recently published (Fabre et al. PLoS One. 2012). This method targeting 72 spacers (variable
sequences within CRISPRs) by using a bead-based liquid hybridization assay (Luminex
) has two major advantages: its

rapidity and low cost. The CRISPOL method was retrospectively applied on 2,209 human clinical isolates (one per
patient) addressed to the French National Reference Center for Salmonella in 2011. These isolates represented 54.1%
(2,209/4,074) of all the Typhimurium and monophasic isolates received during the year. Of the 2,209 isolates CRISPOL
typed, 1103 were of serotype Typhimurium (57% of the annual number) and 1106 were monophasic (51% of the annual
number). We have also analyzed 311 non-human isolates (102 of serotype Typhimurium and 209 monophasic) from the
French Agency for Food Environmental and Occupational Health and Safety. These non-human isolates represented
30% of the isolates collected in 2011. We found 201 distinct CRISPOL types (CT) among clinical isolates and 51
among non-human isolates. All CTs observed in non-human isolates were also present in clinical isolates. The major
CTs, both in human and non human isolates, were CT1 associated with the emerging monophasic 1,4,[5],12:i:- variant,
and CT21 and CT30, both associated with the MDR DT104 serotype Typhimurium clone. During this period a steady
increase in the prevalence of CT1 was observed which corresponded to documented outbreak by consumption of dried
pork sausage. Furthermore, some new CTs (currently more than 700 in our database) have allowed us to link food
poisonings to the contaminated food. We could also attribute some CTs with specific non-human sources. Subtyping
information obtained in real-time is very interesting for the most prevalent serotypes. This will be undoubtedly helpful for
surveillance, outbreak investigation and for tracking particular strains.
INTRODUCTION
Salmonellosis is one of the most common causes of foodborne diarrheal disease worldwide. An
efficient surveillance system for salmonellosis is therefore crucial. Laboratorybased approaches
are a key component of monitoring strategies in developed countries. They require a network of
clinical laboratories covering the population and referring isolates or information to a central
public health reference laboratory. The speed with which public health laboratories obtain
information after the onset of symptoms and the regular sharing of information between public
healthlaboratoriesandepidemiologistsarecriticalforthesuccessfuluseofinformationtodetect
outbreaks early and to identify their source. The basic information currently provided by
laboratories is the serotype of the isolates. Typhimurium and Enteritidis, are highly prevalent
worldwide and account for most outbreaks. The sensitivity of serotyping for the detection of
outbreaksinvolvingthesecommonserotypes,evenwiththeuseofclusterdetectionalgorithms,is
thereforeunsatisfactory.Amongtheexistedsubtypingmethods,anewhighthroughputmethod
based on the polymorphisms of the CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) regions, named CRISPOL, for subtyping serotype Typhimurium and its monophasic
variant 1,4,[5],12:i: have been recently published (Fabre et al. PLoS One. 2012) [1]. This bead
based liquid hybridization assay, both rapid and easy, was carry out on human clinical isolates
addressedtotheFrenchNationalReferenceCenterforSalmonellain2011.
WeaimtodemonstratethatCRISPOLsubtypingissuitableforuseinpublichealthlaboratories.

85
MATERIALANDMETHODS
NationalSurveillanceSystemofHumanSalmonellaInfectionsinFrance
In France, the human Salmonella surveillance system is a voluntary laboratorybased network
headed by the National Reference Centre for Salmonella (NRC) based in Paris. Participating
laboratories(1,392(62%)in2011)sendaround8,000SalmonellaisolatestotheNRCperyear.The
NRCperformstheserotypingofallhumanSalmonellastrainsreceivedusingtheWhiteKauffmann
Le Minor scheme and runs weekly outbreak detection algorithms, notifying exceeded thresholds
to the French Institute for Public Health Surveillance (Institut de Veille Sanitaire InVS) [2]. The
NRCalsosignalsinrealtimetotheInVSanysuspectedclustersbasedonobservationsofserotying
results in the course of analysis. During outbreaks, serotyping results are notified to the InVS in
realtime. In 2008, it was estimated that the Salmonella surveillance system detects 66% of
confirmedhumanSalmonellainfectionsinFrance[3].
HighthroughputmethodforsubtypingserotypeTyphimuriumoritsmonophasicvariantinreal
time:theCRISPOLassay.
Based on the CRISPR1CRISPR2 combined alleles identified previously [1], we developed a bead
basedliquidhybridizationassay(Luminex
technology),CRISPOL(forCRISPRpolymorphism).A25
to 32 bp capture probe was designed for each of 72 of the 79 spacers identified. Each capture
probe was coupled to a defined xMAP bead. We used thermolysates as the DNA template and a
single primer pair (including a biotinylated primer) hybridizing to DR sequences to amplify the
spacercontentofthetwoCRISPRlocirapidly.ThePCRmixturewashybridizedwiththe72probe
coupled beads and incubated with streptavidinphycoerythrin for detection. The Luminex
platformwasthenusedtomeasurethefluorescenceassociatedwitheachbead(correspondingto
auniqueprobe/spacer).Thismethodgaveahighlyrobustreadout,withmeanfluorescencesignals
of709to5,707inthepresenceofthespacer,andof52to193intheabsenceofthespacer.The
positive/negativeratioswerebetween13(forabeadforwhichcouplingwasnotoptimal)and92
(meanof50.8).ItwasalsoeasytoidentifythefourSNPvariantspacers.
Bacterialisolates
The CRISPOL method was retrospectively applied on 2,209 human clinical isolates (one per
patient)addressedtotheFrenchNationalReferenceCenterforSalmonellain2011.Theseisolates
represented54.1%(2,209/4,074)ofalltheTyphimuriumandmonophasicisolatesreceivedduring
the year. Of the 2,209 isolates CRISPOL typed, 1103 were of serotype Typhimurium (57% of the
annualnumber)and1106weremonophasic(51%oftheannualnumber).Wehavealsoanalyzed
311 nonhuman isolates (102 of serotype Typhimurium and 209 monophasic) from the French
Agency for Food Environmental and Occupational Health and Safety. These nonhuman isolates
represented30%oftheisolatescollectedin2011.
RESULTS
We found 201 distinct CRISPOL types (CT) among clinical isolates and 51 among nonhuman
isolates. All CTs observed in nonhuman isolates were also present in clinical isolates. The major
CTs,bothinhumanandnonhumanisolates,wereCT1associatedwiththeemergingmonophasic
1,4,[5],12:i: variant, and CT21 and CT30, both associated with the MDR DT104 serotype
Typhimurium clone. During this period a steady increase in the prevalence of CT1 was observed
which corresponded to documented outbreak by consumption of dried pork sausage.
Furthermore, some new CTs (currently more than 700 in our database) have allowed us to link
foodpoisoningstothecontaminatedfood.
DISCUSSION
A previous study demonstrated that the assessment of CRISPR content is a robust, highly
discriminatory and practical method for typing Salmonella isolates. The CRISPR method can be
86
used for simultaneous typing and subtyping. The first CRISPOL assay developed is focused on
Typhimurium and its variants (monophasic or nonmotile) populations. It provides an excellent
alternative to PFGE (see Poster CadelSix et al), being cheaper, less technically demanding and
yieldeddatathatareeasytointerpretandexchange.
The retrospectively detection and/or the confirmation of human salmonellosis outbreaks in 2011
due to rare CT of serotypes Typhimurium or its monophasic variants indicates its interest in
optimizing surveillance. The possibility to obtain results more rapidly enlarge its use in realtime.
Thiswillbeundoubtedlyhelpfulforsurveillance,outbreakinvestigationandfortrackingparticular
strains.
An approach based on the initial use of the CRISPOL assay, followed by MLVA for genetically
homogeneous populations, such as the DT104 clone (CT21) or the emerging monophasic strain
(CT1)wouldbehighlyeffective.
REFERENCES
1. FabreL,ZhangJ,GuigonG,LeHelloS,GuibertV,AccouDemartinM,deRomansS,LimC,RouxC,Passet
V, Diancourt L, Guibourdenche M, IssenhuthJeanjean S, Achtman M, Brisse S, Sola C, Weill FX. CRISPR
typing and subtyping for improved laboratory surveillance of Salmonella infections. PLoS One.
2012;7(5):e36995.
2. WeillFX,LeHelloS.CentreNationaldeRfrencedesSalmonella:Rapportd'activitannuel2011.
3. JourdanDa Silva N, Le Hello S. Salmonelloses en France, 20022010: tendances en pidmiologie
humaine, mergence de la souche monophasique, principaux aliments impliqus dans les dernires
pidmies.BulletinEpidmiologiqueHebdomadaire2012;Horssrie:2528.

87
May 27-28-29, 2013
Saint-Malo France

Session 2


Chairpersons:

Posters

88
Geneticbasisoflackofexpressionofphase2flagellarantigen
inItalianisolatesofS.4,[5],12:i:
A.LONGO
1
,L.BARCO
1
,A.A.LETTINI
1
,E.RAMON
1
,C.SACCARDIN
1
,M.C.DALLAPOZZA
1
,
E.CORTINI
1
andA.RICCI
1
1
IstitutoZooprofilatticoSperimentaledelleVenezie,NationalReferenceCentre
ofSalmonellosis,LEGNARO(PD),Italy
INTRODUCTION
Salmonellaserotypeisdeterminedbythesomatic(O)andflagellar(H)antigenspresentonthecell
wall.TheOantigensdeterminetheserogroup,whiletheHantigensdefinetheserotypeidentityof
aSalmonellastrain.MostofSalmonellaserotypesarebiphasic,meaningthattheycanexpresstwo
distinctflagellar(phase1andphase2)antigens.Flagellarphasevariationdependsontwoflagellin
genes, fliC and fljB, that are expressed alternatively. This phenomenon is controlled by the
recombinase Hin, which enables the reversible inversion of a DNA segment containing the
promoterofthefljABoperon,encodinganegativeregulatorofthefliCgeneforthefirstflagellar
antigen(fliA),andthesecondphaseantigen(fljB),respectively.
S. 4,[5],12:i: is an emergent monophasic variant of S. Typhimurium for which a sharp rise in
prevalencehasbeenreportedinthelastdecade(5).Thesetworelevantserovarsareantigenically
similarandgeneticallycloselyrelated,althoughthemonophasicvariantlacksexpressionofphase
2 flagellar antigen. Despite some studies, characterizing isolates of S. 4,[5],12:i:, have described
differentprofilesofdeletionofgenesincludedinthefljABoperonandothercloselylocatedgenes
(4,5),thismonophasicserovarisusuallysimplyassociatedwiththedeletionofthefljBgene.
In order to provide new insights regarding the emergence of S. 4,[5],12:i: and its genetic
repertoire, a selection of Italian isolates belonging to the most common phagetypes was
investigated.DeletionsofthegenesincludedinthefljABoperonandup/downstreamgeneswere
characterized and the correlation between the profiles of deletion and the phagetypes was
evaluated. Moreover, the gene repertoire detected was compared to the previously described
data concerning both the Spanish and the American clones of S. 4,[5],12:i: (3,4,5) in order to
identifydifferentialmarkerswhichcouldbeusefulforsurveillancepurposes.
MATERIALS&METHODS
Ninetythree strains of S. 4,[5],12:i: isolated in Italy from various animal and food sources,
between 2009 and 2012 were investigated. Twentyfour isolates belonged to phagetype DT193,
24 to U311, 16 to U302, 25 to DT120 and 4 to DT7. The phagetype was defined according to
Andersonetal.(1).PCRprimersetswereusedtocharacterizethedeletionsofthefljABoperonas
well as thepresence or absence ofup/downstream genes.In particular the targetsof these PCR
were: a) STM2757 gene (5), b) fliA gene (5), c) fljB gene (2), d) hin gene (5) and e) iroB gene (5).
Moreover a PCR assay with mixed primers (flanking STM2757 and iroB genes) was set up to
determine if these two genes were consecutive. The structure of these targets is represented in
Figure1.TemplateDNAforPCRwaspreparedbyboilingmethod.
Figure1:structureofthefljABoperoninS.TyphimuriumLT2.

89
RESULTS
6differentdeletotypesoccurringonfljABoperonwereidentified(Table1).Themostcommon
profile of deletion for the 5 targets investigated was characterized by the presence of the genes
STM2757 and iroB and the deletion of fljB, fliA and hin genes. This predominant profile was
identified for 65 out of the 93 investigated strains. Since the genes STM2757 and iroB werent
consecutive,itcanbesupposedthereisaninsertioninthedeletedregionofthefljABoperon,as
previouslydescribed(4,5).Acorrelationbetweenthedeletotypeandthephagetypeswasnot
evidenced.Amongthephagetypesinvestigated,U302isolatespresentedthemostheterogeneous
repertoireofdeletiontypes.
Table 1: Deletotypes detected for the S. 4,[5],12:i: strains investigated. Strains were distributed per
phagetype(VMST:monophasicvariantofS.Typhimurium)
DISCUSSIONANDCONCLUSION
Within the Italian collection of S. 4,[5],12:i: strains several deletotypes were identified including
those previously described and known as the U.S. and Spanish deletotypes, The most prevalent
profileofdeletionreportedamongtheItalianisolatesinvestigatedshowedintermediatefeatures
between the American and the Spanish deletotypes (5). This Italian profile has been already
describedforS.4,[5],12:i:isolatesobtainedinUSAandFrance(3,5).
The present study underlines the importance of the described molecular markers to delineate
genotypesandidentifylineagesamongepidemiologicallyrelevantisolatesofS.4,[5],12:i:.
REFERENCES
1. Anderson, E.S., L.R. Ward, M.J. De Sawe, and J.D.H. De Sa. 1977. Bacteriophagetyping designations of
SalmonellaTyphimurium.J.Hyg.(Lond.)78:297300.
2. Barco L., Lettini A.A., Ramon E., Longo A., Dalla Pozza M.C., Saccardin C., Ricci A. 2011. A rapid and
sensitive method to identify and differentiate Salmonella enterica serotype Typhimurium and Salmonella
entericaserotype4,[5],12:i:bycombiningtraditionalserotypingandmultiplexpolymerasechainreaction.
FoodbornePathogensandDesease.8(6):741743.
3. Bugarel, M., M.L. Vignaud, F. Moury, P.Fach and A. Brisabois. 2012. Molecular identification in
monophasic and nonmotile variants of Salmonella enterica serovar Typhimurium. MicrobiologyOpen 1(4):
481489.
4. Laorden, L., S. HerreraLeon, I. Martinez, A. Sanchez, L. Kromidas, J. Bikandi, et al. 2010. Genetic
evolution of the Spanish multidrugresistant Salmonella enterica 4,[5],12:i: monophasic variant. J. Clin.
Microbiol.48:45634566.
5. Soyer,Y.,A.MorenoSwitt,M.A.Davis,J.Maurer,P.L.McDonough,D.J.SchoonmakerBopp,etal.2009.
Salmonellaentericaserotype4,[5],12:i:,anemergingSalmonellaserotypethatrepresentsmultipledistinct
clones.J.Clin.Microbiol.47:35463556.
VMSTDT193 VMSTU311 VMSTU302 VMSTDT120 VMSTDT7

STM2757+fliAfljBhiniroB+ 19 19 9 15 3
STM2757fliAfljBhiniroB+ 4 4 1 7 1
STM2757+fliAfljBhiniroB 1 1 3 1
STM2757+fliA+fljBhiniroB+ 2
STM2757+fliAfljBhin+iroB+ 1
STM2757fliAfljBhiniroB 2
24 24 16 25 4
90
ValidationofaSalmonellaGenoSerotypingArray(SGSA)
inThreeInternationalLaboratories
ErikaJ.LINGOHR
1
,RoderickCARD
2
,FriederikeTROGNITZ
3
,NikkiMCLAREN
2
,AndreVILLEGAS
1
,
KristynFRANKLIN
1
,StephanieMURPHY
1
,TanjaKOSTIC
3
,AndrewM.KROPINSKI
1
andCatherineYOSHIDA
1
1
LaboratoryforFoodborneZoonoses,PublicHealthAgencyofCanada,GUELPH,Canada;
2
AnimalHealthandVeterinaryLaboratoriesAgency,NewHaw,UnitedKingdom
3
AustrianInstitute
ofTechnology,TULLNANDERDONAU,Austria
INTRODUCTION
Salmonella are among the most common sources of food poisoning in the world, causing
widespreadillnessandmortality.Asmanyofthe2600serotypesofSalmonellavaryinhostrange,
animal reservoirs, ecology and epidemiology, an important early step in the characterisation of
clinical Salmonella isolates is the determination of serotype. Traditional serotyping methods are
basedontheWhiteKauffmannLeMinor(WKL)schemewhichusesantiseraforthedetermination
of the O (lipopolysaccharide), H (flagellar) and Vi (capsular) antigenic epitopes (3). Despite the
usefulness of this scheme, traditional serotyping requires expensive antiserum reagents and
specializedexpertisewithsubjectiveinterpretation.
The Salmonella Genoserotyping Array (SGSA), recently described by Franklin et al., 2011 was
developed and evaluated for use as a molecular alternative to classical Salmonella serotyping
methods for improved diagnostic capabilities. Mimicking the WKL scheme, the SGSA identifies
serotypesbydetectingtheuniqueDNAsequenceswhichencodetheOserogroupandHantigens
preserving continuity with historical surveillance data while maintaining epidemiological
relevance. The SGSA was validated in a large scale trisite study to assess the performance,
repeatability, specificity and sensitivity of the SGSA, and determine its applicability to clinical
microbiology laboratories. The validation assessed known targets representative of the 58 most
commonly reported serovars (Table 1), in addition to nontarget isolates for the assessment of
specificity.
This rapid molecular typing method will increase the efficiency of Salmonella classification and
characterisation thereby reducing the impact of outbreaks and burden of disease. In turn,
supportingsciencebaseddecisionmakingrelatedtofoodbornepublichealthissues.
MATERIALSANDMETHODS
Traditional serotyping was performed within recognized reference laboratories according to the
WKLscheme(1,4).
DNA is extracted from isolated cultures using commercial kits followed by three simultaneous
multiplex PCR reactions amplifying all probe targets present in the given sample. Amplicons are
subsequently dephosphorylated and labelled with biotin using sequence specific end labelled
oligonucleotide(SSELO)primers.Labelledprobesarehybridizedtotheminiaturetubebasedarray
(Alere Technologies, Jena, Germany), and positive signals detected in the ArrayMate Reader
(Alere). An EXCEL macro designed inhouse automates the data analysis and nonsubjectively
identifies the antigenic formula and serovar. Full details on the methodology can be found in
Franklinetal.(2).
RESULTS
Performance was assessed against 704 Salmonella isolates representative of the top 58 globally
reportedserovars.TheSGSAcorrectlyidentified97%ofthesamples.Anadditionalpanelof260
less common serovars was used to assess probe specificity with 95% accuracy. The three
participating laboratories produced similar results, with an average sensitivity of 99%, and a
91
specificity of 100% for S. Enteritidis, S. Typhimurium, and a subset of seven other clinically
important serotypes (Table 1). The repeatability was an average of 94% for 60 replicates
performedonidenticalstrainsateachofthethreelocations.
A questionnaire administered to all researchers involved in the validation identified improved
discrimination of highly homologous serovars, and macro development as key areas for
improvement.
DISCUSSION
The ability to accurately identify predominant Salmonella serovars within a single working day
offersasignificantadvantageovertraditionaltypingmethodologies.TheSGSAlendsitselfwellto
automationforhighthroughputanalysis,reducingtimeandassociatedlabourcosts,andproviding
benefitinoutbreaksituations.
The overall success of the validation has resulted in realtime parallel studies in all three typing
laboratories to assess 800 blind isolates. Additional testing will validate the characterisation of
additionalserotypesinexcessofthetarget58usingexistingOandHprobes.
TheSGSAexceededtheperformancerequirementsdetailedinadvanceofthevalidation.Further
studies will confirm the array is ready for use in diagnostic laboratories as a molecular typing
methodforSalmonella,orasanadjuncttoclassicalserologicaltesting.
ACKNOWLEDGEMENTS
We would like to thank the OIE Reference Laboratory for Salmonellosis (Canada), the Austrian
Agency for Health and Food Safety (Austria), and the Animal Health and Veterinary Laboratories
Agency(UK)fortheirexpertiseandassistanceinconfirmationofSalmonellaidentification.
REFERENCES
1.Ewing,W.H.1986.EdwardsandEwingsidentificationofEnterobacteriaceae.ElsevierSciencePublishing
Co.,Inc.,NewYork,NY.
2. Franklin, K., E.J. Lingohr, C. Yoshida, M. Anjum, L. Bodrossy, C. G. Clark, A.M. Kropinski, and M. A.
Karmali. 2011. Rapid Genoserotyping Tool for Classification of Salmonella Serovars. J. Clin. Micro
49:29542965.
3.Grimont,P.A.D.,andF.X.Weill(ed.).2007.AntigenicformulaeoftheSalmonellaserovars,9thed.WHO
Collaborating Centre for Reference and Research on Salmonella. Institut Pasteur, Paris, France.
http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s000036089.
4.Shipp,C.R.,andB.Rowe.1980.AmechanizedmicrotechniqueforSalmonellaserotyping.J.Clin.Pathol.
33:595597.

92
Table1:The58PrevalentSalmonellaserotypestargetedbytheSGSA(*Additionalisolatesoftheseserovars
weretestedforsensitivityandspecificitycalculations)
S.Abony S.Goldcoast S.Orion(incl.variants)

S.Agama S.Hadar* S.ParatyphiA
S.Agona S.Heidelberg* S.ParatyphiB(incl.Java)
S.Albany S.IIIb61:k:1,5,(7) S.Panama
S.Amsterdam S.Indiana S.Pullorum
S.Anatum S.Infantis* S.Rissen
S.Blockley S.Javiana S.Saintpaul
S.Bovismorbificans S.Kedougou S.Sandiego
S.Braenderup S.Kentucky* S.Schwarzengrund
S.Brandenberg S.Kiambu S.Senftenberg
S.Bredeney S.Kottbus S.Stanley
S.Cerro S.Livingstone S.Stanleyville
S.Chester S.London S.Tennessee
S.Choleraesuis S.Mbandaka S.Thompson
S.Corvallis S.Mississippi S.Typhi
S.Derby S.Montevideo* S.Typhimurium(incl.monophasicandvariants)*
S.Dublin S.Muenchen S.Virchow*
S.Enteritidis* S.Muenster S.Weltevreden
S.Gallinarum S.Newport*
S.Give S.Oranienburg

93
GenomicregionsofdifferencebetweenSalmonellaentericasubsp.enterica
serovarGallinarumbiovarGallinarumandbiovarPullorum
AngeloBERCHIERIJr.,DiegoFelipeAlvesBATISTA,PriscilaDinizLOPES,
AdrianaMariadeALMEIDAandOliveiroCaetanodeFREITASNETO
FacultyofAgricultureandVeterinaryScienceUnivEstadualPaulista,DepartmentofVeterinary
Pathology,LaboratoryofAvianPathology,Jaboticabal,SOPAULO,Brazil
INTRODUCTION
Salmonellaentericasubsp.entericaserovarGallinarumbiovarGallinarum(SG)andbiovarPullorum(SP)
aregeneticallyandphenotypicallysimilarbutarethecausativeoftwodistinctaviandiseases(3).Fowl
typhoidiscausedbySG,producinghighmortalityratesinbirdsofallages.SPisthecausativeofpullorum
disaese which is characterized by mortality mainly in young birds sometimes followed by persistent
infectioninadultswithverticaltransmission(2).SPandSGarewidelydistributedthroughouttheworld
andalthoughmanydevelopedcountrieshaveeradicatedthesebacteriafromcommercialpoultrythey
arestillthecauseofeconomiclossindevelopingcountries(2).Itislikelythatsomedifferencesbetween
pullorumdiseaseandfowltyphoidarecorrelatedwithdifferencesinthegenomesofSPandSG.Inorder
toinvestigatethishypothesisthewholegenomesequencesofSPandSGwerecompared.
MATERIALANDMETHODS
The whole genome sequences of SP (strain RKS5078 ; GenBank : CP003047.1) and SG (strain 287/91 ;
GenBank:AM933173.1)werecomparedusingtheArtemisComparisonTool(ACT)softwareandseveral
genomicregionsofdifference(RODs)wereidentified.Threeofthem,heredesignatedasROD14,ROD22
and ROD37, were chosen to be tested by PCR assay in order to check their conservation among the
isolates.FieldstrainsofSP(16strains)andSG(25strains)wereused.Theprimerssequenceusedwere
ROD14F (5CGCCCTCGCCAGTTCGATCC3), ROD14R (5CCCCAGCCAGAAGCACGCTC3), ROD22F (5
CTCCGCCCCTGCGGCTAAAG3), ROD22R (5GGGACGCGTAAACCCGGCAA3); ROD37F (5
CGGTGGCAGGAATGCTGGGG3)andROD37R(5CGCGCCGGTCGAATAGTCCC3).
The amplicons were analyzed by electrophoresis in a 1.5% (w/v) agarose gel stained with ethidium
bromide.
RESULTS
RODs14and37showedtobeconservedamongthetestedstrains(datanotshown).However,ROD22
waspresentonlyinfewisolatesofbiovarGallinarum(Figure1).
DISCUSSION
Theeraofgenomicanalysishasupdatedourunderstandingaboutgenecontentsofmicroorganisms.As
resulttheknowledgeaboutphysiologyandpathogenesisofbacteriahasbeendrasticallyimprovedinthe
lastdecade.InordertomakeuseoftheavailablegenomicinformationwecomparedthegenomeofSG
287/91andSPRKS5078andsoughtforRODs.AmongseveralRODsidentifiedthreeofthem,whichwere
thelargest,calledourattentionandwerechosentobeanalysed(ROD14and37whicharedeletionsin
SPandROD22whichisadeletioninSG).
RODs 14 and 37 were conserved among all tested strains what suggests that they could play a role in
pathogenesisoffowltyphoidandpullorumdiasease.TheROD14have11lociinside(SG1441SG1453)
and their gene content has similarity with genes from S. Enteritidis P125109 (99% of identity); one of
themencodesaninvasinlikeprotein.TheanalysisofGCcontentofthisRODshowedaGCpercentageof
47.13,similartowhatisfoundinSalmonellaPathogenecityIslands.
TheROD37encompassesfourloci(SG3608SG3611)whicharepartoftheoperontor.Thisoperonis
related to the use of trimethylamine Noxide (TMAO) as terminal electron acceptor in anaerobic
94
conditions (1). Studies in E. coli showed that genes from operon tor are actually expressed during
exponential growth phase under both anaerobic and aerobic conditions, likely to improve the ATP
generation. Furthermore, the activity of the Tor system (reducing TMAO to TMA) could be beneficial
especially in pH homeostasis. The absence of this operon in SP might disturb its survivor in acidic pH
what could be also disadvantageous when competing with other bacteria in environments containing
TMAO(1).
Finally,ROD22havethreelociabsentinSG287/91genome(SPUL_1297SPUL_1299)andtwoofthem
correspondtothemdtIandmdtJgenes.Theproteinsencodedbythesegenes,MdtIandMdtJ,belongto
SMR family (Small Multidrug Resistance) and are important for spermidine excretion. The
overaccumulation this substance is toxic and decrease cell viability by interaction with mRNA and
inhibition of protein synthesis (4). These proteins have been also related to sodium dodecyl sulfate
resistance(4).InterestinglythisRODwaspresentinmajorityofSGisolates(Figure1).Probably,thereis
anotherpathwaytoavoidoveraccumulationofspermidineoncethedeletionofthesegenesinsomeSG
isolatesseemsnotaffecttheproteinsynthesis.
CONCLUSIONS
TheconservationofRODs14and37inallSGandSPtestedstrainssuggeststhattheycouldplayarolein
pathogenesisoffowltyphoidandpullorumdiasease.
REFERENCES
1. Ansaldi, M., Thraulaz, L., Baraquet, C., Panis, G., Mjean, V. Aerobic TMAO respiration in Escherichia coli.
MolecularMicrobiology,Oxford,v.66,n.2,p.484494,2007.
2. Barrow,P.A.,FreitasNeto,O.C.Pullorumdiseaseandfowltyphoidnewthoughtsonolddiseases:areview.
AvianPathology,London,v.40,n.1,p.113,2011.
3. Berchieri Junior,A.;Freitas Neto,O.C.Salmoneloses.In:Berchieri Junior,A.;Silva,E.N.; Fbio,J. D.;Sesti,L.;
Zuanaze,M.A.F.(Eds.).DoenasdasAves.2.Ed.Campinas:FACTA,2009,p.435453.
4. Higashi, K., Ishigure, H., Demizu, R., Uemura, T., Nishino, K., Yamaguchi, A., Kashiwagi, K., Igarashi, K.
Identification of a Spermidine Excretion Protein Complex (MdtJI) in Escherichia coli. Journal of Bacteriology,
Washignton,v.190,n.3,p.872878,2008.
ACKNOWLEDGMENT
TheauthorswouldliketothanksFAPESP,CNPqandCAPESbyfinancialsupport.
FIGURES
Figure 1. ROD22 tested by PCR showing no conservation. M: Ladder 110 bp (Fermentas, US); G+: S. Gallinarum
positivecontrol(287/91);P+:S.Pullorumpositivecontrol;N:Negativecontrol;Numbers1to25:S.Gallinarumfield
isolates;Numbers26to42:S.Pullorumfieldisolates.

95
May 27-28-29, 2013
Saint-Malo France

Session 3


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

96
Session3.Detection,Identification,Quantification
PierreWATTIAU(Belgium)AnneBRISABOIS(France)
This session will deal with various methodological aspects developed for detecting and
characterizing Salmonella isolates. Since their early development in the first half of the 20
th
century, conventional serotyping and phage typing allowed longterm epidemiological

surveillance and characterization of most Salmonella isolates issuing from routine analyses.
Traditional culture methods also proved invaluable in detecting and quantifying Salmonella.
However,withtheeverincreasingqualitycriteriarequestedbythefoodchaincontrolandpublic
health sectors, alternative methodologies popped up aiming at detecting, identifying and
quantifying Salmonella in a fast, robust and traceable way and are continuously evolving.
Molecular characterization methods able to substitute or to complement traditional serotyping
and phage typing literally exploded during the last 20 years. Some of these deliver results
matching fairly well the current Salmonella classification based on serotyping. Some
fingerprinting methods reach or outcompete phage typing in terms of sensitivity, others allow
soundphylogeneticanalysestobeconducted.PCRbasedmethodssignificantlyreducedthetime
required to detect and quantify Salmonella in various raw or preenriched matrices. The use of
such alternative methods is however still precluded in contexts where strict observation of the
reference methodologies and identification rules are imposed.
DETECTION
Surveillance programs that timely detect Salmonella contaminations in the entire food chain
(animalfeed,livinganimals,slaughterhouses,retailsectorandrestaurants)togetherwithsanitary
measures are essential to detect and prevent human Salmonella infections. Therefore,
developmentofrapidandsensitivemethodsforthedetectionandcharacterizationofSalmonella
mayhaveasignificantimpactonthediseaseburdencausedbythispathogen.
For most of them, traditional detection methods start with preenrichment cultures in non
selectiveliquidmedia,possiblymodifiedtocompensateforthepresenceofinhibitorycompounds
in the sample matrix. Preenrichment cultures are then typically subcultured into selective
enrichment media (Rappaport Vassiliadis Soy RVS or MullerKauffmann Tetrathionate
NovobiocinMKTTnbroth)inwelldefinedincubationconditions.Enrichmentculturesarethen
usually inoculated on selective agar media. A number of selective chromogenic agar media
specifically designed for the differentiation of Salmonella colonies are commercially available.
TypicalSalmonellacoloniesonselectiveagarareeventuallysubculturedontononselectivemedia
prior to confirmatory testing. Traditional procedures may last up to 5 days before detection a
Salmonella isolate can be reported. A substantial number of alternative rapid screening methods
have therefore been developed. Many of these are available commercially and have been
successfully validated by official bodies (AOAC, AFNOR). Salmonella rapid test and screening kits
make use of numerous technologies including novel culture techniques, immunemagnetic
separation, enzyme immunoassay (EIA) and enzymelinked immunosorbent assay (ELISA), lateral
flowassays,DNAhybridisationandPCRbasedassays.Somemethodscanbeautomatedforhigh
throughputscreening.Almostallrapidprotocolsincludeaselectiveenrichmentstagefollowedby
detection techniques substituting culture on selective agars and subsequent confirmatory tests.
Most claim to be completed within approximately 2 days or less, depending on the enrichment
protocol. A number of presentations dealing with the setup, evaluation or validation of rapid
detectionmethodsarereportedinthenextpagesofthisconferenceproceeding.
97
IDENTIFICATION
Preliminary identification based on colony appearance on chromogenic and other selective agar
media is traditionally confirmed using classical biochemical and serological testing. Fermentation
ofglucoseanddulcitol,negativeureasereactionandindoletest,positivelysinedecarboxylaseand
optional H
2
S production are some of the numerous biochemical tests available. Many rapid
confirmationandidentificationmethodshavebeendevelopedforSalmonellaandconvertedinto
commercial products. Serological Salmonella spp. confirmation tests typically use polyvalent
antisera for flagellar (H) and somatic (O) antigens. Additional biochemical tests may be
required when serological results are inconclusive. Rapid immunological identification and
confirmation tests based on latex agglutination, EIA and ELISA have been developed as well and
simpletouse lateral flow assays were made commercially available by a number of
manufacturers. Specific DNA hybridisation and PCR assays for the identification of Salmonella
enterica are other technical options to confirm a Salmonella suspicion. These are generally
designed for use as part of a global method for rapid detection and screening rather than for
confirmation. When more specific identification is required, isolates are traditionally analysed by
serotyping in specialised (reference) laboratories where specific antisera are used. Such labs are
usually able to type isolates further using techniques such a phage typing, antimicrobial
susceptibilityandpulsedfieldgelelectrophoresis(PFGE).
Serotyping is a method in which surface antigens are identified based on slide agglutination
reactions with specific antibodies. The serotyping scheme, which is continuously updated as new
serovarsarediscovered,hasgeneratedovertimeadatasetoftheutmostsignificanceallowingfor
the longterm epidemiological surveillance of Salmonella in the food chain and in public health
control. Conceptually, serotyping provides no information regarding the phyletic relationships
inside the different Salmonella enterica subspecies. In epidemiological investigations,
identification and tracking of salmonellosis outbreaks require the use of methodologies that can
fingerprint the causative strains at a level far below the one achieved by serotyping. During the
last two decades, alternative methods emerged that could successfully identify serovars through
DNAbased testing. Molecularbiology based methods were made available to address the
phylogenyandfingerprintingissues.Atthesametime,accrediteddiagnosticbecameincreasingly
generalized, imposing strong methodological requirements in terms of traceability and
measurability. In these new contexts, the handcrafted character of manual serotyping is
challengedalthoughitiswidelyacceptedthatclassificationintoserovarsshouldbemaintained.
Among the numerous molecular typing methods, Pulse Field Gel Electrophoresis (PFGE) was
adaptedtoSalmonellainthe90sanddemonstrateditscapacitytoidentifystrainsattheoriginof
an outbreak. It became rapidly the gold standard for Salmonella molecular fingerprinting. During
the last two decades, surveillance techniques became easier following the development of
alternative methods DNAbased for most of them . These new methods improved the
performanceofthehistoricalmethodsbydisplayinghighersensitivityorfacilitatedtheanalysisby
requiringnospecifictechnicalexpertiseandbymakinguseofcommonlaboratoryreagents.
Molecularmethodsdevelopedasalternativestotraditionalserotypinghavegenerallyprovenvery
successful, though still await thorough validation for most of them. In contexts where strict
observation of the official methodologies is imposed, such alternativemethods often do not find
their way. Paradoxically, situations exist where official methodologies are ineffective in detecting
or identifying some peculiar Salmonella isolates (autoagglutinating or nonmotile) while
alternative nonofficial methodologies are successful. Whenever assessed by traditional or
modernmethodologies,thehistoricalclassificationintoserovarshasdemonstrateditsusefulness
over time and it is generally accepted that this classification should be maintained. Molecular
fingerprinting methods have proven highly valuable in characterizing and differentiating
98
Salmonella strains at a sensitive level for epidemiological studies. Housekeeping genes, virulence
determinants, antibiotic resistance markers, mobile genetic elements, prophage genomes and
sequence repeats have all been used as targets either alone or combined. To evaluate the
discriminationpowerreachedbythesemethods,PFGEisoftenusedasthereferencemethod.For
many of them, this power relies on the number of markers included in the assay and on the
capacity of the chosen platform to analyze them in a single run. In this respect, microarrays
achieved a significant breakthrough by allowing dozens of such markers to be analyzed at once.
Methodologicalchallengesremainformultiplemarkersamplificationandtyping.Besides,analysis
costsoftenprecludetheuseofmanysuchmethodsforroutineuse.
QUANTIFICATION
Official diagnostic does not specifically address the problem of Salmonella quantification.
Determining the number of viable bacterial cells can, however provide useful information for
assessingtheinfectiousriskassociatedtoaSalmonellacontaminatedsample.Suchquantification
data are also useful to establish doseresponse values as recently recommended for the
investigationoffoodbornecommunityoutbreakswhenattemptingtotacklemissinginformation
such as the amount consumed or population exposed [1]. Next to Salmonella positive / negative
detection,quantitativeanalysesmayhelpindirectlyinidentifyingthemostlikelyscenarioleading
to food contamination [2]. High Salmonella amounts in a food product more likely reflects
contamination at the primary production level, bad hygiene practices or cross contamination in
the distribution chain rather than other contamination routes. Quantification methods have to
cope with the very low amounts of Salmonella that are usually observed. MPN (Most Probable
Number) methods and miniaturized MPN methods were developed for this purpose [3]. Using
serialdilutionsofaninoculatedenrichmentbroth,suchmethodsenablethenumberofSalmonella
cells to be estimated for a given sample. Different protocols have been published using either
serologicalorDNAbasedmethodsforSalmonellaquantitativedetection.
CONCLUSION
Detection, quantification and identification methods are available to investigate Salmonella
contamination in almost any kind of sample. During the last two decades, improved
methodologies especially molecularbased enabled to reduce the analysis time, improved
sensitivity and circumvented some important drawbacks of conventional methods. The most
important improvements probably reside in the capacity to assign phenotypicallydefined
serotypesonthebasisofDNAbasedassaysandtofingerprintanyoutbreakorroutinefoodchain
isolate at an unprecedented sensitivity level. Most of these alternative methods still require
thorough validation but some have already shown very promising in achieving fast and reliable
diagnostic. The currently ongoing revision of the ISO 6579 official procedure may be an
opportunity to valorise the detection, quantification and/or identification capacity of the most
promisingmethods.
REFERENCES
1. TeunisTeunis,P.F.M.,Kasuga,F.,Fazil,A.,Ogden,I.D.,Rotariu,O.,Strachan,N.J.C.2010.Doseresponse
modelingofSalmonellausingoutbreakdata.InternationalJournalofFoodMicrobiology144,243249.
2. Tauxe, R.V., Doyle, M.P., Kuchenmller, T., Schlundt, J., Stein, C.E. 2010. Evolving public health
approaches to the global challenge of foodborne infections. International Journal of Food Microbiology
139,S16S28.
3. Fravalo, P., Hascot, Y., Fellic, M.L.E., Queguiner, S., Petton, J., Salvat, G. 2003. Convenient method for
rapid and quantitative assessment of Salmonella enterica contamination: The MiniMSRV MPN technique.
JournalofRapidMethodsandAutomationinMicrobiology11,8188.

99
May 27-28-29, 2013
Saint-Malo France

Session 3


Chairpersons:

Communications
100
Theeffectofpoolingofpoultrymeatsamples
onthedetectionofSalmonella
KirstenA.MOOIJMAN,WendyM.VANOVERBEEKandAnnemariePIELAAT
NationalInstituteforPublicHealthandtheEnvironment(RIVM),CentreforZoonoses
andEnvironmentalMicrobiology(Z&O),EURLSalmonella,BILTHOVEN,theNetherland
INTRODUCTION
According to the EC Regulation on microbiological criteria
7
and its amendment of 2011
8
, poultry
meatshouldbefreeofSalmonellaTyphimurium(includingmonophasicSalmonellaTyphimurium
1,4,[5],12:i:)andSalmonellaEnteritidis,tobetestedin5samplesofeach25gfreshpoultrymeat.
Tosavetimeandresources,laboratorieswouldliketotestthesefivesamplesasapoolinsteadof
analysing individual samples. It was questioned, however, whether pooling would affect the
sensitivity of the method for the detection of Salmonella. To investigate this, a study was
performed,based on a paper of Jarvis
9
and on a protocol for pooling of samples designed byan
ISOworkinggrouponstatisticsin2010.Thepaper,aswellastheprotocoldescribestwowaysof
pooling: dry pooling (pooling of sample units) and wet pooling (pooling of preenriched
cultures).BothwaysofpoolinganditseffectonthedetectionofSalmonellainpoultrymeatwere
tested in the experiments as described below. In these experiments, chicken meat, as well as
turkey meat, with and without skin were tested, to include a large variety of meat samples. The
meat samples were artificially contaminated with a sublethally injured Salmonella serovar, to
mimicreallifesamplesasmuchaspossible.
MATERIALSANDMETHODS
Poultrymeat
Six different batches of each of the following four types of poultry meat were tested: Chicken
meatwithoutskin;chickenbreastskin;turkeymeatwithoutskin;turkeybreastskin.
Before performing the pool experiments, the poultry meat was tested for the absence of
Salmonella, by testing three portions of 25 g per batch, following Annex D of ISO 6579
4
.
Furthermore, the amount of background flora was tested by analysing portions of 1015 g per
batchforthetotalaerobiccountandforthenumberofEnterobacteriaceae,followingrespectively
ISO4833
2
andISO215282
3
.
Salmonellastrainsandinjuryprocedures
Two different strains of each of the following three Salmonella serovars were used to artificially
contaminate the different batches of poultry meat: Salmonella Enteritidis (ATCC 13076 and a
strain isolated from poultry), Salmonella Typhimurium (ATCC 14028 and a strain isolated from
poultry), Salmonella Typhimurium monophasic variant 4,5,12:i: (two different strains, both
isolated from human faeces). For each pool experiment, each strain was cultured in Buffered
Peptone water (BPW) at 37 C, for approx. 18 h. Next tenfold dilutions were made in peptone
salinesolutionandaninjuryprocedurewasappliedtothesedilutions,basedontheinformationas
published in ISO/DIS 161402, Annex C
5
. Three different injury procedures were used, being: 15
min50C;storageat20Cforafewdaysupto2weeks;storageat4Cfor1to8weeks.After
applyinganinjuryprocedure,thedilutionswereplatedonanonselectiveagarmedium(Nutrient
agar:NA)andonaselectiveagarmedium(XyloseLysineDeoxycholateagar:XLD).Thedifference
incolonyformingunits(cfu)onthelogscale:log(cfu/mlonNA)log(cfu/mlonXLD),isameasure
fortheamountofinjury.AccordingtoISO/DIS161402,adifferenceofmorethan0,5log(cfu/ml)
isconsideredassufficientstress.Afterapplyinganinjuryprocedure,approximately110(injured)
cellsperstrainwereusedtoartificiallycontaminate25gofmatrix.

101
Salmonelladetectionmethods
ForthedetectionofSalmonella,theproceduresandthecompositionofthemediaasdescribedin
ISO6579
1
andinAnnexDofISO6579
4
werefollowed.Inshort:preenrichmentinBPWat37Cfor
18h, followedbyselectiveenrichment.ForISO6579,inRappaportVassiliadisSoyabroth(0.1 ml
BPW in 10 ml RVS; 41.5 C for 24h) and in Mueller Kauffmann Tetrathionate with novobiocine (1
ml BPW in 10 ml MKTTn; 37 C for 24h). For AnnexD of ISO 6579, on Modified Semisolid
RappaportVassiliadisagar(0.1mlBPWonMSRV;41.5Cfor24h).Next,thecultureswereplated
on XLD (37 C for 24h) and Brilliance Salmonella agar (Oxoid CM1092b), after which suspect
colonieswerebiochemicallyconfirmed.
Proceduresforpoolingofmeatsamples
Dry pooling: 25 g of meat was artificially contaminated with an injured Salmonella strain at a
level of 110 cfu. This sample was mixed with 4 x 25 g Salmonellafree meat (of the same batch)
and the 125 g pooled meat sample was added to 1125 ml BPW and incubated at 37 C for 18 h.
NexttheproceduresasdescribedinISO6579
1
4
werefollowed.
Wet pooling: 25 g of meat was artificially contaminated with an injured Salmonella strain at a
levelof110cfu,andaddedto225mlBPW.Furthermore,foursamplesof25gofSalmonellafree
meat(ofthesamebatch)wereeachaddedto225mlBPW.TheBPWsampleswereincubatedat
37C,for18h.Afterincubation,5mlwastakenfromeachBPWculture,combinedinatubeand
mixed.Fromthismixture,0.5mlwasaddedto50mlRVS,5mlwasaddedto50mlMKTTnand0.1
mlwasadded,inthreedrops,toaplateofMSRV.NexttheproceduresasdescribedinISO6579
1
4
werefollowed.
Nonpooledcontrolsample:theindividualartificiallycontaminatedsampleof25gwasalsotested
in the normal way (without pooling) for the presence of Salmonella by following ISO 6579 and
AnnexDofISO6579.
Intotal156poultrymeatsamplesweretestedforthepresenceofSalmonellaforallcombinations
ofpoultrymeatbatches,serovars,strainsandinjuryprocedures.Aseachsamplewastestedafter
dry pooling, wet pooling and as individual control sample with each three selective enrichment
media,thisresultedinadatasetof1404results(indicatingpresenceorabsenceofSalmonella).
Statisticalanalysis
DifferencesbetweensampletreatmentswereanalysedusingtheuncorrectedMcNemartestfor
assessing differences between binomial proportions based on paired data
10
. As of the large
numberofpossiblesampletreatmentcombinationsforhypothesistesting(4matrices,3serovars,
6strains,3typesofinjuries,3differentselectiveenrichmentmedia,2typesofpoolingmethods
and one control method), only the laboratory controllable experimental variables were used for
statisticalcomparison.Hereto,drypoolingwascomparedtothenonpooledcontrolsamplesand
wetpoolingwascomparedtothenonpooledcontrolsamples.Thiswasdonefor:
- all matrices, strains, injuries and selective enrichment media (RVS, MKTTn and MSRV)
together;
- all matrices, strains and injuries, but per selective enrichment medium (RVS, MKTTn and
MSRV);
- all strains, injuries and selective enrichment media (RVS, MKTTn and MSRV), but per type
ofmatrix(poultrymeatwithoutskinandpoultrymeatwithskin);
Furthermore, also a comparison was made between the results found with wet or dry pooling of
all matrices, strains and injuries when analysed following ISO 6579
1
(RVS and MKTT) or following
AnnexDofISO6579
4
(MSRV).

102
The BenjaminiHochberg procedure
6
was applied for controlling the false positive rate, i.e.
rejectingthe0hypothesiswhenitistrue(typeIerror),duetomultiplecomparisontesting.
RESULTS
Poultrymeat
Theamountofbackgroundfloravariedpertypeofmatrix(between10cfu/mland10
8
cfu/mlfor
the number of Enterobacteriaceae and between 10
2
cfu/ml and 10
9
cfu/ml for the number of
aerobicbacteria),butwasingeneralhigherforthepoultrymeatwithskin.
Salmonellastrainsandinjuryprotocol
The sensitivity to the injury procedure varied per strain, but could not be related to a specific
Salmonellaserovar.Theamountofstress(log.cfu/mlonNA)log.cfu/mlonXLD))variedlargely
betweenstrainsandinjuryprocedures.ThehighestamountofstresswascausedwhenSalmonella
strainswerestoredat20Cforseveraldays(upto1.3log.cfudifferencebetweencountsonNA
andXLD)orafterheatingat50Cfor15min(upto1.1log.cfudifference).Thelowestamountof
stress was caused by storage at 4C, even if this was done for several weeks (differences varied
between0and0.5log.cfu).
Poolingexperiments
The results of the wet pooled samples, as well as of the dry pooled samples, showed that the
highestnumberofpositivesampleswasfoundafterselectiveenrichmentonMSRV.Theseresults
were close to the number of positives found after analysing the individual nonpooled artificially
contaminatedsamples(controlsamples).Thelowestnumberofpositivesampleswasfoundafter
selectiveenrichmentinRVSincombinationwithdrypooling.
AsummaryofallresultsispresentedinFigure1.
Thestatisticalanalysisshowedsignificantdifferencesforthefollowingcomparisons:
- Dry pooling compared to the control results when testing all samples, serovars and
selective enrichment media together (nonpooled control samples showed significantly more
positiveresults);
- Dry pooling of all samples and serovars when using RVS, compared to the control results
withRVS(nonpooledcontrolsamplesshowedsignificantlymorepositiveresults);
- Drypoolingofthepoultrysampleswithskin(highestamountofbackgroundflora),tested
forallserovars,withallselectiveenrichmentmedia,comparedtothecontrolresults(nonpooled
controlsamplesshowedsignificantlymorepositiveresults);
- WetpoolinganddrypoolingofallsamplesandserovarswhenusingAnnexDofISO6579,
comparedtothesamepoolingexperimentswhenusingISO6579(forbothtypesofpooling,Annex
DshowedsignificantlymorepositiveresultsthanISO6579).
Forallothercomparisons,nosignificantdifferenceswerefound.
Figure1NumberofSalmonellapositiveresultsfoundafterdrypoolingorwetpoolingof5x25g
individual poultry meat samples (where only one individual sample was artificially contaminated
with 110 cfu/25 g) and after analysing the individual, nonpooled, artificially contaminated
samples(control).Intotal156poultrymeatsamplesweretestedforeachpoolingexperiment
103
CONCLUSIONS
TheresultsshowthatpoolingofpoultrymeatsamplesaffectsthedetectionofSalmonella,butthe
effect depends on the type of pooling, the amount of background flora in the meat and the
selective enrichment medium used. When the different variables (matrices, serovars, strains,
typesofinjuries,differentselectiveenrichmentmedia)werecombinedinthestatisticalanalysis,it
wasshownthatdrypoolingresultedinasignificantlylowernumberofpositiveSalmonellaresults
compared to the nonpooled control samples. However, after wet pooling no significant
differences in the number of Salmonella positive results compared to the nonpooled control
samples were seen. Although the selective enrichment medium MSRV is not the prescribed
medium for the detection of Salmonella in meat samples, it still gives the (significantly) highest
number of positive Salmonella results after dry pooling as well as after wet pooling of the meat
samples, when compared to the prescribed procedure of ISO 6579 (selective enrichment in RVS
andMKTTn).
ACKNOWLEDGEMENTS
ThepoultrymeatwaskindlyprovidedbythePlukonFoodGroup,Wezep,theNetherlands.
REFERENCES
1.Anonymous,2002.ISO6579.MicrobiologyoffoodandanimalfeedingstuffsHorizontalmethodforthe
detectionofSalmonellaspp.ISO,Geneva,Switzerland
2. Anonymous, 2003. ISO 4833:2003, Microbiology of food and animal feeding stuffs Horizontal method
fortheenumerationofmicroorganismsColonycounttechniqueat30C.ISO,Geneva,Switzerland.
3. Anonymous, 2004. ISO 215282:2004, Microbiology of food and animal feeding stuffs Horizontal
method for the detection and enumeration of Enterobacteriaceae Part 2: Colonycount. ISO, Geneva,
Switzerland.
4. Anonymous, 2007. ISO 6579: 2002/ Amd1. Microbiology of food and animal feeding stuffs Horizontal
methodforthedetectionofSalmonellaspp.AnnexD:DetectionofSalmonellaspp.inanimalfaecesandin
environmentalsamplesfromtheprimaryproductionstage.ISO,Geneva,Switzerland
5.Anonymous,2013.ISO/DIS161402MicrobiologyoffoodandanimalfeedMethodvalidationPart2:
Protocol for the validation of alternative (proprietary) methods against a reference method. ISO, Geneva,
Switzerland.
6.Benjamini,Y.,2010.Discoveringthefalsediscoveryrate.J.R.Statist.Soc.B72,Part4,405416.
7. EC, 2005. European Regulation EC No 2073/2005 of the European Parliament and of the Council of 15
November 2005, on microbiological criteria for foodstuffs. Official Journal of the European Union L 338:
22.12.2005. http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2005:338:0001:0026:EN:PDF
(visited18022013)
8.EC,2011.No1086/2011EuropeanRegulation(EU)No1086/2011amendingAnnexIItoRegulation(EC)
No 2160/2003 of the European Parliament and of the Council and Annex I to Commission Regulation (EC)
No 2073/2005 as regards salmonella in fresh poultry meat. Official Journal of the European Union L 281:
28.10.2011. http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2011:281:0007:0011:EN:PDF
(visited18022013)
9. Jarvis, B., 2007. On the compositing of samples for qualitative microbiological testing. Lett. Appl.
Microbiol.,45,592598.
10.Newcombe,R.G.,1998.Improvedconfidenceintervalsforthedifferencebetweenbinomialproportions
basedonpaireddata.Statist.Med.17,26352650.

104
Impactofroutineuseoftwonovelrealtimepolymerasechainreaction
assaysforidentificationofSalmonellaentericasubspecies
withinaNationalReferenceLaboratory
TansyPETERS,DavidPOWELL,SteveCONNELL,MartinDAYandElizabethDEPINNA
PublicHealthEngland,MicrobiologyServicesDivision,Colindale,LONDON,UnitedKingdom
ABSTRACT
This study evaluates the turnaround times (TATs) of a large set of samples analyzed with two novel real-time
(RT)-PCR assays and compares the results to a similar set previously subspeciated using traditional
biochemistry. Identification by biochemical methods typically takes 27 days but up to 28 days to perform. This
compares to the RT-PCR assays which allow identification of subspecies I, IIIa and IIIb in approximately 99%
of Salmonella isolates within 2 h of receiving the original culture. Between June 2009 June 2010 the average
TAT for isolates sent to our laboratory for serotyping only was 21.7 days whereas isolates that required phage
typing or a combination of phage and serotyping had TATs of 17.2 days. For this period, serological typing
necessitated subspeciation using traditional biochemical techniques. From July 2011 June 2012 the RT-PCR
assays have been in routine use as a direct replacement for most biochemical tests. We have since seen a
reduction in TATs in a similarly matched sample set. Isolates that are only phage typed have an average TAT
of 6 days pre- and post-PCR implementation while isolates that require a serotyping step now have average
TATs between 16.6 17.5 days dependent on the inclusion or exclusion of a phage typing step. Therefore
implementation of the RT-PCR assays has shown a substantial impact on timely reporting in our laboratory
with the potential for further improvement.
INTRODUCTION
In most reference laboratories accurate subspecies identification of Salmonella is vital for
epidemiological purposes. Currently, the gold standard for Salmonella identification is based on
the differing biochemical phenotypes of each subspecies [Grimont and Wiell, 2007]]. Tubebased
tests are typically used to determine the organisms ability to ferment sugars such as dulcitol,
lactose and salicin, to utilize malonate, to liquefy gelatine, to hydrolyse OnitrophenylD
galactopyranoside(ONPG)ortodecarboxylatelysine,amongotherbiochemicaltests.
However, these tests are labourintensive and timeconsuming procedures often requiring long
incubationperiods.Fewalternativemethodsforsubspeciesidentificationhaveyetbeenpublished
[Dieckmann et al., 2008, Lee et al., 2009 and Park et al., 2009 ] and these often lack extensive
validation of the sensitivity and accuracy of the assay, require specialist equipment, or are
unsuitableforthehighthroughputrequiredinabusyreferencelaboratory.
Here we describe the hierarchical use of two (TaqMan) realtime polymerase chain reaction
(PCR) assays as an alternative to traditional biochemical identification methods. The first assay
(Hopkinsetal,2011)isusedfortheidentificationofS.entericasubspeciesenterica(subspeciesI)
and targets the Salmonella pathogenicity island 1 (SPI1) hilA gene, encoding an invasion gene
transcriptional activator. A second duplex PCR assay (Hopkins et al, 2009) identifies S. arizonae
and diarizonae (subspecies III) and targets the LacZ gene involved in hydrolysis of ONPG by
galactosidase. This duplex assay includes a second primer/TaqMan probe set targeted to the
ttrRSBCA locus, required for tetrathionate respiration. As this has previously been shown to be
suitable for the detection of all Salmonella it is utilised here to act as an internal amplification
controlandconfirmthepresenceorabsenceofSalmonellaDNA.
Both assays were previously validated inhouse on an ABI Prism 7500 RealTime PCR System
(AppliedBiosystems;Warrington,UK)usingalargesetofsamples(n=>2000).
Routinecalculationofturnaroundtimes(TATs)forprocessingandtypingSalmonellaisolatesinour
laboratoryarecalculatedonamonthlybasisandmatchedagainstapublishedtargetinourService
105
User Manual. The TAT is the difference, in days, between the date a sample is received by the
Health Protection Agency and the date that the result of the reference typing is issued. In any
given month, 75% of samples reported are required to meet the target TAT. During the period
prior to implementation of the RTPCR assays the published target TAT for phage typing only,
without biochemical analysis, was 20 days, for serotyping the target TAT was 25 days and for a
combinationofbothteststhetargetTATwas27days.
SincetheimplementationoftheseRTPCRassaysinourlaboratoryinJune2011therehasbeena
substantial impact on timely reporting of isolates with a noticable reduction in the average
turnaroundtimes(TATs)toreporting.Bothassaysarenowinroutineuseasadirectreplacement
formostbiochemicalteststraditionallyusedinSalmonellasubspeciesidentification.
MATERIALANDMETHODS
Boiled cell lysates were prepared by lightly touching a colony/area of growth of suspected
Salmonella from the original culture and carefully emulsifying the material in 8strip PCR tubes
containing 100 l of sterile distilled water. These were boiled for 10 minutes to form the cell
lysates which were used as template DNA. Both assays were performed as per Hopkins et al
(2009, 2011) on an ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems,
Warrington,UK)in25Lvolumereactionscontaining1FastBlueqPCRMasterMixPlusLowROX
(Eurogentec, Seraing, Belgium). PCR products were detected directly by the TaqMan machine
which monitored the increase in fluorescence where a numerical value was assigned i.e. the CT
value(thresholdcycle).Thisindicatedthecyclenumberatwhichmeasuredfluorescenceincreased
abovecalculatedbackgroundfluorescenceandindicatedamplificationoftargetsequences.
The monoplex hilA assay was performed first as this targeted subspecies I, the most common
Salmonellasubspecies.IsolatesthatgavenegativeresultsforthehilAtargetwereretestedwith
the duplex LacZ/ttr assay as subspecies III is the next most common subspecies identified in our
laboratory.
During the period of this analysis the TATs were measured for two workflows: isolates that were
serotypedandisolatesthatwerephagetyped.Alargeproportionofsampleswerebothserotyped
andphagetypedand,ofthese,almostallwerefirstlyserotyped,thenphagetyped.Inthesecases,
where the result of the serotyping component was reported separately (i.e. before) the result of
the phage typing component, the turnaround times for the serotyping and phage typing
componentswerecalculatedseparately.
Furthermore, the workflow for typing Salmonella Typhimurium isolates lengthened significantly
betweenthetwoperiodsoftheanalysis.As21.4%ofsamplesinthepreandpostimplementation
groups were typed as S. Typhimurium. further analysis was carried out, which excluded any
samplesthatwereintheworkflowforS.Typhimurium.
The monoplex assay targeting the hilA gene demonstrates 97.9% specificity and 99.9% sensitivity
foridentificationofSalmonellasubspeciesIwhiletheduplexLacZ/ttrassayshows100%specificity
and 99.6% sensitivity for subspecies IIIa and IIIb. Together these assays allow subspecies
identification in approximately 99% of Salmonella isolates within a few hours of receiving the
original culture. In addition, the assays offer an estimated 70% savings in reagent costs, with no
needtoutiliseawiderangeoftubebasedbiochemicaltests.
Periods before and after the implementation of the RTPCR assay were compared to assess the
impactofthenewmethodontheTATsforsamplesreferredtothelaboratory.Thechosenperiod
for the prePCRimplementation was 1 July 2009 to 30 June 2010, immediately prior to the
implementation of the PCR assays. For this period, serological typing necessitated subspeciation
using traditional biochemical techniques (n=9,341). The period for the postPCRimplementation
group was 1 July 2011 to 30 June 2012 (n=7,547). Samples received between these two periods
106
wereexcludedasthenewmethodwasbeingintegratedintotheroutinelaboratoryworkflow.As
such the TATs for these latter samples may not be reflective of those for the fullyintegrated
method. From midJune 2011 onwards the RTPCR assays have been in routine use as a direct
replacementformostbiochemicaltests.
Theseperiodsabovewerealsochosentobeeasilycomparable;onefullyeareachforthepreand
postimplementation groups. There is a distinct seasonal variation in the number of samples
received for testing and this in itself has an impact on the turnaround times of the laboratory.
Comparison of the overall turnaround times for the analysis periods calculated using the routine
method,showedadecreaseintheturnaroundtimesbetweenthepreandpostimplementation
periods(Table1).
ItisworthnotingthatfromJanuary2012therewasachangeofpracticewithinthelaboratoryfor
the typing of Salmonella Typhimurium isolates. This serogroup previously fell into the subset of
isolates that were only phage typed without the need for biochemical analysis or RTPCR. Since
this date all S. Typhimurium are typed using a combination of phage and serotyping in order to
determinetheirfullantigenicstructureandthuswhichisolatesaremonophasic.Thishasledtoa
considerable increase in the number of isolates that require full serological identification.
However,thepotentialimpactthismayhavehadupontheTATshasnotbeenrealiseddueinpart
to the use of the RTPCR assays. For phage typable isolates that did not require either a
biochemicalsteporanRTPCRsteptherehasbeennosignificantchangeintheirTATsforthepre
andpostPCRimplementationperiods(Table2).
In view of these results, the following revised target TATs were proposed for our Service User
Manualwitheffectfrom1
st
January2013:Salmonellaidentificationandserotyping13days,phage
typingonly8days,andforacombinationofbothteststhetargetTATis21days.
ThereforewithinthelaboratoryworkflowthehierarchicaluseofbothTPCRassaysforSalmonella
subspeciation have demonstrated a real impovement over the previously used traditional
biochemical tests. We have been able to demonstrate a system for more timely reporting within
ourlaboratorywiththepotentialforfurtherimprovement.
REFERENCES
1. R.Dieckmann,R.Helmuth,M.Erhard,B.Malorny.Rapidclassificationandidentificationofsalmonellae
atthespeciesandsubspecieslevelsbywholecellmatrixassistedlaserdesorptionionizationtimeofflight
massspectrometry.Appl.Environ.Microbiol.,74(2008),pp.77677778
2. P.A. Grimont, F.X. Weill . Antigenic formulae of the Salmonella serovars (9th ed.) WHO Collaborating
CentreforReferenceandResearchonSalmonella,InstitutPasteur,Paris(2007)
3. K.L. Hopkins, T.M. Peters, A.J. Lawson, R.J. Owen. Rapid identification of Salmonella enterica subsp.
arizonaeandS.entericasubsp.diarizonaebyrealtimepolymerasechainreaction.Diagn.Microbiol.Infect.
Dis.,64(2009),pp.452454
4. K.L. Hopkins, A.J. Lawson , S. Connell, T.M. Peters, E. de Pinna. A novel realtime polymerase chain
reaction for identification of Salmonella enterica subspecies enterica. Diagn Microbiol Infect Dis. 2011
Jun;70(2):27880.
5. K. Lee, T. Iwata, M. Shimizu, T. Taniguchi, A. Nakadai, Y. Hirota, H. Hayashidani. A novel multiplex PCR
assayforSalmonellasubspeciesidentification.J.Appl.Microbiol.,107(2009),pp.805811
6. S.H.Park,H.J.Kim,W.H.Cho,J.H.Kim,M.H.Oh,S.H.Kim,B.K.Lee,S.C.Ricke,H.Y.Kim.Identificationof
Salmonella enterica subspecies I, Salmonella enterica serovars Typhimurium Enteritidis and Typhi using
multiplexPCR.FEMSMicrobiol.Lett.,301(2009),pp.137146
107
MolecularcategorizationofSalmonellaentericaisolated
frombovinelymphnodes
MarieBUGAREL,KendraK.NIGHTINGALE,SaraE.GRAGG,MindyM.BRASHEARS
andGuyH.LONERAGAN
TexasTechUniversity,LUBBOCK,Texas,UnitedStates
ABSTRACT
It is not uncommon to recover Salmonella from bovine peripheral lymph nodes. Because lymph nodes are
intimately associated with beef, they are frequently incorporated into ground beef and represent a potential
source of human exposure to Salmonella. This study provides a molecular characterization of Salmonella
strains from various bovine lymph nodes in comparison to strains from bovine hides and faeces. Based on the
presence of four genes encoding fimbrial operons, we were able to categorize Salmonella population into 3
clades, A, AB and B. The clades A and B have been previously defined as distinct subpopulations that likely
differ in ecology and transmission characteristics. Clade distributions are similar in lymph nodes and faeces
and hide. The clade A is the most prevalent and represents about 60% of the strains whereas the clade B
represents 30% and 36.2% of the strains isolated from lymph nodes and faeces and hide, respectively.
Furthermore, we found an important diversity in clade distribution especially within serotypes Anatum and
Montevideo. Montevideo is the most prevalent serotype in lymph nodes and seems mainly associated to clade
B. Interestingly, this clade is characterized by a gene repertoire that may facilitate survival among various
ecological niches including insects. The presence of clade B strains in bovine lymph nodes suggests that
insects may represent a transmission route for bovine infection by Salmonella, but the serotype-related
distribution of the clade suggest that the colonization and survival of Salmonella in the cattle lymph nodes may
be associated to serotype-specific characteristics.
INTRODUCTION
Salmonella is an important cause of foodborne disease worldwide. While poultry and
contaminatedfreshproductsarewellcharacterizedvectorsforSalmonellaenterica,undercooked
ground beef and beef products can be sources of sporadic cases and outbreaks of salmonellosis.
Principal sources of S. enterica in beef processing chain are hide and lymph nodes (1). Lymph
nodes have important immune capture functions that include the sequestration of bacteria,
viruses, and other infectious agents to be eventually destroyed by lymphocytes. Cattle possess
numerous lymph nodes located within fatty tissues that are frequently incorporated into ground
beef. Thus lymph nodes if contaminated with Salmonella can be responsible of the
contamination of the final product. The purpose of this study was to provide a molecular
characterizationofSalmonellastrainsisolatedfromvariousbovinelymphnodesincomparisonto
strains isolated from bovine hides and faeces. We investigated the distribution of 4 genes
highlighted by den Bakker and collaborators (2) as relevant markers of two major subdivisions of
Salmonella enterica species. These markers target genes encoding on different fimbrial operons.
These 2 subgroups of Salmonella population differ based on the presence of genes involved in
adhesion, colonization and metabolic capabilities. Therefore these subpopulations differ in
ecologyandtransmissioncharacteristics.
MATERIALANDMETHODS
Strain collection: Three hundred and eightysix strains of Salmonella enterica were investigated.
Three hundred and nine strains were isolated from American cattle; 223 isolates from subiliac
lymph nodes and 86 belonging to faeces samples (3). Seventyseven strains were isolated from
Mexican cattle; 16 from subiliac lymph nodes, 15 from mesenteric lymph nodes, 16 from
mandibular lymph nodes, 14 from hide and 16 from faeces samples. Isolates were serotyped
accordingtoamolecularmethod,followingHerreraLeon(4)protocol.
108
RealtimePCRcharacterization:Toinvestigatethepresenceofthe4geneticmarkers,therealtime
PCR method was employed using a Stratagene Mx3005P (Agilent Technologies, Santa Clara,
California). PCR assays were performed in SYBR Green technology using Brilliant II SYBR Green
QPCRMasterMix(AgilentTechnologies).Standardcyclingconditions(15secat94C,1minat60C
for35cycles)wereperformed.
RESULTS
Determinationoftheclades
Based on the clade characterization performed by den Bakker and collaborators (2), we
investigated the distribution of 4 genes: stfE and lpfB as markers of the clade A, and vep and
sfa1273 as markers of the clade B. The combination of presence/absence of these four markers
identifies7differentpatterns,whichweregroupedinto3clades(Table1).
stfE lpfB vep sfa1273

C
l
a
d
e
A1 +
A2 + +
C
l
a
d
e
A
B
AB1 + + +
AB2 + + + +
AB3 + + +
AB4 + + +
CladeB + +
Table1:Identificationof7differentpatternsaccording
tothedistributionofthestfE,lpfB,vepandsfa1273genes.
CladeAincluded2groups,A1andA2,carryingoneorbothcladeAspecificmarkers.
CladeBpatternharboursthetwocladeBspecificmarkers,vepandsfa1273.
Clade AB is composed of 4 different patterns carrying a combination of 3 to 4 investigated
markers.ThiscladerepresentsanintermediatepatternofcladesAandB.
Distributionoftheclades
Withinourcollection,69.9%(n=270)ofthestrainswereisolatedfromlymphnodes.Withinthese
isolates, 60% were characterized as belonging to clade A, 10% to clade AB and 30% to clade B
(Figure1).ThemostprevalentpatterncarriedbythelymphnodesstrainsistheA2pattern(Table
2).
Figure1:Distributionofthecladesaccordingtotheisolationsource.
About30%(n=116)oftheinvestigatedstrainshavebeenisolatedfromfaecesorhidesamples.Withinthese
strains,60.3%weredefinedasbelongingtocladeA,3.5%tocladeABand36.2%tocladeB(Figure1).The
twomostprevalentpatternsofthestrainsbelongingtofaecesandhideareA2andB(Table2).
109
Prevalence in
lymphnodes
Prevalence in
faecesandhide
A1 25.9% 24.1%
A2 34.1% 36.2%
AB1 5.2% 2.6%
AB2 0.7% 0.9%
AB3 3.7% 0%
AB4 0.4% 0%
B 30% 36.2%
Table2:Distributionofthestrainsisolatedfromlymphnodesorfaecesandhideintothedifferentclades.
Within strains isolated from faeces or hide samples, AB3 and AB4 patterns had not been
recovered.Thesepatternsarealsoinfrequentlyisolatedfromlymphnodes.
StrainsdescribedasbelongingtocladeABrepresentonly8%(n=31)ofalltheinvestigatedstrains,
and10%and3.5%ofthelymphnodes,andfaecesandhidesamples,respectively.
Intraserotypevariability
Serotypes N
Isolation
source
Clades (%)
A AB B
Montevideo 144 LN 22.9 5.6 43.1
F 0.7 27.7
Anatum 88 LN 67.1 3.4 1.1
Fand H 27.3 1.1
Kentucky 40 LN 40
Fand H 60
Meleagridis 20 LN 10 65
F 20 5
Reading 14 LN 85.8
Fand H 14.2
Cerro 8 LN 50
Fand H 50
Mbandaka 8 LN 75
F 25
Ohio
a
8 LN 87.5 12.5
Infantis 5 LN 40
F 60
Lille 4 F 100
Bredeney 3 LN 100
Muenchen 3 LN 33.3
F 66.7
Muenchen
b
3 LN 100
O7:NT 3 LN 100
Nontypeable 3 LN 33.3
Fand H 66.7
Braenderup 2 LN 100
Brandenburg 2 LN 100
Give 2 LN 100
Newport 2 LN 100
Derby 1 F 100
Manhattan 1 F 100
Muenster 1 LN 100
Panama 1 LN 100
Uganda 1 LN 100
Javiana
c
1 LN 100
Table 3: Intraserotype variability. Distribution of the strains according to their clade pattern and their
isolation source within a same serotype. LN means lymph node, F means faeces and H means hide.
IsolatesdesignatedasOhio
a
maybefromserotypesOhio(6,7,14:b:l,w)orThompson(6,7,14:k:1,5);isolates
110
designated as Muenchen
b
may be Muenchen (6,8:d:1,2) or Virginia (8:d:1,2); and isolates designated as
Javiana
c
maybeJaviana(1,9,12:l,z
28
:1,5)orPanama(1,9,12:l,v:1,5).
Within our collection, the serotype Montevideo is the most frequently encountered. Strains
belongingtothisserotyperepresent37.3%oftheinvestigatedstrains.TheserotypeMontevideois
the most prevalent within lymph nodes (41%) and faeces and hide (35.3%) isolated strains,
followed by the serotype Anatum. This serotype is the second most prevalent serotype in our
collection (22.8%), within the lymph node isolates (26%) and within the nonlymph node isolates
(21.6%;Table3).
Withinthesetwomainprevalentserotypes,cladesA,ABandBhavebeenobserved.However,the
serotype Montevideo is mainly associated with the clade B; more than 70% of the Montevideo
strainswithoutdistinctionoftheirisolationsourcearefromcladeB.Incontrast,serotypeAnatum
ismainlyassociatedwithcladeA;morethan94%ofthestrainsofthisserotypecarryoneorboth
typicalmarkersofthecladeA(Table3).
Kentucky is the third most prevalent serotype in our collection. In contrast to the diversity
highlightedwithintheserotypesMontevideoandAnatum,alltheKentuckystrainsareassociated
to clade A; and more precisely these strains carry stfE and lpfB markers, typical pattern of the
cladeA2subgroup.
On the other hand, serotype Meleagridis is the only serotype encountered in this study that is
mainly associated to the clade AB (85%). The typical Meleagridis pattern is positive for stfE, lpfB
andvepmarkers(patternAB1).ThispatternismostlyassociatedtotheserotypeMeleagridis;93%
ofthestrainscarryingthispatternbelongtoserotypeMeleagridis(n=13/14).Theremainingstrain
carryingAB1patternisfromserotypeAnatum.
Except for serotype Ohio, all the other serotypes are related to a single clade. This weak intra
serotypediversitymaybepartlyduetolownumberofisolatedstrainsoftheseserotypes.Forthe
strains characterized as Ohio, the performed serotyping determination didnt provide a clear
antigenicformula.ThesestrainscanbefromserotypesOhioorThompson,whichcanexplainthe
presenceof2cladesinthisgroup.
Finally, within serotype Anatum, 93.7% and 96% of the strains isolated from lymph nodes, and
from faeces and hide, respectively, are associated to clade A. Within serotype Montevideo, 60%
and98%ofthestrainsisolatedfromlymphnodes,andfromfaecesandhide,respectively,belong
to cladeB.These results suggest that theisolation source does not have animportant impacton
theintraserotypecladedistribution.
DISCUSSION
Based on the conclusions of den Bakker, we decided to characterize our strains isolated from
cattle lymph nodes, faeces and hide, in order to identify tendency in the distribution of the
previouslydefinedclades.
We investigated 4 genetic markers, stfE, lpfB, vep and sfa1278, highlighted as closely involved in
the clade categorization. In his study, den Bakker (2) identify 2 major clades, called A and B. The
clade A is mainly defined by the pattern stfE+, lpfB+, vep and sfa1278, whereas the clade B is
mainlydefinedasstfE,lpfB,vep+andsfa1278+.
Inthisstudy,aminorclade,calledTyphiclade,hasbeenhighlightedwithinthemajorcladeA.This
minorcladeisdefinedbythestfE+,lpfB,vepandsfa1278pattern.Inourstudy,thissubcladeis
also a subgroup of the cladeA and has beencalled A1. In den Bakkerstudy (2), this subcladeis
composed of strains belonging to serotypes Typhi, ParatyphiA, Adelaide and Mississippi. In our
characterization study, we did not identify these serotypes, but we identified other serotypes
carrying this pattern. We found few strains of serotype Montevideo (2.1% of all the Montevideo
strains), few Meleagridis strains (5%), most of the Anatum strains (88.7%) and one strain of
serotype Uganda. In den Bakker study (2), one strain of Uganda serotype has been investigated.
111
Basedontheiranalysisof93genomicloci,UgandastrainisclassifiedinthecladeAandnotinthe
Typhi clade, however this strain harbours the stfE+, lpfB, vep and sfa1278 pattern as in our
study.
In the den Bakker clade A, 2 serotypes do not harbour the same typical four genes pattern:
EnteritidisandNewport.ThesestrainscarrythestfE+,lpfB+,vep+,sfa1278pattern,calledinour
studyAB1.Inourstudy,thetwoinvestigatedNewportstrainsdonotharbourtheAB1patternbut
thetypicalA2patternofcladeA.
However, we identified other serotypes carrying the AB1 pattern. We found one Anatum strain
(1.1%ofalltheAnatumstrains),andwehighlightedthestrongassociationofthispatternwiththe
serotypeMeleagridis.Inourstudy,80%oftheMeleagridisstrainscarrytheAB1pattern.
We investigated few other serotypes in common with den Bakker study (2). For strains of
serotypes Kentucky, Give, and Javiana, we found similar patterns. For the serotype Montevideo,
our study highlighted a more important diversity in the pattern distribution. In den Bakker study
(2), the only investigated Montevideo strain is classified in clade B, as 70.8% of our Montevideo
strains. However, by investigating a large number of strains, we were able to identify 6 of the 7
differentpatternswithinthisserotype.
On the other hand, our study highlighted that the clade distribution seems more related to the
serotypethantotheisolationorigin.Thegeneticpatternofcladessuggeststhatinsectsmayplaya
role as alternate hosts for clade B strains (2). Our hypothesis was that the cattle lymph nodes
infectionmightbemediatedbyinsects.However,ourstudyrevealedthatthecladeBpresentsthe
same prevalence in strains isolated from lymph nodes, or faeces and hide. Nevertheless, we
highlighted that 84% of the clade B strains isolated from lymph nodes belong to serotype
Montevideo, which is the most prevalent serotype in the cattle lymph nodes. So maybe the
colonization and survival of Salmonella within cattle lymph nodes is associated with serotype
specific characteristics beyond those described herein. Further studies are needed to better
characterizethesestrainsisolatedfromlymphnodes.
REFERENCES
1. Koohmaraieetal.,JournalofFoodProtection,2012,75:8.
2. denBakkeretal.,BMCGenomics,2011,12:425.
3. Graggetal.,2013,Inpress.
4. HerreraLeonetal.,JournalofClinicalMicrobiology,2004,42.

112
MultiplelociVNTRAnalysis(MLVA)formolecularcharacterization
ofthemonophasicvariantsofSalmonellaentericaserotypeTyphimurium
outbreaksoccurredin2011
SabrinaCADELSIX
1
,BenjaminGLASSET
1
,RenaudLAILLER
1
,SophieGRANIER
1
andAnneBRISABOIS
1
1
Anses,FrenchAgencyforFood,EnvironmentalandOccupationalHealth&Safety.
MaisonsAlfortLaboratoryforFoodSafety,UnitBacterialCharacterizationandEpidemiology,
23avenueduGnraldeGaulle,F94706MAISONSALFORTCedex,France
INTRODUCTION
The genus Salmonella is one of the most prevalent pathogens involved in foodborne infections
worldwide. The Salmonella surveillance system in France detecting Salmonella (especially
serotypes Typhimurium, Enteritidis, monophasic variants etc...) in animals, food, and in the
environment is coordinated in part by the Salmonella network of the ANSES. One of the main
goals of this network is to collect and characterize Salmonella isolates on a national level to
identify and trace them. Several subtyping techniques are employed; among them the Multi
Locus VNTR Analysis (MLVA) is a molecular typing method which targets a variable number of
short tandem repeats. This technique used both at the NRC (National Reference Centre for
Salmonella, Institut Pasteur) and in CEB Unit is known to have an excellent power of
discrimination,lowcosts,reliableandhighlyreproducibility.
TheaimofthisstudywastocomparetheperformanceandtheabilityoftheMLVAandthePFGE
methods for epidemiological investigations of the emerging monophasic variants of S.
Typhimurium strains recovered during two periods of 2011, August/September and
November/December,duringwhichtwofoodborneoutbreakswerenotifiedbytheInVSandNRC
[1].
MATERIALSANDMETHODS
Bacterialstrainsandculturegrowthconditions
A collectionof 210 isolates of monophasic variants of Salmonellaenterica serotype Typhimurium
received at the Salmonella network from July 15 to December 31 2011 was selected . This set of
isolates (different and independent) was obtained after elimination of duplicates from a total of
338isolatesinventoriedin2011.Theepidemiologicaldataespeciallyanimalorfoodsourceswere
enteredintheSalmonellaAccessdatabaseoftheCEBunit(Figure1).
Monophasic variant strains S. 4,[5],12:i: were identified with conventional serotyping by slide
agglutination and confirmed with EFSAbased PCR. Whole strains were conserved either in 3 ml
stockcultureagar(BioRad,MarneslaCoquette,France)atenvironmentaltemperatureorincryo
pearls (Fisher Scientific, Illkirch, France) at 80C. The isolates were cultured on nutrient agar
TSAYE (Biokar Diagnostics, Beauvais, France) and incubated overnight aerobically at 37C before
PFGEandMLVAtyping.
PFGEtyping
Total DNA of the strain was extracted and digested with the enzyme XbaI (Roche, France). PFGE
was performed according to a standardized protocol of PulseNet EU (2009) [2]. Analysis of
genomic profiles was performed using BioNumerics software (Applied Maths, Belgium) and a
comparison of profiles was made by constructing a dendrogram (Dice coefficients, the UPGMA
methodandpositiontolerancesetat1%).

113
MLVAtyping
The InstaGene matrix (BioRad, France) was used to extract the DNA. The MLVA typing was
performed using previously described protocol [3]. The analyses were realized on an AB3500
capillary electrophoresis (Applied Biosystems, France) spectrally calibrated to run filter set G5.
Data were imported into GeneMapper software (Applied Biosystems, France) and normalized
following the instruction proposed by Larsson and Nielsen of Serum Staten Institute of Denmark
(2009) (raw results vs corrected results) [4]. The MLVA profiles were imported into Bionumerics
software version 6.6 (Applied Maths, Belgium) as categorical data. Standard minimum spanning
trees (MST), generated in Bionumerics using the single and double locus variance priority rules,
wereusedtovisualizetherelationshipsbetweenstrains.
Antimicrobialresistancestudy
StrainsweretestedontheirantimicrobialsusceptibilityusingthediscdiffusionmethodonMueller
Hintonagar.InhibitiondiameterswereautomaticallyreadontheOsirissystem.
RESULTS
The diagram in Figure 1 indicates the distribution of the isolates among the animal sectors. The
isolatesofmonophasicvariantofS.Typhimuriumweresignificantlypresentintheporcinesector
(18%), almost as much as in the poultry one (hen and chicken, poultry, turkey and duck). The
majorityoftheisolatescollectedfromJulytoDecember2011weregatheredinthreestepsofthe
food chain: the processing or retail (40%), the farm environment (21%) and the slaughterhouse
(19%).
For10%oftheisolatesnodatawereavailable.
Altogether 32 different PFGE profiles have been found among the 210 isolates of monophasic
variant of S. Typhimurium. The most frequent profile was STYMXB0126 which represent 62% of
theisolatesanalyzedandthesecondonewastheprofileSTYMXB0060for10%oftheisolates.
39differentMLVAprofileshavebeenfoundamongthe210isolatesanalyzed.Themostfrequent
profiles were 31310NA211 and 3139NA211 which represent respectively 19% and 15% of
theisolatesanalyzed.AlltheMLVAprofilesobtainedwerecharacterizedbythelostoftheVNTR10
inaccordancewithHopkinsandhercollaborators(2010)[5].
TheSalmonellaTyphimuriumtandemrepeatlocin5and6(STTR5andSTTR6)showedthehighest
Simpsons diversity index, respectively 0.631 and 0.737 with the highest number of different
tandemrepeatalleles(8and11alleles).
The discriminary power was 0.61 for PFGE subtyping method and 0.91 for MLVA. The two
methods, were not in concordance indeed a specific MLVA profile did not match with a specific
PFGE profile and vice versa, the most frequent PFGE profile STYMXB0126 comprised isolates
characterizedby18differentMLVAprofiles.
The population modeling using minimum spanning tree (MST) method brings out the relations
between the different major MLVA clones. Six major clones were observed (Figure 2). These
cloneswerenotgroupedwithinaspecificPFGEprofile.Fourofthesemajorprofiles(31310NA
211,3119NA211,31311NA211and3149NA211)werecloselycorrelatedtotheepidemic
profile3139NA211foundinpatientstrains.Onthewhole,thesefiveprofilescomprised59%of
the isolates received in the period from July 15 to December 31 (2011). Focusing on two profiles
foundinhumans:31390211and313110211,theseprofilesrepresentedrespectively15%and9%
of the strains analyzed (33 and 19 strains). 313902011 profile was represented by 39% of strains
isolated from dairy products (Figure 3). This data referred to a food alert of contaminated milk
occurredintheCantal(15)in2011.Totracethesourceofcontaminationmanysamplesweresentand
collectedbytheSalmonellanetworkoftheCEBunit.Themonitoringandtraceabilitysystemhasbeen
very effective, and the contaminated products were eliminated. The 313110211 profile was
represented by 79% of strains which have been collected over all steps of the pork industry, from
114
animalsamplingatthefarmleveltoprocessingorretaillevels.Thisprofilewastheonemajorfoundin
strainsisolatedfromdryporksausages[1].
Finally,theantibioticresistanceresultsshowedthat.69%oftheisolatespresentedtheresistance
type AM S SSS TE (Amoxicillin, Streptomycin, Sulphonamides and Tetracycline). This resistance
type was previously described in different European countries [5, 6] and in our study no
correlationtoaspecificPFGEorMLVAprofilehasbeenobserved.Thisresistancetypeshoweda
very well dissemination across all animal sectors in accord with the results recently published by
JourdanDaSilva&LeHello(2012)[7].
Among other resistance types identified in this study, two of them, AM NA S SSS TE (Amoxicillin,
NalidixicAcidStreptomycin,SulphonamidesandTetracycline)andAMCFCTXSSSSTE(Amoxicillin,
Cephalosporin, Cefotaxime Streptomycin, Sulphonamides and Tetracycline) were public health
concern.Thusnalidixicacid(NA)andcefotaxime(CTX),belongtofamiliesofinterestfortreating
Salmonellainfections.
DISCUSSION
AgreatnumberoffoodborneinfectionsworldwidearecausedbythegenusSalmonella,oneofthe
most prevalent pathogens encountered. The nontyphoid Salmonella are still an important
concern forpublic health in both developed and developing countries despite implementation of
food controls and hygiene regulations. Around 94 million salmonellosis cases are reported
worldwide, causing around 155,000 deaths annually [8]. Salmonella Enteritidis and Salmonella
Typhimurium are the two most commonly isolated serotypes around the world, accounting for
between 75% and 80% of the isolates involved in human cases every year [9]. More recently, in
the2000s,amonophasicvariantofserotypeTyphimuriumcarryingantigenicformula1,4,[5],12:i:
was associated with an increasing number of salmonellosis cases worldwide [6]. Emergence of
particular clones underlines the necessity to screen and characterize Salmonella monophasic
variants strains for the surveillance and the monitoring of trends in the frequency of types or
clones within serotypes. In France the NRC reported a gradual increase in the isolation of
Salmonella 4,[5],12 :i : from humans. The serotype moved from ninth to third place in order of
isolation between 2006 and 2010 [7] and a further significant increase was reported in the first
five months of 2010 [10]. It is intended that the monophasic variant of S.Typhimurium becomes
thesecondonemorefrequentlyisolatedfromhumansin2011.Thisyeartwogroupedcaseswith
Salmonella4,[5],12:i:wereobservedbyInVSandtheNRConebetweenthefirstAugustandthe
9 October, with 682 cases reported, and the second between the 31 October and the 18
December with a total of 337 cases identified [1]. In the same period the Salmonella network
inventoried several monophasic variants strains belonging to the same PFGE profile
(STYMXB0126). Therefore, there is a need to identify a performing subtyping method able to
discriminate within the major PFGE type. In comparison with other typing methods, this study
showthattheMLVAmethodismorediscriminatorythanPFGE(thereferencemethod)confirming
that the MLVA typing method is appropriated to study the spread of the monophasic variants
clonesincaseoffoodborneoutbreaks.
Moreover the population modeling using minimum spanning tree (MST) method brings out the
relations between the different major MLVA clones and the epidemiological study underline the
spreaddisseminationofthemonophasicvariantsinFrance,allovertheanimalsectorsandacross
thedifferentstepoftheproduction.
ACKNOWLEDGEMENT
AuthorsthankDr.NielsenandDr.LarssonfromtheSerumStateInstitute(Denmark)forthesetof
referenceSalmonellaTyphimuriumstrainsandthefileofnormalizationforMLVAanalysis.
AthankalsotoDrLeHellofromtheNationalReferenceCenterofPasteurInstitute(France)forthe
exchangeofprofilesfromclinicalisolates.
115
REFERENCES
1. Gossner,C.M.,VanCauteren,D.,LeHello,S.,Terrien,E.,Tessier,S.,Janin,C., etal.(2012). Nationwide
outbreak of Salmonella enterica serotype 4,[5],12:i: infection associated with consumption of dried pork
sausage,France,NovembertoDecember2011.EuroSurveill.17.
2. PulseNetEU,2009link:http://www.pulsenetinternational.org/SiteCollectionDocuments/pfge/
3. ECDC (2011). The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic
AgentsandFoodborneOutbreaksin2009.EFSAJournal9.
4. Larsson, J.T., Torpdahl, M., Petersen, R.F., Srensen, G., Lindstedt, B.A., Nielsen, E.M. (2009).
Development of a new nomenclature for Salmonella Typhimurium multilocus variable number of tandem
repeatanalysis(MLVA).EuroSurveilance14,15.
5. Hopkins, K.L., Kirchner, M., Guerra, B., Granier, S.A., Lucarelli, C., Porrero, M.C., et al. (2010).
MultiresistantSalmonellaentericaserotype4,[5],12:i:inEurope:anewpandemicstrain?EuroSurveill.15,
19580.
6. Switt, A.I., Soyer, Y., Warnick, L.D. and Wiedmann, M. (2009). Emergence, distribution, and molecular
andphenotypiccharacteristicsofSalmonellaentericaserotype4,5,12:i.FoodbornePathog.Dis.6,407415.
7. JourdanDa Silva, N., Le Hello, S. (2012). Salmonelloses en France, 20022010 : tendances en
pidmiologie humaine, mergence de la souche monophasique, principaux aliments impliqus dans les
dernirespidmies.BEH.HS.2528.
8. Majowicz,S.E.,Musto,J.,Scallan,E.,Angulo,F.J.,Kirk,M.,O'Brien,S.J.,etal.(2010).Theglobalburden
ofnontyphoidalSalmonellagastroenteritis.Clin.Infect.Dis.50,882889.
9. Hendriksen,R.S.,Vieira,A.R.,Karlsmose,S.,LoFoWong,D.M.,Jensen,A.B.,Wegener,H.C.etal.(2011).
Global monitoring of Salmonella serotype distribution from the World Health Organization Global
Foodborne Infections Network Country Data Bank: results of quality assured laboratories from 2001 to
2007.FoodbornePathog.Dis.8,887900.
10. Bone,A.,Noel,H.,LeHello,S.,Pihier,N.,Danan,C.,Raguenaud,M.E.,etal.(2010).Nationwideoutbreak
ofSalmonellaentericaserotype4,12:i:infectionsinFrance,linkedtodriedporksausage,MarchMay2010.
EuroSurveill.15.
FIGURES
Figure1:Distributionofisolatesbyanimalsector
Figure2:Populationmodelingusingminimumspanningtree(MST)methodofallisolates.
9%
2%
18%
1%
14%
2%
3% 2%
6%
19%
14%
10%
Bovine Caprine
Porc Petfood
PouleetPoulet Canard
Volaille Dinde
Produitslabors Produitscarns
Produitslaitiers NonApplicable
Bovine
Porcin
HenandChicken
Poulty (others sources)
Ready meal
Milk product
Caprine
Pet food
Duck
Turkey
Meat product
No information
116
Figure3:Populationmodelingusingtheminimumspanningtree(MST)methodoftheMLVAprofilesof210
monophasicvariantofS.TyphimuriumcollectedbytheSalmonellanetworkin2011anddistributedby
animalsector
117
Characterisationofanovel18.4kbgenomicisland
inendemicmonophasicSalmonellaTyphimuriumstrainsfromEurope
SandraSIMON,AntjeFLIEGERandWolfgangRABSCH
RobertKochInstitute,NationalReferenceCentreforSalmonellaandotherEnteric
PathogensandDivisionofBacterialInfections,WERNIGERODE,Germany
ABSTRACT
Recently, a monophasic variant of Salmonella Typhimurium not expressing the second phase flagellar antigene has
become one of the predominant agents causing foodborne infections in humans in Germany and other European
countries. Genome-based analyses displayed an 18.4 kb fragment adjacent to the thrWtRNA locus that is characteristic
for the prevalent German strain type. This element is flanked by direct repeats, contains 27 open reading frames and
exhibits a significantly lower G+C content than the closely related S. Typhimurium LT2 genome. Protein BLAST analyses
revealed several phage-related and uncharacterised gene products. ORF 10 was predicted to encode a type III-effector.
Further, it had been shown that the island is able to form a circular intermediate and conjugational transfer to an
appropriate Salmonella recipient strain had been proven. However, in vitro infection experiments with polarized epithelial
and macrophage-like cells showed no abnormalities regarding the invasion or intracellular replication capacities,
respectively. These results were confirmed in vivo using a streptomycin mouse model. So, although we provide detailed
information about the structure, the dissemination and the transferability of the novel genomic island the aim of our study
is to highlight its relevance in the life cycle of the monophasic Salmonella Typhimurium variant, especially in terms of the
potentially increased virulence or fitness.
INTRODUCTION
SincethemonophasicSalmonellaTyphimuriumvariant4,[5],12:i:firstattractedattentionin2006
it developed to one of the predominant agents causing foodborne infections in humans in
Germany (Figure 1) [1,2]. To this day the same kind of strains has been reported from various
European countries [37]. In spite of minor variations in phage type,PFGE and resistance pattern
within the emerging monophasic strains one type characterised by phage type DT193, PFGE
pattern STYMXB.0131 and at least tetradrug resistance towards antibiotics including ampicillin,
streptomycin, sulfamerazine and tetracycline clearly dominates in Germany. Genomebased
analyses displayed an 18.4 kb fragment adjacent to the thrW tRNA locus in this prevalent strain
type that is composed by 27 potential open reading frames and flanked by direct repeats.
Compared to the closely related S. Typhimurium LT2 genome, the G+C content of the novel
element is significantly lower (47.4% vs. 52.2%), indicating its acquisition via horizontal gene
transfer.Theidentificationofthisnovelislandraisedthequestion,whetherthiselementmightbe
involved in the rapid spread of this particular strain type. Aim of this study was therefore the
structural and functional characterisation of this novel island in order to provide information
about its relevance for bacterial fitness or pathogenicity in the emerging monophasic variant of
SalmonellaTyphimurium.
MATERIALANDMETHODS
Included in this study were human 4,[5],12:i: and 4,[5],12:i:1,2 isolates sent to the National
Reference Centre for Salmonella for typing or obtained from our international cooperation
partners.AllstrainshavebeenserotypedaccordingtotheWhiteKauffmannLeMinorschemeby
slide agglutination with O and Hantigen specific sera (SIFIN Diagnostics). Phage typing was
performed using the extended Anderson phage typing set following the guidelines of the IFEPT.
Pulsedfieldgelelectrophoresis(PFGE)wascarriedoutusingXbaIrestrictionenzymeaccordingto
the PulseNet protocol [8]. PCR reactions were carried out using a 2720 Thermal Cycler from
AppliedBiosystemsandHotStarTaqMasterMixKitfromQIAGEN
,primersweregeneratedusing
the DS Gene 1.5 software (Accelrys Inc.) Sequencing of the 18.4 kb island was performed by
Eurofins MWG GmbH using Genome Sequencer 454 technology. The nucleotide sequence of the
18.4kbfragmenthasbeenenteredinGenBank(accessionnumberGQ478253).RNAwasextracted
118
using pegGOLD RNAPure reagent from PEQLAB Biotechnologie GmbH, according to the
manufacturers instructions. For RTPCR the QIAGEN
OneStep RTPCR Kit was used. For the one

step deletion of the whole island the Datsenko and Wanner method was applied [9]. For cell
infection experiments Madin Darby Canine Kidney (MDCK) polarized epithelial cells (invasion
assay) and RAW 264.7 murine macrophagelike cells (replication assay) were infected with the
monophasic wildtype strain or its islanddeleted mutant (MOI 5). The invasion rate was
determinedby the cfu/ml 2 hours post infection compared to the inoculum while the replication
ratewasdescribedastheresultofthecfu/mlratiofrom18hoursand2hoursp.i.Toconfirmthe
secretion of the 3xFLAGtagged ORF 10 product the bacterial culture supernatant was sterile
filtered through a 0.45 m filter and precipitated with Trichloroacetic acid (TCA) / Acetone,
followed by SDSPAGE and subsequent western blot analysis using FLAGspecific primary (Sigma
Aldrich) and horseradish peroxidase (HRP)conjugated secondary antibodies. The bands were
visualized at a chemiluminescence imager (VILBER LOURMAT) after adding an enhanced
chemiluminescence (ECL) substrate (Thermo Fisher Scientific). The formation of a circular inter
mediate had been shown following isolation of extrachromosomal DNA (GeneJet Plasmid Mini
prep Kit, Fermentas) by PCR using primers that give a product only when both ends of the
fragment are linked to each other. Transfer experiments were carried out by a filter mating
approachusingtheLT2derivativeLB5010[10]astherecipientstrain.
RESULTS
Anovel18.4kbgenomicislandwasdetectedadjacenttothethrWgenebyscreeningthetRNAloci
in a representative of the emerging monophasic S. Typhimurium strains. This element has been
found to be characteristic for the prevalent German strain type. The island is flanked by direct
repeats and harbours 27 potential open reading frames. For several parts of the island
homologous sequences have been found in E. coli and Shigella genomes. Transcripts have been
shown for all but three ORFs. Alignment analyses revealed mainly phagerelated gene products
(includinganintegrase)andseveralhypotheticalproteins.ForORF10furtherinsilicoapplications
predictedaputativelytypeIIIsecretedeffectorandtheproteincouldbeveritablydetectedinthe
bacterialculturesupernatantfraction.Invitroinfectionexperimentswithpolarizedepithelialand
macrophagelike cell lines showed no differences between the wildtype and the islanddeleted
mutant strain regarding the invasion or intracellular replication capacities, respectively.
Nevertheless, it had been shown that the island is able to form a circular intermediate and
thereforecanbemobilizedundercertainconditions(Figure2).Matingexperimentsresultedinthe
successfulconjugationaltransferofthe18.4kbislandfromthedonortotheLT2derivativeLB5010
anditssitespecificintegrationintotherecipientchromosome.
DISCUSSION
The18.4kbelementclearlyresemblesagenomicislandinmanyrespects.Sincemostoftheopen
readingframesencodephagerelatedgeneproductsitmightbeofprophageorigin.Butasallbut
threeopenreadingframesaretranscribed,theelementismostlikelynotjustaphageremnant.In
thiscontextwecouldshowthatthepredictedtypeIIIeffectorencodedbyORF10isreallypresent
in the supernatant fraction of the bacterial cell culture, even if the appropriate secretion system
hasnotbeenidentifiedyet.TheknownT3SSofS.Typhimuriumseemnottomediatetheexportof
the ORF 10 product. Since the cell infection experiments showed no differences between the
wildtype strain and its deletionmutant the island is probably not involved in the invasion of
eukaryotic host cells or the intracellular replication processes. Notably, the creation of an extra
chromosomalcircularformofthe18.4kbislandcouldbeshown,aswellasthelateraltransferof
the element. Since no transfer genes were found it seems unlikely that the island is self
transmissible. The appropriate helper element providing the transfer tools in cis or trans has not
yetrevealed.Finally,thequestionremainsopen,whythismonophasicS.Typhimuriumvarianthas
119
beensosuccessfullyestablishedduringthelastyearswhiletheclassicalbiphasictypeseemsto
berepressed(Figure1).Thisphenomenoncouldbestbeexplainedbyaselectiveadvantageofthe
4,[5],12:i: strains or a better adaption to a special niche or the environmental conditions and is
subjectofongoinginvestigations.
CONCLUSIONS
Here we present substantial data about the structural organisation, the dissemination and
transferabilityofanovelgenomicislandpredominantlyfoundinthemonophasicS.Typhimurium
variant.Thefunctionofthedescribedelementanditspotentialcontributiontotherapidspreadof
theparticularstraintypearepartofcontinuativeinvestigations.
ACKNOWLEDGEMENT
We would like to thank our European cooperation partners for providing us with the 4,[5],12:i:
isolates.
REFERENCES
1. Trpschuch S, Laverde Gomez JA, Ediberidze I, Flieger A, Rabsch W. Characterisation of multidrug
resistantSalmonellaTyphimurium4,[5],12:i:DT193strainscarryinganovelgenomicislandadjacenttothe
thrWtRNAlocus.IntJMedMicrobiol,300(5),279288(2010).
2. HauserE,TietzeE,HelmuthRetal.PorkcontaminatedwithSalmonellaentericaserovar4,[5],12:i:,an
emerginghealthriskforhumans.Appliedandenvironmentalmicrobiology,76(14),46014610(2010).
3. Mossong J, Marques P, Ragimbeau C et al. Outbreaks of monophasic Salmonella enterica serovar
4,[5],12:i:inLuxembourg,2006.Eurosurveillance,12(6),E1112(2007).
4. Dionisi AM, Graziani, C., Lucarelli, C., Filetici, E., Villa, L., Owczarek, S., Caprioli, A., Luzzi, I. Molecular
characterization of multidrugresistant strains of Salmonella enterica serotype Typhimurium and
Monophasicvariant(S.4,[5],12:i:)isolatedfromhumaninfectionsinItaly.FoodbornePathogDis,6(6),711
717(2009).
5. Lucarelli C, Dionisi AM, Torpdahl M et al. Evidence for a second genomic island conferring multidrug
resistance in a clonal group of strains of Salmonella enterica serovar Typhimurium and its monophasic
variantcirculatinginItaly,Denmark,andtheUnitedKingdom.JClinMicrobiol,48(6),21032109(2010).
6. HopkinsKL,KirchnerM,GuerraBetal.MultiresistantSalmonellaentericaserovar4,[5],12:i:inEurope:
anewpandemicstrain?Eurosurveillance,15(22),19580(2010).
7. BoneA,NoelH,LeHelloSetal.NationwideoutbreakofSalmonellaentericaserotype4,12:i:infections
inFrance,linkedtodriedporksausage,MarchMay2010.Eurosurveillance,15(24)(2010).
8. Ribot,E.M.,M.A.Fair,R.Gautom,D.N.Cameron,S.B.Hunter,B.Swaminathan,andT.J.Barrett.2006.
StandardizationofpulsedfieldgelelectrophoresisprotocolsforthesubtypingofEscherichiacoliO157:H7,
Salmonella,andShigellaforPulseNet.Foodborne.Pathog.Dis.3:5967.
9. DatsenkoKA,WannerBL.OnestepinactivationofchromosomalgenesinEscherichiacoliK12usingPCR
products.ProcNatlAcadSciUSA,97(12),66406645(2000).
10. TsaiSP,HartinRJ,RyuJ.TransformationinrestrictiondeficientSalmonellatyphimuriumLT2.Journalof
generalmicrobiology,135(9),25612567(1989).

120
TABLESANDFIGURES
Figure1.HumanS.TyphimuriumisolatesinGermany.Includedaremonoandbiphasicisolatessenttothe
NRCfrom2004to2012(RKISalmoDB).
Figure2.Confirmationofthecircularintermediateofthe18.4kbisland

121
May 27-28-29, 2013
Saint-Malo France

Session 3


Chairpersons:

Posters
122
Studyontheeffectofstorageconditionsontherecovery
ofSalmonellafrombootswabsandhairnets
AndrRABIE
1
,IanMCLAREN
1
,RobinSAYERS
1
,FrancescaMARTELLI
1
,KellyVAUGHAN
1
,
NickKELL
1
andRobDAVIES
1
1
AnimalHealthandVeterinaryLaboratoriesAgency,DepartmentofBacteriologyandFood
Safety,WoodhamLane,NewHaw,Addlestone,SURREY,UnitedKingdom,KT153NB
INTRODUCTION
Samples sent via standard mail could potentially be held at ambient temperatures for extended
periodsoftime,whichcouldcompromisethesurvivaloftargetorganisms.Thereforeastudywas
conducted to investigate the effect of moisture and temperature over time on the ability to
recover Salmonella Enteritidis and Typhimurium from Tyvek boot swabs and hairnets (mob caps
usedasbootswabs).
MATERIALANDMETHODS
Approximately1gofchickenfaecescontaining~10and~100cfu/gofSalmonellawerespreadonto
the swabs.In study 1 boot swabs and hairnets were charged with faeces containing Salmonella
Enteritidis (SE) and Salmonella Typhimurium (ST) and maintained in a dry or moist (1ml distilled
water per four swabs) atmosphere at 5.0C and 20.0C respectively. After 1, 2, 3, 4 and 7 days
fourreplicatesfromeachtestconditionwereexaminedindividuallyforSEorST.
In a second study, boot swabs and hairnets were charged with SE containing faeces only and
maintainedindryormoistatmosphere,butat4.0C,8.2Cand16.6C,withonlyonereplicatefor
each.Inathirdstudy,fivepairsofbootswabswith14cfu/gand132cfu/gfaecesofSErespectively
weresentovernightin acooledcontainertofourdifferentlaboratoriestoassessrecoveryofthe
strain. Samples were incubated in Buffered Peptone Water (BPW) for 18 hours 2 at 37C 1C
andfurtherexaminedusingasensitiveanimal
isolation procedure employing Modified Semisolid Rappaport Vassiliadis (MSRV) and Rambach
agar(basedonISO6579:2002/Amd1:2007[1]).
RESULTS
Logistic regression models were fitted to the proportions of positive samples with categorical
factorsforday,conditions,concentrationandtreatmentforeachroundrespectively.
In the first study, Salmonella recovery was better at 5.0C than 20C in moist conditions with a
linear regression in time (Figure 1 and 2). Boot swabs and hairnets showed similar results
predictedmeans2.352.52and2.502.66respectively.
Inthesecondstudyrecoverywasbestatthehigherconcentrationundermoistconditionsat8.2C
with a downward trend in time (Figure 3, 4 and 5). Time, conditions and concentration were all
highlysignificant(p<0.001)at4.0Cand8.2C.Recoveryat4Cwasbetterthanat16.6C,butnot
asgoodasat8.2C.Time,concentrationandtemperaturewereallsignificant(p<0.001)at16.6C.
Conditionswerenotquitesignificant(p=0.089).
In the third study, all competing laboratories correctly detected 132cfu/g of SE and three out of
fourofthecompetinglaboratoriescorrectlydetected14cfu/gofSEatbothconcentrations.
DISCUSSION
Parish[2]andZhaoetal.[3]havereportedthatSalmonellacansurviveforlongerperiodsoftime
underrefrigerationtemperaturesthanatroomtemperature.Therefore,toensuretherecoveryof
low levels of Salmonella, samples should be kept cool during the period between collection and
boot swabs also benefit from being kept moist before examination after a minimal time delay
123
between sampling and processing. Without temperature and moisture control of the samples
Salmonellarecoverymaybeimpairedafter24hours.
ACKNOWLEDGEMENTS
This project was funded by the Department of Environment, Food and Rural Affairs (Defra)
throughprojectOZ0332.
REFERENCES
1. ISO. Standards catalogue. ISO6579:2002/Amd 1:2007 Annex D: Detection of Salmonella spp. Infaeces
andinenvironmentalsamplesfromtheprimaryproductionstage.
http://www.iso.org/iso/home/store/catalogue_tc/catalogue_detail.htm?csnumber=42109
2. ParishME.Publichealthandnonpasteurizedfruitjuices.Crit.Rev.Microbiol.23(2):10919.(1997).
3. ZhaoT.,M.P.DoyleandR.E.Besser.FateofenterohemorrhagicEscherichiacoliO157:H7inapplecider
withandwithoutpreservatives.Appl.Environ.Microbiol.59(8):252630.(1993).
TABLESANDFIGURES
Figure1Comparisonofconditionsat5C
0
20
40
60
80
100
120
BS
5C
MC
5C
BS
5C
MC
5C
BS
5C
MC
5C
BS
5C
MC
5C
BS
5C
MC
5C
1 2 3 4 7
Day/Temperature/Swab
%
moist high
dry high
moist low
dry low
Figure2Comparisonofconditionsat20C
0
20
40
60
80
100
120
BS
20C
MC
20C
BS
20C
MC
20C
BS
20C
MC
20C
BS
20C
MC
20C
BS
20C
MC
20C
1 2 3 4 7
%
moist high
dry high
moist low
dry low
Figure3Comparisonofconditionsat4.0C
0
20
40
60
80
100
120
4C 4C 4C 4C 4C 4C
DAY 1 DAY 2 DAY 3 DAY 4 DAY 6 DAY 7
%
moist high
dry high
moist low
dry low
0
20
40
60
80
100
120
8.2C 8.2C 8.2C 8.2C 8.2C
DAY 1 DAY 2 DAY 3 DAY 4 DAY 7
%
moist high
dry high
moist low
dry low
0
20
40
60
80
100
120
16.6C 16.6C 16.6C 16.6C
DAY 1 DAY 2 DAY 3 DAY 6
%
moist high
dry high
moist low
dry low

124
Sensitivityofdifferentsamplingmethodsinthedetection
ofSalmonellainflocksoflayinghens
FrancescaMARTELLI,RebeccaGOSLING,IanMCLAREN,RobinSAYERSandRobDAVIES
BacteriologyandFoodSafetyDepartment,AHVLAWEYBRIDGE,UnitedKingdom
INTRODUCTION
Salmonella enterica infections are associated with cases of foodborne illness worldwide and a
significantproportionofthesecasesarerelatedtoeggsandeggproducts.Salmonellacontrolinflocks
oflayinghensiscompulsoryintheEuropeanUnion(CommissionRegulationEC2160/2003).Inorder
toidentifyaflockaspositive,environmentalsamplingofthepoultryhousesisregardedasmorecost
effectivethansamplingindividualbirds.SamplesofchoiceforSalmonelladetectionarepooledfaeces
and dust. Faeces provide an indication of currently active infection whilst dust may also indicate a
previous peak of infection that has passed. As Salmonella shedding is intermittent and the level of
contamination is different across different areas of infected holdings, no sampling method for
Salmonellainpoultryhouseshas100%detectionsensitivity.Testingbothfaecalanddustsamplescan
helpincreasethedetectionsensitivityinsomepoultryhouses(2).
During this study, 20 farms in which Salmonella had already been identified were visited, and
environmentalsampleswerecollectedusingfourdifferentdefinedmethods.
MATERIALANDMETHODS
A total of 20 farms (9 Salmonella Enteritidis, 7 Salmonella Typhimurium and 4 other Salmonella
serovarspositiveflocks)werevisitedwithinthisstudy.Alltheflockshadbeenidentifiedaspositiveby
routine monitoring. The following environmental sampling methods were compared: Method 1 (5
pooled faecal samples and 2 dust samples), Method 2 (20 point faeces and 20 point dust samples),
Method 3 (1 Dust and 1pooled faecal sample taken as 5 replicates) andMethod 4 (1 pooled faeces
sampletakenas5replicates).ForMethods1,3and4faecalsamplesconsistedofbootswabsfornon
cage houses or naturally pooled faeces for cage houses. For Methods 1, 3 and 4, two pairs of boot
swabs were added to 450 ml of Buffered Peptone Water (BPW) for preenrichment. For faeces
collection,inMethod1eachsamplewasmixedand200gwasaddedto200mlofBPW.Afterletting
themixturestandfor15minutes,asubsampleof50gramswastakenandaddedto200mlofBPW.
For both Methods 3 and 4, each faecal sample was mixed and then 300g was weighed out. A
subsampleof25gwasthenaddedto225mlofBPW.EachMethod1styledustsamplewasmixedand
50gwasaddedto50mlofBPW.Afterlettingthemixturestandfor15minutesandfurthermixing,a
subsample of 50 grams was taken and added to 200 ml of BPW. For Methods 3 and 4 each dust
sample was dry mixed and 25g was weighed out into 225ml of BPW. All samples in BPW were
incubated at 37C for 1620 hours and 0.1 ml of incubated broth was inoculated onto modified
semisolidRapaportVassiliadismedium(MSRV).MSRVplateswereincubatedat41.5Candexamined
after24and48hoursforsuspectSalmonellagrowth.FromallMSRVplatesa0.1mlloopwastaken,
and streaked onto Rambach agar. Inoculated plates were incubated at 37C for 24 hours, and
presumptiveSalmonellacolonieswereidentified.WhenSalmonellawasidentifiedafterthefirst24hrs
inRambachplates,nofurtherplatingfromMSRVwascarriedout.
AflockwasidentifiedaspositiveifSalmonellawasisolatedfromatleastonesampleinanysampling
method.Differencesbetweensamplingmethodsweretestedbyttest.
RESULTS
Significantstatisticaldifferenceswereidentifiedbetweenthedifferentmethods.Thedifferencesinthe
performancesofeachsamplingmethodaredetailedinTable1.Thebestperformingoverallsampling
methods were Method 2 and Method 1 (85.1% and 70.2% of positive flocks detected respectively).
When considering only the first replicate, Methods 3 and 4 performed significantly less well, with
125
55.7% and 30.9% of positive flocks detected. However, when the results for all five replicates were
considered,Method3wasabletodetect95.5%ofthepositiveflocks,andMethod472.3%..
DISCUSSION
Arangeofenvironmentalsampleswerecollectedduringthisstudy.Differentsamplingmethodswere
explored,toidentifypossibledifferencesinsensitivitybetweenfoursamplingmethods.
TherelationshipbetweenSalmonelladetectionandthenumberofsamplestakenhasbeenreported
before.EFSAreportedthatifthenumberofsamplestakenintheEUbaselinesurveyin2005hadbeen
decreased from 7 to 4, the observed Salmonella prevalence would have been significantly lower (3).
Theresultsofthecurrentstudyshowthatincreasingthenumberofsamplescollectedfrom1to5in
both Methods 3 and 4 would be likely to result in a significant increase in the number of flocks
confirmed as positive, although the current study was only carried out in known or suspectpositive
layinghouses.
Theinclusionoftwosampletypesinseparatetestswasalsoshowntoincreasethenumberofflocks
detectedaspositiveinthisstudy.Forexample,Method4(faecesonly)wasabletodetect30.9%ofthe
positiveflocks,whilstMethod3(faecesanddust)detected55.7%ofthepositiveflocks.
Method 2 was reported to be at least as sensitive as Method 1 (1). In this study, we showed that
Method 2 was able to detect more positive flocks than Method 1 (85.1% cf. 70.2%) even if this
differencewasnotstatisticallysignificant(p=0.059).Whenconsideringthecombinedresultsofthefive
replicatesthesensitivityofbothMethods3and4increasedsignificantly,suggestingthatcollectinga
largernumberofsampleswouldleadtoconfirmationofahighernumberofpositiveflocks,especially
in cases where the level of environmental Salmonella contamination is low. Including the testing of
faecal and dust samples, especially for cage flocks, results in increased sensitivity. However multiple
pooledfaecalorbootswabsamplescouldprovideasensitivealternativeoptionincaseswheredustis
difficulttocollect.
ACKNOWLEDGEMENTS
REFERENCES
1. CarriqueMas, J. J., M. Breslin, L. Snow, M. E. Arnold, A. Wales, I. McLaren, and R. H. Davies. 2008.
Observations related to the Salmonella EU layer baseline survey in the United Kingdom: followup of positive
flocksandsensitivityissues.EpidemiolInfect136:15371546.
2. CarriqueMas, J. J., and R. H. Davies. 2008. Sampling and bacteriological detection of Salmonella in poultry
andpoultrypremises:areview.RevSciTech27:665677.
3. EFSA.2007.ReportoftheTaskForceonZoonosesDataCollectionontheAnalysisofthebaselinestudyon
theprevalenceofSalmonellainholdingsoflayinghenflocksofGallusgallus.TheEFSAJournal97:184.
TABLESANDFIGURES
Tableone:Sensitivityofthedifferentsamplingmethodstestedinthisstudy.
Samplingmethod Description Predicted%+vefarms(95%cl)
Method1 5pooledfaeces/BSand2dustsamples 70.2(51.189.3)
Method2 20faecesand20dusts 85.1(70.999.3)
Method3(1streplicates) 1pooledfaeces/BSand1dust 55.7(31.879.6)
Method3(replicates) 5pooledfaeces/BSand5dusts 95.5(76.299.3)
Method4(1streplicate) 1pooledfaeces/BS 30.9(8.753.1)
Method4(replicates) 5pooledfaeces/BS 72.3(53.890.9)

126
ImprovedIsolationofSalmonellafromPoultryEnvironmentalSamples
usingdirectTetrathionateEnrichmentfollowedbyMSRV
DougWALTMAN
GeorgiaPoultryLaboratoryNetwork,OAKWOOD,Georgia,USA
INTRODUCTION
In the United States of America there are two methods approved by the National Poultry
Improvement Plan (NPIP) for isolating Salmonella from poultry environmental samples. The first
method involves directly enriching the samples in tetrathionate (TT) followed by delayed
secondaryenrichment(DSE)(TT/DSE).Thesecondmethodinvolvesdirectlyenrichingthesamples
in buffered peptone water (BPW) followed by enriching in TT and Rappaport Vassiliadis (RV) or
ModifiedSemisolidRappaportVassiliadis(MSRV).
This study compares a modification of the two methods using naturally contaminated poultry
environmentalsamples.
MATERIALSANDMETHODS
Thefirstmethodinvolveddirectlyenrichingthesampleintetrathionate(TT)enrichmentbrothfor
24 hrs at 37 C and plating onto selective media, such as brilliant green agar supplemented with
novobiocin(BGN)andxyloselysinetergitol4(XLT4)agars.Afterplating,theenrichedculturewas
leftatroomtemperaturefor57days,atwhichtime1mlwastransferredtoatubecontaining10
ml of TT enrichment broth (delayed secondary enrichment, DSE). After 24 hrs incubation the
culturewasplated.
Thesecondmethodinvolveddirectlyenrichingthesampleinbufferedpeptonewater(BPW)for24
hoursat37Candthentransferring1mlinto10mlofTTenrichment(BPW/TT),0.1mlinto10mls
ofRVenrichment(BPW/RV),and0.1mlintoaplateofMSRV(BPW/MSRV).Eachenrichmentwas
incubatedat42Cfor24hoursbeforeplatingontoBGNandXLT4agar.
Themodifiedmethodutilizeddirectlyenrichingthesamplesat37Cfor24hoursinTTfollowedby
transfer of 0.1 ml into a plate of MSRV (TT/MSRV). The MSRV was incubated for 24 hours and
inoculated onto Brilliant green agar supplemented with novobiocin (BGN) and Xylose Lysine
Tergitol4(XLT4)agarifazoneofmigrationwasobserved.Ifnozoneofmigrationwasobserved
theMSRVplatewasincubatedanother24hoursat42CandplatedontoBGNandXLT4agars.
RESULTS
Study1.
The modified method (TT/MSRV) was compared to the first NPIP method (TT/DSE) using 2246
routinely submitted poultry house and hatchery samples. A total of 956 of these samples were
positiveforSalmonellabyoneorbothmethods(Table1andFigure1).Subsequentanalysisofthe
datasetselectingforsamplesthatwerepositiveforSalmonellaEnteritidis(SE)revealedthat261
ofthesampleswerepositiveforS.E.TheresultsoftheanalysisareshowninTable2andFigure1.
Study2.
A second study compared the TT/MSRV method with both the first (TT/DSE) and the second
(BPW/TT,BPW/RV,BPW/MSRV)NPIPmethods.Thesamplesweresplittoallowdirectenrichment
inbothTTandBPW.Eighthundredninetyeightpoultryhouseandhatcherysamplesweretested
and 407 were positive for Salmonella by one or several methods (Table 3 and Figure 2.
Subsequent analysis of the data set selecting for samples that were positive for SE revealed that
128 of the samples were positive for SE. The results of the analysis are shown in Table 4 and
Figure2.
127
DISCUSSION
Study1.
The comparison of the TT/MSRV and TT/DSE methods showed that the TT/MSRV enrichment
procedure isolated 15.3% more Salmonella and 41.8% more S. E than the established TT/DSE
procedure.
Study2.
The comparison of the TT/MSRV and TT/DSE methods showed that the TT/MSRV enrichment
procedure isolated 13.8% more Salmonella and 49.3% more S. E than the established TT/DSE
procedure, which was comparable to the results found in Study 1. The comparison of TT/MSRV
with the preenrichment procedures showed and increased isolation of 40.3 % over BPW/TT,
56.0% over BPW/RV and 4.4% over BPW/MSRV. Even when the results of the BPW/TT and
BPW/RV are combined, the TT/MSRV isolated 17.9% more Salmonella. For the isolation of S. E
specifically, TT/MSRV isolated 53.2% more than BPW/TT, 56.3% more than BPW/RV and 9.4%
morethanBPW/MSRV.
CONCLUSION
The TT/MSRV procedure was more sensitive for the isolation of Salmonella in general and SE in
particularthantheconventionalTT/DSEorBPW/TTandBPW/RVmethods.
Table1.ComparisonofthedirectTTfollowedbyDSEmethod(TT/DSE)withdirectTTfollowedbyMSRV
method(TT/MSRV)fortheisolationofSalmonella
Source No.Samples
No.
Positive
No.Salmonellaisolated
TT TT/DSE TT/MSRV
Chickpapers 396 170 85 131 157
Fluff 385 296 153 219 287
House 1425 485 298 386 437
Other 40 5 3 4 5
Total 2246 956 539 740 886
%+ 56.4 77.4 92.7
Table2.ComparisonofthedirectTTfollowedbyDSEmethod(TT/DSE)withdirectTT
followedbyMSRVmethod(TT/MSRV)fortheisolationofSE
Source
No.
Samples
No.SEisolated
TT TT/DSE TT/MSRV
Chickpapers 84 25 48 80
Fluff 154 40 65 138
DS 23 14 14 18
Total 261 79 127 236
%+ 30.2 48.6 90.4
Figure1.ComparisonofthedirectTTfollowedbyDSEmethod(TT/DSE)withdirectTTfollowedbyMSRV
method(TT/MSRV)fortheisolationofSalmonellaandSalmonellaEnteritidis(SE)
56,4
77,4
92,7
30,2
48,6
90,4
0
50
100
TT TT/DSE TT/MSRV
%
i
s
o
l
a
t
e
d
Salmonella SE
128
Table4.ComparisonofthedirectTTfollowedbyDSEmethod(TT/DSE)andthepreenrichmentfollowed
byselectiveenrichmentmethodwithdirectTTfollowedbyMSRVmethod(TT/MSRV)fortheisolationof
SE
Source No.
No.SEisolated
TT
TT/
DSE
TT/
MSRV
BPW/
TT
BPW/
RV
BPW/
MSRV
BPW/TT
BPW/RV
Chickpapers 61 16 21 53 30 29 52 32
Fluff 57 3 19 46 4 3 35 5
DS 10 1 1 5 2 0 5 2
Total 128 20 41 104 36 32 92 39
%+ 15.6 32.0 81.3 28.1 25.0 71.9 30.5
Figure2.ComparisonofthedirectTTfollowedbyDSEmethod(TT/DSE),preenrichmentfollowedby
selectiveenrichmentmethod,withdirectTTfollowedbyMSRVmethod(TT/MSRV)fortheisolationof
SalmonellaandSE
51,1
72,7
86,5
46,2
30,5
82,1
50,6
15,6
32
81,3
28,1
25
71,9
30,5
0
20
40
60
80
100
TT37 TT37/DSE TT37/MSRV BPW/TTH42 BPW/RV42 BPW/MSRV BPW/TTH42
BPW/RV42
%
i
s
o
l
a
t
e
d
Salmonella SE
Table3.ComparisonofthedirectTTfollowedbyDSEmethod(TT/DSE)andthepreenrichmentfollowed
by enrichment method, with the direct TT followed by MSRV method (TT/MSRV) for the isolation of
Salmonella
Source
No.
Samples
No.
Positive
No.Salmonellaisolated
TT
TT/
DSE
TT/
MSRV
BPW/
TT
BPW/
RV
BPW/
MSRV
BPW/TT
BPW/RV
Chick
papers
182 76 28 38 68 35 35 64 40
Fluff 127 88 25 64 80 29 25 67 34
House 558 243 155 194 204 124 64 203 132
Other 31 0 0 0 0 0 0 0 0
Total 898 407 208 296 352 188 124 334 206
%+ 51.1 72.7 86.5 46.2 30.5 82.1 50.6
129
Validationofselectiveenrichmentbrothsandchromogenicmedia
forSalmonelladetectioninhighlycontaminatedchickencarcassrinse
JiyeonHYEON
1
andKunhoSEO
2
1
DivisionofVaccineResearch,KoreaNationalInstituteofHealth,OSONG,SouthKorea;
2
KUCenterforFoodSafety,HwaYangDong,SEOUL,SouthKorea
INTRODUCTION
Salmonellosis is one of the most widespread infectious diseases in the world and is a common
cause of gastrointestinal food poisoning. Raw and processed meat products, especially chicken
meats remain the principal reservoir of Salmonella. The aims of this study were to determine
prevalence of Salmonella from retail chicken carcass from three types of grocery stores and to
compared sensitivity and specificity of selective enrichment broth and selective plate media for
SalmonelladetectionfromnaturallycontaminatedchickencarcassesaccordingtoISOmethod.
MATERIALANDMETHODS
Atotalof180chickencarcasseswerecollectedfromthreetypesofgrocerystores,andSalmonella
isolation was performed by the culture method according to the United States Department of
Agriculture(USDA).Thechickencarcasseswereasepticallyrinsedwith400mlofsterileBPWinto
thecavityofthecarcassforoneminute.Thesamplerinsefluids(25ml)weremixedwith25mlof
sterile BPW followed by incubation at 37C for 24 h. The enriched BPW culture (0.1 ml and 1ml)
were transferred into 10 ml of RV and MKTTn and incubated at 37C and 42C for 24 h,
respectively, for selective enrichment. A loopful of RV and MKTTn enrichment culture was
streaked onto XLD and Chrom ID Salmonella (bioMrieux). The presumptive colonies with a
positive result were confirmed as Salmonella with VITEK2 (bioMrieux) and Salmonellaspecific
realtimePCR.
RESULTS
Salmonella were isolated from 118 samples (65.5 %) and Salmonella Enteritidis were 67 of 118
samples (56.8 %). Significantly fewer Salmonellapositive samples were recovered with MKTTn
(63.5%)thanwithRVS(98.3%).
Theselectivitywas81.7%inRVSand39.0%inMKTTn.ThesensitivitiesofSMID2andXLDwere
93.2%and88.1%inRVS,56.8%and36.4%inMKTTn,andtheselectivitywere88.6%and81.6%
inRVS,54.9%and45.3%inMKTTn,respectively.ThecombinationofselectiveenrichmentinRVS
and SMID 2 was showed the highest sensitivity and specificity to detect Salmonella with fewer
falsepositivecolonies.
DISCUSSION
The selective enrichment in RV gave greater recovery of Salmonella than selective enrichment in
TT. In addition, using ChromID for selective isolation resulted in numerically greater recovery of
Salmonella than XLD when selectively enriched in either RV or TT. This observation coupled with
numericallygreaterrecoveryafterselectiveenrichmentinRVsuggeststhatuseofRVforselective
enrichment and ChromID for selective isolation may be effective to determine the presence of
Salmonellaonchickencarcass.
REFERENCES
1.Dusch,H.andM.Altwegg.1993.ComparisonofRambachagar,SMIDmedium,andHektoenEntericagar
forprimaryisolationofnontyphisalmonellaefromstoolsamples.JClinMicrobiol31:4102.

130
AcomparisonbetweenRambachagar,RapidSalmonellaCompleteagar
andBrillianceSalmonellaagarfordetectionofSalmonella
AndrRABIE
1
,IanMCLAREN
1
,FrancescaMARTELLI
1
andRobDAVIES
1
1
AnimalHealthandVeterinaryLaboratoriesAgency,DepartmentofBacteriologyandFoodSafety,
WoodhamLane,NewHaw,Addlestone,SURREY,UnitedKingdom,KT153NB
INTRODUCTION
A H
2
S negative, lactose fermenting Salmonella Senftenberg was isolated from poultry house dust
andspreadprofuselyinModifiedSemisolidRappaportVassiliadis(MSRV)agar,butdidnotdisplay
Salmonellalike colonies on Rambach, Xylose Lysine Desoxycholate (XLD) or BGA+Novobiocin
(BGAN)agars.Theinabilityofcommonlyusedagarstodetectsuchstrainsisaconcernandthusit
was decided to compare Rambach, XLD and BGAN to Rapid Salmonella Complete (RSC) and
Brilliance Salmonella (BS) agars for detection of such strains and Salmonella in general. RSC is a
chromogenicagarwhichidentifiesSalmonellawiththeaidoftheenzymeC8esterasetoproduce
magenta coloured Salmonella colonies [1]. BS identifies Salmonella spp. from clinical and food
sampleswiththeaidofenzymehydrolysis[2].
RSC and BS agar were thought likely to be able to detect H
2
S negative, lactose fermenting
Salmonellastrainsduringroutinetestingofartificiallyandnaturallycontaminatedsamples.
MATERIALSANDMETHODS
Rambach,RSCandBSweretestedinparallelusinganartificiallyinoculatedfaecalmatrixaswellas
withnaturallycontaminatedfaecalandenvironmentalmaterialfromsixfarmsthattestedpositive
for various strains of Salmonella, including S. Senftenberg, S. Typhimurium, monophasic S.
Typhimurium,S.Newport,S.Panama,S.AnatumandS.London.
Two more rounds of testing were done with Rambach, BGAN, XLD, RSC and BS with S.
Senftenberg,S.Enteritidis,monophasicS.Typhimurium,S.NottinghamandvaccinestrainsGallivac
SEandAviproE,TandETatvariousdilutions.
RESULTS
In the first round, in faecal matrix S. Senftenberg was detected on RSC and BS, but not on
Rambach.Intotal,245comparativeculturesoffieldsampleswerecarriedout.Therewereatotal
of 165 true positive samples based on isolation of Salmonella on any medium counting as a true
positive.Rambachdetected158(64.5%)positives,RSC155(63.2%)andBS161(65.7%)(Figure1).
Nobiochemicallyatypicalstrainswereencounteredduringthetrial.
In the second round RSC and BS detected all the strains as well as the atypical S. Senftenberg,
whereas Rambach, XLD and BGAN did not detect this. The Gallivac and Avipro vaccine strains
producedtypicalSalmonellalikecoloniesonRambach,XLDandBGAN,butappearednegativeon
RSC and BS. In both rounds BS agar detected more samples and their dilutions than the other
media(Figure2).
DISCUSSION
Despitegoodperformance,thetwoalternativemediaarenotassimpletointerpretasRambach,
BGANorXLDagar.Thisisespeciallytrueforindividualswhoarecolourblind,sinceitisdifficultfor
themtodistinguishbetweenpositive(magenta)andnegative(blue)colonies.
Thedifferenceinpriceshouldalsobetakenintoconsideration.Bothofthealternativemediaare
more expensive to prepare than Rambach agar. The preparation of Rambach is less labour
intensive since it requires no weighing out of the agar powder. Elimination of weighing also
minimises the risk of operator error in the preparation of the agar which may influence results
Alternatively, it is possible to presumptively identify the presence of motile Salmonella at the
131
MSRV selective enrichment stage by incorporating a double filter paper disc saturated with
polyvalent Salmonella flagella antiserum in the plate. This selectively inhibits the growth of
Salmonellaaroundthedisc.
ACKNOWLEDGEMENTS
throughprojectFZ2000.
REFRENCES
1. BioRad. Rapid Salmonella Agar 3563961. 3564705. v6_03.08.10_US. (2010). http://www.bio
rad.com/webroot/web/pdf/fsd/literature/3563961_4705_RAPIDSalmo_TechSheet_V6_030810_US.pdf
2. Thermo Fisher Scientific. Rapid Methods from Oxoid, Thermo Fisher Scientific Microbiology. J Cloke.
(2010).http://www.nmkl.org/NordVal/Symposium_2010/NordVal%20jonathan%20cloke.pdf
TABLESANDFIGURES
Figure1:Round1ComparisonofRambach,BSandRSC
62.00%
62.50%
63.00%
63.50%
64.00%
64.50%
65.00%
65.50%
66.00%
Rambach BS RSC
Agar type
%
Rambach
BS
RSC
Figure2:Round2ComparisonofRambach,XLD,BGAN,RSCandBSagar
0
10
20
30
40
50
60
70
80
90
100
Rambach XLD BGA RSC BS
Agar type
%
Rambach
XLD
BGA
RSC
BS

132
PerformanceofmediaplatesfordetectionofSalmonella
MartineDENIS,FranoiseLEGALL,SandraROUXEL,StphanieBOUGEARD
andMarianneCHEMALY
Anses,HygieneandQualityofPoultryandPigProductsUnit,BP53,sitedesCroix,
22440Ploufragan,France
INTRODUCTION
The goal of this study was to evaluate the performance of 6 media plates for the detection of
Salmonella.
MATERIALANDMETHODS
Strains.20strainsofSalmonellaincludingserotypesTyphimurium,Enteritidis,Hadar,Infantisand
Virchow were tested. The other serotypes were chosen for their atypical characteristics on
Rambach,orXLT4orXLDusedinthelaboratory.
Media. Six media plates were compared: XLD, XLT4, RAMBACH, chromID, COMPASS, and RAPID
Salmonella.
Productivityanddescriptionofthestrainsonthemedia.AfterenrichmentinTSBYe,strainswere
enumerated from a tenfold serial dilution on the 6 media plates and on TSAYe (non selective
media). The productivity was calculated from the 6 dilution. The colonies were described for
checkingwhetherdescriptioncorrespondstothatexpected.
Detection from artificially contaminated samples. Dropping and neck skin from poultry were
artificially contaminated with the 20 strains. After enrichment in BPW at 37C during 182H, a
second enrichment was realized on MKTTn, RVS and MSRV at 41.5C during 243H. Each
enrichmentwasthenstreakedonthe6mediaplates.Presenceoftypicalcoloniesasdescribedby
the suppliers was checked after 24h and also 48h at 37C. A total of 120 streakings per type of
mediaplatewerefinallyconsidered.
RESULTS
From pure strains, chromID was the most efficient for productivity but among the 20 strains, 6
strains were not typical. The other media plates had similar productivity (table 1) except for
COMPASS with the lowest productivity. However, RAPIDSalmonella was the one for which 19
strainsamongthe20hadtheexpectedcolonydescription.
Fromartificiallycontaminatedsamples,RAPIDSalmonellawasthemostefficientforthedetection
ofSalmonella.Typicalcoloniesweredetectedon85.7%ofthe120streakingsdoneonthismedia
plate.Forallthemediaplates,areadingafter24hofincubationwassufficient(figure1).

133
DISCUSSION
ThisstudyshowedadifferenceinperformancebetweenmediafordetectingSalmonella.
For the 6 tested media, RAPIDSalmonella had the best productivity combined with a better
identification on a large collection of Salmonella. RAPIDSalmonella could be recommended as
second media plate for the NFU 47100 and NF EN ISO 6579 methods. This study is undergoing
withothersmediaandserovars.
Figure1:Percentageoftypicalcolonies(CC)ornontypicalcolonies(CNC)oneachmediaafter24or48h.
REFERENCES
NFU47100:Juillet2007;NFENISO6579:Dcembre2002;NFENISO6579/A1:Octobre2007
(AnnexeD)

134
Table1:Productivityoftheserotypesandcharacteristicsofthecoloniesonthemedia.
Productivity:numberofcoloniesonmedia/numberofcoloniesonTSAYe;0:nogrow;+:0<P<0,5;++:0.5P<1;+++:P1
Serotype Rambach XLD XLT4 RAPID'Salmonella COMPASS ChromID
S.Typhimurium +++ fuschia ++translucentrim,blackcenter ++translucentrim,blackcenter +magenta 0magenta +++mallow
S.Enteritidis ++ fuschia +++translucentrim,blackcenter +++ translucentrim,blackcenter +++magenta + magenta ++ mallow
S.Hadar + fuschia +translucentrim,blackcenter + translucentrim,blackcenter +magenta 0magenta ++mallow
S.Infantis + fuschia ++translucentrim,blackcenter ++translucentrim,blackcenter +magenta + magenta ++mallow
S.Virchow ++fuschia ++translucentrim,blackcenter +translucentrim,blackcenter ++magenta +magenta ++mallow
S.Enteritidis ++salmonpink ++translucentrim,blackcenter ++ translucentrim,blackcenter ++magenta + magenta +++mallow
S.Heidelberg ++ fuschia ++lightblackcenterat24hmore
markedat48h
+++translucent ++magenta +magenta ++mallow
S.Choleraesuis ++salmonpink ++lightblackcenterat24h,
translucentrim,blackcenterat48h
0nogrowth ++magenta 0translucents<1mm
dediameter
+++ mallowmorepale
S.Montevideo ++ fuschia ++translucentrim,blackcenter ++translucentrim,blackcenter +++magenta +magenta ++mallow
S.Llandoff ++pink ++translucentrim,blackcenter ++ translucentrim,blackcenter ++magenta +++magenta +++mallow
S.salamae +++fuschia ++translucentrim,blackcenter ++translucentrim,blackcenter ++magentawithbluecenter + magenta ++mallow
S.arizonae 0 blue/purple ++yellowat24h,translucentrim,
blackcenterat48h
0nogrowth ++magenta +magenta 0 translucentat24h,
mallowat48h
S.diarizonae +palegreen ++translucentrim,blackcenter ++ translucentrim,blackcenter.
Redmedia
++magenta + magenta ++mallow
S.Sofoa ++ fuschia ++translucent 0 translucent + magenta 0moreclearmagenta +++ palemallow
S.Dublin +fuschia +lightblackcenterat24h,
translucentrim,blackcenterat48h
0translucent 0magenta 0 translucents<1mm
dediameter
+small,translucentat
24h,purplishat48h
S.Panama ++fuschia +++yellow ++ yellowat24h,sometranslucent
rim,blackcenterat48h
+++magenta ++magenta +++mallow
S.Panama +fuschia ++translucentrim,blackcenter ++yellowat24h,sometranslucent
+magenta +magenta ++mallow
S.Senftenberg ++khaki ++yellow +yellowat24h,sometranslucent
+ magenta + magenta ++ colorlessirregular
rim
S.AbortusOvis 0translucent,
colorless
0translucent 0 nogrowth 0 translucenttopalemallowat
24h,palemallowat48h
0translucents<1mm
dediameter
+small,translucent
S.Typhimurium ++ fuschia ++translucentrim,blackcenter +translucentrim,blackcenter +magenta +magenta ++mallow
Globalproduct. 33+ 38+ 28+ 32+ 17+ 42+

135
StateofplayofinternationalstandardisationofSalmonellamethods
KirstenA.MOOIJMAN
andEnvironmentalMicrobiology(Z&O),EURLSalmonella,BILTHOVEN,theNetherlands
INTRODUCTION
For the detection of Salmonella spp. in food, animal feed and samples from the primary
production stage, currently three international standard methods exist. This concerns ISO
6785:2001
1
for milk and milk products, ISO 6579:2001
2
for (other) food and animal feed samples
and Annex D of ISO 6579:2007
3
for samples from the primary production stage. At the 5 years
review of ISO 6579 (in 2007), committees in the European (CEN) and International (ISO)
standardisation decided to start the revision of the document and to try to merge the three
aforementioned standards. Additionally it was agreed to start the standardisation process of
proceduresforenumerationandserotypingofSalmonella.Asthesethreeprocedures,allconcern
methods for analysing Salmonella spp., it was agreed to publish them as three different parts of
ISO6579withthegeneraltitle:MicrobiologyoffoodandanimalfeedHorizontalmethodforthe
detection, enumeration and serotyping of Salmonella. The titles of the three parts will become:
Part1: Horizontal method for the detection of Salmonella; Part2: Enumeration by a miniaturized
MostProbableNumbertechnique;Part3:GuidelinesforserotypingofSalmonellaspp.
ISO65791,DetectionofSalmonella
TherevisionofISO6579:2002
2
,ondetectionofSalmonella,startedin2009.ThecommentstoISO
6579 indicated to consider the following items: 1) Description of the detection of S. Typhi and S.
Paratyphi in an annex; 2) Comparing different formulations of tetrathionate broths and including
the most optimal formulation in the revised version; 3)Comparing the use of MSRV with RVS for
foodanalysisforconsideringtheuseofMSRVintherevisedversion;4)Comparing24hand48h
ofincubationoftheselectiveenrichmentmediaanddecideontheincubationtimeintherevised
version; 5) Retaining XLD as mandatory isolation medium and give clearer direction on suitable
media for the second plate; 6) Investigation to the possibility of refrigerating the enrichment
media before subculturing; 7) Making the plating stage less prescriptive; 8) Making the
confirmation stage less prescriptive; 9) The nonselective medium for purification of colonies
should be left for choice; 10) Allowing parallel biochemical testing and purity check; 11)
Investigationtotheusefulnessofsomebiochemicaltests.
Bytheendof2009,anISOworkinggroupwasraised(WG9)todealwiththerevisionofISO6579.
In 2011 this work was moved from ISO to CEN (for political reasons) becoming a Technical
Advisory Group (TAG8). A first draft version of ISO 65791 was prepared in May 2010. Currently
thefifthdraftversionisunderpreparationandwilllikelybelaunchedforvotingasISODIS(Draft
InternationalStandard)beforesummer2013.
ConcerningtheabovementioneditemsthestateofplayofdraftISO65791isasfollows:
1) Detection of S. Typhi and S. Paratyphi is described in Annex D, with additional selective
enrichmentinSeleniteCystine(SC)brothandplatingoutonBismuthSulphite(BS)agarandXylose
LysineDeoxycholate(XLD)agar.2)Differentformulationsoftetrathionatebrothswerecompared
forthegrowthofpureculturesofdifferentSalmonellaserovars,aswellasforthesuppressionof
nonSalmonella strains. The results showed that the current formulation of Muller Kauffmann
Tetrathionatenovobiocin(MKTTn)brothgavethebestresults,andthusMKTTnshouldberetained
as second selective enrichment medium. 3) Information was obtained from seven (validation)
studies in which the detection of Salmonella by using Modified Semisolid Rappaport Vassiliadis
136
(MSRV)agarwascomparedtotheuseofRappaportVassiliadisSoya(RVS)broth(whetherornotin
combination with MKTTn). In all studies, it was shown that MSRV gave equal results to RVS (and
MKTTn), except in one study for one type of milk powder, where significantly lower results were
foundwithMSRV.ItwasagreedtoleavethechoicefortheusertouseeitherRVSorMSRVasfirst
selective enrichment medium. Only for some specific products it will be indicated that RVS (milk
powder) or MSRV (primary production samples) is preferred. 4) Literature was reviewed for the
incubation times as well as studies were performed. It was concluded that for the majority of
matrices, 24 h of incubation of the selective enrichment medium is sufficient. Only for a few
products (some dairy products and primary production samples) it may be necessary to incubate
48h.5)XLDwasretainedasmandatoryfirstisolationmediumandtableswereaddedtoAnnexE,
giving information on the indicator systems of different isolation media. 6) Literature was
reviewed and a few studies were performed showing the possibility of storage of incubated
(pre)enriched cultures at 5 C for a maximum of 72 h. 7) The plating stage is made less
prescriptive.Itisonlyindicatedthatwellisolatedcoloniesneedtobeobtained.Howthisisdoneis
leftfortheuser8),9)and10).Theconfirmationstageismadelessprescriptive,thenonselective
medium is left for choice and the possibility for parallel biochemical testing and purity check is
indicated11).Threemediaforbiochemicaltestsareretainedasprescribed(Triplesugar/iron(TSI)
agar,UreaagarandLysinedecarboxylation(LDC)medium),theothertestshavebecomeoptional
(galactosidaseandindole),ordeleted(VogesProskauer).
ISO/TS65792EnumerationofSalmonella
At the ISO meeting in 2007 it was agreed to prepare an ISO document for the enumeration of
SalmonellaandtobaseitontheprocedureasdescribedbyFravaloetal.
4
,includingaminiMPN
procedure.Forthisprocedurethepreenrichment(inBPW)aswellastheselectiveenrichment(on
MSRV) is performed in 12 well microtitre plates. The final version of this ISO document was
publishedinNovember2012.
ISO/TR65793SerotypingofSalmonella
At the ISO meeting in 2009 it was agreed to prepare an ISO document for the serotyping of
Salmonella.ISOworkinggroup,WG10,wasraisedandafirstdraftdocumentwasprepared,based
on the WhiteKauffmannLe Minor scheme
5
, in 2010. It was agreed to give this document the
status guidance document (Technical Report) instead of a full ISO standard. Currently the sixth
draftversionisunderpreparationandwilllikelybelaunchedforfinalvotinginspring2013.
REFERENCES
1.Anonymous,2001.ISO6785.MilkandmilkproductsDetectionofSalmonellaspp.ISO,Ch.
2.Anonymous,2002.ISO6579.MicrobiologyoffoodandanimalfeedingstuffsHorizontalmethodforthe
detectionofSalmonellaspp.ISO,Ch.
3. Anonymous, 2007. ISO 6579: 2002/ Amd1. Microbiology of food and animal feeding stuffs Horizontal
methodforthedetectionofSalmonellaspp.AnnexD:DetectionofSalmonellaspp.inanimalfaecesandin
environmentalsamplesfromtheprimaryproductionstage.ISO,Switzerland
4.Fravalo,P.,etal,2003.ConvenientmethodforrapidandQuantitativeassessmentofSalmonellaenterica
contamination:theminiMSRVMPNtechnique.J.RapidMeth.Autom.Microbiol.,11,2003,pp.8188
5. Grimont, P.A.D. and Weill, FX., 2007. Antigenic formulae of the Salmonella serovars, 9
th
ed. WHO
Collaborating Centre for Reference and Research on Salmonella. Institute Pasteur, France.
http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s000036089(visitedFeb.2013)

137
Performanceofreferencelaboratoriesininterlaboratorycomparisonstudies
onserotypingofSalmonellaspp.
WilmaJACOBSREITSMA,IrenePOLHOFSTADandKirstenMOOIJMAN
NationalInstituteforPublicHealthandtheEnvironment(RIVM),CentreforZoonosesand
EnvironmentalMicrobiology(Z&O),EURLSalmonella,BILTHOVEN,theNetherlands
INTRODUCTION
Incasesofhumansalmonellosis,serotypingisstillthephenotypictypingmethodusedasthebasis
for surveillance, outbreak detection, and linkage to suspected sources. Consequently, correct
serotypinginreferencelaboratoriesisakeypriorityinEUcountriesandworldwide.
TheEuropeanUnionReferenceLaboratory(EURL)forSalmonella,situatedattheNationalInstitute
for Public Health and the Environment RIVM, Bilthoven, The Netherlands, annually organises an
interlaboratory comparison study to test the performance of the approximately 30 European
NationalReferenceLaboratories(NRLs)onserotypingofSalmonella.
In2008,2009and2010,alsothereferencelaboratoriesoftheECDCFoodandWaterborneDiseases
and Zoonoses surveillance network, were joining the same interlaboratory comparison studies on
Salmonella serotyping. Within this network, 3234 laboratories (FWDs) from all over the world,
thoughwithamajorityfromEurope,participated.
MATERIALANDMETHODS
Each study contains a set of 20 Salmonella strains. H antigens, O antigens and serovar names
accordingtothemostrecentWhiteKauffmannLeMinorscheme(GrimontandWeill,2007)haveto
bereported.Since2007,theperformanceoftheNRLlaboratoriesisclassifiedaccordingtoasystem
ascribing penalty points to incorrect serotyping. A distinction is made between the five most
importantSalmonellaserovars(asindicatedinEUlegislation)andallotherstrains:
4 penalty points: Incorrect typing of S. Enteritidis, S. Typhimurium (including the monophasic
one), S. Hadar, S. Infantis or S. Virchow or assigning the name of one of these five serovars to
anotherstrain.
1penaltypoint:IncorrecttypingofallotherSalmonellaserovars.
The total number of penalty points is determined for each participant. The criterion of Good
Performanceis<4penaltypoints.
AllNRLsofEUMemberStatesnotmeetingthecriterionofGoodPerformancehavetoparticipate
inafollowupstudy,inwhich10additionalstrainshavetobeserotyped.
RESULTS
Table 1 shows an overview of the results obtained for the serotyping studies for all participants in
thestudies,bothwithintheEUandworldwide.
Thepercentagesofcorrectlytypedstrainsremainquitestableovertheyears(Figure1),withusually
a slightly better performance for the Oantigens than for the Hantigens (Table 1). The relatively
large number of 56 penalty points in 2009 (Table 1) was mainly due to the results of one nonEU
NRL,participatingforthefirsttime.ThenumberoflaboratoriesnotmeetingtheGoodPerformance
criteriondecreasedfrom7in2007to2in2012.
REFERENCES
1.JacobsReitsma,W.F.,etal.SixteenthEURLSalmonellainterlaboratorycomparisonstudy(2011)ontyping
ofSalmonellaspp.RIVMReport330604027(www.eurlsalmonella.eu/Publications/).
2. JacobsReitsma, W.F., et al. Interim Summary Report on the 17th EURLSalmonella interlaboratory
comparison study (2012) on typing of Salmonella spp., dated 122013
(www.eurlsalmonella.eu/Publications/).
138
3. Grimont, PAD and Weill, FX, 2007. Antigenic formulae of the Salmonella serovars, 9th ed. WHO
CollaboratingCentreforReferenceandResearchonSalmonella.InstitutePasteur,Paris,France
(http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s000036089).
4. PolHofstad, I.E., et al. European Centre for Disease Prevention and Control. Third external quality
assuranceschemeforSalmonellatyping.ISBN9789291933426.Stockholm:ECDC;2012.
TABLESANDFIGURES
Table1.Overviewoftheresultsobtainedintheserotypingstudiesforallparticipants(bothwithintheEU
andworldwide).
Laboratory type NRLs NRLs FWDs NRLs FWDs NRLs FWDs NRLs NRLs
Study XII XIII XIII XIV XIV XV XV XVI XVII
Year 2007 2008 2008 2009 2009 2010 2010 2011 2012
N participants 26 29 34 31 32 33 32 36 31
N strains evaluated 20 20 20 20 20 19 19 19 20
O-antigens correct, all strains
510/520
98%
568/580
98%
665/680
98%
603/620
97%
631/640
99%
616/627
98%
482/494
98%
670/684
98%
612/620
99%
H-antigens correct, all strains
497/520
96%
568/580
98%
659/680
97%
581/620
94%
599/640
94%
598/627
95%
460/494
93%
657/684
96%
605/620
98%
Names correct, all strains
493/520
95%
560/580
97%
646/680
95%
578/620
93%
596/640
93%
593/627
95%
456/494
92%
586/612
96%
597/620
99%
All O-antigens correct/lab
18/26
69%
22/29
76%
25/34
74%
23/31
74%
26/32
81%
29/33
88%
25/32
78%
31/36
86%
24/31
77%
All H-antigens correct/lab
15/26
58%
21/29
72%
25/34
74%
14/31
45%
17/32
53%
22/33
67%
21/32
66%
25/36
69%
19/31
61%
All names correct/lab
14/26
54%
17/29
59%
20/34
57%
15/31
48%
16/32
50%
20/33
61%
19/32
59%
25/36
69%
17/31
55%
Total number of Penalty Points 36 34 n.a. 56 n.a. 37 n.a. 41 20
Total number of labs non-Good
Performance
6 4 n.a. 5 n.a. 4 n.a. 4 2
Total number of labs non-Good
Performance after Follow-up
0 0 n.a. 0 n.a.
0
(n=3)
n.a.
1
(n=3)
in process
n.a.=notapplicable
Figure1.Thepercentageofstrainsthatwerecorrectlyserotyped(serovarnames)bytheNRLandFWD
laboratoriesfrom2007onwards.

139
MicroVal/ISO16140validationofaPCRmethodforSalmonelladetection
inpowderedinfantformula,probioticculturepowdersandingredients,
afterafirstscreeningforEnterobacteriaceae
WilmaJACOBSREITSMA
1
,MarkWUITE
1
andBenjaminJUNGE
2
1
MicroValExpertLaboratory,
NationalInstituteforPublicHealthandtheEnvironment(RIVM),
BILTHOVEN,theNetherlands
2
BIOTECONDiagnosticsGmbH,POTSDAM,Germany
INTRODUCTION
Certain types of food products, like powdered infant formula, need to be tested on absence of
EnterobacteriaceaeaswellasSalmonella.Thiscanefficientlybedonebycombinedtestingonthe
samesamplepreparationasdescribedbelow:
FollowingpreenrichmentinBPW,DNAfromdeadbacterialcellsisinactivatedwithReagentDto
avoidfalsepositivePCRresults.ThefoodproofStarPrepOneKitisusedforrapidpreparationof
DNA. Presence of Enterobacteriaceae is tested by realtime PCR (foodproof Enterobacteriaceae
plus E. sakazakii Detection Kit). Enterobacteriaceaepositive samples only are next tested by a
SalmonellaspecificrealtimePCR(foodproofSalmonellaDetectionKit).
This alternative method for Salmonella detection (BIOTECON Diagnostics) was evaluated in an
independentMicroValvalidationstudyaccordingtoISO16140:2003.
MATERIALANDMETHODS
The alternative method was evaluated against the reference method ENISO 6579:2002 for
detection of Salmonella spp. in Powdered Infant Formula (PIF), probiotic Culture Powders (CP),
andIngredients(Ingr)forinfantformulalikestarch,vitaminsandsucrose.
RESULTS
MethodComparisonStudy:
Testswereperformedin2011/2012on180samples.Nonaturallycontaminatedsamplescouldbe
tested, due to the nature of the sample types. A total of 114 samples were artificially
contaminated and 66 samples were noncontaminated. Calculations of the relative accuracy,
relativesensitivityandrelativespecificityaregiveninTable1.Relativedetectionlevel(LOD)tests
were performed on the 3 combinations of sample types/strains as described in Table 2. The
samples were analysed 6 times with each of the 2 methods, at 5 levels of contamination. The
LOD
50%
, being the estimation of the level of contamination enabling positive detection in 50% of
cases(Wilrich&Wilrich,2009),isgiveninTable2.Forinclusivitytesting,all50Salmonellastrains
gave a Salmonellapositive result with the alternative method. For exclusivity testing, all 30 non
SalmonellastrainsgaveaSalmonellanegativeresultwiththealternativemethod.
Interlaboratory Study: The interlaboratory study was conducted in February 2012, involving 15
laboratories from 4 different countries. Analyses were carried out on 100 grams portions of
powderedinfantformulasamples,artificiallycontaminatedwithaSalmonellaSenftenbergstrain.
Each collaborator received 8 replicate samples for each of 3 levels of contamination. Results are
showninTable3andTable4.
CONCLUSION
The validation study concluded that there was no significant difference between the alternative
method and the ISO 6579 reference method for the detection of Salmonella in powdered infant
formula,probioticculturepowders,andingredients.
Thecertificateofcompliance(2011LR39)isavailableonwww.microval.org.
140
REFERENCES
1. Anonymous,ISO6579:2002MicrobiologyoffoodandanimalfeedingstuffsHorizontalmethodforthe
detectionofSalmonellaspp.
2. Anonymous,ISO16140:2003MicrobiologyoffoodandanimalfeedingstuffsProtocolforthevalidation
ofalternativemethods.
3. Wilrich, C., and P.T. Wilrich, 2009. Estimation of the POD function and the LOD of a qualitative
microbiologicalmeasurementmethod.JAOACInt.92,(6)17631772.
TABLES
Table1.Calculationoftherelativeaccuracy,relativesensitivityandrelativespecificity.
Category PA NA ND PD
Sum
Relative
Accuracy
AC(%)
N+
Relative
sensitivity
SE(%)
N
Relative
specificity
SP(%)
N
100 x
(PA+NA)/N PA+ND
100 x
PA/N+ NA+PD
100 x
NA/N
PIF 28 30 2 0 60 96,7% 30 93,3% 30 100,0%
CP 29 29 1 1 60 96,7% 30 96,7% 30 96,7%
Ingr 29 30 1 0 60 98,3% 30 96,7% 30 100,0%
TOTAL 86 89 4 1 180 97,2% 90 95,6% 90 98,9%
Table2.RelativedetectionlevelLOD50%withconfidenceinterval(cfu/sample).
Category Sampletype Strain(origin)
Reference
method
Alternative
method
PIF
Infant formula
(100gram)
S.Infantis
(milkpowder)
0,46[0,250,83] 0,46[0,250,83]
CP
Culturepowder
(25gram)
S.Livingstone
(infantformula)
0,65[0,331,29] 0,65[0,331,29]
Ingr
Starch
(100gram)
S.Senftenberg
(cocoa)
0,23[0,120,44] 0,23[0,120,44]
Table3.ResultsInterlaboratoryStudy.
Contamination level
persample(100g)
Number of
samples
analysed
Number of
results
evaluated
1
Number of
negativeresults
Numberof
positiveresults
Ref Alt Ref Alt
L0(blank) 120 104 103 103 1 1
L1(approx.5cfu) 120 104 5 5 99 99
L2(approx.30cfu) 120 104 104 104 104 104
1
Two laboratories reported several positive results for some of the blank samples, but they also
mentionedleakageoroverflowofthesamplebagsduringanalysis.Thedatasetsfromthesetwo
laboratorieswerethereforeexcludedfromthestatisticalevaluationofthestudy.
Table 4. Accordance (Accor.), Concordance (Concor.) and Concordance Odds Ratio (COR) values for both
methods.
Contamination level
persample(100g)
Referencemethod Alternativemethod
Accor. Concor. COR Accor. Concor. COR
L0(blank) 98.3% 98.1% 1.12 98.3% 98.1% 1.12
L1(approx.5cfu) 91.6% 90.8% 1.10 91.6% 90.8% 1.10
L2(approx.30cfu) 100% 100% 1.00 100% 100% 1.00

141
AOACRIValidationofRAPIDSalmonellamethod,
aSingle18hSelectiveEnrichmentandChromogenicMediaDetection
ofSalmonellaspp.fromFood
YannickBICHOT,FrdricMARTINEZ,WendyLAUER,MaryseRANNOU,DanileSOHIER,
RebeccaDIEVARTandChristopheQUIRING
BioRadLaboratories,3BdRaymondPoincar,92430MARNESLACOQUETTE,France;
BioRadLaboratories,2000AlfredNobelDrive,HERCULES,CA94547,USA;
ADRIADveloppement,ZACrachGwen,29196QUIMPER,France
INTRODUCTION
Current reference methods for Salmonella detection recommended by the USDA and the FDA
involvenumeroussteps,andareconsequentlycostlyandtimeconsumingdeliveringresultswithin
timeframesuptofivedays.
The RAPID'Salmonella method offers a more convenient One Broth One Plate shortened
protocol.Resultsareobtainedwithintwodaysafter(Figure1).
TypicalmagentacoloniesontheplatesarepresumptiveforSalmonellaandshouldbeconfirmed.
The RAPID'Salmonella method has been tested for validation according to the AOAC Research
Instituterequirements.
MATERIALANDMETHODS
AllexperimentswereperformedbyAdriaDevelopmentlaboratory.
InclusivityandExclusivityStudies
Inclusivity and exclusivity were investigated with, respectively, 100 Salmonella isolates
representative100differentserotypesand31otherGram()bacteria.
MethodComparisonStudy
TheRAPID'Salmonellamethodwascomparedwiththeappropriatereferencemethod:USDAMLG
4.05formeatproductsandFDABAMChapter5forothertypesoffoodproducts(2,3).
Fourfoodswereselected:rawgroundchicken,shelleggs,cantaloupeandpeanutbutter.
Twenty samples of each product were spiked with a Salmonella strain (table 1), targeting 15
cfu/sampleandtestedbyboththereferencemethodandtheRAPID'Salmonellamethod.Fivenon
spikedsampleswerealsoanalyzedascontrol.
Theproportionofpositivesamplesforthereferencemethodwascomparedtotheproportionof
positive samples for RAPID'Salmonellamethod using theMantelHaenszel ChiSquare analysis for
unpaired samples. A X
2
value of less than 3.84 was indicative of a no significant difference at the
5%probabilitylevel(4).
RuggednessandStabilityStudies
The ruggedeness study was designed to test four minor procedural variables: plate incubation
temperature(35C,37Cand39C),plateincubationtime(21h,24hand27h),platesmadefrom
dehydratedmediumagainstreadytouseplates.Platesmadefromdehydratedmediumwerealso
testedonday0,day1andday2ofstorage.
Thestabilitystudywasperformedwiththreekitstestedatdifferentpointsintheshelflife:anew
lot,alotatthemiddleofshelflifeandalotclosetotheendofshelflife.
Samestrainshavebeenusedforthessstudies:SalmonellaEnteritidisATCC13076andSalmonella
Typhimurium ATCC 13311 as positive strains and Klebsiella pneumoniae ATCC 13883 as negative
controlwereusedforthesestudies.
Fivereplicatesofeachstrainweretested.Ruggednesswasperformedintriplicate.
142
RESULTS
InclusivityandExclusivityStudies
NinetysevenstrainsofSalmonellawerescoredaspositive.Thethreenegativestrainsbelongedto
Paratyphiserotypeswhichareunusualinfoodproducts.Inclusivityratewas97%.
Among the 31 nonSalmonella strains tested 28 strains were negative. Three strains, one
Escherichia hermanii, one Citrobacter diversus and one Serratia marcescens produced cross
reactions.Otherstrainsfromthesamespeciesweretestedwithnegativeresult.
MethodComparisonStudy
For all foods tested, the X
2
value was less than 3.84 (Table 2). No significant difference was
observed between the RAPID'Salmonella method and the reference methods for raw ground
chicken,eggs,cantaloupeandpeanutbutter.
RuggednessandStabilityStudies
All strains of Salmonella gave positive results with the addition of minor modification into the
procedure. Klebsiella pneumoniae remained negative (Table 3). Plates made from dehydrated
mediumgavethesameresultsafterday0,day1andday2ofstorage.
The three readytouse lots tested (new, mid and at the end of the shelf life) performed also as
expected(Table4).
CONCLUSION
WhencomparedtotheUSDAandFDAreferencemethods,RAPID'Salmonellawasshowntobean
effectivealternativemethodforthedetectionofSalmonella.Themethodprovidesgoodinclusivity
and exclusivity results. Ruggedness study varying format used, incubation time and incubation
temperaturedidnotaffecttheperformancesofthemethod.Stabilityofthemediumwasproven
by testing readytouse plates and different point in the shelf life and plates made from the
dehydratedmediumafterthreedaysofstorage.
Consequently, the RAPID'Salmonella method was successfully validated, greatly shortening the
time to result over reference methods. Its convenience should be a key attractive criterion for
foodchainoperatorsandcontributetothecontrolofthismajorpublichealthconcern.
REFERENCES
1. United States Department of Agriculture, Food Safety and Inspection Service. Microbiology Laboratory
Guidebook Chapter 4.05. Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg
andCatfishProducts.
2. United States Food and Drug Administration, Center for Food Safety and Applied Nutrition.
BacteriologicalAnalyticalManualChapter5Salmonella.
3. Mantel,N.andHaenszel,W.1959.JournaloftheNationalCancerInstitute,22,719774.

143
TABLESANDFIGURES
Figure1RAPIDSalmonellaprotocol
25g+225mlBPW
supplementedwiththeRAPIDSalmonellacapsule
Stomach2min
Incubate16h(16h20h)at41.5C
Streak10lonRAPIDSalmonellaplate
Incubate24h+/1hat37C1C
Examineplatesforpresenceoftypicalcolonies:
RAPIDSalmonella:magentacolonies,round,regular,diameterfrom1to2mm
Confirmtypicalcoloniesaccordingtothereferenceprotocol
Table1MethodComparisonInoculumStrains
Matrix InoculumStrain Origin
RawGroundChicken SalmonellaBraenderup ATCCBNA664
Eggs SalmonellaEnteritidis CIP82.97
Cantaloupe SalmonellaGrumpesis CIP105621
PeanutButter SalmonellaTyphimurium ATCC14028
Table2MethodComparisonStudyResults
Matrix Inoculation
(MPN/25g)
#
samples
RAPID'Salmonella
confirmed
Referencemethod
positive
X
2
RawGroundChicken 0 5 0 0
0.7 20 10 14 1.63
Eggs 0 5 0 0
2.3 20 11 12 0.10
Cantaloupe 0 5 0 0
1.42 20 10 15 3.66
PeanutButter 0 5 0 0
2.3 20 14 12 0.43
Table3RuggdenessStudyResultsComparingFormulation,IncubationTemperatureandIncubationTime
RAPID'SalmonellaPositiveReplicate1*
Formulation Incubation
Temperature
IncubationTime S.Enteritidis
ATCC13076
S.Typhimurium
ATCC14028
K.pneumoniae
ATCC13883
Readytouse
plates
35C/37C/39C 21h/24h/27h 5/5 5/5 0/5
Dehydrated
medium
35C/37C/39C 21h/24h/27h 5/5 5/5 0/5
*:Resultswerethesameforreplicate2andreplicate3.
Table4LottoLot/StabilityResultsComparingNew,MidandEndShelfLifeMediaRAPID'Salmonella
positive
PointinShelfLife S.Enteritidis
ATCC13076
S.Typhimurium
ATCC14028
K.pneumoniae
ATCC13883
New 5/5 5/5 0/5
Mid 5/5 5/5 0/5
End 5/5 5/5 0/5

144
VIDASUPSALMONELLA(VIDASSPT)todetectSalmonella
inPrimaryProductionSamples:Certificationaccording
totheISO16140Standard
ChristineAguilhon
1
,ClineDomingos
2
,ClaudieLeDoeuff
3
,JeanLouisPittet
1
,
MaryseRannou
3
andDanileSohier
3
1
Biomrieux,ChemindelOrme,69280MARCYLETOILE,France;
2
ChemindelOrme,69280MARCYLETOILE,France;
3
ADRIADveloppment,Z.A.CrachGwen,29196QUIMPER,France
INTRODUCTION
Even if no clinical symptom is usually observed, Salmonella infections in primary production are
majorfoodsafetyconcerns,sincetheseareimportanttransmissionroutesofSalmonellathrough
the food chain to humans. The ISO and CEN Committees have thus decided a few years ago to
describestandardizedprotocolsforthedetectionofsuchzoonoticagents,whichareoftenfound
inlownumbersinprimaryproductionsamples.TheAnnexDoftheENISO6579waspublishedin
2007,andismainlybasedonSalmonellamobilitydetectioninMSRVbeforerunningstreakingon
selective agars and appropriate confirmation tests. An AFNOR standard is also available, the NF
U47100method,whichincludesasecondselectivestepinMKTTnbroth.
MATERIALANDMETHODS
The VIDAS
Up Samonella (VIDAS SPT) method is an automated immunoassay including a

selectiveenrichmentinSX2brothfor6h24h41,5C1CafteranovernightcultureinBPW.
TheconfirmationtestsaredonebystreakingtheSX2brothonselectiveagar(s)beforerunningAPI
galleriesontypicalcolonies.
RESULTS
AnENISO16140validationstudywasrecentlyperformedinordertocomparetheperformances
of the alternative method to both standards. 94 primary production samples (bootstocks, faeces
rectal, dust samples, hygiene swabs, drinker waters, litters, hatchery samples, etc.) were
analysed in the relative accuracy, sensitivity and specificity study, which concludes to equivalent
performances between the compared methods. The relative detection limits of both standard
methodsvaryfrom0,6and1,5CFU/25g,thoseoftheVIDASSPTmethodfrom0,5to1,5CFU/25g.
The selectivity and specificity of thealternativemethod was assessed by testing 50 target strains
and30nontargetstrains.
DISCUSSION
TheVIDASSPTisareliablealternativemethodforSalmonellasppdetectioninprimaryproduction
samples,andoffersimportanteconomicsavingsbyreducingtimetoresultandhandlingtime.
REFERENCES
1. EN ISO 6579:2002/Amd 1:2007 Annex D: Detection of Salmonella spp. in animal faeces and in
environmentalsamplesfromtheprimaryproductionstage.
2. EN ISO 16140 (2003) Microbiology of food and animal feeding stuffs Protocol for the validation of
alternativemethod(ISO/FDIS16140:2000(E))
3. NFU47100Mthodesd'analyseensantanimaleRechercheparl'isolementetidentificationdetout
srovaroudesrovar(s)spcifi(s)desalmonellesdansl'environnementdesproductionsanimales

145
EvaluationofaRapidLateralFlowMethod
fortheDetectionofSalmonella
ChristineAGUILHON
1
,LaurePUTHOD
1
,JinSHI
2
,AmparoSANJUAN
2
andJeanLouisPITTET
1
1
BioMrieux,MARCYL'ETOILE,France;
2
BioMrieuxShanghaiBiotech,SHANGHAI,China
INTRODUCTION
The bioNexia
TM
Salmonella assay is a rapid qualitative lateral flow device for the detection of
Salmonellainenrichedfoodandenvironmentalsamples.
The objective of this study was to evaluate the new assay for detection of Salmonella in
environmental surfaces according to AOAC guidelines (1). The comparative study for 3 surfaces
wasundertakentoprovidedataontheperformanceofthebioNexiamethodcomparedtotheFDA
BAM(2)referenceculturemethodfordetectionofSalmonella.
MATERIALANDMETHODS
Three surfaces (plastic, stainless and ceramic) were included in the study. For each matrix, five
samplesinoculatedathighlevel,twentysamplesinoculatedatalowlevel(target0.22cfu)and5
uninoculated samples were tested using the lateral flow assay and the FDA/BAM reference
method.Sampleswerepreenrichedinbufferedpeptonewater(BPW)for1624hoursat37Cand
then enriched in the selective SX2 broth for 624 hours at 41,5C. Samples were boiled for
5minutes before testing. The boiled broth was then cooled to room temperature, mixed by
vortex, and 100 5 l was transferred into the sample well on the bioNexia cassette Positive
resultsvisualizedasabluelinewereobtainedin20minutes.
BioNexia assay : bioNexia Salmonella is an immunochromatographic assay for the detection of
Salmonellaantigens.Duringtesting,theSalmonellaantigenpresentinthesamplereactswiththe
Salmonellareceptors.Theantigenreceptorcomplexmigratesupthemembranetothetestline(T)
by capillary action. If the sample contains a sufficient quantity of Salmonella antigen, a
conjugate/antigencomplexformsandbindswiththeSalmonellareceptorspresentinthetestline
region(T).Acoloredline(blueofvaryingintensity)willforminthetestlineregion(T)
IfthesampledoesnotcontainSalmonellaantigen,nolinewillappearinthetestlineregion(T).In
bothcases,abluelinemustalsoappearinthecontrollineregion(C).Thecontrollineservesasa
proceduralcontrolandindicatesthattestmigrationhasbeencorrectlyperformed.Itistheresult
of the action between the conjugate and theantimouse IgG antibody present in the control line
region(C).
Confirmation of positive results : The SX2 broth was streaked onto BS, XLD and HE agars and on
thechromogenicmediumchromID
TM
Salmonella(SM2).Plateswereincubatedat35Cfor242
h.OnetypicalcolonywasstreakedtoTSIandLIAslopesandincubatedfor242hat35C,prior
tobiochemicalconfirmationusingAPI20E(OfficialMethod978.24).Thesomatic(O)andflagellar
(H)testswerealsoperformed
RESULTS
Stainlesssteel:Forthestainlesssteelsurfaceatthelowinoculationleveltherewere13confirmed
positives for the bioNexia method at 6h and 24h and 9 confirmed positives for the FDA BAM
referencemethod.Forthehighinoculationleveltherewere5confirmedpositivesforbioNexia(6
and 24h) and 4 confirmed positives for reference method. All 5 uninoculated samples were
negativewithbothmethods.Therewerenopresumptivefalsepositiveorfalsenegativeresultsfor
the bioNexia method. Confirmation using SM2 media showed exact agreement with traditional
146
media. Although the bioNexia method detected an additional 4 confirmed positives at the low
level, and one additional confirmed positive at the high level, this was not significant at the 5%
levelusingPODanalysis.
Plastic (polypropylene) : For the plastic surface at the low inoculation level there were 16
confirmed positives for the bioNexia method at 6 and 24h and 9 confirmed positives for the FDA
BAM reference method. For the high inoculation level there were 5 confirmed positives for
bioNexia(6and24h)and5confirmedpositivesforreferencemethod.All5uninoculatedsamples
were negative with both methods. There were no presumptive false positives or false negatives
forthebioNexiamethod.ConfirmationusingSM2mediashowedexactagreementwithtraditional
media. The bioNexia method detected an additional 7 confirmed positives this was significant at
the5%levelusingPODanalysis.
Ceramic : For the ceramic tile (sponge) at the low inoculation level there were 14 confirmed
positives for the bioNexia method at 6 and 24h and 9 confirmed positives for the FDA BAM
referencemethod.Forthehighinoculationleveltherewere5confirmedpositivesforbioNexia(6
and 24h) and 5 confirmed positives for reference method. All 5 uninoculated samples were
negativewithbothmethods.Therewerenopresumptivefalsepositiveorfalsenegativeresultsfor
the bioNexia method. Confirmation using SM2 media showed exact agreement with traditional
media. Although the bioNexia method detected an additional 5 confirmed positives at the low
levelthiswasnotsignificantatthe5%levelusingPODanalysis
DISCUSSION
Forall3surfacesthebioNexiamethodgavemoreconfirmedpositivesthanthereferencemethod.
POD analysis showed that the difference in recovery was significant at 5% level for the plastic
surface only. There was exact agreement between results for SX2 broths tested at 6h and 24h.
There wereno false presumptive positive or negative presumptive results for any of the samples
usingthebioNexiamethod.ConfirmationresultsforchromogenicSM2mediaagreedexactlywith
resultsfromthetraditionalselectivemedia.
ThebioNexiamethod,easytoperformandtoreadandrequiringnospecialisedinstrumentation,it
cananswertothedemandofasimpleandrapidmethodfordetectionofSalmonella.Anextday
resultwasavailablewhentheSX2brothwastestedafter6hincubation.Asthepositivecontrolis
incorporated into the strip, it should be suitable for manufacturing facilities where Salmonella
culturesarenotdesirable.
The use of the chromogenic media SM2 for confirmation gave identical results to traditional
selective media. Chromogenic agar may have an advantage for detection of biochemically
atypicalstrainsforexampleH
2
Snegativeorlactosefermentingstrains.
REFERENCES
1. AOAC International Method Committee Guidelines for Validation of Microbiological Methods for Food
andEnvironmentalSurfaces.18Jan12prepublicationdraft
2.FDABAM
onlinehttp://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBA
M/UCM070149December2007Edition).
147
NFValidationExtensionStudyofanImmunoassayMethod
forTheDetectionofSalmonellain375gfoodSamples
1
MelindaMAUX,
1
AlicePEPLINSKI,
2
ChristineAGUILHONand
2
JeanLouisPITTET
1
EurofinsIPLNord,LILLE,France;
2
bioMrieux,MARCYLETOILE,France
INTRODUCTION
TheVIDASUPSalmonella(SPT)assayisaspecificphageproteinligandassayperformedinthe
automated VIDAS instrument associated with a 1step enrichment procedure.The method has
previously been ISO 16140 certified by AFNOR Certification for detection of Salmonella in all
25gfoodsamples.
The objective of this study was to extend the application of the alternative method to 375 g
samplesizeofrawbeef,drymilkpowderandmilkingredients,chocolateandcocoaproducts.
MATERIALANDMETHODS
375gsamples,dilutedinprewarmedsupplementedBufferedPeptonewater(rawbeefand
milk powder) or in prewarmed supplemented non fat dry milk (chocolate and cocoa), were
enrichedfor2226hoursat41.51C.Forcocoapowderandsodiumcaseinatea1/10dilution
wasrequired.Forthecategoryrawbeefandrawveal,twoenrichmentprotocolsweretested,
the first using the VIDAS SPT specific selective supplement and the second using vancomycin.
After incubation, samples were boiled for 5 1 minutes before performing the VIDAS assay.
The VIDAS SPT test is an enzymelinked fluorescent assay (ELFA) using a novel recombinant
phage protein based technology for use with the automated VIDAS instruments for the
specificdetectionofSalmonella.Attheendofthetest,theresultsareanalyzedautomatically
bythesystem,whichgivesatestvalue(TV)foreachsample.Thisvalueiscomparedtointernal
references(thresholds)andeachresultisinterpreted(positive,negative).
All presumptive positive samples were further confirmed after streaking of the enrichment
broth on a chromogenic agar plate. Plates were incubated at 37C for 24 2 h. One typical
colonywasfurtheridentifiedusinganAPI20Emicrogallery.
The validationwasconducted basedon theENISO 16140(2003) standard incomparisonwith
theENISO6579:2002referencemethodaspartoftheNFValidationprocess.
RESULTS
In the food comparative study 252 products were tested including 127 raw beef and veal
samples, 65 milk powder and ingredients samples and 60 chocolate and cocoa samples.
Artificial contaminations were achieved by using stressed bacterial suspensions, the stress
treatment and efficiency of which have been determined before spiking. Overall 127 positive
samples and 125 negative samples were obtained. The comparison showed a concordance of
98.4% between the two methods and a sensitivity for the alternative method equal to 96,9%.
Allthepositivesampleswereconfirmedandnofalsepositivewereobtainedwiththeunspiked
samples showing a specificity of 100% for the VIDAS assay. No statistically significant
differencewasobservedbetweenbothmethods.
The 50% relative detection level was determined using spiked samples at different
contamination levels with a strain of S. Ohio (raw beef), S. Enteritidis (milk powder) and S.
Anatum (chocolate). The 50% relative detection level, determined using the Spearman Krber
method, was found to be between 0.2 and 0.9 CFU/375g for both methods. Results for each
foodcategoriesareshownintheTable1below.

148
Table 1 : 50% relative detection level (CFU/375g). Raw beef 1 refers to the enrichment protocol using the
VIDASSPTspecificselectivesupplement.Rawbeef2referstotheenrichmentprotocolusingvancomycinas
thesupplement.
Product ISOMethod Alt.Method

Rawbeef1 0,5[0,30,9] 0,5[0,30,8]
Rawbeef2 0,5[0,30,9] 0,4[0,20,7]
Milkpowder 0,5[0,30,9] 0,5[0,30,9]
Chocolate 0,4[0,20,7] 0,4[0,30,7]
DISCUSSION
OnthebasisofalltheresultsobtainedaccordingtotheENISO16140(2003)standard,thescope
oftheVIDASSPTNFVALIDATIONwasextendedtothedetectionofSalmonellaspp.in50to375g
foodsamplesofrawbeefandveal,chocolateandcocoaandmilkpowderandingredients.
The VIDAS SPT demonstrated consistency as a rapid alternative method to the traditional
reference methods for detecting Salmonella in less than 24 hours in 375g food samples. The
dilutionofthematrixintotheenrichmentbrothisasignificantadvantageintermofcost,weight
forthetechnicianandspaceintotheincubator.

149
SimultaneousdetectionofSalmonellaenterica
serovarsTyphi,Enteritidis,InfantisandTyphimurium
bymultiplexPCR
RezaRANJBAR
1
,SeyyedMOJTABAMORTAZAVI
1
,AliMEHRABITAVANA
2
,FatemehPOURALI
1
,
AliNAJAFI
1
,ZahraSAFIRI
1
andCaterinaMAMMINA
3
1
MolecularBiologyResearchCenter,BaqiyatallahUniversityofMedicalSciences,
TEHRAN,Iran;
2
HealthManagementResearchCenter,BaqiyatallahUniversityofMedicalSciences,
TEHRAN,Iran;
3
DepartmentofSciencesforHealthPromotionG.DAlessandro,UniversityofPalermo,
PALERMO,Italy
INTRODUCTION
Gastroenteritis and diarrheal diseases remain as the most important health problems in all parts
of the world especially in developing countries. The infections caused by Salmonella spp. are
consideredasthemostcommonfoodborneillnessesworldwide.Typhoidfeveraloneisoneofthe
most important health problems in worldwide, especially in developing countries. Among the
serotypes of non typhoidal Salmonella spp, S. Typhimurium, S. Infantis, and S. Enteritidis are
amongst the most prevalent serotypes in Iran. Traditional procedures such as culture are time
consuming resulting in delay in diagnosis, treatment andcontrol of Salmonella infections. Hence,
thereisaneedtoprovideanddevelopmolecularmethodsforrapidandsimultaneousdetectionof
mostcommonpathogenicserovars.
There has been a general move toward molecular methods of Salmonella detection and typing,
whicharebasedlessonphenotypicfeaturesandmoreonstablegenotypiccharacteristics.PCRhas
become a potentially powerful alternative in microbiological diagnostics due to its simplicity,
rapidity,reproducibility,andaccuracy.
Theaimofthisstudywastodevelopamultiplexpolymerasechainreactionforthesimultaneous
detectionofthemostcommonSalmonellaspeciesinIran.
MATERIALANDMETHODS
Four sets of primers were designed to amplify the target genes of four Salmonella serovars
includingTyphi,thecauseofentericfever,andthethreemostprevalentnontyphoidalSalmonella
serovars,inIran,i.e.Enteritidis,InfantisandTyphimurium.Serovarspecificregionsofthegenome
wereconsideredtodesigntheprimerstoavoidcrossreactivitywithineachotherserovarorother
Enterobacteriaceae.
Standard and clinical Salmonella strains were used in the study. Clinical Salmonella strains were
recovered from fecal and blood specimens of the patients admitted to different hospitals in
Tehran.Bacterialisolationwascarriedoutbystandardbacteriologicalmethods.Anuniplexversion
ofthePCRassaywasfirstevaluatedusingbacterialcoloniesisolatedfrompureculturesofclinical
strainsofeachSalmonellaserovar.
The targets genes were Sequence STY4669 hypothetical protein for Typhi; Sequence STMO159
putativerestrictionendonucleaseforTyphimurium;SequenceSEN1383predictedphageprotein
for Enteritidis and modification metilase (M.SinI) and restriction endonuclease for Infantis. Then
multiplex PCR was optimized for the simultaneous detection of these four Salmonella serovars.
Sensitivity and specificity assays were investigated by testing different Salmonella serovars and
otherenterobacterialstrains.

150
RESULTS
Allthesampleswereconfirmedbytraditionalculturemethodsforeachindividualserovars.
TheuniandmultiplexPCRassaysproducedtheexpectedfragmentsof489bp,304bp,224bp,and
104bp for Typhi, Enteritidis, Typhimurium and Infantis, respectively. Each serovar specific primer
pair did not show any crossreactivity when tested on other Salmonella serovars and other
enterobacterialstrains.
DISCUSSION
We aimed in this study to design and optimize a multiplex PCR for rapid and simultaneous
detection of the most prevalent Salmonella serovars in Iran. High consistency was obtained
between the results of multiplex PCR and traditional methods including culture and serological
tests. Molecular weight of PCR products were consistent with expected DNA band sizes and no
falsepositiveandfalsenegativeresultsoccurredduringtheassay.
Our results show that the multiplex PCR using four primers sets had a good specificity and can
provideanimportantmethodfortherapidandsimultaneousdetectionofthefourmostprevalent
serovarsofSalmonellainIran.
Figure1:MultiplexPCR:Lanes14areuniplexPCRofS.Infantis,S.Enteritidis,S.TyphimuriumandTyphi,
respectively.Lane5isthemultiplexPCRforthefourSalmonellaserovars.
ACKNOWLEDGEMENTS
This research was supported in part by a grant from Molecular Biology Research Center,
Baqiyatallah University of Medical Sciences, Tehran, Iran. The authors would like to thank Dr.
Haghi Ashtiani and Mrs. Mina Abedini from Microbiology Laboratory of the Childrens Medical
Centerfortheirkindcooperation.

151
REFERENCES
1. Kumar S, Balakrishna K, et al. Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective
amplificationofinvA,viaB,fliCdandprtgenesbypolymerasechainreactioninmutiplexformat.LettAppl
Microbiol..2006;42:14954.
2. BejAK,MahbubaniMH,BoyceMJ,AtlasRM.DetectionofSalmonellaspp.inoystersbyPCR.
3. ApplEnvironMicrobiol.1994;60:36873.
4. HerreraLen S, McQuiston JR, Usera MA, Fields PI, Garaizar J, Echeita MA. Multiplex PCR for
distinguishing the most common phase1 flagellar antigens of Salmonella spp. J Clin Microbiol. 2004 ;
42:25816.
5. Echeita MA, Herrera S, Garaizar J, Usera MA. Multiplex PCRbased detection and identification of the
mostcommonSalmonellasecondphaseflagellarantigens.ResMicrobiol.2002;153:10713.
6. AminiK,ZahraeiSalehiT,NikbakhtGh,RanjbarR,AminiJ.MoleculardetectionofinvAandspvvirulence
genesinSalmonellaenteritidisisolatedfromhumanandanimalsinIran.AfrJMicrobiolRes.2010;4:2202
10.
7. Ranjbar R, Giammanco GM, Farshad S, Owlia P, Aleo A, Mammina C. Serotypes, antibiotic resistance,
and class 1 integrons in Salmonella isolates from pediatric cases of enteritis in Tehran, Iran. Foodborne
PathogDis.2011;8:54753.
8. Ranjbar R, Giammanco GM, Aleo A, Plano MR, Naghoni A, Owlia P, et al. Characterization of the first
extendedspectrum betalactamaseproducing nontyphoidal Salmonella strains isolated in Tehran, Iran.
FoodbornePathogDis.2010;7:915.
9. Naghoni A, Ranjbar R, Tabaraie B, Farshad S, Owlia P, Safiri Z, et al. High prevalence of integron
mediatedresistanceinclinicalisolatesofSalmonellaenterica.JpnJInfectDis.2010;63:41721.
152
RealtimePCRfordetectionofSalmonellainenvironmentalsamples
fromemptycleanedanddisinfectedpoultryhouses
EkaterinaSODOLEVSKY
1
,CinthiaSATUCHNE
1
,ElinorYECHEZKEL
1
,IritMOSERI
1
,GiladAYALI
2
,
OritMAMAN
1
,ElyakumBERMAN
3
,MichaelPIRAK
1
1
PoultryHealthLaboratories,EggPoultryBoard,POB443,KiryatMalachi,Israel;
2
Poultryfieldveterinarian;KiryatMalachi,Israel
3
IsraelVeterinaryServices,BeitDagan,Israel
INTRODUCTION
Foodproductsofanimalorigin,inparticularpoultrymeatandeggs,areconsideredtobeamajor
source of human Salmonella infections. Poultry flocks, are often infected through horizontal
transmission from the environment including contaminated poultry houses. Therefore, it is
importanttouseareliableandrapidmethodtoevaluateeffectivenessofcleaninganddisinfection
of the poultry farm before a new flock is placed. PCR technique is a good alternative to labor
intensive and timeconsuming bacteriological methods. Realtime PCR method for the specific
detectionofSalmonellawasdevelopedbyMalornyetal.[5].TheassaytargetsthettrRSBCAgene
complexofSalmonellaandincludesaninternalamplificationcontrol,whichcoamplifiedwiththe
same primers pair as the Salmonella DNA and indicates presence of PCR inhibitory substances in
tested samples. The method was validated for various food, animal feed and poultry faecal
samples [25]. We used that system for detection of Salmonella in environmental samples from
emptycleanedanddisinfectedpoultryhouses.
MATERIALANDMETHODS
The sampling was carried out during the down time in broiler houses following cleaning and
disinfection.Floor,walls,drinkingtroughs,feedingandventilationsystemsandaccesspathswere
sampledwithsterilegauzedragswabsimpregnatedwithskimmilk.Theswabswerepreenriched
in buffer peptone water for 182 hours. DNA was extracted using PrepMan Ultra sample
preparation reagent according to the manufacturer's instructions and analyzed by PCR as
previously described [5]. In parallel, all samples were analyzed by reference standard method
according to ISO 6579:2002/ Amd 1:2007 [1]. PCR efficiency was evaluated using preenriched
sterile drag swabs artificially inoculated with S. Typhimurium ATCC 14028 in concentration range
of10
6
CFUto1CFUperPCRreaction.
RESULTS
StandardcurveofrealtimePCRfromartificiallycontaminatedsamplesshowsalinearrelationship
between the log cell number and the Ct values with high correlation coefficient (R
2
= 0.9948) and
generatesaslopeof3.6thatcorrespondtoPCRefficiencyof90%(Figure1).
Inthecomparativetrial1001samplesfrom172housesweretested.Therelativeaccuracy,relative
sensitivity and relative specificity of the realtime PCR method were determined to be 98.4%,
98.2% and 97.5% respectively. Cohen's kappa analysis gave a "very good degree of agreement"
betweenthetwomethods(=0.95)(Table1).
DISCUSSION
ttrRSBCArealtimePCRhasbeenreportedforthedetectionofSalmonellainvariousfoodandfeed
samples,aswellasinpoultryfaecalsamples[25].OurresultsshowthatttrRSBCArealtimePCRis
suitable for quick and accurate detection of Salmonella in environmental samples of empty
cleaned and disinfected poultry houses. The method supplies results within 24 hours from
submission to the laboratory and allows poultry growers to quickly respond to detection of
153
Salmonellacontaminationandtoorganizeanadditionaldisinfectionprocedurebeforeanewflock
isplacedonthefarm.
REFERENCES
1. InternationalOrganisationforStandardization,2007.ISO6579:2002/Amd1:2007.AnnexD:Detectionof
Salmonellaspp.Inanimalfaecesandinenvironmentalsamplesfromtheprimaryproductionstage.Geneva,
Switzerland.
2. Lfstrm, C., Hansen, F., Hoorfar, J., 2010. Validation of a 20h realtime PCR method for screening of
Salmonellainpoultryfaecalsamples.Vet.Microbiol.144,511514.
3. Lfstrm,C.,Hoorfar,J.,2012.Validationofanopenformula,diagnosticrealtimePCRmethodfor20h
detectionofSalmonellainanimalfeed.Vet.Microbiol.158,431435.
4. Lfstrm, C., Krause, M., Josefsen, M. H., Hansen, F., Hoorfar, J., 2009. Validation of a sameday real
timePCRmethodforscreeningofmeatandcarcassswabsforSalmonella.BMCMicrobiol.9,85.
5. Malorny, B., Paccassoni, E., Fach, P., Bunge, C., Martin, A., Helmuth, R., 2004. Diagnostic realtime PCR
fordetectionofSalmonellainfood.Appl.Environ.Microbiol.70,70467052.
Figure1.Standardcurveofa10foldseriallydilutedcultureofS.TyphimuriumATCC14028withaveraged
resultsfrom3repeatedrealtimePCRexperiments.
Table 1. Results obtained for the drag swab samples from poultry houses in the comparative trial by the
realtimePCRandreferencemethod(selectiveenrichmentonMSRV)[1].
samplename
number
of
samples
PA NA FN TP FP AC SE SP
Floor 174 156 15 1 98.3 99.4 100.0 0.96
Accesspaths 168 137 27 1 3 99.4 99.3 90.0 0.92
Walls 166 63 97 2 1 3 98.2 98.5 97.0 0.92
Ventilators 165 64 99 1 1 99.4 98.5 99.0 0.97
Drinkingtroughs 163 51 107 3 2 98.2 94.4 98.2 0.93
Feedingsystem 165 81 77 4 1 2 97.0 96.5 97.5 0.92
Total 1001 552 422 12 2 11 98.4 98.2 97.5 0.95
PApositive agreement, NAnegative agreement, TPtrue positive, FNfalse negative, FPfalse positive, AC
relative accuracy, SErelative sensitivity, SPrelative specificity, Cohen's kappa coefficient. The results
wereconfirmedbyrepeatedplatingonMSRVandrepeatedDNAextractionandrealtimePCR.
154
TwonovelrealtimePCRmethodsforrapiddetection
ofSalmonellaEnteritidisandTyphimurium
HlneFRENKIELLEBOSS
1
,VirginieLAURENT
1
,AngelinaRIGAUDEAU
2
,
MarieAndreBRIFFAUD
2
,CcileOGERDUROY
1
,JeanPhilippeTOURNIAIRE
1
,ClineMAZURE
1
,
JeanPierreFACON
1
,SophiePIERRE
1
andJeanFranoisMOUSCADET
1
1
BioRadLaboratories,3BdRaymondPoincar,92430MARNESLACOQUETTE,France
2
ResalabLabovetanalysesZACLaBuzenireBP53985505LESHERBIERS,France

INTRODUCTION
In the USA, regulations require environmental monitoring of S. Enteritidis in poultry houses, prior to
theeggtesting.InEurope,foodsafetyregulationwasreinforcedtopreventpoultrymeatsadulterated
witheitherS.EnteritidisorS.Typhimuriumfrombeingmarketed.Whilethecurrentstandardmethods
require4to5daystoassessthepresenceofthesepathogens,BioRadhasdevelopedtwoalternative
realtime PCR methods, allowing their simultaneous and specific detection within 24 hours. They
include common enrichment and DNA extraction steps according to the BioRad iQCheck
Salmonella II method and can be used either as a rapid serotyping step after the detection of
Salmonellaspp.orasdirectscreeningmethods.
MATERIALANDMETHODS
PartoftheexperimentswasperformedbyLabovetlaboratory.
Testsonroutinematrices
Eggsandavarietyofpoultryandmeatsamples,aswellasbootswabs,wereartificiallyspikedwithlow
amountsofSalmonellaEnteritidisorTyphimuriumandtestedwiththerealtimePCRassays(SEorST).
Protocols are shown onfig. 1. Results were comparedto those obtained withthe AFNOR and AOAC
validatediQCheckSalmonellaspp.*(SPP)realtimePCRdetectionmethod.
Detectionlimits
Theintrinsiclimitsofdetection(LD)wereevaluatedasfollows:serialdilutionsofanexponentialphase
cultureofSEATCC13076andofSTATCC14028weresubmittedto10independentDNAextractions
andPCR.TheLDistheconcentration(CFU/mL)atwhichthestrainisdetected9timesoutof10.
Specificitystudiesonpurestrains
DNA was extracted according to the kit protocol, either from pure cultures grown in BPW (37C) or
fromisolatedcolonies.
RESULTS
Testsonroutinematrices
ResultsforSEandSTtestsarereportedanddiscussedonrespectivelytables1and2.
Detectionlimits
ForbothSEandST,LDswereestimatedaround250CFU/mL(tables3and4).
Specificitystudiesonpurestrains
TheSEkitshowed100%inclusivity,and94%exclusivity(=(21612)/216,seetable5).Itisimportantto
notethatthisisbynomeansameasureofthefalsepositiverateonroutinetesting.Somestrainsof7
serovarsrarelyassociatedwithpoultryandeggsyieldedpositivesresults,whileotherstrainsfromthe
sameserovarsyieldednocrossreaction.
The ST kit showed 100% inclusivity, (including monophasic S. Typhimurium variants), and 93%
exclusivity (= (20414)/204, see table 6). Some strains of 10 serovars yielded positive results: while
otherstrainsfromthesameserovarsyieldednocrossreaction.Formostofthecrossreactingstrains,
thetargetCqwashigherthantheCqresultingforSalmonellaspp.testing.Itisinterestingtonotethat
alargedifferencebetweentheseCqscanhinttothepresenceofanonSTcrossreactingstrain.
155
CONCLUSION
These novel realtime PCR assays for detecting S. Enteritidis and S. Typhimurium showed a perfect
inclusivityandgoodLDlevelsat250CFU/ml.Alongwiththe24hourstimetoresult,shorterthanthe
one of standard methods, these performances allow an efficient release of products free of
contamination. Eggs or meat poultry producers may thus better assess the contamination risk and
decreasecosts.Additionalevaluationsarecurrentlyongoingindifferentlaboratories.
All together, the performances of these kits should be a key attractive criterion for food chain
operatorsandforcontributingtothecontrolofthismajorpublichealthconcern.
TABLESANDFIGURES
Figure1TherealtimePCRiQCheckrealtimeprotocol
Table1MatricesforSE
Sample
Spikinglevel
(CFU/25g)
Ntested
PCRpos.
SE SPP
Wholeeggs
0 9 0 0
1 2 2 2
10 10 10 10
Eggyolk
0 7 2 2
10 7 7 7
Poultrymeat
0 21 1
(1)
0
5 10 7
(2)
7
(2)
8 8 8 8
Bootswabs
0 21 0 2
(3)
10 10 6
(4)
8
(4)
100 10 10 10
1000 10 9 10
(4)
(1) Verylatepositive
(2) The3negativeextractscomefromsamplesthateventuallygavenegativeresultsbythemicrobiological
referencemethod
(3) S.SenftenbergandS.Montevideowereidentified
(4) 2sampleswerenegativebybothPCRandreferencemicrobiologicalmethods,2sampleswerepositivewith
SPPandnegativewithSE,andwereidentifiedwithS.IndianaandS.Senftenberg.

156
Table2MatricesforST
Sample
Spikinglevel
(CFU/25g)
Ntested
PCRpos.
ST SPP
Diversefood
0 36 0 0
510 33 33 33
Poultrymeat
0 10 0 0
5 10 3
(1)
5
(1)
Bootswabs
0 15 0 2
(2)
1 10 2
(3)
4
(3)
10 10 9
(4)
10
(4)
100 10 10 10
(1) Outofthe2SPPpositivesamplesthatarenegativewithST,oneisSTpositiveafterseconddetermination(not
shown),bothshowlateCqwithSPP.
(2) S.MbandakaandS.Indianawereidentified.
(3) Out of the 2 SPP positive samples that are negative with ST, one sample found ST positive after second
determination,theotheryieldedaS.Indianaidentification.
(4) ThemissingposisSTpositiveafterseconddetermination
Table3LDforSE
CFU/mL Replicates PCRpos.
2600 10 10
260 10 9
26 10 2
Table4LDforST
CFU/mL Replicates PCRpos.
470 10 10
235 10 9
47 10 3
Table5SpecificityforSE
Nstrains
tested
PCRpos.
S.Enteritidis 45 45
Salmonella non
Enteritidis
216 12
NonSalmonella 61 0
Table6SpecificityforST
Nstrains
tested
PCRpos.
S.Typhimurium 84 84
Salmonella non
Typhimurium
204 14
NonSalmonella 33 0

157
Molecularidentificationinmonophasicandnonmotilevariants
ofSalmonellaentericaserovarTyphimurium
MarieBUGAREL,MarieLoneVIGNAUD,FrdriqueMOURY,PatrickFACH
andAnneBRISABOIS
ANSES,FrenchAgencyforFood,EnvironmentalandOccupationalHealth&Safety,
MaisonsAlfortLaboratoryforFoodSafety,MAISONSALFORT,France
INTRODUCTION
In most Salmonella enterica subsp. enterica serovars, the antigenic formula is composed of two
flagellarphases.ThefirstflagellarphaseisencodedbythefliCgeneandthesecondoneisencodedby
thefljBgene.ExpressionoffliCandfljBisregulatedthroughaphasevariationmechanismmediated
by a DNA invertase, Hin, involved in the reversible inversion of the H segment. The fljA gene, which
encodes a negative regulator of fliC expression, is located downstream of fljB. Monophasic variant
lacking second flagellar phase S.1,4,[5],12:i: emerged worldwide a few years ago and have since
becomeoneofthemostfrequentlyisolatedserovarsinmanycountries.Inhumancasesaswellasin
food and animal products, isolation rates have increased 10fold from 2005 to 2010 (NRCSalm and
ANSES Salmonella Network data). Furthermore, two other variants of S.Typhimuriumlike not
frequentlyencounteredhavebeendescribed,onelackingthefirstflagellarphaseS.1,4,[5],12::1,2and
thenonmotilevariant,S.1,4,[5],12::.DuetotheincreasingprevalenceofmonophasicS.1,4,[5],12:i:
isolates,theEuropeanFoodSafetyAuthority(EFSA)recommendedtheconfirmationoftheserological
identificationofmonophasicS.1,4,[5],12:i:strainsusingaPCRprotocolbasedonthedetectionofthe
fliABintergenicregionspecificofserovarTyphimuriumanditsvariantsandthefljBgene[1].
MATERIALANDMETHODS
Bacterialstrains:TwohundredandfiftysixSalmonellaentericastrainsofserovarTyphimuriumandits
monophasic and nonmotile variants collected in France between 2001 and 2010 through the
SalmonellaNetworkattheANSESLaboratoryforfoodsafetywereanalyzed.Isolateswererecovered
from various animal and food sources, mainly from poultry, cattle and pig, pork, chicken and dairy
products. Furthermore, the reference strain LT2 was used as a positive control for the investigated
markerandasingleisolateofeachfollowingserovarsFarsta,GloucesterandLagoswasinvestigatedas
negativecontrols.Theantigenicformulaeofstrainswereserologicallydeterminedusingagglutination
tests with antisera according to an inhouse method [2]. Finally, strains of serovar S. 1,4,[5],12:i:
(n=90),S.1,4,[5],12::1,2(n=25),nonmotileS.1,4,[5],12::(n=17)andserovarTyphimurium(n=124)
werestudied.
PCR detection: The seven markers listed in table 1 were detected either with conventional PCR
method:fliABintergenicregionandfljBgeneorwithrealtimePCRmethod:mdh,STM2757,fljA,fliC,
hin genes). Moreover, presence of ttrC gene, marker of the Salmonella genus was checked. Primers
usedarelistedintable1.
RESULTS
ThreemarkersSTM2757,mdhandfliABwerecommonlydetectedinserovarTyphimuriumexceptfor
asingleTyphimurumstrain(fliAB)andinallvariantstrainsexceptfor9strainsofmonophasicstrains
outofthe115tested(STM2757).MonophasicS.1,4,[5],12:i:weregenotypicallyconfirmedwiththe
absenceofthefljB,fljAandhingenes.Nevertheless13ofthem(14.5%)werepositivefortheselast
three genes, revealing monophasic strains called inconsistent as previously described [3]. All non
motile1,4,[5],12::strainshadthefliC,fljA,fljBandhingenes.ThefliCgenewasdetectedin88%of
monophasic S.1,4,[5],12::1,2 strains. The combination of the seven markers detection enables to
recognizeatotalofelevendifferentgenotypesamongwhicheightgenotypeswithintheS.1,4,[5],12:i:
158
collection (table 2). The Spanish and the US clones previously described for S.1,4,[5],12:i: could be
distinguishedandassignedtoagenotype[4].
DISCUSSION
Basedonthismolecularapproach,71%oftheFrenchS.1,4,[5],12:i:collectioncouldbeattributed
to the Spanish clone (patterns P5 and P8) whereas only 2%were assigned to the US one (P7 and
P9).Thisstudyhighlightstheusefulnessofthesemolecularmarkersandgenotypesforidentifying
lineages,especiallyamongtheepidemiologicallyimportantmonophasicS.1,4,[5],12:i:variant[5].
ACKNOWLEDGEMENT
The authors wish to thank all the participant laboratories to the Salmonella Network for sending
theirSalmonellaisolatesandthetechnicianteamfortheirworkinconventionalserotypingallthe
investigatedstrains.
REFERENCES
1. European Food Safety Authority (EFSA) Panel on Biological Hazards (BIOHAZ). Scientific Opinion on
monitoringandassessmentofthepublichealthriskofSalmonellaTyphimuriumlikestrains.EFSAJournal.
2010;8(10):1826
2. http://www.efsa.europa.eu/en/efsajournal/doc/1826.pdf
3. Danan, C., Fremy, S., Moury, F., Bonhert, M.L. and Brisabois, A. (2009). Determining the serotype of
isolated Salmonella strains in the veterinary sector using the rapid slide agglutination test
Recommendations for regulated serotypes in the poultry sector. Les cahiers de la rfrence de l'AFSSA
Cahiern2(dcembre2009).
4. Hopkins, K.L., Kirchner, M., Guerra, B., Granier, S.A., Lucarelli, C., Porrero, M.C., et al. (2010).
Multiresistant Salmonella enterica serovar 4,[5],12:i: in Europe: a new pandemic strain? Euro Surveill. 15,
19580.
5. Soyer, Y., Moreno Switt, A., Davis, M.A., Maurer, J., McDonough, P.L., SchoonmakerBopp, D.J., et al.
(2009). Salmonella enterica serotype 4,5,12:i:, an emerging Salmonella serotype that represents multiple
distinctclones.J.Clin.Microbiol.47,35463556.
6. BugarelM.,VignaudM.L.,MouryF.,FachP.,BrisaboisA.(2012).Molecularidentificationinmonophasic
and nonmotile variants of Salmonella enterica serovar Typhimurium. Microbiology Open. doi: 10.
1002/mbo3.39.

159
TABLESANDFIGURES
Table1.Primersequencesdesignedforthesevenmarkersamplification
Target
name Primersequences(53)
fliAfliB
FFLIB
RFLIA
CTGGCGACGATCTGTCGATG
GCGGTATACAGTGAATTCAC
fljB
Sense59
Antisense83
CAACAACAACCTGCAGCGTGTGCG
GCCATATTTCAGCCTCTCGCCCG
fliC
Sense60
Antisensei
ACTCAGGCTTCCCGTAACGC
ATAGCCATCTTTACCAGTTCC
fljA
fljAF
fljAR
TCCGAAGCCAGAATCAAATTTTCC
TACGTTTTAATGATATCCCTGTTCG
hin
hinF
hinR
CGCCCCGGCCTGAAACGA
CGACTAATCTGTTCCTGTTCATGTT
mdh
mdhF
mdhR
TGCCAACGGAAGTTGAAGTG
CGCATTCCACCACGCCCTTC
STM
2757
STM2757F
STM2757R
AACCGTACAGGGTTTATACGCC
TTATCGTGCCGCCGAATTATGG
ttrC
ttrCF
ttrCR
CTCACCAGGAGATTACAACATGG
AGCTCAGACCAAAAGTGACCATC
Table2.DifferentgenotypeswithinthemonophasicS.1,4,[5],12:i:strains(N=90),(SP):Spanishclone,(US):
Americanclone,(IC):Inconsistentvariant
Genotype
(rate%)
STM
2757
mdh fliAB fliC fljB fljA hin
P5(SP)
(64.5%)
+ + + +
P1(IC)
(14.5%)
+ + + + + + +
P8(SP)
(6.7%)
+ + +
P3
(4.4%)
+ + + + + +
P4
(4.4%)
+ + + + +
P6
(3.3%)
+ + + + + +
P7(US)
(1.1%)
+ + + + +
P9(US)
(1.1%)
+ + + +

160
CharacterizationoftheemergingSalmonella4,[5],12:i:
inDanishanimalproduction
HectorARGUELLO
*1
,GitteSRENSEN
2
,AnaCarvajalUREA
1
,DorteLAUBAGGESEN
2
,
PedroRUBIO
1
andKarlPEDERSEN
2
1
InfectiousDiseasesandEpidemiologyUnit,DepartmentofAnimalHealth,Faculty
ofVeterinaryScience,UniversityofLen,Len,Spain
2
DanishTechnicalUniversity,NationalFoodInstitute,Blowsvej27,
DK1790COPENHAGENV,Denmark
INTRODUCTION
S. 4,[5],12:i. serotype has emerged in the last decade as one of the main serotypes related to
human salmonellosis[1]. Various studies have been carried out in Europe and other parts of the
world[2],toimprovetheknowledgeaboutthecirculatingpopulationsofthisserotypeindifferent
countries. The purpose of this study was to investigate Danish isolates of the S. 4,12:i: and S.
4,5,12:i: coming from Danish farm animals and with well defined farm identification, i.e. DSCP
isolates,freshmeatsurveillanceprogrammeorEUbaselinestudies,makinganevaluationoftheir
clonalitybyphagetypingandMLVA,togetherwiththeirantimicrobialsusceptibilityprofiles.
MATERIALANDMETHODS
107 isolates from Danish animal production system were selected from the laboratory database
forfurtherstudies.Tobechoseneachstrainhadtobeserotypedas4,[5],12:i:.
The selected isolates were serotyped by slide agglutination according to Grimmont & Weill [3].
ThefliBfliAintergenicregionandfljBgenewerecheckedafterserotyping.
Phagetyping: Performed according to according to the International Federation for Enteric Phage
Typing scheme (IFEP, Laboratory of Enteric Pathogens, Health Protection Agency, Colindale,
London,UK).
MLVA:TheselectedisolateswereanalysedbyMLVAaccordingtotheprotocolofTorphdaletal.,
[4].
Antimicrobial resistance: Antimicrobial susceptibility testing was performed to establish by MIC
valuesbyabrothmicrodilutionprocedureinaccordancewithCLSIandDANMAPguidelines,using
thesemiautomaticSensiTitresystem(TrekDiagnosticSystemsLtd.,UK).
RESULTS
Fromtheoverall107isolatesincludedinourstudy,66hadtheantigenicformulaS.4,5,12:i:while
41 were S. 4,12:i:. All the isolates tested amplified a 1kb product corresponding to the fliBfliA
intergenic region. Seventy of the isolates (65.4 %) were negative for the fljB allele amplification,
which means that they are true S 4,[5],12:i:. The remaining 37 (34.6 %) presented the fljB allele
(1389bpfragment).
The absence of the fljB gene was more associated with 4,5,12:i: isolates (77 %) than 4,12:i.
isolates(46%).
ThemostprevalentphagetypewasDT193,61isolates(57.0%);twelveisolates(11.2%)wereDT
120 and another eight were DT U302. Other phage types were also found. MLVA typing divided
the isolates collection in 28 different profiles. The six most prevalent are reported in Table 1.
Thesesixtypesgroupedatotalnumberof67isolates(62.6%oftheoverall).
The antimicrobial susceptibility analysis showed that a 97.2 % of the isolates were resistant to at
least one compound with only four strains being pansusceptible. Of the 107 isolates, 76
presentedmultiresistanceprofiles(71%),definingmultiresistanceasresistancetofourormore
antimicrobials.SixtysixisolateshadtheantimicrobialpatternAMPSTRSMXTET,whichis86.8%
of the multidrugresistance isolates and 61.7 % of the overall collection. Furthermore, 72 strains
161
presentedthispatternassociatedtootherantimicrobialresistances,whichmeansa95.7%ofthe
multidrugresistancestrains.
DISCUSSION
Theabsence/presenceofthefljBgeneisthecriterionfordistinctionbetweentruemonophasicS.
4,[5],12:i: and S. Typhimurium according to the EFSA guidelines [4]. Seventy of the isolates
selectedforthisinvestigationwereconfirmedasmonophasicS.TyphimuriumbylackingfljBgene
and the other thirtyseven should be included in the S. Typhimurium group according to the
guidelines cited above. It is also worth noting that the absence of the fljB gene was more
associated with 4,5,12:i: isolates (77 %) than 4,12:i. isolates (46 %). Thus, the 4,5,12:i: strains
were predominantly true monophasic, lacking the fljB gene, whereas the 4,12:i: strains were
almost evenly distributed between true monophasic strains without the fljB gene and
phenotypicallymonophasicstrainspossessingthefljBgenebutseeminglyunabletoexpressit.
Several different clones were demonstrated using molecular typing methods, with a major clone
ofDT193isolates,whichisalsoknownfromothercountries.Theisolateswerecharacterizedbya
high level of antimicrobial resistance, most notably to ampicillin, streptomycin, sulphonamides,
andtetracyclines.
The fact that just four strains were pansusceptible to the antimicrobials tested shows the high
level of resistance in monophasic S. Typhimurium. Resistance to classical antimicrobials like
ampicillin, tetracyclines, streptomycin or sulphonamides was common and what is more, the
association of resistances in multiresistant types was seen in 71 % of the strains selected. The
mostcommonRtypefoundwasAMPSTRSMXTET.
Twoofthesefivemainprofiles3129NA211and31310NA211thatrepresented8%and11.3
% of theisolates, respectively, are the most commonly found in otherEuropean studies [4]. The
Danish main profiles, 3139NA211 and 3149NA211, were only separated by a single
repetitioninlocusSTTR5andmostofthesewerealsofljB(),RtypeAMPSTRSMXTET.
REFERENCES
1. Guerra B., et al., (2000) Molecular characterisation of emergent multiresistant Salmonella enterica
serotype[4,5,12:i:]organismscausinghumansalmonellosis.FEMSMicrobiolLett.190:341347.
2. Hopkins K.L., et al., (2010). Multiresistant Salmonella enterica serovar 4,[5],12:i: in Europe: a new
pandemicstrain?EuroSurveill.3;15,(22):19580.
3. Torpdahl M., et al., 2007 Tandem repeat analysis for surveillance of human Salmonella Typhimurium
infections.EmergInfectDis.13:388395.
4. EFSA, (2010). Scientific Opinion on monitoring and assessment of the public health risk of Salmonella
Typhimuriumlikestrains.EFSAJ.,8:1826.
5. Hauser E. et al., (2010). Pork contaminated with Salmonella enterica serovar 4,[5],12:i:, an emerging
healthriskforhumans.ApplEnvironMicrobiol.7646014610.
TABLESANDFIGURES
Table 1. Characteristics of the six most representative MLVA profiles found in the analysis of the
107S.4,[5],12:i:isolatesincludedinthestudy.

162
MonitoringofmonophasicandnonmotilevariantsofserotypeTyphimurium
amongnonhumanstrainscollectedbytheFrenchSalmonellaNetwork
RenaudLAILLER,FrdriqueMOURY,JoelGROUT,CatherineLAPORTE,VivianeMOREL,
ClaudeOUDART,ChristinePIQUETandAnneBRISABOIS
MaisonsAlfortLaboratoryforFoodSafety,UnitBacterialCharacterizationandEpidemiology,MAISONS
ALFORT,France
INTRODUCTION
Emergence of monophasic Salmonella Typhimurium strains, harboring the antigenic formula
1,4,[5],12:i:,hasbeenhighlightedinEuropeoverthelasttwodecades.Since2004,thisserotypehas
been among the top ten human serotypes collected by the French National Reference Centre for
Salmonella.In2010,itrepresented15%ofcollectedhumanstrains.Thestatisticalsurveillancesystem
setupbytheLaboratoryforFoodSafety(Anses,MaisonsAlfort)hasrepeatedlyidentifiedanumberof
reportedisolatesgreaterthantheexpectednumber,since2008.Strainslackingexpressionofthefirst
flagellar phase or both flagellar antigens (respectively 1,4,[5],12::1,2 and 1,4,[5],12::) are also
identified but uncommon, as reported by the European Food Safety Authority in a recent published
opinion[1].
Regardingthissituation,itseemedimportanttoestablishamonitoringatanationalleveltoestimate
the presence of these variants of Salmonella Typhimurium in the various channels throughout the
agrofoodchain.
MATERIALANDMETHODS
In the framework of the Salmonella network and in agreement with the French authority
(DGAL/SDSSA/N20108059 memorandum), the Laboratory for Food Safety (Anses, MaisonsAlfort)
collected707SalmonellaTyphimuriumlikestrainsfrom140partnerlaboratoriesin2011and2012,
mainlydistributedintheporkandpoultrychannels(Table1).
Uponreceipt,eachstrainserotypewasconfirmedbyconventionalmethod(glassslideagglutination).
A multiplex PCR, based on EFSA recommendations [1] and Bugarel et al. [2], was implemented to
confirmrelatednesstoS.Typhimurium.ThefirstPCRtargetedfljBgeneandtheintergenicregionfliA
fliB.ThesecondPCRtargetedthemdhgene,specificfromserovarTyphimurium[2]andfliCgene.
Moreover,therobustnessofCheck&Trace
Salmonellamethod[3]wasinvestigatedagainstapartof
thispanel(n=31).
RESULTS
Amongthe694strainsfromantigenicformulaS.1,4,[5],12:i:,654strains(94.2%)weremonophasic
variants confirmed from serovar Typhimurium, 38 strains (5.4%) were inconsistent (fljBpositive)
variants and only two strains (0,03%) were monophasic variants confirmed from another serovar
(Table2).
AllthestrainsS.1,4,[5],12::1,2(n=8)collectedin2011and2012bytheSalmonellanetworkwerefliC
positivemonophasicvariantfromserovarTyphimurium.
Among the five strains from antigenic formula S.1,4,[5],12::, one strain was a monophasic variant
confirmed from serovar Typhimurium, and four strains were monophasic variants confirmed from
anotherserovar.
AperfectconcordancewasobservedbetweentheresultsobtainedbyCheck&Trace
Salmonellaand
bymultiplexPCR(Table3).Moreover,resultswerealsoconcordantwiththeonesobtainedbyother
molecularmethods(PFGE,MLVA)(datanotshown).

163
DISCUSSION
The multiplex PCR implemented allowed us to characterize the French panel of Salmonella
Typhimuriumlike strains, collected during the last two years by the Salmonella network. The
proportion of monophasic variants confirmed as serovar Typhimurium is derived from a very large
majority.
This accurate characterization of these Salmonella Typhimuriumlike strains is important for
monitoringtheadequacyofregulatoryactionswithpublichealthtargets.
REFERENCES
1. European Food Safety Authority (EFSA) Panel on Biological Hazards (BIOHAZ). Scientific Opinion on
monitoringandassessmentofthepublichealthriskofSalmonellaTyphimuriumlikestrains.EFSAJournal.
2010;8(10):1826http://www.efsa.europa.eu/en/efsajournal/doc/1826.pdf
2. BugarelM.,VignaudM.L.,MouryF.,FachP.,BrisaboisA.(2012).Molecularidentificationinmonophasic
and nonmotile variants of Salmonella enterica serovar Typhimurium. Microbiology Open.
doi:10.1002/mbo3.39.http://onlinelibrary.wiley.com/doi/10.1002/mbo3.39/pdf
3. Wattiau P., Van Hessche M., Schlicker C., Vander Veken H., Imberechts H. Comparison of Classical
Serotyping and PremiTest Assay for Routine Identification of Common Salmonella enterica Serovars.
JournalofClinicalMicrobiology,2008b;46,pp.40374040.
TABLESANDFIGURES
Table 1: Distribution of the origin among the 707 Salmonella Typhimuriumlike strains collected in 2011
and 2012, in the framework of the French Salmonella network managed by Anses lab (MaisonsAlfort,
France)
serovars
S
.
1
,
4
,
[
5
]
,
1
2
:
i
:
S
.
1
,
4
,
[
5
]
,
1
2
:
:
1
,
2
S
.
1
,
4
,
[
5
]
,
1
2
:
Feed 23 / 2
Ecosystem 27 4 1
Animal health and
productionincluding
cattle
poultry
pigs
23
3
48
16
0
13
4
Food including
beef
poultrymeat
pork
40
7
39
13
13
3
/ /

164
Table 2: Results obtained by multiplex PCR on strains collected in 2011 (n=312) and 2012 (n=395) by the
SalmonellaNetwork,accordingtoantigenicformula.
Targetedmarkers
S
.
I
1
,
4
,
[
5
]
,
1
2
:
i
:
S
.
I
1
,
4
,
[
5
]
,
1
2
:
:
1
,
2
S
.
I
1
,
4
,
[
5
]
,
1
2
:
fliC
fliAfliB
fljB
mdh
+ 1000bp + 654 1
+ 1000bp + + 38 8
+ 250bp 2 4
Table3:ResultsobtainedbyCheck&TraceSalmonella,forapanelof31strainscollectedbytheSalmonella
Networkin2011and2012,accordingtoantigenicformulaandPCRresults.
[codes of genovar: 2570: not defined, 2717: S1,4,[5],12:i:, 10909: Typhimurium, 14909:
Schwarzengrund(S.I1,4,12,27:d:1,7)orGrumpensis(S.I1,13,23:d:1,7)]
Targetedmarkers
S
.
I
1
,
4
,
[
5
]
,
1
2
:
i
:
S
.
I
1
,
4
,
[
5
]
,
1
2
:
:
1
,
2
S
.
I
1
,
4
,
[
5
]
,
1
2
:
fliC
fliAfliB
fljB
mdh
1000bp
23
(2717)
+
1000bp
3
(10909)
1
(10909)
+
250bp
2
(2570)
1
(14909)
1
(14622)

165
StabilitystudyoftheMLVAprofiles
SabrinaCADELSIX
1
,BenjaminGLASSET
1
,FrdricAUVRAY
2
andAnneBRISABOIS
1
1
Anses,FrenchAgencyforFood,EnvironmentalandOccupationalHealth&Safety,
MaisonsAlfortLaboratoryforFoodSafety,BacterialCharacterizationandEpidemiologyUnit,
23avenueduGnraldeGaulle,F94706MAISONSALFORTCedex,France;
2
Anses,FrenchAgencyforFood,EnvironmentalandOccupationalHealth&Safety,Ecophysiology
andBacterialDetectionUnit,23avenueduGnraldeGaulle,
F94706MAISONSALFORTCedex,France
INTRODUCTION
At today the MLVA (MultiLocus VNTR Analysis) is more and more used to subtype foodborne
isolatesofSalmonella.Somequestionsstillexistinthescientificcommunityabouttheinstabilityofthe
VNTRs(VariableNumberofTandemRepeats)andthetraceabilityoftheMLVAprofilesoverthetime.
Indeed, the main disadvantage of the MLVA method, comparing with PFGE (the gold standard sub
typing method), is thattherate of mutation of the VNTR(and consequently of MLVA profiles) is still
notwellknown.
We present here an in vitro study about the stability of some MLVA profiles over time and stress
conditions. 670 samples from 8 different strains of monophasic variants of Salmonella enterica
serotype Typhimurium were analyzed. Several conditions such as storage techniques, different
temperatures of growth and different media, effect of food matrix and stress were tested. For
traceabilitysconcerns,andtocheckthattheMLVAanalysisitselfhadnotaholduponthestabilityof
theresults,theprotocolofMLVAanalysisusedwasalsocheckedout.
MATERIALandMETHODS
Bacterialstrains
To study the stability of MLVA profiles, 8 strains were chosen in reference to the diversity of their
PFGE,MLVAprofiles,theirantibioticresistanceandtheepidemiologicaldata(Table1).Moreovertwo
ofthesestrains(11CEB838SALand11CEB4971SAL)werechosenbecausetheypresentedtheepidemic
MLVAprofile(profilefoundinpatientstrains)duringthetwoperiodsofalertsinAugust/September
andNovember/December2011duetotheemergingmonophasicvariants[1].
MLVAanalysisandTestsofthestability
The InstaGene matrix (BioRad, France) was used to extract the DNA and the MLVA typing was
performedusingpreviouslydescribedprotocol[2].
ThestabilityoftheMLVAprofileswerestudiedalongthemanipulationsdescribedintheseprotocols
(reception of the strains, DNA extraction and MLVA analysis). Moreover the modes of conservation
(stockcultureagartubes,BioRadandcryopearltubes,FisherScientific),thelifestimeofthestrains
at4C(Day+4,andDay+20),thethermalshock(at20Cand56C)andthegrowthofthestrainsinthe
matrix (sausage meat and dried sausage) were tested. 10 colonies/plate were collected, DNA
extractedandMLVAanalysesrealizedasexplainedbeforeforeachsample.
RESULTS
On the 670 samples tested, 11 variations of one or several repeated units (UR) were observed. The
variationfrequencywas1.64%.
NovariationswereobservedwithintheMLVAprofilesofthesamplestestedtoevaluatetheconditions
employed in the protocol used for MLVA analyses (from the reception of the strains to the MLVA
analysis). On 123 samples (96 from cryotubes and 27 from stock culture agar tubes), no changes on
theprofilesoccurred.
166
SecondarythelifestimeofthestrainsonTSAYEat4Cwastestedat4andmorethan20days.The
TSAYE is the growth medium used in the Bacterial Characterization and Epidemiology (CEB) Unit for
the Salmonella. Consequently it was interesting to test the stability of the MLVA profiles of strains
conservedseveraldaysonthismedium.On97samples,variationswereobservedin2samples(forthe
strain11CEB5898SAL)after20daysonTSAYEat4C.Thisrepresentedavariationfrequencyequalto
2.06%.
Thethermalshockprotocoltestedconsistsinstressthestrains3daysat20Candimmediatelyafter,
15min at 56C. A counting before and at the end of the experiences to verify that the stress had an
effectonthebacteriagrowthwererealized.On160samples,only3changedtheirMLVAprofiles(2for
the11CEB4538SALand1for11CEB4791SALstrains).Thevariationfrequencywas1.87%.
Finally, the tests performed on food matrix were realized with the strain 11CEB4791SAL on sausage
meatanddriedsausage.Thesametestswereusedonthepureculturestrainascontrol.Sixvariations
were observed on 290 samples. Four variations were observed on the dried sausage matrix and 2
variationsonthecontrolwithrespectivelyavariationfrequencyof3%and2%.Thefacttotransplant
the strain several times (3 or 4 times) and in different culture media (TSAYE, TS, XLD, and peptone
water)probablyhadanimpactontheDNAduplicationevents.
The11variationsoccurredonlyonthelociSTTR5andSTTR6.Themodificationsvariedfrom2to3UR
onlocusSTTR5andfrom1to5URonlocusSTTR6.Thesetwolociareclosedtoproteinsinvolvedin
the interaction with the host: respectively yohM (a nickel/cobalt efflux protein) and gogB (an anti
inflammatoryeffector)[3][4]andareknowntohaveaelevatediversityindex.
DISCUSSION
This study on the stability of the MLVA profiles in vitro is the first one realized on strains of
monophasique variants with so many conditions tested. Altogether, 11 samples varied their MLVA
profiles. Altogether 11 variations of one or several repeated units (UR) were observed. These
variationsappearedinsamplesleftinthesamecultureplateat4Cmorethen20days,stressedbya
thermal shock, or when several culture media were implied. Nevertheless the variation frequency
registeredisparticularlylow(1.64%)inallstressconditionstestedandzerointheconditionsdescribed
bythecurrentprotocolusedintheCEBunittoobtainMLVAresults.
REFERENCES
1. Gossner,C.M.,VanCauteren,D.,LeHello,S.,Terrien,E.,Tessier,S.,Janin,C., etal.(2012). Nationwide
outbreak of Salmonella enterica serotype 4,[5],12:i: infection associated with consumption of dried pork
sausage,France,NovembertoDecember2011.EuroSurveill.17.
2. ECDC (2011). The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic
AgentsandFoodborneOutbreaksin2009.EFSAJournal9.
3. Pilar AV, ReidYu SA, Cooper CA, Mulder DT, Coombes BK. GogB Is an AntiInflammatory Effector that
LimitsTissueDamageduringSalmonellaInfectionthroughInteractionwithHumanFBXO22andSkp1(2012).
PLoSPathogensVolume8,Issue6:114.
4. Luo Y, Kong Q, Yang J, Mitra A, Golden G, Wanda SY, Roland KL, Jensen RV, Ernst PB, Curtiss R.
Comparative Genome Analysis of the High athogenicity SalmonellaTyphimurium Strain UK1(2012). PLoS
ONE,Volume7,Issue7:113.
Table1.StrainsanalyzedforthestabilitystudyoftheMLVAprofiles

167
TheApplicationofMultiLocusVariablenumbertandemrepeat
Analysis(MLVA)fortheepidemiologicalsubtyping
ofSalmonellaentericaSerovarTyphimurium,inScotland
DerekBROWN,AngelaMURRAY,KirstenLOCKHART,HenryMATHERandJohnCOIA
ScottishSalmonella,Shigella&ClostridiumdifficileReferenceLaboratory,
GLASGOW,Scotland,UK

INTRODUCTION
S.Typhimuriumisanimportantcauseoffoodbornediseaseworldwidetogetherwithmonophasic
group B Salmonellae. Molecular subtyping of isolates is essential for outbreak investigation.
While PFGE is the GoldStandard for subtyping, certain PFGE profiles predominate limiting the
epidemiologicalsignificance.Inthisstudy,wehaveevaluatedmultilocusvariablenumbertandem
repeat analysis (MLVA) for routine typing of Typhimurium and monophasic group B Salmonellae
fromhuman,veterinary,foodandenvironmentalisolatesinScotland.
MATERIAL&METHODS
Over 700 isolates were typed including 598 Typhimurium, 80 monophasic group B (4,12:i: or
4,5,12:i:),and29ofothergroupBserotypes(Agona,Derby,Heidelberg,Java,Saintpaul,Stanley)
submitted to SSSCDRL for routine typing. Six hundred of these were serotype Typhimurium or
monophasic group B (4,12:i: or 4,5,12:i:). Phage typing
1,2
, PFGE
3
and MLVA
4
were carried out
usingstandardmethods.
RESULTS
Typhimurium could be differentiated into 32 phage types (PT), 180 PFGE types, and 321 MLVA
patterns. By phage typing, 0.8% of isolates were untypable and 55 (9.2%) did not conform to
recognisedtypes(RDNC).
TyphimuriumandmonophasicgroupBisolatescoclusteredinseveralmajorMLVAclusters(Fig1).
Other group B serovars amplified up to 3 of five MLVA loci used but did not cluster with
Typhimurium. Phage types DT120 and 193 also clustered together but the major MLVA clusters
couldbesubtypedbyPFGEandbyPT(Fig2)
CONCLUSION
Typhimurium MLVA is rapid and effective. There is not 100% correlation between PT, PFGE and
MLVA. If used as a single molecular typing scheme, single MLVA types, including common types,
maycomprisemultiplePTsandorPFGEtypes.
Figure 1: MLVA minimum spanning tree of Typhimurium (dark grey), 4,12:i: (white) and 4,5,12:i: (light
grey)isolatedinScotlandin2011/2012.

168
Figure2:MLVAMinimumspanningtreeofDT193andDT120isolatesshowingcoclusteringby
a.Phagetypeb.PFGEprofile
REFERENCES
1.AndersonES,WardLR,deSaxeMJ&deSaJDH(1977).J.Hyg.(Lond.)78:297300.
2.CallowBR(1959).J.Hyg.(Lond.)57:346359.
3.PulseNetInternational(2008)
http://www.pulsenetinternational.org/SiteCollectionDocuments/pfge/5%201_5%202_5%204_PNetStand_E
coli_with_Sflexneri.pdf
4.TorpdahlM,SrensenG,LindstedtBA,NielsenEM(2007).Emerg.Infect.Dis.13:388395.

169
PulsenetMLVAprotocolforSalmonellaentericaserovarEnteritidis
appliedtoSalmonellaentericaserovarDublin
JeanOCONNOR,NiallDELAPPEandProf.MartinCORMICAN
NationalSalmonella,ShigellaandListeriaReferenceLaboratory,Dept.ofMedicalMicroBiology,
GalwayUniversityHospital,GALWAY,Ireland
INTRODUCTION
Thedevelopmentofnewsubtypingmethodstolinkhumanisolatestofoodoranimalsourcesor
tohumanisolatesisextremelyimportant.(1)(2)(3)
MultilocusVNTRAnalysis(MLVA)isanewsubtypingmethodwhichisbasedontheVariablecopy
NumbersofTandemRepeats(VNTR).
MLVA provides greater discrimination among nonepidemiologically linked isolates than PFGE or
phage typing. As S. Enteritidis and S. Dublin are genetically closely related and have similar
antigenicstructuresthismethodwasappliedtoS.Dublinisolatestoseeifitwouldbeofvalue.(4)
METHODS
The Laboratory Standard Operating Procedure as described by the CDC for Pulsenet MLVA of
SalmonellaEntericasubtypeEnteritidisusingtheBeckmanCoulterCEQ
TM
8000wasperformedon
S.Dublinisolates.
RESULTS
Fifty one S.Dublin isolates were provided by the National Salmonella , Shigella and Listeria
ReferenceLaboratoryIreland(NSSLRL).
All S. Dublin isolates received by the NSSLRL in 2011 (2 human and 8 food or animal) ,2010 (7
humanand18foodoranimal)andallhumanisolatesfrom2009(6),2008(3),2007(4)and2006
(3)wereanalysedbytheCDCsMLVAS.Enteritidisassay.
Of the 25 human isolates ,7 were from blood,15 from faeces, 1 joint fluid,1 urine and 1 from a
psoasabscess.Noforeigntraveldetailswerelistedforanyofthehumanisolates.
Ofthe26animalorfoodisolates,22werebovine,1fromabapmixer,1fromapeahen,1chicken
and1frompoultrybootswabs.
Twentyninedistinctpatternsemerged.
AntimicrobialSusceptibilityTesting(AST)wasdoneonallisolatesagainstapanelof14antibiotics
(Ampicillin,Chloramphenicol,Streptomycin,Tetracycline,Trimethoprim,Nalidixicacid,Kanomycin,
Ciprofloxacin, Cefpodoxime, Ceftazidime , Gentomycin, Minocycline and Cefotaxime). Three
isolateswereresistanttoNalidixicacidand2wereresistanttoStreptomycin,asallotherisolates
weresensitivetoallantibiotics,ASTdidnotprovidegreatdiscrimination.
Thirtythree of the 51 isolates had the same pattern 3411023XX , for the first 6 loci, with
variationonlyinwiththelastalleleXX(seeTable1below).
Itisproposedtosequencethelastallele.
Ten clusters were present involving 35 of the 51 isolates. Within each cluster animal and human
isolatespreviouslyunlinkedcouldthenbecheckedforepidemiologicallinks
DISCUSSION
While this method while not designed to study S. Dublin could provide valuable data linking
isolates.

170
REFERENCES
1.BoxrudD,PedersonGulrudK,WottonJ,MedusC,LyszkowiczE,BesserJ&BartkusJM(2007)Comparison
of multiplelocus variablenumber tandem repeat analysis, pulsedfield gel electrophoresis, and phage
typingforsubtypeanalysisofSalmonellaentericaserotypeEnteritidis.JClinMicrobiol45:536543.
2. Lindstedt BA, Vardund T, Aas L & Kapperud G (2004a)Multiplelocus variablenumber tandemrepeats
analysisofSalmonellaentericasubsp.entericaserovarTyphimuriumusingPCRmultiplexingandmulticolor
capillaryelectrophoresis.JMicrobiolMethods59:163172.
3. HyytiaTrees E, Smole SC, Fields PA, Swaminathan B & Ribot EM (2006) Second generation subtyping: a
proposed PulseNet protocol for multiplelocus variablenumber tandem repeat analysis of Shiga toxin
producingEscherichiacoliO157(STECO157).FoodbornePathogDis3:118131.
4.LauraBetancor,LucaYim,AracMartnez,Maria
Fookes, Sebastian Sasias, Felipe Schelotto, Nicholas Thomson, Duncan Maskell, and Jose A
Chabalogoity.(2012) Genomic Comparison of the Closely Related Salmonella enterica serovars Enteritidis
andDublin.OpenMicrobiology.J.6:513
Table 1. MLVA patterns generated following the application of the CDC protocol for Pulsenet MLVA of
SalmonellaentericasubtypeEnteritidistoSalmonellaentericasubtypeDublinisolates.
ASTMLVApatternSourceSpecimen
none 341102310HumanFaeces
Na 341102310HumanFaeces
none 341102310BovineFaeces
none341102310BovineLive
Na 341102310BovineLive
none341102312HumanFaeces
none341102312BovineMilk
none341102312BovineMilk
none 341102312HumanBlood
none 341102312BovineMeat
none 341102313HumanFaeces
none 341102313BovineLive
none341102313BovineLive
none=sensitivetoall
Na=Naladixicacidresistant

171
Pulsenet Multi-locus VNTR Analysis MLVA protocol
for Salmonella enterica subtype Enteritidis compliments phage typing

Jean OCONNOR, Niall DELAPPE and Prof.Martin CORMICAN
National Salmonella, Shigella and Listeria Reference Laboratory, Dept. of Medical Micro Biology ,
Galway University Hospital, GALWAY, Ireland

INTRODUCTION
S. Enteritidis and S. Typhimurium are two of the most important salmonella serotypes resulting in
salmonellosis transmitted from animals to humans(1)(2). Current sub-typing methods used to type
salmonella isolates include serotyping, antimicrobial sensitivity testing (AST) pulse-field gel
electrophoresis (PFGE) and phage typing.
The development of new sub-typing methods to link isolates to their food source or to other
outbreaks isolates is extremely important (3)(4).
Multi-locus VNTR Analysis (MLVA) is a new sub-typing method which is based on the Variable copy
Numbers of Tandem Repeats (VNTR).
MLVA provides greater discrimination among non-epidemiologically linked isolates than PFGE or
phage typing (5).

MATERIALS AND METHODS
The Laboratory standard operating procedure as described by the CDC for Pulsenet MLVA of
Salmonella enterica subtype Enteritidis using the Beckman Coulter CEQ
TM
8000 was used to
analyse 108 S. Enteritidis isolates.

RESULTS
One hundred and eight S. Enteritidis isolates from the National Salmonella, Shigella and Listeria
Reference Laboratory, Ireland (NSSLRL) were analysed.
Sixty seven isolates from 2011, then groups of known clusters of PT1, PT8, PT6, PT4, PT14b and
PT21 from previous years.
In total one hundred and four human isolates and four animal or food isolates were analysed.
Within the clusters, 12 patterns were seen while 30 patterns were seen overall between all the
2011 isolates and the clusters.
Sixteen S. Enteritidis PT1 isolates were analysed, six isolates from 3 known clusters from different
years were studied. Three distinct MLVA patterns emerged corresponding to the three clusters
(Table 1). Ten S. Enteritidis PT1 isolates from 2011 with no known links generated 6 patterns.
Similar results were seen within each of the other phage type clusters.

DISCUSSION
This method provides extra discriminatory power within a particular phage type.
As 60% of isolates were fully sensitive to 14 antibiotics (Ampicillin, Chloramphenicol,
Streptomycin,Tetracycline,Trimethoprim, Nalidixic acid, Kanomycin, Ciprofloxacin, Cefpodoxime,
Ceftazidime, Gentomycin, Minocycline and Cefotaxime) and 30% resistant to Nalidixic acid, AST
did not provide great discrimination .
As PFGE does not offer great discrimination of S. Enteritidis (3) the CDC protocol for Pulsenet
MLVA of Salmonella enterica subtype Enteritidis provides an added means of discriminating
between strains of S. Enteritidis.

172
REFERENCES
1. Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, OBrien SJ, et al. (2010).The global burden of
nontyphoidal Salmonella gastroenteritis. Clin Infect Dis. 50(6):882-9.
2. European Food Safety Authority (EFSA). (2007).The community summary report on trends and sources of
zoonoses, zoonotic agents, antimicrobial resistance and foodborne outbreaks in the European Union in
2006. EFSA Journal.;130.
3. Boxrud D, Pederson-Gulrud K,Wotton J,Medus C, Lyszkowicz E,Besser J & Bartkus JM (2007) Comparison
of multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and phage
typing for subtype analysis of Salmonella enterica serotype Enteritidis. J Clin Microbiol 45:536543.
4. Lindstedt BA, Vardund T, Aas L & Kapperud G (2004a) Multiple-locus variable-number tandem-repeats
analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor
capillary electrophoresis. J Microbiol Methods 59: 163172.
5. Hyytia-Trees E, Smole SC, Fields PA, Swaminathan B & Ribot EM (2006) Second generation subtyping: a
proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-
producing Escherichia coli O157 (STEC O157). Foodborne Pathog Dis 3: 118131

Table 1. Three clusters of Human faecal isolates of S. Enteritidis PT1 generated using the CDC MLVA
protocol.

AST MLVA pattern Cluster FT
Na 5-4-1-10-3-3-10 P/09 Spain *
Na 5-4-1-10-3-3-10 P/09 Spain *
None 5-5-1-10-3-3-10 D/08 -
None 5-5-1-10-3-3-10 D/08 -

Na 5-5-1-10-3-3-9 I/06 Portugal
Na 5-5-1-10-3-3-9 I/06 Portugal

AST =Antimicrobial Sensitivity Testing
none =sensitive to all 14 antibiotics
Na =Nalidixic acid resistant
FT =Foreign Travel
* =Animal contact with Tortoise

173
Multiplelocusvariablenumberoftandemrepeatsanalysis
ofSalmonellaentericasubsp.entericaserovarDublin
MarianneKIRSTINEKJELDSEN
1,2
,MiaTORPDAL
1
,KarlPEDERSEN
2
andEvaMLLERNIELSEN
1
1
FoodbornepathogensandBacterialTyping,DepartmentofMicrobialSurveillanceand
Research,StatensSerumInstitut,Artillerivej5,2300COPENHAGEN,Denmark;
2
ZoonosisCenter,NationalFoodInstitute,TechnicalUniversityofDenmark,
MrkhjBygade26,2860SBORG,Denmark
ABSTRACT
Salmonella Dublin is the most frequently isolated organism in clinical cases of cattle and infection causes
diarrhoea, abortion, decreased milk yield and fatality. In humans, the serotype causes severe invasive disease
and high mortality rates have been reported. Epidemiological investigation of S. Dublin benefits from molecular
typing tools but is complicated as more methods needs to be combined in order to obtain sufficient diversity.
The method multiple locus variable number of tandem repeats (MLVA) has been successfully used for
surveillance of S. Enteritidis and S. Typhimurium.
In this study we developed a MLVA protocol for S. Dublin. The polymorphism of nine potential VNTRs was
evaluated with a panel of 40 diverse isolates. Four VNTRs were selected for a MLVA analysis and the
discriminatory power of these was evaluated on 272 veterinary and human isolates and compared with that of
PFGE. The four VNTRs exhibited 100% in vitro stability and contained only true repeats. When analysing 105
isolates MLVA obtained a total of 58 genotypes and displayed a high level of discrimination (DI of 0.98)
compared to PFGE (DI of 0.57) grouping the isolates into 10 genotypes. MLVA divided the 272 isolates into
103 genotypes and successfully identified isolates from an epidemiological confirmed outbreak. A big overlap
of MLVA types was observed between veterinary and human isolates indicating that that the same clones may
be responsible for animal and human cases in Denmark.
The proposed MLVA provided a high discriminatory power, was simple to perform, straightforward in genotype
identification and can be recommended to be used in routine subtyping of isolates for outbreak investigations
and disease surveillance. The method may provide valuable additional information that can improve the
effectiveness of epidemiological investigations of S. Dublin infections and contribute to the efforts to reduce
transmission of infection.

174
Anewimmunoenzymaticstrategy
fortherapidandselectivegrowthofSalmonella
IsabellePONSARD
1
,PierreWATTIAU
2
andPatriceSOUMILLION
1
1
InstitutdesSciencesdelaVie,UniversitcatholiquedeLouvain,LOUAINLANEUVE,Belgium
2
FoodborneBacterialzoonosesandAntibioticResistance,Veterinary&Agrochemical
ResearchCentre(CODACERVAVAR),BRUSSELS,Belgium
In detection methods of bacteria, preenrichment is a preliminary step that is generally required

for providing sufficient amount of material for the subsequent microbiological, immunological or
PCRbasedassay.Selectivemediaareusedforthispurposebuttheirstringentcompositionresults
in slow growth. In a medical or foodprocessing environment, minimizing the time is of prime
importance but the efforts have been concentrated on the improvement of assays sensitivities
andveryfewmethodsaredevelopedforspeedingupthetimeconsumingpreenrichmentstep.In
this report, we simply brought selectivity to a rich non selective medium by the use of two
additives: a lactam antibiotic that kills most of the bacteria in the sample and a lactamase
antibodyconjugatethatselectivelyprotectstheSalmonella(Fig.1).
Figure1.Immunoenzymaticprincipleofconferringaspecificpenicillinresistancetoabacteriumbyaddinga
betalactamaseantibodyconjugate(ASB)intheculturebroth.
A commercial mouse monoclonal IgG to Salmonella typhimurium and a purified TEM1

lactamase hwere individually biotinylated and then assembled into a conjugate by premixing
them with streptavidin in a 1:1:1 ratio. In the mixture, all the biotinylated proteins were
conjugated to the streptavidin and the betalactamase lost only 20% of its original activity. The
conjugate is a mixture of different compounds (A
x
SB
y
with x and y between 0 and 4 and x+y 4)
andwasusedassuch.
The ASB conjugate was used as an additive together with ampicillin in a classical lysogeny broth
(LB) supplemented with 1% BSA for growing Salmonella (ATCC29631 strain). The quantity of ASB
conjugate was adjusted to hydrolyze the ampicillin in the first hour of culture. Ampicillin was
added5minaftertheconjugate.AsshowninFigure2,SalmonellawasfullyprotectedbytheASB
conjugate in the initial presence of 400 mg/l of ampicillin. Replacing the ASB conjugate by
lactamase alone did not provide any protection to the bacteria. Similar results were obtained
using a three fold increase in both ampicillin and ASB conjugate concentrations. As an additional
negativecontrol,theASBconjugatewasunabletoconferampicillinresistancetoacloselyrelated
bacteriumsuchasE.coli.

175
Figure2.GrowthcurveofSalmonellainLBbrothsupplementedwith1%BSA,ampicillin(400g/ml)andthe
ASB,andcontrolexperimentwiththenonconjugatedenzyme.
ThedetectionandselectiveenrichmentofSalmonellastartingfromamixtureofbacteriawasthen
tested. Cultures of 20 ml inoculated with various amount of cfu of Salmonella and a kanamycin
resistant E. coli strain were grown for 24h at 37C with or without addition of the ASB/ampicillin
cocktail. The titers were calculated by plating serial dilutions on Petri dishes with or without
kanamycin. As shown in Table 1, the cocktail afforded the quasi absolute selective growth of
Salmonellastartingfromasfewasabout10cfumixedwith1000cfuofE.coli.
Inoculum
(cfu)
ASB+
ampicillin
%Salmonella
t=0h t=24h
10
8
1 5
10
8
+ 1 >99
10
7
18 17
10
7
+ 18 >99
10
4
+ 1 >99
10
3
+ 1 98
Table1.SelectiveenrichmentfromE.coli/Salmonellamixedcultures
TheASBconjugatewasalsousedasadetectiontoolinanELISAlikeexperimentonbacteriausing
nitrocefin, a chromogenic substrate of the betalactamase. A positive signal was detected after
only6hoursofselectivegrowthusingonetenthofacultureinitiallyinoculatedwitharound10cfu
ofSalmonellaand10
6
cfuofE.coli.Similarresultswereobtainedusinganartificiallycontaminated
liquidyogurtsample.Ourworkdemonstratesthatlactamresistancecanbeselectivelyprovided
to Salmonella by fastening a lactamase to its surface via a specific antibody. Using such a
conjugate/antibiotic cocktail in a rich broth affords the rapid and selective growth of the target
bacteria. The method is very simple and should find useful applications in medical and food
processing environment where the detection of pathogens must be as fast as possible. Using a
single growth step, we were able to detect a few cfu of Salmonella in less than a working day
whereas all the commercial kits that we are aware of for the detection of Salmonella have
protocols requiring at least two days because of the necessity of a more or less selective pre
enrichmentstepfollowedbyarapidenrichmentstep.Withourmethod,anyintrinsicallyampicillin
resistantbacteriainthesamplewillalsogrowinthemediumandpotentiallyreducethetimefor
ampicillin hydrolysis but, for preenrichment purpose, this may not be a problem if the resistant
bacteria titre is lower than the Salmonella titre. Using what we know about the betalactamase
content in a resistant E. coli strain transformed with pBR322, we estimated that we would need
more than ten thousand resistant germs per ml to really affect the assay at 1,200 mg/ml
ampicillin. A solution to that potential problem will be to use a conjugate with an extended
spectrum betalactamase (ESBL) together with a second or third generation betalactam
compound. The method is also applicable whenever a temporary and non genetic antibiotic
resistanceisrequired.Forexample,Salmonellaisbeingdevelopedasananticancervector(2)but
the strains must be antibioticsensitive because of biosafety regulations (1). It is however
potentiallyinterestingtobeabletouseantibioticsandthebacteriasimultaneouslyforavoidingor
combatingpossibleinfectionsduringtreatment.
ACKNOWLEDGMENTS
ThisworkwassupportedbyUCBS.A.,Brussels,Belgium.WethankThomasR.Wardforproviding
thestreptavidinandJacquesFastrezforadviceandassistancewiththiswork.
176
REFERENCES
1. Low KB, Ittensohn M, Luo X, Zheng LM, King I, Pawelek JM, Bermudes D. 2004. Construction of
VNP20009: a novel, genetically stable antibioticsensitive strain of tumortargeting Salmonella for
parenteraladministrationinhumans.MethodsMol.Med.90:4760.
2.PawelekJM,LowKB,BermudesD.1997.TumortargetedSalmonellaasanovelanticancervector.Cancer
Res.57:45374544.
177
AComparisonofsubtypingmethods
forDifferentiatingSalmonellaentericaserovarEnteritidisIsolates
ObtainedfromFoodandHumanSources
JiyeonHYEON
1
andKunhoSEO
2
1
DivisionofVaccineResearch,KoreaNationalInstituteofHealth,OSONG,SouthKorea
2
KUCenterforFoodSafety,Hwayangdong,SEOUL,SouthKorea
INTRODUCTION
Salmonella typing technologies are essential for bacterial source tracking and to determine the
distribution of pathogens isolated from ill people. Traditional typing methods based on their
phenotypictraits,suchasbiotyping,antibioticsusceptibilityprofiles,serotyping,andphagetyping
provideinsufficientinformationforepidemiologicalpurposes.Molecularsubtypingmethodshave
revolutionized the fingerprinting of microbial strains, but most of them have not been
internationallystandardized.Inthisstudy,weevaluatedtheabilitiesoftwophenotypicsubtyping
methods (phage typing and antimicrobial susceptibility) and three genotypic subtyping methods
(PFGE, repPCR, and MLST) to distinguish among a collection of S. enterica serovar Enteritidis (S.
Enteritidis)isolatesthatwerecollectedfromfoodandhumansources.
MATERIALANDMETHODS
WedeterminedsubtypesofSalmonellaEnteritidis(S.Enteritidis)isolatedfromfoodproducts(n=
10) and human clinical samples (n = 10) between 2009 and 2010 in Seoul using five subtyping
methods and evaluated the abilities of these subtyping methods. These methods included phage
typing,antimicrobialsusceptibility,PFGE,RepPCR,andMLSTmethods.Wecomparedtheabilities
of three different subtyping methods to distinguish Salmonella enterica serovar Enteritidis
(S.Enteritidis)bycalculationofSimpson'sdiversityindex.
RESULTS
Amongthe 20isolatestested,thereweresixantimicrobialsusceptibilitypatterns,threedifferent
phage types, four different PFGE profiles, seven RepPCR patterns, and one MLST type. Food
isolateswereconsiderablymoresusceptibletoantibioticsthanhumanisolates.Wewerebestable
to discriminate among S. Enteritidis isolates using RepPCR, and obtained the highest Simpsons
diversity index of 0.82, while other methods produced indices that were less than 0.71. PFGE
pattern appeared to be more related to antimicrobial resistance profiles and phage types of S.
Enteritidis isolates than repPCR. MLST revealed identical alleles in all isolates at all seven loci
examined,indicatingnoresolution.
DISCUSSION
The results of this study suggest that RepPCR provided the best discriminatory power for
phenotypically similar S. Enteritidis isolates of food and human origins, while the discriminatory
abilityofMLSTmaybeproblematicduetothehighsequenceconservationofthetargetedgenes.
REFERENCES
Healy M, Huong J, Bittner T, et al. Microbial DNA typing by automated repetitivesequencebased PCR. J
ClinMicrobiol2005Jan;43(1):199e207.

178
FastSalmonellaTyphimuriumsubtypingmethodbased
onCRISPRpolymorphisms
MurielMARAULT
1
,SimonLEHELLO
2
,SabrinaCADELSIX
1
,TiffanyMALEK
1
,
FranoisXavierWEILL
2
andAnneBRISABOIS
1
1
LaboratoryforFoodSafety,UnitCaractrisationetEpidmiologieBactrienne,
MAISONSALFORT,France;
2
InstitutPasteur,UnitdesBactriesPathognesEntriques,CentreNationaldeRfrence
desSalmonella,WHOCollaboratingCentreforReferenceandresearchonSalmonella,
PARIS,France
INTRODUCTION
The genus Salmonella is one of the most prevalent pathogens involved in foodborne infections.
Even if multiple measures of control have been implemented, salmonellosis continues to be a
health problem worldwide. Therefore, rapid detection, identification and characterization of
Salmonella subtypes , as well as timely response are crucial to avoid or control their global
spread. Conventional slide agglutination serotyping method is the basic information currently
providedbylaboratoriesbutitsdiscriminatorycapacityisquestionedforsomeserotypessuchas
Typhimurium, emerging monophasic variants and Enteritidis highly prevalent worldwide and
accountformostoutbreaks.Differentiationbetweenisolateswithinthemostcommonserotypes
requires the use of subtyping methods. The PulseField Gel Electrophoresis (PFGE) method is the
currently gold standard method for this purpose, this technique allowed refining and improving
the controls with a suitable level of discrimination [1]. Nevertheless, PFGE method has some
limitations especially time consuming and handlingcost; therefore an original method based on
the polymorphism of the CRISPR (Clustered Interspaced Short palindromic Repeats) for
characterizingSalmonellaisolateshasbeendeveloped[2].Theproposedworkaimstoexplorethe
performance of this CRISPOL method (CRISPR Polymorphism) for subtyping serotype
TyphimuriumandmonophasicvariantincomparisonwithresultsobtainedwithPFGE.
MATERIALANDMETHODS
Bacterial strains: A panel of 545 isolates from various food and animal origins was selected
through the collection of the Salmonella network. The food origin was in majority of pork and
poultry (29 and 27%), 10% are from the bovine sector, other isolates were from various other
sources(ovine,caprine,milkproducts,delicatessen)Table1.
Westudied169strainsofserovarTyphimuriumand376ofmonophasicvariantsmainlybelonging
totheemergingmonophasingvariant:S.I4,[5],12:i:,andalsoafewnumberofisolatesbelonging
toS.I4,12::1,2,ortothenonmotilevariantS.I4,12::.Themajorityofthesestrains(85%)were
isolatedin2010and2011,someothersstrainswerecollectedsince2007.
Methods: PFGE was performed according to a standardized protocol [3]. CRISPOL subtyping was
performedbasedontheprotocoldevelopedattheNRCforSalmonella(InstitutPasteur)targeting
72 spacers located on two CRISPR regions and revealed with a direct hybridization of the
biotinylated amplified products to specific beads detected by flow cytometry on the xMAP
Luminexplatform[2].

179
RESULTS
On the total collection of studied strains, 77 different CRISPOLtypes (CT) were observed out of
which 45 were detected only in serovar Typhimurium and 24 in the emerging monophasic
variants. The three main CTs found in serovar Typhimurium were CT21, CT 30 and CT34 whereas
CT1andCT9weremainlyencounteredintheemergingmonophasicvariants(Table2).Noanimal
originsorfoodsourceswerespecificallyrelatedtoCTsandnospecificgeographicalCTdistribution
wasobserved.
During the 20102011 period, some isolates were studied in the frame of foodborne outbreak
investigations. The CRISPOL method was able to trace the suspected food isolates and link them
with the clinical human isolates. In parallel, PFGEtyping identified 58 types for serovar
Typhimurium out of which two of them represented around 32% of the strains. For the
monophasic variants, 62 different PFGEtypes were identified out of which one of them (PFGE
type 126) for the half of the strains (Table 2). CRISPOL method was able to differentiate these
PFGEtype126strainsin13differentCTs.Finally,appliedtoTyphimurium,thediscriminarypower
was very similar for the both methods, 0.82 for the CRISPOL and 0.86 for the PFGEtyping. The
discriminary power of the PFGE method for the monophasic variant strains was of 0.72 higher
than the CRISPOL (0.64). Nevertheless, CRISPOL method was able to discriminate in several CTs,
theemergingmonophasicstrainsbelongingtothemainPFGEtype126.
DISCUSSION
For the studied panel, some CTs are mainly found within Typhimurium strains or in monophasic
variants, on the 77 CRISPOLtypes, only 7 of them were common. Nevertheless, these common
CRISPOLtypes were the most frequently encountered either preferentially in Typhimurium or in
themonophasicvariants.Indeed,CT1wasdetectedfor59%ofvariantsstrainsandonlyfor5%of
Typhimuriumstrains.
For the emerging monophasic variants, 31 different CTs were observed among the 376 studied
strains. In spite of the clonality of these strains, discrimination between strains belonging to the
samePFGEtypewaspossible.Givenitscontentofimplementationanditsanalyticalpossibilities,
CRISPOL method is fully adapted to monitor a large collection of Salmonella serotype
Typhimuriumanditsvariants;itallowsaquickandeasydetectionofclonesemergingfromclinical
isolates, as well as veterinary, food and environmental ones. This technique is therefore a useful
toolbothforsurveillanceandinvestigationofoutbreaksinadditiontoorreplacementofPFGE.
REFERENCES
1. KrouantonA,MaraultM,LaillerR,WeillFX,FeurerC,EspiE,Brisabois.A.PulsedfieldGel
electrophoresissubtypingdatabaseforfoodborneSalmonellaentericaserotypediscrimination.
2. FoodbornePathogDis.2007Fall;4(3):293303.
3. FabreL.andal,CRISPRTypingandSubtypingforImprovedLaboratorySurveillanceofSalmonella
Infections.PlosOne,May2012,Volume7,Issue5,e36995).
4. PulseNetEU,2009link:http://www.pulsenetinternational.org/SiteCollectionDocuments/pfge/
180
Table1
Animalandfoodsourcesofthestudiedstrains
Origin
Numberof
strains %
Others 6 1,1
Bovine 54 9,9
Caprine 5 0,9
Equine 6 1,1
Ovine 1 0,2
Pork 158 29,0
Poultry 149 27,3
Delicatessen 58 10,6
Readymeatfood 26 4,8
Milkproducts 43 7,9
Seafood 2 0,4
AnimalFood 15 2,8
Ecosystem 22 4,0
Total 545 100,0
Table2:ComparisonoftheabilityofdifferenciationoftheCRISPOLandPFGEmethodsonTyphimuriumand
variantstrains.
Typhimurium Variants
n=169 n=376
Numberofdifferent 52 31
CRISPOLtypes ID=0,93 ID=0,64
MainCRISPOLTypes CT21,CT30 CT1andCT9
andCT34
Numberofdifferent 58 62
PFGEtypes ID=0,92 ID=0,74
MainPFGEtypes
STYMXB0007
and STYMXB0126and
STYMXB0063 STYMXB0060
TABLES
181
AnInterlaboratoryproficiencytrialforSalmonellaenumeration
MariemELLOUZE
1
,ValrieMICHEL
2
,AnneGIROUD
3
,ClnieSAGE
3
,CatherineDENIS
4
,
ValrieSTAHL
5
andJeanChristopheAUGUSTIN
6
1
Ifip,MeatQuality&Safety,MAISONSALFORT,France;
2
Actilait,SanitaryDepartment,LAROCHESURFORON,France;
3
ENILBIO,MicrobiologyDepartment,POLIGNY,France;
4
AdriaNormandie,MicrobiologyDepartment,VILLERSBOCAGE,France;
5
Aerial,MicrobiologyDepartment,ILLKIRCH,France;
6
ENVA,HIDAOAUnit,MAISONSALFORT,France
INTRODUCTION
Salmonellaisamajorfoodbornepathogenthatcaused99020toxiinfectioncasesin27countries
in 2010 [1]. Quantitative data about this pathogen is scarce mainly because of the absence of a
standardized quantification method, however, it is essential to make use of such a method to
better understand the dissemination pathways of Salmonella through the food chain. A method
basedontheworksofFravalo[2]fortheenumerationofSalmonellausingtheMPNminiaturized
technique will be soon available as an ISO/TC standard [3]. However, this method is time
consuming,notcosteffectiveandafewlaboratorieshavetheexpertisetoperformit[4].Methods
based on direct plating on chromogenic media are commonly used by laboratories and are
thereforeworthinvestigatingasalternativemethods.
MATERIALANDMETHODS
A. Samples preparation. Seven types of cheeselike samples were prepared according to table 1
toinvestigatetheseveralfactorsoftheexperimentaldesign.Priortothetriallaunch,
Table1.Presentationoftheexperimentaldesign
Modalityn Salmonellastrain [Salmonella] [E.coli]
1 Enteritidis 10 10000
2 Typhimurium 10 10000
3 10000
4 Enteritidis 1000 10
5 Typhimurium 1000 10
6 Enteritidis 10 10
7 Typhimurium 10 10
a study was conducted to investigate weather the samples would be homogenous and stable.
Double blind samples were then randomly numbered, packed and sent to the participants by an
expressprofessionalcarrier(Ciblex).
B. Methodstobeused.TwotypesofmethodswereusedinthisPT,achromogenicmediabased
method(M1)andaMostProbableNumber(MPN)method(M2).ForM1,andintheabsenceofa
specificmethodanadaptedprotocolwasproposedinwhichthesampledilutionsandthenumber
of petri dishes to be used were optimized to lower the detection limit (10 CFU/g). This protocol
was applied simultaneously for three different chromogenic media (M
1.1
, M
1.2,
M
1.3
) by all
participatinglaboratories.Onlytwolaboratorieswithpreviousknowledgecarriedouttheanalyses
fortheM2method.
C. Statistical analysis. Mandels h and k statistics [5] were used to evaluate the homogeneity of
the data as well as the performance of the participants in terms of precisions for each method.
The individual zscore [6] was also calculated to assess the individual trueness performance of
laboratories.
182
RESULTS
Table2isanexampleofresultsobtainedforthe4methodsforonemodality(n2Table1).
Table2.Statisticalparametersestimatedformodalityn2oftheexperimentaldesignforthedifferent
methods
Method
Nbr
labs
Assigned
valuem
(log10CFU/g)
Reproducibility Repeatability
Std
Repro
(log10CFU/g)
LimitR
(logCFU/g)
Std
Repet
(log10CFU/g)
Limitr
(logCFU/g)
M
1.1
8 0.966 0.224 0.626 0.224 0.626
M
1.2
8 0.937 0.255 0.715 0.255 0.715
M
1.3
8 1.022 0.138 0.387 0.091 0.254
M2 2 1.164 0.530 1.483 0.530 1.483
Table 2 shows that the repeatability (between the labs) and the reproducibility (in the same lab)
obtained in this study are satisfactory for the chromogenic methods M
1.1
, M
1.2
and M
1.3
since the
standard deviation for reproducibility and repeatability are lower than the standard deviation
associatedwiththePoissonlaw(here0.137logCFU/g)andalsolowerthan0.5logCFU/gwhichis
a threshold commonly used in the colonycounting techniques. However, with method M2, the
resultsofrepeatabilityandreproducibilityarelesssatisfactory.
Fig1.kvaluesofthelaboratorieswithmethodsM
1.1
()M
1.2
()M
1.3
()andM2(x).
Figure 1 presents the k values calculated for the 8 laboratories and shows that a good precision
wasobtainedbythelaboratoriesforalltestedmethodssincethekvaluesremainlowerthanthe
1% critical limit. Figure 2 shows the z score for the chromogenic methods, all laboratories
exhibited satisfactory performances for the trueness (|z scores|<3), only one laboratory had a
warningsignal.
Fig2.zscoresforM
1.1
()M
1.2
()andM
1.3
()
DISCUSSION
The figures obtained in this PT trial showed satisfactory results in terms of reproducibility and
repeatability for the direct plate chromogenic methods whereas less satisfactory results were
obtained for the MPN method. To compare the results of the two types of methods,
reproducibility standard deviations obtained by all participating laboratories for each modality of
theexperimentaldesign(table1)arediscussed.
183
Figure 3.
Reproducibility
for the different modalities of the experimental design for methods M
1.1
(dark grey)
M
1.2
(white),M
1.3
(lightgrey)andM2(black).
Figure 3 confirms the tendency previously observed in Table 2 for modality 2. In fact, in the
present study, the reproducibility standard deviations for the M2 method are often (except
modality n 5) higher than those observed for the chromogenic methods. Nevertheless, these
conclusions need to be confirmed as the study was carried out on a relatively simple matrix
(cheeselikefood)andMPNmethodwasonlyrealisedbytwolaboratoriessincetheothersdidnot
showpreviousexperiencewithM2.
CONCLUSION
Thisstudyshowedthatsatisfactoryresultsareobtainedbytheparticipatinglaboratoriesinterms
of repeatability and trueness when enumerating Salmonella using direct plate chromogenic
methods. Less satisfactory results were obtained when MPN based method was used. Further
workisplannedtotestthesemethodsonotherfoodmatrices.
ACKNOWLEDGMENT
This study was financed by ACTIA, the French association for coordination of the agrofood
industry.
REFERENCES
1. EFSAJournal2012;10(3):2597.
2. ISO/TS 65792:2012. Microbiology of food and animal feed Horizontal method for the detection,
enumerationandserotypingofSalmonellaPart2:Enumerationbyaminiaturizedmostprobablenumber
technique.
3. Fravalo,P.etal,2003.ConvenientmethodforrapidandquantitativeassessmentofSalmonellaenterica
contamination:theminiMSRVMPNtechnique.J.RapidMethodsAutomat.Microbiol.11:8188.
Ellouze,M.etal,2013.Besoinsetpratiquespourlaquantificationdessalmonelles,poster.SFM,1
8/2/2013,Lille,France.
4. NF EN ISO 16140/A1 Octobre 2011. Microbiologie des aliments Protocole pour la validation des
mthodesalternativesAmendementA1.
5. ISO135.ISO13528:2005.Mthodesstatistiquesutilisesdanslesessaisd'aptitudeparcomparaisons
interlaboratoires.
0,00
0,10
0,20
0,30
0,40
0,50
0,60
0,70
0,80
0,90
Modality 1 Modality 2 Modality 4 Modality 5 Modality 6 Modality 7
Reproducibility
184
DevelopmentofMPNrealtimePCR
forenumerationofSalmonellafromporkmeat
AuroreFABLET,MegROUXEL,MartineDENIS
Anses,HygieneandQualityofPoultryandPigProductsUnit,BP53,sitedesCroix,
22440PLOUFRAGAN,France
INTRODUCTION
Salmonella is one of the commonest foodborne pathogens transmitted to humans and
primarily by contaminated food. Pork is regarded as a major source of Salmonella infection
(Gossner et al., 2012). Around 1020% of human Salmonella infections in the EU may be
attributable to the pig reservoir as a whole (EFSA, 2010). Quantitative data are needed for
risk assessment. In this work, a method for quantification of Salmonella by MPNreal time
PCR was developed and compared to the MPNMSRV method; method based on mobility of
thebacteria.
MATERIALANDMETHODS
Samples. Artificial contaminations were realized on fresh pork meat with 3 quantities of
bacteria (around 1, 10
2
and 10
4
CFU/g) and by 5 different serotypes, 1 S. Derby and 1 S.
Typhimurium (the most prevalent Salmonella serovars in French pig production), 1 non
motile S. Typhimurium and 2 monophasic variants of S. Typhimurium (I 4,12:i: and I 4,12:
:1,2).
EnrichmentinPeptoneWaterbroth.Porkmeatwas1:10dilutedinBPW.Then,2.5mlwere
distributedinthefirst3wellsofa12wellmicrotiterplate.A1:5serialdilutionwasdoneon
thefollowingwells.After18h2at37C,quantificationwasdoneby2methods.
Quantification by MPNMSRV. 20l from each well were put on a 12well microtiter plate
containing MSRV. After 24 to 48h at 41.5C, halos of migration were streaked on XLD for
Salmonellaconfirmation.
QuantificationbyMPNrealtimePCR.
DNA extraction was realized from 1 ml per well of enriched BPW with Instagene matrix kit
(Biorad).RealtimePCRwasdoneinatotalvolumeof25lcontaining12.5lofSYBRGreen
Mix (Sigma), 0.6M of ST11 and ST15 (Aabo et al., 1996), and 2 l of a 1:10 diluted DNA.
AmplificationconditionswereasdescribedbySoumetetal.,(1999).
NumberofUFC/g.
ThenumberofUFC/gwasdeterminedbyMPNcalculatorwithintervalconfidenceat95%.
RESULTS
Finally, a total of 81 tests were realized (table 1). 65 samples gave similar enumeration
results for the 2 methods. Five samples had lower numeration with MPNreal time PCR
because Salmonella was not detected by PCR in some wells but difference with MPNMSRV
was not significant. 11 tests with the non motile S. Typhimurium were countable with MPN
realtimePCRmethodandnotwiththeMPNMSRVmethod.
DISCUSSION
This MPNreal time PCR method gave enumeration of Salmonella in 2 days against 45 days
with the NPPMSRV method. Moreover, this method allowed detection of no motile
Salmonellaserotype.The2methodshadalimitofdetectionof1.3UFC/g.
While1:10dilutionofDNAextractavoidsPCRinhibition,developmentofaninternalcontrol
forPCRisundergoinginordertoconfirmnegativewells.
185
REFERENCES
1. Aabo S., et al., (1993) Salmonella identification by the polymerase chain action. Molecular and cellular
Probes(1993)7,171178
2. EFSA, (2010). Scientific Opinion on a quantitative microbiological risk assesment of Salmonella in
slaughterandbreederpigs.EFSAJournal8,180
3. Gossner, C.M., et al. (2012). Nationwide outbreak of Salmonella enterica serotype 4,[5],12:i: infection
associated with consumption of dried pork sausage, France, November to December 2011. Euro
Surveillance17(5).
4. Soumet C., et al., (1999) Evaluation of a Multiplex PCR assay for simultaneous identification of
Salmonella sp., Salmonella Enteritidis and Salmonella Typhimurium from environmental swabs of poultry
houses.LettersinAppliedMicrobiology1999,28,113117.
S.Derby S.Typhimurium
CFU/g 10
2
CFU/g CFU/g
CFU/g 10
2
CFU/g CFU/g
MPNMSRV
>710[190;2700] 13[4,3;39] <1,3[0,22;11] >710[190;2700] 59[19;180] <1,3[0,22;11]
MPNPCR
>710[190;2700] 13[4,3;39] <1,3[0,22;11] >710[190;2700] 59[19;180] <1,3[0,22;11]
MPNMSRV
>710[190;2700] 31[10;99] <1,3[0,22;11] >710[190;2700] 41[13;130] <1,3[0,22;11]
MPNPCR
>710[190;2700] 31[10;99] <1,3[0,22;11] >710[190;2700] 41[13;130] <1,3[0,22;11]
MPNMSRV
>710[190;2700] 41[13;130] <1,3[0,22;11] >710[190;2700] 41[13;130] <1,3[0,22;11]
MPNPCR
>710[190;2700] 41[13;130] <1,3[0,22;11] >710[190;2700] 41[13;130] <1,3[0,22;11]
MPNMSRV
>710[190;2700] 21[6,8;66] <1,3[0,22;11] >710[190;2700] 31[10;99] <1,3[0,22;11]
MPNPCR
>710[190;2700] 21[6,8;66] <1,3[0,22;11] >710[190;2700] 31[10;99] <1,3[0,22;11]
MPNMSRV
>710[190;2700] 13[4,3;39] <1,3[0,22;11] >710[190;2700] 4,9[1,4;17] <1,3[0,22;11]
MPNPCR
>710[190;2700] 8,9[2,9;27] <1,3[0,22;11] >710[190;2700] 4,9[1,4;17] <1,3[0,22;11]
MPNMSRV
>710[190;2700] 12[4;36] <1,3[0,22;11] >710[190;2700] 61[20:180] <1,3[0,22;11]
MPNPCR
>710[190;2700] 12[4;36] <1,3[0,22;11] >710[190;2700] 5,9[1,8;20] <1,3[0,22;11]
S.TyphimuriumI4,12:i: S.TyphimuriumI4,12::1,2
CFU/g 10
2
CFU/g CFU/g
CFU/g 10
2
CFU/g CFU/g
MPNMSRV
>710[190;2700] 21[6,8;66] 1,6[0,22;11] >710[190;2700] 110[35;350] 13[4,3;39]
MPNPCR
>710[190;2700] 13[4,3;39] 1,6[0,22;11] >710[190;2700] 41[13.130] 1,6[0,22;11]
MPNMSRV
>710[190;2700] 31[10;99] <1,3[0,22;11] >710[190;2700] 110[35;350] <1,3[0,22;11]
MPNPCR
>710[190;2700] 31[10;99] <1,3[0,22;11] >710[190;2700] 110[35;350] <1,3[0,22;11]
MPNMSRV
>710[190;2700] 96[31;300] <1,3[0,22;11] >710[190;2700] 45[15;140] <1,3[0,22;11]
MPNPCR
>710[190;2700] 96[31;300] <1,3[0,22;11] >710[190;2700] 45[15;140] <1,3[0,22;11]
MPNMSRV
>710[190;2700] 41[13;130] <1,3[0,22;11] >710[190;2700] 31[10;99] 1,6[0,22;11]
MPNPCR
>710[190;2700] 41[13;130] <1,3[0,22;11] >710[190;2700] 31[10;99] 1,6[0,22;11]
MPNMSRV
>710[190;2700] 41[13;130] <1,3[0,22;11] >710[190;2700] 31[10;99] <1,3[0,22;11]
MPNPCR
>710[190;2700] 41[13;130] <1,3[0,22;11] >710[190;2700] 31[10;99] <1,3[0,22;11]
NonmotileS.Typhimurium
CFU/g 10
2
CFU/g CFU/g
MPNMSRV
<1,3[0,22;11] <1,3[0,22;11] <1,3[0,22;11]
MPNPCR
>710[190;2700] 170[51;540] <1,3[0,22;11]
MPNMSRV
<1,3[0,22;11] <1,3[0,22;11] <1,3[0,22;11]
MPNPCR
>710[190;2700] 41[13;130] 1,3[0,17;11]
MPNMSRV
<1,3[0,22;11] <1,3[0,22;11] <1,3[0,22;11]
MPNPCR
>710[190;2700] 170[51;540] 5,1[1,5;18]
MPNMSRV
<1,3[0,22;11] <1,3[0,22;11] <1,3[0,22;11]
MPNPCR
>710[190;2700] 66[22;200] <1,3[0,22;11]
MPNMSRV
<1,3[0,22;11] <1,3[0,22;11] <1,3[0,22;11]
MPNPCR
>710[190;2700] 66[22;200] <1,3[0,22;11]
Table1:NumerationbyMNPMSRVandMPNPCRartificiallycontaminatedby5serovarswith3differentquantitiesof
Salmonella.
Inbold:sampleswithlowernumerationwithMPNrealtimePCR;
Ingrey:nonmotileS.TyphimuriumcountablewithMPNrealtimePCRmethodandnotwiththeMPNMSRVmethod.
Inbracket:intervalconfidenceat95%

186
DeterminationofMinimalInhibitoryConcentration(MIC)values
forclinicalisolatesfromthepoultrypractice
usingamicrodilutionantimicrobialsusceptibilitytestingprocedure
DanielWINDHORST,DavidTARAS,FranciscoMONSALVEandHansC.PHILIPP
LohmannAnimalHealthGmbH,ScienceDepartment;HeinzLohmannStrasse4,
27472CUXHAVEN,Germany
The prudent use of antibiotics in veterinary medicine requires rapid and reliable results from
clinical microbiology providing the basis for diagnosis and treatment of bacterial infections. For
poultry veterinary medicine, specifically adapted microdilution plates are useful tools for clinical
routinework.
The format of the AviPro
PLATE facilitates the standardized and quality assured microdilution

antimicrobial susceptibility testing procedure in poultry veterinary diagnostic laboratories. The
easy and valid determination of Minimal Inhibitory Concentration (MIC) values is of particular
importance for clinical microbiology and antimicrobial therapy. In addition to diagnostics
accompanyingthetherapyoffarmanimals,thedetectionofzoonoticpathogensaspotentialfood
contaminants has moved into the focus of laboratory diagnostics. Salmonella are very important
poultryassociated zoonotic bacteria which are regularly isolated from clinical specimens,
environmental samples, feedstuff and food. For Salmonella isolates from poultry origin the
differentiation of field and vaccine strains is a common diagnostic problem. Discriminatory
antibiotic markers allowing the identification of potentially reisolated live vaccines are included
on the AviPro
PLATE. The plate layout focuses on antibiotic compounds with clinical and
regulatoryrelevanceforpoultry.ScreeningfunctionsforMRSAandESBLareavailablethroughtest
wellswithoxacillinandapanelofcephalosporins.Tomonitorsuccessfulvaccinationortoconfirm
the identity of Salmonella isolations, the AviPro
PLATE allows the discrimination of field

Salmonella from Salmonella metabolic drift mutants as AviPro
SALMONELLA VAC E and AviPro
SALMONELLAVACT.
Moreover the method to correctly identify both strains in parallel which is necessary in case of
AviPro SALMONELLA DUO is discussed. Details on the use of Rifampicin resistance as a reliable
markeraregiven.Inadditiontothis,theanalysisofCampylobacterisolatesisdiscussedaswellas
anadditionalfeaturewhichbecomesmoreimportantwiththerisingCampylobacterawareness.
187
Establishment of a multiplex PCR-Lateral Flow Assay (mPCR-LFA)
for the detection of DNA from S. Typhi and S. Paratyphi A

Amalina NOR, Rahman FAIZUL and Aziah ISMAIL
Email: aziah@kck.usm.my;
INFORMM, Health Campus, 16150 KELANTAN, Malaysia

Abstract:
Enteric fever remains a public health problem with 25 million cases annually mainly among
children. The disease is caused by Salmonella enterica serovar Typhi and Salmonella enterica
serovar Paratyphi which are transmitted via food handlers who are carriers. Multiplex PCR-
lateral flow assay (mPCR-LFA) was developed in this study to overcome the drawback of
culture method that is time consuming, laborious, and require highly skilled personnel. The
mPCR-LFA was established to detect S. Typhi and S. Paratyphi, genus Salmonella (as a control
for Salmonella serovars) in the presence of internal amplification control with the amplicon
sizes of 70bp, 93bp, 146bp and 123bp respectively. The primers were shown to be specific to
the target genes without any cross-reaction after validating with 75 bacterial isolates. The
analytical sensitivity of mPCR-LFA for S. Typhi and S. Paratyphi was determined as 101 and
102 cfu/ml, respectively. The mPCR-LFA was able to show five positive results out of 85 stool
samples collected from food handlers and suspected carriers. Therefore we conclude that
the mPCR-LFA is a potential assay to detect the presence of S. Typhi and S. Paratyphi A in
stool samples of possible carriers.

188
May 27-28-29, 2013
Saint-Malo France

Session 4

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

189
Antimicrobialresistanceinagenomiceragoingfromphenotypetogenotype
ReneHENDRIKSEN(Denmark)
ABSTRACT
Carbapenemase producing Gram negative bacteria in humans have emerged around the world. In 2013,
carbapenemase producing Salmonella Infantis harbouring the VIM-1 gene were reported from livestock farms in
Germany. The data also revealed difficulties detecting those genes using conventional phenotypic antimicrobial
susceptibility testing. This calls for better methodologies for detection such as genotyping of antimicrobial resistance
genes. The answer to this demand might be the use of Next Generation Sequencing which is rapidly advancing due to
decreasing costs and better platforms. However, there is also a need for bioinfomatic tools to interpret the genome data.
The Danish Center for Genomic Epidemiology has developed an antimicrobial resistance gene finder; ResFinder, which
is a database consisting of more than 1800 resistance genes. The ResFinder, tool along with other bioinformatic tools,
was used as a proof of concept project to evaluate if bioinformatic tools could replace the current conventional methods.
Two Extended Spectrum -lactamase producing Salmonella isolates from Zambia were investigated using tools to
detect the species, the MLST, the plasmid replicon, the antimicrobial resistance profile and the phylogenetic SNP tree.
The data reveal them as extreme multi-drug resistant Salmonella Senftenberg harbouring an arsenal of resistance genes
including several extended spectrum -lactamases. Those data were surprising and initiated a whole range of new
questions such as why Salmonella need several genes conferring resistance to the same drug class and how much each
gene contributes to the MIC. As a proof of concept for replacing phenotypic resistance monitoring, the study compared
resistance phenotype predicted by ResFinder with the phenotypic MIC profile resulting in an overall concordance of
100% for Salmonella Typhimurium. The outcome of the study was that genotypic monitoring should be feasible for
surveillance. In conclusion, time has matured to use NGS to detect antimicrobial resistance genes.
INTRODUCTION
Third and fourthgeneration cephalosporins and carbapenems are critically important
antimicrobials as classified by the WHO (www.who.int). In fact, carbapenems are lastline clinical
antibiotics against infections caused by multidrugresistant Gramnegative bacteria (1, 2). During
the last decade, the prevalence of carbapenem resistance in Enterobacteriaceae has increased
worldwideaffectedhospitalsandthecommunity(13).
Recently, the occurrence of carbapenemasecarrying commensal Escherichia coli isolated from
livestock and their environment has been reported (1), and this could be the beginning of a new
eraintheantibioticresistancefield.
In Germany, several studies have been initiated, collecting potential ESBLcarrier organisms from
Germanfarms.
From 221 isolates collected during 2011, three of them were described as Salmonella enterica
serotype Infantis (4). The three Salmonella isolates were obtained from two pigfattening farms
and one broiler farm. The MIC values for some carbapenemase producers can be lower than the
currentlyrecommendedbreakpoints,andtheresultsofthecarbapenemsusceptibilitytestscanbe
influenced by the genetic background (1, 2, 5). The S. Infantis isolates showed decreased
susceptibility to these antimicrobials [nonwildtype by the EUCAST epidemiological cutoff
(ECOFF),but susceptible or intermediate according to the CLSI clinical breakpoint. The three
isolates carried both the AmpCencoding gene; bla
ACC1
and the carbapenemase gene; bla
VIM1
.
Since the real prevalence of carbapenemaseencoding genes present in zoonotic bacteria or
commensals is unknown, livestock carbapenem producers should be included in monitoring
programmes. Unfortunately there is still a debate about the methodology for detection (best
detection medium, antibiotic and concentration) (5). Due to the importance of carbapenems for
human treatment, the presence of these genes located on mobile genetic elements in livestock,
and the possibility of further transmission via food or direct contact in the communityand
hospitals,isapublichealthconcernanddeservessurveillance.
190
The cost of Next Generation Sequencing (NGS) technologies is dropping precipitously, and the
mostimmediatebottlenecksresultfromalackoftoolscriticalforInternetbaseddataanalysis(6).
New sequencing technology will provide the opportunity to create a global system of linked
databases for identification and detailed genetic characterization of all microorganisms. This
would enable us to circumvent the need for expansion of existing expensive, cumbersome, and
unreliablesystemswherenotfullydevelopedandtopartiallyorwhollyreplacethoseinexistence.
Suchaglobalsystemwouldresultinareductionincharacterizationtime(e.g.,hours/daysinstead
of weeks). It would also strengthen local, national, and international surveillance of infectious
diseasesbyensuringmoreappropriatetechnologyforcommunicablediseasesurveillance.
Alargenumberofdifferentgenescanberesponsibleforantimicrobialresistance.Identificationof
these genes is important to understand resistance epidemiology, for verification of non
susceptible phenotypes and for identification of resistant strains, when genes are weakly
expressed in vitro. Detection of resistance genes has typically been performed using PCR (7) or
microarrays(8).
The Center for Genomic Epidemiology (www.genomicepidemiology.org) aims at providing the
bioinformatic and scientific foundation for processing and handling NGS information in a
standardized way useful for outbreak investigation, source tracking, diagnostics and
epidemiologicalsurveillance.Theservicesarepublicallyavailablethroughwebserversspecifically
designedtobeuserfriendlyandalsoforinvestigatorswithlimitedbioinformaticsexperience.
OnesuchtoolsistheResFinder.Thetoolwasdevelopedbycollectingdataonacquiredresistance
genes from databases (http://faculty.washington.edu/marilynr/, http://ardb.cbcb.umd.edu/ and
http://www.lahey.org/Studies/) and published papers including reviews (9, 10). All sequences
were collected from the NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore/) and
used to build the ResFinder database (11). To our knowledge, we have created the largest
collection and database of acquired antimicrobial resistance genes converting this into an online
free of charge bioinformatic tool to detect antimicrobial resistance genes. The ResFinder was
evaluated by identifying the acquired resistance genes in the 1862 GenBank files from which the
databasesoriginalwerecreatedwithanIDof100%.
The ResFinder tool along with other bioinformatic tools provided by The Center for Genomic
Epidemiology was used as a proof of concept project to evaluate if bioinformatic tools could
replacethecurrentconventionalmethods(12).TwoextendedspectrumlactamaseproducingS.
Senftenberg isolates from Zambia were investigated using bioinformatic tools of The Center for
GenomicEpidemiology.Thegenomesoftheisolatesweresequencedtodeterminethemultilocus
sequencetype(MLST)andtoinvestigatetheoccurrenceandgeneticmechanismsofantimicrobial
resistance, plasmid replicons, and genetic relatedness by singlenucleotide polymorphism (SNP)
analysis. On 18 January 2012, a 34 year old male from Mazabuka, Zambia (72 km south of the
capital, Lusaka), was admitted to the Mazabuka District Hospital. Based on medical examination,
thepatientwasdiagnosedwithgastroenteritisandtreatedwithciprofloxacinandcotrimoxazole.
Two days later, the patient was discharged, with continuing treatment on cephalexin and co
trimoxazole, but was readmitted with epistaxis and occipital pulsatile headache and treated with
adrenalineandvitaminK.Thepatientwasdischarged6dayslaterandscheduledtobereviewed.
On6FebruarythepatientwasreferredtotherenalunitoftheUniversityTeachingHospital(UTH)
in Lusaka, as he was pale, dehydrated, afebrile, tachycardic, with a scaphoid abdomen, and later
healsodevelopedureamicencephalopathy.Thistime,thepatientwasdiagnosedwithsepsisand
chronic renal failure. Among other tests, which were all negative, a renal ultrasound was normal
butthestoolcultureyieldedSalmonella(isolate588).Threedayslater,thepatientwasunableto
eat and was fed through a nasogastric tube and intravenous fluids. The patient was transfused 4
days after admission, but no dialysis was initiated. The patient died the morning of 11 February
2012.Asecondpatient,a30yearoldmalefromtheChibolyacompound(2kmwestofLusakaand
191
74kmawayfromcase1),hadspentmostofhistimeinthecompound.Thepatientwasadmitted
to the UTH and diagnosed with gastroenteritis with tuberculosis (TB), after having been referred
from a local clinic on 9 March. 2012. Three months prior to admission the patient had been
treatedwithantimicrobialsduetosexuallytransmittedinfections.Priortoadmissionon9March
2012,thepatientcomplainedofaheadache,chills,fever,diarrhea,andgeneralweakness.On13
March, a stool sample was collected, and it yielded Salmonella (isolate 1028). The patient was
reported to have consumed vegetables bought from the local market. Based on chest X ray, the
patientwasdiagnosedwithextrapulmonaryTBandtreatedwithrifampin,isoniazid,pyrazinamide,
and ethambutol. On 16 March, the patient was also diagnosed with HIV and received
emtricitabine, tenofovir, efavirenz, and cotrimoxazole. The Salmonella isolates were shipped to
the Technical University of Denmark (DTU) for further characterization. The isolates were
serotyped,followedbyMICdeterminationsaspreviouslydescribed,includingthetigecyclineMIC
(13). Both isolates belonged to S. Senftenberg and had an almost identical antimicrobial
susceptibility pattern, conferring resistance to amoxicillin plus clavulanic acid (MIC, 16 mg/l),
ampicillin(MIC,32mg/l),cefepime(MIC,16mg/l),cefotaxime(MIC,64mg/l),cefpodoxime(MIC,
32 mg/l), ceftazidime (MIC, 128 mg/l), ceftiofur (MIC, 8 mg/l), ceftriaxzone (MIC, 128 mg/l),
chloramphenicol (MIC,64 mg/l), ciprofloxacin (MIC, 4 mg/l), gentamicin (MIC, 16 mg/l), nalidixan
(MIC,64mg/l),neomycin(MIC,32mg/l),spectinomycin(MIC,256mg/l),streptomycin(MIC,128
mg/l),sulfamethoxazole(MIC,1,024mg/l),tetracycline(MIC,32mg/l),andtrimethoprim(MIC,32
mg/l). In addition, isolate 588 was also resistant to florfenicol (MIC, 64 mg/l). The isolates were
susceptible to apramycin, cefoxitin, colistin, imipenem, meropenem, and tigecycline. However,
one could question if those antimicrobials would be used for treatment, since florfenicol and
apramycin are only approved for animal usage, colistin is difficult to administer and has renal
toxicity (14), cefoxitin is grouped with extendedspectrum cephalosporins and would most likely
nothaveanyeffect,duetotheisolatesalreadybeingresistanttobroadspectrumcephalosporins
(15),andcarbapenemsaretooexpensive,consideringthatthesepatientswererequiredtocover
thehospitalexpensesthemselves.Treatmentwithtigecyclinemaybeeffectivetowardnontyphoid
Salmonella, but clinical trials need to be conducted to further investigate the full potential of its
useforhumantreatmentofinfectionscausedbymultidrugresistantnontyphoidSalmonella.The
isolates were sequenced on the Ion Torrent PGM system (Life Technologies) following the
manufacturers protocols for 200bp genomic DNA (gDNA) fragment library preparation (Ion
Xpress Plus gDNA and Amplicon Library Preparation), template preparation (Ion OneTouch
system), and sequencing (Ion PGM 200 sequencing kit). The sequence data were assembled and
analyzed using the pipeline available on the Center for Genomic Epidemiology website
(www.genomicepidemiology.org)(11,16).
The isolates belonged to MLST ST14. The Resfinder tool (11) detected the following resistance
genespresenteitherinbothorinoneoftheisolates,aswellastwomutationsingyr(A)andone
mutation in par(C) responsible for highlevel fluoroquinolone resistance (Table 1). Both isolates
containedanincH12plasmidreplicon,andisolate588containedanincA/Cplasmidreplicon. The
genetic relatedness of the two isolates was examined and identified 93 highquality SNPs (the
informativeSNPsweredeterminedbasedonaminimumcoverageof20times,basecallingquality
of30,andaminimumdistanceof10bpbetweeneachSNP)betweenthetwoisolates,usingtheS.
Senftenberg SS209 reference genome (Bio project number PRJEA82547) (17) and 530 and 521
SNPs between isolates 1028 and 588 and the reference genome. There are currently insufficient
data on the nucleotide diversity between clonally related and unrelated Salmonella isolates to
determine whether this was indicative of separate or clonally related strains. NGS studies on
Salmonella have indicated an accumulation rate of 1 to 2 SNPs per year (18). Thus, the 93 SNP
differences observed here in combination with the differences in resistance profiles and genes
may suggest that the isolates have an unrelated origin. S. Senftenberg has also previously been
192
reported as the cause of serious human infections (1921). S. Senftenberg is well recognized as
being common among poultry (22), but it has also been associated with infant formula (23),
mussels(24),andvegetables(21,25,26).Itisnoteworthythatoneofthepatientsclaimedtohave
consumedvegetablespriortoonsetofsymptoms.S.Senftenberghastheabilitytoadheretoplant
leaves, perhaps contributing to infections in such cases (25). A similar case of one resistant S.
Senftenberg isolate was recently reported for a traveler returning from Egypt, indicating the
importanceofthisresistantserovarinAfrica(27).
WehavereportedherecasesfromZambiaofextremelydrugresistantS.Senftenbergisolatesthat
caused severe human infections and for which there were very few treatment options. We
question why these extreme multidrug resistant S. Senftenberg harbouring an arsenal of
resistancegenesincludingseveralextendedspectrumlactamases,whySalmonellaneedseveral
genes conferring resistance to the same drug class and how much each gene contributes to the
MIC. The ResFinder tool (11) was also used as a proof of concept for replacing phenotypic
resistance monitoring to identify resistance genes, predict a resistance phenotype and compare
thepredictedresistancephenotypewiththephenotypicMICprofile.
In 1995, Denmark was the first country to establish an integrated surveillance of antimicrobial
resistance based on susceptibility testing of indicator and pathogenic bacteria and to compare
isolates from food animals, food and humans (28, 29). Several other countries have since
established similar programmes (30), and recommendations for antimicrobial agents to be
included, methodology to use and suggestions for epidemiological cutoff (ECOFF) values and
clinicalbreakpointshavebeenpublished(3133).Theresultsobtainedindifferentlaboratoriesare
not entirely comparable and problems with phenotypic testing continue to result in mistakes
(34,35). Current routine surveillance programmes are often accompanied by a need for further
geneticcharacterizationofisolates(36),suchassubtypingandidentificationofresistancegenes,
oftenrequiringtheinvolvementofspecializedorreferencelaboratories.However,especiallywith
regard to antimicrobial susceptibility, there is insufficient knowledge regarding the concordance
betweenphenotypictestingandthepresenceofresistancegenesindifferentisolates.
Therefore, two hundred isolates originating from Danish pigs, covering four bacterial species, S.
Typhimurium (n:50), E. coli (n:50), Enterococcus faecalis (n:50) and Enterococcus faecium (n:50),
were included in the study. All isolates were collected randomly during the first half of 2011.
GenomicDNAwaspurifiedfromtheisolatesandsequencedontheIlluminaplatform(pairedend
reads). Reads were assembled de novo prior to prediction of the resistance profiles. N50 is the
lengthofthesmallestcontiginthesetthatcontainsthefewest(largest)contigswhosecombined
lengthrepresentsatleast50%oftheassembly.N50valuesoftheassemblywereusedforquality
controloftheDNAsequences.
TheResFinderwebserver(11)wasusedtoidentifyacquiredantimicrobialresistancegenesinthe
NGS data, using a threshold of 98.00% identity (ID). ResFinder will detect the presence of
resistance genes, but not functional integrity and expression or resistance due to acquired
variation in housekeeping genes. Based on the ResFinder results, a predicted phenotype was
determined using phenotypes from original published studies of the genes found. The predicted
resistanceswerecomparedwithphenotypicantimicrobialsusceptibilitytesting(MICsdetermined
by the microdilution method, as previously described) (34). The isolates were tested for
susceptibility to 1417 different antimicrobial agents depending on the species. Results were
interpreted using current EUCAST (www.eucast.org) ECOFF and European Food Safety Authority
(EFSA) epidemiologic breakpoints (31), Of the 200 isolates included in the study, three were
excluded:oneS.TyphimuriumbecauseofpoorqualityoftheNGSdata(N50:197),andtwoE.coli,
which turned out to be E. fergusonii. The E. coli and S. Typhimurium isolates were tested for
susceptibility to 16 and 17 different antimicrobial agents belonging to 7 different antimicrobial
classes,E.faecalisforsusceptibilityto14antimicrobialagentsbelongingto9differentclassesand
193
E. faecium for susceptibility to 15 antimicrobial agents belonging to 10 different classes. In total,
3051differentphenotypictestswereperformedfortheentiredataset.For23
testsin20isolatestherewasdisagreementbetweenthephenotypicandpredictedsusceptibility.
Repeating the susceptibility tests for these isolates led to a match between predicted and tested
resistancefor16ofthese.
Fortheentiredataset,482ofthe3051testsshowedresistance,whiletheremaining2569showed
susceptibility.ExcludingdataforlactamsinenterococciandciprofloxacinandnalidixicacidinS.
Typhimurium and E., disagreements between tested and predicted susceptibility were only
observed in seven cases (six E. coli and one E. faecalis, all false positive), corresponding to a
concordance of 99.74%. For the S. Typhimurium and E. faecium isolates complete agreement
betweentestedandpredictedsusceptibilitywasobserved.
To our knowledge, this study describes the first large comparison of NGS and phenotypic
susceptibility testing for the prediction of antimicrobial susceptibility. We observed a very high
concordance between predicted and observed phenotypes. Especially for the 49 S. Typhimurium
isolates, complete agreement was found between the result of phenotypic susceptibility testing
and that predicted from the observed genes. Overall, the concordance between phenotypic
testing and susceptibility predicted by ResFinder was 99.74%. This is a much higher concordance
than results previously obtained during phenotypic performance testing and ring trials (external
quality assurance exercises), even involving national reference laboratories, where performances
aslowat90%correctresultshavebeenconsideredtobeacceptable(34,35,37).Withourcurrent
knowledge it is not advisable to completely replace phenotypic susceptibility testing with NGS in
theclinicalsetting.However,ourresultsdosuggestthatthismightalreadybeafeasibleprocedure
for surveillance purposes. Another benefit of NGS is that it will provide a lot of additional
information. In this study we only performed MLST of the isolates, which can yield important
information on changing patterns of bacterial populations, but NGS might also give a continuous
background of data for detection of outbreaksor for comparison in putative outbreak situations.
In conclusion, this study showed a very high concordance between phenotypic antimicrobial
susceptibility and that predicted from NGS data. This suggests that NGS might eventually replace
or be used with great benefit in combination with phenotypic methods initially for surveillance
purposes,buteventuallyalsoforrapidclinicaldiagnosis.Overall,timehasmaturedtouseNGSto
detectantimicrobialresistancegenes.
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196
Table1.DistributionofantimicrobialresistancegenesinSalmonellaSenftenbergisolatesfromZambia.
Resistancegeneclasses,resistance
genes,andresistancemutations.
IsolateID
588 1028
Aminoglycoside aac(6)IIc aac(6)IIc
aac(6)Iy aac(6)Iy
aadA2
aac(6)Ibcr
strA
strB
aph(3')Ic
aac(6)Ibcr
strA
strB
Betalactam bla
TEM1
bla
TEM1
bla
CTXM15
bla
CTXM15
bla
OXA10
bla
OXA30
Lowlevelfluoroquinolone aac(6)Ibcr aac(6)Ibcr

Highlevelfluoroquinolone gyrA(S83YandD87G) gyrA(S83YandD87G)
parC(S80I). parC(S80I).
Macrolide/lincosamide/streptogramin ereA ereA
Phenicol catB3
catA2
cmlA1
catB3
catA2
floR
Rifampicin ARR2
Sulphonamide sul1 sul1
sul2 sul2
Tetracycline tetA tetD
tetD
Trimethoprim dfrA14
dfrA18
dfrA23
dfrA18

197
May 27-28-29, 2013
Saint-Malo France

Session 4

Chairpersons:

Communications
198
Regulation of the AcrAB multidrug efflux pump
in Salmonella enterica serovar Typhimurium

Kunihiko NISHINO
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, OSAKA
567-0047, Japan

ABSTRACT
One important mechanism that gives rise to multidrug resistance is the active efflux of drugs by multidrug transporters. In
Gram-negative bacteria, multidrug transporters belonging to resistance-nodulation-cell division family such as AcrAB are
especially effective in generating this resistance. A number of multidrug transporter genes are tightly regulated by
transcriptional activators and/or repressors. In several pathogens, the RamA global transcriptional activator participates
in multidrug resistance by activating the expression of the AcrAB. Abouzeed et al. identified, directly upstream of ramA,
the ramR gene coding for a protein of the TetR family of transcriptional repressors. RamR is an important factor of
multidrug resistance because various types of mutations in ramR or in the ramR-ramA intergenic region were identified
in multidrug-resistant strains of S. Typhimurium, other S. enterica serovars, and K. pneumonia, which result in increased
expression of ramA and increased efflux-mediated multidrug resistance. Here, we report that several compounds and
conditions, such as indole and an Escherichia coli conditioned medium induced AcrAB in S. Tyhimurium through the
RamR/RamA pathway. The RamA binding site was located in the upstream regions of acrAB and RamR/RamA was
required for indole induction of acrAB. Other regulators of acrAB such as MarA, SoxS, Rob, SdiA, and AcrR did not
contribute to acrAB induction by indole. Indole activated ramA transcription, and overproduction of RamA caused
increased acrAB expression. Our results suggest that RamR/RamA controls the Salmonella AcrAB-TolC multidrug efflux
system in response to external signals. In conclusion, we have made an effort to extend our knowledge of the
transcriptional regulation mediated by RamR/RamA, a regulator of multidrug resistance in Salmonella.

INTRODUCTION
Emerging resistance to antibiotics in Salmonella has been found in both humans and animals and
is a potentially serious public health problem. High-level fluoroquinolone resistance in S. enterica
serovar Typhimurium phage type DT204 has been reported to result from multiple target gene
mutations and active efflux by the AcrAB-TolC multidrug efflux pump (2, 3). Multidrug efflux
pumps have important physiological functions, including transport of drugs, bile salts, toxins, and
environmental compounds. In bacteria, drug resistance is often associated with multidrug efflux
pumps that decrease cellular drug accumulation. Recent studies have shown that S. enterica
serovar Typhimurium has nine functional drug efflux pumps (7, 13 ). Many multidrug pumps have
overlapping substrate spectra, and it is intriguing that bacteria, with their economically organized
genomes, harbor large sets of multidrug efflux genes.
The key to understanding how bacteria utilize these pumps lies in the regulation of their
expression. Currently available data indicate that multidrug efflux pumps are often expressed
under precise and elaborate transcriptional control. For example, expression of acrAB, which
encodes the AcrAB pump, may be subject to multiple levels of regulation (14). In Escherichia coli, it
is modulated locally by the repressor AcrR(9). At a more global level, it is modulated by stress
conditions and by regulators such as MarA, SoxS, and Rob. Oliver et al.(16) reported that mutation
in acrR contributes to over-expression of acrAB in Salmonella and increases resistance to multiple
drugs. Eaves et al. (5) reported that acrB, acrF, and acrD are coordinately regulated and that their
expression influences expression of transcriptional activators marA and soxS. Furthermore,
integration of IS1 and IS10 elements into the upstream region of the acrEF operon has been
reported to cause increased expression of acrEF (15). These examples illustrate the complexity and
diversity of the mechanisms regulating bacterial multidrug efflux pumps. However, few data are
available on signals that induce multidrug efflux genes in Salmonella.
Previously, it was reported that indole induces the acrD, acrE, cusB, emrK, mdtA, mdtE, and mdtH
multidrug efflux pump genes in E. coli (6). They also reported that indole induction of acrD and
mdtA is mediated by the BaeSR and CpxAR systems. However, the effect of indole on the AcrAB-
199
TolC multidrug efflux pump, which plays a major role in antibiotic resistance, remains unknown.
Very few signals inducing multidrug efflux pumps in Salmonella have been identified so far. Here,
we report on induction of acrAB in Salmonella via the RamR/A pathway in response to indole, bile,
and an E. coli conditioned medium.

RESULTS and DISCUSSION
In E. coli, indole is produced from tryptophane by tryopthophanase and is excreted from the cell.
However, Salmonella does not produce indole because it lacks the tnaA gene encoding
trypthophanase. Indole has also been reported to auto-regulate multidrug efflux genes in E. coli
(6). We postulated that Salmonella multidrug efflux genes may respond to indole. Indole
significantly induced expression of the acrAB, emrAB, acrD, and mdtABC efflux genes in
Salmonella. A survival assay using benzalkonium showed that indole enhanced drug tolerance of
Salmonella.
Among the multidrug efflux pumps, AcrAB plays a major role in the intrinsic resistance of
Salmonella. It was reported that the baeSR and cpxAR signal transduction system genes are
required for indole induction of multidrug efflux pumps in E. coli (14). To identify the regulatory
elements that induce acrAB in response to indole in Salmonella, we constructed a mutant that
lacked baeSR and cpxAR. In the baeSR cpxAR mutant, the expression of acrAB was not
significantly different from that in the wild-type (WT) strain; however, indole induction of acrD
was significantly lower in the mutant compared to the WT strain. The result indicates that the
BaeSR and CpxAR signal transduction systems are not involved in indole induction of acrAB
whereas they are required for acrD induction. Other regulators, marA, soxS, rob, sdiA, and acrR,
have been previously reported to control acrAB expression in E. coli (14). With the exception of
ramA, none significantly altered the indole induction of acrAB in Salmonella. The stimulatory
effect of indole on acrAB expression was completely eliminated in the ramA mutant. The results
indicate that the RamA regulator is required for indole induction of acrAB in Salmonella.
The AcrAB pump is reported to export bile salts and play a role in bile resistance in E. coli and
Salmonella. Also, acrAB is reportedly induced by bile in a Rob-dependent manner in E. coli (18).
Although acrAB is also induced by bile in Salmonella, the induction mediating regulator is
unknown. Prouty et al. (17) further reported that acrAB activation by bile is independent of MarA,
Rob, PhoP/PhoQ, and RpoS. We investigated the possibility that RamA controls acrAB expression
in response to bile. Bile salts, cholic acid, and deoxycholic acid significantly induced acrAB
expression in Salmonella. When ramA was deleted, acrAB induction was eliminated. These
findings indicate a RamA-dependent pathway for bile-mediated regulation of the AcrAB efflux
pump in Salmonella, different from that observed in E. coli.
Indole accumulates and MdtEF is induced in stationary phase cultures of E. coli, but experiments
with a tnaAB mutant showed that indole partially contributes to this induction. These results
indicate that E. coli produces indole as well as other efflux pump inducers. Therefore, we
investigated whether an E. coli conditioned medium would induce multidrug efflux pumps in
Salmonella. Conditioned medium, prepared from E. coli MG1655, significantly induced eight
Salmonella multidrug efflux pumps, including acrAB and the outer membrane protein gene tolC.
Inductions of acrAB and tolC were significantly decreased in ramA, indicating that the E. coli
conditioned medium induced acrAB and tolC via the RamA regulator.
The aforementioned results indicate that RamA plays a major role in inducing acrAB in response to
environmental signals such as indole and bile. To understand the regulation of acrAB by RamA,
electrophoretic mobility shift assays (EMSA) with the RamA protein were performed. Plasmids
encoding the histidine-tagged RamA or the N-terminus truncated RamA proteins were
constructed. Since it was reported that RamA overproduction was related to increased AcrAB
expression in clinical K. pneumonia isolates (19), we investigated the effect of histidine-tagged
200
RamA on acrAB expression. Overproduction of histidine-tagged RamA significantly induced the
expression of acrAB; however, the truncated RamA did not induce acrAB and was used as a
negative control in subsequent EMSA. Upstream regions of acrA were amplified by PCR and the
fragments were incubated with RamA or truncated RamA protein. RamA bound to the upstream
region of acrA whereas truncated RamA did not. RamA did bind to the upstream region of tolC
whereas the truncated RamA did not. These results indicate that RamA directly controls the
expression of acrAB and tolC.
Abouzeed et al. reported that RamR is the local repressor of ramA (1). Therefore, we examined
whether indole induces acrA and ramA via RamR. We first investigated the effect of the ramR
deletion on acrA induction in response to indole. acrA expression increased in the presence of
indole, but was absent in the ramR strain. This result suggests that acrA induction in response to
indole is RamR dependent. We next investigated the effect of ramR deletion on ramA induction in
response to indole. ramA expression increased in the presence of indole (4.9-fold increase
compared to that in the absence of indole), but it decreased in the ramR strain and only slight
ramA induction in response to indole was observed (1.4-fold increase). This suggests that RamR is
required for the complete ramA induction in response to indole (11), and that there may be an
additional RamR-independent regulatory pathway for ramA induction in response to indole.
In this study, we found that indole, bile, and an E. coli conditioned medium induced several
multidrug efflux genes in Salmonella (10, 12 ). We found that acrAB induction by these three signal
sources is dependent on RamA, indicating that RamA plays a major role in inducing acrAB. The
ramA gene appears to be specific for Salmonella serovars and is absent in many other Gram-
negative microorganisms; notable exceptions are Klebsiella pneumoniae and Enterobacter spp. (4,
8, 21). The results of genomic comparison indicate that the gene organization surrounding ramA
gene and the corresponding region in E. coli are similar, with two exceptions: the absence of ramA
and the presence of Yi81-2 in E. coli. We suggest that the AcrAB induction pathway in Salmonella is
different from that in E. coli. Bile induces AcrAB in both Salmonella and E. coli. In E. coli, the
transcriptional factor Rob plays a major role in inducing acrAB expression in response to bile.
However, our data indicate that bile induction of acrAB in Salmonella is completely dependent on
RamA, not Rob. Other regulators, including MarA, SoxS, SdiA, and AcrR, are not involved in AcrAB
induction by indole and bile. These results suggest that RamA is a master regulator of Salmonella
acrAB and may mask the contributions of any other acrAB regulators. In E. coli, it was reported
that multiple regulators, including MarA, Rob, SoxS, and SdiA work coordinately in controlling
acrAB expression in response to acrAB inducers. This may be related to the lack of RamA in E. coli.
Indeed, overproduction of RamA has induced the drug resistance level of E. coli (20-22). A recent
report suggests that RamA and RamR, not SoxS and MarA, are involved in AcrAB-mediated
multidrug resistance in Salmonella (1). Based on our results and these other studies, RamA
appears to be the master regulator of acrAB in Salmonella.
Indole and bile are found in various internal human environments, especially in the intestine.
Indole is produced by many enteric bacterial species, and bile is often present at high
concentration in the intestinal tract. Therefore, RamR/RamA may be required for Salmonella to
detect environmental signals and for subsequent induction of the AcrABTolC system, resulting in
excretion of toxic compounds by Salmonella into surrounding environments.

ACKNOWLEDGEMENTS
We thank Sylvie Baucheron for the assay protocols and discussions and comments; Axel
Cloeckaert and Etienne Giraud for discussions and comments. This work is supported in part by
grant LS080 (NEXT Program).

201
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2434.
2. Baucheron, S., E. Chaslus-Dancla, and A. Cloeckaert. 2004. Role of TolC and parC mutation in high-level
fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT204. J Antimicrob Chemother
53:657-659.
3. Baucheron, S., H. Imberechts, E. Chaslus-Dancla, and A. Cloeckaert. 2002. The AcrB multidrug
transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar
typhimurium phage type DT204. Microb Drug Resist 8:281-289.
4. Chollet, R., J. Chevalier, C. Bollet, J. M. Pages, and A. Davin-Regli. 2004. RamA is an alternate activator of
the multidrug resistance cascade in Enterobacter aerogenes. Antimicrob Agents Chemother 48:2518-2523.
5. Eaves, D. J., V. Ricci, and L. J. Piddock. 2004. Expression of acrB, acrF, acrD, marA, and soxS in Salmonella
enterica serovar Typhimurium: role in multiple antibiotic resistance. Antimicrob Agents Chemother
48:1145-1150.
6. Hirakawa, H., Y. Inazumi, T. Masaki, T. Hirata, and A. Yamaguchi. 2005. Indole induces the expression of
multidrug exporter genes in Escherichia coli. Mol Microbiol 55:1113-1126.
7. Horiyama, T., A. Yamaguchi, and K. Nishino. 2010. TolC dependency of multidrug efflux systems in
Salmonella enterica serovar Typhimurium. J Antimicrob Chemother 65:1372-1376.
8. Komatsu, T., M. Ohta, N. Kido, Y. Arakawa, H. Ito, T. Mizuno, and N. Kato. 1990. Molecular
characterization of an Enterobacter cloacae gene (romA) which pleiotropically inhibits the expression of
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9. Ma, D., M. Alberti, C. Lynch, H. Nikaido, and J. E. Hearst. 1996. The local repressor AcrR plays a
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11. Nikaido, E., I. Shirosaka, A. Yamaguchi, and K. Nishino. 2011. Regulation of the AcrAB multidrug efflux
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12. Nikaido, E., A. Yamaguchi, and K. Nishino. 2008. AcrAB multidrug efflux pump regulation in Salmonella
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203
Epidemiologicalandgeneticfeaturesassociated
withtheinternationalspreadofmultidrug
resistantSalmonellaKentuckyST198
S.LeHello
1
,B.Doublet
2
,L.Fabre
1
,A.Bekhit
1,3
,A.Cloeckaert
2
,K.Holt
1,4
andF.X.Weill
1
1
InstitutPasteur,UnitdesBactriesPathognesEnt
riques,CentreNationaldeRfrencedesSalmonella,
WorldHealthOrganization(WHO)CollaboratingCentreforReferenceandResearchonSalmonella,Paris,
France
2
InstitutNationaldelaRechercheAgronomique(INRA),UMR1282,InfectiologieetSantPublique,Nouzilly,
France
3
Biochemistrydepartment,FacultyofPharmacy,Miniauniversity,Egypt
4
DepartementofMicrobiology&Immunology,UniversityofMelbourne,Australia
ABSTRACT
Salmonella genomic island 1 (SGI1) is a 43 kb integrative mobilizable element that harbors a great diversity of multidrug
resistance gene clusters described in numerous Salmonella enterica serovars and also in Proteus mirabilis. Recently,
the international spread of a ciprofloxacin-resistant S. enterica serovar Kentucky Sequence-Type 198 (ST198) strain
containing SGI1-K variants has been reported. A continuous surveillance at international level has revealed an extension
of antibiotic resistance in Kentucky strains (including carbapenems), the widening of countries of acquisition (whole of
Africa, Middle-East, South-East Asia and Europe) and various sources for human contamination. Commercial poultry
production and poultry feedstuffs have been suspected to disseminate this clone. Here, we characterized 48
representative ST198 serovar Kentucky strains isolated worldwide between 1969 and 2011 from humans and non-
humans, displaying a range from fully susceptible to extensively antibiotic resistant profiles. We revealed that two
subpopulations independently and exclusively acquired the SGI1 during the 1980s and 1990s. Unlike the ST198-X1
African epidemic subpopulation, the ST198-X2 subpopulation, mainly from Asia, harbors variants of the SGI1-J. A next
generation sequencing approach has been performed on this representative subset of Kentucky ST198 strains, allowing
a deeper understanding of the phylogenetic relationships.
INTRODUCTION
Despite substantial progress has been made in preventing foodborne diseases, new multidrug
resistant (MDR) Salmonella have emerged, and some have spread worldwide decade by decade
[1]. Their treatment in both animals and humans has become more difficult and foodborne
infectionsandoutbreakswithMDRSalmonellaarealsoincreasinglyreported[2,3].Oneexample
ofthemistheglobalspreadofamultidrugresistantS.TyphimuriumphagetypeDT104inanimals
andhumansinthe1990s[4].WhilethespreadofDT104mayhavebeenfacilitatedbytheuseof
antimicrobials,internationalandnationaltradeofinfectedanimalsisthoughttoplayamajorrole
ininternationalspread[5].
Morerecently,anemergingSalmonellaKentuckyST198X1 clonehasbeendescribed[68].It has
accumulated various chromosomal resistance determinants since the mid 1990s with the
integrationoftheSalmonellagenomicisland1,a43kilobasegenomicislandinitiallydescribedin
DT104 [9], encoding resistance to multiple antimicrobials including amoxicillin, gentamicin, and
sulfonamides [10], followed by cumulative mutations in the gyrA and parC genes leading to
resistancetonalidixicacidthentociprofloxacinin2002(KentuckyCIPR)[6,7].Thispopulationwas
mostly detected in Egypt before 2005, but has now rapidly spread throughout Africa, the middle
East, Europe, North America, Indian subcontinent and Southeast Asia [68 and personal data].
Another matter of concern is the widening livestock reservoir of this Salmonella Kentucky CIPR
strain: initially identified in autochthonous poultry but then found in various animals and food
(contaminatedspicesinFranceandtheUS,turkeyflocksinGermanyandPoland,wildanimals.)
[7,1113].Ofgreatconcern,theseisolatesarenowproducersofvariouscarbapenemasesand/or
cephamycinaseand/orESBLs[14].
We propose here a fine molecular epidemiologic study (PFGE, MLST, QRDR mutations, CRISPR
profiling and whole genome sequencing) on a human and nonhuman collection of Salmonella
204
Kentucky ST198X1 strains (n=48), isolated worldwide between 1969 and 2011 and displayed a
rangefromfullysusceptibletoextensivelyantibioticresistantprofiles.
MATERIALANDMETHODS
Bacterialisolates
Through the continuous surveillance, the present working group has been able to identify
Kentucky CIPR strains from different origins (animals, food, humans) in new countries of
acquisitionsincethelaststudyledin2009[8].
Atotalof50S.entericaserotypeKentuckyisolateswereextensivelycharacterizedasfollows:38
humanisolatesfromtheAfrican(n=28),MiddleEast(3),India(4)andSouthEastAsia(3)isolated
during 1961 to 2011; 12 nonhuman isolates from 1937 to 2011 (8 animals and 4 from
food/environmentalsamples).
Microbiologicalinvestigations
All human Salmonella strains received by the FNRC are serotyped using the WhiteKauffmannLe
Minorscheme.
Antimicrobial susceptibility testing (AST) was performed on all Salmonella isolates, by the disk
diffusion method with a panel of 32 antimicrobial agents (BioRad, MarnesLaCoquette, France).
The MICs of ceftriaxone, ceftazidime, imipenem, ciprofloxacin, azithromycin were determined by
Etests (AB Biodisk, Solna, Sweden). Results were interpreted with the Antibiogram Committee of
the French Society for Microbiology (CASFM) (www.sfmmicrobiologie.org/) breakpoints. In
particular, susceptible isolates were defined as having a MIC 0.5 mg/L for ciprofloxacin, and
resistantisolatesweredefinedashavingaMIC>1mg/Lforciprofloxacin.
Moleculartyping
PulseNetstandardpulsedfieldgelelectrophoresis(PFGE)ofXbaIdigestedchromosomalDNAand
multilocussequencetyping(MLST)wereperformedaspreviouslydescribed[7].
Determinationofresistancemechanisms
The presence of betalactam resistance genes, plasmidmediated quinolone resistance genes,
macrolideresistancegenes,aminoglycosideresistancegenes,class1integrongenecassettesand
Salmonellagenomicisland1(SGI1)wasassessedbyPCR,aspreviouslydescribed[7,14].
The quinolone resistancedetermining region (QRDR) of gyrA, gyrB, parC and parE (encoding
subunitsoftheDNAgyraseandthetopoisomeraseIV)wassequenced,aspreviouslydescribed[7].
Thenucleotideanddeducedaminoacidsequenceswereanalysedandcomparedwithsequences
available from the National Center for Biotechnology Information website
(http://www.ncbi.nlm.nih.gov).
CRISPRcontentsandpolymorphism
AfamilyofDNArepeatsnamedCRISPR(clusteredregularlyinterspacedshortpalindromicrepeats)
ishighlypolymorphicinSalmonella[15].WeanalysedthecontentsofthetwoCRISPRlociinallthe
50Kentuckystrains.
WholeGenomeSequencing(WGS)
The isolates supplied have been sequenced through services provided by a DNA sequencing
platform, using both 454 and illumina for two strains (for the reference 98K and one
representative X1ST198 Kentucky CIPR strain) and using illumina for the 48 remaining isolates.
Theresultingsequenceshavebeenassembledandcompared(withthehelpofabioinformatician
team).Comparativeanalysisofthegenomeshasbeenperformed.

205
RESULTS
ClonalityoftheKentuckyCIPRpopulation
Genetic relatedness of 50 Kentucky isolates was studied using PFGE, MLST, CRISPR contents and
WGS.AllKentuckyCIPR(n=34)wereclusteredintoahomogeneousgroupdisplayingoneX1PFGE
group, a unique ST198 clonal complex, one CRISPR content complex and one lineage by WGS.
Interestingly, 8 additional strains belonging to the same lineage displayed no resistance to
ciprofloxacin(buttonalidixicacidortooldantiobioticsorsusceptibletoall),suggestingprecursor
strainsoftheepidemicKentuckyCIPR.
Ciprofloxacin resistance in all the 34 Kentucky CIPR isolates tested was due to gyrA and parC
mutations. All contained a double mutation in gyrA (Ser83 and Asp87) and a single parC
substitution (Ser80 encoding an isoleucine residue). No isolates had gyrB or parE mutations. In
gyrA, all isolates contained a change at codon Ser83 to phenylalanine, whereas mutations in the
Asp87 codon resulted in substitutions to asparagine, tyrosine, or glycine residues depending on
thegeographicoriginoftheisolates.All3mutationsinAsp87codonwerefoundinisolatesfrom
Egypt,whereasisolatesfromnorthAfricaandtheMiddleEasthadthemutationthatresultedinan
asparagine residue; those from east Africa had the mutation that resulted in a tyrosine residue;
and those from west Africa had the mutation that resulted in a glycine residue. This distribution
seemedtobeconfirmedbyaWGSapproach.
Noplasmidmediatedquinoloneresistancegenes,suchasqnr,aac(6)Ibcr,andqepA,havebeen
foundinCIPRKentuckyisolates.
A genomic island that harbors a multidrugresistant gene cluster was integrated between the 2
chromosomalgenesthdFandyidYinall42testedST198X1S.entericaserotypeKentuckyisolated
since 1996, regardless of their susceptibility to ciprofloxacin. The extensive genetic
characterizationof28isolatesidentifiedseveralvariantsoftheSGI1thatbelongedtotheSGI1Ks,
Ps, and Qs subgroups. Insertion sequences (ISs) IS26 and ISVch4 were always inserted in the
upstreamS044andS010regionsoftheSGI1,respectively.Highlydiversegeneticrearrangements
mediated by various ISs and transposons were ob served in the multidrugresistance region of
these SGI1Ks, Ps, and Qs variants, accounting for resistance profile diversity among Kentucky
CIPR.
WealsodistinguishedanotherST198subclonewithisolatesrecoveredinpatientsreturningfrom
China, India, or Thailand with decreased susceptibility to CIP. These strains clustered into theX2
group,possessedtheSGI1Jsandbelongedtoanotherlineage(byCRISPRcontentandWGS)[16].
DISCUSSION
Salmonella Kentucky ST198X1 is a particularly successful strain that has accumulated various
chromosomalresistancedeterminantssincethemid1990s,withtheintegrationoftheSalmonella
genomic island 1 (encoding resistance to multiple antimicrobial drugs, including amoxicillin,
gentamicin, and sulfonamides), followed by cumulative mutations in the gyrA and parC genes,
leading to resistance to nalidixic acid, and then to ciprofloxacin, in 2002. This strain was mostly
identifiedinEgyptbefore2005[7]buthassincespreadrapidlythroughoutAfricaandtheMiddle
East, Indian subcontinent, Southeast Asia [7, 14]. The diversity of the recently acquired b
lactamases suggests that the growing Salmonella Kentucky ST198X1 CIPR
strain has been

collectinggenesforresistancetoESCsorcarbapenems.Thisepidemicstrainwasassociatedwith
alivestock(autochthonouspoultry)reservoirinAfricabutthenfoundinvariousanimalsandfood
worldwide.
To understand by an evolutionary genetic approach the different steps that lead to successful
MDRSalmonellaserotypeKentuckypopulation,aglobalisolatescollectionfromaroundtheworld
coveringalongtimespan(includingpreantibioticeraisolates),variouslivestockspecies,andwild
206
life (possible reservoirs) has been gathered. Chromosomal markers (SNPs, indels, prophage,
genomic islands from WGS) can allow to reconstruct the evolutionary history of these emerging
pathogens.Putativenewtargetgeneticelementswillbeexplored.Furthermore,newtools(qPCR)
will be developed for rapid detection and identification of these MDR Salmonella, as timely
responseandcontrolarecrucialcapacitiestoavoidorstop/limittheirglobalspread.
In conclusion, recent experience with multidrugresistant S. enterica serotype Typhimurium DT
104 demonstrates the potential for global spread of resistant Salmonella infection. In our study,
multinationalsurveillanceallowedpromptidentificationoftheepidemicST198X1CIPRKentucky
cloneataninternationallevel.Heightenedawarenessbynationalandinternationalhealth,food,
and agricultural authorities is necessary to implement measures to monitor and limit spread of
thisstrain.
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10. Doublet B, Praud K, Bertrand S, Collard JM, Weill FX, Cloeckaert A. Novel insertion sequence and
transposonmediated genetic rearrangements in genomic island SGI1 of Salmonella enterica serovar
Kentucky.AntimicrobAgentsChemother.2008Oct;52(10):374554.
11. Beutlich, J., Guerra, B., Schroeter, A., Arvand, M., Szabo, I., Helmuth, R. 2012. [Highly ciprofloxacin
resistantSalmonellaentericaserovarKentuckyisolatesinturkeymeatandahumanpatient].Berl.Munch.
Tierarztl.Wochenschr.125,8995.
12. Wasyl, D., Hoszowski, A. 2012. First isolation of ESBLproducing Salmonella and emergence of
multiresistantSalmonellaKentuckyinturkeyinPoland.FoodRes.Int.45,958961.
13. Mnch S, Braun P, Wernery U, Kinne J, Pees M, Flieger A, Tietze E, Rabsch W. Prevalence, serovars,
phage types, and antibiotic susceptibilities of Salmonella strains isolated fromanimals in the United Arab
Emiratesfrom1996to2009.TropAnimHealthProd.2012Oct;44(7):172538.
14. LeHelloS,HarroisD,BouchrifB,Sontag
L,ElhaniD,Guibert
V,ZeroualiK,WeillFX.Highlydrugresistant
Salmonella Kentucky ST198X1 in the Mediterranean basin: a microbiological study. Lancet Infect Dis. In
press.
207
15. FabreL,ZhangJ,GuigonG,LeHelloS,GuibertV,AccouDemartinM,deRomansS,LimC,RouxC,Passet
V, Diancourt L, Guibourdenche M, IssenhuthJeanjean S, Achtman M, Brisse S, Sola C, Weill FX. CRISPR
typing and subtyping for improved laboratory surveillance of Salmonella infections. PLoS One.
2012;7(5):e36995.
16. Le Hello S, Weill FX, Guibert V, Praud K, Cloeckaert A, Doublet B. Early strains of multidrugresistant
Salmonella enterica serovar Kentucky sequence type 198 from Southeast Asia harbor Salmonella genomic
island 1J variants with a novel insertion sequence. Antimicrob Agents Chemother. 2012 Oct;56(10):5096
102
208
DramaticincreaseofAmpCandESBLproducingdTartratepositive
SalmonellaentericaserotypeParatyphiBfrombroilersinBelgium,20082010
BenotDOUBLET
1
,KarinePRAUD
1
,AxelCLOECKAERT
1
,andPatrickBUTAYE
2
1
InstitutNationaldelaRechercheAgronomique(INRA),UMR1282,InfectiologieetSantPublique,
NOUZILLY,France;
2
DepartmentofBacteriologyandImmunolgy,VeterinaryandAgrochemicalResearchCentre,VAR
CODACERVA,Groeselenberg99,B1180UKKEL,Belgium

Salmonella isolates from animal production and their antimicrobial resistances are monitored by
theCODACERVAinBelgium.Since2008,theproportionofdTartratepositiveSalmonellaenterica
serotypeParatyphiB(S.ParatyphiBdT+)isolatedfrompoultryhasincreasedsignificantly(8.8%in
2008 to 36.5% in 2010). The great majority of the isolates were from broilers. The complete
antimicrobial susceptibility profile and the presence of ESBLs and AmpCencoding genes, were
determined. Genetic relationship between isolates were analysed by PFGE. Conjugation was
performed and plasmids were characterized by PCRbased Replicon Typing (pBRT) and RFLP
profiling.
Among the S. Paratyphi B dT+ isolates studied, thirtyfive per cent were resistant to third and
fourgeneration cephalosporins. The ESBL genes bla
CTXM1
, bla
CTXM2
, bla
TEM52
and the AmpC
cephalosporinase genes bla
CMY2
were detected on different selftransferable plasmids. pBRT and
RFLP profiling revealed the occurrence of bla
CTXM1
, bla
TEM52
and bla
CMY2
genes on different
conjugative IncI1 plasmids (n=24), underlining the epidemic success of this type of plasmids.
Interestingly, and for the first time to our knowledge, the bla
CTXM2
gene was located on a large
multirepliconplasmidcontainingbothIncHI2andIncP(IncHI2/P)replicons.Inaddition,twostrains
carrying IncI1 and IncHI2/P plasmids with either bla
CMY2
or bla
CTXM2
genes, respectively, were
detectedinthisstudy.
Furtherresearchesareanabsolutenecessitytounderstandtheepidemiologythereofandtobe
abletoproposeinterventionstolimitthespreadofcephalosporinandmultidrugresistant
Salmonellaspp.
.
209
NationwidehumanoutbreakofcephalosporinresistantSalmonella
serotypeTyphimuriuminFranceassociatedwithconsumption
ofunpasteurizedcheese,alinkwithawarmbloodhorsereservoir?
SimonLEHELLO
1
,SophieA.GRANIER
2
,JackieTapprest
3
,CorinneDanan
2,6
,PalomaCARRILLO
SANTISTEVE
4,5
,VirginieDUSCH
6
,VroniqueGUIBERT
1
,ClaireLAUGIER
3
,
NathalieJOURDANDASILVA
4
,WeillFX
1
andAnneBRISABOIS
2
:bothauthorsequallycontributedtothiswork
1
InstitutPasteur,UnitdesBactriesPathognesEntriques,
CentreNationaldeRfrencedesE.coli,Shigella,Salmonella,PARIS,France
2
Anses,LaboratoiredeScuritdesAliments,MAISONSALFORT,France
3
Anses,LaboratoiredePathologiequinedeDozul,MAISONSALFORT,France
4
FrenchInstituteforPublicHealthsurveillance,SAINTMAURICE,France
5
EuropeanCentreforDiseasePreventionandControl;Stockholm,STOCKHOLM,Sweden
6
DirectionGnraledelAlimentation,Ministredelagriculture,delagroalimentaireetdelafort,
PARIS,France
Early 2010, the ANSESSalmonella Network identified, through routine surveillance,a cluster of 6
MDRSalmonellaentericaserotypeTyphimuriumstrainsfromfourequinesamples,onefrommilk
and one from unpasteurized cheese (producer A) in the Normandy region. The strains were
resistant to ampicillin, streptomycin, kanamycin, tobramycin, gentamicine, sulphamethoxazole,
trimethoprime, tetracycline, chloramphenicol and expandedspectrumcephalosporins, including
cefotaxime, ceftazidime, cefepime and cefoxitine by producing both CTXM1 and CMY2
lactamases (phenotype called MDR1). One month later the first human cases with a S.
Typhimurium with the MDR1 resistance profile were reported by the French National Reference
Center(NRC)forSalmonellatotheFrenchInstituteforPublicHealthsurveillance.
An outbreak investigation was set up and simultaneously epidemiological, microbiological and

veterinary and food investigations were launched to define the extend of the outbreak and
identifythevehicleoftransmissioninordertotakeappropriatecontrolmeasures.
HumancaseswereindividualswithanisolationofMDR1S.TyphimuriumbetweenMarch,8
th
and
April, 19
th
2010 reported to the French NRC for Salmonella. Thirtyfive human cases were
detected,mainlyintheNorthwestofFrance.Medianagewas36years(range:190years),55%of
patients were hospitalized, none died. Among 29 interviewed cases, the consumption of
unpasteurized cheese was the mainly reported exposure (24 of 29, 82.7%). We revealed also 2
caseswithclosecontactwithhorses.
A total of 40 human and 28 nonhuman (milk, unpasteurized cheese, horses and their
environment) MDR1 S. Typhimurium isolateswere collected between January and July 2010. The
strains were positive for both ESBL(bla
CTXM1
) and cephalosporinase (bla
CMY2
)
genes and belonged

totheXTYM136PFGEtype.TheyhadidenticalMLVAType(3149NA211)andCRISPOLtype(CT
62).Plasmidanalysisrevealedthepresenceoftwoconjugativeplasmids:oneof90kbIncI1bearing
bla
CMY2
andoneof250kbIncHI1bearingbla
CTXM1
.
Eveniftherouteofthecontaminationbetweenhorsesandcowshadntbeendirectlytraceddue
todifficultiesininvestigationofthebovinesector,ithastobenoticedthatinthisarea,horsesand
cows commonly pasture on together. The hypothesis of cases contamination from the
consumptionofunpasteurizedcheeseproducedbyproducerAwithanundetectedcontamination
couldnotbecompletelyrejected.
210
Thisreporthighlightstheimportantrolethatroutineantimicrobialsusceptibilitymonitoringplays
inearlydetectionandfollowingdisseminationofextensivelydrugresistantSalmonellaisolatesin
unexpectedsourceviaunexpectedrouteofcontamination.

211
HighlyciprofloxacinresistantSalmonellaentericaserovarKentuckyisolates
ofhumanandanimalorigininGermany
JanineBEUTLICH
1
,BeatrizGUERRA
1
,AndreasSCHROETER
1
,ErhardTIETZE
2
,IstvanSZABO
1
andReinerHELMUTH
1
1
FederalInstituteforRiskAssessment,GermanNationalReferenceLaboratoryforSalmonella,
BERLIN,Germany
2
RobertKochInstitute,NationalReferenceCentreofSalmonellaandotherenterics,
WERNIGERODE,Germany
INTRODUCTION
Multidrugresistant(MDR)Salmonella(S.)isolatesconstituteagrowingthreattohumanhealthas
they are an increasing source for human infections and outbreaks (Alcaine et al., 2007). Food
producing animals are one of the main reservoirs for nontyphoid MDR Salmonella. These
pathogensaremainlytransmittedbytheconsumptionofcontaminatedfoodproducts(Crayetal.,
2000;Piresetal.,2009).Multidrugresistancetoclinicallyimportantantimicrobials,inparticularto
fluoroquinolones like ciprofloxacin (CIP), may disable therapies for patients with systemic course
ofdiseaseorriskdisposal(Anonymous,2007).TheoccurrenceofS.Kentuckyinhumanswasonly
rarelyobservedintheEUandhasbeennormallylinkedtoforeigntravel(Weilletal.,2006;Collard
et al., 2007). According to the European Food Safety Authority (EFSA) and European Centre for
DiseasePreventionandControl(ECDC)in2009S.Kentuckywasforthefirsttimereportedamong
the ten most frequent serovars causing human salmonellosis (EFSA and ECDC, 2011). In recent
yearsnationalsurveillancesystemsinFrance,England,Wales,DenmarkandtheUSAregisteredan
increasedoccurrenceofMDRS.Kentuckydisplayinghighlevelresistancetofluoroquinolones(CIP,
MIC > or = 4 mg/l). These isolates were ascribed to clone ST198X1, which caused between 2000
and 2008 in the countries mentioned above around 500 human infections and was presumably
transferred by poultry (Le Hello et al., 2011). To determine whether this clone is also present in
Germanyinnonhuman sources, the National Reference Laboratory for Salmonella (NRLSalm) at
theFederalInstituteforRiskAssessment(BfR)analysedthetrendofS.Kentuckyisolatesreceived
over the past years. Human isolates collected within the same period were also included in the
studyforcomparisonpurposes.
MATERIALANDMETHODS
Inthisstudyatotalof62S.Kentuckyisolateswereanalysed.Theisolateswerechosenpendingon
theirCIPresistancephenotype.Intotal49isolatesdisplayedhighlevelCIPresistance(MICvalues
of > or = 4 mg/l). One isolate showed lowlevel CIP resistance (MIC = 0,25 mg/l). As a reference
group 11 isolates susceptible towards all previous routinely tested antimicrobials were included,
as well as two isolates displaying MDR to sulphonamides, tetracycline, and trimethoprim, and to
ampicillin,andsulphonamides,respectively.
Thirtytwo isolates were selected from the NRLSalm internal strain collection from animal, food,
andnonhumansources(23isolatesshowinghighlevelCIPresistancewithMICvaluesof>or=4
mg/l; 8 susceptible isolates). One Polish human isolate originated from the MedVetNet WP21
straincollection(CIP,MIC=0,25mg/l).Inaddition,30humanisolatesfromtheNationalReference
Centre of Salmonella and other enterics at the Robert Koch Institute (RKI) were analysed (25
isolates displaying highlevel CIP resistance with MIC values of > or = 8 mg/l; 5 CIP susceptible
isolates).
S.KentuckyisolatedfromdifferentsourcesinGermanybetween2003and2011wereanalysedin
thepresentstudy.
All isolates were subjected to phenotypic analyses. Serotyping was done following the White
KauffmannLe Minor scheme (Grimont and Weill, 2007). All isolates were tested by broth
212
microdilutionfortheirantimicrobialsusceptibility,accordingtotheguidelinesoftheCLSI(Clinical
andLaboratoryStandardsInstitute,2009)usingcustomdefinedmicrotiterplates(TrekDiagnostic
Systems, United Kingdom). From 20002007 the plate format NLMV1A with 17 antimicrobial
agents was applied. Since 2008 the European plate format EUMVS with 14 antimicrobial
substances has been used (Federal Institute for Risk Assessment, 2010). Evaluation of the results
was performed according to the cutoff values set in Decision 2007/407/EC. These are based on
thecutoffvaluessetbytheEuropeanCommitteeofAntimicrobialSusceptibilityTesting(EUCAST,
2001;www.eucast.org).
WithinmolecularanalysisthefluoroquinolonetargetscodinggenesgyrAandparCwereamplified
by PCR and subsequently sequenced (Qiagen, Hilden, Germany). The detection of the
presence/absenceofthechromosomalresistanceregionSalmonellaGenomicIsland1(SGI1)was
alsoperformedbyPCR(Amaretal.,2008).MoreoverallisolatesweretestedbyPCRamplification
forthepresenceof30differentresistancegenesaswellasclass1integrons(Beutlichetal.,2011).
GenomicDNAfromall62S.Kentuckyisolateswassubjectedtomacrorestrictionanalysisusingthe
endonucleases XbaI and BlnI according to the standardized PulseNet protocol
(http://www.pulseneteurope.org).
Themainpartoftheisolates(53of62isolates,85,5%)originatedfromGermany,butalsostrains
fromEthiopia(2),Morocco(3),Belgium(1),Egypt(1),Peru(1),andPoland(1)wereincluded.
For further analysis Multi Locus Sequence Tying (MLST) was applied according to Kidgell et al.
(2002) and the MLST protocol for Salmonella enterica of the MLST database hosted by the
University College Cork, Ireland
(http://mlst.ucc.ie/mlst/dbs/enterica/documents/primersEnterica_html).
RESULTS
Between1998and2012theNRLSalmidentifiedamongallreceivedisolates(N=67.953)atotalof
176S.Kentuckyincluding122isolatesfromGermany.Sixtytwoisolatesrevealedresistancetotwo
ormoreantimicrobialsubstances.FromtheGermanisolates,40(32,8%)wereMDRandoriginated
mainly from poultry samples. A total of 25 from the 122 German S. Kentucky isolates (20,5%)
showed an MIC value of more than 8 mg/l to CIP and were considered both by EUCAST (clinical
breakpointMHK>1mg/l;ECOFF>=0.064)andCLSI(MHK>=4mg/l)ashighlyresistant(EUCAST,
2011;CLSI,2008).
The resistance pattern most frequently detected included resistance to ampicillin, gentamicin,
streptomycin, tetracycline, and sulphonamides, and was recorded in 28 of the 62 isolates. This
profilewasdetectedinisolatesoriginatingfromvarioustypesofmatrices.Themostfrequentones
were isolates originating from turkey (10 isolates), humans (9) and fish meal (3, imported to
GermanyfromMoroccoandPeru).
Sequence analysis identified mutations in the genes gyrA and parC as underlying mechanism for
the CIP resistance. Fortynine isolates displayed high level CIP resistance and at molecular level
showed double mutation in gyrA (serine83 and aspartic acid87) with an amino acid change in
codonSer83tophenylalanine(Phe,49isolates)andincodonAsp87totyrosine(Tyr,24isolates),
asparagine(Asn,16isolates),orglycine(Gly,9isolates),aswellasapointmutationinparC(Ser80)
to isoleucine (Ile). The one isolate displaying low level resistance to CIP only showed a single
mutationingyrA(Phe83).
Fortynine CIP resistant isolates tested positive for the presence of the MDR conferring region
SGI1.
MLST revealed that all CIP resistant isolates belonged to sequence type (ST) 198, independentof
their origin and isolation matrix. From the reference group (13 isolates), nine isolates were
assignedtoST314,onetoST393,onetoST696,andtwotoST198.PFGEanalysisgeneratedforthe
50CIPresistantisolates19closelyrelatedXbaIprofiles,and25relatedBlnIpatterns.

213
DISCUSSION
In recent years, in different European countries human salmonellosis infections caused by the
highlyCIPMDRS.KentuckycloneST198X1havebeenincreasinglyregistered.Thefirstinfections
described in Europe, were associated to Egypt travels in 2002, but meantime the clone has
geographically wide spread. From the public health aspect, this type of infections seemed to be
travel associated. However other routes of transmission, such as the consumption of
contaminatedfoodproductsmaybepossible(LeHelloetal.,2011).
InthepresentstudywedescribetheoccurrenceandincreasingprevalenceofhighlyCIPresistant
S. Kentucky isolates observed since 2010 in Germany. Molecular analyses revealed that all CIP
resistant isolates belonged to the Kentucky clone ST198X1 described by Le Hello et al. (2011).
These results show that especially poultry derived food items traded in Germany, in particular
turkey meat, may serve as important infection vehicle for this clone. In fact, from all the isolates
registered in the data base of the NRLSalm (Friedrich et al., 2011), from the 50 ascribed to the
clone, 10 were isolated from turkey meat. However we also found for the very first time in
Germany,oneisolatecollectedfromturkeyfaecesthatshowedtheCIPresistanceandbelongedto
the clone. Similar isolates have been also found in turkey primary production in other countries
like Poland (Wasyl andHoszowski, 2011). Interesting was also the detection of clone ST198X1 in
fish meal samples intended for animal feed. Moreover the clone was also detected in 25 human
urin or stool samples collected between 2008 and 2011 in Germany. The possibility of travel
associated infection cannot be discarded. However, in 2011 a highly CIP resistant human isolate
was sent to the NRLSalm for molecular analysis by the Hesse State Health Office (Dillenburg,
Germany;Beutlichetal.,2012).Thepatientsufferingfromgastroenteritishadnotravelanamnesis
withinthelastfourweeksbeforediseaseoutbreak,sothatitwasassumedthattheinfectionhad
beenacquiredinGermany.Bythisfindingonecanhypothesizethatthisclonemightestablishalso
inGermanyasahumaninfectioncausingpathogen(Beutlichetal.,2012).Itmightbeconceivable
that the spread of the highly CIP resistant clone ST198X1 in Germany could be related to the
import of contaminated food items or animal feed. Since WHO considers fluoroquinolones to be
drugsofcriticalimportanceinhumanandveterinarymedicine(WorldHealthOrganization,2001),
andsincetheyarestillagentsofchoiceintheclinicaltreatmentofsystemicSalmonellainfections
(Anonymous, 2007, Helmuth and Hensel, 2004), the spread of ST198X1 S. Kentucky demands
specialattention.Theimplementationofmitigationstrategiesforthishighlyresistantcloneseems
tobeurgentlyrequired.
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andvirulencedeterminantsinEuropeanSalmonellaGenomicIsland1positiveSalmonellaentericaisolates
fromdifferentorigins.ApplEnvironMicrobiol77:56555664.
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Wochenschr125:8995.
214
6. Bundesinstitut fr Risikobewertung (2010): Deutsche AntibiotikaResistenzsituation in der
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k.pdf
7. Clinical and Laboratory Standards Institute (2008): Performance standards for antimicrobial
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ed.(M07A8),vol.29,no.2.CLSI,Wayne,PA.
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Struelens M, Bertrand S (2007): Travelacquired salmonellosis due to Salmonella Kentucky resistant to
ciprofloxacin, ceftriaxone and cotrimoxazole and associated with treatment failure. J Antimicrob
Chemother60:190192.
10.CrayPJF,GrayJT,WrayC(2000):Salmonellainfectionsinpigs.In:WrayC,WrayA(Hrsg.),Salmonellain
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11. EUCAST (2011): Breakpoint tables for interpretation of MICsand zone diameters. Version 1.3, January
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Granier SA, JourdanDa Silva N, Cloeckaert A, Threlfall EJ, Angulo FJ, Aarestrup FM, Wain J, Weill FX
(2011): International spread of an epidemic population of Salmonella enterica serotype Kentucky ST198
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215
AntibioticresistancetrendsinSalmonella,20082012
DariuszWASYL,MagdalenaZAJCandAndrzejHOSZOWSKI
NationalVeterinaryResearchInstitute,DepartmentofMicrobiology,PUAWY,Poland
ABSTRACT
Monitoring of antimicrobial resistance in Salmonella has been introduced in Poland based on EFSA recommendation.
Minimal Inhibitory Concentrations of 14 compounds were tested on 3795 random isolates representing 241 serovars and
interpreted according to epidemiological cut-off values. Prevalence of microbiological resistance was calculated with
95% interval-based approach and temporal trends were assessed in regression analysis performed on qualitative and
quantitative data. Resistance level was related to Salmonella serovar. S. Virchow and S. Enteritidis were usually
susceptible to all antimicrobials tested besides quinolones. Resistance to these compounds was found in nearly all
isolates of the first serovar while in S. Enteritidis it raised from 29% in 2008 up to 51% in the following year. Interestingly,
the resistance level was stable within S. Enteritidis originating from layers, whereas raised up in broiler isolates.
Significant increase of resistance to several compounds was observed in S. Infantis and S. Newport. Resistance trend in
S. Typhimurium was affected by epidemiological change: pentaresistant clone (ACSSuT) is being replaced with less
resistant monophasic variant occurring mostly in pigs and cattle as well as foods of thereof. In 2009 clonally related S.
Kentucky showing resistance to wide range of antimicrobials emerged in poultry production, mostly turkey. Emergence
of resistant clones seems to be an important factor in spread of resistant Salmonella. Long-term epidemiological
observation of S. Mbandaka with virtually no resistance might prove that antimicrobial usage and possible overuse is not
the only driving force for antimicrobial resistance spread in Salmonella. Taking the above under consideration we
conclude that random selection of 170 isolates per study population for antimicrobial resistance monitoring and
evaluation of temporal trends, as it is recommended by EFSA, might be less effective than targeted testing of the
isolates within the serovars most prevalent in the country.

INTRODUCTION
Theintroductionofantimicrobialsfordiseasecontrolinhumansandanimalsprovidedfavourable
conditionsforselection,spreadandpersistenceofresistantbacteria.Overthelastdecade,there
has been an increase in awareness of the potential problems that selection of antimicrobial
resistance (AMR) in foodproducing animals could cause serious consequences for public health.
For reliable estimation of AMR scope standardized monitoring scheme was needed [1]. The
requirements of Decision 2007/407/EC have been implemented in Poland in 2008, and over the
study period monitoring has included consecutive poultry production sectors covered with
national Salmonella control programmes (NSCPs). Herewith, the results of AMR monitoring in
Salmonellaisolatedfrompoultry,aswellasotheranimals,foodsandfeedwereanalysed.
MATERIALANDMETHODS
Bacterial isolates: Approximately 11 500 Salmonella were isolated at or submitted to NRL for
reference testing. A subset of random 3795 isolates was selected for antimicrobial resistance
monitoring: starting from 346 in 2008, 429 in 2009, and approximately 1 000 in the following
years. They originated from NSCPs in Gallus gallus breeders (N=143), layers (N=703), broilers
(N=769),andfatteningturkeys(N=131),butalsofromotherresearchprojectsinanimalsincluding
reptiles(N=538),andhygienechecksinfood(N=514)andfeed(N=143).
Salmonellaserovars:Mostof241serovarsidentifiedwererepresentedbysingleisolates,but71%
belonged to 7 top frequent serovars tested over the study period: S.Enteritidis (N=1360),
S.Infantis(N=374),S.Typhimurium(N=330;includingmonophasicvariant),S.Mbandaka(N=213),
S.Newport(N=173),S.Virchow(N=133),andS.Kentucky(N=111).
MIC testing: Resistance to 14 antimicrobials was tested with microbroth dilution method
(Sensititre, TREK D. S.). The MIC values were interpreted according to EUCAST epidemiological
criteria (Table 1). Nonwild type MICs above cutoff values were classified as microbiological
resistance and the isolate was considered to carry relevant resistance determinant. Only
216
phenotypic resistance was considered and resistance patterns were used to give possible insight
intocoresistanceandcrossresistance.
Statisticsandtrendanalysis:Theprevalenceofresistancewascalculatedasafractionofisolates
with microbiological resistance, within 95% confidence interval. Temporal trends were assessed
on both qualitative andquantitative results using regression analysis [2]. Qualitative results were
analysedinlogisticmodel,whereaslinearregressionwasappliedforlog2MICdata.Thepvalues
(p[Q]andp[MIC],respectively)lowerthan0,05indicatedrelevanttrendsgraphicallydisplayedon
graphs.
RESULTS
MIC distributions and prevalence of microbial resistance (% NWT) to all tested compounds were
showninTable1.Resistancetoatleastoneantimicrobialwasfoundin56,9%oftheisolatesand
its level varied between the serovars. One hundred fifty resistance patterns covering up to 11
substancesfrom8antimicrobialclasseswerenoted(datanotshown).
S. Enteritidis was usually susceptible to all tested antimicrobials besides quinolones. That
resistanceincreasedsignificantlyoverthestudyperiod,mostlyduetoarisefrom29%in2008up
to51%inthefollowingyear.Itwasobservedinbroilerisolates,butnotintheonesobtainedfrom
laying hens (Figure 1). In S.Infantis a rising number of isolates was resistant to nalidixic acid,
ciprofloxacin, streptomycin, tetracycline and sulfamethoxazole (Figure 2.). Resistance patterns
including at least those five mentioned antimicrobials were observed in 35% of all tested S.
Infantis and 18% NSCPs isolates. The major S.Typhimurium resistance continuously embraced
ampicillin, phenicols, streptomycin, sulfamethoxazole, and tetracycline related to pentaresistant
profile. It was often supplemented with quinolone resistance. The frequency of resistance to a
givencompounddiminishedbyapproximately20%overthestudyperiod(Figure3),althoughthe
decline was not seen in NSCPs isolates (Figure 4). Resistance profiles containing at least
pentaresistant core were observed in 37% of all Typhimurium tested, and up to 58% NSCPs
isolates. Antimicrobial resistance in S.Newport varied and increased dramatically even by 60%
(nalidixic acid or tetracycline in 2009 versus 2012; Figure 5). The prevalence of ciprofloxacin
resistance was definitely higher than nalidixic acid. No temporal trends were observed in the
remaining predominating serovars: resistance in S. Mbandaka remained low, whereas 78% of
tested S. Kentucky was multidrug resistant (AmpStrGenTcySmxNal Cip). Essentially all S. Virchow
isolates were resistant to quinolones (data not shown). Resistance in isolates representing other
serovarvariedfromincidents(cephalosporins,0,4%)to40%(quinolones,streptomycin),nomatter
ifallsourcesorNSCPsisolateswereconsidered.
DISCUSSION
Antimicrobial resistance monitoring has been successfully implemented in Salmonella isolated
from control programs in poultry [1], as well as from other animal sources, foods and feeds.
Althoughnumerousisolatesweretested,temporaltrendscouldhavebeenassessedonlyforafew
serovars represented with statistically sound number of isolates. Random selection of 170
Salmonellaisolatesperproductionsectormightnotbethemostcostbenefitefficientapproach.
Alternatively, the selection of a defined number of isolates representing the most prevalent
serovarsmightprovidemoreapplicableinformationonAMR.
That approach was further supported with microbiological data from our study demonstrating
strictrelationsofantimicrobialresistanceandSalmonellaserovars.Thechangeinepidemiological
situation, such as emergence or vanishing of a serovar at given geographical location or animal
productionsectorwillsurelyaffectantimicrobialresistanceoftheisolates.Itisnotonlyrelatedto
antimicrobialusageoroveruseandselectivepressure,butreferssimplytospreadofaclonewith
resistance determinants [3,4,5]. Quinolone resistance is the major issue in the most prevalent S.
Enteritidis, S. Virchow and some other serovars. Being chromosomally encoded [6], it does not
217
spreadhorizontally,butonlyvertically.Infectionwithresistantclonemightexplaintheincreaseof
resistance level observed in broiler isolates in 2009. The differences between broiler and layer
isolatesmightresultfromdifferentmanagementpractices(Figure1).Theintroductionandspread
of multidrug resistant clones were presumably responsible for increased resistance in S. Infantis
(Figure 2), and S.Newport (Figure 5). Ciprofloxacin resistance level higher than observed for
nalidixic acid was explained by the emergence of S. Newport carrying qnrS gene [6]. The
emergence of S.Kentucky, including multidrug resistant clone, were discussed elsewhere [4,7].
Previously we have reported the change in S.Typhimurium epidemiology: pentaresistant clone
beingreplacedbymonophasicvariant[3].Sincemonophasicisolateswerelessfrequentlyresistant
toantimicrobialsandfoundmostlyinpigsandcattleandfoodofthereof,thechangewasnotseen
in NSCPs isolates (Figure 4), but due to reduction of the number of pentaresistant isolates, the
resistance in overall S.Typhimurium was declining (Figure 3). S.Mbandaka was introduced into
poultryproductioninPolandinmid1990ties[8].Sincethen,theserovarhasbeenrankedwithin
thetopfrequentnotedinthecountry,althoughtheresistanceremainslow.
Calculated pvalue proved to be efficient and understandable mean for evaluation of resistance
trend [2]. They showed tremendous dynamics in current epidemiological situation and the
conclusion drawn are coherent with epidemiological typing data on clonal spread of Salmonella.
Even if prevalence of resistance is low and seems to be stable (i.e. florfenicol, 1,9 5,5% of all
Salmonella tested) the regression analysis can pinpoint MIC values shifts within both susceptible
(WT)andresistant(NWT)population.
ACKNOWLEDGEMENTS
ukasz Bocian (Department of Epidemiology, NVRI) is thanked for performing the regression
analysisusedincurrentstudy.
REFERENCES
1. Bronzwaer S, Aarestrup F, Battisti A, Bengtsson B, Piriz Duran S, Emborg HD, et al. Harmonised
monitoring of antimicrobial resistance in Salmonella and Campylobacter isolates from food animals in the
EuropeanUnion.ClinMicrobiolInfect.2008;14(6):52233.
2. EFSA. Technical specifications for the analysis and reporting of data on antimicrobial resistance in the
EuropeanUnionSummaryReport.EFSAJournal.2012;10(2(2587)):153.
3. Wasyl D, Hoszowski A. Occurrence and characterization of monophasic Salmonella enterica serovar
Typhimurium(1,4,[5],12:i:)ofnonhumanorigininPoland.FoodbornePathogDis.2012;9(11):103743.
4. Wasyl D, Hoszowski A. First isolation of ESBLproducing Salmonella and emergence of multiresistant
SalmonellaKentuckyinturkeyinPoland.FoodResInt.2012;45(2):95861.
5. Wasyl D, Zajc M, DomanskaBlicharz K, Hoszowski A. Ad hoc study on Salmonella Stanley outbreak
strains. Proceedings of the International Symphosium on Salmonella and Salmonellosis; 2013; Saint Malo,
France.
6.WasylD,ZajcM,HoszowskiA.Prevalenceandcharacterisationofquinoloneresistancemechanismsin
Salmonella isolated in Poland. Proceedings of the International Symphosium on Salmonella and
Salmonellosis;2013;SaintMalo,France.
7. Zajc M, Wasyl D, Hoszowski A, Szulowski K. Genotypic diversity of Salmonella Kentucky isolated from
reptiles, poultry food, feed and environmental samples in Poland. Proceedings of the International
SymposiumonSalmonellaandSalmonellosis;2013;SaintMalo,France.
8. Hoszowski A, Wasyl D. Typing of Salmonella enterica subsp. enterica serovar Mbandaka isolates. Vet
Microbiol.2001;80(2):13948.

218
Table1.Antimicrobialdilutionrange(whitebackground),interpretationcriteria(verticallines),MIC
distributionandprevalenceofmicrobialresistance(%NWT)
Antimicrobial
NumberofisolatesperMIC(mg/L)
%NWT
0
,
0
0
8
0
,
0
1
6
0
,
0
3
2
0
,
0
6
4
0
,
1
2
5
0
,
2
5
0
,
5
1
6
3
2
6
4
1
2
8
2
5
6
5
1
2
1
0
2
4
>
1
0
2
4
Ampicillin(AMP) 1257 1590 302 11 2 1 2 630 16,7%

Ceftazidime(CAZ) 2695 1006 75 3 2 4 2 8 0,4%
Cefotaxime(CTX) 1703 1812 236 29 2 1 12 0,4%
Gentamicin(GEN) 454 1774 1321 133 7 10 77 19 3,0%
Kanamycin(KAN) 3660 81 3 1 3 7 40 3,6%
Streptomycin(STR) 234 1038 504 1041 334 295 178 171 25,8%
Nalidixicacid(NAL) 2260 117 53 7 53 1305 36,0%
Ciprofloxacin(CIP) 45 920 1277 49 141 860 246 140 18 8 57 34 39,6%
Sulfamethoxazole(SMX) 75 134 941 1567 360 43 3 19 653 17,8%
Trimethoprim(TMP) 3634 58 6 1 3 93 2,6%
Colistin(COL) 2774 114 5 4,1%
Chloramphenicol(CHL) 502 2642 462 28 1 9 151 4,2%
Florfenicol(FLR) 1097 2327 212 45 70 17 27 3,0%
Tetracycline(TET) 2594 502 26 3 38 166 181 285 17,7%
Figure 1. Quinolone resistance in S. Enteritidis (N=1340), including layers (N=414) and broiler (N=443)
isolates

Figure2.IncreaseinantimicrobialresistanceinS.InfantisisolatedfromNSCPs(N=191)

219
Figure3.DecreaseofantimicrobialresistanceinS.Typhimurium(N=301)
Figure4.StableantimicrobialresistanceinS.TyphimuriumisolatedfromNSCPs(N=86)
Figure5.IncreaseinantimicrobialresistanceinS.Newport(N=173)

220
May 27-28-29, 2013
Saint-Malo France

Session 4

Chairpersons:

Posters
221
InvestigationofSalmonellainpigslaughterhousesinSouthernBrazil
andcharacterizationofphenotypicandgenotypicantimicrobialresistance
oftheisolates
GracielaVOLZLOPES
1,2
,CarolinePISSETTI
2
,GeovanaBRENNERMICHAEL
1
,StefanSCHWARZ
1
andMarisaCARDOSO
2
1
FriedrichLoefflerInstitut(FLI),InstitutfrNutztiergenetik,NEUSTADTMARIENSEE,Germany;
2
UniversidadeFederaldoRioGrandedoSul(UFRGS),DepartamentodeMedicinaVeterinriaPreventiva,
PORTOALEGRE,Brazil
INTRODUCTION
Salmonella is the most common cause of foodborne illness and pork has been reported as an
importantvehicleforthedisseminationofthispathogen.Theaimsofthisstudyweretodetermine
the occurrence of Salmonella in slaughterhouses and characterize the phenotypic and genotypic
antimicrobialresistanceoftheisolates.
MATERIALANDMETHODS
Atotalof270sampleswascollectedinthreeslaughterhousesinSouthernBrazil,eachofthemwas
sampled twice, during the period of June 2010 and March 2011. Faecal samples from lairage
(n=18) and carcasses in the precooling stage (n=252) were subjected to Salmonella isolation
according to the ISO6579 protocol. A single isolate from each positive sample was used for
antimicrobial susceptibility testing by agar disk diffusion according to CLSI recommendations.
Multiresistant isolates were investigated for the presence of antimicrobial resistance genes and
class1integronsbyPCRassays.
RESULTS
Salmonella enterica subsp. enterica (S.) was found in 83.3% (15/18) of the lairage samples and
26.6% (67/252) of the carcasses. Among the 82 Salmonella isolates tested twelve serovars were
identified with Typhimurium and Derby as the most common serovars. Twelve isolates (14.6%)
were susceptible to all antimicrobial agents tested and 33 (40.2%) were multiresistant. Thirteen
antimicrobialresistancepatternswereobservedwiththetrimethoprimsulfonamidetetracycline
ampicillinnalidixic acid resistance pattern being the most frequent pattern. The following
resistance genes were detected: sul1, sul2, and sul3 (sulfonamides), aadA variants
(streptomycin/spectinomycin), strA (streptomycin), aphA1 (kanamycin), tet(A) and tet(B)
(tetracycline),bla
TEM
(ampicillin),catA1(chloramphenicol),andfloR(chloramphenicol/florfenicol).
TheintegrasegeneintI1fromclass1integronswasidentifiedin27.3%(9/33)isolates.
CONCLUSIONS
ThehighoccurrenceofSalmonellainthelairageunderlinestheriskofcrosscontaminationinthe
slaughterhouseandthemultiresistantisolatesrepresentanadditionalrisktofoodsafety.

222
Phenotypicandgenotypicprofilesofantimicrobialresistance(AMR)
inSalmonellaspp.isolatesfromfaecesofdifferentcaninepopulations
ofNorthernandCentralItaly
DanielaPASOTTO,MicheleDRIGO,GiorgiaDOTTO,AntonioFRANGIPANEdiREGALBONO,Alessandra
MONDINandMarcoMARTINI
UniversityofPadua,DepartmentofAnimalMedicine,ProductionandHealth,vialedellUniversit,16,
Legnaro,PADUA,Italy
INTRODUCTION
Antimicrobialresistance(AMR)isaverycomplexissueworldwideinvolvingvariousbacterialspecies,
resistancemechanismsandreservoir.
The number of companion animals, especially dogs and cats, has substantially increased in the
modern societies together with the attention devoted to pet health and welfare. Because of these
changes, antimicrobials are frequently used in small animals. These molecules often belong to the
same classes used in human medicine, especially broadspectrum agents such as aminopenicillins,
cephalosporinsandfluoroquinolones.
This veterinary practice contributes to the diffusion of antimicrobial resistance and poses risks to
humans because of transmission of zoonotic and commensal resistant bacteria through different
pathwayssuchasenvironmentalexposureanddirectanimalcontact[4].
Duetothepotentiallyrelevantimpactonpublichealthofdogsasreservoirofzoonoticagentsandof
antimicrobialresistancegenes,thisstudywasaimedto:
a) Evaluate the occurrence of Salmonella spp. in canine faecal samples collected from different
populations(owned/kenneldogs)andgreenpublicgrounds;
b) DeterminethephenotypicAMRprofiles;
c) StudythegeneticmechanismsofAMRamongstrains,investigatingthepresenceofintegronsand
geneslocatedonplasmids(plasmidmediatedquinoloneresistance,PMQR)and/orofchromosomal
pointmutationsinthequinoloneresistancedeterminingregions(QRDRs)ofgyrases(gyrAandgyrB)
and/oroftopoisomeraseIV(parCandparE)conferringalowsensitivityorresistancetoquinolones.
MATERIALSANDMETHODS
Isolates
From March 2012 to September 2012, fresh faecal samples of 511 dogs (237 collected from
practitionersintheirclinics,193fromkennelsand81frompublicrecreationalareas)weretestedto
detect the presence of Salmonella spp. using the classical bacteriological culture procedure.
Salmonellaisolateswereconfirmed,serotypedandphagotypedbytheOIEReferenceLaboratoryfor
Salmonellosis(IZSVe,Legnaro,Italy).
Antimicrobialsusceptibilitytesting
Antimicrobialsusceptibilityofisolateswasperformedbytheagardiskdiffusionmethodaccordingto
the Clinical and Laboratory Standards Institute [3]. The antimicrobial agents tested were: colistin
(CT10g),sulphamethoxazole/trimethoprim(SXT25+75g),kanamycin(K30g),gentamycin(CN10
g), neomycin (N 30g), cefotaxime (CTX 30g), ceftazidime (CAZ 30g) amoxicillin/clavulanic acid
(AMC 30g), nalidixic acid (NA 30g), tetracycline (TE 30g), ampicillin (AM 10g), streptomycin (S
10g), sulphonamide compounds (SSS 0,25g), chloramphenicol (C 30g), cephalothin (CF 30g),
enrofloxacin(ENR5g),ciprofloxacin(CIP5g)andmarbofloxacin(MAR5g).
223
AllSalmonellaisolateswerescreenedforthepresenceofclass1andclass2integrons,PMQRgenes
(qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA and oqxB) and plasmids as previously described [125].
PointmutationsingyrAwereinvestigatedbyPCRandsequenceanalysis[5].
RESULTS
Salmonellaspp.wereisolatedfrom14(2,7%)faecalsamples,collectedfromownedandkenneldogs(3
and 9 strains, respectively), and green public areas (2 isolates). Serotyping of Salmonella isolates
showed the presence of S. Napoli in the 9 samples collected from kennels and S. Bredeney in the
environmentalfaeces,whilethe3onesisolatedfromprivateowneddogswereS.Abony,S.Veneziana
and a monophasic variant of S. Typhimurium (phagotype DT7) respectively. None of the isolates
showedfullsusceptibilitytoallthedrugstested.Absenceofsusceptibilityrangedfromaminimumof1
drugtoamaximumof14drugs.AlltheisolateswereresistanttoNandSandallbutonetoKandSSS.
AbouttwothirdsofthestrainsshowedresistanceoralowsusceptibilitytoNA,ENRandonethirdof
the isolates to AM, C, CF, CAZ, CTX and CIP. SXT, MAR and AMC acid were the most effective
antimicrobialswithmorethan93%oftheisolatessusceptible.
Molecular analyses showed the absence of PMQR determinants and class 1 and class 2 integrons.
However,themonophasicvariantofS.Typhimurium,theonlystrainshowingacompleteresistanceto
NAtogetherwithalowsusceptibilitytoENR,showedamutationinaminoacidposition87(AsptoAsn)
in gyrA. Additionally, molecular typing of its plasmid content by PCRbased replicon typing (PBRT),
showedthepresenceof2plasmidtypes,IncPandIncI1.
DISCUSSIONANDCONCLUSIONS
Inthisstudy,theoccurrenceofSalmonellaspp.recoveredfromdogsinItalyandtheirphenotypicand
genotypicAMRpatternswereexamined.Theresults,inaccordancewithotherstudies,showedalow
prevalence, of Salmonella spp. in healthy dogs and suggest that the close contact between humans
and pets can lead to the exchange of pathogenic bacteria, including those carrying AMR. Moreover,
therelevantpercentageofAMRprofilesdetectedinthisstudysuggeststhattheuseofantimicrobial
agentsintheveterinarypracticepromotestheselectionandspreadofdrugresistantstrains.Therapid
development and spread of resistance especially to sulphonamides and aminoglycosides are often
attributetothehorizontaltransferamongbacteriaofgeneticelementssuchasintegrons.
On the other hand, the rapid diffusion of fluoroquinolone resistance has been ascribed to PMQR
determinants, since they canconferlowlevelresistance to quinolonesand promote the selection of
highlevelresistantstrainswithmutationsonthechromosomeinQRDRofgyrA,gyrB,parCandparE
genes.However,theresultsofthisstudyshowedtheabsenceofthesetransmissiblegeneticstructures
anddeterminantsinSalmonellastrainsofpetoriginandonlyonestrain,themonophasicvariantofS.
Typhimurium isolated from a private owned dog, showed a high resistance to NA and a single
chromosomemutationinQRDRingyrAgene.
Thismutationisresponsibleforanaminoacidsubstitutioninpositionof87conferingresistancetofirst
generations quinolones. Further investigations on the plasmid content of this strain showed the
presenceof2IncI1andIncPplasmidtypes.IncI1usuallycarriesAMRdeterminants,especiallyPMQR
genes and those codifying for ESBLs, and is limited to Enterobacteriaceae. Whereas, plasmids
belonging to the IncP incompatibility group are broadhostrange plasmids, widely described among
several genus of bacteria generally associated with AMR determinants. This finding suggests that
further spread of AMR to other strains, both commensal and pathogens, could be enhanced by this
genetic elements, since genes linked to IncP plasmids have a large host range, including
EnterobacteriaceaeandPseudomonasspp.andevengrampositivebacteria.
ThefindingofemergingAMRmechanismsinpathogensfrompetsisofgreatvalueandclaimsfurther
surveillancestudiesandepidemiologicaltracingofthepotentialanimalreservoirofrelevantresistance
determinants.
DetectionofAMRdeterminantsandplasmidtyping
224
REFERENCES
1. JMicrobiolMethods.2005;63:21928.
2. AntimicrobAgentsChemother.2012June;56(6):342327.
3. CLSI.2006;M100S17Vol.27No.1;replacesM100S16,Vol.26No.3.
4. JAntimicrobChemother.2012;67(1):17481;5.JClinMicrobiol.2012;50(8):263138.
225
Salmonella spp. prevalence and antimicrobial resistance patterns
(phenotypic profiles and presence of genetic AMR determinants)
of serotypes isolated from pet reptiles in Northern Italy

Daniela PASOTTO, Giorgia DOTTO, Emanuela SCARSATO,
Alessandra MONDIN and Marco MARTINI
University of Padua, Department of Animal Medicine, Production and Health,
viale dellUniversit, 16, Legnaro, PADUA, Italy

INTRODUCTION
In the last decades, the interest in exotic animals, especially reptiles, as pet animals has greatly
increased. Salmonella spp. is known to be associated with free-living and captive reptiles and
usually has been detected at high prevalence rates. Salmonella infection and associated
antimicrobial resistance (AMR), especially multidrug resistance (MDR), represent a crucial public
health issue and reptiles seem to play an important role in their transmission to humans [4]. The
diffusion of AMR is of global concern and has been attributed to the indiscriminate use of
antimicrobials both in humans and animals.
Many studies have highlighted the crucial role played by the horizontal transfer of genetic
elements, such as integrons, in the acquisition and spread of MDR in Salmonella and in other
gram-negative bacteria. These structures, especially class 1 integrons, are largely widespread
among the Typhimurium serotype. Additionally, their diffusion is increasingly found in other
emerging serotypes (such as S. Newport, S. Kentucky and S. Virchow) and in non-Typhimurium
Salmonella isolates of different geographical origins [2-3]. This study is aimed to: a) evaluate the
occurrence of Salmonella spp. in apparently healthy reptiles; b) determine the phenotypic and
genotypic AMR profiles.

A total of 324 cloacal swabs were collected from reptiles kept at pet animals import centres (48
samples), pet shops (103 samples), zoological parks (71 samples) and private owners (102
samples) located in Northern Italy. Animals were sampled once. The isolation of Salmonella spp.
was performed according to classical bacteriological culture procedure. Genus confirmation of the
suspected positive isolates was obtained using biochemical macro-method characterization tests.
All the isolates were serotyped by the OIE Reference laboratory for salmonellosis (IZSVe Legnaro,
Italy). The evaluation of susceptibility of Salmonella isolates to different classes of antimicrobials
was performed using the agar disc diffusion method, according to the specifications of the Clinical
and Laboratory Standards Institute [1]. The antimicrobial agents tested were: colistin (CT 10 g),
sulphamethoxazole /trimethoprim (SXT 25+75 g), kanamycin (K 30 g), gentamycin (CN 10 g),
neomycin (N 30 g), cefotaxime (CTX 30 g), ceftazidime (CAZ 30 g), amoxicillin/clavulanic acid
(AMC 30 g), nalidixic acid (NA 30 g), tetracycline (TE 30 g), ampicillin (AM 10 g), streptomycin
(S 10 g), sulphonamide compounds (SSS 0,25 g), chloramphenicol (C 30 g), cephalothin (CF 30
g), enrofloxacin (ENR 5 g), ciprofloxacin (CIP 5 g) and marbofloxacin (MAR 5 g).
The isolates were also screened for the presence of class 1 and class 2 integrons and AMR genes
(tetA, tetC, floR, cmlA, strA, strB, aadA, sulI, sulII and ampC) as previously described [5].
Characterization of integrons was investigated by sequencing their genic content.

RESULTS
Samples were collected from apparently healthy snakes (n = 147), lizards (n = 85) and turtles (n =
92). Salmonella spp. was isolated from 205 (63.3%) cloacal swabs. Prevalence data differed
significantly (
2
=55.91; P<0.001) among snakes (76.9%), lizards (74.1%) and turtles (31.5%).
Salmonella strains were isolated from pet animals of import centres (62,5%), pet shops (62%),
226
zoological park (67,6%) and private owners (52,5%) Serotyping showed the presence of S. enterica
subsp. enterica (53.2%), S. enterica subsp. diarizonae (29.8%), S. enterica subsp. houtenae (10.2%),
S. enterica subsp. salamae (6.3%) and S. enterica subsp. arizonae (0.5%). Among the 109 S.
enterica subsp. enterica strains, 40 different serotypes were identified, including S. Typhimurium,
S. Enteritidis, S. Kentucky, S. Newport, S. Virchow and S. Infantis.
These are part of the 10 most common serovars isolated from human cases of salmonellosis in UE
in 2010-2011 [3].
Out of 205 isolates, only 12 (5.8%) showed full susceptibility to all the drugs tested and 95 (46.3%)
showed MDR, equally distributed among the Salmonella subspecies isolated and the animal origin.
Absence of susceptibility concerned a minimum of 1 drug (61 isolates) to a maximum of 16 drugs
(3 isolates). Most of the isolates showed resistance to streptomycin (34.1%), tetracycline (54%),
triple sulfas (59%) and neomycin (64.4%).
The most effective antimicrobials were: cephalothin (92.2% susceptible isolates), nalidixic acid
(79.5%), enrofloxacin (83.4%), ciprofloxacin (89.7%), marbofloxacin (99%), colistin(88.3%),
sulphamethoxazole /trimethoprim (96.6%), gentamicin (90.7%), kanamycin (86.8%), ampicillina
(86.8 %), amoxicillin/clavulanic acid (91.7%), cefotaxime (87.3%), ceftazidime (100%), and
chloramphenicol (81%). None but one of the isolates carried integrons.
Only a S. Typhimurium strain, isolated from a snake, was identified as having a class 1 integron
carrying aadA2 gene that confers resistance to streptomycin.
Additionally, molecular analysis of its genetic AMR content showed the presence of sulI and floR
genes conferring resistance to sulphonamides and phenicols. These data confirmed the
phenotypic AMR profile.
DISCUSSION AND CONCLUSIONS
In accordance with other studies, our results showed a high prevalence of Salmonella spp. in
apparently healthy pet reptiles, confirming that these animals could act as reservoir of zoonotic
microorganisms, including those carrying AMR/MDR.
Additionally, most of the isolates belonged to S. enterica subsp. enterica. This subspecie is usually
detected in worm-blooded animals. Interestingly, out of the 40 different serotypes identified,
there were the 10 most widespread Salmonella serovars isolated from clinical human cases of
infection in UE (S. Typhimurium, S. Enteritidis, S. Kentucky, S. Newport, S. Virchow and S. Infantis).
The high percentage of AMR and/or MDR profiles detected suggests that pets may act as source of
antimicrobial resistant strains which could be transmitted to humans and spread into the
environment.
The emergence and diffusion of resistance, especially MDR, are often attribute to the horizontal
AMR gene transfer among bacteria, usually associated with integrons.
However, in this study only one strain, the MDR S. Typhimurium isolated from a private owned
snake, carried an integron and AMR genes conferring resistance to aminoglycosides,
sulphonamides and phenicols.
In conclusion, this study highlights the potential risk for public health represented by pet reptiles,
which are increasing in the modern society.
Further investigations are needed to better elucidate the occurrence of AMR among different pet
reptile populations.
REFERENCES
1. CLSI, 2006. M100-S17-Vol. 27 No. 1; replaces M100-S16, Vol. 26 No. 3.
2. J Appl Microbiol 2012; 112(6):1113-22.
3. EFSA Journal, 2012: 10:2597.
4. Res Vet Sci, 2012: 92(2):187-90.
5. Int J Antimicrob Agents, 2007: 29(3):254-62.
227
MECHANISMSOFANTIMICROBIALRESISTANCEINBIOFILMASSOCIATED
SALMONELLATYPHIMURIUM
DovSCHLISSELBERGandSimaYARON
Technion,FacultyofBiotechnologyandFoodEngineering,LaboratoryforMolecularBiologyof
Pathogens,HAIFA,Israel
INTRODUCTION
Salmonella is capable of forming biofilms on a variety of biotic and abiotic surfaces which are
almost impossible to eradicate from the product using conventional antimicrobials. Several
mechanisms may explain the resistance phenotype of the biofilm. One of them is the hypothesis
thatbiofilmcellsexpressspecificprotectivefactorstoimprovetolerance.
To date, most of the methods for targeting response mechanisms to antimicrobials depict the
medianoftheproteinsormRNAlevelsintheentirepopulation.Inbiofilm,however,themedianis
based on sum of the expression in many small heterogeneous environments. We propose that
response mechanisms of clusters of cells in the biofilm may be a key factor resulting in a
phenotypeofanelevatedresistance.
MATERIALANDMETHODS
WeinvestigatedtheexpressionoftheentiregenomeofSalmonellaentericaserovarTyphimurium
(STM)inbiofilminresponsetociprofloxacin(CIP)onasinglecellresolutionusinghighthroughput
methods. Screening and identification of genes activated by antimicrobials in biofilms was
performed with the modified Differential Fluorescence Induction (DFI) approach. This method is
basicallyanenrichmentstrategywhichusessmallrandomfragmentsofSTMgenomicDNAcloned
upstream to a promoterless gfp gene. The promoter activity is monitored and sorted with a
fluorescenceactivated cell sorter (FACS). The result of this method is a pool of activated
promoters.
RESULTS
We sequenced this pool using 454pyroseqeuencing and identified about 50 possible promoters
which are induced in the biofilm following exposure to CIP. In silico
analysis showed that the induced genes encode for proteins such as stress regulators, ABC
exporters,membraneproteins,secretedeffectorproteinsandhypotheticalproteins.
Next, we chose six of those suspected promoters to investigate their impact on the higher
resistance biofilm phenotype. In order to validate promoter upregulation during CIP treatment
wemonitoredthebiofilmcellscarryingthepromotersgfpfusionsonasinglecellresolutionusing
flowcytometry. For example, we found and increase of 9.3% of induced cells with the blc (a
lipoprotein)promoterfollowing6hrofCIPtreatment(seefigure1).Wealsorevealedthatoutof
theotherfivepromoterstested;fourshowedanincreaseof1.2%7.4%ofinducedpositivecellsof
inthebiofilm.
DISCUSSION
We developed a highthroughput method to identify upregulated promoters inside the
heterogenic biofilm community on the entire STM genome. Currently we are examining the
influenceonSTMbiofilmCIPresistanceofnullmutationsofthesixgeneschosen.

228
GFP-A
C
o
u
n
t
-10
2
10
2
10
3
10
4
10
5
0
18
35
53
70
pos
Figure 1: Fluorescence histogram of biofilmassociated Salmonella containing the blc promoter fused to a
gfp gene. Black histogram biofilm associated cells without any induction. Red histogram biofilm
associated cells following 6 hr ciprofloxacin induction. Bar represents induced cells (30% in the treated
biofilmand18%inthecontrol).
229
Monitoring3
rd
generationcephalosporinresistance
inFrenchnonhumanSalmonella:20102012overview
SophieA.GRANIER,CorinneDANAN,SylvineFREMY,JolGROUT,ClaudeOUDART,ChristinePiquet,
CatherineLAPORTE,VivianeMOREL,RenaudLAILLERandAnneBRISABOIS
Anses,LaboratoryforFoodSafety,BacterialCharacterizationandEpidemiologyUnit,
MAISONSALFORT,France
Antimicrobial resistance of Salmonella isolated from the agrofood sector has been monitored in
France by Anses (formerly AFSSA) for now more than 20 years. With regards to its Public health
importance,thissurveillanceespeciallyfocusesonthirdgenerationcephalosporin(3GC)resistance
detection.
Eachyear,approximately3000nonduplicateisolatesaretestedfortheirantimicrobialresistance
phenotypebydiskdiffusionmethod.
Afterdetectionofapotential3GCresistancephenotype,theresistancemechanismisassessedby
theuseofcommercialDNAmicroarrays(CheckMDRCT101,CheckPoints,TheNetherlands).
Thismicroarraycandetectbla
KPC
,bla
FOX
,bla
DHA
,bla
MOX
,bla
ACT
,bla
AAC
,bla
MIR,
groupsofbla
CTXM
and
delineatesESBLfromnonESBLforbla
TEM
andbla
SHV
.
Duringthis3yearperiod,thenumberof3GCdetecteddidnotsignificantlyincrease(Table1).
Table1:3GCResistantSalmonellaspp.detectedbytheFrenchSalmonellaNetwork
Year Numberof
Antibiograms
Numberof1GC
Rstrains
ESBLand/orcephalosporinasepositivestrains
2010 3312 64 33
2011 3289 38 20
2012 3380 26 7
Legend:1GC=1stgenerationcephalosporin;R=resistant
Figure 1: Proportions of animal species providing 3GC resistant Salmonella spp. to the French Salmonella
Network,20102012
Nevertheless, a shift in the lactamase genes encountered can be noticed. bla

CMY2
, the only
pAmpC gene identified, was first detected in the French agrofood sector in 2009. Since then, it
has been repeatedly encountered over the past 3 years, combined or not with an ESBL encoding
gene(Figure2).
Serotypes as well as geographic distributions of 3GC resistant Salmonella do not follow any
scheme.
21%
32%
44%
1% 2%
Miscellaneous
Poultry
Horses
Pigs
Sheep
230
Figure 2: Repartition of ESBL and plasmidic AmpC (pAmpC) detected through the French Salmonella
Network,20102012
If bla
CMY2
remains the only pAmpC gene identified, diversity is observed in genes responsible for
ESBLphenotypes(Figure3).bla
CTXM1group
isthe
mostfrequentlydetected.
Figure3:diversityofESBLgenesdetectedthroughtheFrenchSalmonellaNetwork,20102012
Overall, detection of 3CG resistant Salmonella in the French agrofood sector remains still a rare
event.Constantmonitoringof3GCresistantSalmonellaappearsnowtobeessentialtofollowthe
circulationof3GCresistantstrains.
ACKNOWLEDGEMENTS
Authors would like to thank the laboratories participating to the French Salmonella Network for
providingstrainsalongwiththerelevantepidemiologicalinformation.
55%
15%
30%
ESBL pAmpC ESBL+pAmpC
66%
14%
2%
8%
10%
CTXM1group
CTXM2group
CTXM9group
TEMESBL
SHVESBL
231
Emergence of the ASSuT Multidrug Resistant Profile
in Salmonella enterica serotype 4,[5],12:i:- in Canada
Michael MULVEY, Greg HORSMAN, Rita FINLEY, Melissa MCCRACKEN, Stacie LANGNER,
Vanessa ALLEN, Lei ANG, Sadjia BEKAL, Sameh EL BAILEY, David HALDANE and Linda HOANG
Public Health Agency of Canada, 1015 Arlington St., R3E 3R2 WINNIPE,G Canada
Abstract:
Objective: Over the last decade the monophasic Salmonella enterica 4,[5],12:i:- has become
one of the most prevalent Salmonella serotypes in Canada. We report on the emergence of
multidrug resistance in this serotype.
Methods: Human isolates of S. 4,[5],12:i:- were identified by provincial laboratories from
2003-2010 as part of the Canadian Integrated Program for Antimicrobial Resistance
Surveillance (CIPARS). MIC values were determined by the broth microdilution using the
Sensititre. Serotyping and phage typing were performed by standardized methodologies.
PCR was used to determine the presence of ampicillin (A), streptomycin (S), sulphonamide
(Su), and tetracycline (T) resistance genes. Multilocus sequence typing was performed on a
selected number of isolates.
Results: A total of 25,310 Salmonella were collected as part of the CIPARS program over the
study period. Of those isolates, 767 (3.0%) S. 4,[5],12:i:- were identified and the prevalence
increased from 1.5% (n=46) in 2003 to 4.8% (n=164) in 2010. PT 191/191a was the most
common phage type observed (n=322; 42.0%). The ASSuT phenotype was observed in 12.0%
(n=92) of S. 4,[5],12:i:- isolates and it increased from 2 isolates in 2003 to 35 isolates in 2010.
MLST conducted on a sample of the ASSuT isolates (n=24; 37%) revealed all were ST34. PCR
confirmed the presence of blaTEM1/2, strA-strB, tet(B), and sul2 in all ASSuT isolates.
Conclusions: S. 4,[5],12:i:- has steadily increased from 2003 to 2010 and is now the fifth
most common serotype reported in Canada. The presence of ST34 taken together with the
ASSuT resistance genes suggests the presence of a genomic island similar to isolates
reported in Europe.
232
The Emergence of Ciprofloxacin-Resistant Salmonella enterica
Serovar Kentucky, in Canada
MICHAEL MULVEY, David BOYD, Rita FINLEY, Ken FAKHARUDDIN, Stacie LANGNER, Vanessa
ALLEN, Lei ANG, Sadjia BEKAL, Sameh EL BAILEY, David HALDANE and Linda HOANG ,
Public Health Agency of Canada, 1015 Arlington St., R3E 3R2 WINNIPE,G Canada
Abstract:
Objective: We describe the emergence of ciprofloxacin-resistant (Cip-R) Salmonella enterica
serovar Kentucky from human cases in Canada from 2003-2011.
Methods: From 2003 to 2011, provincial public health laboratories submitted human clinical
isolates as part of the Canadian Integrated Program on Antibiotic Resistance Surveillance
(CIPARS). MIC values were determined by broth microdilution using the Sensititre. PFGE
was performed on all Cip-R isolates and MLST was conducted on a selected number of
isolates. PCR was used to determine the presence of Salmonella Genomic Island1 variants.
Results: A total of 90 S. Kentucky isolates were identified out of 26,600 non-typhoidal
Salmonella human isolates submitted for susceptibility testing to CIPARS. S. Kentucky
comprised 67% (32/48) of all Cip-R isolates identified since 2003. Most Cip-R isolates
displayed a core resistance phenotype of ampicillin, gentamicin, nalidixic acid, sulfonamide
and tetracycline resistance (n=22; 78%). PFGE analysis revealed a majority of Cip-R isolates
and one multidrug resistant (MDR) ciprofloxacin susceptible (Cip-S) isolate clustered
together with a percent similarity of >80% (pattern A), whereas three other MDR Cip-S
resistant isolates did not belong to this cluster (patterns B and C). MLST of isolates of PFGE
pattern A were ST198. Isolates of PFGE pattern A were found to contain SGI1-K, SGI1-Q, and
SGI1-P. Novel SGI1 variants were also identified. CIPARS animal and retail meat surveillance
has not identified Cip-R. Eleven cases had travel history: Morocco (4), Egypt (3), Africa (3)
and Libya (1).
Conclusion: Cip-R ST198 MDR isolates have been described in Europe and Africa. The data
strongly suggests Cip-R isolates are a result of travel to countries where resistance is
endemic. Travel history should be collected in prospective studies to determine this
hypothesis.
233
Effect of ramR mutations on efflux genes expression
and on fluoroquinolone susceptibility
in Salmonella enterica serotype Kentucky ST198
Axel CLOECKAERT
1
, Sylvie BAUCHERON
1
, Simon LE HELLO
2
, Benoit DOUBLET
1
,
Etienne GIRAUD
1
and Franois-Xavier WEILL
2

1
INRA, UMR1282 Infectiologie et Sant Publique, 37380 NOUZILLY France;
2
Institut Pasteur, Unit des Bactries Pathognes Entriques 28 rue du Docteur Roux,
75724 PARIS France
Abstract:
Efflux is a mechanism that has been previously reported to increase fluoroquinolone (FQ)
resistance levels in clinical isolates of Salmonella enterica mainly of serotype Typhimurium.
In this study efflux genes were investigated in the emerging FQ-resistant epidemic S. enterica
serotype Kentucky ST198 clone.
Among a representative panel of thirty serotype Kentucky strains from east Africa with
decreased FQ susceptibility, three strains overproducing the AcrAB-TolC efflux system were
detected and studied. Genetic relatedness was determined by XbaI-pulsed field gel
electrophoresis and multilocus sequence typing. The ramRA, soxRS, marOR loci and acrR,
acrS genes were sequenced. Wild type strains, ramR mutants and ramR mutants
complemented with a wild-type ramR gene were analysed by qRT-PCR for gene expression
of regulatory and efflux genes and by MIC determinations of quinolones, FQ and florfenicol
as other substrate of AcrAB-TolC.
All Kentucky strains studied were X1-ST198, excepted the susceptible reference strain 98K
which was X4-ST198. The three strains overproducing AcrAB-TolC presented three different
mutations in the ramR gene in comparison to two FQ-resistant strains and the sucsceptible
strain with a basal expression level of AcrAB-TolC. All other efflux regulatory genes were not
affected. The three detected mutations (deletion of 91 bp, insertion of 1 bp or 4 bp) resulted
in frame shift of the ramR gene. As confirmed by complementation, all three mutations were
responsible for increased expression of ramA and acrAB. Increased expression of tolC and
acrEF genes was observed in 2 out of the 3 strains. All three mutations were shown to
increase two-fold the MICs of FQ and florfenicol. Various novel ramR mutations, responsible
for increased efflux, were detected in the emerging epidemic serotype Kentucky ST198
clone. However, ramR mutations seem to be sporadic as previously reported for other FQ-
resistant strains and contribute only to a little extent to the decreased FQ susceptibility.
234
Plasmidmediatedquinoloneresistance
indomesticandimportedfood
andinfoodproductionanimalsinFinland,20032010
HenryKURONEN
1
,KaroliinaJUNTTILA
1
,SuviNYKSENOJA
1
,TarjaPOHJANVIRTA
1
andSinikkaPELKONEN
1
1
Evira,FinnishFoodSafetyAuthority,VeterinaryBacteriologyandMicrobiologyResearchUnitsFinland
INTRODUCTION
Plasmidmediatedquinoloneresistance(PMQR)inGramnegativebacteriaisincreasingworldwide
andhasalsobeenobservedsince2003inFinnishtravelersreturningbackhome(3,6).ThePMQR
isolatestypicallyexpressnonclassicalquinoloneresistancephenotypewithreducedsusceptibility
(MIC 0.122 g/ml) to ciprofloxacin, but are either susceptible or show only lowlevel resistance
(MIC32g/ml)tonalidixicacid(2,3).ToevaluatetheriskofPMQRinFinland,weanalyzedthe
presenceofPMQRinsalmonellaisolatesfromtheFinnishfoodchainandimportedfoodstuffs.
MATERIALANDMETHODS
Salmonella isolates included in the study were collected during 20032010 in the National
salmonella reference laboratory. The study included isolates from domestic food production
animals,representingallthesalmonellapositivefarmsdetectedintheFinnishSalmonellaControl
Program (7), and all isolates obtained from domestic and imported food samples for salmonella
serotyping(Table1).
The salmonella isolates were tested for resistance to quinolones using antibiotic disc method
including discs for enrofloxacin/ciprofloxacin (5 g) and nalidixic acid (30 g), and with VetMIC
TM
method to determine MICvalues (1). The isolates with reduced susceptibility to

ciprofloxacin/enrofloxacin(MIC0.122g/ml)wereanalyzedforPMQRgenesqnrA,qnrB,qnrSand
aac(6)IbcrusingPCR(4,5).
RESULTS
Altogether 103 isolates showed reduced susceptibility (MIC 0.122 g/ml) to ciprofloxacin or
enrofloxacin (Table 1) and were analyzed for PMQR genes. Four of these isolates were from
domesticcattlefarms,theremainingonesfromforeignfoodstuffs.
Most isolates (88.5 %) showed reduced susceptibility both to nalidixic acid and
ciprofloxacin/enrofloxacin,withMICvalues128g/mltonalidixicacid.Onlytwelveisolates had
the nonclassical quinolone resistant phenotype with reduced susceptibility to
ciprofloxacin/enrofloxacin,butwereeithersusceptibleorshowedonlylowlevelresistance(MIC
32 g/ml) to nalidixic acid. The source of these 12 isolates was either imported broiler or turkey
meat,andtheirSalmonellaserovarsincludedAnatum,Enteritidis,Give,Indiana,Infantis,Saintpaul
andssp.I(4,5,12:i:).
The qnrB gene was detected in four isolates. Three of these had the nonclassical quinolone
resistance phenotype, while one isolate was also resistant to nalidixic acid with MIC value 128
g/ml(Table2).AllfourisolatesfromtheFinnishcattlefarmswerenegativeforPMQRgenes.
DISCUSSION
ResistancetofluoroquinoloneswasoverallrareamongsalmonellaisolatedfromtheFinnishfood
production chain. Only four out of 271 (1.5 %) Salmonella isolates had reduced susceptibility to
fluoroquinolones,andalltheseisolateswereresistanttonalidixicacid.Theresistancemechanism
islikelytoresideinthechromosomalmutation,asnoPMQRgenesweredetected.
235
AmongtheSalmonellaisolatesfromimportedfoodstuffs,therewere99/376(26.3%)isolateswith
reduced susceptibility to fluoroquinolones. All of these isolates originated from broiler or turkey
meat and the countries of origin included twelve countries from South America, Far East and
Europe.ThenonclassicalquinoloneresistantphenotypesuggestiveofPMQRwasobservedonlyin
12/99 (12 %) of the isolates with reduced susceptibility to fluoroquinolones. However, a plasmid
mediatedgene,qnrBwasdetectedonlyinthreeoftheseisolates.
REFERENCES
1. CLSI (2008), Performance Standards for Antimicrobial Disk and Dilution Susceptility Tests for Bacteria
IsolatedfromAnimals,ThirdEdition
2. FINRESVet 20072009 Finnish Veterinary Antimicrobial Resistance Monitoring and Consumption of
AntimicrobialAgents,EviraPublications1/2011,5859
3.Gunelletal.2009,AntimicrobAgentsChemother.2009,53:38323836.
4.ParkC.H.,RobicsekA.,JacobyG.A.,SahmD.andHooperD.C.(2006).PrevalenceintheUnitedStatesof
aac(6)lbcr Encoding a CiprofloxacinModifying Enzyme. Antimicrobial Agents and Chemotherapy, Nov.
2006,p.39533955
5. Robicsek A., Strahilevitz J., Sahm D.F., Jacoby G. A. and Hooper D. C. (2006). qnr Prevalence in
CeftazidimeResistant Enterobacteriaceae Isolates from the United States. Antimicrobial Agents and
Chemotherapy,Aug.2006,p.28722874
6. Strahilevitz J., Jacoby G. A., Hooper D. C. and Robicsek A. (2009). PlasmidMediated Quinolone
Resistance:aMultifacetedThreat.ClinicalMicrobiologyReviews,Oct.2009,664689
7.ZoonosesinFinland,in20002010,Salmonellosisp.4257,TheZoonosisCentre2012
TABLE1.Salmonellaisolatesofthestudy,andthosewithreducedsusceptibility(MIC0.122g/ml)to
ciprofloxacin/enrofloxacinselectedforPMQRanalysis.
Sourceofisolate No.of
isolates
No.of
different
serovars
Countryoforigin No.isolatesselected
forPMQRanalysis
(MIC0.122g/ml)
cattle 95 12
Finland
4
pig 55 10
Finland
0
poultry 88 12
Finland
0
beef,porkand
poultrymeat
33 6
Finland
0
broilermeat 136 21
Argentina,Belgium,Brazil,Denmark,
France,Germany,Latvia,Lithuania,
Poland,Thailand
50
turkeymeat 112 21
Brazil,TheNetherlands,France,
Germany,Hungary,Poland
49
otherimportedfood 128 32
19differentcountries
0
Total 647 68
21differentcountries (excl.Finland)
103
Table2.OriginandphenotypiccharacteristicsofqnrBpositiveSalmonellaisolates.
Serovar
(Year)
PMQR
gene
Food/animal Countryof
origin
Cip/Enro
MIC
(g/ml)
Nal
MIC
(g/ml)
Cip/enro
5gdisc
(mm)
Nal30
gdisc
(mm)
No.
isolates
S.Agona
(2006)
qnrB Turkeymeat Brazil >1 >128 15 6 1
S.Anatum
(2009)
qnrB Turkeymeat Brazil 1 32 10 11 1
S.Anatum
(2010)
qnrB Turkeymeat Brazil 0,5 32 17 11 1
S.Give
(2007)
qnrB Turkeymeat Brazil 0,5 32 16 11 1
236
Prevalenceandcharacterisationofquinoloneresistancemechanismsin
SalmonellaisolatedinPoland
DariuszWASYL,MagdalenaZAJCandAndrzejHOSZOWSKI
INTRODUCTION
The introduction of quinolones into clinical use was believed a marvellous measure for infectious
diseasescontrol.Themythofcompoundswithnoknownnaturalresistancewasdepletedshortlyafter,
whenarandomsinglepointmutationinabacteriumunderquinoloneselectivepressurehasresulted
in the occurrence of resistant flora. Chromosomally encoded resistance results only in treatment
failuresandcanbetransferredonlyvertically.Ittookfewdecadestodiscovertransmissiblequinolone
resistancedeterminants.
Quinolones, being considered critically important for human medicine [1], were included in
antimicrobial resistance monitoring in Salmonella. The aim of current study was to assess the
prevalence of quinolone resistance and characterise resistance mechanisms in Salmonella recently
isolatedinPolandfromveterinarysources.
MATERIALANDMETHODS
Minimalinhibitoryconcentration(MIC)ofnalidixicacidandciprofloxacin(Sensititre,TrekD.S.,UK)was
testedin 2680 Salmonella isolated from animals, foodand feedfrom 2008 to 2011. In a selection of
isolates representing ciprofloxacin MIC ranging from 0,125 to 4 mg/L, Quinolone Resistance
Determining Regions (QRDR) of the genes encoding for gyrase subunits A and B (gyrA, gyrB) and
topoisomerase IV subunits C and E (parC, parE) were amplified and sequenced. Plasmid Mediated
Quinolone Resistance (PMQR) determinants (qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac(6)Ib) were
tested in isolates with MIC
Cip
0.125 mg/L and nalidixic acid MIC ranging from 4 to 32 mg/L. The
relevant amplicons were sequenced and intended for Genbank deposition. XbaIPFGE was run
according to the PulseNet protocol to assess genetic similarity on a subset of quinolone resistant
isolates.
RESULTS
Microbiological resistance to nalidixic acid and ciprofloxacin was found in, respectively, 37,7% and
40,8% of isolates. Of 1093 ciprofloxacin nonwild type isolates, 61% showed MIC
Cip
=0,25 and other
valueswererepresentedlessfrequently.TwelveQRDRmutationpatternswereobservedin43isolates
representing12serovars(Table1).Pointmutationsresultingintherelevantaminoacidsubstitutions
havebeenfoundingyrA(Ser83andAsp87)andparC(Thr57,Ala141).Singleirrelevantmutationwas
found in gyrB and nonein parE. Thesame mutations were observed in different serovars, but some
patternsseemedtoberelatedtogivenserovar.
PlasmidMediatedQuinoloneResistancewassuspectedin75(2,8%)isolatesrepresenting8serovars,
mostly Newport (N=44), Typhimurium (N=9) and Enteritidis (N=9). The genes were identified as
qnrS1/S3 (N=44), qnrS2 (N=1), qnrB10/B19 (N=2) or yet unspecified qnrB (N=20). Two isolates were
bothQRDRmutantsandPMQRpositive.PFGErevealedindistinguishableprofiles(datanotshown)in
isolatesharbouringthesamesubstitutionsinQRDRregionsorcarryingthesamePMQRdeterminants
(i.e.S.Newport).Inafewcases(i.e.S.EnteritidisqnrS2gyrAAsp87Tyrmutant)thePFGEprofilewas
sharedwithfluoroquinolonesusceptibleisolates.
DISCUSSION
Alikeotherstudies,quinoloneresistancewasfoundathighprevalence(seecommunication#1323249)
duetothepresenceofvariousresistancedeterminants[2,3].Resistancemechanismsdonotseemto
be serovar related, but some of them might disseminate with clonal spread of isolates harbouring
237
givenresistancemechanism.ItcanbeexemplifiedwithS.NewportclonessharinggyrASer83Tyr,parC
Thr57Sermutationpatternandplasmiddeterminant(qnrS1/S3)(Table1),butalsoS. Stanley(poster
#6879198)orhighlyciprofloxacinresistantS.Kentuckyclone[4,5].
Quinolone resistance in Salmonella is concluded to be a complex and diverse issue resulting from
spontaneous mutations of chromosomal genes, spread of clones already harbouring resistance
determinants,aswellasacquisitionofplasmidresistancemechanismsfromenvironmentalreservoirs.
REFERENCES
1. Collignon P, Powers JH, Chiller TM, AidaraKane A, Aarestrup FM. World Health Organization ranking of
antimicrobialsaccordingtotheirimportanceinhumanmedicine:Acriticalstepfordevelopingriskmanagement
strategiesfortheuseofantimicrobialsinfoodproductionanimals.ClinInfectDis.2009;49(1):13241.
2. Hopkins KL, Davies RH, Threlfall EJ. Mechanisms of quinolone resistance in Escherichia coli and Salmonella:
recentdevelopments.IntJAntimicrobAgents.2005;25(5):35873.
3.VeldmanK,CavacoLM,MeviusD,BattistiA,FrancoA,BotteldoornN,etal.Internationalcollaborativestudy
ontheoccurrenceofplasmidmediatedquinoloneresistanceinSalmonellaentericaandEscherichiacoliisolated
from animals, humans, food and the environment in 13 European countries. J Antimicrob Chemother.
2011;66(6):127886.
4.WasylD,DomanskaBlicharzK,HoszowskiA.QuinoloneresistancemechanismsinSalmonellaKentuckywith
highlevel ciprofloxacin resistance. Proceedings of the 3rd ASM Conference on Antimicrobial Resistance in
ZoonoticBacteriaandFoodbornePathogensinAnimals,HumansandtheEnvironment;20122629June2012;
AixenProvence,France.2012.p.101.
5. Wasyl D, Hoszowski A. First isolation of ESBLproducing Salmonella and emergence of multiresistant
SalmonellaKentuckyinturkeyinPoland.FoodResInt.2012;45(2):95861.

238
Table 1. Gyrase (gyrA) and topoisomerase VI (parC, parE) genes substitutions within Quinolone Resistance
DeterminingRegionbyCiprofloxacinMICvaluesandSalmonellaserovars:SAAdelaide,SAgAgona,SE
Enteritidis, SH Hadar, SI Infantis, SL Lexington, SM Mbandaka, SN Newport, SS Saintpaul, STe
Tennessee, ST Typhimurium, SV Virchow. Plasmidmediated quinolone resistance (qnrS1/3, qnrS2)
presencewasindicated.NorelevantsubstitutionswerefoundinparE.
gyrA* gyrB* parC* NumberofisolatesbyMIC
Cip
(mg/L)
total
Ser83
Asp87 Leu470 Thr57 Ala141 0,125 0,25 0,5 1 2 4

Asn
1

4
(ST3,SS1)
4
Tyr
2

2
(SE,SV)
5
(SE2,SV2,SS1)
1
(SE,
qnrS2)
8
Phe
3

1
(SV)
1
(SV)
2
(SE2)
4
Tyr
4

1
(SE)
3
(SE)
4
Asn
1
Ser
5

1
(SH)
1
(SH)
2
Gly
6
Ser
5

1
(SN)
1
Tyr
2
Ser
5

5
(SH,SA2,STe2)
5
Leu
7
Asn
1

1
(SV)
1
Phe
3
Ser
5
3(SN2,SM) 3
Tyr
4
Ser
5

1
(SN)
3
(SAg1,SI2)
3
(SI2,SN1)
1
(SN)
1
(SN,
qnrS1/3)
9
Ser
5
Ser
8
1
(SL)
1
Tyr
4
Met
9
Ser
5
1(SI) 1
No.oftestedisolates 5 22 8 5 2 1 43
1
AAC;
2
TAC;
3
TTC;
4
TAC;
5
AGC;
6
GGC;
7
TTG;
8
TCG;
9
ATG(irrelevantforquinoloneresistance
239
May 27-28-29, 2013
Saint-Malo France

Session 5

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

240
SalmonellacontrolinfoodanimalsintheEU:successes,threatsandfuturechallenges
RobDAVIES(UnitedKingdom)
ABSTRACT
Salmonella has a long history of research and successful legislative and voluntary control, ranging from the virtual
eradication policy of the Nordic countries to biosecurity and vaccine based interventions for serovars of greatest public
health significance in most of Europe. Both approaches have led to a substantial reduction in human Salmonella
infections across many EU countries. This has followed strict legislative control associated with obligatory culling or
marketing related penalties in breeding poultry and laying hens. Eradication of certain serovars has been proposed as
having the potential to leave a niche for the emergence of others, e.g. the rise in S. Enteritidis (SE) followed the
eradication of S. Gallinarum/Pullorum in many countries, and in Hungary multi-drug resistant (MDR) S. Infantis emerged
in broiler production after control of SE. Other epidemic strains such as S. Typhimurium DT104 and monophasic S.
Typhimurium DT193/120 have emerged in cattle and pigs respectively, but the reasons for these cyclical events are not
fully understood. The rise in resistance to multiple antibiotics in the enteric flora of food animals, including transferrable
resistance to extended spectrum cephalosporins, fluoroquinolones and carbapenems, makes transfer of resistance
genes, often associated with virulence genes, more likely. Recently, antibiotic resistant strains of S. Kentucky and S.
Stanley have emerged in turkeys in the EU and have been responsible for International outbreaks of human cases.
Molecular typing initiatives have been important in the identification of these outbreaks. The rapid progress being made
towards routine high throughput whole genome sequencing and associated high capacity data bases and analytical
tools, as well as initiatives to introduce harmonised molecular typing methodology organised by EFSA and ECDC, offer
great promise for improved future surveillance. As the barriers to international trade are reduced and there are more
imports of breeding animals, meat and eggs from third countries, such tools will become increasingly important in the
early identification of new strains with epidemic potential.
Background
Salmonellosis is the second most commonly reported bacterial cause of foodborne illness in the
EUandmostofthedevelopedworld,rankingafterCampylobacterintermsofthenumberofcases
and lower in terms of total burden to society, as measured by total disability adjusted life years
(DALYs) or severity in terms of DALYs per case (Havelaar et al., 2012). Salmonella has a long
history in terms of its definitive identification, research and control initiatives, which have been
hugely facilitated by the early development of phenotypic typing schemes. These have been
based on serotyping according to the White KauffmannLe Minor scheme, first developed in the
1930s(Grimont&Weill,2007).Thishasbeensupplementedbyvariousphagetypingschemesfor
specific serovars in several countries and the use of antibiograms as an indication of different
clonallineswithinserovars.Manyserovarshaveadistinctepidemiology,e.g.anassociationwith
feed ingredients or certain food animal or wildlife species,or often establishmentas a persistent
residentorhousestrainwithincertaincompaniesorfarms,feedprocessorsorfarms.Theability
to track sources amongst more than 2,600 serovars and multiple phenotypic variants within
serovars enabled rapid progress to be made on identification of dissemination routes, so when
human cases began to increase following the increasing industrialisation of food animal
productionitwaspossibletointroducelegislativeorvoluntarycontrolofinfectionusingevidence
based measures based on sound science. In the Nordic countries the focus was on control of
almostallserovarsinmostfoodanimalspecies(Hoppetal.,1999),butinthemajorityofcountries
thefocuswasonthemainserovarsassociatedwiththehighestnumberofhumancases,typically
S. Typhimurium (ST) and S.Enteritidis (SE). In some EU Member States, S. Infantis has also been
prominentinpigsandpoultry(Wegener&Baggesen,1996)andhasbeentheonlyotherindividual
serovarthathasbeenconsistentlyresponsibleformorethan1%ofreportedhumancasesinthe
EU(EFSA,2010a).
In some countries antibiotics, particularly fluoroquinolones such as enrofloxacin, were regularly
usedtoreducetheoccurrenceofSEinthepoultrybreedingandproductionpyramid(Edel,1994)
andthis,togetherwithmedicationforotherproductionanddiseaseproblems,isthoughttohave
241
resultedintheselectionandpersistenceofmutantresistantstrainsofSalmonella,Campylobacter
and E. coli. Such medication, ideally combined with a move to clean accommodation and two
dosesofcompetitiveexclusiontreatment,wasonlymoderatelysuccessfulinreducingSalmonella
infection,largelyonlyreducingtheabilitytodetectinfection(Reynoldsetal.,1997).Vaccination
wasalsoimportantinmanycountriesforcontrolofSEandintheUKanalhydrogeladjuvantedSE
bacterin, grown in iron limitation conditions to increase antigenicity, was important in gaining
controloverinfectioninbroilerparentflocksintheearlymid1990s.Laterinthe1990s,itsusein
commercial laying flocks as part of the Lion quality assurance scheme was instrumental in
achievingthelargereductionineggassociatedhumancasesobservedfrom1998until2010(Ward
etal.,2000;OBrien,2013).FurtherdeclinesinSEfollowedtheintroductionofliveoralvaccines
which were perceived as easier to administer and so were used by a greater number of egg
producers. Also instrumental in driving further improvements was the EU baseline survey for
Salmonella in laying hens carried out in 20045. This used sensitive sampling and testing
methodology and led to the identification of an unexpected high prevalence of infection in most
countries,particularlyinlargecagedflocks(EFSA,2007).ThiswasfollowedbylegislationintheEU
thatintroducedharmonisedoperatorandofficialmonitoringfrom2008andrestrictionsonsaleof
fresh eggs from infected flocks from 2009. The threat of restrictions acted as a strong incentive
for further improvements in vaccination programmes, vaccine administration techniques, farm
hygiene, especially rodent control, and cleaning/disinfection between flocks. The legal
requirement to replace conventional cage systems with colony cages or alternative production
systems also offered an opportunity to eliminate pests and residual contamination in laying
housesaspartoftherefurbishmentprocess.
Numbers of cases of ST in people have also reduced gradually over time, largely in parallel with
the unexplained decline in ST DT104 (EFSA, 2012). It is likely that the periodic emergence,
international dissemination, predominance and decline of certain ST phage types/clonal groups
(Rabsch et al., 2002) is related to an ability to initially evade protective immune responses
associated with prior exposure to unrelated ST strains, occurrence in breeding animals that are
tradedinternationallyandpresenceofmultipleresistancetocommonlyusedantimicrobialswhich
may aid their selection in medicated animals. Regression of such strains may be due to
development of herd immunity across animal populations, reduction in virulence or persistence
abilityamongststrainsandtheirreplacementbyotherstrains.
The contribution of poultry to human ST is variable according to National prevalence and
consumptionpatterns,butisthoughttoberelativelylowinmostcases(EFSA,2012b),withthepig
reservoir contributing most cases. EU legislation (EC, 2003) has introduced minimum harmonised
monitoringandcontrolmeasuresforbreedingandproductiongenerationsofchickensandturkeys
to be applied across the EU, as well as by third country operators supplying the EU, and it was
anticipated that similar controls for pigs would be introduced in 2012. However, the known
difficultiesofcontrollinginfectioninpigherdsanditshighcost,sinceinmostcountrieselimination
of infection from holdings is not economically feasible, so a continuous and ongoing cost of
infection reduction measures is incurred, make effective control much more challenging than in
thepoultrysector.TheemergenceofmonophasicstrainsofST(mST)withtheantigenicformula
1,4,(5),12:i:wasafurtherdevelopment,firstlyinSpainwithU302strainsandfrom2006inmost
of the rest of the EU, this time involving DT193/120 strains with a characteristic tetra resistance
pattern(EFSA,2010b).Insomecasesresistancegenesmaybelinkedwithvirulencegenesonthe
sametransferablegeneticelements.IntheUSA,differentmSTstrainshaveemergedoverasimilar
timescaleandthereasonforthisconcurrentemergenceofmultipleclonesisnotknown(Bugarel
et al.,2012). Loss of the phase 2 flagella antigens has been postulated as a mechanism by which
organismsmaypartiallyevadetheinitialcytokineresponseinhostanimals(Crayfordetal.,2011).
AfurthershiftinvolvinganincreaseintheproportionofmSTstrainslackingtheO5antigenisalso
242
thought to provide similar immunological advantages to the organism. The timing of the
emergence of mST corresponds with the withdrawal of antibiotic growth promoters and
increasing use of zinc oxide supplements in feed to help suppress bacterial overgrowth in the
intestines of weaned pigs. mST DT193 strains have two genomic islands, one of which codes for
both the tetraresistance and resistance to heavy metals. Use of heavy metals in pigs may
therefore have been involved in the preferential selection and proliferation of mST mutants
(Gebreyes,2011).
Currentandfuturechallenges
There are now some signs that the decline in the numberof human SE cases andoutbreaks may
beslowing,andthenumberofcaseshasnotyetreturnedtopre1980slevels(Anon,2012).Inthe
UK, the vast majority of human SE cases involve strains with phage types and antibiograms that
are different from those historically or currently found in British poultry. The majority of SE
outbreaks still involve imported eggs and it is important to ensure that implementation of
legislationanddetectionofinfectedflocksiscarriedouteffectivelyacrosstheEU.InGB,alllaying
flocksinwhichSEorSThasbeenconfirmedhavebeenslaughtered.Thismeansthatthesourceof
infection is removed and there is extended downtime in the house during which pests and
environmental contamination can be eliminated. This is not always possible in situations where
the infected flocks remain but eggs are sent for heat treatment, making persistence of infection,
whichmayremainundetectedinsomecases,morelikely(Dewaeleetal.,2012).
The ongoing rise in mST in pigs and people requires additional measures to address both the
contaminationofpigmeatproductsandthepigreservoir,whichcontributescontaminatedwaste
to the environment and leads to spillover infections in other animals, particularly cattle, dogs
andhorses.Sincebiosecuritybasedcontrolonmostpigfarmsisdifficultandcostlyandthereis
nocommercialsourceofSalmonellafreereplacementbreedingstockthereisanurgentneedfor
simple intervention measures such as vaccination. A live attenuated ST vaccine is available in
Germany and has produced encouraging preliminary results when used in experimental studies
(Leymanetal.2012)andinfieldcasestudiescarriedoutintheUK.ThereiscurrentlynotanEU
widelicenceforthisvaccineandithaslargelyonlybeenusedintheUKincaseswheretherehas
beenclinicalsalmonellosisinweanedpigs.Forfullimplementationofmeasurestocontainpublic
health threats in primary production there needs to be a financial incentive, such as the penalty
system used in the Danish pig industry or the EU wide restrictions on table eggs from infected
layinghenflocks.Alternatively,ifavaccinesuchasamultiagentvaccineinaSalmonellavectoror
other control measures could be conclusively demonstrated to have a positive economic benefit
for farm productivity, it should be possible to more readily encourage voluntary uptake of such
controls.
Theemergenceofresistancetoextendedspectrumcephalosporinantibioticsfollowingtheuseof
offlabeltreatmentsbyinjectionorsprayofhatchingeggsordayoldchicksinthebreedingand
production pyramid to avoid hatcheryassociated infections and excess firstweek mortality has
ledtotheselectionanddisseminationofmultidrugresistance(MDR)plasmidsamongsttheenteric
flora of poultry in some countries. Where the prevalence and selection pressure is high, these
have sometimes transferred to Salmonella (Hasman et al., 2005), especially strains such as
S.Paratyphi B variant Java, which is prominent in broiler production in some European countries.
It is likely that recent agreements in the EU to stop both the offlabel use of cephalosporins in
poultry and routine use of fluoroquinolones for newly placed chicks will favourably influence the
resistance situation in Salmonella, as occurred after temporary withdrawal of such treatment in
Quebec(Dutiletal.,2010),buttheregressionofresistantstrainsofE.coliwhentheproportionof
resistant strains is high and plasmid addiction systems are present is less certain. Restrictions in
the use of cephalosporins will also be important to limit the emergence of Salmonella with
243
carbapenem resistance in farmed livestock. This has been reported as being increasingly widely
distributedinE.coliinfoodanimalsandhasrecentlybeenreportedfromS.Infantisinbroilersand
pigs in Germany (Seiffert et al., 2013). There is a danger that cephalosporins may be used as
treatments of convenience in cattle and pigs rather than as a last resort because of their wide
spectrum, lack of milk withhold and short meat withhold periods, and the availability of low
volume single dose longacting formulations. In poultry, ceftiofur was used because it could be
mixedinthesameinjectionastheMareksdiseasevaccinewithoutdamagingthevaccine.Thereis
a lincomycinspectinomycin product available that can also be used in this way if hatcherybased
treatmentisrequired,butimprovementsinmanagementofbreedingflocks,hatcheryhygieneand
commercialfarmmanagementisapreferableapproach.
The reduction of major Salmonella serovars has sometimes been followed by the emergence of
others and it is postulated that a Salmonella niche may exist in animal production that is more
likely to be filled by emergent strains than if the original strains persisted (Rabsch et al., 2000).
This is specifically suspected in relation to the elimination of S. Gallinarum (SG) biovar Pullorum
(SP) from commercial scale poultry production in many countries, which may have provided an
immunologically nave niche for the emergence of SE. Since 2005/6 there have however been
several reports of the occurrence of SG in large biosecure commercial caged laying flocks as well
as in smaller backyard flocks. The source of infection has not been identified but it appears to
affectmoregeneticallysusceptiblemainlybrownbirdsandclinicalinfectionoftenbeginsincages
close to the entry points to houses or where visiting workers have been stationed. Transfer of
infection via red mites as well as persistence within infected mites between flocks is a strong
possibility and with the increased use of colony cages and associated dry cleaning programmes
thereisariskofgreaterpopulationsofredmitesdeveloping.
Another example of emergence of new Salmonella clones is that of MDR S. Infantis in Hungary,
whichproliferatedinbroilerproductionafterthecontrolofSE(Nogradyetal.,2012).Thishasalso
caused significant levels of infection in people and has been suspected to be involved in some
internationaltradeinbreedingstock.
OtherrecentlyemergedsalmonellaewithsubstantialpublichealthsignificancewithintheEUhave
occurredinturkeys.AMDRhighlyciprofloxacinresistantstrainofS.KentuckyemergedinPoland
andneighbouringcountriesin2010andalthoughsimilarMDRstrainshavebeenfoundinpoultry
in the African continent and people who have travelled there, the origin of the particular turkey
strainisunclear(Wasyletal.,2012).Acaseinvolvingthisstrainhasrecentlybeenreportedfroma
doginGBandthisissubjecttofurtherinvestigation.AsimilarsituationoccurredwithS.Stanleyin
turkey production in Hungary, Austria and Germany (Anon, 2012) and in this case international
outbreaks of infection appeared to be associated with consumption of frozen turkey products.
These serovars are not included in statutory harmonised control programmes in the EU and in
countries where all isolates are not fully serotyped the early stages of their emergence may be
missed.
MDR, cephalosporin (AmpC mediated) resistant strains of S. Newport, S. Heidelberg and S.
Typhimurium have been reported from cattle, pigs and poultry in the USA, Canada and South
America.ThesestrainsdonotappeartohavesignificantlyspreadtoEurope,buttherehavebeen
reportsoflimitedimportationofMDRNewportviahorsemeatfromUSAandMDRHeidelbergand
DerbyinbreedingboarsfromCanada(Aarestrupetal.,2004).Thepotentialforincreasedtradein
eggs and food animal products with the USA in future years indicates the need for caution in
obtainingmaterialfromsourceswithuncertainSalmonellastatus.
Futurechallengesandopportunities
If more than a limited number of Salmonella serovars are to be controlled, to reduce the risk of
the emergence of new epidemic strains, more attention will need to be focussed on control of
244
contamination of animal feed and importation of live animals and hatching eggs, which
substantially increases the cost of control programmes but may have other benefits in terms of
generalbiosecuritywithinthelivestockindustry.
InitiativesbyEFSAandECDCtointroduceharmonisedmoleculartypingprogrammesacrosstheEU
offertheopportunitytoidentifyoutbreaksamongstSalmonellacasesthatmightotherwiseappear
tobesporadicandtotakeactiontolimittheirsizeandduration.ProposalsbyECforharmonised
antimicrobial monitoring of Salmonella and other pathogens from the major food animal sectors
would also help identify localised and generalised resistance issues, but it is desirable to further
extend harmonisation beyond the minimum number of isolates suggested and the programme,
usingcommonmicrobrothtestingequipmentmaystimulatewiderutilisationasthecostreduces.
Nextgeneration,highthroughputwholegenomesequencingoffersunparalleledopportunitiesto
evaluate and compare strains (Holt et al., 2008), but it is essential that accurate epidemiological
data is provided to allow the genetic information to be fully interpreted and powerful databases
and analytical capacity is available to facilitate efficient data mining. This will allow replacement
of much of the current phenotypic and molecular strain typing methodology by a single process,
leadingtocostsavings.Geneticcharacterisationalsoneedstobelinkedwithepigeneticstudiesof
thepathogenandthehostresponsetofullyevaluatethecapabilityofstrainstodevelopepidemic
potential(Soleimanietal.,2012).
For best control of future Salmonella and other foodborne zoonotic problems, multidisciplinary
teamsofveterinarians,medicalspecialists,bacteriologists,moleculargeneticists,epidemiologists,
mathematical modellers, social scientists, risk managers and industry stakeholders should work
together in a task force approach so that difficult issues can be addressed in an holistic way and
seenthroughtocompletion.
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247
May 27-28-29, 2013
Saint-Malo France

Session 5

Chairpersons:

Communications
248
Salmonellainsoybeanmeal,
whatisthesourceofinfection?
PerHGGBLOM
1
,CelinaSOARES
2
andEdnaOLIVEIRA
2
Email:per.haggblom@sva.se;
1
NationalVeterinaryInstitute,Travvgen22,S75189UPPSALASweden;
2
Embrapafoodtechnology,AvenidasdasAmericas29501,23020470RIODEJANEIROBrazil
ABSTRACT
Oil seed meals are considered a high risk material for Salmonella when used in compound feed manufacturing because
of frequent contamination with the pathogen. In Europe, soybean and rape seed meal are the vegetable proteins most
often reported positive. The aim of the present work was to trace where in the feed chain Salmonella could be detected
focusing on crushing plants where soybean meal is produced. For two months, sampling for Salmonella was carried out
in the environment of crushing plants and in a harbor where the meal was stored before being exported to various
continents. Salmonella was isolated from the samples using the Modified Semisolid Rappaport Vassiliadis (MSRV)
medium and serotyping was according to Kaufmann-White. A total of 600 environmental samples were investigated and
the results showed that 14% of the samples from the meal storage facilities at the crushing plants and 16% of the
samples from the export harbor were positive for Salmonella. Eighteen Salmonella serotypes were identified where
several were identical between the crushing plant and export harbor. The data shows that the soybean meal was heavily
contaminated already at the crushing plant despite high temperatures used in the processing. Cross-contamination after
the heat treatment is the most likely reason for the meal contamination.
INTRODUCTION
Thefeedchainisacomplexnetworkofstakeholderstradinglargeamountsofvegetableoranimal
feedrawmaterialsondomesticand/orglobalmarkets.Coproductsfromoilproductionsuchas
soybean, rapeseed or sunflower meals are important protein sources in animal feed worldwide.
Accumulated prevalence data have shown a higher contamination with Salmonella in processed
oilseedsparticularlyrapeseedandsoybeanmealcomparedtootherplantrawmaterials(Binteret
al., 2010 ; Wierup & Haggblom, 2010 ; Kwiatek et al., 2008 ; EFSA, 2008). The reason why
processedoilseedsshowahigherprevalenceforSalmonellaispresentlyunknownastemperatures
and processing conditions used in the oil extraction would efficiently destroy bacterial cells.
Several hypotheses have though been proposed such as contamination during transport of the
bulkcommoditiesorcontaminationfromrodents,birds,previouscargoetc.Thelowwateractivity
of the meal does not however, support the growth of salmonella and for that reason extensive
contaminationofaconsignmentisnotlikelytheresultofasinglecontaminationevent.Datafrom
DenmarkhaveshownthatinsomecrushingplantscertainSalmonellaserotypesmaypersistinthe
premisesforseveralyears(BrnnumPedersen,2007).
Recently, the European Food Safety Authority (EFSA) concluded that control of animal feed is an
essential element in an integrated control strategy for Salmonella (EFSA, 2010). Other authors
have also concluded that feed raw materials are important vectors for Salmonella dissemination
causinganimalinfections(Crumpetal.,2002;sterbergetal.,2006;Haggblom;2009,Moritaet
al.,2006).
Aim: The aim of the present work was to trace the source of contamination of soybean meal,
more specifically to investigate if Salmonella could be detected in the processing environment of
crushingplantsforsoybeansandalsoinnewlyprocessedsoybeanmeal.Wewerealsointerested
tofindoutifthemealwascontaminatedwithSalmonellainharborwarehousesusedforexportof
soybeanmeal,inthatcasesupportingthehypothesisthatthemealwascontaminatedsoonafter
production.
249
MATERIALSANDMETHODS
Environmental and meal samples from two soybean crushing plants and one export harbor in
Brazilwerecollectedonaweeklybasisduringatwomonthsperiod.Thesamplingplanfocusedon
a) environmental samples from the warehouse of processed soybean meal (dust samples from
walls,spillage,aggregatedmaterial)aswellasthestoredmealb)theloadingareawherethemeal
was loaded on trucks (transfer augers, elevators and outloading gantries) and c) environmental
samples from warehouses at the exporting harbor for soybean meal. Dust, spillage and meal
samples (550g) were collected using disposable gloves, surface samples were collected using
sterile gauze and put in sterile plastic bags. Approximately 80 samples were collected each week
andsenttothemicrobiologylaboratoryatEmbrapa,RiodeJaneiroforanalysis.
Salmonella isolation was performed using the Modified Semisolid Rappaport Vassiliadis (MSRV)
(ISO 6579, 2002) medium after pre enrichment in buffered peptone water at 37
0
C for 18 h.
PresumptiveSalmonellaobtainedfromXyloseLysineDesoxycholate(XLD)agarandBrilliantGreen
agar(BG)wassubmittedtobiochemicaltestsincludingAPI20E(Biomeriux).Isolatedsalmonella
strainswereserotypedusingtheKauffmannWhitescheme.
RESULTS
In crushing plant A and B, respectively, 136 and 120 samples were collected during the sampling
period from the warehouse of the processed soybean meal (Table 1 and 2). Seventeen samples
werepositiveforsalmonellafromplantA(12.5%),whileatplantB9samples(7.5%)werepositive.
At the loading area for finished soybean meal a total of 104 samples were collected in plant A
where 20 (19.2%) samples were positive. At plant B 18 positive samples (18.8%) were detected
from the 96 collected in this area. At the harbor warehouse that received soybean meal from
processingplantA54sampleswerecollectedwith7(15.5%)beingpositiveforsalmonella.
Table1ResultsofsalmonellamonitoringatcrushingplantAandassociatedexportharbor*
Soybeanmeal Environment
Warehouse 96(14) 40(3)
Loadingarea 48(13) 56(7)
Exportharbor 45(7)
*numberofanalyzedsamples(numberofpositivesamples)
Table2ResultsofsalmonellamonitoringatcrushingplantB*
Soybean
meal
Environment
Warehouse 80(6) 40(3)
Loadingarea 48(15) 48(3)
*numberofanalyzedsamples(numberofpositivesamples)
Eighteen different Salmonella serotypes were isolated in the study. A total of 14 Salmonella
serotypes were detected in crushing plant A and 11 in plant B. Seven serotypes were isolated in
bothplants(S.Alachua,S.Cerro,S.Cubana,S.Livingstone,S.Rissen,S.TennesseeandS.enterica
subsp.I,O:13,23).
In plant A, four identical serotypes were identified in the warehouse for soybean meal and the
loadingarea(S.Anatum,S.Alachua,S.TennesseeandS.Livingstone).S.Rissenwasisolatedinthe
loadingareaandexportharborwhileS.Anatumwasisolatedinthewarehouse,loadingareaand
exportharbor.S.MbandakaandS.Orionwereonlyisolatedfromtheharborwarehouses.
In plant B, S. Tennessee, S. Cubana and S.Orion were detected both in the meal warehouse and
loadingareaofthecrushingplant.
250
At both plants a larger number of serotypes were detected in the loading area compared to the
warehouse. A larger number of serotypes were also detected in the finished meal compared to
environmentalsamples.
DISCUSSION
ItiswelldocumentedthatproteinrichfeedmaterialsareonriskofSalmonellacontaminationand
may introduce the bacteria in compound feed and subsequently into the feed and food chains
(EFSA2008;Hoszowskietal.,2008;Malmqvistetal.,1995).Forthatreasonitisessentialtotrace
whereinthefeedchainthecontaminationtakesplace(Binteretal.,2010).
Because soybean meal is used as a valuable protein source in several food producing animals
worldwideitisimportanttocontrolSalmonellaatanearlystageinthefeedchain.
Environmental sampling, as was previously used in Salmonella monitoring of feed mills (Wierup
and Haggblom, 2010) were used in the present study as a sensitive bacteriological monitoring of
thecrushingplants(DaviesandWales,2010;Whyteetal.,2003;RickardssonandJones,2004).
Recently, comparative studies were performed on cultural and molecular isolation methods for
salmonellaindifferentfeedmaterials.ItwasshownthattheMSRVmethodwasequivalenttothe
ISOmethodinisolatingsalmonellafromfeed.Thedetectionlevelsinsoybeanmealthatresultin
50% detection probability was 110 CFU/ 25 g (Koyuncu et al., 2010 ; Koyuncu and Haggblom,
2009)
TherepeateddetectionofSalmonellaintheprocessedmealduringthesamplingperiodshoweda
continuous presence of Salmonella despite the heat treatment during processing of the beans.
The data clearly indicate that crushing plants may, for extended periods, continuously produce
contaminated soybean meal that subsequently will be detected at later stages of the feed chain.
The reasons why crushing plants for soybeans are often contaminated may be explained by the
presenceofsalmonellaindustparticlesfromthebeans(WierupandHaggblom,2010).However,
thesourceofcontaminationofthebeansisstillunknownandneedsfurtherinvestigations.
It is well known that e.g. the coolers may remain persistently contaminated with Salmonella for
extended periods because of contaminated dust particles in the cooling air that inoculate the
oftenmoistandwarmcoatingsinside(DaviesandWray,1997).Contaminatedwarehousesforthe
finished meal may also cause recontamination of the processed meal. It is also important that
nonheated soybean hulls should not be mixed with the processed soybean meal because of
possiblecontaminationofthesoybeans.
The isolated Salmonella serotypes from the crushing plants and the harbor warehouse were
identicaltoserotypesdetectedinsoybeansandcompoundfeedlaterinthefeedchainindicating
that the contamination at the crushing plants is important to control (Wierup and Haggblom,
2010). Similarly, common serotypes in food producing animals such as S. Enteritidis and S.
Typhimuriumwerenotdetectedinthissurvey.
This is the first screening study showing the risk for contamination with Salmonella in soybean
mealatanearlystageofthefeed chain.Futureworkshouldbedirectedtofindingthesourceof
contaminationandalsotoimprovethehygienicconditionsatcrushingplantsinordertoimprove
theSalmonellastatusofthisimportantfeedprotein.
CONCLUSIONS
Theworkrevealedthatcrushingplantsforsoybeansandtheprocessedsoybeanmealisfrequently
contaminated with salmonella. Cross or recontamination of the processed meal within the
crushing plant is the most likely explanation for the soybean meal contamination. Further work
shouldbedirectedtominimizerecontaminationofthemealatcrushingplantsinordertoprevent
furthertransmissioninthefeedchain.
251
ACKNOWLEDGEMENTS
The work was supported by the European Unionfunded interated project BIOTRACER (contract
036272)underthe6
th
RTDframework.
REFERENCES
1. Binter, C., Straver, J.M., Hggblom, P., Bruggeman, G, Lindqvist, P.A, Zentek, J., Andersson, G. 2010.
Transmission and control of Salmonella in the pig feed chain: A conceptual model. Int. J. Food
Microbiol.145Suppl1:S717.
2. BrnnumPedersen,T.2007.Occurrenceandpersistenceofsalmonellaandcoliformbacteriainpoultry
feed,feedprocessingandproductionenvironments.PhDthesis,Univ.ofCopenhagen,Denmark.
3. Crump,J.A.,Griffin,P.M.,Angulo,F.J.2002.Bacterialcontaminationofanimalfeedanditsrelationship
tohumanfoodborneillness.FoodSafety35:859865.
4. Davies, R. H., Wray, C. 1997. Distribution of Salmonella contamination in ten animal feedmills.
VeterinaryMicrobiology57:159169.
5. Davies R.H., Wales A.D. 2010. Investigations into Salmonella contamination in poultry feedmills in the
UnitedKingdom.J.ofAppliedMicrobiology109:14301440.
6. EFSA 2008. Scientific opinion of the panel on biological hazards on a request from the health and
consumer protection, directorate genereal. European Commission on microbiological Risk assessment in
feedingstuffsforfoodproducinganimals.EFSAJ.720:184.
7. EFSA 2010. EFSAPanel on biological hazards: scientific opinion on a quantitative microbiological risk
assessmentofsalmonellainslaughterandbreederpigs.EFSAJ.8(4)1547.
8. Hggblom, P. 2009. The feed borne outbreak of Salmonella Tennessee in Finland in the spring of 2009.
NationalVeterinaryInstitute,Uppsala,Sweden,pp120.www.mmm.fi/.../.
9. Koyuncu, S. , Hggblom, P. 2009. A comparative study of cultural methods for the detection of
Salmonellainfeedandfeedingredients.BMCVeterinaryResearch5:6.
10. Koyuncu, S., AndersonnN, M.G., Hggblom, P. 2009. 2010. Accuracy and sensitivity of commercial PCR
based methods for detection of Salmonella enterica in feed. Applied and Environmental Microbiol. 76:
28152822.
11. Kwiatek,K.,Kukier,E.,WasylD,Hoszowski,A.2008.Microbial qualityoffeedinPoland.(InPolishwith
anEnglishSummary)MedycynaWeterynaryjna68:183188.
12. Morita,T.,Kitazawa,H.,Iida,T.,Kamata,S.2006.PreventionofSalmonellacrosscontaminationinanoil
mealmanufacturingplant.JApplMicrobiol101:46473.
13. Osterberg,J.,Vagsholm,I.,Boqvist,S.,Lewerin,S.S.2006.FeedborneoutbreakofSalmonellaCubanain
swedish pig farms: risk factors and factors affecting the restriction period in infected farms. Acta
VeterinariaScandinavica,47:1322.
14.RichardsonK.E.,Jones,F.T.2004.Salmonellaincommerciallymanufacturedfeeds.PoultryScience,83,
384391.
14. Whyte,P.,McGill,K.andCollins,J.D.A.2003.SurveyoftheprevalenceofSalmonellaandotherenteric
pathogensinacommercialpoultryfeedmill.JournalofFoodSafety23:1324.
15. Wierup,M.,Hggblom,P.2010.Anassessmentofsoybeansandothervegetableproteinsassourceof
salmonellacontaminationinpigproduction.ActaVeterinariaScandinavica,52:15.
252
SalmonellainwildbirdsinIsrael:Surveillancestudies
inhealthyanddeadbirds
AvishaiLUBLIN
1
,SaraMECHANI
1
,YigalFARNOUSHI
1
,ShimonPERK
2
,RoniKING
3
andIgalHOROWITZ
4
1
DivisionofAvianandFishDiseases,KimronVeterinaryInstitute,and
2
VeterinaryServices
andAnimalHealth,POBox12,BETDAGAN50250,Israel;
3
IsraelNatureandParks ProtectionAuthority,AmVe'Olamo3JERUSALEM95463,Israel;
4
WildlifeHospital,ZoologicalCentreTelAvivRamatGan,POBox984,
RAMATGAN52109,Israel
INTRODUCTION
Wild birds, due to their great geographic mobility, play an important role in mechanical and
biological transmission of Salmonellae to poultry farms (Soliman et al., 1988) or other domestic
animals (Vico, 2011). A relationship had been demonstrated between environmental
contamination with Salmonellae and their incidence in wild birds (Cizek et al., 1994). Israel, as a
way station on the path of over 500 million migratory birds twice a year or as a final seasonal
stationforotherspecies,hasaspecialroleinthespreadofSalmonellaethroughwildbirds.Israel
has also its own permanent population of wild birds. Reitler (1953) was the first to report on
salmonellosis in feral birds of Israel, groupB Salmonellae in starlings (Sturnus vulgaris). The
percentages of infected birds in a survey of Singer et al. (1977a), ranged from 2% in house
sparrows (Passer domesticus) and collared doves (Streptopelia decaocto) to 20% in European
starlings (Sturnus vulgaris). All the serovars found in wild birds can be found also in humans and
domestic animals. Moreover, the same serovars isolated from pigeons (Columba livia), doves
(Streptopeliaspp.)andcattleegrets(Bubulusibis)werefoundinpoultry.Althoughthecarriageof
Salmonellaebyapparentlyhealthywildbirdshasbeenwidelyreported(Abulreeshetal.,2007),no
comprehensive survey in wild birds of Israel has been published since the reports of Singer et al.
(1977a,1977b).InthepresentpaperwepresenttheresultsofasurveyofSalmonellainover3500
healthywildbirdsinIsrael,andover1500wildbirdsfounddeadorinjured.
MATERIALANDMETHODS
Methodology
Atotalof3550healthywildbirdsand1550
Healthybirds,19801999
Outofca.3400specimens,551(16.2%)wereSalmonellapositive.Splittingofthe20yearperiod
intofourperiods,revealedacontinuousdecreaseinprevalence,from29.8%during19801984to
15.9%during19851989,11.9%during19901994,and7.7%during19951999.Thetrendofdead
wild birds were tested for infectivity by Salmonella spp. in several periods between 1980 and
2012. All the surveyed birds belonged to species whose capture for birdringing or surveying,
eitherhuntingorcollectingdeadbirds,waslegallypermitted.
Species
Thesurveyincludedspeciesof13differentorders:Passeriformes,Coraciiformes,Strigiformes,
Falconiformes,Ciconiiformes,Anseriformes,Pelecaniformes,Columbiformes,Charadriiformes,Lariformes,
Gruiformes,GalliformesandProcellariiformes.
Laboratorytechniques
Diagnosis of Salmonella was performed from intestines or visceral organs by common culturing
methodsofenrichmentandconfirmationonselectivemedia(triplesugarironagar)(Mallinson&
253
Snoeyenbos, 1989). Serological classifications of positive cultures were performed with a rapid
slide agglutination test kit (Institute of Vaccines and Antisera, Central Laboratories, Ministry of
Health,Jerusalem)ofantiseraforserogrouping.Furtherserovarandphagetypeclassificationwere
carriedoutattheInstituteofVaccinesandAntiseraoftheCentralLaboratories,MinistryofHealth,
Jerusalem.
Statisticalanalysis
Statistical analysis was carried with the SAS software (Alice, 1985) to analyze effects of bird
migrationandseasononSalmonellainfectivity.Forbirdmigrationstudiesallspeciesareclassified
asresidentormigratory.Seasonsweredefinedas"cold",fromNovembertoApril,and"hot",from
May to October, or as "peakcold", during DecemberFebruary, and "peakhot", during June
August.
The distribution of serovars, was not well correlated with that found in poultry in the parallel
period (Central Laboratories, Ministry of Health, Jerusalem), in which other serovars were more
abundant.
Decreaseinprevalenceislogarithmicasfollow:
Log10prevalence=3.76(5yrperiod)X.43(R=.99,P<.02)
These changes are not correlated with the situation in poultry and in humans as reported by the
Israel Ministry of Healths National Salmonella Center (Central Laboratories, Ministry of Health,
Jerusalem),probablymakingthewildbirdpopulationofIsraelnotthemostsignificantsourceof
Salmonellainfectiontopoultryorhumans.
Speciesandecologicalgroupeffects
A total of 57 species were surveyed, and Salmonella was diagnosed and isolated in 27 of them.
PrevalenceofthepathogeninthevariousspeciesandecologicalgroupsofbirdsisshowninTable
1. As seen in the table, birds inhabiting plantation and groves have the highest prevalence of
Salmonellaewhilebirdslivingnearorinwaterhavethelowest.Oneof theinterestingfindingsis
the high prevalence found in birds that are hunted and whose meat is eaten by people, such as
partridges (Alectoris chukar) with 33% prevalence. This has to be taken into consideration in
processingthismeatforhumanconsumption.
Serogroup,serovarandphagetypeeffects
ThepredominantSalmonellaserogroupinhealthywildbirdsofIsraelwasC,53.0% ofallpositive
cases,thanBwith42.4%,D12.7%,andE3.0%(over100%duetobirdswithseveralserovars).
The predominant serovar was S. Typhimurium var. copenhagen (23.1% of the positives).
SalmonellaTyphimuriumcontributed13.5%,S.Infantis9.3%,S.Kottbus5.8%,andlessthan5%
foreachoftheothers.Phagetypewasdeterminedin89oftheisolatesofS.TyphimuriumandS.
Typhimurium var. copenhagen. R6 was the predominant (31.5%), than phage type 1 (15.7%) and
phage type 2b (10.1%). Prevalence were significant (24.0 and 1.7%, 25.9 and 4.0%, P<.001 for
both).
Comparison between seasons for the various serovars, revealed interserovar differences, like
higher percentage of S. Typhimurium var. copenhagenpositives in the cold season when many
migratory species winter in Israel, and other serovars which are typical of resident species, were
isolatedmainlyinthehotseason.Someoftheserovarsweredetected
Migrationeffect
The four phenological groups that were considered are: resident birds, passingthrough birds,
wintering birds and summering birds. Prevalence of the two predominant groups, resident and
wintering birds, is not statistically different. Also, the difference between resident and migratory
(allothergroups)birdsisnot statisticallysignificant.The onlystatisticallysignificantdifferencein
254
Salmonella prevalence was between resident and passingthrough birds, those birds that their
staying in Israel on their way between Europe and Africa is the shortest (17.1% and 6.2%,
respectively,P<.005).ThepredominantserogroupinresidentspecieswasB(7.9%),withCthenext
(6.9%).Inwinteringbirds,thepredominantserogroupwasC(7.0%).
Bird migration affected also the distribution of serovars. While S. Typhimurium var. copenhagen
was the predominant serovar in resident birds (4.4%), in wintering birds the predominant was S.
kottbus (1.5%). The next are S. Typhimurium (2.1%) and S. Infantis (1.4%) in resident birds but S.
Agonainwinteringbirds(.9%).PrevalenceofS.Typhimuriumvar.copenhagenwasmuchhigherin
resident than in migratory birds. In general, the variety of serovars in migratory species was
greater.
Seasoneffect
Statisticalcomparison betweencoldandhotseasonsdid notrevealanyinterseasonaldifference
in prevalence of Salmonella, but comparison between the peaks of the seasons showed
significantlyhigherprevalenceinthepeakcoldperiod(P<.001).Whenonlyresidentspecieswere
included, there was always a significant difference (P<.001 for both). In Columbiformes and
Passeriformes inhabiting urban areas, differences between DecemberJanuary and JulyAugust in
Salmonellasampledmanyspecimensofspecieswithhighprevalence,likeTurdusmerula,Columba
livia,Passerdomesticus,Passermoabiticus,SturnusvulgarisandBubulcusibis.Mostoftheisolates
(52%)belongedtoserogroupB,thanC(20%)andD(8%),with18%unclassified.
ThecommonserovarsinthatsurveyincludedTyphimurium,S.4.12:i(groupB)andEnteritidis(D),
butserovarslikeIsangiandinoneoftheseasons,e.g.,S.Senftenbergwasfoundonlyinthecold
season,andS.Kottbus,onlyinthehotseason.
Thetrendof"winterSalmonella infectivity"inresidentbirdsisinterestingInhumans,mostfatal
salmonellosis occur in the hot months of the year (Shears & Wright, 1995), but, a few reports
indicate an opposite trend (Shi, 1990; Davies & Wray, 1996). These findings must be taken into
consideration in the planning of monitoring and control programs against the spread of
salmonellosisviatheferalavifaunaofIsrael.
HealthybirdstrappedinEilat,2005
InthesecondsurveyinhealthymigratorybirdsmostlypasserinesandraptorstrappedinEilat,only
1 out of 44 birds, a Common buzzard (Buteo buteo), shed Salmonella in the cloaca (serovar
Nottingham),while11outof146(7.5%)werecontaminatedonlyontheirfeathers,whichmeans
mechanical transmission of Salmonella by feathers. Out of these, about half were Common
buzzards and the rest Blackcups (Sylvia atricapilla). Except for serovar Nottingham, all others
inhabited on feathers serovar S.4.12:i of group B. Phage type of most isolates was R9, the rest
group2andR13.
Deadbirds,20052012
Between 2005 and 2012, ca. 1550 wild birds, mostly dead or injured, that were submitted for
pathologicalexamination,weresampledalsoforSalmonellae.Ofthese, 50isolates ofSalmonella
hadbeenfound(about3.2%),amuchlowerratethanknowninintensivepoultryraisingorinthe
previousdescribedstudyinhealthybirds.Thereasonforthediscrepancywiththeprevioussurvey
isprobablythedifferenceinthebirdgroupsthatweresampled.InthefirstsurveywereKentucky
(bothofgroupC)werealsofound.OnecaseofS.entericasubspeciesArizonaewasdetectedina
deadinnest 2monthold chick of a Griffon vulture. Disease syndromes that were observed in
Salmonellainfected wild birds were weakness, diarrhea, impaired development and mortality. In
several of the cases the bacterium was isolated from granuloma or from skin nodules. In some
255
cases it was combined with other significant avian pathogens like velogenic Newcastle Disease
Virus.Insomecasesnoapparentclinicalsignscouldbedemonstrated.
REFERENCES
1. Abulreesh, H.H., Goulder, R. & Scott, G.W. (2007). Wild birds and human pathogens in the context of
ringingandmigration.Ringing&Migration,23,193200.
2. Alice,A.(1985).SASUser'sGuide:Statistics.NorthCarolina:SASInsituteIncorporation.
3. AnnualReportsoftheNationalSalmonellaCenter.Jerusalem,CentralLaboratories,MinistryofHealth.
4. Cizek, A., Literak, I., Hejlicek, K., Treml, F. & Smola, J. (1994). Salmonella contamination of the
environmentanditsincidenceinwildbirds.ZentralblattfurVeterinarmedizinReiheB,41,320327.
5. Davies, R.H. & Wray, C. (1996). Seasonal variations in the isolation of Salmonella typhimurium,
Salmonella enteritidis, Bacillus cereus and Clostridium perfringens from environmental samples.
ZentralblattfurVeterinarmedizineReiheB,43,119127.
6. Mallinson, E. T. & Snoeyenbos, G.H. (1989). Salmonellosis. In H. G. Purchase et al. (Eds), A laboratory
manual for the isolation and identification of avian pathogens (pp. 311). Kendall: Hunt publishing
company.
7. Reitler, R. (1953). Proceedings of the VI Congress Internationale di Microbiologia, Vol. III Roma, Italy,
961962.
8. Shears,P.&Wright,A.(1995).CommunityInfectionsamongchildreninanurbanenvironment:a2year
prospectivestudyinLiverpool,U.K.JournalofInfection,30,173177.
9. Shi, J. (1990). A survey of nosocomial infection by Salmonella typhimurium. ChungHua Liu Hsing Ping
HsuehTsaChih,11,284287.
10. Singer, N., Weisman, Y., Schechter, I. & Cahane, D. (1977a). Salmonella isolations from wild birds in
Israel.RefuahVeterinarith,34,6368.
11. Singer, N., Weissman, Y., YomTov, Y. & Marder, U. (1977b). Isolation of Salmonella hessarek from
starlings(Sturnusvulgaris).AvianDiseases,21,117119.
12.Soliman,A.,Mousa,S.,Bayoumi,A.H.,Gad,N.&Atia,M.(1988).Theroleplayedbyfree flyingbirdsin
the transmission of avian pathogens II. Mycoplasma and Enterobacteriaceae. Assiut Veterinary Medicine
Journal,20,184188 .
13. Vico, J.P. (2011). Salmonellosis in wild birds and its relationship with the infection in finishing pigs.
SafePork264267.

256
Table 1: Prevalence (%) of Salmonellae out of all positive cases in various species and ecological groups
(onlywithn>15).
Species Prevalence(%)
Alectorischukar 18 33.3
Bobulcusibis 252 32.1
Ciconiaciconia 24 16.7
Columbalivia

(inc.vardomestica)692 18.2
Corvuscorone 508 9.4
Corvussplendens 30 0.0
Fulicaatra 121 8.3
Garrulusglandarius 67 25.4
Gypsfulvus 21 4.8
Larusridibundus 431 11.8
Passerdomesticus 122 15.6
Passermoabiticus 72 41.7
Pelecanusonocrotalus 45 2.2
Pycnonotusxanthopygos 52 7.7
Streptopeliasenegalensis 67 13.4
Sturnusvulgaris 722 15.7
Turdusmerula 48
18.8
Ecologicalgroup
Birdsofurbanareas 2573 16.6
Shorebirds(salt&freshwater) 450 11.8
Waterbirds(deepfreshwater) 175 10.9
Birdsofopenandmountainousareas116 16.4
Plantationandgrovebirds 83 39.8
257
PolyphasiccharacterizationofSalmonellaEnteritidisisolates
onpersistentlycontaminatedlayerfarms
duringtheimplementationofanationalcontrolprogram
withobligatoryvaccination:alongitudinalstudy
KoenDEREU
1*
,GeertruiRASSCHAERT
1
,ChristaWILDEMAUWE
2
,HildeVANMEIRHAEGHE
3
,
MiaVANROBAEYS
4
,EvelyneDEGRAEF
3
,LieveHERMAN
1
,RichardDUCATELLE
5
,
MarcHEYNDRICKX
1,5
andIsabelleDEWAELE
6
1
InstituteforAgriculturalandFisheriesResearch(ILVO),TechnologyandFoodScienceUnit,
Brusselsesteenweg370,9090MELLE,Belgium;
2
ScientificInstituteofPublicHealth(WIVISP),Engelandstraat642,1180BRUSSELS,Belgium;
3
Formerlyat:AnimalHealthCareFlanders(DGZ),Industrielaan29,8820TORHOUT,Belgium;
4
AnimalHealthCareFlanders(DGZ),Industrielaan29,8820TORHOUT,Belgium;
5
GhentUniversity,FacultyofVeterinaryMedicine,DepartmentofPathology,
BacteriologyandPoultryDiseases,Salisburylaan133,9820MERELBEKE,Belgium;
6
FormerlyatILVO;nowPoulpharm,PrinsAlbertlaan111,8870IZEGEM,Belgium
*
Correspondingauthor:Koen.Dereu@ilvo.vlaanderen.be
ABSTRACT
Since 2007, a national Salmonella control program including obligatory vaccination has been ongoing in Belgium. In this
context, the aim of the present study was to investigate the diversity of Salmonella Enteritidis (SE) isolates on five
persistently contaminated Belgian layer farms and to examine the potential sources and transmission routes of SE
contamination on the farms during successive laying rounds. A collection of 346 SE isolates originating from the
sampled farms were characterized using a combination of multilocus variable number of tandem repeat analysis (MLVA)
and phage typing (PT).
On each farm, one or two dominant MLVA-PT types were found during successive laying cycles. The dominant MLVA
type was different for each of the individual farms, but some farms shared the same dominant phage type. Isolates
recovered from hens feces and ceca, egg contents, eggshells, vermin (mice, rats, red mites and flies) and pets (dog and
cat feces) had the same MLVA-PT type also found in the inside henhouse environment of the respective layer farm.
Persistent types were identified in the layer farm inside environment (henhouse and egg collecting area). Furthermore,
this study demonstrated cross-contamination of SE between henhouses and between the henhouse and the egg
collecting area. Additional isolates with a different MLVA-PT type were also recovered, mainly from the egg collecting
area. A potential risk for cross-contamination of SE between the individual layer farms and their egg trader was
identified.
INTRODUCTION
The aim of the current longitudinal study, performed on five persistently SE positive layer farms,
sampled during subsequent laying rounds, was to (i) investigate whether the contamination on
these layer farms is maintained by one or several persisting strains and/or caused by repeatedly
introduction by occasional strains possibly originating from external sources and (ii) identify
factors contributing to the maintenance and/or introduction of SE in the environment of laying
hens.
MATERIALANDMETHODS
FiveBelgianlayerfarms(farmsA,B,D,EandG(Dewaeleetal.,2012a),witharecentpreviousor
current SE positive status in the national monitoring and control program were visited. All flocks
werevaccinatedagainstSEduringrearing.Farmsweremonitoredduringtwoorthreesuccessive
laying cycles at end of lay, after cleaning and disinfection (C&D), beginning and middle of lay.
Duringeachsamplingevent,20to26sitesineachhenhouseand8to11sitesintheeggcollecting
areaweresampledbeside200freshlylaideggsandcecaof50hens.Salmonellawasisolatedfrom
all samples according to the ISO6579: 2002 Annex D protocol. The eggshell and egg content was
analyzed as described by (De Reu et al., 2006a ; De Reu et al., 2006b). The serogroup was
258
determined by the Poly AI Vi test (222641, Becton Dickinson). A specific PCR targeting the SdfI
region was applied to confirm the isolates belonging to the Dserogroup as serotype SE
(Botteldoorn et al., 2010). Phage typing of the SE isolates was performed according to the phage
typing scheme of Ward (Ward et al., 1987) at The National Phage Typing Centre (Scientific
Institute of Public Health WIVISP, Brussels, Belgium). MLVA was performed as described by
Dewaeleetal.(2012b).
RESULTS
General
Thedifferenttypes(MLVAPTcombination)presentoneachfarmaregivenindetailinthepaper
ofDewaeleetal.(2012c).OnfarmsA,B,EandG,onedominantMLVAPTtypewaspresent.Two
dominantMLVAPTtypeswerefoundonfarmD.ThedominantMLVAtypewasdifferentforeach
oftheindividualfarms;however,somefarmssharedthesamedominantphagetype,i.e.,PT4bon
farms B and D (75.8% and 50.5%, respectively); PT6c on farms E and G (74.6% and 100%,
respectively). Besides the dominant type, other MLVAphage types were found at lower
prevalenceonfourofthesampledfarms.
SEisolatesfromfarmA
Atotalof96SEisolatesfromfarmAwerecharacterized.Detailsofthosecharacterizationresults
canbefoundinDewaeleetal.(2012c).Insummary,attheendofthefirstlayinground,onemain
type(4129287/PT8PT28)waspresentintheinsideenvironmentofhenhouses1,2,3,theegg
collectingarea,onmobileequipmentandoutsideenvironment(crates,thedrain,thehygienemat
andboots).ThistypewasstillisolatedafterC&Dandduringtheentiresecondlayingcycle.Floor,
wall and feed trough in the henhouse and floor, pallet truck and egg collector/sorter in the egg
collectingareawerecontaminatedwiththistypeduringthesuccessivelayingcycles. Thecatand
dogfecescontainedthistype,aswellasthehensfecesfoundcontaminatedduringthefirstand
the second sampled laying cycle. Salmonella was not detected in the hens ceca. However, two
poolsofeggshellsfromhenhouse2(aftermoltinginlayinground2)containedthetype41292
87 / PT23, which was also found on the floor of henhouses 2 and 3 (overshoes) during the first
layingroundandincracks/gapsinthewallinhenhouse1duringthesecondlayinground.Inother
words,atypethatwasnotfrequentlyisolatedfromthehenhousewasrecoveredfromeggshells.A
closely related type (4139287 / PT828) was isolated from boots outside the henhouses. In
henhouse 1, a different type (689287 / PT51) was isolated from cracks/gaps in the wall and
fromthefeedhopper.
Twocompletelydifferenttypes(withdifferencesin5of6VNTRsandadifferentphagetype)were
also found in the egg collecting area: 5118383 / PT21c, recovered from a dust pan and from
thecontrolpaneloftheconveyorand568384/PT7a,isolatedfromtheeggtrayconveyor.A
thirddifferenttype(4109286/PT828)wasisolatedfromeggstraysintheeggcollectingarea.
SEisolatesfromfarmB,D,EandG
The characterization results of the SE isolates from farm B, D, E and G are in detail discussed in
Dewaeleetal.(2012c).
DISCUSSION
AdetaileddiscussionofallresultscanbefoundinDewaeleetal.(2012c).Insummarythepresent
study showed the occurrence of one or two dominant SE types, spread over the henhouses and
eggcollectingareathatpersistedduringseverallayingcycles.
The C&D procedure was not able to eliminate the persistent type in the henhouse. In addition,
completelydifferenttypes(with
259
adifferenceintandemrepeatcopynumbersinmultipleVNTRsandadifferentphagetype),canbe
present on persistently SEcontaminated layer farms, which indicates previous and/or current
additional SE contamination. Furthermore, more closely related types can also be present on
persisting layer farms. Results suggest that the environment within the henhouse and vermin
present on layer farms may constitute a major reservoir for SE strains and that laying hens and
eggs (internally and externally) may become SE contaminated with strains present in the
henhouse environment. Some indications were also noted for risk of crosscontamination
betweenindividualfarmsandtheeggtradingcompanies.ThecharacterizationofSEstrainsinthe
present study resulted in a better understanding of the factors (e.g., vermin, the floor, the egg
collecting area) which contribute to the maintenance of SE contamination on persistently
contaminated farms and demonstrated that various additional measures will be necessary to
reducethepersistentcontaminationandtoimprovetheSalmonellastatusofthelayerfarms.
ACKNOWLEDGMENTS
We gratefully thank the farmers, Galluvet, Degudap Veterinary Practices and Luc Ver Eecke for
participating in this study. Also many thanks to Bruno Vandevelde and Mieke Geerinckx who
helped take the samples. Thanks to Rik Lenaerts, Thierry Fevery, Tilly Loncke and Ann Van de
WalleforexcellenttechnicalassistanceandtoMiriamLevensonforEnglishlanguageediting.This
research is funded by the Belgian Federal Public Service for Health, Food Chain Safety and
Environment(RF6195).
REFERENCES
1. BotteldoornN.,E.VanCoillie,J.Goris,H.Werbrouck,V.Piessens,C.Godard,P.Scheldeman,L.Herman
and M. Heyndrickx. (2010). Limited Genetic Diversity and Gene Expression Differences between Egg and
NonEggRelatedSalmonellaEnteritidisStrains.ZoonosesPublicHealth57:345357
2. De Reu, K., K. Grijspeerdt, M. Heyndrickx, W. Messens, M. Uyttendaele, J. Debevere and L. Herman.
(2006a). Influence of eggshell condensation on eggshell penetration and whole egg contamination with
SalmonellaentericaserovarEnteritidis.J.FoodProt.69:15391545.
3. De Reu, K., K. Grijspeerdt, W. Messens, A. Heyndrickx, M. Uyttendaele, J. Debevere and L. Herman.
(2006b). Eggshell factors influencing eggshell penetration and whole egg contamination by different
bacteria,includingSalmonellaEnteritidis.Int.J.FoodMicrobiol.112:253260.
4. Dewaele, I., H. Van Meirhaeghe, G. Rasschaert, M. Vanrobaeys, E. Degraef, L. Herman, R. Ducatelle, M.
HeyndrickxandK.DeReu.(2012a).PersistentSalmonellaEnteritidisenvironmentalcontaminationonlayer
farms in the context of an implemented National Control Program with obligatory vaccination. Poult. Sci.
91:282291.
5.DewaeleI.,RasschaertG.,BertrandS.,WildemauweC.,WattiauP.,ImberechtsH.,HermanL.,Ducatelle
R., De Reu K., Heyndrickx M. (2012b). Molecular Characterization of Salmonella Enteritidis: Comparison of
an Optimized MultiLocus VariableNumber of Tandem Repeat Analysis (MLVA) and PulsedField Gel
Electrophoresis.FoodbornePathogensandDiseases,vol.9,885895.
6.DewaeleI.,RasschaertG.,WildemauweC.,VanMeirhaegheH.,VanrobaeysM.,DeGraef,E.,HermanL.,
DucatelleR.,HeyndrickxM.DeReuK.(2012c).PolyphasiccharacterizationofSalmonellaEnteritidisisolates
on persistently contaminated layer farms during the implementation of a national control program with
obligatoryvaccination:alongitudinalstudy.Poult.Sci.91:27272735.
7. Ward, L.R., J.D.H. Desa and B. Rowe. (1987). A PhageTyping Scheme for Salmonella Enteritidis.
Epidemiol.Infect.99:291294.

260
Cattle,Beef,ConsumersandSalmonella:
ANovelPathwayofConsumerExposure
andaNewUnderstandingoftheAgentHostEcology
GuyLONERAGAN
1
,SaraGRAGG
1
,HattieWEBB
1
,MarieBUGAREL
1
,KendraNIGHTINGALE
1
,
MindyBRASHEARS
1
,TomEDRINGTON
2
andDaynaBRICHTAHARHAY
3
1
TexasTech,MS42141,79409LUBBOCKUSA;
2
USDA/ARS,2881F&BRoad,77845COLLEGESTATIONUSA;
3
USDA/ARS,Box166,StateSpur18D,68933CLAYCENTERUSA
ABSTRACT
Little if any reduction has occurred in key U.S. metrics of Salmonella or salmonellosis in ground beef or the human population,
respectively. We conducted studies to a) evaluate the burden of Salmonella within peripheral lymph nodes (LNs); and b)
explore the Salmonella diversity within LNs of cattle presented for harvest. Subiliac LNs were collected from 1,501 carcasses
of feedlot-finished cattle and 1,826 carcasses of cows presented for slaughter for human consumption. Salmonella was
recovered from 8.0%of LNs; after accounting for clustering, prevalence was estimated at 3.0%(95% CL 1.7, 5.2%).
Salmonella was 8.7 times more likely to be recovered from LNs from feedlot cattle than cows. Salmonella was recovered more
frequently from LNs of feedlot cattle in summer/autumn (14.6%) compared to winter/spring (1.3%). Salmonella was 2.3 times
more likely to be recovered in southern relative to northern abattoirs. 34%of LNs harbored Salmonella at 3.0 log10 cfu/LN or
greater. Montevideo and Anatum were the most common serotypes observed and 86%of Salmonella were pansusceptible.
In addition, feces, hide swabs, and mandibular, mediastinal, mesenteric and subiliac LNs were collected from 68 carcasses.
Salmonella was recovered from 94.1, 100, 55.9, 7.4, 91.2 and 76.5%of samples, respectively. Substantial diversity was
observed within animals. Evidence of serotype dependency across sample type (LNs versus feces/hides) was observed.
However, the likelihood of recovering Salmonella from one sample within a carcass appeared independent of recovery from
other samples. Salmonella harbored within LNs are protected from usual microbial interventions used on beef carcasses and
may be a source of Salmonella contamination of ground beef. We believe it uncommon that Salmonella spreads to peripheral
LNs systemically from the GIT in healthy animals. Rather, we believe that a transdermal route of infection, possibly via biting
flies, contributes to a significant proportion of the Salmonella recovered from peripheral LNs.
INTRODUCTION
Despite sustained and coordinated efforts between publichealth agencies and food production
industries,thehumanincidenceofsalmonellosishasnotappreciablydecreasedoverhelast10to15
years in the USA. Furthermore, production controls that were expected to control foodborne
pathogens do not seemthat have had thedesired effect on Salmonella. Forexample, from 2001to
present,thelikelihoodofrecoveringE.coliO157fromrawgroundbeefhasdecreasedmorethan90%
because of abattoir microbial process control. Yes the likelihood of recovering Salmonella from
groundbeefhasnotchangedoverthesameperiodoftime.
Theseresultsaresomewhatparadoxicalasinlaboratorystudies,Salmonellaonbeefsurfacesactsin
the same manner as E. coli O157 in response to interventions (such as hot water or lactic acid).
Preliminary work indicated that Salmonella harboured within lymph nodes of cattle might be a
possible explanation for these observations. More specifically, because peripheral lymph nodes are
consideredbeefwhenincludedinproductattheirusualproportions,iftheycontainSalmonella,then
theymaybeasourceofSalmonellaingroundbeef.Ifso,thisrouteofgroundbeef(akamincedbeef)
contaminationwouldevadetheusualsurfaceinterventionsappliedtocarcassesintheUSA.
The objectives of the studies described herein were to a) evaluate the burden of Salmonella within
peripherallymphnodes(LNs);andb)exploretheSalmonelladiversitywithinLNsofcattlepresented
forharvest.
261
MATERIALANDMETHODS
Aseriesofstudieswereconductedtoachievetheobjectivesofourwork.
Study 1 an initial exploration was performed to evaluate the burden of Salmonella within lymph
nodes of cattle. Subiliac (aka prefemoral) lymph nodes were collected from cattle at harvest. The
populationsofinterestwerecattledeliveredto7abattoirs.Thesecattleoriginatedfromfeedlotsor
were adult cattle (cows) removed from breeding herds for productivity reasons (such as failure to
conceive).
Study 2 a yearlong surveillance program was undertaken to describe the season, region, and
populationofcattleatriskofcarriageofSalmonellawithinlymphnodes.Subiliaclymphnodeswere
collectedfromcattleatharvest.Thepopulationsofinterestwerecattledelivered12abattoirs.These
cattleoriginatedfromfeedlotsorwereadultcattle(cows)removedfrombreedingherds.
Study3aninitialexplorationwasperformedtoevaluatewithinanimalSalmonelladiversity.Feces,
hide swabs, and subiliac, mandibular, and mesenteric LNs were collected from cattle presented for
slaughterinaMexicoabattoir.
Study4asystematicevaluationofwithinanimalSalmonelladiversitywasperformed.Afecalswab
and6lymphnodeswerecollectedfrom100carcassesoffeedlotcattlepresentedforharvestataUSA
abattoir.
Similar methods were used for all studies in that lymph nodes were cultured for Salmonella using
selective enrichment, immunomagnetic capture, and plating on to selective agar as described (1).
Quantitative estimates were generated. Salmonella isolates were subjected to susceptibility testing,
and serotype determination. For isolates from samples collected in Mexico, pulsedfield gel
electrophoresis was performed. DNA was extracted from isolates from Study 4 and a region of the
ClusteredRegularlyInterspacedShortPalindromicRepeats(CRISPR)wasamplifiedandsequenced.
RESULTS
Study13,327lymphnodeswerecollectedfromaconveniencesamplefrom7abattoirs.Salmonella
was recovered from 8.0% of subiliac lymph nodes. Prevalence varied by season, region, and animal
type.Salmonellawas8.7timesmorelikelytoberecoveredfromLNsfromfeedlotcattlethancows.
Salmonella was recovered more frequently from LNs of feedlot cattle in summer/autumn (14.6%)
compared to winter/spring (1.3%). Furthermore, Salmonella was more likely to be recovered from
lymphnodescollectedfromfeedlotcattleinsouthernabattoirs.Medianprevalenceinlymphnodesof
feedlot cattle (11.8%) was greater than that observed in nodes from cattle removed from breeding
herds(0.65%).
Study2Salmonellawasrecoveredfrom6.0%of4,764lymphnodescollectedin12abattoirsinthe
USA.ResultsweresimilartoStudy1.Interestingly,whileadistinctseasonalvariationisevidencefor
lymph nodes collected from feedlot cattle in southern abattoirs, no such seasonal effect is observed
amonganimalsremovedfrombreedingherds(typicallyadultcows;Figure1).

262
0
20
40
60
80
100
FEB-
MAY
J UN-
J UL
AUG SEP-
OCT
NOV
P
r
e
v
a
l
e
n
c
e

(
%
)
Adult cattle (979)
Feedlot (1,049)
Figure1.PrevalenceofSalmonellainlymphnodesofhealthycattlepresentedforharvest.
Inbothstudies1and2,S.Montevideowasthemostcommonserotyperecoveredandaccountedfor
approximately 40% of isolates. The vast majority of isolates (~85%) were pansusceptible to all
antimicrobialdrugstested.Geometricmeanconcentrationwas1.75log
10
cfu/gLN;however,~20%of
LNs contained Salmonella at greater than 4 log
10
cfu/LN. Moreover, we have observed some
estimates greater than 6 log
10
cfu/LN. No meaningful association of LN weight and likelihood or
Salmonellarecoveryorconcentrationwasdetected.
Study3Salmonellawasisolatedfrom100%(hideswabs),94.1%(feces),and91.2%,76.5%,55.9%,
and7.4%ofmesenteric,subiliac,mandibularandmediastinalLN,respectively(2).S.Meleagridiswas
more likely recovered from lymph nodes than feces or hides whereas S. Kentucky was more likely
recoveredfromfecesandhidesthanfromlymphnodes.Moreover,serotypeandPFGEdiversitywas
observedamonglymphnodeswithinanimals.Insomeinstances,adifferenceserotypewasidentified
ineachsampletype.
InStudy4,Salmonellawasrecoveredfrom32and75%lymphnodesandfecalsamples,respectively.
Assamplingcontinuedintheautumn,theprevalencedeclined(Table1).
Table1.ObservedprevalenceofSalmonellainLNofhealthycattlepresentedforharvest.
Visit Carcasses
sampled
Fecal
prevalence
LN
prevalence
Positive
in 1 or
moreLN
Positive
inall6LN
1 15 80%
(n=15)
58.9% 100% 20%
2 30 100%
(n=6)
56.1% 96.7% 23.3%
3 20 95%
(n=20)
15.8% 50% 5%
4 35 47.8%
(n=23)
37.1% 37.1% 0%
A significant time by LN type interaction was observed in that during visits 1 and 2, prevalence was
greater in subiliac LNs than in prescapular LNs which in turn was greater than the prevalence in the
poplitealLNs(P<0.05forallcomparisons).However,duringthefinal2visitswhenoverallprevalence
haddecreasednodifferencesinprevalencewereobservedbetweenLNtypesintermsofprevalence.
263
DISCUSSION
SalmonellacanberoutinelyrecoveredfromtheLNsofhealthycattlepresentedforharvestparticularly
insouthernpartsoftheUSduringcertaintimesoftheyear.SimilarregionalvariationinSalmonella
prevalence is observed in feces (3, 4). Because peripheral LNs are widespread and intimately
associated with lean and adipose tissue, they are typically incorporated into minced beef. As such,
SalmonellawithinLNsofcattlelikelycontributestoSalmonellawithinmincedbeef.
The traditional paradigm is that Salmonella escapes the GIT and disseminates systemically. Yet we
believethisviewpointisinsufficienttoexplaintheobservedecologyanddistributionofSalmonellain
peripheral LNs. Saliently, if Salmonella was spread systemically, one would expect dependence
betweenlymphnodeswithinacarcassintermsofthelikelihoodofdetectionaswellastheserotype
recovered across LNs. This is, however, rarely observed. If we explore the data from Study 3, for
example, if sample types were indeed independent from one another within a carcass, the joint
probabilitythatallsampleswouldhavebeenpositivewas2.7%whereastheobservedprevalencewas
similarat2.9%.Moreover,frequentlyadifferentserotypewasobservedwithineachLNexamed.
We propose an alternative hypothesis namely that the primary route of infection for Salmonella
observed in the peripheral LNs in our studies was transdermal. More specifically, skin abrasions or
diseasesoftheintegument(suchasinterdigitaldermatitisorotherconditionscausedbyinvertebrates)
provides an avenue for Salmonella to enter the host and subsequently drain to the regional lymph
node. If so, we further believe that these data indicate a broader exploration of the ecology of
Salmonellainanimalpopulationsiswarranted.Indeed,simplefecaloralmodelsofthespreadofsome
serotypeswithincattlepopulationsisinadequatetoexplaintheobservedecology.
REFERENCES
1.Graggetal.FoodbornePathogDis.Inpress.
2.Graggetal.ApplEnvironMicro.Inreview.
3.Kunzeetal.ApplEnvironMicro.2008;74(2):345351.
4.Loneraganetal.FoodbornePathogDis.2012;9(6):549555.
264
EstimatingtheprevalenceofReptileacquiredsalmonellosis
inEnglandandWales
usinganoveldemographicbasedattributionmodel
ChantilSINCLAIR,ChrisLANEandTansyPETERS
GastrointestinalandEmergingZoonoticInfectionsDept,HealthProtectionAgency,UK
ABSTRACT
The proportion of UK households keeping exotic pets has grown considerably in recent years. Reptile acquired
salmonellosis (RAS) is a well-documented source of infections in humans; predominantly in children. Reptiles are known
to carry Salmonella, and the husbandry of these pets allows for both direct contact and environmental cross-
contamination of the home.
In England and Wales routine laboratory-based surveillance of Salmonella infections is insufficient to ascertain the
source of infection in sporadic cases. While previous UK studies have estimated the proportion of RAS to be 1%, this is
significantly lower than studies within other countries.
This study aimed to develop an attribution model which will improve the estimate of RAS in the UK using existing
surveillance data; based on serovars known to be associated with reptiles and case demographics seen in previous
outbreaks.
An outbreak associated with reptile feeder mice between 2008 and 2011 caused infections in over 530 cases; 51% were
children under 10 years of age. Similarly, laboratory surveillance of subspecies III salmonellae reports, known to have
reptile links, indicates 57% in this age group.
Indigenous Salmonella reports between 2007 and 2011 were analysed by serovar and age group. All serovars
representing greater than 40% in children were defined as Reptile types. These were compared against a list of
serovars found in reptiles identified in scientific and epidemiological literature. The correlation between the two lists was
used to determine the sensitivity of correctly identifying a serovar as Reptilian, and used to determine the number of
infections reported by those serovars each year.
The attribution model indicated an increasing trend in RAS between 2007 and 2011 reflecting the increase in exotic pet
ownership. We determined that up to 7.5% of indigenous salmonellosis may be reptile acquired, supporting the findings
of RAS in other countries as reported in the literature.
INTRODUCTION
Reptile acquired salmonellosis (RAS) is a welldocumented source of infections in humans;
predominantly in children. Reptiles are known to carry Salmonella, and the husbandry of these
pets allows for both direct contact and environmental crosscontamination of the home. The
proportionofUKhouseholdskeepingexoticpetshasgrownconsiderablyinrecentyears,withthe
latest figures suggesting that eight million reptiles and amphibians were being kept as pets in
2008,comparedwithonlyfivemillionin2004[4],andcurrentindicationssuggesttheproportion
isincreasing.
Routine laboratorybased surveillance of Salmonella infections is insufficient to ascertain the
sourceofinfectioninsporadiccases.WhilepreviousUKstudieshaveestimatedtheproportionof
RAStobe1%[1]thisismarkedlysignificantlylowerthanthe36%estimatedinstudiesconducted
intheUSAandCanada[3,5].
An outbreak of Salmonella Typhimurium DT191a in England and Wales associated with reptile
feedermicebetween2008and2011causedinfectionsin536cases;49%werechildrenunder10
years of age (37% were children under 5 years of age). A matched casecontrol study found a
strongassociationbetweenillnessandkeepingreptiles(mOR16.82,95%CI2.78)[2].Similarly,
laboratorysurveillanceofsubspeciesIIISalmonellareports,knowntohavereptilelinks,indicates
59% of cases occur in children below the age of 10 years compared to only 22% of S. Enteritidis
and 26% of S. Typhimurium (excluding DT 191a) cases, the two predominant serotypes reported
fromsubspeciesI.
265
MATERIALANDMETHODS
ThisstudyaimedtodevelopanattributionmodelwhichwillimprovetheestimateofRASintheUK
usingexistingsurveillancedata;basedonserovarsknowntobeassociatedwithreptilesandcase
demographicsseeninpreviousoutbreaksknowntohavereptileassociation.Thehypothesisbeing
investigatedis:theattributionofRAScanbedefinedbyademographicmodelwheregreaterthan
40%ofinfectionsforanygivenserovararewithinchildrenagedlessthan10years.
Five years of Salmonella reports (2007 to 2011) were extracted from the laboratory surveillance
system. All typhoidal cases and those where a history of foreign travel was recorded were
excludedfromtheanalysis.
Thedatasetconsistedofbasicdemographicvariablessuchasage,gender,sampledateandresult
information, including serovar, Salmonella enterica subspecies group and the date which the
resultwasuploadedfromthelaboratorysystem.
All reports were then assigned two additional variables to determine their potential reptile
associationbasedonthehypothesisunderinvestigation(hypotheticalreptileassociation=HRA)or
known reptile association through the literature (literature reptile associated = LRA). If a serovar
wasrepresentedbygreaterthan40%intheunder10yearagegroup,thentheserovarwascoded
asHRA
+
,iftheproportionwasbelow40%theserovarwascodedasHRA
.Ifaserovarwascitedin
the literature search as being isolated from reptiles either in case studies or reptile surveys, the
serovar was defined as LRA
+
; if a serovar was not cited as being associated with reptiles in the
literatureitwascodedasLRA
Although some phagetypes of S. Enteritidis and S. Typhimurium have been found in literature to
be associated with reptiles the majority are not. Therefore all cases of S. Enteritidis and S.
TyphimuriumwererecodedasLRA
.Knownoutbreaksassociatedwithreptileswerealsoremoved
(S.TyphimuriumDT191a).AllanalysiswascarriedoutusingStata12.Thecorrelationbetweenthe
twolistswasusedtodeterminethesensitivityofcorrectlyidentifyingaserovarasReptilian,and
usedtodeterminethenumberofinfectionsreportedbythoseserovarseachyear.Themodelwas
thenusedtoestimatetheproportionofindigenoussalmonellosiswhichmaybereptileacquired.
RESULTS
There were 234 different serovars of Salmonella reported between 2007 and 2011 which were
identified with a >40% proportion in children and 205 serovars were defined in the available
literatureasReptileassociatedwithacorrelationof0.37betweenthetwolists.
When comparing the hypothetical case reptilian status with the reptile status assigned by the
literature,thesensitivitywas14.7%(95%CI14.415.1)whilethespecificitywasmuchhigherat
98.0% (95% CI 97.9 98.2). Both the positive and negative predictive values fell between 73.4%
and75.3%.WhenconsideringonlysubspeciesIisolatesthesensitivitydecreasedto10.2%(95%CI
9.910.5)butincreasedto98.8%(95%CI98.099.7)whensubspeciesIisolateswereexcluded
(Table1).
The attribution model indicated an increasing trend in RAS between 2007 and 2011, from 3.8%
(95%CI3.44.2)to7.4%(95%CI6.78.0)reflectingtheincreaseinexoticpetownership(Figure
1). We determined that up to 7.5% of indigenous salmonellosis may be reptile acquired,
supportingthefindingsofRASinothercountriesasreportedintheliterature.

266
DISCUSSION
Prior to the development of this model, only 1% of reported cases of salmonellosis were
attributed to reptile sources (based on calculations from a study carried out between 2004 and
2007). This would equate to approximately 90cases in 2011. The suggested model indicates that
7.4% of indigenous, nontyphoidal Salmonella isolates in England and Wales in 2011 may be
attributable to reptile sources. The proportion associated with this source is increasing with the
growing trend and popularity of keeping reptiles as pets. In 2011, 5,956 human cases of
indigenous nontyphoidal Salmonella were reported to the Health Protection Agency. Based on
thesefigures,theattributionmodelpotentiallyassociatesbetween399and476caseswithReptile
sources. Whilst travel reporting is known to be under ascertained [6], the extent of under
ascertainment can be addressed. Extrapolating from the findings of Zenner et al the number of
cases potentially attributable to RAS accounting for travel underascertainment is reduced to
between258and308casesofindigenous,nontyphoidalSalmonellainfectionsin2011,or2.9%
3.4% of all Salmonella reports in that year. Whilst these figures are considerably greater than
previousstudiesinEngland,theyremainclosertothelevelsseeninEuropeanStatesandtheUSA,
andmaytakeintoaccountthechangingtrendinreptileownershipsincethemid2000s.
The increased sensitivity in correctly identifying a true reptile serovar in nonsubspecies I
Salmonella is expected given the host adaptive origins of many of these serovars to the reptile
world.Whilsttherewasalimitedcorrelationbetweenthelistofserovarsnotedasreptilianinthe
wider literature, and those indicated by the hypothetical demographic model, this may be a
reflection of the underascertainment on human isolates. Not all serovars isolated from reptiles
will cause human infections; many may in fact be truly host adaptive with no human
pathogenicity.
All attribution models incur some level of ascertainment bias, either through generalisation or
excessive assumptions. There are a number of limitations to this study; firstly it was not possible
tovalidatethismodelusingcurrentroutinelaboratorybasedsurveillance.Insteadthemodelwas
developedusingthedemographicsofpreviousoutbreaks,casestudiesandpublishedliteratureon
RAS.Theresultswerethencomparedwithfindingsreportedinstudiesfromothercountries.
Theresultantestimatesfromthemodelassumethatallcasesofagivenstrainwheregreaterthan
40% of cases are under 10 years old, are associated with RAS, including those cases in adult age
groups, resulting in potential over ascertainment. Equally, serovars with greater than 40%
representationinunder10yearoldsmaypotentiallybeattributabletofoodsspecifictothatage
group, although the authors believe these events would have been detected and investigated as
outbreakswerethisthecase.
Serovars commonly associated with RAS tend to be infrequent in their reporting; 153 serovars
withbetween1and718cases(accountingforatotalof10,051cases)overthe5yearperiod.The
inclusion of serovars with small numbers reported (<5 cases) means that HRA status may have
been incorrectly assigned resulting in some over estimation. The belief that general practitioners
are more likely to take stool specimens from children suggests that this could cause an over
estimation of RAS through misclassification of a serovar as Reptile acquired. Equally, adults are
lesslikelytoattendaphysicianforgastroenteritis,leadingtounderestimationbythemodel.
Anotherlimitationwasthatanumberoftheresearchstudiesusedintheliteraturesearchusedto
identify whether serovars had been associated with reptiles did not include phage typing.
Therefore, it was not possible to split serovars such as Enteritidis and Typhimurium into reptile
and nonreptile types. Because of this, and because it is widely accepted that most phage types
267
are associated with exposures other than reptiles, all cases of Enteritidis and Typhimurium were
recoded as nonreptilian. Likewise, because further typing for cases of subspecies III salmonella
wasnotalwaysavailable,itwasnotpossibletobreakthesedownfurtherintoreptilianandnon
reptiliantypes.Asthesetypesarepredominantlyassociatedwithreptiles,allcaseswerecodedas
reptilian.
Further analysis of the determinants used in the model described, including accounting for and
removing the adults misrepresented above, and the application of a cutoff value to account for
infrequently reported serovars need to be carried out. However the existing model provides a
more balanced and up to date representation of potential RAS infections in the UK that can be
appliedtofuturesurveillance.
For validation purposes the only approach would be to select and interview random cases of the
humanserovarsdefinedasreptilianwithatooldesignedtopickupbothreptilianandfoodbased
exposures. Such an approach, though valid, would place heavy demands on already reduced
humanresources,butisneverthelessasuggestionforfurtherresearch.
REFERENCES
1. Aiken, A. M., Lane, C., & Adak, G. K. (2010). Risk of Salmonella infection with exposure to reptiles in
England,20042007.Eurosurveillance.
2. Harker, K. S., Lane, C., De Pinna, E., & Adak, G. K. (2011). An outbreak of Salmonella Typhimurium
DT191aassociatedwithreptilefeedermice.Epidemiology&Infection.
3. Mermin,J.,Hutwagner,L.,Vugia,D.,Shallow,S.,Daily,P.,Bender,J.,etal.(2004).Reptiles,Amphibians,
and Human Salmonella Infection: A PopulationBased, CaseControl Study. Clinical Infectious Diseases,
S25361.
4. Newman,C.(2013)ReptileExoticPetTradeAssociation(REPTA).PersonalCommunication.
5. Woodward, D. L., Khakhria, R., & Johnson, W. M. (1997). Human Salmonellosis Associated with Exotic
Pets.JournalofClinicalMicrobiology,27862790.
6. Zenner, D., Gillespie, I. (2011). Travelassociated salmonella and campylobacter gastroenteritis in
England: estimation of underascertainment through national laboratory surveillance. J Travel Med. 2011
NovDec;18(6):4147

268
TABLESANDFIGURES
Table1:SensitivityandSpecificityoftheattributionmodel
a) Allcases
LRA+ LRA Total
HRA+ 1,480 497 1,977 PositivePredictiveValue 74.86(74.8675.31)
HRA 8,571 24,679 33,250 NegativePredictiveValue 74.22(73.7774.68)
Total 10,051 25,106 35,227 Sensitivity 14.72(14.3515.09)
Specificity 98.03(97.8898.17)
b) Group1casesonly
LRA+ LRA Total
HRA+ 970 457 1,427 PositivePredictiveValue 67.97(67.4868.47)
HRA 8,565 24,659 33,224 NegativePredictiveValue 74.22(73.7674.68)
Total 9,535 25,116 Sensitivity 10.17(9.8510.49)
Specificity 98.18(98.0498.32)
c) NonGroup1casesonly
LRA+ LRA Total
HRA+ 510 40 550 PositivePredictiveValue 92.73(90.6194.85)
HRA 6 20 26 NegativePredictiveValue 76.92(73.4880.36)
Total 516 60 Sensitivity 98.84(97.9699.71)
Specificity 33.33(29.4837.18)
Figure1:ThepredictedproportionofReptileAssociatedSalmonella
0.0
2.0
4.0
6.0
8.0
10.0
2007 2008 2009 2010 2011
P
r
o
p
o
r
t
i
o
n

o
f

R
e
p
t
i
l
e

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May 27-28-29, 2013
Saint-Malo France

Session 5

Chairpersons:

Posters
270
Alongitudinalstudy,fromweaningtocarcass,
todeterminetheprevalence
ofSalmonellaonpigfarmsinNorthernIreland
ElizabethMAGOWAN
1
,ElizabethBALL
1
,RobertMADDEN
2
,GintareBAGDONAITE
3
andAlanGORDON
4
1
Agrifood&BiosciencesInstitute,Agriculture,HILLSBOROUGHBT266DR,NorthernIreland;
2
AgriFood&BiosciencesInstitute(AFBI),FoodMicrobiology,BELFASTBT5PX,NorthernIreland;
3
AFBI,VeterinarySciencesDivision,BELFASTBT43SD,NorthernIreland;
4
AgriFood&BiosciencesInstitute(AFBI),Biometrics,BELFASTBT5PX,NorthernIreland
INTRODUCTION
As part of an industry sponsored investigation into the prevalence of salmonellas in Northern
Irelandpigherds,astudywasundertakentoinvestigatethepresenceofsalmonellasonpigfarms.
InvestigationsmadeuseoftheELISAtestingroutinelyundertakenonpigsatslaughter,theresults
ofwhichwerefedbacktothefarmer.Inadditionbloodsamplingofpigsonfarmwasundertaken,
aswasfaecalsampling.
MATERIALANDMETHODS
Bloodsamplesweretakenfrompigsfromfiveherds(n=250)everythreeweeksfromweaningto
slaughter (22 weeks of age). All herds had a high prevalence of antibodies to salmonella (using
meat juice ELISA testing). Faeces samples were also taken from the pens in which the pigs were
housed, to correspond with the blood samples. Blood samples were also taken from the sows
(n=50)atweaning.
RESULTS
Mostofthesows(60%)hadantibodiestoSalmonellaandthepredictedchanceofpigletbloodat
weaninghavingapositiveantibodyresponsewashigh,91%,(P<0.01)ifthemothersfaecestested
positive for Salmonella. On all farms Salmonella prevalence in faeces was highest just before
slaughter (21%). S. Typhimurium was the predominant serovar found, with S. Derby being the
second most common. Blood antibody responses immediately pre slaughter averaged 37%
positive.
DISCUSSION
Overall,fewstatisticallysignificantrelationshipswerefoundbetweenthepresenceofSalmonella
infaeces,andbloodantibodystatus,acrossthesevensamplingperiods.
Overall, 45% of the pigs studies carried Salmonella in their large intestine at slaughter. If the
faeces sample taken from the preslaughter pen was positive for Salmonella, there was a strong
chance(95%,P<0.05)thatfaecesinthegutoftheanimal,sampledafterslaughter,wouldalsobe
positive. However, there was also a 35% chance (P<0.05) of postmortem gut faeces being
positive when faeces sampled just before slaughter was negative. This marked increase in
Salmonellaprevalencefromthefarm,totheabattoir,suggeststhatinNorthernIrelandtransport
andlairagehaveasignificantimpactofonSalmonellaprevalenceinthegutofanimalsatthepoint
of evisceration. This is despite the relatively short journeys undertaken, in a moderate climate.
Furtherstudywillberequiredtodefinethesourcesofcontamination,andprophylacticmeasures.
271

Salmonella in pig farms in Reunion Island :
Prevalence assessment and identification of risk factors

Claire TESSIER, Laura ATIANA, Martine DENIS and Eric CARDINALE
Email: laura.atiana@gmail.com;
France, Ile de la Runion
Abstract:
Salmonella is the second cause of foodborne diseases in France; and pork and pork products
are regularly incriminated. In Reunion Island, Salmonella is also a public health burden. The
pig industry will be concerned, soon or later, by a new European regulation regarding
Salmonella; and that is why it is necessary before its implementation, to better understand
the ways of infection in the pig farms to propose adequate specific control measures. Our
epidemiological study aims (i) to determine Salmonella prevalence of fattening pigs, (ii) to
identify the main risk factors for infection and (iii) to identify the main origins of infection by
comparing genetic (by PFGE) and antibiotic susceptibility profiles of the isolates.
Fifty farms (farrow-to finish and multiplier) were randomly selected and visited 4 times: pre-
slaughtering of previous batch, after cleaning and disinfection, beginning and end of the
fattening period for the studied batch. A questionnaire was submitted to the farmers. Pools
of fresh faeces, gauze socks and gauze swabs were sampled to assess the bacteriological
status of these pig farms. Samples from rodents and cockroaches were also collected.
To date, we observed a massive contamination (76%7%) of the pig farms, maybe related to
unthorough decontamination procedures (68% of the farms were positive at this stage).
Moreover, 20% of the rats and 27% of cockroaches were also positive to Salmonella. The
main serovars isolated are Salmonella Typhimurium, 4,[5],12,i:-:, Derby, Livingstone, Rissen,
Newport and Weltevreden.
The present ongoing analyses (molecular and statistical) will highlight the risk factors and
potential sources of contamination; these results will confirm the measures to be
immediately taken to lower such a high prevalence.
Controlling infection at the farms will finally reduce the overall contamination of the pork
products and then reduce the risk for the consumer to be infected with Salmonella.

272
LongitudinalinvestigationofSalmonellaspp.
fromfarmtoforkinthepigindustryofReunionIsland
ClaireTESSIER
1,2
,LauraATIANA
1
,AlexisLEHARDY
3
,MartineDENIS
3
andEricCARDINALE
1
1
CIRAD,BIOS,UMRCMAEECRVOI,SAINTDENIS,Runion
2
CooprativedesProducteursdePorcsdelaRunion,SAINTPIERRE,Runion
3
Anses,HQPAP,PLOUFRAGAN,France
INTRODUCTION
Our epidemiological study aims to identify the pathways of Salmonella contamination along the
pigchainindustryofReunionIsland.
MATERIALANDMETHODS
ThreeSalmonellapositivefarmsA,BandCweremonitoredfromfarrowingtotheporkcuts.Three
sows and 5 piglets by sow were selected from one batch per farm. At farm level, Salmonella
excretion was detected by individual samples of feces, after farrowing and throughout each
growing stage. At slaughterhouse level, gauze swabs from pigs entering and from carcasses after
polishing, splitting and chilling, caecal contents and pork cuts were sampled. Environmental
sampleswerecollectedatbothfarmandslaughterhouselevels.Theywerecollectedateachvisit
infarmsandallalongtheslaughteringandcuttingprocessatthebeginningoftheday,beforethe
slaughterofthefollowedpigbatch.
ThemicrobiologicalculturewascarriedoutaccordingtoanadaptedmethodfromISO6579Annex
D.Atotalof900sampleswereanalyzed.OneisolateperpositivesamplewasgenotypedbyPFGE
usingXbaIenzymeandoneisolatebypulsotypewasserotyped.
RESULTSDISCUSSION
The 301 collected isolates were divided into 51 XbaI pulsotypes and 13 serovars: monovariant of
Typhimurium 4,12:i: (125), Rissen (63), Livingstone (32), Typhimurium (29), Derby (18), London
(12), Weltevreden (12), Senftenberg (3), Bredeney (2) , Newport (2), Blockley (1), Durban (1) and
Give(1).Someserovarswerepredominantinsomefarms:4,12:i:infarmAandB,RisseninfarmB
andTyphimurium/LivingstoneinfarmC.
DistributionofpulsotypesinfarmBfromfarrowingtoporkcutsisillustratedinFigure1.
Waterandfeedsamplesreturnallnegative.
Although floor of maternity and corridor were positive for Salmonella, neither sows nor piglets
excreted Salmonella until the end of nursing (2/3). Passive maternal immunity of sows may have
protectedpreweanedpigsfromSalmonellacontamination(4,6).
Piglets started to excrete Salmonella a few weeks after the beginning of postweaning (PW).
Pulsotypes were common to the ones found in the room after cleaning and disinfection (C&D),
and in corridor before the beginning of PW stage (2/3). These results confirm that horizontal
transmissioninPWmaybeduetoresidualcontaminations(1,2,4).
Afterwards,untiltheendofpigfattening,pulsotypeswereidenticaltotheonespreviouslyfound
duringPWorinfarmenvironment(includingotheranimalproductions).
For one of the 3 farms, pigs were excreting Salmonella at a late fattening stage whereas, in the
otherones,thenumberofcontaminatedpigsdecreasedduringthefatteningperiod.
Loading bay (2/3), truck (3/3) and lairage room at slaughterhouse (3/3) were contaminated with
newpulsotypes.
Beforeslaughteringandcutting,equipmentandtoolswerenegativeexceptknives(1/3).However,
thispulsotypewasnotdetectedinanyothersample.
273
Atslaughterhouse,justpriortoslaughter,pigswerecarryingbothpreviouslydetectedpulsotypes
and new ones. These results confirm that transport and lairage are important stages of cross
contamination(3,5).Somepulsotypesweredetectedfromcarcassesafterpolishing(3/14forfarm
B) and after splitting (2/14 for farm B and 2/15 for farm C). However, all samples collected after
splitting steps were negative. This suggests that slaughter process reduce Salmonella
contamination,notablyafterchilling.
REFERENCES
1.Berendsetal.Int.J.FoodMicrobiol.1996;30:3753
2.Funketal.J.Vet.Diagn.Investig.2000;12:412418
3.Hurdetal.Appl.Environ.Microbiol.2002;68:23762381
4.Krankeretal.J.Clin.Microbiol.2003;41:22822288
5.VieiraPintoetal.J.FoodMicrobiol.2006;110:7784
6.Walesetal.Vet.Rec.2009;165:648657
Figure1:DistributionofSalmonellapulsotypesdetectedalongtheinvestigationinfarmBfromfarrowingto
porkcuts
Postweaning Fattening Farrowing Nursing
Farrowing
+4days
After
disinfection
After
disinfection
After
disinfection
End
+1
month
End Weaning Entering
+1
month
+1
month
+2
weeks
Slaughterhouse Cuttingplants
Lairage Evisceration Loading
Transport
Polishing Splitting
2nd
cutting
Sows
Pigs
Pigs*
Caeca*
Carcasses*
Pork cuts*
Room
Corridor
Adjacent
environment
Other
productions
Truck
Lairage room
Equipment
Chilling
Loading bay
1st
cutting
/

/

/ X06 X06 /
X06
X09
X12
X06
X02
X15 X06

X05 X06 X06 X06 X08 X06 X15 X02 X06
X01 / / / X06 / /

/ / / X06
/ / / / / / / / / / / X11
/ / X02 / / X02 / / X11 X13

/ / X03/X04

/ / / /

X06 X06/X11
/
X06/X18/
X21/X22/X25
X02/X06/X18/
X23/X26
X15/X16/X26 / X24

/

X16
X18/X19
X20(knife) / /

/, not done. , negative sample. *, sampled after step indicated in the arrow.
, sampled before
stepindicatedinthearrow.PulsotypesX01,X03,X05,X06,X09,X10,X11,X15,X17,X21,X22and
X23areS.Typhimurium;X02,X12andX24areS.Livingstone;X16andX18areS.Derby;X19and
X25 are S. London; X20 is S. Bredeney and X26 is S. Rissen. X06 (in bold) is a predominant
pulsotype. X16 and X18 (underlined) are pulsotypes identified from truck and lairage room and
detectedoncarcasses.
274
Trackingintrucks,lairage,slaughterlineandquartering
asausefultooltostudySalmonellaprevalence
inafreerangepigprocessingplant
JaimeGMEZLAGUNA
1
,ManuelaHERNNDEZ
1
,LucaREGUILLO
1,2
,InmaculadaLUQUE
2
,
SilviaHERRERALEN
3
,AlfonsoMALDONADO
2
andRafaelJ.ASTORGA
2
1
CICAPAgrifoodResearchCenter,14400Pozoblanco,CRDOBA,Spain;
2
AnimalHealthDepartment,VeterinaryFaculty,UniversityofCordoba,14071CRDOBA,Spain;
3
NationalCenterofMicrobiology,InstituteofHealthCarlosIII,MADRID,Spain
INTRODUCTION
Salmonellaisconsideredasthesecondmostimportantfoodbornepathogenworldwide,justafter
Campylobacter spp., and is commonly recovered from poultry and pigs and their products in the
EuropeanUnion(1).Focusingonpigandporkproduction,Spainoccupiessecondplaceinbothpig
andporkproductionandisconsideredasthehighestporkconsumerinEurope(2).
Pigs may acquire Salmonella infection from different sources at the final stages of the pig
production with transport and lairage identified as critical points. The aims of this study were to
determine the prevalence of Salmonella spp. in Trucks, Lairage, Slaughter line and Quartering
(TLSQ) along a freerange pig production plant and to genetically characterize the Salmonella
isolatesrecoveredinordertodeterminecarcasscontamination.
MATERIALANDMETHODS
During2009and2010asystematicsamplingfromeightdifferentfreerangepigproductionunits
wascarriedout(TLSQ1 toTLSQ8assays).Tenfatteningpigsperherdweresampled.Sixdifferent
stagesoftheproductionchainweretestedineachTLSQassay(Table1).
Allsampleswerecollectedintosterilecontainerorplasticbagwithspongesandtransportedfrom
the slaughterhouse to the laboratory to be processed on the same day according to ISO 6579:
2002procedureforSalmonellacultureandisolation.
Salmonella isolates were typed by means of agglutination techniques (Statens Serum Institut,
Denmark), and a Phage's panel (International Reference Laboratory of Phage typing, Colindale,
London, England). These procedures were carried out at the National Center of Microbiology
(InstituteofHealthCarlosIII,Madrid,Spain).
ThePulsedFieldGelElectrophoresis(PFGE)techniquewasperformedasoutlinedbythePulseNet
protocolbyusingCHEFDRIIISystem(BioRadLaboratories,Hercules,CA,USA).
TheroleofeachphaseasariskfactorwasevaluatedbymeansofOddsratio/CI
95
and
2
estimate
(Winepi,VeterinaryMedicineFaculty,UniversityofZaragoza,Spain).
RESULTS
Atotalof126Salmonellaisolatesfrom1160samples(10.86%)wererecovered(Table1).
The distribution of serotypes and phage types observed from trucks, lairage, environmental and
pig samples was: Bredeney (37 strains), Rissen (35), Derby (25), Typhimurium (11), Montevideo
(4),Israel(4),Anatum(2),Emek(2),MonophasicSalmonellaTyphimurium(mST)(1),Choleraesuis
v. Kunzendorf (1), Durban (1), Kentucky (1), London (1) and Sandiego (1). S. Typhimurium phage
typesU311(8strains),193(1),104b(1)andUT(1)wereidentified.Monophasicstrainbelongedto
U311phagetype.
PFGE was used to characterize the genotype of strains when the same Salmonella serotype was
isolatedfrompigandenvironmentalsamplesinaTLSQsampling(TLSQ1,TLSQ2andTLSQ4).TLSQ1
Derby strains showed three different genotypes with a similarity coefficient value range of 45
100%.TLSQ2Rissenstrainsshowedfourdifferentgenotypeswithageneticsimilaritycoefficientof
86.6100%.Finally,TLSQ4Bredeneystrainsshowedanindistinguishablegenotype.
275
Statisticalanalysisresultsshowedthatscaldingphasewasaprotectionfactorinthechain.
DISCUSSION
In the present study, several uncommon serotypes and phage types were obtained from carcass
and environment samples (Montevideo, Israel, Emek, Durban, Kentucky and Sandiego).
Interestingly, these serotypes are different from those previously recovered from pigs reared in
intensiveandoutdoorsystemsfromthesamegeographicalarea(3,4).
S. Derby, S. Rissen andS. Bredeney serotypes,were isolated from trucks to quartering, or from
slaughterlinetoquarteringinTLSQ1,TLSQ2andTLSQ4,respectively,pointingtoapotentialcross
contaminationbetweenenvironmentalandpigsamples.Thiscrosscontaminationwasconfirmed
by means of PFGEXbaI for the three TLSQs. Pulsotypes from TLSQ1 and TLSQ2 showed same
clonesorgenotypesforS.DerbyandS.Rissenintonsil,ileocecallymphnodeand/orcecalcontent
ofthesamepig,highlightingpigsasacarrierwiththeabilitytocontaminatemultiplecontacts(5).
Several authors have described that the presence of Salmonella in the intestinal tract of pigs
representsanincreasedriskforcarcasscontaminationsinceitisalsoassociatedwiththepresence
of the pathogen in the tonsils (6,7). PFGEXbaI from TLSQ4 showed an indistinguishable banding
pattern for all the strains analyzed pointing to the same epidemiological origin and confirming
cross contamination. In the remaining TLSQ assays different serotypes were obtained from each
stagesampled,assumingdifferentepidemiologicalorigins.
CONCLUSION
This study showed the isolation of different serotypes of Salmonella spp. from both pigs and
environmental samples, which constitutes a great risk for the contamination of pork from free
rangepigsbothpriorandpostslaughter.TheHazardAnalysisandCriticalControlPoints(HACCP)
atthepigslaughterhousemustincludeC+Dfortrucksandlairage,anintensiveC+Dprograminthe
preslaughterenvironmentaswellastheinclusionofnewchemicalagentswhichallowdecreasing
oreliminatingtheriskofSalmonellaspp.infectionorrecontaminationfromtheenvironment.
ACKNOWLEDGEMENTS
This study has been recently published in International Journal of Food Microbiology 162 (4854)
(2013).
REFERENCES
1. EFSA,EFSAJournal(2012)
2. Marquer,StatisticsinFocus.Eurostat(2010)
3. Astorgaetal.,J.FoodProt.(2007)
4. GmezLagunaetal.,Vet.J.(2011)
5. Kichetal.,SafeporkProceedings(2011)
6. Blahaetal.,SafeporkProceedings(1997)
7. VieiraPintoetal.,I.J.Food.Microbiol.(2006)
276
Table1.NumberofSalmonellaisolatesandprevalencepercentageineachTLSQassayintheexaminedfree
rangepigprocessingplant(TLSQ1toTLSQ8assays)
TLSQ T1 T2 L1 L2 S1 S2 S3 S4 S5 To Ln C E Q Total
1 2 1 2 0 11 0 0 0 0 2 6 7 2 0 33
2 2 0 0 1 3 0 0 0 0 6 6 4 0 1 23
3 0 0 3 0 0 0 2 0 0 0 0 2 8 1 16
4 1 1 0 1 4 6 0 0 0 0 0 1 8 1 23
5 0 0 0 0 0 0 0 0 1 1 0 1 0 0 3
6 0 3 0 0 5 0 0 0 0 0 0 1 0 0 9
7 1 1 0 0 6 1 0 0 0 5 1 1 0 0 16
8 1 0 1 1 0 0 0 0 0 0 0 0 0 0 3
Total of S.
isolates
7 6 6 3 29 7 2 0 1 14 13 17 18 3 126
Total of
samples
24 32 32 32 80 80 80 80 80 80 80 80 320 80 1160
Prevalence 29.17 18.75 18.75 9.38 36.25 8.75 2.50 0.00 1.25 17.50 16.25 21.25 5.63 3.75 10.86
T1,truckpriorC+D;T2,truckafterC+D;L1,lairagepriorentryofthepigs(cleanedanddisinfected);L2,lairageafterexitofthepigs;
Slaughterline(carcassessamples):S1(prescalding),S2(postscalding),S3(postflaming),S4(postevisceration),S5(postwashing);
To, tonsils; Ln, ileocecal lymph nodes; C, cecal contents; E, environmental samples from slaughter line and quartering plant
(scaldingandsterilizationwater,knivesandaxes,tables,meshgloves);Q,quarteringsamples(ham,shoulderandloin).
277
PrevalenceofSalmonellainNorthernIrelandpigsatslaughter
RobertH.MADDEN
1
,GintareBAGDONAITE
2
,ElizabethBALL
3
,MalcolmTAYLOR
1
andElizabethMAGOWAN
3
1
AgriFood&BiosciencesInstitute(AFBI),FoodMicrobiology,BELFASTBT5PX,NorthernIreland;
2
AFBI,VeterinarySciencesDivision,BELFASTBT43SD,NorthernIreland;
3
AFBI,Agriculture,HILLSBOROUGHBT266DR,NorthernIreland
INTRODUCTION
The prevalence of Salmonella being carried by pigs into abattoirs in Northern Ireland was
determined,basedonastudyof120localherds,attwoabattoirs.Thenumberofherdssampled
representsapproximately71%ofherdswhichkeepover20sows.
MATERIALANDMETHODS
Faecalsamplesfromfivepigsineachofthe120herdsweretakenfromthedistalendofthelarge
intestine shortly after evisceration, and collected in the gut room below the slaughter line to
whichallintestinesweredispatched.Sampleswereplacedinindividualcontainersandheldunder
refrigerationbeforebeinganalysedforSalmonellathefollowingdayusingmethodsbasedonISO
BSEN12824:1998.
Foreachherdonecompositesamplewaspreparedfromthefiveindividualsamples,andanalysed,
givingaprevalenceresultperherd.
RESULTS
Overall, Salmonella was found to be present in 66 herds (55%). The main serovars found were S.
Derby(28herds),S.Rissen(14herds)andS.Typhimurium(13herds).
DISCUSSION
When the Zoonoses National Control Programme (ZNCP) score of each of the 120 herds was
related to the presence or absence of Salmonella in the herd it was found that as ZNCP score
increased so did the proportion of herds that were found to be positive for Salmonella: 89% of
herdswithaZNCPscore>70%hadSalmonellawhereas53%ofherdswithaZNCPscore<10%had
Salmonella.Therefore,whilsthigherZNCPscoreswereassociatedwithSalmonellapositiveherds,
therelationshipwasnotaclosecorrelation.
ComparisonoftheresultswiththosefromCanada(1),andtheEU(2,3)indicatethattheyarenot
atypical, and that higher levels of prevalence can reflect infection acquired during transport and
lairage.AssociatedwithSalmonellapositiveherds,therelationshipwasnotaclosecorrelation.
REFERENCES
1. EFSA. 2009. Analysis of the baseline survey on the prevalence of Salmonella in holdings with breeding
pigsintheEU,2008.PartA:Salmonellaprevalenceestimates.EFSAJournal2009;7(12):1377.
2. Korsak,N.,Benoit,J,Groven,B.,Etienne,G.,Ghafir,Y.andDaube,G.2003.Salmonellacontaminationof
pigsandporkinanintegratedpigproductionsystem.JournalofFoodProtection,66:11261133.
3. Wilkins, W., Waldner, C., Raji, A., McFall, M., Muckle, A., MainarJaime, R.C. 2010. Comparison of
bacterial culture and realtime PCR for the detection of Salmonella in growfinish pigs in western Canada
usingaBayesianapproach.ZoonosesandPublicHealth57:115120.

278
OccurrenceandgeneticdiversityofSalmonella
inorganicandconventionalpigproductionsinFrance
AnnalleKEROUANTON,ValrieROSE,MaximeEVEN,EmmanuelleHOUARD
andMartineDENIS
ANSES,HygieneandQualityofPoultryandPigProductsUnit,BP53,sitedesCroix,
22440Ploufragan,France
INTRODUCTION
The objectives of this study were 1) to assess the occurrence of Salmonella in organic and
conventionalpigproductions,2)toevaluatethegeneticdiversityofstrainsisolatedfromthesetwo
productions, and 3) to estimate the crosscontamination on slaughter line between conventional
pigsandorganicpigs.
MATERIALANDMETHODS
26 organic herds and 31 conventional herds were considered in one slaughterhouse. Two pigs per
herdsweresampledifpossible.Foreachpig,Salmonelladetectionwasrealizedfromcoloncontent
andfromswabsofcarcassusingtheNFENISO6579method.Twoisolatesperpositivesampleswere
serotyped and genotyped by PFGE using XbaI enzyme (1). PFGE profiles were analysed with
BioNumercissoftware(AppliedMaths).
RESULTS
Atotalof227sampleswereanalyzed:114coloncontentsand113swabsofcarcass.Prevalenceof
Salmonellaincoloncontentwashigherfororganicpigs,37,9%
IC95%
[25.551.6],thanforconventional
pigs, 32.7%
IC95%
[19.544.5], but the difference between these two productions was not significant
(2; p= 0,563). Salmonella prevalence was lowest on carcasses and very close between the two
productions: 10.7%
IC95%
[4.021.8] for organic carcasses and 10.3%
IC95%
[3.921.2]) for conventional
carcasses.
Onehundredandfourisolateswerecollectedanddistributedin7serovars:Derby(48),Brandenburg
(18), Typhimurium (13), 2 types of monophasic variant of serovar Typhimurium 4,12:i: (11) and
4,5,12:i:(10),Infantis(2)andMbandaka(2).
Sixteen PFGE profiles were obtained (figure1): 1 per serovar for Mbandaka, Infantis, and
Brandenbrug, 2 for monophasic variant 4,5,12:i:, 3 for Derby, 4 for Typhimurium and 4 for
monophasicvariant4,12:i:.SevenPFGEprofiles(84%ofthestrains)werecommonbetweenorganic
andconventionalpigs.Amajorprofilegathered79%oftheS.Derbystrains.S.Brandenburgstrains
presentedonlyonePFGEprofilewhichwasdetectedin5differentherds.
With the 20 strains isolated from 12 carcasses, it has not been possible to show with certainty
Salmonellacrosscontaminationbetweenorganicandconventionalpigsduringtheprocess.Indeed,
onlyoneS.TyphimuriumPFGEprofilewasevidencedfororganicandconventionalcarcassesduring
thesamesamplingday.
DISCUSSION
In this study, the prevalence in colon is highest in organic pigs. As organic pigs are slaughtered
before conventional pigs, they could contribute to contamination of the conventional carcasses
during process. This can explain similar prevalences on carcasses thereafter. But PFGE profiles
analysis of carcass isolates didnt permit to conclude to a transfer of Salmonella between both
productions at the time of the slaughter. Predominance of the serovar Derby in organic pigs was
observed as for conventional pigs (2). Monophasic variants of S.Typhimurium were particularly
isolated,withahighgeneticdiversityfor4,12:i:.Thesevariantsareknowntoincreaseinthehuman
salmonellosiscasestheselastyears(3).
279
AKNOWLEDGMENT: This study was conduct in the frame of a ERA NET CORE organic II project
Safeorganic.Theauthorsthanktheresponsibleoftheslaughterhouse.
REFERENCES
1. Ribotetal.FoodbornePathogDis2006;3:5967.
2. Valdezateetal.,EmergInfectDis2005;11:6948.
3. ReportfromtheFrenchNRCforSalmonella2011.
Figure1:PFGEprofilesobtainedforthe104strainsisolatedinorganicandconventionalpigsatslaughter
fromcoloncontentsandswabsofcarcass.

280
Observationsontheoccurrenceandpersistence
ofmonophasicSalmonellaTyphimurium(S.4,[5],12:i:)
inpoultryfarms
RobDAVIESandDorisMUELLERDOBLIES
AnimalHealthandVeterinaryLaboratoriesAgency(AHVLA),BacteriologyandFood
SafetyDepartment,NewHaw,Addlestone,SURREY,KT153NB,UK
INTRODUCTION
Since2006,monophasicSalmonellaTyphimurium(mST)(S.4,[5],12:i:)DT193/DT120withresistance
toacoresetofantimicrobials(ampicillin,streptomycin,sulphonamidesandtetracycline)hasemerged
acrossEurope,predominantlyinpigandhumanpopulations(EFSA,2010).InUK,therehasalsobeen
significant involvement of cattle, especially dairy cattle, but there have also been a small number of
cases in chicken and turkey flocks. In contrast to mammalian species, the avian cases have
predominantly involved strains lacking the somatic antigen O5; i.e. so called Copenhagen strains.
Most of these reports were from the period between 2009 and 2010 before mST was formally
includedinEUSalmonellacontrollegislation.
MATERIALSANDMETHODS
Notifications of mST obtained from routine surveillance monitoring were followed up by visits to
gather background data and to take samples to assess the distribution of Salmonella. In the case of
broilerandturkeyflocks,theimplicatedflockshadalreadybeenslaughteredbythetimethefirstvisits
could be carried out. Samples were taken either with boot swabs or large gauze chiffonette hand
held swabs and were collected into Buffered Peptone Water (BPW: Merck) on site. Subsequent
isolation involved BPW enrichment (18hr, 37C; MSRV (Mast) selective enrichment (24/48hr, 41.5c)
andplatingonRambachagar(Merck)(24hr,37c).Suspectcolonieswereconfirmedserologically.
RESULTS
In free range farm A, mST
(M)
was not found, suggesting either spontaneous clearance of S.
TyphimuriumDT193
(T)
S.Bovismorbificans
(B)
andS.London
(L)
werealsofoundinassociationwith
thepigs.Table3showspersistenceofmSTontherangeat4monthsafterremovalofaninfected
turkeyflock,butnomSTwasfoundafterrotavationofland,reseedingandrestocking10months
later.Inthiscasetherewerenonearbypigsorcattle,butconventionalrearingfarmCappearsto
have been the potential source of mST for both flocks, as well as harbouring S. Kottbus
(K)
and S.
Senftenberg,whichwereknowntobeofhatcheryorigininthiscase.
DISCUSSION
Thesecases,andothersthatarenotdescribedindetailhere,illustratetherisksoftransmissionof
mSTfrompigsorpurchasedcattletopoultry.Freerangeflocksappeartobeatparticularriskand
often involvement of rodents has been noted on positive farms. Persistence of contamination of
soilandsurfacewaterintherangeareaisaparticularhazard,aswithotherSalmonellainfections
(Davies & Breslin, 2003). mST appears to have only been detected in nonvaccinated poultry
sectors, such as turkeys or broilers, or in laying flocks where SE but not ST vaccination has been
used.
CONCLUSIONS
The risk of transmission of Salmonella from pigs and cattle to poultry should be given greater
attention on mixed holdings. In some cases the original positive sample can not be confined by
intensive sampling, suggesting more care in collection of samples, especially boot swabs, may be
warranted. In cases involving laying flock, or dust only positives, failure to confirm mST may
281
suggestrapidresolutionofinfection,butforflocksatincreasedriskvaccinationforbothSEandST
isrecommended.
ACKNOWLEDGMENTS
ThisworkwasfundedbyDefraprojectOZ0342
REFERENCES
1. Davies, R.H. and Breslin, M. (2003). Persistence of Salmonella Enteritidis PT4 in the environment and
arthropodvectorsonanemptyfreerangechickenfarm.EnvironmentalMicrobiology5,7984.
2. EFSA (2010). EFSA Panel on Biological Hazards (BIOHAZ); Scientific Opinion on monitoring and
assessment of the public health risk of 'Salmonella Typhimuriumlike' strains. EFSA Journal, 8, (10), 1826
1874
Table1:CommercialFreerangeLayingFarms
(No.samplespositiveforSalmonella/No.samples
taken(%))
FarmA FarmB Visit2
(Newflock)
HouseInteriorfaeces 5/37(13.5)
VS
0/75 0/20
HouseInteriordust 0/20 3/20(15.0)
M
2/10(20.0)
S
Mousefaeces/carcasses 0/6 0/2

RangeArea 7/20(35.0)
S
0/20 0/20
Wildbirdfaeces 1/4(25.0)
M
0/4
Cattlehousing 16/20(80.0)
M
0/55
Table2:FreerangeBroilerFarm
Visitone(aftercleaninganddisinfection) Visittwo(afterrestocking)
Floor 5/20(25.0)
M
Litter/faeces 0/160
Structureandequipment 2/100(2.0)
M
Anteroom 1/5(20.0)
M
0/11
Driveway 9/14(64.3)
M
4/9(44.4)
MB
Range 20/20(100.0)
MT
18/20(90.0)
M
Pighousing 8/60(13.3)
MT
10/73(13.7)
BL
Table3:FreerangeTurkeyFarms
FarmA FarmB RearingFarmC

Visit1
House Range House Range Disinfected
houses
Occupied
houses
10/14(74.4)
M
6/6(100.0)
M
0/14 6/6
(100.0)
M
NS NS
Visit2(postclean)
2/40
(5.0)
M
14/20
(70.0)
M
0/40 7/40
(17.5)
M
3/120
(2.5)
M
28/40
(70.0)
KS
Visit3(Postrestock)
1/1
K
0/60 NS 15/60(25.0)
K
NS NS

282
AstudyofthedynamicsofSalmonellainfectioninturkeybreeding,
rearingandfinishinghouseswithspecialreference
toelimination,persistenceandintroductionofSalmonella
DorisMUELLERDOBLIESandRobertH.DAVIES
BacteriologyandFoodSafetyDepartment,AHVLAWeybridge,WoodhamLane,NewHaw,
ADDLESTONE,KT153NB,UnitedKingdom
INTRODUCTION
Until recently, little was known about the importance of turkey meat as a source of Salmonella.
ThefirstEUwidedataonSalmonellaprevalenceinturkeyflocksweregatheredduringabaseline
survey in 2006/07 (SANCO/2083/2006), and from the survey data, the UK flock level prevalence
for Salmonella spp. was estimated as 32.2% for fattening flocks and 4.4% for breeding flocks
(EuropeanFoodSafetyAuthority,2008).Thequantitativecontributionofturkeystotheburdenof
human salmonellosis across the EU was estimated recently and resulted in the conclusion that
2.6% of human salmonellosis cases may be attributable to turkeys (Efsa Panel on Biological
Hazards,2012).However,thisnumbermayvarysignificantlydependingontheindividualsituation
inthedifferentmemberstates.
In the UK, the number of Salmonella serovars of major zoonotic significance from turkey
productionhasdeclinedoverthepasttenyears(Papadopoulouetal.,2009),butotherSalmonella
serovarsarestillfoundfrequentlyinturkeyflocksinrearingandfinishinghousesandlessoftenin
breeding houses. Since the introduction of the National Control Programme in 2010, the
prevalenceoftheregulatedserovars(i.e.S.EnteritidisandS.Typhimurium)hasbeengoingdown
acrosstheBritishturkeyindustry,andin2010,theUKflockprevalenceforfatteningflocksunder
the NCP was 16.49% for all serovars and 0.24% for regulated serovars (Animal Health and
VeterinaryLaboratoriesAgency,2012).
Infection of a turkey flock with Salmonella may be: 1) the result of residual contamination inside
thehouseorinthefreerangearea(inthecaseoffreerangeflocks)originatingfromapreviously
infected flock followed by ineffective cleaning and disinfection (C&D); 2) due to the presence of
infected vectors inside or around the houses, such as rodents or litter beetles; 3) the result of
spread from other flocks on the holding due to poor biosecurity procedures; 4) acquired via
contaminatedfeed,water,beddingorequipmentor5)importedalongwiththereplacementbirds
themselves.
In this descriptive study, the dynamics of Salmonella infection of turkey flocks were investigated
by taking samples from the followon flocks housed in buildings where Salmonella had being
detected before, with the aim of identifying the most common scenarios involved in elimination,
persistenceandintroductionofSalmonellainthedifferentbranchesoftheUKturkeyindustry.
MATERIALANDMETHODS
A total of 62 houses on 34 farms (breeding, rearing and finishing farms) were enrolled in this
study.Thestartingpointwasalwaystheidentificationofapositiveflock.Atotalof117followon
flocks (32 breeding, 36 rearing, 49 finishing flocks) were sampled and tested for the presence of
Salmonella.
Thenumberoffollowonflockssampledperhouserangedfromonetofive.Everyconfirmationof
aserovarorthedisappearanceofaserovarinafollowonflockwasclassifiedasanincidenti.e.
eithercarryoverofthesameserovar,clearanceofaserovarortheintroductionofanewserovar.
In some cases, two incidents per followon flock were recorded, for example when two different
serovars were isolated from the same flock. Therefore, the number of incidents was higher than
thenumberoffollowonflocks.
283
Samples taken from occupied houses usually consisted of boot swabs and/or dust swabs, and in
some cases, individual faecal samples were taken in addition to the boot swabs. The sampling
method using boot swabs and/or dust swabs has been described previously (MuellerDoblies et
al.,2009).
Whenever possible, a postcleaning and disinfection (PostC&D) visit was carried out between
flocks to assess the quality of the cleaning and disinfection procedure and to determine the
predictive value of the postC&D result. Samples taken at postC&D visits consisted of handheld
swabs from several areas of the houses, including floor, walls (including posts, partitions, ledges
andwindows),drinkers,feeders,anteroom(ifany)andnestboxes(ifany).Ahousewasconsidered
positiveifoneormoresampleswerepositiveforSalmonella.ThepostC&Dsamplingprotocolhas
beendescribedpreviously(CarriqueMasetal.,2008).
RESULTS
35.5% of incidents were carryover incidents of the same serovar. Even though carryover was
seen in all production types, it was more frequent in breeding and rearing houses compared to
finishinghouses.Mostcarryovereventsweretheresultofinsufficientcleaninganddisinfection.
Clearancewasseenin40%ofallincidentsandintroductionofanewserovarwasseenin24.5%of
incidents. A difference was seen between serovars, with S. Typhimurium being cleared about
three times more often than carried over, whereas other serovars were more often carried over
thancleared.
ThemostinterestingoutcomeofapostC&Dvisitisthepotentialstatusofthefollowonflock,i.e.
whether the followon flock is likely to get infected with the same serovar or not. For this, we
definedtheoutcomeofthefollowonflockasthegoldstandardandcalculatedthepositiveand
negativepredictivevalueofapostC&Dvisit.Thepositivepredictivevalueforallproductiontypes
was52.5%,whichmeansmorethanhalfofallpostC&DvisitswhereSalmonellawasisolatedwere
followed by carryover of the infection into the next crop. The negative predictive value was
calculatedas76.5%forallproductiontypesand100%forfinishersandrearers,whichmeansthat
a negative postC&D visit will in 76.5% of cases be followed by a negative followon flock. In our
study,allfourflockswherecarryoverwasobservedafteranegativepostC&Dvisitwerefromone
particular farm with a resident mouse population which was likely tobe the source of carryover
despitesuccessfulcleaninganddisinfectionofthehouses.
90%offollowonflocksbeingplacedinhouseswithmorethan9%positivesamplesaftercleaning
and disinfection had a carryover of infection, thus showing that the proportion of positive
samples obtained after cleaning and disinfection has a high predictive value for the followon
flock. The percentage of positive samples obtained from a postC&D proved to be the better
predictiveofapossiblefollowonflockcomparedtothelocationwherethepositivesampleswere
foundwithinthehouse.
DISCUSSION
ThefactthatS.Typhimuriumwasmorelikelytobeeliminatedthanotherserovarscanbedueto
several reasons, like more stringent cleaning and disinfection after the isolation of S.
Typhimurium, greater environmental survival by certain serovars or different management
practicesinindividualcompanies.
The reasonably good correlation between the number of positive samples obtained at the post
C&Dvisitandtheprobabilityofcarryoveroftheserovarintothefollowonflockshowsthatgood
cleaninganddisinfectionisacrucialstepineliminatingresidualcontaminationandpreventingthe
carryoverofinfectionintothenextflock.
Toachievethis,cleaninghastobeperformedtoahighstandard,asuitabledisinfectanthastobe
used at the right concentration and reintroduction of Salmonella from the environment of the
284
houseshastobepreventedbysuccessfulcontrolofpestssuchaslitterbeetleandrodentsandby
applyingeffectivebiosecuritymeasuresbetweendisinfectionandrestockingofthehouses.
REFERENCES
1. AnimalHealthandVeterinaryLaboratoriesAgency,2012.SalmonellainlivestockproductioninGB2011.
http://vla.defra.gov.uk/reports/rep_salm_rep11.htm.
2. CarriqueMas, J.J., Breslin, M., Snow, L., Arnold, M.E., Wales, A., McLaren, I., Davies, R.H., 2008.
Observations related to the Salmonella EU layer baseline survey in the United Kingdom: followup of
positiveflocksandsensitivityissues.EpidemiologyandInfection136,15371546.
3. EfsaPanelonBiologicalHazards,2012.Scientificopiniononanestimationofthepublichealthimpactof
settinganewtargetforthereductionofSalmonellainturkeys.EFSAJournal10,26162616.
4. European Food Safety Authority, 2008. Report of the Task Force on Zoonoses Data Collection on the
AnalysisofthebaselinesurveyontheprevalenceofSalmonellainturkeyflocks,intheEU,20062007Part
A:Salmonellaprevalenceestimates
http://www.efsa.europa.eu/en/efsajournal/pub/134r.htm
5. MuellerDoblies, D., Sayers, A.R., CarriqueMas, J.J., Davies, R.H., 2009. Comparison of sampling
methodstodetectSalmonellainfectionofturkeyflocks.JournalofAppliedMicrobiology107,635645.
6. Papadopoulou, C., Davies, R.H., CarriqueMas, J.J., Evans, S.J., 2009. Salmonella serovars and their
antimicrobialresistanceinBritishturkeyflocksin1995to2006.AvianPathology38,349U332.
285
EpidemiologicalinvestigationofSalmonellaentericasubsp.entericaisolatedfrom
chickencarcassesandenvironmentatslaughterinReunionIsland:prevalence,
geneticcharacterizationandantibioticsusceptibility
*
IsabelleHENRY
1
,SophieGRANIER
2
,ClineCOURTILLON
3
,FranoiseLALANDE
3
,
MarianneCHEMALY
3
,GillesSALVAT
3
andEricCARDINALE
4
1
CrtedOrEntreprise,4aveMichelDebr,97427ETANGSALE,Runion;
2
AnsesCEB,23aveGnraldeGaulle,94706MAISONSALFORTCedex,France;
3
AnsesUnitHQPAP,Sitedescroix,22440PLOUFRAGAN,France;
4
CIRADUMRControlofanimalexoticandemergingdiseases,CRVOI,
2rueMaximeRivireBios,97490STECLOTILDE,Runion
INTRODUCTION
Salmonella is a leading cause of bacterial foodborne disease outbreaks in developed countries
(Hue et al., 2011) and is also a public health concern in tropical countries. Chicken meat
production is only 9 000 t a year (in 2010) but it is also the first source of animal protein
(32kg/inh/yr) and approximately 25% of chicken is consumed in the form of processed meat
products(sausagesparticularly).
The aim of the study was to show the distribution of Salmonella in chicken carcasses and the
environment in slaughterhouse and to compare their genetic and antibiotic susceptibility profiles
tohighlightanypotentialcrosscontamination.
MATERIALANDMETHODS
A total of 71 broiler chicken flocks, randomly chosen, were studied between October 2007 and
January2009.
Isolationprocedure
Samples were analyzed for Salmonella according to ISO6579 standard. All Salmonella isolates
wereserotypedaccordingtotheKauffmannWhiteschemeusingslideagglutinationtestaccording
tothediagnosticPasteur,Paris,France.
Antimicrobialsusceptibilitytesting
Antimicrobial susceptibility was tested by the disk diffusion method following the CLSI guidelines
(ClinicalandLaboratoryStandardsInstitute,2008).
Moleculartyping
ThefollowingharmonizedprotocolforPFGEwasusedforthestudyasdescribedpreviously
(Kerouantonetal.,2007).
RESULTS
Salmonella spp. was isolated from 40 of 71 (56% [4567]) broilerchicken flocks at slaughter. The
most prominent serovars were S. Blockley (31%), S. Typhimurium and S. Brancaster (14%), S.
Hadar (10%), S. 1,4,[5],12:i: and S. Virchow (8%). At the farm, 27% of the broilerchicken flocks
tested positive for Salmonella spp. Salmonella spp. could be isolated from 124 of 497
environmental samples (25%). Strains belonging to the four most commonly isolated S. enterica
serovars (Blockley, Typhimurium, Brancaster and Hadar) were further characterized by Xba1
macrorestriction.ForS.Blockley,thePFGEofXba1digestedDNAfrom77isolatesgavestableand
reproducible patterns consisting of 1418 fragments. We obtained 4 patterns with Xba1 enzyme.
PatternB2wasthepredominantgenotypeaccountingfor95%ofthestrains.ForS.Typhimurium
(50 isolates) and S. 1,4,[5],12:i:, (15 isolates), macrorestriction with Xba1 yielded 8 patterns
consisting of 14 18 fragments. B54 was the predominant profile with 35% of the strains. The
dendrogram showed three clusters with 82, 86 and 85% similarity respectively; the first cluster
(23% of the isolates) comprised 2 profiles from seven chicken strains and eight environmental
286
strains; the second one (31%) 3 profiles from nine chicken strains and eleven environmental
strains and the third one (46%) from 17 chicken strains and 13 environmental strains. S.
1,4,[5],12:i:wasintegratedwithintheanalysisbecauseitexhibitedthesamegeneticprofiles(B73
and B69). Antimicrobial resistance pattern and PFGE pattern for S. Blockley, S. Typhimurium, S.
1,4,[5],12:i:, S. Brancaster and S. Hadar isolates are shown in table 1. The most common
resistance pattern was a sevenresistance profile A,Amc,Cf,Enr,Na,S,T accounting for 46% of the
isolates.
DISCUSSION
Fifty six percent of the broilerchicken flocks at slaughter tested positive for Salmonella spp. The
presence of S. Typhimurium in poultry (live broiler chickens as well as chicken carcasses) is of
considerable importance from the standpoint of public health. The high level of S. Typhimurium
contaminationcouldbeattributedtothespecificconditionsinReunionIsland;mostoftheisland
is covered by sugarcane fields that are natural habitats of rodents and rodents are known to be
vehicles of S. Typhimurium (Meerburg and Kijlstra, 2007). This study highlighted the primary
source of Salmonella was the farm of origin and downstream stages in processing could not
remedytobutamplifythisSalmonellacontamination.
ThepredominantSalmonellaserovarsweresusceptibletomostofthetestedantibioticdrugsbut
S. Hadar exhibited high levels of resistance. S. Hadar emerged in Reunion island as particularly
resistant to nalidixic acid (81%) and this resistance was also associated with reduced efficacy of
fluoroquinolones, such as enrofloxacin (81% also). This worldwide overuse of antimicrobials has
created enormous pressure for the selection of antimicrobial resistance among bacterial
pathogens; this explained why the same pulsotype (B37) did not exhibit the same antibiotic
resistanceprofilewhichisdirectlyrelatedtotheuseofdrugsatthefarmlevel(Useraetal.,2002).
The comparison between the Salmonella serovars (and their pulsotypes) isolated from chicken
carcassesandthosefromlivebroilersconfirmedthatbroilerchickensareinfectedprimarilyatthe
farm.Inourstudy,mostofthechickenflocksatslaughter(30%)becameinfectedafterleavingthe
farm. Contamination of Salmonellanegative flocks occurred either during transport with
contaminated crates or during processing when the plant was contaminated. Again, the strains
isolatedonchickencarcassesattheendofprocessingwereidenticalphenotypically(serovar)and
genotypically (pulsotype) with those isolated on transport crates or in the slaughtering plant.
Improving hygiene management during transport of broilers and in slaughterhouses can
significantlyreducetheriskofSalmonellacontaminationofpoultrymeatandthentheriskoffood
poisoninginReunionIsland.
REFERENCES
1. Hue,O.,etal.2011.FoodControl22,11581164.
2. Meerburg,B.G.andKijlstra,A.,2007.JournalofFoodProtection65.
287
TABLESANDFIGURES
Table1:AntimicrobialresistancepatternandPFGEpatternforS.Blockley,S.Typhimurium,S.4,12:I,
S.BrancasterandS.Hadarisolates(ReunionIsland,20072009,185isolates).
Broilerchicken Environment

SalmonellaSerovars PFGEPattern Antibioticresistancepattern PFGEPattern Antibioticresistancepattern
S.Blockley
(n=77)

B1(2) B1(0)
B2(48) B2(25)
B3(0) B3(1)
B4(1) B4(0)

S.Typhimurium(n=50) B50(2) ASSuT(1) B50(4)
B54(15) SSuT(15) B54(8) SSuT(7)
B60(0) B60(1)
B62(5) B62(3) SuT(1)
B73(1) ASSuT(1) B73(3) ASSuT(3)
B74(1) ASSuT(1) B74(0)
B77(2) T(2) B77(5) T(5)

S.4,12:I
(n=15)
B69(1) ASSuT(1) B69(3) ASSuT(3)
B73(6) ASSuT(6) B73(5) ANaSSuT(1)
ASSuT(4)

S.Brancaster
(n=17)
B5(14) S(10)
T(1)
B5(2) S(2)

B6(0) B6(1)
S.Hadar
(n=26)
B37(7) AAmcCfEnrNaST(4)
AAmcCfNaST(1)
EnrNaST(1)
ST(1)
B37(13) AAmcCfEnrNaST(8)
AAmcCfNaST(1)
EnrNaST(4)
B39(0) B39(2) AKST(1)

S(1)
B40(2) AKST(1)
ST(1)
B40(2) AKST(2)

288
Salmonellaoccurrenceandantimicrobialresistanceinnestlings
oftwoseagullspecies,LarusmichahellisandLarusaudouinii
inEbroDelta(NESpain)
NoeliaANTILLS
1
,SergioLPEZSORIA
1
,JacobGONZLEZSOLS
2
,MartaCERDCULLAR
1,3
1
CentredeRecercaenSanitatAnimal(CReSA),UABIRTA,
CampusdelaUniversitatAutnomadeBarcelona,08193Bellatera(CerdanyoladelValls),
BARCELONA,Spain;
2
InstitutdeRecercadelaBiodiversitat(IRBio)andDepartamentdeBiologiaAnimal,
FacultatdeBiologia.UniversitatdeBarcelona,Av.Diagonal645,08028BARCELONA,Spain;
3
InstitutdeRecercaiTecnologiaAgroalimentries(IRTA),BARCELONA,Spain
INTRODUCTION
Salmonella spp. is one of the leading agents of zoonotic bacterial enteric infections worldwide.
Seagulls are one of the most documented carriers of Salmonella, which is presumably related to
theiruseofrefusedumps.However,theprevalenceandantimicrobialresistanceofthispathogen
among gull species with different feeding habits is poorly documented. In the present work, we
determined the occurrence and antimicrobial resistance of Salmonella spp. in nestlings of the
Audouins and the yellowlegged gull (Larus audouinii and Larus michahellis) in the Ebro Delta
(Spain).
MATERIALANDMETHODS
Duplicatecloacalswabswereobtainedfrom251nestlingsoftheAudouinsand270oftheyellow
legged gull from 2009 to 2011 in Ebro Delta (NE Spain). Swabs were placed in Amies transport
medium with charcoal, transported to the laboratory and cultured using standard methods to
isolate Salmonella. Serotyping of the isolates was performed at Departament dAgricultura,
Ramaderia, Pesca, Alimentaci i Medi Natural (Generalitat de Catalunya). Antimicrobial
susceptibility(discdiffusionmethod)wastestedtoapanelof18antimicrobialscommonlyusedin
human or veterinary medicine. Diversity of strains was assessed by genotypic analyses (ERICPCR
andPFGE)oftheisolates[1,2].
RESULTS
Yellowlegged gull prevalences of Salmonella ranged from 4.76 % to 25% and showed a high
diversity of Salmonella serotypes, being the most abundant S. Typhimurium and S. Hadar. In
Audouins gulls, Salmonella was not detected in 2009 but prevalences rose to 1.14% in 2010 and
to13.51%in2011(Table1).
Table1.FrequencyofSalmonellaaccordingtogullsspecies
* npositive/ntotal(%ofprevalence).
Year Larusmichahellis Larusaudouinii
2009 0/52(0%)* 4/84(4.76%)
2010 1/88(1.14%) 25/100(25%)
2011 27/111(24.32%) 14/86(16.28%)
289
Over 65% of the isolates showed resistance to at least one antimicrobial. The main resistances
found were to amoxicillin, followed by some other lactams, streptomycin and tetracycline.
Multiresistance,asindicatedbyresistancetomorethan4drugs,wasdetectedinstrainsisolated
frombothgullspecies.GenotypinganalysesshowedthatSalmonellapositivebirdsusuallycarried
asinglestrainbutoverallthetwogullspeciesshowedahighdiversityofstrains.
DISCUSSION
DespiteyellowleggedgulluserefusedumpsinmuchgreaterproportionthanAudouinsgull,both
gull species act as important reservoir of Salmonella serotypes. Also, although exposure to
antibiotics is expected to be rare in this seabird population, a high prevalence of Salmonella
showingresistancetoanantimicrobialwasdetected.
CONCLUSION
BothseagullspeciesserveasareservoirofSalmonellaresistantstrainsandcandisseminatethem
through their droppings, contaminating the environment. Overall, results indicate that both gull
speciesareofpublichealthconcern.
ACKNOWLEDGEMENTS
Authors are grateful to the personnel from Delta Ebro Natural Park for the sampling program
coordinationandsupport.AuthorsarethankfultoTeresaAyats(CReSA)fortechnicalsupport.This
work was funded by grant FAU200800012C0201 from INIA. Noelia Antills is a recipient of a FI
fellowshipfromtheGeneralitatdeCatalunya.
REFERENCES
1. CerdCullar, M., Naranjo, J.F., Verge, A., Nofrarias, M., Cortey, M., Segales, J., Aragon, V. 2010. Sow
vaccination modulates the colonization of piglets by Haemophilus parasuis. Veterinary Microbiology. Oct.
26;145(34):31520.
2. Ribot, E.M., Fair, M.A., Gautom, R., Cameron, D.N., Hunter, S.B., Swaminathan, B., Barrett, T.J. 2006.
Standardization of PulsedField Gel Electrophoresis protocols for subtyping of Escherichia coli O157:H7,
SalmonellaandShigellaforPulseNet.Foodbornepathogensanddisease.Volume3.Number1.

290
PrevalenceofSalmonellaspp.onduckproductsattheretaillevel
TyphainePOZEVARA,FranoiseLEGALL,MarianneCHEMALY*
Anses,LaboratoiredePloufraganPlouzan,UnitHygineetQualitdesProduitsAvicoles
etPorcinsBP5322440PLOUFRAGAN,France
INTRODUCTION
Salmonella is a major zoonotic pathogen and the cause of numerous outbreaks worldwide each
year.Poultryandeggsremainthemajorsourceofinfectionindevelopedcountries.TheEuropean
and national surveys conducted since 2004 allowed to estimate the prevalence of Salmonella in
layinghens(HuneauSalanetal.,2009),ineggs(Chemalyetal.,2009),broilers(LeBouquinetal.,
2010) and turkeys (Aury et al., 2010). This was in line with the implementation of the regulation
2160/2003 on the control of Salmonella and other zoonotic agents present in the food chain.
However,nostudywascondusctedtoassesstheprevalenceofSalmonellaintheduckproduction.
The objective of this work is to estimate the prevalence of Salmonella on ducks from retail
products.
MATERIALANDMETHODS
102 fillets originating from 13 different plants and from 87 batches were sampled. The detection
of Salmonella was carried out according to the standard NF EN ISO 6579. All the samples were
subjected to enumeration following the MPN method on miniMSRV. Genotyping of isolates was
performed by macrorestriction using two enzymes XbaI and BlnI, and Pulsed field gel
electrophoresis(PFGE).
RESULTS
11samplestestedpositiveforSalmonella,leadingtoaprevalenceof5.4%(3.9%positivemuscles
and 6.9% positive skins). These samples were from 9 different batches and 6 plants. Two fillets
only were found positive on the skin and the muscle. Positive samples presented all an
enumerationlevelbelowthethresholdofthemethod(>1.6CFU/g),indicatingthattheseproducts
present a low contamination level. The serotyping of the strains allowed the identification of 4
serovars: S. Typhimurium, S. SaintPaul, S. Kottbus and S. Indiana. In two different plants, two
different Salmonella strains were found simultaneously showing a multiple contamination within
the plant. Moreover, in these same plants the same strain of Salmonella Indiana was found at
different times of sampling indicating a possible residual contamination. Genotyping of isolates
confirmedtheresultsbecausethestrainssharedthesamepattern(figure1)
291
Figure 1. PFGE patterns of Salmonella isolates obtained by macrorestriction using two enzymes XbaI and
BlnI.
This study allowed the estimation of Salmonella prevalence on duck products at the retail level.
Theproportionofcontaminatedproducts(5.4%)ofducksissubstantiallyequivalenttothatfound
onproductsofturkeys(7.4%in2010)orofbroilers(3.6%in2009).Althoughtheprevalencewas
low, this investigation highlighted weaknesses in the hygienic process because of possible multi
contaminationwithinoneplantandresidualcontaminationacrossthetime.
REFERENCES
1. Aury K., Chemaly M., Petetin I., Rouxel S., Picherot M., Michel V., Le Bouquin S. 2010. Prevalence and
riskfactorsforSalmonellaentericasubsp.enterica contaminationinFrenchbreedingandfatteningturkey
flocksattheendoftherearingperiod.PreventiveVeterinaryMedicine.94,8493.
2. Chemaly M., HuneauSalaun A., Labb A., Houdayer C., Petetin I., Fravalo P. 2009. Isolation of
Salmonella enterica in laying hen flocks and assessment of eggshell contamination in France. Journal of
FoodProtection.72:20712077.
3. HuneauSalan A., Chemaly M., Le Bouquin S., Lalande F., Petetin I., Rouxel S., Michel V., Fravalo P.,
Rose N. 2009. Risk factors for Salmonella enterica subsp. enterica contamination in 519 French laying hen
flocksattheendofthelayingperiod.PreventiveVeterinaryMedicine.89:5158.
4. LeBouquinS.,AllainV.,RouxelS.,
,
PetetinI.,PicherotM., MichelV.,Chemaly M.2010.Prevalenceand
risk factors for Salmonella spp. contamination in French broilerchicken flocks at the end of the rearing
period.PreventiveVeterinaryMedicine.97,245251.
292
Salmonellacontaminatedeggsinflocksidentifiedaspositive
bytheNationalControlProgrammeforSalmonellaintheUK
FrancescaMARTELLI,BeckyGOSLING,IanMCLAREN,AndrRABIEandRobDAVIES
INTRODUCTION
Salmonella Enteritidis (SE) is the serovar most frequently associated with egg contamination and
responsible for the majority of egg related human salmonellosis outbreaks. Salmonella
Typhimurium(ST)andotherSalmonellaserovars(SO)mayinfectflocksandcancontaminateeggs,
buttoalesserextentthanSE.InEurope,thetwoSalmonellaserovarsthatareregulatedinlaying
flocksareSEandST.SEhasareportedlyuniqueabilityforlongtermcolonizationofovary/oviduct
andcontaminationofformingeggs,andhasbeenthemostfrequentserovarassociatedwithegg
relatedfoodborneoutbreaksinEuropesincethemid1980s(4).FlocksinfectedwithSEproducea
variableproportionofcontaminatedeggs,dependingonstrain,levelofcontaminationoftheflock
and time of the production period in which the eggs are laid. Several studies have reported
experimental infections of laying hens with ST, and in these shortterm, high dose challenge
studiesnoconsistentdifferencecouldbedeterminedbetweenSEandSTintheirabilitytocolonize
the reproductive tract and consequently contaminate eggs. The role of ST in egg related
foodborneinfectionsappeartobelesssignificantthanthatofSE(5).SOarepreferentiallyisolated
fromeggshellratherthancontentsincommercialtableeggs,butcertainserovars(e.g.S. Infantis
andS.Heidelberg)havealsobeenisolatedfromeggcontents(2).
IntheUK,flocksonholdingsofmorethan350layinghensaresubjecttocontrolsaccordingtothe
National Control Programme (NCP) for Salmonella in layers (Gallus gallus). The programme
involvessampling(bootswabsorfaeces)every15weeksduringtheperiodofproductionofeggs
of each holding, supplemented by official sampling of one flock per holding of more than 1000
birds per year, using the same method plus one additional dust or faecal/boot swab sample.The
percentageofflocksthatareidentifiedaspositiveintheUKisverylow(0.17%oftheeligibleadult
flockswerepositiveforSEorSTin2011).Duringthisstudyholdingsconfirmedaspositiveduring
theroutineNCPtestingwerevisited,andeggsandbirdswerecollected,toinvestigatetheextent
ofeggcontaminationinflocksoflayinghensthatwerenaturallyinfectedwithSE,STandSO.
MATERIALANDMETHODS
Atotalof19flocks(8SE,7STand4SOpositiveflocks)werevisitedwithinthisstudy.Theseflocks
wereidentifiedasSalmonellapositivewithintheNCPtestingscheme.Foreachflock,100hensand
4000eggswerecollected.Shellsandcontentswerepooledseparatelyingroupsofsixforculture
and preenriched in 225 and 333 ml of Buffered Peptone Water (BPW) respectively. The hens
collectedonfarmweresubjectedtopostmortemexamination,whereovaries/oviductandcaeca
wereasepticallyremovedandculturedseparatelyas25gsamplesin225mlofBPW.
The samples in BPW were incubated at 37C for 1620 hours and 0.1 ml of incubated broth was
inoculated onto modified semisolid Rapaport Vassiliadis medium (MSRV). MSRV plates were
incubatedat41.5Candexaminedafter24and48hoursforsuspectSalmonellagrowth.Fromall
MSRV plates a 0.1 ml loop was taken, and streaked onto Rambach agar. Inoculated plates were
incubated at 37C for 24 hours, and presumptive Salmonella colonies were identified. The
estimated prevalence in eggs and egg contents (estimated from pooled samples) was measured
againsttheprevalenceinovaries(basedonpostmortemof100birds).
RESULTS
Salmonella was isolated from eggs from 12 of the 19 flocks sampled. The mean six egg pool
prevalenceforshells,contentsandeggsthatwerecontaminatedontheshellsandinthecontents
293
0
0.5
1
1.5
2
2.5
M
e
a
n

e
g
g

p
o
o
l

p
r
e
v
a
l
e
n
c
e

(
%
)
Shells Contents Shells and
Contents
Location within the egg
Salmonella serovars and eggs
SE
ST
SO
areshowninFigure1.Theproportionofcontaminatedcontentswasgenerallylow.Eggsfrom2of
the SO positive farms had no contaminated egg contents. There was no statistically significant
differencebetweentheestimatedeggcontaminationrate(shellsorcontents)betweenSE,STand
SO. The SO positive group (n=4) showed a high proportion of contaminated eggs (mainly on
eggshells),butthiswasmainlyduetoonefarm(mixedSOinfection)thatpresentedanunusually
highnumberofpositiveeggshells(50/666tested).ThehenscollectedfromSEpositiveflockshad
thehighestpercentageofpositiveovariesandcaeca(13.4%comparedwith11.1%and8.9%ofST
andSOpositiverespectively).
TherewasahighlysignificantpositiveassociationbetweentheprevalenceofSalmonellainovaries
andineggcontents(r=0.82,p<0.001),butnotforwholeeggs(r=0.44,p=0.066).
DISCUSSION
The overall egg contamination rate was low, especially for egg contents, in most of the flocks
sampled, confirming that flocks infected with Salmonella typically yield a low number of
contaminated eggs (1). All the flocks had been vaccinated for SE, and most had also been
vaccinated for ST, which may have limited the degree of eggtransmission. Salmonella was not
isolatedatallfromtheeggsof7ofthe19positiveflocks.
Different serovars were associated with similar levels of egg contamination, suggesting that that
thepredominanceofSEineggrelatedoutbreaksmaynotbesolelyrelatedtoaparticularabilityof
SE in contaminating eggs, but to the fact that in the last three decades SE has been
epidemiologically more successful than other serovars in infecting large scale egg production
farms(4).
A strong association between the recovery of Salmonella in ovaries and in egg contents was
demonstrated, confirming the correlation between invasive strains and the production of
contaminated eggs. These findings support those of a recent study that has observed a
relationship,althoughnotstatisticallysignificant,betweenthenumberofSalmonellaorganismsin
ovariesandoviductandthefrequencyofcontaminatedeggs(3).
ACKNOWLEDGEMENTS
REFERENCES
1. Chemaly, M., A. HuneauSalaun, A. Labbe, C. Houdayer, I. Petetin, and P. Fravalo. 2009. Isolation of
Salmonella enterica in layinghen flocks and assessment of eggshell contamination in France. J Food Prot
72:20712077.
2. Martelli, F., and R. H. Davies. 2011. Salmonella serovars isolated from table eggs: an overview. Food
ResearchInternational45:745754.
3. Renu, Y., Tripathi, Shing. 2011. Salmonella occurrence in chicken eggs and environmental samples and
theirseroprevalenceinlayinghens.IndianJournalofAnimalSciences81:10871088.
4. Thorns,C.J.2000.Bacterialfoodbornezoonoses.RevSciTech19:226239.
5. Wales, A. D., and R. H. Davies. 2011. A critical review of Salmonella Typhimurium infection in laying
hens.AvianPathol40:429436.
Figure1:PercentageofpositiveeggsproducedbyflocksinfectedwithSalmonellaEnteritidis(SE),S.
Typhimurium(ST)andotherSalmonellaserovars(SO).

294
FateofSalmonellaentericaserovarInfantis,
thepredominantserovarinIsrael,intableeggs
AvishaiLUBLIN
1
,IlanaMALER
2
,SaraMECHANI
1
andShlomoSELA(Saldinger)
3
1
DivisionofAvian&FishDiseasesand
2
ThelaboratoryofFoodMicrobiology,
KimronVeterinaryInstitute,POBox12,BetDAGAN50250,Israel;
3
DepartmentofFoodQualityandSafety,InstituteofPostharvestTechnology
andFoodScience,VolcaniCenter,AgriculturalResearchOrganization,POBox6,
BETDAGAN50250,Israel
INTRODUCTION
Contaminatation of table eggs by serovars of Salmonella enterica is considered a major source for
foodborne salmonellosis. In the last couple of years, a single clone of S. enterica serovar Infantis
became the most prevalent strain isolated from poultry in Israel and from human cases of
salmonellosis. Since, no information is availble regarding the fate of this clone in table eggs, the
objectiveofthisstudywastoevaluatethepotentialoftableeggstoserveasaroutefortransmission
ofthepathogenfrompoultrytohuman.Consequently,theabilityofS.Infantistosurviveoneggshell
orwithintheeggcontentduringrefrigerationandatroomtemperaturewastested.
MATERIALANDMETHODS
Salmonella cells (ca. 10
5
cfu/egg) were inoculated on the surface of intact eggs, or injected into the
eggyolk.Theeggswerestoredat5.50.3C,oratroomtemperature(25.50.1C)forupto10weeks.
Atbothtemperatures,thenumberofSalmonellacellsontheshelldeclinedby2logupto4weeksand
remainedconstantthereafter.YolkinoculatedSalmonellacountsatcoldstoragedeclinedbyonelog
upto4weeksandremainedconstant,whileroomtemperaturestoragesupportedthegrowthofthe
pathogentoalevelof10
8
colonyformingunitspermloftotaleggcontent,asearlyas4weekspost
inoculation. Examination of egg content following surfaceinoculation revealed the presence of
Salmonella in a portion of the eggs at both temperatures up to 10 weeks, suggesting that this clone
can also penetrate through the shell and survive within the egg. These findings imply that serovar
Infantis is capable of survival and growth both on the exterior and interior of shell eggs. In order to
prevent Salmonella multiplication to high levels during storage at room temperature and to secure
consumershealth,promptrefrigerationofeggsisindispensable.
REFERENCES
1. Braden,C.R.,2006.SalmonellaentericaserotypeEnteritidisandeggs:AnationalepidemicintheUnited
States.ClinInfectDis43:512517.
2. CDC. 2010. Investigation Update: multistate outbreak of human Salmonella Enteritidis infections
associatedwithshelleggsDecember2,2010.
3. Cox, N.A., M.E.Berrang and J.A. Cason. 2000. Salmonella penetration of egg shells and proliferation in
broilerhatchingeggsareview.PoultrySci79:15711574.
4. GalMor, O., L. Valinsky, M., Weinberger, S. Guy, J. Jaffe, Y.I. Schorr, A. Raisenfeld, V. Agmon, and I.
Nissan. 2010. MultidrugResistant Salmonella enterica serovar Infantis, Israel. Emerg Infect Dis 16:1754
1757.
5. Gast,R.K.,R.Guraya,J.GuardBouldin,P.S.Holt,andR.W.Moore.2007.Colonizationofspecificregions
of the reproductive tract and deposition of different locations inside eggs laid by hens infected with
SalmonellaEnteritidisorSalmonellaHeidelberg.AvianDis51:4044.
6. Humphrey,T.J.1994.ContaminationofeggshellandcontentswithSalmonellaEnteritidis:areview.IntJ
FoodMicrobiol21:3140.
7. Lublin,A.,andSela,S.2008.Theimpactoftemperatureduringthestorageoftableeggsontheviability
ofSalmonellaentericaserovarsEnteritidisandVirchowintheeggs.PoultrySci87:22082214.
295
8. Messens,W.,K.Grijspeerdt,andL.Herman.2005.EggshellpenetrationbySalmonella:areview.World
PoultryScij61:7185.
9. Schoeni,J.L.,K.A.Glass,J.L.Mcdermott,andA.C.L.Wong.1995.GrowthandpenetrationofSalmonella
Enteritidis,SalmonellaHeidelbergandSalmonellaTyphimuriumineggs.IntJ.FoodMicrobiol24:385396.
296
TheprevalenceofSalmonellaentericasubspeciesdiarizonae
serovar61:(k):1,5,(7)inSwedishsheepherds
KaisaSRN
1
,HeleneWAHLSTRM
1
,ErikERIKSSON
1
andLennartMELIN
1
1
NationalVeterinaryInstitute,SE75189UPPSALA,Sweden
INTRODUCTION
InSweden,NorwayandFinland,theprevalenceofSalmonellainfoodproducinganimalshasbeen
very low for decades, due to rigorous control programs. However in Norway, an exception from
the otherwise low prevalence of Salmonella is the endemic presence of S. diarizonae 61:(k):1, 5,
(7) in sheep herds (1). In Sweden, a slaughterhouse survey in 1998 indicated a prevalence of S.
diarizonae 61:(k):1, 5, (7) of 0.5% in sheep carcasses (2). From 1998 to 2010 the serovar was
isolated from eleven sheep herds (3),
and since 2006, when sampling of sheep carcasses at

slaughter according to EUlegislation (EC 2073/2005) was initiated it has also been detected in
approximately1.8%ofsheepcarcasses.However,anassessmentoftheprevalenceofthisserovar
atherdlevelhasbeenlacking.InordertoestablishtheprevalenceofS.diarizonae61:(k):1,5,(7)
in Swedish sheep herds, a study was conducted by the National Veterinary Institute during the
winter2012.
MATERIALANDMETHODS
Data on sheep herds (n=16478) was obtained from the Swedish Board of Agriculture. The herd
size distribution was scewed, 79.9% of the herds having 30 sheep or less and 20.1% having
between 31 and 1425 sheep. The sample size was calculated to detect a herd prevalence of 1%
with 95% confidence, and was then increased with 50% since a large falling off in sampling was
expected.Samplingmaterialwassentto474randomlyselectedherds,stratifiedbysize(30or>
30 animals) in order to avoid that most sampling would be done in small herds. The study was
conductedanonymously,andtheonlydataavailableabouteachherdwasthenumberofanimals
intheherdandtheCountyinwhichtheherdwassituated.Samplesconsistedofcompositefecal
samples picked by the animal owner from the bedding, each sample representing at least 15
animals. One and two pooled samples were taken in herds with 38 and > 38 animals,
respectively.About50%oftheanimalsinaherdwereassumedtobeadult(>1year).Thissample
size was considered sufficient to detect a prevalence of 10% in adult animals assuming a test
sensitivity of 1. Each sample (25 g) was analyzed for presence of Salmonella using the MSRV
enrichment method (ISO 6579:2002/Amd 1:2007 Annex D) and any detected Salmonella was
typed biochemically and serotyped by agglutination of Oantigen and flagellar antigen according
to the WhiteKauffmanLe Minor scheme in order to confirm that it was S. diarizonae 61:(k):1, 5,
(7).TheoverallherdprevalenceofS.diarizonae,p
h
,wascalculatedbyweightingtheprevalencein
small herds and large herds, p
h
= 0.799 x p
sh
+ 0.201 x p
lh
, where
p
sh
and p
lh
is the prevalence
in
smallandlargeherdsrespectively.
RESULTS
Sampleswerereceivedfrom262herds.Afterexclusionof18herdswherei)informationonherd
size was missing and ii) wrong number of samples were taken a total of 244 herds remained for
analysis.Atotalof40of100(40%,95%C.I.30.350.2)oflargeherds(>30animals)and17of144
(12%, 95% C.I. 7.018.2) of small herds ( 30 animals) were positive. The overall prevalence was
17.5%(95%C.I.12.822.1).S.diarizonae61:(k):1,5,(7)wasdetectedinallCounties(n=21).
297
DISCUSSION
TheresultsshowedthatS.diarizonae61:(k):1,5,(7)isendemicinSwedishsheepherds.Itismore
common in large herds (>30 animals) and not limited to certain parts of the country. During the
past years, it has been discussed whether to exclude findings of S. diarizonae 61:(k):1, 5, (7) in
sheep from the regular control measures taken in animal herds in Norway when Salmonella is
diagnosed(1).TheresultsofthepresentstudyhaveleadtosimilardiscussionsinSweden.
CONCLUSION
S.diarizonae61:(k):1,5,(7)isendemicinSwedishsheepherdsandnotlimitedtocertainpartsof
thecountry.
ACKNOWLEDGEMENTS
The authors wish to thank the Swedish AnimalHealth Service for promoting this study amongits
members,aswellasallthefarmerswhocontributedtothesampling.
REFERENCES
1. Vitenskapskomiteen for mattrygghet (2008). Salmonella diarizonae hos dyr i Norge konsekvenser for
dyrochmennesker,ISBN:9788280822659.
2. Engvall, A. (1998). Prevalensstudien fr zoonotiska patogen vid slakt hsten 1998. Rapport Statens
veterinrmedicinskaanstalt.
3. Sternberg Lewerin, S. and Elvander, M. (2012). Salmonella diarizonae i svenska frbesttningar. Svensk
veterinrtidning89(64):1115.

298
AlongitudinalstudyonthepersistenceofnonavianSalmonellaEnteritidis
oncattlefarmsintheUK
RebeccaGoslingandRobDavies
DepartmentofBacteriologyandFoodSafety,AnimalHealthandVeterinaryLaboratoriesAgency,
NewHaw,Addlestone,SURREY,KT153N,UK
INTRODUCTION
Salmonella Enteritidis (SE) is normally associated with poultry, however it is also occasionally
found in cattle in Great Britain and throughout Europe
1
. In Great Britain there is no routine
monitoring of Salmonella in cattle, therefore the majority of isolates are identified from clinical
cases.ThemainSEphagetypesisolatedincattlein2011werealsowithinthetoptenphagetypes
identifiedincausingSEoutbreaksinhumans
2
.NonavianSEcasesdonotappeartobereducingin
line with the large reductions seen in poultry after the introduction of harmonised control
regulationsintheEU
2
.Theaimofthecurrentstudywastoconductadetailedinvestigationofthe
distributionandpersistenceofSEoninfectedcattlefarms.
METHODS
Four farms were recruited after being identified through routine surveillance. Individual and
pooled faecal samples and environmental hand swab samples were collected at each visit.
Samples were tested for Salmonella by a BPW/MSRV/Rambach agar based method and a
selectionofisolateswerefullyserotypedandphagetyped.
RESULTS
Onedairyfarm(Farm1)andtwobeeffarms(Farms3and4)wererecruitedafterreportsofclinical
outbreaks.Thephagetype(PT)differedbetweentheclinicaloutbreaksoneachfarmwithPT13a,
PT8 and PT11 found respectively. Two of the three farms were found to be positive at the first
longitudinal visit, with SE being isolated from environmental samples only on Farm 1, where S.
Mbandaka was extremely widespread at both visits. This may have masked SE to an unknown
extent.The concentration of SE in the positive individual faecal samples ranged from 1x10
1
to
1x10
8
cfu/gfaeces.SEPT11wasnotisolatedagainaftertheinitialsingleclinicaldiarrhoeacase.SE
PT13a persisted in pooled water up to visit 3. Farm 2 was initially positive for monophasic
SalmonellaTyphimurium,underadifferentstudy,butbecamepositiveforSEPT8bythe2ndvisit,
with SE isolated from individual and pooled faecal samples and environmental samples. PT8 is a
strainthatisoftenfoundinhumaninfectionsandmaybelinkedtothepresenceofhumanwaste
contaminatingtheenvironment.
DISCUSSION
This investigation to date has demonstrated that non avianSE appears to regress spontaneously,
howevercareshouldbetakentoavoidinfectionofpeoplethatareincontactwiththecattle.
ACKNOWLEDGEMENTS
ThisworkwasfundedbydefraprojectOZ0342.
REFERENCES
1. ANON (2010) Scientific Report of EFSA: The Community Summary Report on trends and sources of
zoonoses, zoonotic agents and foodborne outbreaks in the European Union in 2008, EFSA Journal; 2010
8(1):1496.
2. ANON (2012) Salmonella in Livestock Production in Great Britain 2011 http://www.defra.gov.uk/ahvla
en/publication/salm11/Accessed30/01/2013
299
TABLESANDFIGURES
Figure1:NumberofSalmonellaEnteritidispositiveenvironmentalfaecalsamplescomparedtototal
samplescollectedperfarm.
0
10
20
30
40
50
60
70
80
90
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
R
o
d
e
n
t
s
W
i
l
d

b
i
r
d
s
P
o
o
l
e
d

w
a
t
e
r
G
e
n
e
r
a
l
Visit 1 Visit 1 Visit 2 Visit 1 Visit 1 Visit 2 Visit 3 Visit 4
Farm1 Farm2 Farm3 Farm4
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
Salmonella Enteritidis
positive
Total samples taken
Figure2:NumberofSalmonellaEnteritidispositiveindividualandpooledfaecalsamplescomparedtototal
samplescollectedperfarm.
0
50
100
150
200
250
300
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
I
n
d
i
v
i
d
u
a
l
P
o
o
l
e
d
Visit 1 Visit 1 Visit 2 Visit 1 Visit 1 Visit 2 Visit 3 Visit 4
Farm1 Farm2 Farm3 Farm4
N
u
m
b
e
r

o
f

s
a
m
p
l
e
s
Salmonella Enteritidis
positive
Total samples taken

300
AnalysisofsalmonellosisepidemicsituationintheRFin20082012
OlgaPRUNTOVAandNatalyaSHADROVA
FGBIFederalCenterforAnimalHealth(FGBIARRIAH),Yur`evets,600901VLADIMIRRussia
INTRODUCTION
Salmonellosis morbidity poses a serious problem in many countries of the world. WHO global
monitoring of foodborne infections demonstrated that 47% of outbreaks were caused by
Salmonella,and34%outofthemresultedfromchickenmeatconsumption(3).
"National Laboratory Veterinary Residue Control of Banned and Harmful Substances in Live
Animals, Animal Products and Feeds in the Territory of the Russian Federation" has been
implemented in the Russian Federation since 2004. In accordance with the monitoring plan
biological materials (organs, tissues and sera) collected from farm animals, food raw materials
(milk,meat,offal)andfinishedproductsareregularlysubmittedfortestingtotheFederalCentre
forAnimalHealth(FGBI"ARRIAH").
The aim of this work is the analysis of test results of sera, food raw materials and food products
over the period from 2008 to 2012, as well as of the Rosselkhoznadzor data on salmonellosis
epidemicsituationinfarmanimals(4).
MATERIALANDMETHODS
Test samples were collected and submitted by the representatives of the Rosselkhoznadzor
Administrations for Vladimir, Ivanovo and Kostroma Oblasts in accordance with the monitoring
plan. Microbiological criteria were determined in compliance with the requirements of SanPiN
2.3.2.107801andGOSTR528142007.
Chicken and porcine sera samples were tested for antibodies against Salmonella bacteria by
indirectELISAkitdevelopedintheFGBI"ARRIAH"(1).TheELISAtestswereconductedaccordingto
theroutineprotocol.Theseraweretestedatonedilution1:400withABTS(2,2'azinodi(3ethyl
benzthiazolinesulphonicacid)asasubstrate.Thereadingofresultswasperformedbythevertical
pathwayspectrophotometer,TECANAustria,at492nm.
Serologicaltestsofchickenserasamples(51036samples)demonstratedthatpositivesamplerate
withintheobservationperiodvariedfrom21%in2010to27.70%in2012.
When testing food raw materials and food products (7628 samples in total) for microbiological
criteria Salmonella bacteria were detected. Salmonella contaminated sample rate had the
following variations 0.41% in 2009; 1.7% in 2010; 0.7 % in 2011; 1.5 % in 2012. The majority of
positive samples referred to products containing chicken meat, and that is consistent with the
reportsofotherauthors(3).SalmonellaofsuchserovariantsasS.Enteritidis(70.8%to81.8%),S.
Dublin(2.7%to6.9%)weredetectedmostcommonly.
S. Infantis, S. Moscow, S. Nigeria were isolated in less than 1% of the total amount of samples
contaminatedbySalmonella.
In samples of pork and pork skin contaminated by S. Choleraesuis, S. Typhimurium, S. Dublin, S.
Enteritidisserovariantswereisolatedpredominantly.
Itshouldbenotedthatinthecourseoftheanalysisofserologicalandfoodmonitoringresultsover
theperiodof5yearsnotendencytowardsthedecreaseinSalmonellacontaminationofbiological
materials and food raw materials was observed whereas according to the data on salmonellosis
epidemicsituationinfarmanimalsthedecreaseinmorbidity(Table1)andreductioninnumberof
affected sites in the Russian Federation (Table 2) are noted. Data on salmonellosis epidemic
situationaredevelopedbytheRosselkhoznadzorInformationandAnalysisCentreonthegrounds
ofveterinaryreportsandarepostedontheRosselkhoznadzor'swebsite(4).
301
86
146
103
79
58
114 114
93
52
27
0
20
40
60
80
100
120
140
160
2008 2009 2010 2011 2012
Year
N
u
m
d
e
r

o
f

a
f
f
e
c
t
e
d

s
i
t
e
s
Chickens
Pigs
CONCLUSION
Analysis of epidemic situation and National Laboratory Veterinary Monitoring over the period from
2008 to 2012 demonstrates that notwithstanding the decrease in salmonellosis morbidity in farm
animals and reduction in number of affected sites the Salmonella contamination level of food raw
materials and animal food products varied from 0.41% to 1.7% within the course of observation
periodbutnotendencytowardsdecreaseinSalmonellacontaminationwasobserved.
REFERENCES
1. Kapuskina Yu.V., Pruntova O.V. Production of Salmonella enterica subspecies enterica antigens // Vet.
Pathology.2007.4(23).P.7681.
2. PruntovaO.V.,Rusaleyev V.S.,GnevashevV.M.UsingELISAfordetectionofsalmonellosisantibodiesin
pigs//Veterinariya.2001.12.P.1820.
3. Avian salmonellosis epidemic situation in Russia / S. S. Yakovlev, S.V. Lenev, N.A. Drogalina et al. //
Veterinaria.2008.6.p.1213.
4. www.fsvps.ru/fsvps/iac/rf.html.
TABLESANDFIGURES
Table1.NumberoffoodproductssamplespositiveforSalmonellain20082012.
Year Numberof
samples
%samplespositive
forSalmonella
2008 1255 3(0,24%)
2009 1206 5(0,41%)
2010 1308 16(1,7%)
2011 1428 9(0,7%)
2012 2431 23(1,5%)
Figure1.SalmonellosismorbiditydynamicsinchickensandpigsintheRussianFederationin20082012.
40282
4766
4080
2204
1484
6555
15024
13000
7092
8133
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
2008 2009 2010 2011 2012
Year
N
u
m
b
e
r

o
f

s
i
c
k
e
n

a
n
i
m
a
l
s
Chickens
Pigs
Figure2.ReportedSalmonellacasesforfarmchickensandpigsintheRussianFederationin20082012.

302
GenotypicdiversityofSalmonellaKentuckyisolatedfromreptiles,
poultryfood,feedandenvironmentsamplesinPoland
MagdalenaZAJC,DariuszWASYL,AndrzejHOSZOWSKIandKrzysztofSZULOWSKI
NationalVeterinaryResearchInstitute,DepartmentofMicrobiology,PULAWY,Poland
INTRODUCTION
Spread of antimicrobial resistance among Salmonella become significant public health factor.
Multidrug and highlevel ciprofloxacin resistant Salmonella Kentucky has recently gained
epidemiological importance in several European countries causing infections in turkey, food and
humans[1].InPolanditoccurredinautumn2009,andspreadsmostlyinturkey[2].
Many studies show high occurrence of Salmonella infection in reptiles and indicate on possibility
of transmission to human [3, 4]. In general, reptile related serovars are susceptible for most of
antimicrobials.Nowadays,Salmonellafoundinreptilesoftenbelongstoisolatescharacteristicfor
warmbloodedvertebrates[4].
The aim of the research was to define relationship among reptile S. Kentucky and their
epidemiologicalrelationstotheisolatesfrompoultry,food,andenvironment.
MATERIALANDMETHODS
Twenty four isolates from reptile samples collected in 20102012 were identified as Salmonella
entericaserovarKentucky.Theyderivedfromsnakes(N=8),geckos(N=7,includingonegeckoeggs
sample), chameleons (N=4), surface swabs of reptile environment (N=3), agamas (N=1), lizard
(Varanus)(N=1).Theywerecomparedwith40isolatesoriginatingmainlyfromturkeyflock(N=15),
turkey farm environment (N=9), turkey meat (N=8), poultry meat (N=5) and feed (N=2). One
isolatewasobtainedfromsewagesludge.Someofthoseisolateshavebeendescribedpreviously
[2] PFGE typing was carried out according to the PulseNet protocol following DNA digestion with
XbaI. Antimicrobial resistance profiles were defined based on minimal inhibitory concentrations
interpretedwithepidemiologicalcutoffvalues[2].
RESULTS
Reptile isolates have been clustered into 7 indistinguishable profiles showing 67,3% relatedness
withisolatesfromturkey,meat,feed,sewagesludgeandenvironmentalsamplesshowed(Figure
1). Twenty reptile isolates represented 5 profiles with 83,4% similarity. Basically those isolates
showedno antimicrobialresistance.Oneoftheprofileswasobservedin11isolatesfromgeckos,
chameleonsandreptileenvironment,mainlyfromthesameregionofPolandTheothergathered
five isolates from snakes and agama originated from the different places in different sampling
dates.
Two snake isolates and one obtained from surface swabs after reptile exhibition showed
indistinguishableprofilewithtwoturkeymeatandturkeyflockisolates.AnotherS.Kentuckyfrom
lizardsharedprofilewithturkeymeatisolate.Thoseisolatesweremultidrugresistantandfoundin
diamondpythonsandNilemonitor.
An arbitrary defined 80% cutoff similarity value allowed identification of 3 clusters gathering
isolatesfromdifferentsourcesandresistanceprofiles.Oneofthemembracedmostlysusceptible
isolatesfromreptiles(mostlyfromsnakes,agamas,geckosandchameleons)whiletheotherafew
feed isolates The biggest cluster collected multidrug resistant isolates from turkey, food and
environment,butalsotheonesfrompythonsandlizard.
DISCUSSION
Highly ciprofloxacin resistant and multidrug resistant S. Kentucky has recently gained
epidemiological attention [1, 2]. In the current study, three isolates obtained from pythons and
303
Dice (Opt:0.50%) (Tol 1.5%-1.5%) (H>0.0%S>0.0%) [0.0%-100.0%]
PFGE-XbaI
1
0
0
9
5
9
0
8
5
8
0
7
5
7
0
100
96.3
100
95.8
94.8
100
94.2
100
100
95.7
92.8
100
96
87.2
96.3
77.6
100
96.6
100
95.2
88.2
100
83.8
.3
PFGE-XbaI
0158/2011
0506/2010
0956/2010
1190/2010
1272/2010
1290/2010
1424/2010
1601/2010
1628/2010
1652/2010
1719/2010
2163/2010
2185/2010
2225/2010
2238/2010
2284/2011
2343/2010
2344/2010
2537/2011
1637/2010
0133/2012
0979/2011
1246/2011
1248/2011
1281/2011
1693/2011
1695/2011
2339/2010
1476/2010
1478/2010
2189/2011
1189/2010
1653/2010
1294/2010
1739/2010
2164/2010
0234/2012
0712/2012
0734/2012
1262/2011
0240/2012
1643/2010
2095/2010
2181/2010
1497/2010
1651/2011
1931/2011
0451/2012
0459/2012
0721/2010
0820/2012
1501/2010
0533/2010
0698/2010
1934/2011
1937/2011
1938/2011
1939/2011
1940/2011
1942/2011
2101/2011
2102/2011
2124/2011
2144/2011
3014
3207
1001
0212
0801
0809
0419
2807
0810
3207
3210
0809
2814
3207
3214
3210
0809
0809
0801
3210
0809
2814
1610
1610
0809
2814
2814
0809
1465
1465
0663
0212
3207
2862
1061
0809
0809
2807
2815
3064
2807
0809
1002
2803
1465
0609
2476
0617
0617
0663
1465
1465
0609
0617
2476
2408
2408
2408
2408
2408
2408
2408
2408
2408
AmpNalCipTetStrGenSmx
AmpNalCipTetStrGenKanSmx
AmpNalCipChlFlrTetStrGenKanSmx
susceptible
AmpNalCipStrGenTcySmx
AmpNalCipKan
AmpNalCipKan
AmpNalCip
NalCipStrGenTcySmx
AmpNalCipStrKanSmx
NalCipStrGenTcySmx
AmpNalCipStrGenKanTcySmx
NalCipStrGenTcySmx
AmpNalCipStrCtxCazKanGenTcySmx
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
Str
susceptible
susceptible
susceptible
susceptible
susceptible
susceptible
food (poultry meat)
turkey flock
turkey flock
turkey meat
turkey flock
turkey flock
turkey flock
food (turkey neck skin)
food (turkey neck skin)
turkey flock
turkey flock
turkey flock
turkey flock
turkey flock
turkey flock
turkey - farm environment
food (poultry neck skin)
food (turkey meat)
turkey flock
food (broiler meat)
food (poultry neck skin)
snake
snake
reptile envirnoment
food (turkey meat)
turkey flock
food (poultry meat)
sewage sludge
turkey flock
food (turkey meat)
food (turkey meat)
lizard
food (turkey meat)
turkey flock
feed (components)
feed
snake
snake
reptile envirnoment
snake
snake
agama
snake
snake
gecko
gecko
reptile envirnoment
chameleon
chameleon
chameleon
chameleon
gecko (eggs)
gecko
gecko
gecko
gecko
monitor lizard sharing indistinguishable patterns with turkey and food isolates might indicate
foodborne infection with poultryrelated clones of highly resistant isolates as well as possible
publichealthconsequencesforreptileowners.Suchroutofinfectionhasalreadybeendescribed
for reptileassociated S. Typhimurium outbreak [5]. Most of the isolates belonging to reptile
associated clones are separate group and suggest distinct origin and independent rout of reptile
infection. The same PFGE profiles in isolates from distinct places might result from long distance
tradeofanimalsinfectedwithspecificS.Kentuckyclones.
In conclusion, carnivorous reptiles should be taken into account as possible vector of infection
with multidrug resistant S. Kentucky. Public awareness and education is necessary to prevent
potencialSalmonellainfectionsinreptilebreeders,distributorsandowners.
REFERENCES
1. Le Hello, S., et al. International spread of an epidemic population of Salmonella enterica serotype
KentuckyST198resistanttociprofloxacin.TheJournalofinfectiousdiseases,2011.204(5):p.67584.
2. Wasyl, D. and A. Hoszowski, First isolation of ESBLproducing Salmonella and emergence of
multiresistant Salmonella Kentucky in turkey in Poland. Food Research International, 2012. 45(2): p. 958
961.
3. Bertrand, S., et al. Salmonella infections associated with reptiles: the current situation in Europe. Euro
Surveill,2008.13(24).
4. Zajc, M., et al. Prevalence of Salmonella isolates in exotic reptiles in Poland. in International
SymposiumonSalmonellaandSalmonellosis.2013.SaintMalo,France.
5. Fuller, C.C., et al., A multistate Salmonella Typhimurium outbreak associated with frozen vacuum
packedrodentsusedtofeedsnakes.ZoonosesPublicHealth,2008.55(810):p.4817.
Figure1.RelationshipofS.Kentuckyisolatedfromreptiles,poultry,food,feedandenvironmentsamplesin
Poland

304
PrevalenceofSalmonellaisolatesinexoticreptilesinPoland
MagdalenaZAJC,DariuszWASYL,AndrzejHOSZOWSKI,MagdalenaSKARYSKA,
AnnaLALAK,IlonaSAMCIK,DanutaWNUKandKrzysztofSZULOWSKI
INTRODUCTION
Reptiles are considered as a potential reservoir of many pathogenic bacteria. This coldblooded
vertebrates are known to be asymptomatic carriers of Salmonella often shedding the pathogen
intermittently.
Simultaneously, reptiles have become popular companion animals in many countries. This
increasingpopularityhasledtoanincreasenumberofreptileassociatedSalmonellainfectionsin
human[1,2].
TheaimofstudywastoinvestigatetheoccurrenceandserovardistributionofSalmonellaisolates
inexoticreptilesinPoland.
MATERIALANDMETHODS
Salmonellastrains were isolated from faecal samples orcloacal swabsof healthy animals. A total
of 694 samples were collected from snakes (357), lizards (275), tortoises (60) and crocodiles (2)
from 2010 to 2012. Most samples were collected from reptile farms, zoological shops, reptile
shelter, zoological gardens and private owners or reptile exhibitions. The testing was run
according to EN ISO 6579:2003 standard. Problematic strains were confirmed by PCR tests (i.e.
identificationoffljB,lacZ,iroB,STM3357,STM4057genes).
RESULTS
Salmonella was detected in 86% (598/694) of tested samples, and in 38% of positive cases two
(30%) or more (8%) different serovars per sample were found. Salmonella was found in 92,4%,
84,4% and 60% samples, respectively, taken from snakes, lizards and tortoises. Both crocodile
samples were negative. Single S. bongori isolate was found whereas the remaining 880 strains
belonged to Salmonella enterica including subspecies enterica (66%), salamae (15%), diarizonae
(11%), arizonae (4%) and houtenae (4%). A total of 209 different serovars was noted. The most
frequentwereS.Oranienburg(n=53),S.Tennessee(n=44),S.Agona(n=43),S.Muenchen(n=41),
S. Fluntern (n=39) and S. enterica subsp. salamae 30:l,z28:z6 (n=36). Interestingly, serovars
commoninfood,animalsandhumanwerealsofoundinreptiles,withS.Infantis(n=16),S.Hadar
(n=10),S.Enteritidis(n=10),andS.Typhimurium(n=6)beingthemostprevalent.
All S. enterica subsp. arizonae and more than 93% of S. enterica subsp. diarizonae strains were
isolated from snakes. S. enterica subsp. salamae was the frequent in lizards (65,5%), followed by
snakes(24%).IsolatesofS.entericasubsp.entericaoccurredinallgroupsofreptiles.
DISCUSSION
Similar to other studies, a high prevalence of Salmonella in reptiles was found particularly in
snakes and lizards [3]. Of all studied reptiles, the occurrence of Salmonella was the lowest in
tortoises. One of the possible explanation could be seasonal effects like a hibernation what is
congruent with other research [4]. The majority of serotypes belonged to S. enterica subsp.
enterica. The isolates were characterized with high diversity and many of the serovars had not
beenpreviouslyfoundinPoland.Isolatesbelongingto S.entericasubsp.arizonaeandS.enterica
subsp. diarizonae seemed to be snake related. The frequency of infection was not related to
sampling place. Serovar distribution do not seem to be animal species correlated, but some of
themmightbeconnectedwithfoodintake.Serovarscommoninpoultry,likeS.EnteritidisandS.
Typhimurium, were detected only in carnivorous reptiles like snakes and lizard (Varanus).
305
Interestingly,S.Kentucky,whichanincreasingnumberofisolateshavebeenobservedsince2009
inprimaryproductioninPoland[5]alsowasfoundinsnakesandlizards.
Reptiles should be considered an important reservoir and possible vector for Salmonella
transmissiontohumanhosts.
REFERENCES
1. Woodward, D.L., R. Khakhria, and W.M. Johnson. Human salmonellosis associated with exotic pets.
JournalofClinicalMicrobiology,1997.35(11):p.278690.
2. Zajc, M., et al. Salmonella in reptiles: epidemiology of infection and public health aspect. Medycyna
Weterynaryjna,2011.67(6):p.376379.
3. Geue,L.and U.Loschner.SalmonellaentericainreptilesofGermanandAustrianorigin.VetMicrobiol,
2002.84(12):p.7991.
4. Corrente, M., et al. Isolation of Salmonella strains from reptile faeces and comparison of different
culturemedia.JApplMicrobiol,2004.96(4):p.70915.
5. Wasyl, D. and A. Hoszowski. First isolation of ESBLproducing Salmonella and emergence of
multiresistantSalmonellaKentuckyin turkeyinPoland.FoodResearchInternational,2012.45(2):p.958
961.

Table1.PrevalenceofSalmonellaserovarsinexoticreptilesinPoland
Host
Numberof
samples
investigeted
Salmonella
positive
(%)
Number
ofisolates
Determined
Salmonella
subspecies
ThemostfrequentSalmonellaserovars(n)
lizards 275
232
(84,3%) 349
I(224)
Tennessee(30),Fluntern(23),Kentucky(12),Oranienburg(11),
Infantis(11),Muenchen(10),Pomona(8),Ago(8),Hadar(6),
Newport(3),Enteritidis(1)
II(93) 30:l,z28:z6(23),40:g,m,t:(18),58:a:z6(10),16:m,t:(6)
IIIa(1) 42:z4,z24:(1)
IIIb(15) 50:k:z(4),61:z52:z53(2)
IV(15) 44:z4,z23:(5),42:z36:(2),11:z4z23:(2)
V(1) 48:z65:(1)
snakes 357
330
(92,4%) 486
I(326)
Agona(43),Oranienburg(38),Muenchen(30),Newport(16),
Fluntern(13),ParatyphiBv.Java(12),Enteritidis(9),Kentucky
(8),Typhimurium(6),Rosslyn(2)
II(25) 30:l,z28:z6(11),40:g,m,t:(4),16:m,t:(3)

IIIa(36)
41:z4,z23:(10),13,23:z4,z23,z32:(6),44:z4,z23,z32:(4),
48:z4,z24:(2),44:z4,z32:(2)

IIIb(82)
53:z10:z35(15),47:k:z35(7),14:z10:z(6),57:k:enxz15(4),
59:z52:z53(4)
IV(17) 38:z4,z23:(4),43:z4,z23:(2)
tortoises 60 36(60%) 46 I(36)
4,5:b:(5),Oranienburg(4),Tennessee(3),Fluntern(3),
Teddington(2)
II(9) 1,40:g,m,t:(2),30:l,z28:z6(2)
IIIb(1) 47:r:z53(1)
crocodiles 2 0(0%)
total 694
598
(86,2%) 881
306
PrevalenceandantimicrobialresistanceofSalmonellaspp.inhealthyreptiles
GudrunOVERESCH,SandraZUMWALD,NadiaROBERT,AndreasandTHOMANN
InstituteofVeterinaryBacteriology,VetsuisseFaculty,UniversityofBern,BERN,Switzerland
INTRODUCTION
Salmonellosis is an important zoonosis worldwide. Transmission via the foodborne route for
Salmonella (S.) enterica subspecies enterica was extensively described in the last decades,
whereas for other routes, including reptiles, limited information are available. Increasing
popularity of reptiles may lead to more human cases of reptileassociated salmonellosis in the
future. Therefore data on Salmonella species colonizing healthy reptiles and information about
theirsusceptibilitypatterntoantibioticsareneeded.
MATERIALANDMETHODS
From different Swiss public zoos and a commercial pet shop a total of 210 cloacal or fecal swab
samples from healthy tortoises, turtles, snakes and saurians were collected. Detection of
Salmonella spp. was performed using an enrichment method and subculture onto two different
selective agar plates. Up to eight suspicious Salmonella colonies per sample were phenotypically
identified. Identification and serotyping was done according to the KauffmannWhiteLe Minor
sheme. For susceptibility testing, minimum inhibitory concentration (MIC) using microdilution
method was performed for ciprofloxacin, nalidixic acid, ampicillin, tetracycline, neomycin,
gentamicin, sulfamethoxazole/trimethoprim, and florfenicol according to the guidelines of the
ClinicalandLaboratoryStandardsInstitute(CLSI).
RESULTS
Salmonellaspp.weredetectedin113animals,resultinginatotalprevalenceof53.8%(95CI47.0
60.4%). Substained differences were seen within the animal species. Only 27.5% of tortoises and
turtles(n=80)harbouredSalmonellaspecies,but>60%belongingtoSalmonellasubgenusI.More
than50%ofallsaurians(n=52)werepositiveforSalmonellaspecies,butSalmonellasubgenusIIIb
was predominant and only in 20% Salmonella subgenus I was detected. In snakes 94.7% of the
samples were positive for Salmonella spp., Salmonella subgenus IIIb was followed by Salmonella
subgenusI.AllSalmonellaspp.isolatesfromsaurians,tortoisesandturtlesweresusceptibletothe
antibiotics tested, except for one multiresistant S. Pomona strain, exhibited resistance to
ampicillin, fluoroquinolones and tetracycline. Six Salmonella spp. isolates from snakes displayed
resistance to ciprofloxacin, seven to nalidixic acid. Two strains were resistant to ampicillin and
tetracyclineandonestrainshowedresistancetoflorfenicol.
DISCUSSION
HealthyreptilesareanimportantreservoirofSalmonellaspp.andespeciallyturtles,tortoisesand
snakes harbour S. enterica subsp. enterica. Resistances to fluoroquinolones in Salmonella spp.
fromsnakesunderlinedtheimpactonhumanhealth.Resultsshouldbetakenintoaccountforrisk
assessmentandeducation,especiallyforcontactpersonslikeownersandzookeepers.
ACKNOWLEDGEMENTS
DanielKunzisacknowledgedfortakingthesamplesanddoingbacterialculture.Weliketothank
thestaffofzoosandthepetshopforthereagreementtosupportthisstudy.
307
TABLESANDFIGURES
Figure1:DetectionofSalmonellaspp.inreptiles

308
Arefreelivingturtlesanimportantsource
ofCampylobacterandSalmonella?
SantiagoVEGA
1
,SofaINGRESACAPACCIONI
1
,SaraGONZLEZ
1
,AnnaAGUILAR
1
,
NriaPONCE
1
,MaraAuroraCOLVE
1
,IsabelCALVO
1
,ZoraidaMOLL
1
,
FranciscoMARCOJIMNEZ
2
andClaraMARN
1

1
InstitutodeCienciasBiomdicas,DepartamentodeProduccinAnimal,
SanidadAnimalyCienciayTecnologadelosAlimentos,FacultaddeVeterinaria,UniversidadCEU
CardenalHerrera,AlfaradelPatriarca,VALENCIA,Spain;

2
InstitutodeCienciayTecnologaAnimal,UniversidadPolitcnicadeValencia,
VALENCIA,Spain
INTRODUCTION
Inrecentyears,thenumberofexoticreptileshasbeenrisinginpopularityaspets,andhasledtoan
increaseinthenumberofreptileassociatedsalmonellosisinfections,especiallyinvulnerablepatients
such as infants, young children, the elderly or immunocompromised adults (CDC, 2007). In this way,
turtles represent a special risk, as they are commonly kept as pets for children (Hernndez et al.,
2012). Results of prevalence studies in turtles have shown that the incidence of Salmonella in free
livingturtlesisrangedfrom0%to15.4%(Richardsetal.,2004;HidalgoVilaetal.,2007:2008;Readel
etal.,2008:2010).InthiscontexttheaimofthisstudywastoassesstheprevalenceofSalmonellain
freelivingnative(Emysorbicularis)andexoticturtles(Trachemysscriptaelegans)locatedin11natural
pondsareainEasterSpain(ValenciaRegion).
MATERIALANDMETHODS
Two hundred freeliving turtles were captured from July to October 2012. After capture, every
individual was housed singly in plastic containers with 2 liters of sterile water in order to prevent
bacterialtransmissionamongthem.Watersamplesweretakenaftertwodayskeeping.Thisstudywas
made within the framework of a program for eradication of exotic turtles. Therefore native turtles
were returned to their habitat after sampling, while exotic turtles were euthanized with sodium
pentobarbitalinjectionbeforetakingthesamples(Dolethal,Vtoquinol,E.V.S.A).Foreachindividual,a
cloacal sample was taken using cotton sterile swabs (Cary blair transport swabs sterile, DELTALAB
).
Water samples were collected after 2 days in the container. Moreover, for exotic turtles, after
euthanized, 2 cm of large intestine were collected and the content was homogenized. All samples
were analyzed for Salmonella isolation according to ISO 6579: 2002 (Annex D). The prevalence of
Salmonellaaccordingtothespeciesofturtlestestedandthenaturalpondoforiginwerecomparedby
aChisquareTest.Statisticalanalyseswereperformedusingacommerciallyavailablestatisticspackage
(StatgraphicsPlus,Version5.1,STSCInc.,Rockville,MD,USA).
RESULTS
Overall517sampleswereexaminedforSalmonellaisolation,200fromwaterfromthecontainer(117
fromexoticand83fromnativeturtles),200fromcloacalswabs(117fromexoticand83fromnative),
and 117 from intestinal content (only from exotic). With independence of the species of turtle
analyzed,the12.5%oftheturtlestestedwerepositive.Moreover,outofexoticturtlessampledand
out of the native turtles sampled, 15.4% and 8.4% were positive, respectively. Salmonella was
detectedinexoticandnativeturtlesofeightoftheelevennaturalpondsinvestigated.Theprevalence
of Salmonella in the different natural ponds from Valencia Region was ranged from 4.5% to 60.0%.
309
DISCUSSION
In this study, the prevalence of Salmonella has been tested in two species of freeliving turtles from
Valencia Region. The native threatened species coexist with colonies of the exotic species in many
natural ponds of Valencia Region. Colonies of exotic turtles are the result of the pet trade of
Trachemys scripta elegans, which is forbidden since 1997 in the European Union (Commission
Regulation(EC)No.2551/97,of15December1997).Despitethisfact,manyexoticturtleshavebeen
abandonedinnaturalpondsandhaveestablishedcoloniesthataredisplacingthenativespecies.The
main prevalence detected was moderate (12.5%) and similar to those registered in previous studies
(Harwood et al., 1999; HidalgoVila et al., 2008; Readel et al., 2010). However, results in literature
appear to be contradictory. Several authors reported medium and high prevalence of Salmonella
(Brionesetal.,2004;ChambersandHulse,2006;Gaertneretal.2008;SanchezJimenezetal.,2011),
whileotherstudiesrevealedalowprevalenceofthiszoonoticagentinfreelivingturtles(Mitchelland
McAvoy,1990;Harwoodetal.,1999;Richardsetal.,2004;Saelingeretal.,2006;Readeletal.2008;
HidalgoVilaetal.,2008).Toourknowledge,littleisknownabouthealthstatusoffreelivingexoticand
native turtles in Valencia Region. Results of this study show that these animals are a significant
reservoir of Salmonella in our region. Moreover, there are differences between the prevalence of
Salmonellainturtlesfromtheelevennaturalpondstested.ThehigherprevalenceofSalmonellawas
found in natural ponds where only exotic turtles inhabit. Salmonella shedding in reptiles may be
intermittentandstressinduced,thereforefreelivingturtlesarebelievedtoshedSalmonellaatlower
rates than captive turtles because they are less or not even expose to stress factors, that increase
sheddingrates,orbecausetheyarenotnaturalcarriersofthebacteria(Richardsetal.,2004;Saelinger
et al., 2006). Nevertheless, this study showed that freeliving turtles may be considered as an
important source for Salmonella, and therefore as a risk factor for Salmonella infection. The health
statusoftheseanimalsisapotentialPublicHealthconcernforhumansbecausemostoftheseanimals
are usually in close contact with humans and also a health risk to other native species living in the
sameenvironment.
We thank the staff of the Consellera de Infraestructura, Territorio y Medio Ambiente for their
assistanceandfinancialsupportofthisproject(Life09Trachemys,Resolucin28/02/12CITMA).
REFERENCES
1. Briones, V., S. Tllez, J. Goyache, C. Ballesteros, M. P. Lanzarot, L. Domnguez, and J. F. Fernndez
Garayzbal, 2004: Salmonella diversity associated with wild reptiles and amphibians in Spain. Environ.
Microbiol.6,868871.
2. Centers for Disease Control and Prevention, 2007: Turtleassociated Salmonellosis in humans United
States,20062007.Morb.Mortal.WklyRep.56,649652.
3. Centers for Disease Control and Prevention, 2010: Multistate outbreak of human Salmonella
typhimuriuminfectionsassociatedwithpetturtleexposureUnitedStates,2008.Morb.Mortal.WklyRep.
59,191196.
4. Chambers, D. L., and A. C. Hulse, 2006: Salmonella Serovars in the Herpetofauna of Indiana County,
Pennsylvania.Applied&EnvironmentalMicrobiology.72,37713773.
5. Gaertner, J. P., D. Hahn, J. Jackson, M. R. J. Forstner, and F. L. Rose, 2008: Detection of Salmonellae in
CaptiveandFreeRangingTurtlesUsingEnrichmentCultureandPolymeraseChainReaction.J.Herpetol.42,
223231.
6. Harwood, V. J., J. Butler, D. Parrish, and V. Wagner, 1999: Isolation of fecal coliform bacteria from the
diamondbackterrapin(Malaclemysterrapincentrata).Appl.Environ.Microbiol.65,865867.
7. Hernndez, E., J. L. Rodriguez, S. HerreraLen, I. Garca, V. de Castro, and N. Muniozguren, 2012:
SalmonellaParatyphiBvarJavainfectionsassociatedwithexposuretoturtlesinBizkaia,Spain,September
2010toOctober2011.Euro.Surveill17.
8. HidalgoVila, J., C. DazPaniagua, C. de FrutosEscobar, C. JimnezMartnez, and N. PrezSantigosa,
2007:Salmonellainfreelivingterrestrialandaquaticturtles.Vet.Microbiol.119,311315.
310
9. HidalgoVila, J., C. DazPaniagua, N. PrezSantigosa, C. de FrutosEscobar, and A. HerreroHerrero,
2008: Salmonella in freeliving exotic and native turtles and in pet exotic turtles from SW Spain. Res. Vet.
Sci.85,449452.
10. Readel,A.M.,C.A.Phillips,andT.L.Goldberg,2008:AbsenceofCloacalSheddingofSalmonellainwild
redearedsliders(Trachemysscriptaelegans).Herpetol.Rev.39,427430.
11. Readel, A. M., C. A. Phillips, and T. L. Goldberg, 2010: Prevalence of Salmonella in Intestinal Mucosal
Samples from Freeranging RedEared Sliders (Trachemys scripta elegans) in Illinois. Herpetological
ConservationandBiology5,207213.
12. Richards, J. M., J. D. Brown, T. R. Kelly, A. L. Fountain, and J. M. Sleeman, 2004: Absence of detectable
SalmonellacloacalsheddinginfreelivingreptilesonadmissiontothewildlifecenterofVirginia.J.ZooWild
Med.35,562563.
13. Saelinger, C. A., G. A. Lewbart, L. S. Christian, and C. L. Lemons, 2006: Prevalence of Salmonella spp in
cloacal, fecal, and gastrointestinal mucosal samples from wild North American turtles. J. Am. Vet. Med.
Assoc.229,266268.
311
AGFPpromoterfusionlibraryforthestudyofSalmonellabiofilmformation
andthemodeofactionofbiofilminhibitors
Stijn
ROBIJNS
1
,Stefanie
ROBERFROID
1
,Sandra
VANPUYVELDE
1
,Bart
DEPAUW
1
,
ElenaUCEDASANTAMARA
1
,AmiDEWEERDT
1
,DavidDECOSTER
1
,KimHERMANS
1
,
SigridDEKEERSMAECKER
1
,JosVANDERLEYDEN
1
andHansSTEENACKERS
1
1
KULeuven,departmentofMicrobialandMolecularSystems,CentreofMicrobial
andPlantGenetics,KasteelparkArenberg20,B3001LEUVEN,Belgium
INTRODUCTION
Salmonella,animportantfoodbornepathogen,formsbiofilmsinmanydifferentenvironments[1].
The composition of these biofilms differs depending on the growth condition and their
developmentishighlycoordinatedintime.Todevelopefficienttreatmentsitisthereforeessential
that biofilm formation and its inhibition is understood in different environments and in a time
dependent manner. Many currently used techniques, like transcriptomics or proteomics, are still
expensive and thus limited in their application. Moreover, several highthroughput techniques
determineanaverageprofileforthetotalbiofilm,notprovidinginsightintobiofilmheterogeneity.
Therefore a GFPpromoter fusion library with ~100 important Salmonella biofilm genes was
developed, covering i.a. genes involved in biofilm regulation, matrix production, quorum sensing,
metabolism, motility and cdiGMP synthesis and degradation. This library is a fast, inexpensive
and easytouse tool and can therefore be conducted in different experimental setups in a time
dependentmanner.Here4possibleapplicationsarehighlightedtoillustrateandvalidatetheuse
of this reporter fusion library: mode of action study of biofilm inhibitors, localization of gene
expressioninbiofilms,identificationofantibiofilmtargetsandevaluationofdifferencesbetween
differentSalmonellastrains.
MATERIALANDMETHODS
A GFPpromoter fusion library of important Salmonella biofilm genes was constructed. The
selectedgeneswereidentifiedfromliteratureorbasedoninhouseknowledgeandcovermostof
the processes that have been shown or are expected to be important for Salmonella biofilm
formation. Currently the library contains ~100 reporter fusions, and is being extended according
tonewknowledgethatbecomesavailable.Thepromotersequencesofthegeneswereamplified
out of S. Typhimurium ATCC14028 and cloned into the pFPV25 vector (Ap
r
), which contains a
promoterlessgfpmut3genethatproducesaverystableGFP,withahalflifeinE.colireportedto
bewellover24h[2].Allconstructswereverifiedbysequencingandsubsequentlyelectroporated
intoSalmonellastrainsATCC14028andSL1344.
RESULTS
TostudythemodeofactionofSalmonellabiofilminhibitors,theireffectontheGFPexpressionof
all the reporter fusions was measured in time, using a multiwell plate based assay. As such we
could quickly identify the effect of the compounds on specific biofilmrelated processes. Results
with one of our biofilm inhibitors indicate that this compound inhibits the expression of csgD,
encoding the master regulator of Salmonella biofilms, and its downstream genes adrA and csgB.
FurthermorewewereabletopinpointanumberofupstreamregulatorsresponsibleforthecsgD
downregulation.
Biofilms are known to be heterogeneous. Therefore we used the gene reporter library to study
spatial differences in expression within rdar colonies by fluorescence imaging and FACS analysis.
Clear location dependent differences in gene expression were found amongst others for csgD,
fimAandflhD.
312
Since in situ biofilms can exist of more than one microbial species, it is essential for the future
identification of new inhibitors to identify antibiofilm targets which are of importance both in
monospecies and multispecies biofilms. Therefore we compared by FACS analysis the expression
of the promoter fusions in monospecies Salmonella biofilms and multispecies biofilms containing
Salmonella. Some genes showed a high and similar expression in monospecies and multispecies
biofilms(withoutbeingexpressedinfreelivingcultures)andthereforeformgoodtargetsfornew
antibiofilmstrategies.
RemarkabledifferencesinthebiofilmformationcapacityofS.Typhimuriumstrainsexist.Weused
the reporter fusion library to comparethe gene expression in biofilms of strains ATCC14028 and
SL1344.WecouldexplainthelowerbiofilmformationcapacityofSL1344byalowerexpressionof
themlrAgene([3]andattributethistoalowerexpressionofrpoS.
DISCUSSION
Thestudiesaboveshowthatthereporterfusionlibraryisaversatiletooltostudygeneexpression
in(Salmonella)biofilmsinmanydifferentexperimentalsetups. Thereforethelibrarycanbeused
toquicklygatherexpressionprofilesinmanydifferentenvironmentsforbothfundamentalbiofilm
studies and applied mode of action studies of biofilm inhibitors. This library is not limited to
applications on abiotic surfaces in vitro, but can also be used to study the (biofilm related) gene
expression on e.g. epithelial celllines or in vivo (e.g. in the roundworm Caenorhabditis elegans)
and could also prove useful to study the role of biofilm formation in the infection processes of
Salmonella. After all, the link between biofilm formation and virulence in Salmonella is still not
fullyunderstood(Steenackersetal.2012).
Based on new knowledge becoming available (either in literature or by own experiments) this
library is constantly being extended and is therefore becoming an evolving tool to quickly gather
timelapse expression profiles in Salmonella biofilms. This not only delivers valuable direct
informationoncellularprocessesinvolved,butcanalsobeusedtodeterminethemostinteresting
andimportanttimepointsforadditionalhighthroughputstudies(transcriptomics,proteomics,).
Whilethislibraryhasaclearbiastowardsbiofilmrelatedprocessesandisunabletodeliverafull
pictureofthegeneexpression,theeaseofuseenablestoquicklygatherinformationoninvolved
biofilmprocessesinmanydifferentenvironments.
REFERENCES
1. Steenackers,H.,etal.,FoodResearchInternational,2012.45(2):p.502531.
2. Andersen,J.B.,etal.,ApplEnvironMicrobiol,1998.64(6):p.22406.
3. Garcia,B.,etal.,MolMicrobiol,2004.54(1):p.26477.
313
MonitoringofSalmonellainfood,feedandveterinarysamples
bytheFrenchSalmonellanetwork
FrdriqueMOURY,RenaudLAILLER,CatherineLAPORTE,VivianeMOREL,
ClaudeOUDART,ChristinePIQUETandAnneBRISABOIS
LaboratoryforFoodSafety,UnitBacterialCharacterizationandEpidemiology,
MAISONSALFORT,France
INTRODUCTION
Despite many measures of prevention and control in France, Salmonella are still the leading cause of
notified foodborne bacterial diseases in human (1). Thus, the implication of Salmonella in large
outbreaks and the importance of the salmonellosis associated to large economic and public health
consequencesrequestthemonitoringofSalmonellaisolatesfromallsectorsoforigin.Formanyyears,
the MaisonsAlfort Laboratory for Food Safety(AnsesLSAl) has been collecting non human Salmonella
strains with epidemiological information concerning sample origin, in collaboration with 150 public or
private veterinary or foodhygiene laboratories throughout France and organized in a network since
1997. Overthepastyears,15000to 20000 data peryearhavebeencollected.Salmonellaserotyping
resultsarepublishedinanannualreport(2).ThetwomainobjectivesoftheSalmonellanetworkareto
bringalongascientificandtechnicalsupportforSalmonellaserotypingandtostudythespatiotemporal
trends of the nonhuman Salmonella isolates at a national level in terms of serovars, animal and food
sourcesandgeographicorigins.
MATERIALANDMETHODS
TwokindsofSalmonelladataarecollectedthroughtheSalmonellanetwork:
- Salmonellaisolatessenttotheserotypingcentreforserotyping.
- Resultsofserotypingcarriedoutbysomepartnerlaboratoriesthemselves.
In each case, epidemiological information concerning isolates are transmitted. Confirmation of
Salmonella strains is checked by biochemical characterization tests and then serotyping is performed
using agglutination technique with polyvalent and monovalent antisera against somatic and flagellar
antigen according to an internal method in reference to the WhiteKauffmannLe Minor scheme (3).
Resultsofserotypingandepidemiologicalinformation,especiallygeographicaloriginandsamplenature,
are entered in an Access software database, allowing the analysis and classification of strains. For
epidemiologicalanalysis,strainsareclassifiedintothreemajorsectorsaccordingtotheirorigins:animal
healthandbreedingenvironment,foodhygieneincludingenvironmentoffoodproduction,andnatural
ecosystem.
RESULTS
During theyear 2011, 18 815 Salmonella data werecollectedfrom non human origins. Analysis of the
distributionoftotalstrainshighlightedthepredominanceofstrainsfromtheanimalsector(74.2%).Food
hygienerepresented24%ofstrains,outofwhich3.3%wereisolatedfromfeedstuff.Finally1.8%came
from natural environment. The collected strains were distributed into 272 serovars, most of them
(97.1%) belonging to the species enterica subspecies enterica (I). The most frequently encountered
serovars were Senftenberg, followed by Typhimurium and Indiana. These three serovars together
represent 39.8% of the analysed strains. Variations in the serovar frequencies were observed:
Senftenberg,Typhimuriummonophasicvariant"S.I1,4,[5],12:i:"andRegentincreased,whereasDerby,
LivingstoneandAnatumwereindecreasing.

314
Animalspeciesandbreedingsector
13 961 animal strains were collected during this period and were divided into three major animal
sources: 92.5% were recovered from poultry, 5.1% from cattle and 1% only from swine. The most
prevalent serovars in this sector were Senftenberg followed by Typhimurium and Indiana (all three
mainlyisolatedinpoultrystrains).Theselasttwoserovarswereinrelativedecreasecomparedtoother
serovarssuchasMbandaka,Regent,andMontevideowhichwereinhighincreaseamongpoultrystrains.
Some serovars were specifically associated to poultry species such as Indiana, Regent and Kottbus in
duck, Senftenberg and Newport in turkey, Senftenberg, Mbandaka and Typhimurium in chicken.
MontevideoandMbandakawerethemainserovarsisolatedfrombovineandDerbyfromswine.
Foodhygienesector
Among the 4 523 isolates from food hygiene sector, 630 came from feedstuff and 3 893 from food
products.Analysisoffoodorigindistribution,showedthatalargepartofthemwereisolatedfrompork
meat, meat products and food processing environment of pork (21.3%). Strains also were mainly
recovered in food products such as: poultry meat (15.3%), beef and veal meat (3.6%), delicatessen
(11.9%), milk and milk products (18.7%), eggs and egg products (1.3%). The other sources were
represented by sea food products and ready to eat meals (7.7%) or by food processing environment
(2.2%). The first identified serovars in human food products were: Typhimurium (particularly in meat
productsanddelicatessen),followedmostlybyDerby,Dublin(inincreasefollowingtoareinforcementof
the self inspection), the Typhimurium monophasic variant "S.I 1,4,[5],12:i:" (increasing linked to
foodborne cases), Indiana, Rissen. Some predominant serovars are strongly associated to a product
category : Indiana and Kottbus in poultry, Enteritidis in eggs and egg products, Derby and the
Typhimuriummonophasicvariant"S.I1,4,[5],12:i:"inporkproducts,"S.IIIb61:k:1,5,7"indairyproducts.
MontevideoandMbandakawerethemainserovarsfoundinfeedstuff.
Naturalecosystemsector
331strainsmainlyrecoveredfromwater,sewageandmudswerecollectedduringthisperiod.Theyare
representedbyalargediversityofserovars.Typhimuriumwasthemostprevalentserovarfollowedby
Mbandaka.
CONCLUSION
TheseresultsarethesynthesisofdatacollectedfromtheSalmonellanetworklaboratories.Since1997,
thecreationofthenetworkhasimprovedtheexchangeofisolateswithepidemiologicaldata.Although
data are transmitted on a voluntary basis, and therefore are not exhaustive, Salmonella network
performance is stable. Thus, quantitative and qualitative importance of the collection linked to
epidemiological information is valuable to describe the current situation at a national level and to
evaluate trends in terms of serovars, sectors and geographical origin for many years. The
implementation of regulation in poultry sector, by targeting certain channels, some serovars and the
food surveillance plan, constitutes a selective pressure for the rise of the information. Thus, the
informationrelativetoSalmonellanotcoveredbytheregulationsshouldbeunderestimate.
ThesedescriptiveresultsfromtheglobalSalmonellanetworkmaybecompiledwithothermorespecific
networksmonitoringanimalSalmonella.Moreover,theepidemiologicaldatacouldbealsocomparedto
human Salmonella monitoring data performed by the National Reference Centre (NRC), especially in
case of epidemiological investigation of Salmonella foodborne outbreaks, in collaboration with the
InstitutdeVeilleSanitaire(InVS)andtheDirectionGnraledelAlimentation(DGAl).Thesesurveillance
datacontributetothecontrolandpreventionoffoodbornediseases,thustotheimprovementofrisk
assessment.
315
ACKNOWLEDGEMENTS
The Salmonella network team thanks the partner laboratories for their active collaboration to this
surveillance.
REFERENCES
1. Anonymous. Surveillance des toxiinfections alimentaires collectives Donnes de la dclaration obligatoire,
2010.Availableonthewebsite:http://www.invs.sante.fr.
2. LaillerR.,FremyS.,OudartC.,PiquetC.,PiresGomesC.,DananC.,GranierS.,MouryF.,BrisaboisA.(2012):
Inventaire des Salmonella dorigine non humaine Rseau Salmonella 2010. Available on the web site:
http://www.anses.fr.
3. Danan C., Fremy S., Moury F., Bohnert M., Brisabois A. (2009): Determining the serotype of isolated
Salmonellastrainsintheveterinarysectorusingtherapidslideagglutinationtest.Euroreference,n2.Availableon
thewebsite:http://www.anses.fr.
316
SetupofaNationalmoleculartypingdatabaseforSalmonellasurveillance
SophieROUSSEL
1
,CorinneDANAN
1
,BenjaminFELIX
1
,SabrinaCADELSIX
1
,MurielMARAULT
1
,
SergeBORREL
2
,AnneBRISABOIS
1
andCaroleFEURER
3
1
Anses,FrenchAgencyforFood,EnvironmentalandOccupationalHealth&Safety,
MaisonsAlfortLaboratoryforFoodSafety,UnitBacterialCharacterizationandEpidemiology,
23avenueduGnraldeGaulle,F94706MAISONSALFORTCedex,France;
2
Anses,FrenchAgencyforFood,EnvironmentalandOccupationalHealth&Safety,Information
TechnologyServices,23avenueduGnraldeGaulle,F94706MAISONSALFORTCedex,France;
3
Ifip,FrenchPorkInstitute,7avenueduGnraldeGaulle,
F94704MAISONSALFORTCedex,France
INTRODUCTION
IFIP, the French Pork Institute, has since 2006 been developing a database of epidemiological
information,serotypingdataandPFGEprofilesofmorethan850representativeSalmonellastrains
specifically isolated from the pig and pork industry. This database aims at establishing how
Salmonella strains circulate from pig farming to finished products in order to better control the
hazard,andtodevelopamonitoringtoolfortheporksector.
Moreover, since 1997, through the Salmonella network, the MaisonsAlfort Laboratoty Food
SafetyofAnseshasbeendevelopinganationaldatabaseforstrainsisolatedfromtheentireagro
food chain. This database includes the most frequently detected Salmonella serotypes and
contains about 3700 PFGE profiles of strains isolated from food, animals and the environment.
Thisdatabaseisregularlyconsultedbypublichealthandfoodauthoritiesduringinvestigationsand
outbreaks.
The consolidation of capability for standardized typing by PFGE (1) and the need for exchanging
typingdatabetweenAnsesandIfipresultedintherecentcreationofamoleculartypingdatabase.
Thisdatabase,setupintheframeoftheTERESAjointtechnologicalunit,anationalcollaborative
project, will be shared between the French technical institutes involved in the national
surveillance of Salmonella. This article describes in a first part the activities of typing
standardization which have led to the setting up of the database, and in a second part the
differentstepsinvolvedinthedevelopmentandfunctioningofthedatabase.
1) Consolidationofcapabilitiesforstandardizedtyping
Ifip has organized, in 2011 and 2012, two PFGE proficiency test trials (PT trials), for the
participants, including different Anses laboratories and three French technical institutes
ACTILAIT, AERIAL and ADRIA Dveloppement. These PT trials provided a valuable
opportunity to facilitate and to stimulate exchanges of reproducible PFGE profiles between the
collaborativepartners.
Moreover, Ifip has been trained by Anses to a PFGE profile interpretation standard operating
procedure (SOP) which reduces operatordependent subjectivity. These activities enabled to (i)
consolidate typing capabilities, (ii) harmonize PFGE typing methods, and (iii) improve the
reproducibilityandinternalcommunicationbetweenpartners.
2) DevelopmentandfunctioningoftheDatabase
ThedatabaseishostedbytheMaisonsAlfortLaboratotyFoodSafetyofAnses.
Anseswasinchargeofthesetupofthedatabaseincludingtypingresults(conventionalserotyping
and PFGE data) as well as epidemiological information related to strains isolated from food,
environmentaloranimalorigins.
317
Anses is administrator and curator of this database. The administrator is responsible for the
organizationofthedatabase.Healsomanagestheparticipantsconnections(loginandpasswords)
andisresponsibleformaintenance.
Validatedprofilesareaddedtothedatabaseandidentifiedincompliancewiththenomenclature.
ThecuratorisalsoinchargeoftroubleshootingandtechnicaladvicetopartnersonPFGEprotocol
and gel analysis aspects. After each profile submission, the curator reports back to the partners
(acceptance or rejection of the profile(s) submitted). This report is transmitted to the participant
throughthecommunicationmailbox.ThetechnicalskillsofthecuratorforPFGEgelinterpretation
is regularly checked through an internal habilitation process describing the test for curator
qualification.
The user can consult the database by matching its profile against database existing entries or by
consultingdirectlythedatabaseaccordingtoepidemiologicalcriteria.Participantscriteriarequired
to access the database include the successful participation to a PFGE PT trial. Participants must
complywiththememorandumofunderstanding(MoU)whichregulatesthedatabaseuse.
Thedatabaseismanagedbyasteeringcommitteecomposedofonememberforeachparticipant.
It is supported by a data processing platform, based PulseNet communication script with
agreementofUSCenterforDiseaseControlandPrevention.
The PFGE profile nomenclature is based on codification previously used during the SalmGene
project (2). The epidemiological classification was defined with regards to Salmonella risk,
accordingtoEFSArecommendations.
Up to date, 4 partners were trained to the use of the database. The communication scripts
developedandtestedatAnseshavebeentransferredtothepartnersatthebeginningoftheyear
2013andtestsarecurrentlyongoing.
This database is designed to be opened to (i) the integration of alternative molecular typing
methodsand(ii)communicationwithotherdatabasessuchasthosecontaininghumanstrains.
CONCLUSIONS
Used in combination with collaborative epidemiological investigations, the multisector database
should improve the surveillance of Salmonella in the food chain by (i) better allowing the
detection of contamination clusters, (ii) optimizing the detection of emerging strains potentially
pathogenicforconsumersand(iii)suggestinglinkswithpotentialsourcesofcontamination.Such
linking between food and human strains would represent an efficient tool for epidemiological
investigations, would allow a better assessment of the importance of certain foods and animal
populations as sources of Salmonella foodborne infections and outbreaks, through casebycase
source attribution studies, and finally would allow a better understanding of the ecology of this
pathogeninthefoodchain.

REFERENCES
1. Feurer C, et al. 2010. Setting up a French molecular subtyping database for Salmonella surveillance in
thepigandporkindustry.I3S.SaintMalo(France)2830June2010.
2. Peters, T.M et al. 2003. The Salmgene project a European collaboration for DNA fingerprinting for
foodrelatedsalmonellosis.Eurosurveillance.Vol8,n2,4650.
ACKNOWLEDGEMENTS
The authors would like to thank ACTIA (Technical Coordination Association for the Food and
AgricultureIndustry)foritsfinancialsupport.

318
May 27-28-29, 2013
Saint-Malo France
Session 6
Chairpersons:
Marcel ZWIETERING (the Netherlands) and Jean-Christophe AUGUSTIN (France)
319
Riskassessmentandcontrol
MarcelH.ZWIETERING(TheNetherlands)
ABSTRACT
Food safety management has changed in the last century from end product criteria, to HACCP and use of quantitative
risk assessments. Looking towards the future these approaches need to be extended to cover the whole food chain, and
given all the interconnections even food webs.
End-product testing is in no way a good method to control food safety, neither is end-of-line harsh treatments the
ultimate best control option. Optimal food safety control goes from seed and feed, through primary production, food
processing and storage and ultimately consumer handling and consumption. All dynamics of hazards throughout a whole
food chain will be difficult to determine accurately, however first global analysis of dynamics of prevalences and
concentrations can show important bottlenecks and direct more detailed analysis, for example including variability. This
is a better approach than neglecting that food safety is influenced from farm to fork. Finally the whole food chain should
be analysed and controlled with a HACCP like system, where the critical control points are placed in the part where they
are most efficient. Furthermore for a record of safety the dynamics of the hazards need to be estimated and validated.
The ICMSF equation for the prevalence and levels of microorganisms from the initial contamination (H0), reduction ( R),
growth and recontamination ( I), and factors influencing these are considered throughout food production until
consumption, and in their role in meeting the FSO.
Of course end product data can and should be used for verification if the overall safety system is working properly and
as intended, and to detect deviations. The combined information of specifications (design), critical processes and their
validation (control), intermediate and end product testing data (verification) together comprises the record of safety that
turns into a license to produce.
Sincethe1920sprocesscriteriaforheattreatmentsinfoodprocessinghavebeenestablishedand
integratedinlaws.Sincethe1960smicrobialcriteriahavebeenestablished,mainlythroughCodex
Alimentarius.Sincetheendingofthelastcentury,HACCPhasbeenenteredinfoodlaws,andmore
and more quantitative microbiology models and quantitative risk assessments have been carried
out and used for legislation. Currently the new ALOP and FSO metrics are developed for setting
microbialandprocessstandards.
Modelscanbesubdividedinprimary,secondaryandtertiarymodels.Primarymodelsdescribethe
dynamics over time, for example growth or inactivation models. Secondary models describe the
effectofproductandprocessvariablesongrowthandinactivationrates.Tertiarymodelsintegrate
primary and secondary models, and possibly other models like heat transfer, to predict the
behaviourofmicroorganismsinprocesses.
Initially deterministic quantitative microbiology model were developed, but through the years
more and more variability and uncertainty have been integrated in models and in risk
assessments. Full quantitative risk assessments over the whole food chain from primary
production until consumption are often very extended, complex, containing many assumptions
anddifficulttointerpret.
Overallmuchprogresshasbeenachievedonmodellinggrowthandinactivationofmicroorganisms
in food, however it should be realised that for a proper chain analysis various factors are of
relevance that have had relatively less attention, like the initial level, recontamination, and lag
phasesunderrealisticconditions.Furthermoretherearerelevantfactorslikestrainvariability,that
cannot be controlled. Often doseresponse relations are generally known with little accuracy,
contributing much to uncertainty in the overall analysis. Also variability in consumer behaviour,
like for instance refrigerator temperature and storage time can result in even greater variability
than the effects of biological variability of the microbes. Therefore inaccuracies in growth and
320
inactivation models are in many cases not the most relevant factors to obtain more information
on.
For an overall overview of the dynamics and changes in prevalence and concentration over the
wholefoodchain,theFSOconceptcanbeausefultool.Afterafirstdeterministicanalysisalsoin
thisconceptvariabilitycanbeincludedtomaketheanalysismorerealistic.Byusingthisapproach,
food safetycontroloverthechaincanbevalidated.Validationisthecollectionandevaluationof
scientific and technical information to determine if the processes in a food chain, when properly
applied,willeffectivelycontrolthemicrobiologicalhazard,orinotherwords,iftheprocesscriteria
can reliably deliver a specified performance or food safety objective. The advantage of the FSO
concept is that for every stage, initial levels, inactivation, increases due to growth and
recontamination have to be evaluated and estimated. The approach can therefore help to
structurally evaluate all relevant phenomena in every stage of the chain, helping not to overlook
relevantphenomena.Thisthencanfurthermorehelptodetectthemostrelevantphenomenaand
derive critical control points in the whole chain and performance objectives (intermediate
specifications)foreverystageinthefoodchain.Inevaluatingalltheseaspectstherewillbefactors
thatwillintroduceuncertaintylikeinitiallevels,recontamination,consumerhandlingetc.andthis
candirectfurtherdataneedsornecessityforcontrol.
The approach gives the possibility to perform risk management over the whole food chain,
including primary production and the consumer stage, not only focussing on the stage of food
productionandstorage.Thisthencanhelptodeterminebalancedoptimalinterventions,atthose
locationsinthefoodchainwheretheyaremostefficient.Furthermorenotineverystagehazards
need to be controlled, they need to be controlled (at least) somewhere in the chain. The overall
procedureisstructuredandtransparent,isriskbased,andforcestoputnumbers.TheHACCPlike
system,basedonPerformanceObjectivesinallstages,andintegratingallincreasesanddecreases
in every stage, with validation data of these phenomena, and data of the critical points, ensures
the correct functioning of the chain. Finally this can result, together with verification data, based
onforexampleendproducttesting,inaRecordofSafetyoralicencetoproduce.
Integrating dynamics of phenomena over the whole chain, including prevelances and
concentration and variation therein, can give important clues for control, since for robust food
safety,thelevelneedstobecontrolled,butalsothevariabilitythereof.
321
May 27-28-29, 2013
Saint-Malo France

Session 6

Chairpersons:

Communications
322
Changingthepresentationofpigsfeed:
acosteffectivesolutiontoreduceSalmonellaspp.excretion
PhilippeLEBEL
1
,PhilippeFRAVALO
1
,JessieLONGPRE
1
,BenotLAPLANTE
2
,DanielMASSE
3
andAnnLETELLIER
1
1
ChairedeRechercheenSalubritdesViandes,FacultdeMdecineVtrinaire,
UniversitdeMontral,STHYACINTHE,Qc,Canada;
2
F.Mnard,LANGEGARDIEN,Qc,Canada;
3
AgricultureCanada,SHERBROOKE,Qc,Canada
ABSTRACT
If some studies have attempted to mitigate the Salmonella spp. excretion in pigs by feed related interventions, none
clearly demonstrated the impact of the presentation (mash or pellet and particular size). Thus this study aimed to
determine if the modification of the pigs feed presentation alone can lower the Salmonella spp. excretion. To do so, 144
eight week aged piglets, previously confirmed as homogeneously in contact with Salmonella, were given diets that varied
only by the particle size (500, 750 or 1250m) and the texture (mash or pellet). They were individually sampled on day
0, 21 and 88 after feed transition for weight, blood and feces collection. After only 21 days, significantly less pigs
excreted Salmonella spp. in the 500m mash (1/24), the 1250m pelleted (5/24), and 1250m mash (5/24) (P<0.05)
than in the 500m pelleted (15/24) reference group. These differences should be consider in the perspective of a greater
production (P<0.05) of butyric and propionic acids measured in colon contents from the 1250m mash group
(respectively 2179 and 3050mg/L) than in the 500m pelleted group (respectively 1260 and 2493mg/L) while other
short-chain fatty-acids concentration (acetic acid) didnt vary. Further analyses, qPCR tests, will allow determining any
changes in the intestinal Lactobacilli, E. coli and Bifidobacteriumpopulations. Finally, it should be noticed that for the two
groups with significantly lower Salmonella spp. excretion (500m mash and 1250m pelleted) the feed conversion was
equivalent to the 500m pelleted group, most commonly used in the industry. In conclusion, this study is the first one to
demonstrate and suggest how to mitigate Salmonella spp. excretion by only modifying the texture or the feed particle
size without raising the production cost in swine.
INTRODUCTION
Modifying the pigs feed to mitigate the presence of Salmonella spp. in herds at the farm level
while reducing the use of antibiotics is gaining in popularity over the years. Many solutions have
been explored but none resulted in the elimination of the pathogen. These strategies are
associated with beneficial modifications of the gut microbiota and its production of antibacterial
element such has volatile fatty acids (Mountzouris, 2006). One promising strategy is the use of
mashfeedinsteadofthecommonpelletedfeed(LoFoWong,2004).Inherreviewcomparingfeed
management practices and feed characteristics associated with Salmonella prevalence, Oconnor
et al. (2008) came to similar conclusions based on serological data. Unfortunately, none
demonstrated a Salmonella spp. excretion reduction and many variables were present between
thecompareddiets(composition,particlesize,texture,heating,etc.).Itisalsoimportanttonotice
thatwhilepromisingontheSalmonellabasistheuseofmashfeedoftencomeswitharaiseinthe
production cost. The goal of this study was to isolate the different factors possibly involved in a
reduction of Salmonella spp. excretion on the farm when only the feed presentation is modified
(mash/pellet/particlesize).
MATERIALANDMETHODS
Onfarm
Nine hundred 5 weeks old piglets known to have been in contact with Salmonella spp. at the
nurseryweresplit(10perpen)into6groups.Eachgroupreceivedadifferentdietsvaryingonlyby
theirparticlesize(500,750or1250m)ortexture(mashorpellet).Pellet500beingthereference
groupfromtheindustry.Outofthese,144pigs(24fromeachdiet,2perpen)wereweightedon
days: 0, 21, 46, 86. Individual blood and feces sample were taken as well as gut content at
323
slaughter. An aliquot of the feces was put into liquid nitrogen and later kept at 80 C for
molecularbiologyanalysis.
Bloodanalysis
Salmonella spp. seroconversion was followed by a commercial ELISA kit (Maxivet, StHyacinthe,
Quebec, Canada) developed to detect serological response to more than 95% of Salmonella
serotypes commonly found in Canada (Letellier, A, 2009). Seras were analysed by the diagnostic
serviceoftheFacultdeMdecineVtrinaireoftheMontrealUniversity.
Salmonellaspp.detection
WeusedamodifiedversionoftheISO6579annexeDforthedetectionofSalmonellaspp.infeces
andenvironment(BPW1824h,MRSV48h,isolationonBGSandXLDfollowedbybiochemicaland
seroagglutinationconfirmation).
RealtimePCR
DNA extraction (mechanical lysis followed by a phenolchloroform extraction) was performed on
all samples. Realtime PCRs, using the parameters shown in table 1, were performed on the
samples of interest using an Eco Illumina realtime PCR with EvaGreen qPCR mix as
recommendedbythemanufacturer.Forlactobacilliandenterobacteria,15ngofDNAandafinal
primerconcentration1Mwasused.FortheBifidobacteriumgenus,theamplificationsreactions
consisted of 10 ng of DNA with a final primer concentration of 0.25 M. The standard curves for
each reaction were obtained by diluting the precipitate of PCR product realized in the same
conditions with the following reference strains L. acidophilus ATCC314, E. coli 25922 and B.
LongumATCC15707.Resultswereexpressedaslogofcopies/10ngofDNAused.
Volatilefattyacids(VFA)
Atslaughter,ileal,caecalandcoloncontentsfrom165pigsweresampledandstoredat20Cfor
analysis of VFA. Samples were diluted with distilled water and centrifuge at 18000 rpm for 30
minutes. Supernatants were retained and Sulfuric acid (0.5M) was added. Samples were
centrifuged at 13000 rpm for 15 minutes and the supernatants were used for further analysis.
Internalstandard(2ethylbutyric)andresinwereadded.Sampleswerefilteredandthevialswere
keptat4Cuntiltheywereanalysedwiththechromatograph.
Statisticalanalysis
AllnonparametricdataweretestedwithalogisticregressionandparametricdatabyStudentsT
test.StatisticalanalysesweremadewithSAS9.2.
RESULTS
Salmonellaspp.excretion
Asshownintables2and3,atday0therewasnodifferenceofexcretioninbetweenthegroups.
After only 21 days of specific diets, three groups (pellet 1250 m, mash 500 m and mash 1250
m)showedsignificantlylessSalmonellaspp.excretion(P<0.05)thanthereferencegroup(pellet
500m)(Table2).
Table1.RealtimePCRparameters.
Group Primers Lenght Cycles
Lactobacilli
(Castillo,2006)
LBF:GCAGCAGTAGGGAATCTTCCA(Tm=57 C)
LBR:GCATTYCACCGCTACACATG(Tm=57C)
200
pb
Init.Den:10m@95C
40cycles:15s@95C
1m@60C
Enterobacteria
(Castillo,2006)
EntF:ATGGCTGTCGTCAGCTCGT(Tm=60C)
EntR:CCTACTTCTTTTGCAACCCACTC(Tm=60C)
364pb Init.Den.:10m@95C
40cycles:15s@95C
1m@60C
Bifidobacteriumspp.
(Matsuki,2004)
BifF:CTCCTGGAAACGGGTGG(Tm=53 C)
BifR:GGTGTTCTTCCCGATATCTACA(Tm=53C)
550pb Init.:5m@94C
40cycles:20s@94C
20s@55C
50s@72C
324
When comparing all the mash feed groups to the pellet feed groups (regardless of their particle
size), the mash feed groups had significantly less Salmonella spp. excretion on three different
dates:day21(P=0.012),88(P=0.002)andinthecoloncontentatslaughter(P=0.026)(Table3).
Table2.NumberofpigspergroupexcretingSalmonellaspp.ondays0and21.Legend:Datawith(a)notice
meaning:significantlydifferent(P<0.05)thandatawith(b)notice.
Diets Day0
(n=24)
Day21
(n=24)
500pellet 13 15
(a)
750pellet 11 10
1250pellet 12 5
(b)
500mash 10 1
(b)
750mash 10 10
1250mash 11 5
(b)
Table 3. Total of pigs in the mash or pellet groups excreting Salmonella spp. on days 0, 21, 46, 88 and
positivecoloncontentatslaughter.Legend:Datawith(a),(b)or(c)noticearedifferentfromeachother.
Day Mash
(n=72)
Pellet
(n=72)
0 31 36
21 16
(a)
30
(a)
46 9 14
88 5
(b)
19
(b)
Coloncontent 10
(c)
21
(c)
Seroconversion
Salmonella spp. seropositive pigs were only detected after 88 days of specific diet. No statistical
differencewasobservedbetweenthegroups(totalaverageof25.7%positive).
Table4.Feedconversionattheendofthefatteningperiod.Legend:Ratioofkgfeed/kgweightgain.Ratio
forpellet500m(a)(referencegroup)isonlydifferentfromthemash1250m(b)ratio.
Pellet Mash
500 3.04
(a)
3.16
750 2.95 3.4
1250 2.99 3.45
(b)
Feedconversion
Feed conversion was not statistically different between pellet 500 (reference commercial feed)
and mash 500. Only mash 1250 was significantly higher in comparison to reference group (Table
4).
Volatilefattyacids
Propionic acids concentration was significantly higher (P = 0.0012) in the colon content of pigs
from the mash feed group (2750.62) in comparison to the pellet group (2389.91). The same
observationweremadeforbutyricacid(1823.38vs.1537.47,P=0.0008).Moreover,butyricacid
was present in significantly higher concentration in the mash 1250 (2179.07) than in any other
group(P<0.05).
RealtimePCR
Thelactobacilliandenterobacteriagroupsandtheirratio(lactobacilli/enterobacteria)weresimilar
from group to group. On the other side, more Bifidobacterium spp. were detected in the mash
feedgroupscomparedtothepelletfeedgroups(allparticlesizetogether)andhighervaluesinthe
mash1250mcomparedtothe3othergroupstestedonday21(P<0.05)werenoted.
325
Table5.LogofcopiesofBifidobacteriumspp.16SRNAgene/10ngofDNAfrompigfecesonday0and21.
Legend:Datawith(a)or(c)noticedaresignificantlyhigherthandatawithrespectively(b)or(d)notice.
Diet Day0 Day21
Pellet500 3.52 3.25
(b)
Pellet1250 3.62 3.32
(b)
Mash500 3.40 3.39
(b)
Mash1250 3.52 4.23
(a)
Totalpellet 3.57 3.29
(d)
Totalmash 3.46 3.81
(c)
DISCUSSION
Salmonellapositivepigs
Many published article related that the use of mash feed instead of pellet feed reduces the
numberofseropositivepigsinaherd(LoFoWong,2004;Farzan,2009).Incontrast,nodifference
in seroconversion was observed between the groups in our study. On the other hand it is to our
knowledge the first study to demonstrate the reduction of the number of pigs excreting
Salmonella spp. by only modifying the feed presentation. Our results indicate for the first time
that a simple modification of the particle size or the texture can reduce pigs Salmonella spp.
excretion. In addition, since the only diet to have a significantly higher conversion ratio than the
reference group (pellet 500 m) is the mash 1250 group, it would be possible to only vary one
parameter such as particle size or texture and have a Salmonella spp. benefit while being costly
effective.
Volatilefattyacids
Atslaughter,thereductionofthepresenceofSalmonellaspp.wasobservedwhereanincreaseof
propionic and butyric acids concentration in the colon was also seen. These Short Chain Fatty
Acids(SCFA)areknowntodownregulatetheexpressionofSPI1apathogenicityislandnecessary
tocellinvasion(VanImmerseel,2006).Similarobservationandhypotheseshavebeenmadewith
biggerparticlesizefeedandcoliformcountinotherstudies(Mikkelsen,2004).
Microbiota
Bifidobacteriumspp.beingproducersofbutyricacid,alinkcanbemadebetweenitsincreasesin
the same groups where higher butyric and propionic concentrations were found (Mountzouris,
2006).
Conclusion
To this date our data have provided us with a cost effective solution (mash 500 m) to cure
Salmonellaspp.fromthefarmlevelofthepigproduction.Moreover,thedataobtainedfromthe
mash1250mandthemashvs.pelletregardlessoftheparticlesizehavegivenusgoodclueson
themechanismbywhichasimplepresentationchangeinthepigsfeedcanreduceSalmonellaspp.
excretion in the herd. Further analysis of the microbiota at different time and with different
targets bacteria will give us an extended look to the real impact of the feed presentation on the
pigsguthealthanditsbeneficialimpactonthereductionofSalmonellaspp.
ACKNOWLEDGEMENT
Thanks to F. Mnard for the experimental design and help on the farm (special thank to Guy
MaynardandMartineMessierfortheirhelp).ThankstoallthestudentsontheCRSVteamforthe
helponthefarmandinthelaboratories.

326
REFERENCES
1. Castillo, M. 2006. Quantification of total bacteria, enterobacteria and lactobacilli populations in pig
digestabyrealtimePCR.VeterinaryMicrobiology.Vol.114.165170.
2. Farzan,A.,Friendship,R.M.,Dewey,C.E.,Warriner,K.,Poppe,C.,Funk,J.2009.EvaluationoftheRisk
Factors for Shedding Salmonella with or without Antimicrobial Resistance in Swine Using Multinomial
RegressionMethod.ZoonoseandPublicHealth.Vol.57.8593.
3. Letellier,A.,Beauchamp,G.,Guvremont,E.DAllaire,S.Quessy,S.RiskFactorsatSlaughterAssociated
withPresenceofSalmonellaonHogCarcassesinCanada.JournalofFoodProtection.Vol.72.No.11.2326
2331.
4. LoFoWong,D.M.A.,Dahl,J.,Stege,H.,vanderWolf,P.J.,Leontides,L.,vonAltorock,A.,Thorberg,B.
M. 2004. Herdlevel risk factors for subclinical Salmonella infection in European finishingpig herds.
PreventiveVeterinaryMedicine.Vol.62.253266
5. Matsuki,T.Etal.2004.QuantitativePCR16srRNAGeneTargetedSpeciesSpecificPrimersforAnalysisof
HumanIntestinalBifidobacteria.AppliedandEnvironmentalMicrobiology.Vol.70.No.1.167173
6. Mikkelsen,L.L.,Naughton,P.J.,Hedemann, M.S.,Jensen,B.B.2004.EffectsofPhysicalProperties of
FeedonMicrobialEcology,andSurvivalofSalmonellaentericaSerovarTyphimuriuminPigGastrointestinal
Tract.AppliedandEnvironmentalMicrobiology.Vol.70.No.6.34853492.
7. Mountzouris, K. C., Balaskas, C. Fava, F., Tuohy, K. M., Gibson, G. R., Fegeros, K. 2006. Profiling of
compositionandmetabolicactivitiesofthecolonicmicrofloraofgrowingpigsfeddietssupplementedwith
prebioticoligosaccharides.Anaerobe.Vol.12.178185.
8. OConnor, A.M. et al. 2008. Feeding management practices and feed characteristics associated with
Salmonella prevalence in live and slaughtered marketweight finisher swine: A systematic review and
summationofevidencefrom1950to2005.PreventiveVeterinaryMedicine.Vol.87.213228.
9. VanImmerseel,F.,Russel,J.B.,Flythe,M.D.,Gantois,I.,Timbermont,L.,Pasmans,F.,Haesebrouck,F.,
Ducatelle, R. 2006.Theuseoforganicacids tocombatSalmonellainpoultry:amechanisticexplanationof
theefficacy.AvianPathology.Vol.35.No.3.182188.
327
DecreasingprevalenceinDutchdairyherdsundersalmonellosissurveillance
MaartenWEBER,HenrietteBROUWER
1
,JanVELING
1
,PetraA.KOCK
1
,RoloandoA.M.MONTESSORI
2
and
GerdienSCHAIK
1
Email:h.brouwer@gddeventer.com;
1
GDAnimalHealthService,POBox9,7418EXDEVENTER,TheNetherlands;
2
DutchDairyAssociation,P.O.Box165,2700ADZOETERMEER;TheNetherlands
ABSTRACT
Since 2008, bulk milk samples of all Dutch dairy herds are tested three times a year for antibodies against Salmonella
enterica subsp. enterica serogroups B and D. Farmers with persistently positive bulk milk results are stimulated to
control the infection. This programme aims to reduce the prevalence of Salmonella infections among Dutch dairy cattle.
The goals of this study were to determine the apparent Salmonella prevalence over time and the risk factors associated
with positive bulk milk results. Risk factors were studied by multilevel logistic regression with a random herd effect.
Subgroup analyses were performed for herds in low, average and high prevalence areas, respectively.
The results showed that the moving annual average of the apparent herd-level Salmonella prevalence in Dutch dairy
herds decreased from 11.5% in 2009 to 7.6% in 2011.
Factors positively associated with a positive bulk milk result in all herds (irrespective of the risk area) were: the nearby
presence of dairy herds (<500m.), introduction of cattle from other herds, larger herd size, low milk production, keeping
pigs, soil types clay and bog and surface water area (> 2%).
Subgroup analyses showed that Salmonella infections persisted longer in infected herds in high prevalence areas. This
may be associated with different management practices.
Soil type and surface water area were significant risk factors in high prevalence but not in low and average risk areas.
However, keeping pigs was an important risk factor in low and average prevalence areas.
In conclusion, the results indicate a substantial decrease of the herd-level Salmonella prevalence in dairy herds under
surveillance. Both region-specific as well as general risk factors have to be taken into account in advising farmers on the
control of salmonellosis.


328
Evaluationoftheimpactoftherefrigeratedtransportofpigcarcasses
loadedabove7Contheirmicrobialqualityandsafety
MariemELLOUZE
1
,AlainLEROUX
2
,ArnaudBOZEC
2
,PascalGARRY
1
andBriceMINVIELLE
2
1
Ifip,MeatQuality&Safety,MAISONSALFORT,France;
2
Ifip,MeatQuality&Safety,LERHEU,France
ABSTRACT
According to the European Regulation (EC) No 853/2004, pig carcasses must be chilled in the slaughterhouse along a
continuous decreasing chilling curve, to ensure a maximal core temperature of 7C before transport. However, higher
temperatures can be authorized by national competent authorities. The French Ministry of Agriculture has allowed 2
hours duration transports for pig carcasses loaded at a maximum core temperature of 12C. The aim of this study was to
further investigate alternative core temperatures and transport durations while guarantying an acceptable microbiological
quality of the transported pig carcasses. Temperature evolution during cooling and transportation of 183 standardized
carcasses in terms of weight and lean meat percentage were collected from 5 French slaughterhouses and predictive
microbiology models were used to simulate the subsequent growth of Salmonella, Listeria monocytogenes, E. coli and
Pseudomonas. These simulations took also into account the variability related to the pH and water activity of the
carcasses as well as the microbial strain variability. The results of the simulations showed that the increases in the
microbial population caused by the transport of carcasses experimentally loaded at a core temperature above 12C
remained lower than the microbial variability of pig carcass contamination measured in France after slaughter.
INTRODUCTION
In the European Union, carcasses must be cooled immediately after the post mortem inspection
andkeptataconstantcoretemperature,notexceeding7C,evenwhentransported[1].However,
somestudieshavebeenconductedtoassesstheimpactofthetransportofpigcarcasseswithan
internal temperature above 7C. Thus, Moerman [2, 3] showed that such a transport had no
adverseeffectonthehygienicqualityofthemeat.Moreover,theBelgiumScientificCommitteeof
the FASFC, recently used predictive microbiology models to indicate that the transport of pig
carcasseswasacceptableforamaximumof2hoursiftheywerepreviouslycooledtoamaximum
surface temperature of 9C surface associated to a maximum core temperature of 16C, or for 3
hours at a surface temperatures of 7C and a core temperature of 12C [4, 5]. In France, the
transport of pig carcasses loaded at a maximum core temperature of 12C during 2 hours was
allowed in 2009 [6]. The present study was conducted to further investigate alternative loading
temperatures and transport durations with respect to acceptable microbiological quality of the
carcasses. Core and surface temperature kinetics of carcasses were collected in slaughterhouses
during cooling and transportation and were used along with predictive microbiology models to
simulatethegrowthofrelevantbacteriaonthecarcasses.Simulationstookalsointoaccountthe
variability related to the pH and water activity of the carcasses as well as the strain variability
relatedtothemicrobialbehavior.
MATERIALANDMETHODS
A. Temperaturekinetics
Data on the temperature evolution during cooling and transportation of 183 pig carcasses,
standardized in terms of weight and lean meat percentage, were collected in 5 slaughterhouses,
with different cooling technologies, representative of the slaughtering activity in France. The
temperaturesweremeasuredfromthechillingstepthroughthecarcasscoolingandstorageatthe
slaughterhouse cold room, or during the loading and transport, on the ham, the anatomic site
wherethecoolingkineticistheslowest.Thecoretemperature(usedinregulation)aswellasthe
surface temperature (where contamination occurs) were monitored using data loggers (Testo,
France, Oceasoft, France). Transports were organized in several campaigns with a targeted
carcasses core temperature at loading of 18C, 15C or 12C. 908 surface temperature kinetics,
329
representingthenaturalvariabilityduringthecarcasscoolingandtransportwerethusobtained.A
resampling procedure among the set of kinetics of each slaughterhouse was performed to
equilibratetheircontributionandtogenerateatotalnumberof10000kinetics.
B. Simulationofmicrobialbehaviourforalternativetransportconditions
Listeria monocytogenes and Salmonella as major hazards in the pork meat industry as well as
PseudomonasandE.coliasspoilerswerechosentosimulatethemicrobialbehaviouronthemeat
andontherindsidesofpigcarcasses.
TheprimarymodelofRosso[7]withoutlagtimewasusedtosimulatethemicrobialconcentration
withtime.Asecondarycardinalmodel,basedonthegammaconcept[8],wasusedtosimulatethe
effectoftheenvironmentalfactorsonthegrowthrateofthemicroorganisms
max
:
with
and
whereX
min
,X
opt
,andX
max
arethecardinalvaluesofthemicroorganism,
opt
theoptimalvalueof
themaximumspecificgrowthrate(h
1
)whenX=X
opt
andnashapeparameter(n=2fortemperature
and n=1 for pH). The values of the parameters used in the simulations were either fixed or
sampledamongthenormaldistributionspresentedinTable1.
Table1Parametersusedingrowthsimulations.
N(m;s)Normaldistributionwithitsmeanmandstandarddeviations
For each microorganism, 10

4
simulations were performed on both type of carcass surface (rind
and meat). These simulations were used to take into account the natural variability of the
microbialparameters(sampledintheirrespectivenormaldistributions)aswellasthevariabilityof
Parameter Pseudom. E.coli Salmonella L.mono.
N
0
(logcfu) 0 0 0 0
N
max
(logcfu) N(10.4;0.1) N(8.0;0.1) N(8.5;0.1) N(9.0;0.1)
opt
(h
1
) N(1.1;0.1) N(3.3;0.4) N(1.7;0.2) N(1.2;0.2)
T
min
(C) N(6.4;2.3) N(5.9;1.4) N(4.7;0.6) N(1.3;1.1)
T
opt
(C) N(27.5;1.0) N(41.2;1.3) N(39.6;0.7) N(38.2;0.7)
T
max
(C) N(31.8;2.8) N(46.7;2.3) N(45.9;0.5) N(43.3;1.1)
pH
min
N(5.0;0.4) N(3.8;0.1) N(3.2;0.3) N(4.2;0.1)
pH
opt
7 7 7 7
pH
max
14pH
min
14pH
min
14pH
min
14pH
min
aw
min
N(0.94;0.01) N(0.96;0.01) N(0.94;0.01) N(0.92;0.01)
aw
opt
0.997 0.997 0.997 0.997
aw
max
0.997 0.997 0.997 0.997
pH
r
N(8.6;0.1) N(8.6;0.1) N(8.6;0.1) N(8.6;0.1)
pH
m
N(6.3;0.5) N(6.3;0.5) N(6.3;0.5) N(6.3;0.5)
aw 0.995 0.995 0.995 0.995
) ( ). ( ). ( .
1 2 max
aw SR pH CM T CM
opt
=
| |
>
< <
+

s
=

max
max min
min opt max opt opt min opt
1
min opt
min max
min
n
, 0
,
) )( ( ) )( ( ) (
) )( (
, 0
) (
X X
X X X
nX X X X X X X X X X X
X X X X
X X
X CM
n
n
s <
s
=
opt min
min opt
min
min
,
-
, 0
) (
X X X
X X
X X
X X
X SR
330
the temperature kinetics, during the cooling at the slaughterhouse and during the carcasses
transport. Finally, the increases in the microbial populations obtained for carcasses loaded at
different core temperatures and transported for several durations were compared to those
obtainedforequivalentcarcassescooledinthesameconditionsandmaintainedatthecoldroom
of the slaughterhouse. This comparison made it possible to evaluate the acceptability of several
transportscenarios.
RESULTS
Loading core temperatures of 12, 15 or 18C were targeted, however, measured core
temperatures showed a great variability. Table 2 is a summary of the measured temperature
kinetics.
Table2Presentationofthetemperaturekineticscharacteristics
Slaughterhouses A B C D E Total
Numberofkinetics 145 195 56 260 252 908
MeanloadingcoreT (C) 14.7 19.
9
13.4 14.
6
18.
1
17.2
MeanloadingsurfaceT(C) 9.5 13.
0
9.6 10.
0
11.
0
11.0
Meancoolingtime (h) 11.4 5.8 7.4 2.7 5.1 5.7
Meantransporttime (h) 13.6 10.
4
22.0 15.
1
12.
1
12.9
Meankinetictime(h) 25.0 16.
2
29.4 17.
7
17.
3
19.7
Twoscenarioswereconsidered.Inthefirst(Referencescenario),thecarcassesweremaintainedin
the cold room. In the second (transport scenario), equivalent carcasses were first cooled in the
coldroombutthentransportedatacoretemperatureabovetheregulatory7C.
Fig.1Evolutionofthesurfacetemperatureofthecarcassesduringcoolinginthecoldroomandduringthe
transport.
Figure 1 depicts an example of the evolution of the surface temperature of a carcass with time
when cooled in the cold room (bold line) or when cooled in the cold room up to a surface
temperatureof13C(coretemperature18C)thenloadedandtransportedduring6hours.
Theeffectofbothscenariosonmicrobialbehaviorwasassessedforthe10
4
temperaturekinetics,
onthemeatandrindpartsofthecarcasses.Thedistributionofthedifferencesinconcentrations
obtained for Salmonella between the reference scenarios and the transport scenarios are
presentedin
Figure2.
331
Fig.2DistributionofthedifferencesinSalmonellaconcentrationbetweenreferenceandtransportscenarios
onmeatsurface.
Figure 2 shows that the Salmonella concentration (log CFU/ arbitrary unit) in the transport
scenariosremainveryclosetothoseobservedinthereferencescenarios,sincethedistributionis
peaked around the value 0. Results show that the median increase observed between the
referencescenarioandthetransportscenarioisestimatedto0.07logCFUonthemeatsideandto
0.06 log CFU on the rind surface (Table 3). The results of the 8.10
4
simulations obtained for the
four tested microorganisms on both rind and meat surfaces of the carcasses are presented in
Table3.
Table 3. 5
th
, 50
th
and 95
th
centiles of the distributions of the differences in the microbial contamination
obtained between reference scenarios and transport scenarios for the four microorganisms on the meat
andrindsideofthecarcasses.
This table confirms the results obtained for Salmonella thus indicating that the increases in the
microbial populations caused by the transport of carcasses experimentally loaded at a core
temperature ranging from 9 to 29C for the several studied durations were in 50% of the cases
lowerthan0.2logCFU/arbitraryunit.
DISCUSSION
The simulated increases in the microbial populations caused by the transport of carcasses
experimentally loaded at a core temperature ranging from 9 to 29C for several durations were
compared to the standard deviation observed for the natural contamination of pig carcasses and
cuts [10], estimated to of 0.9 log CFU. This comparison shows that the natural variability
associated with pig carcasses (0.90 log CFU) is well above the difference between the microbial
contaminationobtainedbetweenreferencescenariosandtransportscenariosforthefourmicro
organisms and particularly for Salmonella where in 95% of the cases, this difference is estimated
to 0.32 log CFU on the meat side and 0.27 log CFU on the rind side (Table 3). An analysis of
variance was ultimately conducted to identify the most important factors influencing the
differences between the reference scenario and the transport scenario. This analysis showed
similar results for four microorganisms. Particularly for Salmonella, two categories of important
factors were identified : factors on which the operator cannot act such as the microbial
characteristics(T
min
,
opt
),themeatorrindpH;andfactorsonwhichtheoperatorcanactsuchas
-1.5 -1 -0.5 0 0.5 1 1.5
0
1000
2000
3000
4000
5000
6000
7000
p
Centiles L.mono. Salmonella E.coli Pseudom.
Meatside
5 0.06 0.04 0.05 0.10
50 0.10 0.07 0.06 0.15
95 0.43 0.32 0.44 0.75
Rindside
5 0.04 0.04 0.04 0.04
50 0.07 0.06 0.05 0.04
95 0.31 0.27 0.35 0.36
Log CFU
Samplesize
332
the core temperature at loading and the transport duration, the latter one being the most
important.
CONCLUSION
The results of this study indicate that the increases in the microbial population caused by the
transport of carcasses experimentally loaded at a core temperature ranging from 9 to 29C for
several durations remain low compared to the corresponding storage in cold rooms, (maximum
mediandifferenceof0.15logCFU)andacceptablewithregardstothenaturalmicrobialvariability
of pig carcass contamination observed after slaughter in France. Complementary investigations
are being conducted to propose acceptable maximal transport durations for pig carcasses for
differentmaximumtemperaturesatloading.
ACKNOWLEDGMENT
ThisstudywasfinancedbyINAPORC,theFrenchInterprofessionalPorkCouncil.
REFERENCES
1. Anon, 1964. Council Directive 64/433/EEC of 26 June 1964 on health problems affecting intra
Communitytradeinfreshmeat
2. MoermanPC(1983)Thechillingofpigcarcassesduringtransport.29thEur.Congr.MeatRes.Workers,
Salsomaggiore,Italy,14p.
3. Moerman PC (1992) Chilling and transport of pork. ICoMST Proc. vol. 4, 38th International Congress of
MeatScienceandTechnology,ClermontFerrand,France,1992,pp695698
4. Anon (2004) Avis 2004/01 Problmatique du transport de viande non compltement refroidie
(transportchaud)(dossier2003/25).FASFC,Belgium,3p.Availableatwww.afsca.be
5. Anon(2008)Advice312008oftheScientificCommitteeoftheFASFContheuncooledtransportofpig
carcasses.FASFC,Belgium,12p.Availableatwww.afsca.be
6. Anon(2009)Arrtdu18dcembre2009relatifauxrglessanitairesapplicablesauxproduitsdorigine
animale et aux denres alimentaires en contenant. Journal Officiel de la Rpublique Franaise, 29
dcembre2009,34p.
7. Rosso,L.(1994).Modlisationetmicrobiologieprvisionnelle:Elaborationd'unnouveloutilpourl'agro
alimentaire.Thse:UniversitClaudeBernard,Lyon1.pp190.
8. Zwietering, M., Wijtzes, T., DeWit, J.C. et Van'tRiet, K. (1992) A decision support system for the
predictionofthemicrobialspoilageinfoods.JournalofFoodProtection,55,973979.
9. Rosso, L., Lobry, J.R., Bajard, S. et Flandrois, J.P. (1995) Convenient model to describe the combined
effectsoftemperatureandpHonmicrobialgrowth.AppliedandEnvironmentalMicrobiology,61,610616.
10. Augustin,JC.,Minvielle,B.(2008)Designofcontrolchartstomonitorthemicrobiologicalcontamination
ofporkmeatcuts.FoodControl,19(1),8297.
333
AcomparisonofSalmonellatestresultsfrommicrobiologicaltesting
undertakenbyFoodBusinessOperatorsandthatOfficialyundertaken
JohnEGAN,SharonDUGGANandMontserratGUTIERREZ
NationalReferenceLaboratorySalmonella(Food,FeedandAnimalHealth),
BackwestonCampus,Celbridge,Co.KILDARE,Ireland
ABSTRACT
The primary responsibility for the safety of food rests with Food Business Operators (FBOs) and they undertake the vast
bulk of microbiological testing required to underpin food safety systems. Results of FBO Salmonella testing in private
laboratories over a three year period, 2007-2009, were compared with those of official testing. Data was available on
253,754 Salmonella tests of which 8,861 (3.5%) corresponded to official samples and 244,893 (96.5%) to FBOs. Over
90% of testing was associated with raw of cooked foods with the remaining associated with monitoring Salmonella
controls at primary poultry production. For food samples, results show consistently higher recovery rates from official
testing compared with FBO testing. No significant differences were apparent for cooked products but overall isolations
rates were low and the numbers of official samples small. For primary poultry production samples, the FBO testing
showed a higher Salmonella recovery when compared with official testing for each of the three years. A total of 159
Salmonella isolates (13 serotypes) were available to the NRL from official testing of meat and meat product samples
compared with 1,108 (50 serotypes) from FBO testing. Under poultry regulations a total of 88 isolates (12 serotypes)
were available from official sampling compared with 1,076 (31 serotypes) from FBO testing. Factors that might contribute
to variations in results between official and FBO test results are discussed as are their potential implications. The value
of collecting FBO testing data in enhancing food safety controls at very little additional cost is highlighted.
INTRODUCTION
Foodbornediseasesareamajorpublichealthconcernandithasbeenestimatedthatupto30%of
the population in industrialised countries are affected annually. In Ireland the Food Safety
Promotion Board estimated that approximately 8,000 people presented to medical practitioners
every day with symptoms of acute gastroenterititis with illness lasting on average four days.
Based on these data, it was concluded that approximately 35,000 people were suffering from
these symptoms on any day on the island of Ireland (Food Safety Promotion Board, 2005).
Salmonellaspp.isoneofthemostcommonfoodbornepathogensintheEUwithatotalof99,020
confirmedcases of human salmonellosis reported in 2010. It has beenestimated that the overall
economic burden of human salmonellosis could be as high as EUR 3 billion a year. Developed
countries have integrated farm to fork approaches to food safety. Numerous food scares have
demonstratedtheimportanceofhavingcontrolsystemslongitudinallyintegratedwithinthefood
chainsothatsequentialriskreductionmeasurescanbetakenwherepossibleandappropriate.
ResponsibilityforfoodsafetyhasbeenbroadenedsothatFBOsarenowprimarilyaccountablefor
the safety of the food they produce. As a consequence, the role of enforcers of food hygiene
legislation has evolved from primarily one of inspection to an audit function with ongoing
verification to ensure that management systems are effectively implemented and are controlling
pertinent risks. These controls must also be supported with the continued development of
surveillance systems and traceability along the food chain so that early detection and timely
interventionscanbemade.
In 2003, the EU began progressing enhanced control programmes for zoonoses with Salmonella
controlapriority.Regulation(EC)No2160/2003providedforthesettingofEUtargetsforreducing
in animal populations the prevalence of Salmonella serovars with public health significance.
Following on from EUwide baseline prevalence surveys, reduction targets were set for the
reductionofcertainSalmonellaserovarsindifferentpoultrypopulations(e.g.layinghens,broilers,
turkeys) and restrictions imposed on the trade of products from infected flocks. In addition
334
CommissionRegulation(EC)No2073/2005thatcameintoforceinJanuary2006,specifiedcriteria
for Salmonella in several specific food categories entering the market. Systems were also put in
placefortheongoingmonitoringandcollectionofinformationonzoonoses,whichobligeallEUMS
to collect relevant and, where applicable, comparable data on zoonoses and zoonotic agents
(Directive2003/99/EC).
MATERIALANDMETHODS
DataonSalmonellaspp.isolatedfromfoodsofanimalorigininIrelandareavailableprimarilyfrom
theNationalReferenceLaboratory(NRL)Food,FeedandAnimalHealthwheremostofficialtesting
isundertaken.Inaddition,theNRLcapturesandcollatestestinformationfromFBOs.Inthethree
yearperiod(2007to2009)coveredbythiscommunicationresultsonSalmonellatestsundertaken
in25privatelaboratories(22intheRepublicofIrelandand3inNorthernIreland)wererecorded
on the FoodMicro database at the NRL. All Salmonella isolates recovered by private laboratories
weresubmittedtotheNRLforserotyping.
RESULTS
Datawasavailableonatotalof253,754Salmonellatestsundertakenoverthethreeyearperiod,
8,861 (3.5%) resulting from official monitoring and the remaining 244,893 (96.5%) from FBO
testing. Over 90% of testing was associated with raw or cooked foods with the remaining testing
associatedwithmonitoringSalmonellacontrolsatprimaryproductionpoultrysites.
Table 1 shows a breakdown of official and FBO testing undertaken on raw meat and meat
products over the three year period. Although some variations are apparent in Salmonella
isolation rates from individual meat categories the overall data shows a consistently higher
Salmonella recovery rate each year from officially tested samples compared with FBO tested
products.
For samples tested at primary production (Salmonella monitoring on poultry farms) the FBO
testingshowedhigherSalmonellarecoveriesoverofficialtestingforeachofthethreeyears(Table
2). There were some differences in the percentage of sample matrices tested with dust
constituting 84.5% of FBO samples compared with 21.8% for official samples tested. Fluff,
environmental samples and box liners constituted 0.5%, 12.2% and 2.7% of FBO samples tested
comparedwith28.2%,47.2%and2.8%ofofficialtestsrespectively(Table2).
Atotalof159SalmonellaisolateswereavailabletotheNRLfromofficialtestingofmeatandmeat
product samples compared with 1,108 from FBO testing. A total of 13 different serotypes were
isolated from official samples compared with 50 from FBO samples. A similar pattern was
apparentfromcookedmeattestingwith3isolatesavailablefromofficialtestingcomparedwith51
from FBO testing. All the official isolates comprised one serotype compared with 7 for the FBO
testing.WithregardtotheisolationofSalmonellafromprimarypoultryproductionsitesatotalof
88 isolates were available to the NRL from official sampling compared with 1,076 from FBO
testing.Atotalof12differentserotypeswereisolatedonofficialtestscomparedwith31onFBO
testing.
DISCUSSION
The data shown highlights the importance of including FBO data in the overall assessment and
analysis of trends and sources of zoonoses at both MS and EU level. Over 96% of all Salmonella
tests in Ireland were undertaken by FBO in private laboratories. While the results highlight some
differencesintherecoveryratesofSalmonellafrombothofficialandprivatetestsitisdifficultto
make any meaningful assessment as official testing is limited and sample matrices vary to some
extent.Laboratoriestestingsamplessubmittedfromprimarypoultryproductionwererequiredto
335
have tests accredited and were also approved and monitored by the Irish Department of
Agriculture, Food and the Marine (DAFM). However in recent years further requirements are
placedonallFBOtoensureadditionaloversightoftestinglaboratories.
WhileresultsareavailabletoofficialsregulatingorinspectingeachFBOthereisalsoanadditional
requirement in Ireland for laboratories to submit all isolates and data on testing each month to
the NRL. Data produced from testing undertaken by industry owncheck programmes are not
generallycollectedandanalysedcentrallytoenhancefoodsafetycontrolsinmostMSorincluded
intheanalysisoftrendsandsourcesofzoonosesatEUlevel(EFSA/ECDC,2010).Thisisinpartdue
toconcernsoverthereliabilityofthedataparticularlyinrelationtocorrectlycategorisingthefood
products from which isolates are recovered. This problem is recognised in Ireland and efforts
continue to ensure FBOs more accurately categorise the food samples submitted for
microbiological testing. This should facilitate better classification and identification of high risk
products over time. In any case all Salmonella isolates from the DAFM regulated sector must be
submittedtotheNRLirrespectiveofwhetherafoodproductisplacedonthemarketornot.
While it is generally accepted that the true burden of human salmonellosis may be considerably
largerthanthereportedincidencethereisnodoubtthatthelevelofhumaninfectionisdeclining
inmostEUMSincludingIreland.Astestingcostsforthedaytodayimplementationoffoodsafety
programmes are substantial it is essential that regulators optimise and standardise processes
including the collection and analysis of all data which will further improve the effectiveness,
efficiencyandtransparencyofregulationsandreducebudgetcoststoallstakeholders.
REFERENCES
1. EFSA/ECDC(2010)Thecommunitysummaryreportontrendsandsourcesofzoonoses,zoonoticagents
andfoodborneoutbreaksintheEuropeanUnionin2008.EFSAJournal,1496.
2. Food Safety Promotion Board (2005) Acute gastroenteritis in Ireland, North and South: A study of
generalpractitioners.
http://www.safefoodonline.com.
336
Table 1: Crude prevalence rates for Salmonella spp. in raw meat and meat products following official and
FoodBusinessOperatortestingofoverathreeyearperiod
2007 2008 2009 20072009
Total
positive
(%)
Total
tested
Total
positive
(%)
Total
tested
Total
positive
(%)
Total
tested
Total
positive
(%)
Total
tested
Bovine
FBO
25
(0.1%)
35,134
55
(0.2%)
26,975
35
(0.1%)
27,540
115
(0.1%)
89,649
Official
0
(0.0%)
213
1
(0.5%)
217
4
(1.1%)
355
5
(0.6%)
785
Poultry
FBO
328
(4.8%)
6,826
267
(3.9%)
6,810
81
(1.3%)
6,035
676
(3.4%)
19,671
Official
46
(9.7%)
476
55
(12.5%)
440
48
(8.9%)
540
149
(10.2%)
1,456
Porcine
FBO
118
(1.8%)
6,649
142
(2.4%)
5,863
98
(1.1%)
8,876
358
(1.7%)
21,388
Official
7
(3.2%)
216
1
(0.5%)
188
0
(0.0%)
283
8
(1.2%)
687
Other
FBO
34
(0.5%)
7,387
42
(0.5%)
7,915
24
(0.4%)
6,538
100
(0.5%)
21,840
Official
0
(0.0%)
48
0
(0.0%)
46
1
(0.7%)
143
1
(0.4%)
237
Totals
FBO
505
(0.9%)
55,996
506
(1.1%)
47,563
238
(0.5%)
48,989
1,249
(0.8%)
152,548
Official
53
(5.6%)
953
57
(6.4%)
891
53
(4.0%)
1,321
163
(5.2%)
3,165
Table 2: Crude prevalence rates for Salmonella spp. in samples from primary poultry production sites
followingofficialandFoodBusinessOperatortestingofoverathreeyearperiod
2007 2008 2009 20072009
Total
positive
(%)
Total
tested
Total
positive
(%)
Total
tested
Total
positive
(%)
Total
tested
Total
positive
(%)
Total
tested
Dust
FBO
533
(8.5%)
6,277
384
(5.5%)
6,929
56
(1.1%)
4,880
973
(5.4%)
18,086
Official
2
(0.5%)
366
2
(1.0%)
196
0
(0.0%)
208
4
(0.5%)
770
Fluff
FBO
3
(3.8%)
78
0
(0.0%)
13
0
(0.0%)
12
3
(2.9%)
103
Official
42
(7.1%)
591
25
(7.2%)
349
8
(14.0%)
57
75
(7.5%)
997
Environ
ment
FBO
33
(5.7%)
578
13
(1.9%)
681
24
(1.8%)
1,359
70
(2.7%)
2,618
Official
0
(0.0%)
46
8
(1.2%)
684
1
(0.1%)
937
9
(0.5%)
1,667
Boxliners
FBO
0
(0.0%)
149
2
(1.0%)
201
0
(0.0%)
234
2
(0.3%)
584
Official
0
(0.0%)
30
0
(0.0%)
31
0
(0.0%)
39
0
(0.0%)
100
Totals
FBO
569
(8.0%)
7,082
399
(5.1%)
7,824
80
(1.2%)
6,485
1,048
(4.9%)
21,391
Official
44
(4.3%)
1,033
35
(2.8%)
1,260
9
(0.7%)
1,241
88
(2.5%)
3,534
337
ApplicationofforensicevaluationofevidencetothetracingofSalmonella
GunnarANDERSSON
1
,AnnaASPN
1
,CeciliaHULTN
1
,EstelleGREN
1
andGaryBARKER
2
1
NationalVeterinaryInstitute,UppsalaSweden;
2
InstituteofFoodResearch,Norwich,UK
ABSTRACT
Tracing back a Salmonella contamination involves drawing conclusions from a limited amount of information and will
inevitably be associated with a degree of uncertainty. Neither is the isolation of the sero- or genotype of interest a proof
that a certain site is the source nor is a negative sample a proof against its being the source. Reports from trace back
investigations may be used to make decisions of large impact for individuals and companies putting a high demand on
objectivity, transparency and rule of law. In the last decades forensic statistics have become an important tool for
meeting these demands in expert witness reports. A key concept is to express the strength of evidence in relation to
alternative propositions using the likelihood ratio. We show how this framework can be applied to epidemiological
evidence. The investigation of a case of feed-borne Salmonella was represented as a Bayesian belief network (BBN)
where results from typing of isolates and sampling of feeding system and neighboring farms were combined with expert
knowledge on source type association, the relative prior probability of sources, the number of discriminating DNA profiles
and detection probability. Besides well underpinned statements the approach can be used for pre-evaluation of analyses
and identification of research and databases which would support tracing of Salmonella in feed and food chains.
INTRODUCTION
Expertjudgementsconcerningtracinginfoodandfeedchainsinvolvesmakingimportantdecisions
from uncertain information. Forensic experts routinely write statements of witness with a high
demand on objectivity, transparency and rule of law. Microbiologists and epidemiologists write
statementsforcourtslessoften,but,neverthelesstheirreportsneedtobeofequalqualitysince
their judgements may contribute to decisions of large impact for individuals and companies and
have impact on the development and control of an outbreak. In legal cases misunderstandings
relatingtostatisticalinformationandprobabilitieshavecontributedtowardsseriousmiscarriages
of justice (Aitken 2010) and similarproblems could beexpected whendecisions are made during
microbial tracing operations in commercial settings stressing importance of presenting statistical
evidenceintransparent&comprehensiveway.
Microbialforensicshasemergedasanewscientificdisciplinecombiningmicrobiologyandforensic
science and there is an increasing awareness that also data from DNA profiling needs to be
statisticallyevaluatedinthecontextofrelevanthypotheses(Aitken2010).
ThestateoftheartinforensicinterpretationistoevaluateevidenceintheframeworkofBayesian
hypothesis testing (Aitken 2010). The value of evidence (V) is typically expressed as a likelihood
ratio but may be transformed into a qualitative statement using verbal scales (Nordgaard 2012)
e.g. the results speak /to some extent/strongly/ in favor of the main hypothesis ( H1) over the
alternative hypothesis (H2). Bayesian belief networks (BBNs ) have proven a useful tool for
visualizing complex relations between parameters and assessing V accounting for the combined
effect of several uncertainties (Taroni 2006) and to promote transparency and logic (Sjerps
2010).
Intracinginvestigations,bothconcerningSalmonellaandotheragents,therealityisoftentomake
a judgement about source of the infection taking a complex pattern of new information and
previous knowledge into account. The process could therefore benefit from the systematic and
transparentmethodofferedbytheBBNapproach.
Aim:
Theaimoftheworkistodevelopgenericconceptforevaluationofevidenceinmicrobialtracing
basedonforensicstatisticsandtoassessitsapplicabilityonacaseofSalmonellainfectioninapig
herdsuspectedtobefeedborne.
338
Impact:
Theadoptionofareproducibleandaccountabletechniqueforassessingandpresentingthevalue
of evidence from a tracing investigation of Salmonella or other contaminants in food and feed
productioncouldhavesubstantialcommercialimplication.
MATERIALANDMETHODS
Case/scenario:
ThecaseandscenariowereconstructedinthecontextoftheSwedishsalmonellacontrolprogram
and a tracing scenario was constructed from authentic Swedish cases of suspect feedborne
Salmonellainpigherdswhereasdetailsforthespecificcaseweremodified.
FollowingisolationofSalmonellafromapiginthesamplingatslaughterprogramandreisolation
in the pigherd, Salmonella with the same sero and PFGE type was isolated from the production
line of a feedmill. Samples taken from the feedeing system on the farm were negative for
Salmonella.Salmonellawasnotreportedfromothercustomersofthefeedmill.
Resourcesandsoftware:
Thecasewasanalysedinagroupwithparticipantsrepresentingmicrobiology,epidemiology,food
safety,forensicsciencesandstatistics.
ABBNwasconstructedinGeNie2.0(DecisionsSystemsLaboratory,Univ.Pittsburg).
Networkconstructionandaprioriinput:
A priori data for the model were gathered from scientific publications, case reports and expert
opinions.
The main hypothesis to evaluate (H1) is feedmill A is source of Salmonella in the herd. The
alternative hypothesis (H2) feedmill A is not source of Salmonella in the herd is complex and
includes the possibilities that there is another feedsource or that the source is not feed. In the
latter case several alternative sources were identified including birds, rodents, humans and
indirectspreadbyhumansandanimaltransports.
The purpose of the BBN is to evaluate the strength of the evidence and to do this we assigned a
50% prior probability that industrial feed is the source whereas the alternative source types may
be considered approximately equally likely. Based on (hypothetical) information on the feed
supplythepriorprobabilitythatfeedmillAwouldbethesourceofSalmonellacontaminatedfeed
wassetto50%.ThetriggerwasthedetectionofSalmonellainaslaughterhouseandsubsequent
reisolationfrompigsintheherd.
The detection of Salmonella in the production line was the reason for suspecting the feedmill.
Thus this is part of background information. Additional data from sampling and typing was the
evidence to evaluate. Typing information may include serotyping, phagetyping, pulse field gel
electrophoresis(PFGE)andmultiplelocusvariablenumbertandemrepeatanalysis(MLVA).Typing
information can be used to connect two isolates through matching but also to update the
probabilityforaparticulartypeofsource,e.g.feedorbirds.
Inanalogywithacourtcasethejudgementabouttheguiltofthefeedmillwouldbeinfluenced
by design and practices of the farm and feedmill as well as the evidence in form results from
samplingandtyping.Followingtheforensicpracticethedegreeofsuspicionisincludedintheprior
odds whereas the influence of evidence is expressed in the odds ration/value of evidence (V)
(Figure1).
339
Network:
A BBN was constructed that reflects the way an epidemiologist reason using the available
information.Asimplificationofthenetworkisshowninfigure2.Duetothecomplexalternative
hypothesis(H2)extranodes(yellow)wereincludedtoaccountforthesubhypotheses.
In the example the outbreak strain is assumed to be Salmonella enterica serovar Infantis and
genotyping is performed with PFGE using enzyme XbaI. Based on data from Sweden 19931997
(Boqvist, Hansson et al. 2003) the probability that a randomly choosen Salmonella would be of
serovarInfantiswasestimatedtobe1.4%ifthesourcewasindustrialfeedand1.9%ifthesource
was animals others than small birds. The frequency in animals was used as a proxy for
environmentalsourcessuchaswaterandstraw.TheprobabilitythataSalmonellaoriginatingfrom
humanswouldbeserovarInfantiswasestimatedtobeapproximately1%basedon(Hill,Simonset
al.2010).
Insufficient Swedish data was found that could be used to estimate the uniqueness of a PFGE
profile within serovar Infantis. However based on the frequency distribution of PFGE subprofiles
among 76 Australian isolates of Salmonella enterica serovar Infantis (Ross and Heuzenroeder
2008) the probability that two randomly choosen strains of Salmonella enterica serovar Infantis
have matching PFGE profiles was estimated to be approximately 16%. Finally the probability of
obtainingamatchthroughtypingerrorwassetto0.1%forserotypingorPFGEgenotyping.
RESULTS
Valueofevidence:
The calculations in the example results in value of evidence (V) of 76 in favour of the H1.Other
examplestypicallyresultsinVinrange1to100dependingonthesamplingresultsinthefeeding
system,theecologyoftheisolateandtheresolutionofthetypingsystemappliedtotheserovar.
According to scale of the Swedish forensic institute (Nordgaard 2012) this would correspond to
statementsthatresultssupporttosomeextentthat(6<V<100)oratbestresultssupportthat
(100<V<6000). The genotype match was found to be the most significant piece of evidence
whereasnegativesamplingresultsfromthefeedingsystemhadlittleimpactonV.
DISCUSSION
To be useful, a tracing concept should comprise not only mathematical models but also a
transparentframeworkforstructuringproblemsandpresentingresultstodecisionmakers.
In this work we illustrate how the visual BBN framework makes it possible to combine different
sources of information and include information on uncertainty to produce a statement on
certainty. Since priors are partly based on expert opinion the result will be a reflection of the
expertsjudgementsandexperienceincombinationwithexistingscientificdata.
Thestrengthoftheapproachisthatitoffersamechanismforexpressingthestrengthinthebelief
onascalefamiliartolayersandtraceabilityonhowthestatementwasderived.Wearguethatthis
isagreatimprovementovertheuseofvagueexpressionslikeresultsareconsistentwith,could
have originated from, there is a match which are prone to misinterpretation and may or may
notbeunderstoodasestablishingaprovenassociation(Aitken2010).
Therolesofexpertsandriskmanagersinfoodsafetyareanalogoustothoseofforensicinstitute
and court respectively. By explicitly separating prior beliefs and value of evidence in expert
statements roles are clarified and transparency is promoted. By allowing the investigator to
systematically evaluate alternative options for samples to collect and analyses to perform (pre
340
assessment) the approach may also contribute to the design of more cost effective tracing
investigations.
Lessons learned data needs: A cornerstone in forensic evaluation of evidence is the access to
relevant reference samples or reference data to estimate the probability of obtaining the
observed results of the working hypothesis (H1) is right or wrong. When tracing Salmonella the
number of distinct phenotypes (sero + phage) and genotypes in circulation and their relative
frequency has a major impact on the value of evidence for a match. Similar, the expected
probability of detecting Salmonella when sampling a production line or feeding system is crucial
for the value of evidence of a nondetect and frequency of mutation would be important for
evaluatinganonperfectmatch.
At present systematically collected reference data are scarce and the expert would have to
estimatetheprobabilityofevidence(E)fromhisexperience.AlsointhissituationtheBBNisuseful
as a tool for including expert opinion in a transparent way and for communicating the degree of
uncertainty to decision makers. Nevertheless, a mechanism for sharing information on sampling
and typing results and creating more reliable datasets for reference values would contribute to
increasingthevalueofevidencefromsamplingandtypingintracebackinvestigations.
The work presented here is ongoing and the model will be tested on true cases of salmonella
infectiononherdlevel.
REFERENCES
1. Aitken, C., P. Roberts, P., Jackson, G. (2010). Practitioner Guide No 1. Fundamentals of Probability and
StatisticalEvidenceinCriminalProceedings,.R.S.S.s.W.G.o.S.a.t.Law,RoyalStatisticalSociety.
2. Boqvist, S., I. Hansson, etal. (2003). "Salmonella isolated from animals and feed production in Sweden
between1993and1997."ActaVeterinariaScandinavica44(34):181197.
3. Hill, A., R. Simons, et al. (2010). "Quantitative Microbiological Risk Assessment on Salmonella in
Slaughter and Breeder pigs: Final Report." Retrieved Feb, 2013, from
http://www.efsa.europa.eu/en/supporting/doc/46e.pdf.
4. Nordgaard, A., Ansell, R., Drotz, W., Jaeger, L. (2012). "Scale of conclusions for the value of evidence."
Law,probability&risk11(1):124.
5. Ross, I. L. and M. W. Heuzenroeder (2008). "A comparison of three molecular typing methods for the
discriminationofSalmonellaentericaserovarInfantis."FEMSimmunologyandmedicalmicrobiology53(3):
375384.
6. Sjerps, M. J., Berger, C. H. E (2010). "How clear is transparent? Reporting expert reasoning in legal
cases."Law,ProbabilityandRisk0:113.
7. Taroni, F., Aitken, C.,Garbolino, P., Biedermann, A. (2006). Bayesian Networks and Probabilistic
InferenceinForensicScience.Chichester,JohnWiley&SonsLtdChichester.
TABLESANDFIGURES
Figure 1: Evaluation of evidence using Bayes theorem. H1 is the main hypothesis that feedmill A is the
source,whereasH2isthecounterhypothesis,thatthereisanothersource(othersourcetypeORotherfeed
source).Eistheobservedresults,forexamplematchinggenotypesandnpositiveornegativesamples.
341
Figure2.DetailofaBBNforevaluatingevidencewhentracingSalmonella.Therednodisthehypothesisto
be assessed (H). Yellow nodes are supporting hypotheses. Blue nodes are intermediate calculations. Green
nodes represent evidence observed by the user. The evidence considered is sampling results and type
matching from the production line of the mill and the feeding system of the farm. Prior probability for H1
was25%(1notfeed)*(1otherfeedmill).
342
May 27-28-29, 2013
Saint-Malo France
Session 6
Chairpersons:
Posters
343
InvestigationoflowprevalenceSalmonellacontamination
inpoultryfeedmills
RobDAVIES
AnimalHealthandVeterinaryLaboratoriesAgency(AHVLA),Bacteriology
andFoodSafetyDepartment,NewHaw,Addlestone,SURREY,KT153NB,UK
INTRODUCTION
Control programmes for Salmonella Enteritidishave resulted in virtual eradication of this serovar
in broiler production in many countries but infections with other serovars remain within certain
integrations.Themainsourceofthisinfectioniscontaminatedfeed.
Monitoringoffinishedpoultryfeed,whichistypicallyproducedintheformofheattreatedpellets
orcrumbsforcommercialbroilers,orheattreatedmealforsomebroilerparentflocks,islikelyto
underestimate the level of contamination because of the difficulty of obtaining representative
samples from large feed consignments, the testing of composite samples and heat or acid shock
affectingrecoveryofSalmonellaintestsamples.
WhenthepatternofflockinfectionswithfeedrelatedSalmonellaserovarssuch asS.Mbandaka,
S. Ohio or S. Kedougou suggests a feed origin it is necessary to investigate feedmills in detail to
confirmcontaminationandidentifythelocationofresidenthousestrains.
MATERIALSANDMETHODS
TheauthorwasapproachedbypoultrycompanieswhowereexperiencingSalmonellainfectionsin
poultryflocks.TheserovarsinvolvedwereS.Ohioinonecompany,S.Agamainasecondcompany
and S. Kedougou in a third company. Although a feed contamination source was suspected,
internal investigations of the feedmills by the companies concerned had failed to identify
contamination,andnoSalmonellawaseverfoundinfinishedfeedsamples.
The layout and equipment within the mill was demonstrated by the mill manager prior to
sampling. Where necessary complex equipment and product lines were labelled for easy
identificationduringsampling.Millmanagerswereaskednottovacuumupdustfor24hoursprior
tothevisit.Duringtheinspectiontour,likelyareasforsamplingofdustwithinbinsoremanating
from equipment were identified in order to orientate 100 samples across the mill in a
representative way. Up to 100g dust was collected with a gloved hand, sterile scraper or brush
into each of 100 plastic jars. At the laboratory the contents of each jar was dispensed into 4 x
225ml Buffered Peptone water, enriched for 18 hours at 37c then further cultured using MSRV
andRambachagar.Serotypingandphagetyping(ofrelevantserotypes)wasalsocarriedout.
RESULTS
The table shows the main sample types taken in two large commercial feedmills. In mill A the
distribution of Salmonella was consistent with minimal contamination of incoming ingredients
(only3serovarsfoundiningredientprocessingareasbutahighsampleprevalenceofS.Ohiowas
found associated with one of two adjacent pellet coolers and associated post cooling areas. A
similar distribution was found in another mill with S. Kedougou in one of three coolers (data not
shown). In both cases there was little evidence of contamination in the selfcleaning finished
product bins but dust in the cyclone aspiration systems and on beams in the bulk outloading
gantriesproducedindirectevidenceofcontaminationoffinishedfeed.
In mill B there was a wide range of mainly wildlife related serovars (e.g. Agama, Newport,
Typhimium (untypable fully sensitive strains), Kottbus, Durham, Binza) found in ingredient pits,
bins,andaugersbutonlyS.Agamawasfoundinonemealcoolerwhichwasadjacenttotwopellet
coolers.S.Give,asoyaassociatedserovar,wasfoundinapelletshakerandthisserovarwasalso
anintermittentprobleminthecompany.
344
DISCUSSION
The findings of this study support previous suggestions that monitoring for Salmonella in animal
feedcanbedifficult,requiringindirectsamplingofprocessrelatedmaterialsfromitemssuchas
cyclone dust aspiration systems to identify contamination and serotyping or other strain typing
techniquestoconfirmstrainspersistingovertimeascoolerresidentsorasregularcontaminantsin
incoming ingredients. Detailed investigations can then identify the precise source of the
contamination which can then be addressed, although this is difficult in the case of integrated
feedmills because of the extremely short downtime between production runs available for
thoroughclearinganddisinfectionandtheproblemsassociatedwiththeriskofexposureofstaff
to formaldehyde products if these are used. The results also show that only one of several
adjacent coolers is likely to be contaminated, thus countering the suggestion that contaminated
air,ratherthanoccasionalincompleteheattreatmentofcontaminatedingredients,islikelytobe
themainsourceofresidentSalmonella.
CONCLUSIONS
Although the prevalence of contaminated animal feed is reported to be low, the difficulties of
effectivesamplingleadtoanunderestimationandthelargequantitiesoffeedconsumedbyyoung
animalsmakesthisasignificantrisk,requiringimprovedmethodsofmonitoring,investigationand
control. Dust escaping from equipment rather than swabs or scrapings should be used for
assessingfeedmillcontamination,althoughswabsmaybeusefulforareaswherecondensationis
present.Itishoweverimportanttominimisecondensationbyensuringthecoolersarecapableof
returningheattreatedrationstoambienttemperaturebeforeleavingthecooler.
ACKNOWLEDGMENTS
These investigations were funded by Defra under surveillance contract FZ2000 and research
projectOZ0328
REFERENCES
1. Davies, R.H., B, M., Corry, J.E.L., Hudson, W. and Allen, V.M. (2001). Observations on the distribution
andcontrolofSalmonellaintwointegratedbroilercompanies.VeterinaryRecord149,227232.
2. EFSA (2008). Scientific Opinion of the Panel on Biological Hazards on a request from the Health and
Consumer Protection, Directorate General, European Commission on Microbiological Risk Assessment in
feedingstuffsforfoodproducinganimals.TheEFSAJournal,720,184.
345
Salmonellacontaminationintwopoultryfeedmills
(No. samples positive for Salmonella/No. samples taken (%) (Not all sample types taken
shown)
MillA MillB
Inprocesssamples
Intakepit 0/16 2*/24(3.3)(Various)
Intakepitauger 1/12(8.3)(Ohio) 6*/20(30.0)(Various)
Ingredientbins 1/56(1.7)(Mbandaka) 14/40(35.0)(Various)
Weighers 1/16(6.3)(Senftenberg) 4/12(33.3)(Agama)
Grinders 0/16 1/16(6.25)(Agama)
Mixers 0/8 0/8
Presses 0/20 0/16
LineOne LineTwo Lines1&2
Condensation
beneathCoolers
5/8(62.5)(Ohio) NA NA
Cooler1Dust 1/12(8.3)(Ohio)
Cooler2Dust 0/16 0/32 line 3
3/24(12.5)(Agama)
Fatcoater 4/8(50.0)(Ohio) 0/8
Crumbler 4/8(50.0)(Ohio) NA
Shakers 3/8(37.5) 0/4 1/12(8.3)(Give)
Elevators/Augers 4/18(22.2)(line 1 post
cool)
0/18 1/24(4.2)(Agama)
CycloneAspirators 8/10(80.0)(Ohio) 0/10 NS
Wastedust 0/8 0/8
Finished product
Bins
1/68(1.5)(Ohio) 1/54
(1.8)(Agama)
StoreRooms 0/12 NS
Bulk Outloading
Gantry
1/32(3.1)(Ohio) 4
/52(7.6)(Agama)
PigeonFaeces 0/4 NS
PreStartSamplescollectedbymillstaff
Postshut down
ration
0/16 NS
CoolerAggregate 0/24 NS
Overall 31/427(7.3) 44/400(11.0)
* allpositivesamplesfromcerealintake
breederlineonly
NS=Notsampled,NA=notapplicable
346
OrganicacidsforcontrolofSalmonellaserotypesindifferentfeedmaterials
SevincKOYUNCU
1
,GunnarANDERSSON
1
,CharlottaLFSTRM
2
,PanosSKANDAMIS
3
,
AntoniaGOUNADAKI
3
,JrgenZENTEK
4
andPerHGGBLOM
1
1
NationalVeterinaryInstitute(SVA),DepartmentofChemistry,Environment
andFeedhygiene,UPPSALA,Sweden;
2
TechnicalUniversityofDenmark(DTU),NationalFoodInstitute,SBORG,Denmark;
3
AgriculturalUniversityofAthens(AUA),DepartmentofFoodScienceandTechnology,
ATHENS,Greece;
4
FreieUniversittBerlin(FUB),InstituteofAnimalnutrition,Germany
INTRODUCTION
Salmonellacontrolofanimalfeedisanimportantpartinanintegratedcontrolsysteminorderto
protect public health. Application of organic acids is one of the control measures used for
treatmentofSalmonellacontaminatedfeedorfeedingredients.Inthepresentstudy,theefficacy
offormicacid(FA)anddifferentblendsofFA,propionicacid(PA)andsodiumformate(SF)onthe
survival of Salmonella in different feed materials were investigated. Four Salmonella strains
isolated from feed were assayed for their acid tolerance. Also, the effects of acid applications at
lowertemperatures(5Cand15C)werecomparedtoroomtemperature.
MATERIALANDMETHODS
The following strains of Salmonella enterica ssp. enterica isolated from feed, from the culture
collection at the National Veterinary Institute, Uppsala, Sweden, were used in the study (strain
number/isolation year): Salmonella Typhimurium 98/1991, Salmonella Senftenberg 252/1995,
SalmonellaInfantis167/2007,SalmonellaPutten297/2007.
In the experiments with pure acids (FA or FA and PA (80% / 20%)) pelleted compound pig feed,
extracted soybean meal or rapeseed meal were artificially contaminated with the four different
Salmonella strains. The samples were then exposed to 1 % acid for 1, 4, 8, 24, 48 and 120 h at
room temperature. In the experiments with commercial blends of acids compound mash feed or
soybeanmealacidifiedwith0.9or1.5%Amasil(61%FA,20.5%SF,18.5%water)orLuprocid(75%
FA and 25% PA) was used. The feed materials were artificially contaminated with Salmonella
TyphimuriumandSalmonellaInfantisbeforeincubationatroomtemperaturefor0,1,4,7,14and
28days.ForenumerationofSalmonellatrypticsoyagar(TSA)with0.1%sodiumpyruvatefollowed
byoverlaywithxyloselysinedeoxycholate(XLD)agarwasused.
Effectsofreducedtemperatures(5C1Cand15C1C)comparedtoroomtemperature(23C
1C)wasinvestigatedinrapeseedmealandsoybeanmealwith1%FA.
TheresultswerestatisticallyanalysedbyonewayANOVA.
RESULTS
ThestrongestreductioninSalmonellacountswasseeninpelletedandcompoundmashfeed(2.5
log
10
CFU/ml) followed by rapeseed meal (1 log
10
CFU/ml) after 5 days exposure compared to
control.However,insoybeanmealtheacideffectswerelimited(lessthan0.5log
10
CFU/ml)even
after several weeks exposure. In all experiments the survival curves showed a concave shape,
withafastinitialdeathphasefollowedbyreductionataslowerrateduringtheremainingtimeof
theexperiment.
NodifferencewasobservedbetweenFAandtheblendofFAandPA,whereasAmasilwasslightly
moreefficacious(0.51log
10
CFU)thanLuprocidwhencomparedincompoundmashfeed.
Salmonella Infantis was found to be the most acid tolerant strain followed by, S. Putten, S.
Senftenberg and S. Typhimurium. The tolerance of the S. Infantis strain compared with the S.
Typhimuriumstrainwasstatisticallysignificant(p<0.05).
347
ThelethaleffectofformicacidonS.TyphimuriumandS.Infantisinbothrapeseedandsoybean
mealwaslowerat5Cand15Ccomparedtoroomtemperature.At5Cand15Calonger
incubationtimewasnecessarytoobtainthesamelevelofreductioninSalmonellacomparedwith
23C.Therateofthereductionwasapproximatelyproportionaltothetemperature.
DISCUSSION
The efficacy of different acids on reduction of Salmonella varied significantly between feed
materials. Although a prolonged incubation time was applied the effects in soybean meal was
poor. The investigated strains showed a variation in acid tolerance and at lower temperatures
reducedeffectsofacidswereseencomparedtoroomtemperature.
In this study we could confirm previous results that acid treatment of Salmonella in feed is a
matterofreducingthenumberofviablebacterialcellsratherthaneliminatingtheorganism.Many
commercialsuppliersofacidproductsproposea48htreatmentofSalmonellacontaminatedfeed
materials.BasedontheseresultsrecommendationsontheuseofacidsforcontrollingSalmonella
infeedshouldtakeintoaccounttherelativeefficacyofacidtreatmentindifferentfeedmaterials,
the variation in acid tolerance between different Salmonella strains, and the treatment
temperature.
REFERENCES
1. Anonymous: Scientific Opinion of the Panel on Biological Hazards on a request from the Health and
Consumer Protection, Directorate General, European Commission on Microbiological Risk Assessment in
feedingstuffsforfoodproducinganimals.In.:TheEFSAJournal;(2008)720,184
2. Wales AD, Allen VM, Davies RH: Chemical treatment of animal feed and water for the control of
Salmonella.FoodbornePathogensandDisease2010,7(1):315.
348
AssessmentofantiSalmonellaactivityofbootdipsamples
AndrRABIE
1
,IanMCLAREN
1
,MarkBRESLIN
1
,FrancescaMARTELLI
1
andRobDAVIES
1
1
AnimalHealthandVeterinaryLaboratoriesAgency,DepartmentofBacteriology
andFoodSafety,WoodhamLane,NewHaw,Addlestone,Surrey,UNITEDKINGDOM,KT153NB
INTRODUCTION
Theintroductionofpathogensfromtheexternalenvironmentintopoultryhousesbythebootsoffarm
workers and visitors presents a significant risk. The use of boot dips containing disinfectant to help
prevent this from happening is common practice [1]. The efficacy of these boot dips as a preventive
measure can vary. Disinfectants used in the dips may not always be fully active against surface or
organic matter contamination [2, 8] or may be inaccurately measured or diluted to a concentration
otherthanthatspecifiedorrecommended[7].Dipsmaynotbechangedfrequentlyenoughormayhave
been exposed to rain and other environmental elements [3]. The objective of this study was to assess
theantiSalmonellaactivityofbootdipsthatarebeingusedoncommerciallayinghen(layer)farms.
MATERIALANDMETHODS
BootdipsampleswerecollectedfromlayerfarmsinEnglandandWalesandtestedtoassesstheir
antiSalmonellaactivity.AllsamplesweretestedagainstafieldstrainofSalmonellaEnteritidis(SE)
obtainedfromacommerciallayingfarmusedinpreviousstudies[4].Thethreetestmodelsused
were:pureculture,paperdiscsurfacematrixandyeastsuspensionmodel.
The pure culture method involved adding 1ml of ~2 x 10
9
cfu/g SE to 9ml of boot dip with no
addedorganicmatterandholdingitat4Cfor30minutes.
Thepaperdiscmodelutilisedaporouscontaminatedsurfaceplaceddirectlyinto5mlofdip.
Intheyeastsuspensionmethod,0.4mloftheSEchallengestrainwasaddedto9.6mlofa5%killed
yeast suspension (based on the British Standards method BS6734:1986 and 2004 [9,10]). A
volumeof2.5mlofthemixwasaddedto2.5mlofthebootdipsampleandheldfor30minutesat
4C.
All the samples were subjected to a disinfectant neutralisation step by using Nutrient broth +
horseserumandthenincubatedinBufferedPeptoneWater(BPW)for18hours2at37C1C
and further examined using a sensitive isolation procedure employing Modified Semisolid
RappaportVassiliadis(MSRV)andRambachagar.
RESULTS
Resultswereinterpretedasfollows:IfSEwasisolatedfromanydilutionsinmodel1itwasclassed
as a fail. If SE was isolated in model 2 it was classed as a fail. Any SE isolated in four or more
replicatesinmodel3wasconsideredtobeafailedtest.
Ofthe110bootdipsamplestested83.6%wereeffectiveagainstSalmonellainmodel1,37.3%inmodel
2and44.5%inmodel3(Figure1).
DISCUSSION
It appears that disinfectants are not always suitable for surface or organic matter contamination
and are not always made up to the correct concentration by farmers. The dips are not always
changed frequently enough and may have been exposed to rain, organic soil and other
environmentalelements[6].Theremayalsohavebeenevaporationordegradationoftheactive
ingredients after dilution in labile disinfectants. Lack of attention to expiry dates could also be
problematic[3,5].
349
CONCLUSION
This study showed that boot dips that are in use on layer farms are frequently ineffective. Even when
correctlymaintaineditisunwisetorelyonbootdipsaloneandachangeofbootswhenenteringpoultry
housingisalsorecommended.
ACKNOWLEDGEMENTS
throughprojectOZ0332.
REFERENCES
1. AmassSF,VyverbergBD,DowellCA,AndersonCD,StoverJH,BeaudryDJ.(2000):Evaluatingtheefficacy
ofbootbathsinbiosecurityprotocols.SwineHealthandProduction.8(4):169173.
2. Ewart SL. (2001): Disinfectants and control of environmental contamination. In: Smith BL. editor. Large
Animal Internal Medicine: diseases of horses, cattle, sheep and goats. 3rd edition. St. Louis: Mosby. pp.
13711380.
3. McDonnellG,RusseiAD.(1999):Antisepticsanddisinfectants:Activity,actionandresistance.ClinMicrobiol
Rev.1999January;12(1):147179.PMCID:PMC88911
4. McLaren I, Wales A, Breslin M, Davies R. (2011): Evaluation of commonlyused farm disinfectants in wet and
drymodelsofSalmonellafarmcontamination.AvianPathology,40,(1),3342.
5. Moore SL, Payne DN. (2004): Types of antimicrobial agents. pp. 897. In: RUSSELL AD.; HUGO WB.; AYLIFFE
GAJ.;(2004):PrinciplesandPracticesofDisinfection,Preservation&Sterilization.EditedbyAPFraise,PALambert,
JYMaillard.Fourthedition.BlackwellPublishing.
6. Russell AD. (2004): Factors influencing the efficacy of germicides. In: Rutala WA, ed. Disinfection,
sterilizationandantisepsis:Principles,practices,challenges,andnewresearch.WashingtonDC:Association
forProfessionalsinInfectionControlandEpidemiology.16270.
7. Russell AD, McDonnell G. (2000): Concentration:a major factor in studying biocidal action. J. Hosp.
Infect.44:13.
8. StringfellowK,AndersonP,DaldwellD,LeeJ,ByrdJ,McReynoldsJ,CareyJ,NisbetD,FarnellM.(2009):
Evaluation of disinfectants commonly used by the commercial poultry industry under simulated field
conditions.2009PoultryScience88:11511155.doi:10.3382/ps.200800455
9. BSIShop.BS6734:1986
http://shop.bsigroup.com/ProductDetail/?pid=000000000000160048
10. Shop.BS6734:2004http://shop.bsigroup.com/en/ProductDetail/?pid=000000000030102227
TABLESANDFIGURES
Figure1Comparisonofdisinfectantperformanceinthreedifferentmodels
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Model 1 Model 2 Model 3
Test
%Series1
350
Investigationoflaboratorytestingissues
inthecontextoftheSalmonellaNationalControlProgrammeinGB
RebeccaJ.GOSLING
1
,RobinSAYERS
2
andRobH.DAVIES
1
1
DepartmentofBacteriologyandFoodSafety,AnimalHealthandVeterinaryLaboratories
Agency,NewHaw,Addlestone,SURREY,KT153NB
2
SpecialistScientificSupport,AnimalHealthandVeterinaryLaboratoriesAgency,NewHaw,
Addlestone,SURREY,KT153NB
INTRODUCTION
In Great Britain, the National Control Programmes (NCPs) for Salmonella fulfil the requirements
for statutory testing according to EU legislation
2
. Samples are collected regularly by the farmer
(operator)andbytheCompetentAuthorityasverificationofsamplingconductedbytheoperator.
These samples are tested for Salmonella by Defraapproved testing laboratories using a
standardised method (ISO 6579: Annex D
1,2
. In order to confirm that sampling and laboratory
testing is being carried out effectively and to ensure optimum sensitivity in the detection of
Salmonellainpoultryflocksaquestionnairebasedsurveywasconductedamongapprovedtesting
laboratories.
The aim was to identify any complicating factors that the laboratories may encounter during the
processingofsamples.
MATERIALSANDMETHODS
Sixteen approved laboratories for Salmonella testing were sent a questionnaire. Only those
receivingsamplesonaregularbasiswereselectedforinclusion.Questionswerecategorizedinto
2mainsections:thereceiptandconditionofsamples,andtheprocessingofsamples.
RESULTS
Tenoutof16Defraapprovedlaboratoriesreturnedthequestionnaire.Sampleswerereportedto
arrive in good condition and within the specified timeframe after collection (Figure 1). The only
concern was that new clients were occasionally unaware of the required testing protocol or
correctsamplingconsumablestouse.Therewassomevariabilitybetweenlaboratoriesregarding
thesampletestingprocedureused,withdeviationfromtheguidelines(Table1).
DISCUSSION
ISO 6579:Annex D
1,2
provides the methodology required to be followed for the analysis of
SalmonellasamplessubmittedundertheNCP.Failuretofollowthesemayaffectthedetectionof
Salmonella. Submerging the bootswabs in Buffered Peptone Water (BPW) ensures thorough
suspension of the sample, and before subculturing from BPW, the sample should not be
swirled/mixedasthiswillresuspendparticlesandcompetingbacteria.Salmonellaneedssufficient
liquid phase to migrate away from any inhibitory agents in the particulate matrix, therefore re
suspension of particles before culturing may result in a false negative result because particles of
the sample are carried over into the selective enrichment phase
3
. Forty percent of laboratories
reportedthattheydidswirlsamplesafterincubation.
The guideline recommends the use of 10 mg/l Novobiocin in Modified Semisolid Rappaport
Vassiliadis (MRSV)
1
. Previously 20 mg/l or 2% of novobiocin was added
4
; however more recent
studies have reported more Salmonella positive results with 10 mg/L novobiocin rather than 20
mg/l and larger Salmonella migration zones
5
. The variation in the use of different loop sizes
between laboratories may have a direct influence on the outcome of the testing as a larger
inoculum may lead to overgrowth by competing organisms or failure to express typical colony
351
characteristics.Theuseof1lofcultureshouldproducewellisolatedcoloniesonastandardsize
Petridish.
1
Thesefindingssuggestthatfeedbackonlegislativerequirementsandtechniquesalreadyprovided
to laboratories and further ongoing guidance on methodology is likely to be beneficial as sub
optimalmethodsmayaffectthedetectionoflowlevelsofSalmonella.
ACKNOWLEDGEMENTS
REFERENCES:
1. Anon. 2005. Detection of Salmonella spp. in animal faeces and in samples of the primary production
stage.DraftamendmentAnnexDISO6579:2002/DAM.ISO2005
2. Anon, 2006. Commission Regulation (EC) No. 2160/2003 http://eur
lex.europa.eu/LexUriServ/site/en/oj/2003/l_325/l_32520031212en00010015.pdf
3. CarriqueMas,JJ.,Davies,RH.2008.SamplingandbacteriologicaldetectionofSalmonellainpoultryand
poultrypremises:areview.Rev.sci.tech.Off.Int.Epiz.27(3):665677
4. De Smedt, JM., Bolderdijk, RF., Rappold, H., Lautenschlaeger, D. 1986. Rapid Salmonella detection in
foods by motility enrichment on a modified semisolid RappaportVassiliadis medium. Journal of Food
Protection49(7):510514
5. Veenman,C.,Krover,H.,Mooijman,KA.2006.ResearchactivitiesCRLSalmonella.NationalInstitutefor
PublicHealthandtheenvironment,Bilthoven,TheNetherlands.RIVMreport330300010.
TABLESANDFIGURES
Figure1.LaboratoriesperceptionoftheconditionofsamplessubmittedtothemundertheNationalControl
ProgrammeforSalmonella.
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Sample
packages arrive
in good
condition
Faeces samples
look fresh and
moist
Sufficient
weight of layer
faeces sample
(at least 200g)
supplied
Correct number
of bootswabs
for each poultry
type are
supplied
Bootswabs
look well soiled
Bootswabs
look dried out
%

o
f

r
e
s
p
o
n
s
e
s

(
n
=
1
0
)Not applicable
Never
Rarely
Sometimes
Most of the time
Almost always
Always
352
Table 1. Variability in the Annex D sampling method followed by laboratories testing samples under the
NationalControlProgrammeforSalmonella.
Methodstep Technique
%oflabsfollowingcorrect
technique
Bufferedpeptonewater(BPW) Neverusestraightfromthefridge 100
Bootswabs SubmergedinBPW 70
Incubated18h+ 2h 100
Samplenotswirledpostincubation 60
Novobiocininclusioninmigration
media
Atrecommendedconcentration
(10mg/l) 20
Growthplatedout 1ulloop 70
Inoculumtaken
Fromedgeofgrowthzoneawayfrom
theedge 50
Declaringanegativesample Afterchecking5colonies 70
Isolatessentforserotyping Inunder3days 100
353
EscherichiacoliasindicatorofthehumanSalmonellarisk
causedbyconsumptionofpork
AnneMETTEBOLLERSLEV
1
,MaartenNAUTA
2
,TineHALD
2
,TinaBECKHANSEN
1
andSrenAABO
1
TechnicalUniversityofDenmark,Divisionsof
1
FoodMicrobiologyand
2
Epidemiology
andMicrobialGenomics,SBORG,Denmark
INTRODUCTION
Salmonella is widespread in the slaughter pig production in Europe, and Salmonella from pork
constitutes a significant risk for consumers. At slaughter, food chain information do not per se
allow for an effective distinction between pigs from Salmonella positive and negative herds, and
improvementofthegeneralslaughterhygieneistheonlymitigatingtooltouse.Sofartherehave
beennoreportsdescribinghowthehygienelevelatslaughterassociatestoSalmonellarisk.
Wehavecollectedquantitativehygienedata(E.coli)andquantifiedSalmonellaonpigcarcassesat
slaughter. The objective is to establish the correlation between the hygiene level and Salmonella
and to provide the first suggestion for a method to set risk based process hygiene criteria at pig
slaughter.
MATERIALANDMETHODS
Samplecollection
CarcassesfrompigsslaughteredatfiveDanishpigslaughterhousesweresampledintheperiod10
May 2005 to 5 June 2007. Sampling was performed at the slaughterline just before cooling.
Carcass swabs (2800 cm
2
) from 1,906 carcasses were analysed both quantitatively for E. coli and
semiquantitativelyforSalmonella.Atotalof75mlpeptonewaterwasaddedtostomacherbags
with carcass swabs containing approximately 12.5 ml of peptone water and tissue fluid. This
mixturewasstomachedbeforedilution.Onemillilitreof10folddilutionswerespreadonPetrifilm
and subsequently incubated at 41.5 C for 2325 h. The number of E. coli was determined using
Select E. coli Count Plate Petrifilm (3M Microbiology, St. Paul, MN, USA) in accordance with the
suppliersinstructions.CellcountsweredeterminedbyautomatedreadingusingaPetrifilmplate
readerMI6499(3MMicrobiology,St.Paul,MN,USA).Fromatenfolddilutionofthehomogenate,
asemiquantitativeanalysisforSalmonellawasperformed.Allstomachedsampleswereanalysed
forSalmonellausingMSRVagar(ISO6579,AnnexD,Anonymous,2007).
Statisticalanalyses
All statistical analyses were performed with the software R (ver. 2.15.1) and RStudio (ver.
0.96.331). Bacterial counts of E. coli were log
10
transformed to obtain approximately normally
distributed data. Samples in which E. coli was found to be below the detection limit (1 CFU/ml)
wereassignedavalueof0.5CFU/mltoallowlog
10
transformation.
Means and standard deviations were calculated for the log
10
transformed E. coli levels. The
corresponding Salmonella prevalence was calculated after dichotomisation of the results (0 =
Salmonella negative; 1 = Salmonella positive). A boxandwhisker plot was made to illustrate the
correlationbetweentheconcentrationofSalmonellaandE.colifoundinswabsamples.
To determine the association between E. coli and Salmonella, univariable analyses were carried
out.Variableswithp0.25wereincludedinamultivariablelogisticregressionanalysis.Selection
of explanatory variables for the final model was done by stepwise backwards elimination of the
leastsignificantvariableuntilonlysignificantvariablesremained.Intheanalysis,pvaluesloweror
equalto0.01wereconsideredasstatisticallydifferent.Thefinalexplanatoryvariablesweretested
forinteractionandconfounding.
354
Riskmodel
The risk model takes into account both the prevalence of Salmonella on carcasses and the
estimatednumberofSalmonellabacteriapresent.ThenumberofSalmonellabacteriapercm
2
was
estimated from the observed contamination of E. coli on the carcass and the established
regressionbetweennumberofE.coliandnumberofSalmonellabacteriaonthecarcass.Asimple
exposuremodelwasdevelopedassumingthat:1)theconcentrationofbacteriapercm
2
waseven
onthewholecarcass2)thewholecarcasswasconsumedrawin200gramportionsand3)all101
human illnesses associated to pork in 2006 in Denmark could be associated to this. Additionally,
the risk model included three factors: a correction factor, which adjusted the doseresponse
relationshipprovidedbyFAO/WHO(2002);anunderreportingfactor(Havelaaretal.,2012)anda
factoraccountingforpreparationofporktothenumberofregisteredcasesinDenmarkin2006.
RESULTS
The average level of E. coli found on the skin of the carcasses was 0.8 log CFU/cm
2
, from all five
slaughterhouses. The corresponding prevalence of Salmonella was found to be 2.5%. The
correlationbetweentheconcentrationofE.coliandSalmonellaisdepictedinFigure1.
TheoddsofSalmonellabeingpresentonthecarcasswerefoundtoincreaseby1.87foreveryone
log
10
unit increase of E. coli. The risk of Salmonella being present on the carcass varied between
slaughterhouses.
ByapplyingtheobservedE.coliandSalmonelladatatotheriskmodel,itwaspossibletomakean
estimate on the relationship between hygiene level measured by E. coli and the Salmonella
consumerrisk.Table1showthatthenumberofhumancasescouldhavebeenreducedbyapprox.
50%(from101to48.6),iftheE.colilevelatslaughterhadnotexceeded34log
10
CFUper32cm
2
.
CONCLUSION
This is to our knowledge the first report on estimating consumer risk of salmonellosis from the
hygienelevelatpigslaughter.Theperspectiveistheabilitytoestablishriskbasedprocesshygiene
criteriabasedonthisprinciple.
ACKNOWLEDGEMENTS
HardyChristensen,DanishMeatResearchInstitute,DanishTechnologicalInstitute,Denmark.
REFERENCES
1. Anonymous,2007.ISO6579:2002/Amd1:2007,AnnexD:DetectionofSalmonellaspp.inanimalfaeces
and in environmental samples from the primary production stage. International Organisation for
Standardization.
2. Havelaar, A.H., Ivarsson, S., Lfdahl, M., Nauta, M.J. (2012). Estimating the true incidence of
campylobacteriosis and salmonellosis in the European Union, 2009. Epidemiol. Infect., Page 1 to 10,
CambridgeUniversityPress2012.
3. WHO/FAO, 2002. Risk assessments of Salmonella in eggs and broiler chickens. Microbiological risk
assessment series; no. 2. World Health Organization; Food and Agriculture Organization of the United
Nations.
355
Figure 1 Boxandwhisker plot of the level of E. coli stratified by the concentration of Salmonella. The
letters represents the following concentration intervals: K < 0.10 CFU/ml, A: 0.10 0.91 CFU/ml, B: 0.91
10.1CFU/ml,C:10.1101CFU/ml,D:101909CFU/ml,DD>909CFU/ml(theunitCFU/mlcorresponds
toCFU/32cm
2
).
Table 1 Modelled estimation of the total number of human salmonellosis cases in Denmark in 2006
dependingonthemaximumlevelofE.colionpigcarcassesatslaughter.
MaximallevelofE.colioncarcass[log
10
CFU/32cm
2
]
01 12 23 34 45 56
No.ofcases 0.0 6.3 1.6 40.7 0.6 51.8
Accumulatedno.of
cases
0.0 6.3 7.9 48.6 49.2 101.0
356
Assessmentofbiosecurityanduptakeofadviceinchickenlayerfarms
inEnglandandWales
FrancescaMARTELLI,RebeccaGOSLING,AlisonWINTRIP,KarenWHEELER,SamBEECHENER,
RobinSAYERSandRobDAVIES
INTRODUCTION
Biosecurity consists of the measures used to prevent the introduction of diseases causing
organismsand/ortoreducetheeffectsofaninfectioninapopulation(2,3).Biosecuritymeasures
areappliedtoavariableextentacrossthepoultryindustry,withlargerfacilitiesnormallyassumed
tobeimplementingthemcorrectly,butmoreatriskasaresultoftheintensityoftheirproduction
(7). Infectious agents can enter poultry farms through contaminated birds (direct bird to bird
transmission), through indirect routes (e.g. via fomites or the environment), or through vectors
(wildbirds,rodents,insects)(2,5).Salmonellaspp.caninfectpoultryandcertainserovarscanbe
transmitted from poultry to people, through direct contact or consumption of contaminated
poultry products. Salmonella can persist for a long time in the environment and can infect the
resident rodent population. The epidemiology of Salmonella is complex and involves human,
environmentalandanimalinteractions(4).Goodbiosecuritymeasuresarecriticalinthecontrolof
Salmonella in poultry farms (1). The aims of this study were to assess the biosecurity status of a
sample of chicken layer farms in England and Wales, and evaluate changes in this status after
provisionofadvice.
MATERIALANDMETHODS
In this study, we designed a questionnaire targeted on the biosecurity measures that are
particularly relevant for the control of Salmonella (and in particular regulated serovars S.
Enteritidis and S. Typhimurium) in layer farms. The questionnaire designed for this study
contained semi open ended questions, giving the farmers a limited number of choices and the
freedomtoincludeadditionalinformation(6).Tovalidatetheanswersgiveninthequestionnaire,
aparallelvisualassessmentwascarriedoutbytrainedauditors,andadviceprovided.
SixtycommerciallayinghenfarmsinEnglandandWaleswereincludedinthisstudy.Thesefarms
included conventional cage, colony cage, free range and barn systems. All the farms had at least
2000layinghensontheholding.ThefarmswerevisitedtwiceintheperiodbetweenOctober2010
andOctober2011byoneofthreeauditors.Thesameauditorperformedthefirstandsecondvisit
to the same farms. Ten advice cards focussed on differentaspects of biosecurity were produced,
each containing a brief description of the problem and a list of actions that should be taken to
improve biosecurity, in particular for Salmonella control. At the end of the first visit, the main
areas that required improvement of biosecurity were identified by the auditor. A maximum of 3
advice cards were left to the farmer at the end of the first visit. Advice was provided for specific
actionpointsthatshouldhavebeenimplementedonfarm.Asecondvisitwasperformed,andthe
visual assessment was repeated to note any improvements following the advice provided. The
implementationoftheactionpointslistedintherelevantadvicecardswasalsoinvestigated.The
differences in the distributions of scores between the farmer responses (q) and the auditor (v)
from the 1
st
visit were compared by the marginal homogeneity test with exact probabilities that
takesintoaccountthepairingofresponsesbyfarm.FishersExacttestwasusedtodetermineany
associationbetweenmeasuredparameters.StatisticalsignificancewasacceptedinP<0.05.
357
RESULTS
Therewasalowuptakeofparticipationbyfarmerswith312holdingscontactedinordertorecruit
60 (uptake of participation 19.2%). Some of the large egg production companies were not
interestedintakingpartwhichreducedtheavailabilityoflargerrepresentativefarms,and22out
of 87 caged units contacted declined to take part as they were going out of business due to the
requirementtoinstallcolonycages.
For the majority of the questions, there was a good agreement between the farmers and
auditors scores. Significantly different scores were recorded only for question 1.01, Entrance to
site(p=0.002),3.05Handlinganddisposalofdeadbirds(p=0.008),4.02Accumulateddustinhouse
(p=0.039), 7.07 How boot dips are used (p=0.002), 7.08 Cleaning of boots before dipping
(p=0.008). The auditors scores to these questions were generally lower that the ones generated
by the farmers. The Kappa coefficient was calculated for questions 1.01 to 8.13 as a measure of
theagreementbetweenthefarmerresponses(q)andauditorscores(v)atthefirstvisit.Withthe
exception of two questions (3.05, 6.02) the coefficients all fell into the top two categories
indicatinggoodorverygoodagreement.Amaximumof3advicecardswereleftwiththefarmer
at the first visit, and the boot hygiene advice card was the most widely distributed (53
respondents), followed by red mite control (25), hand hygiene (23), entry to the site (18) and
cleaning and disinfection (10). Table 1 summarizes the uptake of advice provided for the top 5
issuedadvicecards,asassessedduringthesecondvisit.
DISCUSSION
There was good agreement between the auditors and the farmers assessment in most of the
biosecurityquestionsasked.Thisshowsthatthefarmersthattookpartinthisstudyareawareof
biosecurityrequirements,andhowtheyareimplementedontheirfarms.Wheredifferenceswere
noted between the farmers and auditors scores the farmer scored themselves higher than the
score of the auditor, suggesting that the farmers may know what they should be doing but in
practicethismaynotalwaysoccur(3).Elementsofgoodbiosecuritypracticewereidentifiedona
numberofsitesbuttherewasscopeforimprovementinoneormoreaspectsonallfarmsvisited.
Theboothygienecardwastheonemostfrequentlydistributed.Designatinghousespecificboots
is frequently recommended, however not changing boots between non cage houses was
frequently observed (this is not so important for cage houses) (3). Hand hygiene and control of
visitorsentrytothesitewerealsoidentifiedasbiosecurityareasthatmightneedimprovementin
previous studies (3). Cleaning and disinfection has been identified as a key procedure to reduce
theSalmonellariskonfarm(4).Mostofthemeasuressuggestedintheadvicecardswereeasyto
implement and not expensive (e.g. accurately measure boot dip chemical, replenish boot dips
regularly, provide overalls for visitors, use disposable gloves) were adopted more readily than
those which requiredan active change of routine (e.g. cleaning boots before dipping, monitoring
red mite with folded cardboard, use of separate boots for different houses, use a vehicle
disinfectantspray).
The development of the auditing tool with targeted prioritised advice proved to be a successful
approach for the layer industry. A significant proportion of the advice offered was implemented,
with a reported intention to maintain this in the long term. The visits were carried out during a
particularlydifficultperiodforthelayerindustry(loweggpriceandhighfeedcostsaswellasthe
conversion from conventional cages to colony or alternative systems) but despite this there was
still significant interest in improvement. The most common barriers encountered by the farmers
wereofafinancialandoperationalnatureandthisisthoughttohavealsoledtothelowerlevels
ofparticipationamongstthemajorcompanies.
358
ACKNOWLEDGEMENTS
REFERENCES
1. Holt,P.S.,R.H.Davies,J.Dewulf,R.K.Gast,J.K.Huwe,D.R.Jones,D.Waltman,andK.R.Willian.2011.
Theimpactofdifferenthousingsystemsoneggsafetyandquality.PoultSci90:251262.
2. Nespeca,R.,J.P.Vaillancourt,andW.E.Morrow.1997.Validationofapoultrybiosecuritysurvey.Prev
VetMed31:7386.
3. Racicot, M., D. Venne, A. Durivage, and J. P. Vaillancourt. 2011. Description of 44 biosecurity errors
while entering and exiting poultry barns based on video surveillance in Quebec, Canada. Prev Vet Med
100:193199.
4. Snow, L. C., R. H. Davies, K. H. Christiansen, J. J. CarriqueMas, A. J. Cook, and S. J. Evans. 2010.
InvestigationofriskfactorsforSalmonellaoncommercialegglayingfarmsinGreatBritain,20042005.Vet
Rec166:579586.
5. Vaillancourt,J.P.,Carver,D.K.1998.Biosecurity:perceptionisnotreality.PoultryDigest31:136143.
6. Vaillancourt, J. P., Martineau, G., Morrow, M., Marsh., W., Robinson, A. 1991. Presented at the
Proceedings of the of the Society of Veterinary Epidemiology and Preventive Medicine, Annual Meeting,
London.
7. Van Steenwinkel, S., S. Ribbens, E. Ducheyne, E. Goossens, and J. Dewulf. 2011. Assessing biosecurity
practices, movements and densities of poultry sites across Belgium, resulting in different farm riskgroups
forinfectiousdiseaseintroductionandspread.PrevVetMed98:259270.
TABLESANDFIGURES
Table1:Analysisoftheimplementationofthetopfiveissuedadvicecards.
Overalluptakeofindividualmeasures
Advicecard Implementedby
2
nd
visit
Tobe
implemented
Willnotbe
implemented
Boothygiene(n=53) 54.8% 12.0% 33.2%
Controlofredmites(n=25) 59.2% 10.2% 30.6%
Handhygiene(n=23) 62.0% 15.2% 22.8%
Controlsatsiteentrance(n=18) 68.0% 6.2% 25.8%
Cleaninganddisinfection(n=10) 79.2% 1.3% 19.5%
359
Assessmentofthesocialscienceaspectofbiosecurityanduptakeofadvice
onchickenlayerfarmsinEnglandandWales
RebeccaJ.GOSLING
1
,FrancescaMARTELLI
1
,AlisonWINTRIP
2
,RobinSAYERS
3
,KarenWHEELER
2
,
SamBEECHENER
2
andRobH.DAVIES
1
1
DepartmentofBacteriologyandFoodSafety,AnimalHealthandVeterinaryLaboratoriesAgency,
NewHaw,Addlestone,SURREY,KT153NB;
2
SustainableLivestockGroup,AnimalHealthandWelfare,ADASUKLtd;
3
SpecialistScientificSupport,AnimalHealthandVeterinaryLaboratoriesAgency,NewHaw,
Addlestone,SURREY,KT153NB
Agency,NewHaw,Addlestone,Surrey,KT153NB
INTRODUCTION
Effective onfarm biosecurity is known to reduce the risk of disease outbreaks
1
; however the
uptakeofadviceandimplementationofbiosecuritymeasuresisdependentonmanyfactors.This
study assessed the uptake of targeted biosecurity advice by a selection of laying hen farmers
provided during two voluntary biosecurity audit visits. Advice was provided as advice cards
focusingonspecificareasidentifiedasinneedofimprovement.
MATERIALANDMETHODS
SixtylayingfarmswererandomlyselectedfromtheBritishPoultryRegisteraccessed2010.Farms
werestratifiedbysizeandtypeofproduction.Anaudittoolcomprisingofninesections(siteentry,
site construction, flock health, hygiene, egg collection, farm pests, operational aspects,
depopulation and general disease knowledge) with a total of 75 questions was developed with
industryconsultation.ThefinalisedaudittoolwasapprovedbytheDefraSurveyControlUnit(No.
17900783)beforethestudycommenced.Trainedauditorsassessedeachstudyfarmandprovided
biosecurity advice to the farmer in the form of advice cards. Each farm was revisited 6 months
later to determine if the advice provided had been implemented. The advice cards covered
rodent,flyandredmitecontrol,vaccination,bootandhandhygiene,sitetidiness,tidinessinside
the houses, site entry procedures, cleaning and disinfection between flocks. Farmers background
knowledge of Salmonella and biosecurity were assessed, and their willingness and intent to
implementadditionalmeasuresevaluated.
RESULTS
Freerangefarmsmadeupthelargestproportionoffarmsrecruited(Table1)whichisinlinewith
the UK egg industry flock type distribution (approximately 50%). More than 50% of farmers had
moderatebackgroundknowledgeofSalmonella,with22%consideredwellinformed,determined
byansweringkeyquestionsonSalmonellacontrolinpoultrycorrectly.Allbutoneofthefarmers
questioned agreed biosecurity could impact on the control of Salmonella and many appeared
willingtoimplementadditionalbiosecuritymeasures.Thefivemostfrequentlyusedadvicecards
were boot hygiene, red mite control, hand hygiene, entrance to the site and cleaning and
disinfection, and uptake of this advice ranged from 5480% depending on the advice card (Table
2).
DISCUSSION
Theuptakeofadvicebythefarmerswasencouraging,especiallyconsideringitwasbeingprovided
bypeopleotherthantheirusualsourceofbiosecurityinformation.Thosewhodidnotimplement
the recommended measures cited cost, difficulty of enforcement and practicality as the main
reasons for not implementing the advice. However, once an action had been implemented there
360
wasagoodchanceofitbeingcontinued,demonstratingthattargetedadviceonasmallnumberof
keyareaswasaneffectivemethodofprovidingbiosecurityinformation.
ACKNOWLEDGEMENTS
REFERENCES
1. Beloeil, P.A., Chauvin, C., Proux, K., Fablet, C., Madec, F., Alioum, A., 2007. Risk factors for Salmonella
seroconversionoffatteningpigsinfarrowtofinishherds.Vet.Res.38,835848.
TABLESANDFIGURES
Table1.Numberoffarmsrecruitedfromeachproductiontype.
Distributionoffarmsbyflocksize
category
Housingtype Smallfarm
a
Largefarm
b
Barn 1 1
Cage/colony 6 8
FR 29 2
Caged/noncaged 4 9
a
<40000noncagedbirdsor<100000cagedbirds.
b
>40000noncagedbirdsor>100000cagedbirds.
Table 2. Percentage of advice cards issued, those implemented by the 2
nd
audit visit and intention to
implementatalaterdate.
Advicecard %ofallfarmersthat
receivedeachadvice
card
Overalluptakeofindividualmeasures(%)
Implementedby2
nd
visit
Tobe
implemented
Willnotbe
implemented
Boothygiene(n=53) 90.0 54.8 12.0 33.2
Controlofredmites
(n=25)
42.0 59.2 10.2 30.6
Handhygiene(n=23) 39.0 62.0 15.2 22.8
Controlsatsite
entrance(n=18)
31.0 68.0 6.2 25.8
Cleaningand
disinfection(n=10)
17.0 79.2 1.3 19.5
Rodentcontrol(n=3) 5.0 60.0 10.0 30.0
Flycontrol(n=3) 5.0 50.0 0.0 50.0
Tidinessinsidehouse
(n=3)
5.0 73.3 27.7 0.0
Sitetidiness(n=1) 2.0 100.0 0.0 0.0
361
EffectoflitteraerationinthespreadofSalmonellainpoultry
SofiaINGRESA
1
,SaraGONZLEZ
1
,InmaculadaHERNNDEZ
1
,ArantxaVILLAGR
2
,
SantiagoVEGA
1
andClaraMARN
1
1
InstituteofBiomedicalSciences,DepartmentofAnimalProductionandHealth,PublicHealth
andVeterinaryScienceandFoodTechnology.VeterinaryFaculty.CEUCardenalHerrera
University,AlfaradelPatriarca,VALENCIA,Spain;
2
AnimalResearchandTechnologyCentreCITAIVIA,Segorbe,CASTELLN,Spain;
Correspondingauthor:sofia.ingresa@gmail.com
INTRODUCTION
Among the different environmental factors involved in the epidemiology of Salmonella, the litter
where the chickens will grow up during the rearing period, plays a important role in the
multiplication and spread of bacteria in poultry farm (Hayes et al., 2006). During the rearing the
accumulationoffaecesresultsintheincreaseofpH,moistureandnitrogencontentaffectingthe
qualityofthelitter(Milesetal.,2004,Meluzzietal.,2008).Consequently,differentmethodshave
been developed to keep the optimal qualities of litter. One of them is Litter Aeration (LA) by
tumbling(vanMiddelkoopetal.,1994).However,thereislittleresearchrelatedtotheeffectthat
thistechniquehasonthespreadingofpathogensofPublicHeathimportancesuchasSalmonella.
In this context, the objective of this study was to determine the epidemiology of Salmonella in
poultryfarms,whenseriallyLitterAerationswereimplementedduringthefatteningperiod.
MATERIALANDMETHODS
This experiment was carried out from October 28
th
to December 9
th
2010 in the poultry meat
facilitiesoftheAnimalTechnologyCentre(CITAIVIA)locatedinSegorbe(Castelln,Spain).Three
identical experimental rooms were used for this purpose. Two rooms were assigned to the litter
aeration treatment, while the other one served as control room. Samples were collected at
differentmomentsoftheflocklifespan:beforethearrivaloftheanimals,thefirstdayofrearing,
thethirdweekoftherearingcycle(theweekbeforeLAstarted)andweeklyafterLA(4lastweeks
ofrearing)(Table1).AllsamplescollectedwereanalyzedaccordingtoISO6570:2002fordetection
ofSalmonella.
RESULTS
Duringthestudy204sampleswerecollectedandanalysedforSalmonellaand34,8%werepositive
for bacterium. The status of the poultry houses before the arrival of the dayold chicks was
negativeinallenvironmentalsamplescollected.
The batch of dayold chicks was diagnosed as positive for Salmonella on arrival at the
experimental farm from the hatchery, deliverybox liners and surfaces of the chicks transport
cagesandmeconiums.
After three weeks of rearing before the LA started, the crosscontamination between chickens
faeces and farm environment has been demonstrated regardless of room sampled or sample
collected.
The results of the evolution of the prevalence of Salmonella along rearing period have showed
that,there were not statistically significant differences of the prevalence of Salmonella between
treatedandcontrolroomsfromthefourLitterAerationsperformed.
362
DISCUSSION
The presence of adverse environmental conditions such as high animal densities, insufficient
ventilation,excessiveaccumulationofdroppingsandhighlevelofmoistureleadingtopoorquality
litter (van de Giessen et al., 2006). Furthermore, litter also has a direct influence on the skin
conditionofthebirds,beingwetlitteramajorriskfactorforcontactdermatitis(DelaRosa,2002).
LA procedures can be used as an alternative to prevent cake formation and, consequently,
mitigate contact dermatitis on pads and hocks (Hayes et al., 2006). The implementation of LA
aimed to control these unfavourable conditions and maintain the optimal qualitythroughout the
rearing period (Allen et al., 1998). However, the breaking up and turning of the litter when
carrying out LA may encourage the multiplication and spread of microorganisms throughout
broiler houses (Allen et al., 1998). However, the results of the study showed that when LA was
performedintreatedroomstherewerenotmoreprevalenceofSalmonellaandcouldcontrolthe
developmentofadverseenvironmentalconditionsandimproveproductiveperformance.
CONCLUSION
Litter Aeration, as litter management technique in poultry broiler production, does not increase
thesheddingorthespreadofSalmonella.Inaddition,itcouldbeaneffectivetechniquetoreduce
theinfectivepressureofthisbacteriumincertainmomentsoftherearingperiodwithmoreriskof
multiplicationandspreadingofSalmonella.
ACKNOWLEDGEMENTS
The authors of this article wish to thank the Animal Research and Technology Centre (CITAIVIA)
fortheircollaborationinthisstudyandforofferingtheirfacilitiesinordertodeveloptheresearch.
Inthesameway,wealsowouldliketothanktotheCEUCardenalHerreraUniversityforfunding
thisstudy.
REFERENCES
1. Allen,W.H.,B.L.Hughes,J.P.Chastain,P.A.Skewes,W.C.Bridges,R.Armstrong,andJ.Thomas.1998.
Aeration of poultry broiler litter to reduce production of odor and hazardous gas. ASAE Annual
InternationalMeeting,St.Joseph,Michigan.
2. De la Rosa, M. C., M. A. Mosso, and C. Ulln. 2002. El aire: hbitat y medio de transmisin de
microorganismos.ObservatorioMedioambiental5:375402.
3. ISO6579:2002(AnnexD)(2002)Microbiologyoffoodandanimalfeedingstuffs.Horizontalmethodfor
thedetectionofSalmonellaspp.InternationalOrganizationforStandardization,Geneve,Switzerland.
4. Hayes, E. T., T. P. Curran, and V. A. Dodd. 2006. Odour and ammonia emissions from intensive poultry
unitsinIreland.BioresourceTechnology.97:933939.
5. Meluzzi, A., Fabbri, C.E., Folegatti and Sirri, F. 2008. Effect of less intensive rearing conditions on litter
characteristics,growthperformance,carcaseinjuriesand meat qualityofbroilers.BritishPoultryScience .
49:509515.
6. Miles,D.M.,Branton,S.L.,andLott,B.D.2004.Atmosphericammoniaisdetrimentaltotheperformance
ofmoderncommercialbroilers.PoultryScience.83:16501654.
7. vandeGiessen,A.W.,M.Bouwknegt,W.D.DamDeisz,W.vanPelt,W.J.Wannet,andG.Visser.2006.
Surveillance of Salmonella spp. and Campylobacter spp. in poultry production flocks in The Netherlands.
Epidemiol.Infect.134:126675.
8. van Middelkoop, K. 1994. New concept in poultry housing: the ventilated litter floor. World Poultry
Misset.10:3233.
363
TABLE
Table 1. Samples collected at different moments of the flock lifespan LA. 1: First Litter Aeration. LA. 2:
SecondLitterAeration.LA.3:ThirdLitterAeration.LA.4:FourthLitterAeration.C.D.B.L:Chickdeliverybox
liners.C.D.B.S:Chickdeliveryboxsurfaces.W.D:WaterDispensers.
Dayoldchickflocks BeforeLitterAeration LA.1,2,3and4
Chicks Room Broilers Room Room
Meconia
C.D.B.L
C.D.B.S
Walls
FeedersW.D
Water
Feed
Blinds Walls
FeedersW.D
Feces
Walls
FeedersW.D
Feces
364
ImportanceofcleaninganddisinfectioninSalmonellapersistence
inpoultryfarms
C.MARN
1
,S.GONZLEZ
1
,I.HERNNDEZ
1
,R.ARLEGUI
2
,M.MATEOS
1
andS.VEGA
1
1
InstituteofBiomedicalSciences,VeterinaryFaculty.CEUCardenalHerreraUniversity,
AlfaradelPatriarca,VALENCIA,Spain;
2
Farmbiocontrol
,PoligonoValdeferrn,n7,50600,Ejeadeloscaballeros,ZARAGOZA,Spain
Correspondingauthor:clara.marin@uchceu.es
INTRODUCTION
Severalepidemiologicalstudiesshowedtheimportanceofcleansinganddisinfection(C&D)inthe
persistence of Salmonella in poultry houses (Rose et al., 1999). In France, a total of 69.8% and
38.4% environmental samples were estimated positive for Salmonella before and after C&D,
respectively. Similar results were obtained by Marin et al. (2011) in Spain, with a total of 41,3%
and 20,0% environmental samples positive before and after C&D, respectively. There are many
hypotheses that explain why Salmonella survives in environment after C&D: an inaccurate C&D,
thepresenceofcontaminatedcarriers(CarriqueMasetal.,2009a),aninefficientconcentrationof
disinfectantsused,incorrectwatertemperatureandwaterhardnessused(Gradeletal.,2004)and
theabilitytodevelopbiofilmbythebacteria(Rameshetal.,2002).Inthiscontext,theaimsofthis
studywere:(i)Toevaluateinvitrothreedisinfectantsbasedonmostcommondisinfectantactive
ingredients used in poultry production (glutaraldehyde, formaldehyde and organic acid with
oxydizing) against Salmonella with or without biofilm development capacity. (ii) To analyze these
disinfectants against Salmonella in an experimental poultry house (3 disinfectants x 5 strains x 2
biofilmornonbiofilmdevelopment).
MATERIALANDMETHODS
The strains used in this experiment (S. Enteritidis, S. Typhimurium, S. Hadar, S. Virchow and S.
Infantis)wereisolatedfrompoultryhouses(Marinetal.,2011).Toevaluatebiofilmdevelopment
a screening method based on the fluorescence of Salmonella colonies on calcofluor agar plates
was used (Solano et al., 2002). Then, each serovar with the capacity to develop or nondevelop
biofilm was tested against glutaraldehyde, formaldehyde and organic acid with oxydizing. The
bacterial survival was measured with LIVE/DEAD (Invitrogen) kit. The proper disinfectant
concentration was applied against each serotype in an experimental poultry house (Centro de
InvestigacinAnimal,IVIA,Segorbe,Castelln).Afterdisinfectionprocedure,allsamplescollected
wereanalizedaccordingwithISO6579:2002(AnnexD).
RESULTS
Formaldehyde and Glutaraldehyde with a concentration of 2% (vol/vol) showed nonviable
Salmonella with independence of the serotype and the capacity to develop or not biofilm.
Moreover, organic acid with oxydizing agents with a concentration higher than 0.5% (vol/vol)
showed nonviable bacteria. Most effective disinfectant concentrations were applied in field
conditions against S. Enteritidis, S. Typhimurium, S. Hadar, S. Virchow and S. Infantis with the
capacity or not to develop biolfilm. In environmental conditions, biofilm formation decrease
disinfectant efficiency. However, no differences between biofilm and nonbiofilm development
capacity of the strains were shown. Thus, all strains were removed with independence of the
disinfectantused(glutaraldehyde,formaldehydeandorganicacidwithoxydizingagents).
DISCUSSION
The control of Salmonella at farm level, is one of the most important aims in poultry production
(Mueller et al., 2009). The results obtained in this study showed that if cleaning and disinfection
365
proceduresarecarryoutproperly,Salmonellacouldberemovedfrompoultryhouses(Daviesand
Breslin,2003).
Cleaningprocedureisessentialtoincreasetheeffectivenessandreducetheconcentrationofthe
disinfectant used; however houses are not normally cleaned before disinfection (Marin et al.,
2011). In addition, other factors such as disinfectant concentration, the temperature, the
presenceoftracesoforganicmatter,timeofapplicationandwaterhardnesscanalsodecreasethe
effectivenessofthedisinfectant(HuneauSalanetal.,2010).
Referring to disinfectants tested in this study, different authors (Wales et al. 2006; CarriqueMas
et al. 2009b) demonstrated that formaldehyde based products are more effective in eliminating
Salmonella than others. Otherwise, formaldehyde products are more expensive and have
carcinogenic properties. Other authors stress the inefficacy of organic acids to eliminate
Salmonella, because in presence of organic matter is rapidly inactivated (Ramesh et al., 2002;
Gradel et al., 2004). On the other hand, bacteria that develop biofilm are 1000 times more
resistant against disinfectant used than those that do not produce biofilm (Moretro et al., 2009).
However, the results obtained in the study demonstrate that applying the disinfectant to the
appropriate concentration is achieved complete inactivation of bacteria, regardless of the active
ingredient used (glutaraldehyde, formaldehyde or organic acids and oxidizing agents), and the
abilitytodevelopbiofilmbythebacteria.
REFERENCES
1. CarriqueMas,J.J.,Breslin,M.,Snow,L.,McLaren,I.,Sayers,A.R.andDavies,R.H.2009a.Persistenceand
clearanceofdifferentSalmonellaserovarsinbuildingshousinglayinghens.EpidemiolInfect,137,837846.
2. CarriqueMas,J.,Marin,C.,Breslin,M.,McLaren,I.andDavies,R.H.2009b.Acomparisonoftheefficacy
of cleaning and disinfection methods in eliminating Salmonella spp. from commercial egg laying houses.
AvianPathol,38,419424.
3. Davies, R.H. and Breslin, M. 2003. Observations on Salmonella contamination of commercial laying
farmsbeforeandaftercleaninganddisinfection.Vet.Rec,152:283287.
4. Gradel, K.O., Chr. Jorgensen J., Andersen J.S., and Corry J.E. L. 2004. Monitoring the efficacy of steam
andformaldehydetreatmentofnaturallySalmonellainfectedlayerhouses.J.Appl.Microbiol,96,613622
5. HuneauSalan, A., Michel, V., Balaine, L., Petetin, I., Eono, F., Ecobichon, F. and Bouquin, S.Le. 2010.
Evaluation of common cleaning and disinfection programmes in battery cage and onfloor layer houses in
France.BritishPoultryScience,51,204212.
6. ISO6579:2002(AnnexD)(2002)Microbiologyoffoodandanimalfeedingstuffs.Horizontalmethodfor
thedetectionofSalmonellaspp.InternationalOrganizationforStandardization,Geneve,Switzerland.
7. Mueller,D., Sayers,A.R., CarriqueMas,J.J.andDavies,R.H.2009.Comparisonofsampling methods to
detectSalmonellainfectionofturkeyflocks.J.Appl.Microbiol,107,635645.
8. Ramesh, N., Joseph, S.W., Carr, L.E., Douglass, L.W. and Wheaton, F.W. 2002. Evaluation of chemical
disinfectants for the elimination of Salmonella biofilms from poultry transport containers. Poultry Sci, 81,
904910.
9. Rose, N., Beaudeau, F., Drouin, P., Toux, J.Y., Rose, V. and Colin, P. 1999. Risk factors for Salmonella
enterica subsp. enterica contamination in French broilerchicken flocks at the end of the raring period.
Prev.Vet.Med,39,265277.
10. Solano,C.,Garcia,B.,ValleJ.,Berasain,C.,Ghigo,J.M.,Gamazo,C.andLasa,I.2002.Geneticanalysisof
SalmonellaEnteritidis.Biofilmformation:Criticalroleofcellulose.Mol.Microbiol,43,793808.
11. Marin, C., S. Bailach, S. Vega, and M. Lainez. 2011. Sources of Salmonella contamination during broiler
productioninEasternSpain.Prev.Vet.Med98:3945.
12. Moretro, T., Vestby, L.K., Nesse, L.L., Hannevik, S., Kotlarz, K and Langsrud, S. 2009. Evaluation of
efficiencyofdisinfectantsagainstSalmonellafromthefeedindustry.JApplMicrobiol,106,10051012.
13. Wales, A., Breslin, M. and Davies, R. 2006. Assessment of cleaning and disinfection in Salmonella
contaminated poultry layer houses using qualitative and semiquantitative culture techniques. Vet
Microbiol,116,283293.
366
ThefirstbivalentSalmonellalivevaccineforchickenandducks
DanielWINDHORST,IlkaSCHRDERandFranciscoMONSALVE
LohmannAnimalHealthGmbH,HeinzLohmannStrasse4,27472CUXHAVEN,Germany
INTRODUCTION
Foodborne infections with Salmonella (S.) serovars Enteritidis and Typhimurium are a serious
publichealthconcern.AftermorethanadecadeofcombatingSalmonellainfections,thisorganism
still represents an important cause of human disease (EFSA, 2009, Newell et al., 2010). Recent
studiesestimate80.3millionannualcasesoffoodbornediseaserelatedtoSalmonellaworldwide
(Majowicz et al., 2010). The consumption of poultry meat and eggs, which represent a major
sourceofanimalproteinofhighnutritivevalueformuchoftheglobalpopulation,isbelievedtobe
themaincauseforSalmonellainfectionsinhumans.Eventodaycontaminatedeggsfrominfected
layersremainthemajorsourceofSalmonellaEnteriditisinfection(Delmasetal.,2006;EFSA,2007;
Korsgaard et al., 2009; Stevens et al., 2009). In a joint effort, the poultry meat and egg industry
and the authorities have made significant progress in reducing the contamination rate of poultry
flocks and products over the past years in Europe. The economic necessities that are connected
with the poultry slaughter process make it much more practical to control Salmonella on the
poultry farm than trying to do that in the slaughterhouse. In many countries, including the EU, a
treatment of table eggs and poultry meat is not allowed, making Salmonella control on the farm
the important intervention point. Vaccination plays an important role in the overall biosecurity
systemonchickenfarmstopreventSalmonellainfections(Temellietal.,2010).Whenvaccination
first arose as a method of combating this organism, inactivated vaccines were developed by
various companies. Due to many reasons, such as ease of application, animal welfare, and
especially efficacy, attenuated live vaccines entered the market with great success a short time
later.
Livevaccinesproducebetterprotectionthankilledvaccines.Killedvaccineshavebeentestedwith
varyingresultsandonlystimulateantibodyproduction(Barrow,1996;Chatfieldetal.,1993).They
can present only those antigens that were induced under the conditions of the fermentation
process (Barrow and Wallis, 2000). Their protective efficacy is additionally restricted by their low
immunogenicityinunprimedhostsandthefactthattheydonotinducecytotoxicTcells(Nagaraja
and Rajashekara, 1999). Furthermore killed vaccines do not elicit secretory IgA responses, which
play an important role in protecting mucosal surfaces (Barrow and Wallis, 2000). On the other
hand, live vaccines reduce the colonisation of the intestine more efficiently. They stimulate a
prevailing Th1 rather than a Th2 response. The Th1 response is assumed to be important for the
eliminationofthebacteriafromthegutorthetissues.Forthisreason,livevaccinesshouldalways
be used in Salmonella control, either alone or in combination with inactivated vaccines. Live
attenuated vaccines derived from S. Enteritidis and S. Typhimurium are widely used as
homologous vaccines against either Salmonella Enteritidis or Salmonella Typhimurium and their
efficacy,easeofuseandexcellentsafetyunderfieldconditionshasbeenproven.
Recently, a new bivalent live vaccine consisting of live attenuated S. Enteritidis and S.
Typhimurium strains obtained marketing authorization in Europe (AviPro Salmonella Duo). Now
for the first time a bivalent Salmonella live vaccine for use in chicken and ducks which provides
homologous protection against S. Enteritidis and S. Typhimurium and protection against the
monophasic S. Typhimurium variant is registered. The active immunization with AviPro
SalmonellaDuoeffectivelyreducesthefecalsheddingandthecolonizationofinternalorganswith
S.EnteritidisandS.TyphimuriumfieldstrainsandtheS.Enteritidiscontaminationofeggs.Inducks
the active immunization with AviPro Salmonella Duo effectively reduces the colonization of
internalorganswithS.Typhimurium.Soonthisnovelbivalentvaccinewillbeofficiallylicensedto
protect breeders and fattening turkeys against Salmonella Enteritidis and Salmonella
367
Typhimurium. The combined vaccine is produced in an innovative cofermentation process. In
vitro studies provide evidence that there are no inhibitory effects of the two vaccine strains. Co
fermentationcouldsuccessfullybeupscaledtoprovideaproductthatissafe,efficaciousandeasy
to use. Equal titers for both vaccine strains were achieved throughout all steps of the
manufacturing process. The presence of two different vaccine strains in the cofermented live
vaccinerequirestheapplicationofadapteddiagnosticprocedurestoclearlyidentifybothstrainsin
parallel and to distinguish them from Salmonella field strains. In conclusion, a third generation
Salmonella vaccine is available with AviPro Salmonella Duo as the first bivalent Salmonella live
vaccineprovidinghomologousprotectionagainstthemainSalmonellaserovarspresentinchicken
eggs and meat: S. Enteritidis or S. Typhimurium, as well as against the emerging monophasic
SalmonellaTyphimurium.Thevaccineisregisteredforchickensandducksandtheregistrationisin
preparationforturkeys.
REFERENCES:
1. Barrow, P.A.. (1996): Immunity to Salmonella and other bacteria, pp. 243263. In: Davidson, T.F., T.R.
Morris, and L.N. Payne (eds.), Proceedings of the Poultry Science Symposium Series, vol. 24, Poultry
immunology,CarfaxPublishingCompany,Oxfordshire,England.
2. Barrow, P.A., and T.S. Wallis. (2000): Vaccination against Salmonella infections in food animals:
rationale, theoretical basis and practical application, pp. 323339. In: Wray, A., and Wray, C. (eds.),
Salmonellaindomesticanimals,CABInternational,Oxford,England.
3. Chatfield, S.N., M. Roberts, P. Londono, I. Cropley, G. Douce, and G. Dougan. (1993): The development
oforalvaccinesbasedonliveattenuatedSalmonellastrains.FEMSImmunol.Med.Microb.7:18.
4. Delmas, G., A. Gallay, E. Espi, S. Haeghebaert, N. Pihier, F. X. Weill, H. De Valk, V. Vaillant, and J. C.
Dsenclos. (2006): Foodborne disease outbreaks in France between 1996 and 2005. B. E. H. 51/52:418
422.
5. EFSA (2007): Reportofthetaskforceonzoonosesdatacollectionontheanalysisofthebaselinestudy
ontheprevalenceofSalmonellainholdingsoflayingflocksofGallusgallus.EFSAJ.97:184.
6. EFSA (2009): The Community Summary Report on trends and sources of zoonoses and zoonotic agents
intheEuropeanUnionin2007.EFSAJ.223:1320.
7. Korsgaard H., M. Madsen, N. C. Feld, J. Mygind, and T. Hald. (2009): The effects, costs and benefits of
SalmonellacontrolintheDanishtableeggsector.Epidemiol.Infect.137:828836.
8. Majowicz, S. E., J. Musto, E. Scallan, F. J. Angulo, M. Kirk, S. J. O`Brien, T. F. Jones, A. Fazil, and R. M.
Hoekstra.(2010):The globalburdenof nontyphoidalSalmonella gastroenteritidis.Clin.Infect.Dis.50:882
889.
9. Nagaraja, K.V., and G. Rajashekara. (1999): Vaccination against Salmonella enterica serovar Enteritidis
infection:dilemmaandrealities,pp.397404.In:Saeed,A.M.(ed.),SalmonellaentericaserovarEnteritidis
inhumansandanimals,IowaStateUniversityPress,Ames,USA
10. Newell, D. G., M. Koopmans, L. Verhoef, E. Duizer, A. AidaraKane, H. Sprong, M. Opsteegh, M.
Langelaar, J. Threfall, F. Scheutz, J. V. van der Giessen, and H. Kruse. (2010): Foodborne diseases The
challenges of 20 years ago still persist while new ones continue to emerge. Int. J. Food Microbiol. 139
(Suppl.1):315.
11. Stevens M. P., T. J. Humphrey, and D. J. Maskell (2009): Molecular insights into farm animal and
zoonoticSalmonellainfections.Philos.Trans.R.Soc.Lond.B.Biol.Sci.364:27092723.
12. Temelli,S.,S.Kahya,A.Eyigor,andK.T.Carli(2010):IncidenceofSalmonellaEnteritidisinchickenlayer
flocks in Turkey: Results by realtime polymerase chain reaction and International Organization for
Standardizationculturemethods.Poult.Sci.89:14061410.
368
TestingoftheimmunogenicityoftheSalmonellaEnteritidislivevaccine
SALMOVACSEagainstnonPT4SalmonellaEnteritidisstrains
andSalmonellaentericasubsp.enterica4,[5],12:i:
SvenSPRINGER,ThomasLINDNERandHansJoachimSELBITZ
IDTBiologikaGmbH,AnimalHealth,ResearchandDevelopment,P.O.Box400214,
D06855DESSAUROSSLAU,Germany
INTRODUCTION
Salmonella Enteritidis (SE) live vaccines based on attenuated SE Phagetype (PT) 4 strains have
been proven successful for many years in controlling SE infections in chicken flocks. The live
vaccine Salmovac SE (IDT Biologika GmbH), also registered under the name Gallivac SE (Merial),
was also licensed for the control of Salmonella Typhimurium (STM) infections. Nevertheless, in
recent years chicken and humans have been infected more frequently by SE nonPT 4 strains.
Fortunately multidrug resistant monophasic STM strains appear only sporadically in chicken
flocks. These strains have often been isolated from pigs. These multidrug resistant strains have
been involved in some large diffuse outbreaks in humans, which are creating an increasing need
for hospitalization. Therefore, the aim of our study was to test the immunogenicity of the above
mentionedlivevaccineagainstselectedSEnonPT4strainsandamonophasicSTMstrain.
MATERIALANDMETHODS
Forthestudydayoldchicks(WL,SPFstatus),8to16pergroup,wereorallyvaccinatedwith1x10
8
CFUoftheSElivevaccineSalmovacSE(GallivacSE).Correspondingnonvaccinatedcontrolgroups
werekeptunderisolatedconditions(Table1).Avaccinatedgroupandacontrolgroupwereeach
challengedorallywith1x10
9
CFUofvirulentnalidixidacidresistantSEstrains(PT1,8and21)or
anampicillin(A),streptomycin(S),sulfamerazine(Su)andoxytetracycline(T)resistantS.enterica
4,[5],12:i: (DT 193) strain (group 7/8). The challenge strain content in liver and caeca was
quantitativelyexamined(log10cfu)ondesoxycholatecitrateagar(LEIFSON)withnalidixicacidor
ASSuT on day 7 (SE) and 7 and 14 (STM) after being challenged. The differences between the
vaccinatedandcontrolgroupsweretestedusingtheWilcoxonMannWhitneyTest.
RESULTS
The results of the challenge strain colonization in liver and caeca are shown in Table 2. In
comparisonwiththecontrolgroupsthevaccinatedgroupsshowedasignificantlowercolonization
(p<0.05)ofthechallengestraininliverandcaeca.
CONCLUSION
ThelivevaccinesSalmovacSEandGallivacSEarelicencedforthecontrolofSEandSTMinfections
in chickens. The proof of the immunogenicity and duration of immunity after challenge with SE
PT4andSTMDT104wasprovidedbySpringeretal.(2011).Theresultspresentedherealsorefer
toanefficacyagainstnonPT4SEstrains.Atthesametimeacrossprotectionagainstamultidrug
resistantmonophasicSTMstrainisshown.
REFERENCES
Springer S, Lindner T, Ahrens M, Woitow G, Prandini F, Selbitz HJ (2011): Duration of immunity
induced in chickens by an attenuated live Salmonella enteritidis vaccine and an inactivated
Salmonella enteritidis/typhimurium vaccine. Berl Mnch Tierrztl Wochenschr 124, 8993
(http://vetline.de/openaccesschickenlivesalmonellavaccinesalmonellazoonosischicken.htm)
369
Table1:Allocationofthegroupsanddatesofthetreatments
Group n Vaccination Challengestrain(Day)
Quantitative
examination
1 8 Day2/Day15
2 8 Notvaccinated(control) SEPT8(Day42) Day49
3 8 Day2/Day15
5 8 Day2/Day15
7 16 Day1
8 16 Notvaccinated(control) STMmonophasic(Day17) Day24/31
Table2:Resultsofthequantitativedeterminationofthechallengestraincontentinliverandcaecaafter
challenge
Dayafter
Challengestraincontent(meanSD)
inCFU/g
Group Challengestrain challenge Liver Caeca
1(vaccinated) 7 1.200.77
a
6.050.42
a
2(control) SEPT8 7 2.850.12 6.960.50

a
5.410.71
a
4(control) SEPT21 7 1.480.66 6.260.63

a
5.420.84
a
6(control) SEPT1 7 2.340.45 6.190.52

a
6.780.28
a
8(control) STM 7 2.060.26 7.270.54

7(vaccinated) monophasic 14 0.120.35
a
5.510.76
a
8(control) 14 0.750.46 6.110.31

a
MannWhitneytestp<0.05
370
ChangeofinterventionsinSwedishdairyherdsunderrestrictions
duetoinfectionwithSalmonellaDublinfarmersviews
EstelleGREN
1
,MySAHLMAN
2
andMariaLUNDH
3
1
NationalVeterinaryInstitute,Departmentofdiseasecontrol,UPPSALA,Sweden;
2
TheFederationofSwedishfarmers,STOCKHOLM,Sweden;
3
SwedishBoardofAgriculture,DepartmentofAnimalWelfareandHealth,
JNKPING,Sweden
INTRODUCTION
In Sweden, all cattle herds detected with Salmonella are put under restrictions until two whole
herd samplings fail to detect Salmonella by culture. While the herd is under restrictions, the
farmer is required to implement hygienic measures aiming at eradicating Salmonella from the
herd. These measures are subsidised by the government. During the last two decades, the
requiredcleaningofstableshasbecomeincreasinglycostly.Thisfocusonthoroughstablecleaning
ratherthanmanagementmeasuresisquestionableinherdswithS.Dublininfection.Therefore,a
modifiedprogrammewithfocusonmanagementandhygienicroutinesisunderdevelopment.The
Swedishanimalwelfarelegislationstatesthatstablesmustbethoroughlycleanedatleastoncea
yearandthislevelwasconsideredsufficientinthemodifiedprogrammeforS.Dublin.
MATERIALANDMETHODS
The suggested 45 measures of the modified programme were presented to seven dairy farmers
andtwobeeffarmers.Theywereaskedtogradehowdifficulttheythoughtthesemeasureswould
be to perform on their farm. A five degree scale was used, where a one was impossible to
perform and a five easy to perform. This was followed up by a group discussion. All
participatingfarmershadpreviouslyhadtheirherdsunderrestrictionsduetoSalmonella.
RESULTS
Three farmers performed the grading and all nine farmers participated in the group discussion.
Ten measures were given a 4 or 5 by all farmers that performed the grading. For nine of the
measuresthegradingspannedfrom14or25.Eightofthemeasureswerecommentedduringthe
group discussion, as possible to perform only if they are subsidised. Examples of such measures
are separate vehicles for handling feed and manure and single boxes for calving. Some routines,
such as cleaning of stables and addition of new straw to deep straw beds was considered
impossibletoachieveonadailybasesbyoneofthefarmers.
DISCUSSION
Onlythreefarmersperformedthegrading,howeverthefollowupgroupdiscussion,whereallnine
farmersparticipated,wasinagreementwiththeresultsfromthegradingfarmers.
WithinherderadicationofSalmonellaDublinisperformedbyimplementingdifferentroutinesand
hygienic measures. This enquiry suggests that the majority of such measures were considered
reasonably easy to perform. For nine of the suggested measures the grading answers differed
greatly between farmers. One likely cause for this is builtin problems, for example on a farm
with a common calving area, achieving calving in single boxes will require reconstructions.
Therefore,theeffortneededtoimplementahygienicprogramwillvarygreatlybetweenfarms.
The results also suggest that a number of the measures are costly. Swedish dairy farmers are
presentlyundergreateconomicpressure.AlthoughS.Dublinhasbeenshowntocausesubstantial
lossofincomeindairyherds(1),itisquestionableifthisknowledgeisenoughtomotivateSwedish
371
farmers to implement the more costly measures in the suggested modified programme, unless
subsidisedbythegovernment.
REFERENCES
1. DahlNielsen,Torben.ConsequencesofSalmonellaDublinonhealthandeconomyinDainishdairycattle
herds.PhDthesis2012,UniversityofCopenhagen.
372
RiskfactorsforSalmonellacontaminationinchickencarcasses
attheslaughterhouse
SaraGONZLEZ
1
,SofaINGRESA
1
,ArantxaVILLAGR
2
,SantiagoVEGA
1
andClaraMARN
1
1
InstituteofBiomedicalSciences,DepartmentofAnimalProductionandHealth,PublicHealth
andVeterinaryScienceandFoodTechnology,VeterinaryFaculty,
CEUCardenalHerreraUniversity,AlfaradelPatriarca,VALENCIA,Spain;
2
AnimalResearchandTechnologyCentreCITAIVIA,Segorbe,CASTELLN,Spain;
Correspondingauthor:sara.gonzalez2@uch.ceu.com
INTRODUCTION
The stringent application of good hygiene practices throughout poultry farms in order to control
the animal infection from Salmonella, has led to a decrease of the prevalence of bacterium.
Consequently, slaughterhouse practices have become an important risk factor for the
contaminationofthecarcasses,asapriorstagetodistributiontotheconsumer(Rasschaertetal.,
2007). The difficulty of controlling Salmonella in the processing line is due to the bacterium
inevitably finds its way to chicken meat surface when carcasses are contaminated with intestinal
contents during processing (Guerin et al., 2010). Added to that, limitations in the design of
processing equipment, the difficulty of washing the abdominal cavity effectively, retentions of
waterbyskinwhichtendstoentrapthebacteriuminthecrevicesandfeatherfolliclesandcross
contamination of carcasses during process are other factors to be considered in the
microbiological quality of the final product (Rosenquist et al., 2006). In this context, the aims of
this study were (i) to determinate the presence of Salmonella in broiler carcasses and the
slaughterhouse facilities, and (ii) to evaluate which are the most risk processing stages in
Salmonellacontaminationofthecarcasses.
MATERIALANDMETHODS
This work was carried out in a commercial broiler poultry processing plant. Over 7 months
(September2011tomarch2012)atotalof7visitswereachievedattheprocessingplant.Ateach
visit,inordertodeterminetheinfluenceoftheslaughterorder,thefirstandlastflockprocessed
in the workday were sampled. When the slaughter truck arrived at the slaughterhouse, two
pooled faeces sampleswerecollected per flock (first and last flock of the workday) to determine
the Salmonella status of the flock. Then, during the processing, 3 carcasses were taken from 3
select points of the processing line. Select points were 1) after bleeding (with feathers), 2) after
defeathering,3)afterchillingandpackaging.Moreover,surfacesampleswerecollectedfromthe
environment in each step. All samples collected were analyzed according to ISO 6570:2002 for
detectionofSalmonella.
RESULTS
During this study a total of 191 samples were collected in different points of the processing lne.
Surfaces samples showed diferente positive percentages of Salmonella depending on the step of
processing, being the defeathering and packaging steps those most contaminated (p<0,05).
Environmentalsamplescollectedattheendoftheprocessingdayarealwaysmorecontaminated
with Salmonella than those collected at the beginning. Finally, 36 skin samples and internal
surfaceswereanalyzedforSalmonellacontaminationandskinsamplesweremorecontaminated
(P=0.03).
373
DISCUSSION
An innacurate cleaning and disinfection procedures in poultry farms and slaughterhouses are an
important way to Salmonella survival in poultry production (EFSA 2010c). The contamination
produced in the slaughterhouse occurs mainly by crosscontamination between batches positive
and negative (Rasschaert et al., 2007) and contaminated surfaces (Heyndrickx et al., 2002;
Rasschaert et al., 2008; Marin and Lainez, 2009). Data from this study reveal thatthe prevalence
of Salmonella in the facilities of slaughterhouse is greater to end of workday, due to cross
contamination with positive flocks (EFSA, 2010b). According with Rasschaert et al. (2007) are the
phasesofscalding,defeatheringandclassificationwhichhaveahigherincidenceofthebacteria.
Severalstudiesrevealedhigherprevalenceinneckskinthaninternalsurfacesbecauseskinismore
exposedtoslaughtersurfacesthanotherpartsofthecarcass(Bayleyetal.,2001).
In conclusion, many efforts from poultry sector are being to control Salmonella at farm level to
avoidcrosscontaminationwhenchickensarrivedtotheslaughterhouse(MarinandLainez,2009).
Therefore, in this final stage of the processing work practices have to be extremely careful,
especially,cleaninganddisinfectingbetweenflocksinthoseareaswherebacteriacanquartered.
REFERENCES
Bayley,J.S.,SternN.J.,FedorkaCray,P.,Craven,S.E.,Cox,N.A.,Cosby,D.E.,Ladely,S.,Musgrove,M.T.
2001. Sources and Movement of Salmonella through Integrated Poultry Operations: A Multistate
EpidemiologicalInvestigation.J.FoodProt,64:16901697.
EFSA (European Food Safety Authority). 2010b. Analysis of the baseline survey on the prevalence of
Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU,
2008.PartA:CampylobacterandSalmonellaprevalenceestimates.TheEFSAJournal8:1503.
EFSA (European Food Safety Authority). 2010c. Analysis of the baseline survey on the prevalence of
Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU,
2008. Part B: Analysis of factors associated with Campylobacter colonisation of broiler batches and with
Campylobacter contamination of broiler carcasses; and investigation of the culture method diagnostic
characteristicsusedtoanalysebroilercarcasssamples.TheEFSAJournal8:1602.
Guerin, MT., Sir, C., Sargeant, JM., Waddell, L., O'Connor, AM., Wills, RW., Bailey, RH., Byrd, JA. 2010.
Houselevel risk factoris associated with the colonization of broiler flocks with Campylobacter spp. In
Iceland,20012004.PoultSci.,89:107084.
Heyndrickx, M., Vandekerchove, D., Herman, L., Rollier, I., Gruspeerdt, K., De Zutter, L. 2002. Routes for
Salmonella contamination of poultry meat: epidemiological study from hatchery to slaughterhouse.
Epidemiol.Infect,129:253265.
MarnC.andLainezM.2009.Salmonelladetectioninfecesduringbroilerrearingandafterlivetransportto
theslaughterhouse.Poultrysci.88:19992005.
Rasschaert,G.,Houf,K.,DeZutter,L.2006.Impactoftheslaughterlinecontaminationonthepresenceof
Salmonellaonbroilercarcasses.J.App.Microbiol.13645072.
Rasschaert,G.,Houf,K.,DeZutter.2007.L.Impactoftheslaughterlinecontaminationonthepresenceof
Salmonellaonbroilercarcasses.J.Appl.Microbiol.103:333341.
Rasschaert, G., Houf, K., Goddard, C., Wiildemauwe, C., PastuszczakFrak, M., De Zutter, L. 2008.
ContaminationofCarcasseswithSalmonelladuringPoultrySlaughter.J.FoodProt.71:146152.
Rosenquist, H., Sommer, H., Nielsen, N., Christensen, B. 2006. The effect of slaughter operations on the
contamination of chicken carcasses with thermotolerant Campylobacter. Int. J. Food Microbiol. 108:226
232.
374
ThelogisticslaughterexperienceinaSpanishslaughterhouse.
Lessonslearnedandimplications
HectorARGUELLO,AlexJARAMILLO,SaraCOSTILLAS,PedroRUBIOandAnaCARVAJAL
InfectiousDiseasesandEpidemiologyUnit,DepartmentofAnimalHealth,
FacultyofVeterinaryScience,UniversityofLen,LEN,Spain
INTRODUCTION
The commission decision EC, 2160/2003 set the basis for the implementation of surveillance and
controlprogrammesforreducingSalmonellaprevalenceinpigsandporkintheEU.Arecentstudy,
conductedtoimprovetheknowledgeaboutharvestsituationinSpain,evidencedhighprevalence
of Salmonella in the slaughterhouse environment and activities as well as on carcasses [1]. The
separation of pigs at slaughtering considering their Salmonella burden, would overcome, at least
partially, the crosscontamination by decreasing the inputs of Salmonella by infected pigs
meanwhilenoninfectedanimalsarebeingslaughtered.Theobjectiveofthisstudywastoorganise
the slaughter by serological farmclassification, and to judge its impact on Salmonella carcass
contaminationattheendoftheslaughterline
MATERIALANDMETHODS
Studydesign:
Batches were slaughtered on three consecutive days, during the first half of the slaughtering,
according to their serological status and samples from the lairage area, several points of the
slaughter line and carcasses at the end of the slaughter line (before chilling) were collected
through each day. The study was conducted throughout three consecutive days Monday (low
seroprevalence farms), Tuesday (moderatehigh seroprevalence farms) and Wednesday (low
seroprevalencefarms).
Samplecollectionandprocessing:
a) Selectedherdswerebledattheendofthefattening(40pigs/herd)andclassifiedaccordingto
thecurrentDanishsurveillance[2].
b) At the slaughterhouse part of the study, the surface of holding pens (before and after pigs
staying),fivepointoftheslaughterline(personnelandtheirtools)and75carcassesattheendof
the line were analysed each sampling day. Samples were processed according to the protocol
describedby[1]bytheAnnexDofISO6579/2002.SerawereanalysedwithIdexxHerdchekSwine
Salmonellakitaccordingtomanufacturersinstructions.
RESULTS
a) Resultsbyslaughterday:TheresultsbyslaughterdayaresummarisedinTable1.Therisktoget
apositivecarcasswasthreetimeshigheroncarcassessampledonDayIIIcomparedtotheother
twodays(
2
=11.57,p<0.001;RR=3.22,95%CI1.636.25).
b) Results by batch: Eleven farms were grouped in Level 1, 11 in Level 2 and one in Level 3.
Salmonellawasisolatedjustonthreeofthe70(4.3%)carcassesanalysedinLevel1batches,while
fromthe167carcassesfromLevels2and3batches,28(16.8%)yieldedapositiveresult.Positive
carcasseswerefourtimeslesslikelytobecontaminatedinbatchesclassifiedinLevel1thaninthe
sumofLevel2andLevel3batches(
2
=5.70,p<0.016;RR=3.91,95%CI1.2312.45).
DISCUSSION
In the present study, results by day of study did not reach the expected effect on carcass
contamination.OnDayIII,therisktohaveapositivecarcasswasthreetimeshigherthantheother
two days and surprisingly the lowest carcass prevalence was detected the day in which
moderate/high seroprevalence batches were slaughtered. A number of studies have reported
375
agreement between serology and carcass results [3]. This unexpected carcass contamination in
seronegative herds was linked to lairage and slaughter line personnel. Salmonella could be
recovered from the floor surface of the eight holding pens where pigs were kept before their
harvest. In another study which took into account herd serological status [4], positive carcasses
from low seroprevalence herds were also linked to holding pens, and lairage was stated as the
mostimportantsourceofcontaminationforcategory1herds.Swanenburgetal.[5]alsoreported
carcass contamination in carcasses from low seroprevalence herds, slaughtered at the beginning
ofthedayintheirlogisticslaughterstudy.
Anotherapproach,analyzingresultsbybatchlevels,wasusedtoexamineresultslookingfornew
insights. Farms serological classification is used by surveillance programmes to categorise pig
herds into different risk levels. By the selected classification system, to find a positive carcass
was four times more likely in levels 2 and 3 batches than in level 1. Considering that using this
classification only one batch was classified in Level 3, the effect on carcass contamination was
mainlyascribedtolevel2herds.Similartoourstudy,Dugganandcolleagues[4]alsoreportedthe
highest carriage of Salmonella in gastrointestinal contents and lymph nodes from category 2
herds.Bothresultshighlighttheriskofthosefarmsclassifiedasmoderatecontaminated.Theaim
of this study was to set up logistic slaughter according to the herd Salmonella status. The
effectiveness of this control strategy was measured by the evaluation of carcass contamination.
Consistent with Danish experts, logistic slaughter can reduce the prevalence but not remove
completelythecontaminationofthecarcassbySalmonella[6].Intheuniquestudyreportedinthe
literature,Swanenburgandcolleagues[5]concludedthatseparateslaughteringofSalmonellafree
herds from infected herds could be useful to decrease Salmonella prevalence on pork. In this
study, they also remarked the role of transport, lairage, house resident flora and cross
contamination from strains of infected herds via slaughter equipment. The experience reported
here, exhibits that to be effective, logistic slaughter should be performed with accurate batch
separation by seroprevalence levels, together with measures such as proper cleaning of lairage
andslaughterhousefacilities
REFERENCES
1. Arguelloetal.,2012.PrevalenceandserovarsofSalmonellaentericaonpigcarcasses,slaughteredpigs
andtheenvironmentoffourSpanishslaughterhouses.FoodRes.Int.45:905912.
2. Alban et al., 2002. The new classification system for slaughterpig herds in the Danish Salmonella
surveillanceandcontrolprogram.PrevVetMed.53:133146.
3. Sorensen et al., 2004. The correlation between Salmonella serology and isolation of Salmonella in
Danishpigsatslaughter.VetMicrobiol.101:131141.
4. Dugganetal.,2010.TrackingtheSalmonellastatusofpigsandporkfromlairagethroughtheslaughter
processintheRepublicofIreland.JFoodProt.73:21482160.
5. Swanenburgetal.2001.Salmonellainslaughterpigs:theeffectoflogisticslaughterproceduresofpigs
ontheprevalenceofSalmonellainpork.Int.J.FoodMicrobiol.70:231242.
6. Goldbach, S.G., and L. Alban. 2006. A costbenefit analysis of Salmonellacontrol strategies in Danish
porkproduction.Prev.Vet.Med.77:114.
TABLESANDFIGURES
Table1.Prevalenceresultsbydayofanalysisinsamplescollectedatlairage,carcassandtheslaughterline
Point of sampling
Day I Day II Day III
Lairage
(No. Holding pens)
8 6 5
Before pigs entry 6 (75% 0 3 (60%)
After pigs stay 2 (25%) 1 (16.7%) 2 (20%)
Carcass
(No. carcass sampled)
80 79 78
Pre-chilling 7 (8.8%) 5 (63.2%) 19 (24.4%)
Slaughter Line
(No. samples collected)
30 30 30
8 (26.7%) 2 (6.7%) 3 (10%)
376
InterandintraserovarvariationininvitropathogenicityofSalmonellaspp.
AngelinaKUIJPERS
1
,OuraniaBRELLA
2
,MariskavanROSMALEN
1
,LucasWIJNANDS
1
,
KirstenMOOIJMAN
1
andEelcoFRANZ
1
1
andEnvironmentalMicrobiology,BILTHOVEN,TheNetherlands;
2
LaboratoryforFoodMicrobiologyWageningenUniversity,WAGENINGEN,TheNetherlands
INTRODUCTION
Food may contain numerous bacteria, harmless, beneficial or pathogenic. Health risks due to a
combinationoffoodproductandapathogenicbacterialspeciescanbeassessedwithaMicrobial
Risk Assessment (MRA). An important part of a risk assessment is hazard characterization, also
referred to as Dose Response (DR) relation. The DRrelation describes the relation between the
doseofingestedpathogensandtheprobabilityofinfection.Currently,DRdataareobtainedfrom
epidemiologicaldata,animalexperimentsorhumanvolunteersstudies.Besidesthelimitationsof
each of these methods, DR relations usually refer to one serotype/serovar or even one specific
strainthuslackingdataonstrainvariationwithinandbetweenserovars/serotypes.
MATERIALANDMETHODS
To assess intra and interserovar variability investigations were undertaken with five Salmonella
serovars. Human and animal strains of Salmonella Typhimurium DT104 (10 each), human and
animal strains of monophasic S. Typhimurium (10 each), and animal strains of S. Derby and S.
Rissen (10 each) were compared using a simulated system for gastrointestinal (GI) passage
including the interaction with differentiated Caco2 cells, mimicking human small intestinal
epithelialcells.ThesurvivalofSalmonellathroughthesimulatedGIpassageandthefinallevelsof
attachmentandinvasiontoCaco2cellsweredetermined.Theprobabilityofonecellresultingin
invasionwascalculated,whichwasusedtoconstructexponentialdoseresponsecurvesandwhich
wasconsideredasameasureforvirulence.
RESULTS
Figure1presentsthedosedependingprobabilityofinfectionoftheSalmonellaserovars.Thedose
expresses the probability of one cell to survive and cause infection after passage of the
gastrointestinal(GI)system.
TheresultsshowthatS.Rissenexhibitedonaveragethelowestvirulencefollowedbymonophasic
S. Typhimurium (animal and human), S. Derby (animal) and S. Typhimurium DT104 (human and
animal). S. Typhimurium DT104 isolates from animals showed the highest virulence. Animal
isolates of monophasic S. Typhimurium were more virulent compared to human isolates of the
sameserovar.
CONCLUSION
ThevirulenceofhumanmonophasicSalmonellaTyphimuriumstrainswascomparabletothatofS.
TyphimuriumDT104,thusendorsingtheopinionoftheEFSApanelonBiologicalHazards
1
onthe
virulenceofmonophasicS.Typhimurium.
377
REFERENCE
1. EFSA Panel on biological Hazards (BIOHAZ); Scientific Opinion on monitoring and assessment of the
publichealthriskofSalmonellaTyphimuriumlikestrains.EFSAJournal2010;8(10):1826.[48pp.]
Availableonline:www.efsa.europa.eu/efsajournal.htm
FIGURE
Figure1.ComparisonoftheprobabilityofinfectionfromtheSalmonellaspecies.
378
RevisedEstimatesoftheBurdenofFoodborneIllnessinCanada
M.KateTHOMAS
1
,ReganMURRAY
1
,LoganFLOCKHART
1
,KatarinaPINTAR
2
,FrankPOLLARI
1
,
AamirFAZIL
2
,AndreaNESBITt
1
andBarbaraMARSHALL
1
Email:Regan.Murray@phacaspc.gc.ca;
1
CentreforFoodborne,EnvironmentalandZoonoticInfectiousDiseases,
PublicHealthAgencyofCanada,GUELPH,ONTARIOCanada;
2
LaboratoryforFoodborneZoonoses,PublicHealthAgencyofCanada,
GUELPH,ONTARIOCanada
INTRODUCTION
Foodborne illness estimates help set food safety priorities and create public health policies. In
2008,thePublicHealthAgencyofCanadaestimatedthat11millionepisodesoffoodborneillness
occureachyearinCanada.Althoughthebestestimateatthetime,itwasdeterminedusingolder
methods and data. The Public Health Agency of Canada recently completed revised estimates of
foodborneillnessforCanada.
PURPOSE
There were two overall objectives: (1) calculate a more accurate estimate of domestically,
acquired foodborne illness in Canada using current data and more robust methods and (2)
identifyknowledgegapsforfurtherresearch.
METHODS
Estimates for 30 known pathogens and unspecified agents using data from Canadian surveillance
systems (for years 20002010), relevant international literature and the 2006 Canadian census
population were calculated. The analysis accounted for underascertainment as public health
surveillance systems are subject to underreporting and underdiagnosis. Estimates on the
proportion foodborne and the proportion travelrelated were incorporated for each pathogen.
Monte Carlo simulations were performed to account for uncertainty using @Risk software
generatingmeanestimatesand90%credibleintervals.
RESULTS
Thereareanestimated4.0millionepisodesofdomesticallyacquired,foodborneillnesseachyear
in Canada (1.6 million episodes from 30 known pathogens and 2.4 million episodes from
unspecified agents). The top four pathogens are (1) norovirus, (2) Clostridium perfringens, (3)
Campylobacterspp.and(4)nontyphoidalSalmonellaspp.
CONCLUSIONS
The revised estimates cannot be compared for trends with the 2008 estimate because different
methods were used. Although lower, the revised estimates are more accurate than the 2008
estimatebecausetheyusecurrentdataandmorerigorousmethods.
Policymakers,industry,academiaandotherorganizationscanusetherevisedestimatestobetter
inform policy, research, food safety risk assessments, education campaigns and other prevention
andcontrolactivitiesultimatelyimprovingthehealthofCanadians.
379
SourceAttributionofSalmonellaentericainIreland
usingtheMicrobialSubtypingmethod
1
NiallDELAPPE,
2
MontserratGUTIERREZ,(jointcoauthors),
1
JeanOCONNOR,
3
SarahJACKSONand
1
MartinCORMICAN
1
NationalSalmonella,Shigella&ListeriaReferenceLaboratory,MedicalMicrobiologydept.,
UniversityHospitalGalway,Ireland;
2
CentralVeterinaryResearchLaboratory,BackwestonComplex,KILDARE,Ireland;
3
HealthProtectionSurveillanceCentre,2527MiddleGardinerStreet,DUBLIN,Ireland
INTRODUCTION
The consumption of contaminated food from food animals is the primary cause of salmonellosis.
The reduction of Salmonella in various farm animals has been the target of public health
interventionsinIreland,asinothercountries.Sourceattributionisausefultoolindeterminingthe
relative contribution of particular food animals to human infections and evaluating efficiency of
targetedcontrolmeasures.
MATERIALANDMETHODS
From 200209 3529 nonenteric Salmonella isolates from humans were referred to the National
Salmonella,Shigella&ListeriaReferenceLaboratory(NSSLRL)fortyping.Inthesameperiod2426
isolatesfromfoodanimals(poultry=1332,swine=844,bovine
=250)weretypedintheCentralVeterinaryResearchLaboratory(CVRL).Themicrobialsubtyping
method using the European Food Safety Authority (EFSA) Source Attribution Modelling software
was used. Data was divided into 4year blocks, i.e. 200205 and 200609 and the subtype
properties were serotype and phage type. Isolates from patients with recent foreign travel were
removedandjustoneisolateperpointsourceoutbreakwasincluded.
RESULTS
Twentyfive percent of human isolates had a history of recent foreign travel. S. Enteritidis (n =
1229) was the most common serotype and 36.8% of these were associated with foreign travel
while S. Typhimurium (n = 891) was more commonly associated with domestically acquired
infection (14% foreign travel). Poultry and pork accounted for the majority of domestically
acquiredinfectionswhilethenumberofcasesassociatedwithbovinesourceswaslow.
DISCUSSION
This is a first attempt at applying the EFSA SAM to Salmonella attribution in Ireland. Work is
ongoing on subdividing poultry into broilers, layers, ducks and turkey, discriminating between
domesticallyproducedandimportedfoodandalsoaddingotherfoodsources.Preliminaryanalysis
showsthecontributionofovineandfishsourcestohumaninfectionsislow.
CONCLUSION
Use of the EFSASAM software should provide a useful tool in monitoring the causes of human
salmonellosisinIrelandandhelpinevaluatinghoweffectivemeasurestakentoreduceSalmonella
infoodanimalsimpactonhumanhealth.Thepublicationofthesourceattributionsoftwareonthe
EFSAwebsiteisusefulforstandardisationandmayallowcomparisonofdatabetweencountries.
380
ACKNOWLEDGEMENTS
IwishtothankTineHaldinDTUforhelpwiththesoftware,laboratoriesinIrelandthatreferred
isolatestotheNSSLRLandCVRLaswellasourcolleaguesinpublichealthforprovidingforeign
traveldata.
REFERENCES
1. Callow,B.R.(1959).AnewphagetypingschemeforSalmonellaTyphimurium.J.Hyg(Lond).57,346359.
2. Hald,T.,andLund,J.(2012).DevelopmentofauserfriendlyinterfaceversionoftheSalmonellasource
attributionmodel.
3. Hald, T., Vose, D., Wegener, H., and Koupeev, T. (2004) A Bayesian Approach to Quantify the
ContributionofAnimalFoodSourcestoHumanSalmonellosis.RiskAnalysis,Vol.24,No.1.
381
May 27-28-29, 2013
Saint-Malo France
Session 7
Chairpersons:
Bob TAUXE (USA) and Nathalie JOURDAN DA SILVA (France)
382
Changes and Challenges: Salmonellosis in the 21st Century
Robert V. Tauxe, MD, MPH
Deputy Director, Division of Foodborne, Waterborne, and Environmental Infections
National Center for Zoonotic and Emerging Infections
Centers for Disease Control and Prevention
Atlanta, Georgia
Abstract
Salmonella infections are an important challenge to public health, whose epidemiology remains incompletely
understood. Although important progress has been made in controlling the global pandemic of Salmonella
Enteritidis in egg flocks, the diversity of serotypes, reservoirs and pathways for infection means that one single
method alone is not sufficient to control Salmonella.
The incidence, sources, and success in controlling Salmonella vary widely from country to country. Reported
incidence depends on the health care system, the nature of surveillance, and the actual local epidemiological
situation. Eggs and poultry are the predominant sources, particularly in countries where serotype Enteritidis
predominates. Outbreaks and models for attributing the fraction of salmonellosis to different sources indicate
the importance of other foods and reservoirs as well. Pre-harvest interventions on-farm have been
successful at reducing infections from broiler breeders and layer flocks, with results are visible in human
disease surveillance.
In the US, where salmonellosis has not decreased over 15 years, Enteritidis has decreased from to only 19%
of reported Salmonella infections, while other serotypes have increased. Plant-derived foods (e.g. tomatoes,
cantaloupes, and sprouts) now account for an estimated 44% of salmonellosis. Salmonella may not be passive
on plants, but can internalize and persist, colonizing the edible parts, where they may reach the next
herbivore. Is a two host herbivore-plant cycle another way that Salmonella can transmit beyond the simple
fecal oral route, like transovarian transmission?
Detecting dispersed outbreaks helps define gaps in foods safety systems. Salmonella serotyping, supported
by other subtyping systems has been the key to detecting such outbreaks. In North America, the PulseNet
system has dramatically increased the identification of dispersed multistate and multinational outbreaks. Like
reportable disease surveillance, subtype-based surveillance depends on bacterial isolation to confirm cases,
and to determine subtypes. New diagnostic technologies that are culture-independent isolate threaten current
systems of surveillance, outbreak detection and control. Meeting that challenge means developing new
diagnostic strategies that provide critical data to public health and to the clinician. Such a diagnostic tool offers
hope for systematic realtime global subtyping.
Salmonella is likely to remain with us for years to come, exploiting a range of reservoirs and pathways, and
making surprising appearances in our food supply.
383
Salmonella infections are an important challenge to public health, whose epidemiology remains
incompletely understood. Although important progress has been made in controlling the global
pandemic of Salmonella Enteritidis in egg flocks, the diversity of serotypes, reservoirs and
pathways for infection means that one single method alone is not sufficient to control Salmonella.
National incidence varies
Salmonellosis is largely reported from the developed world, where it is likely to be diagnosed.
Reported incidence of diagnosed salmonellosis varies, depending on the nature of the health care
system of the country, and the reporting requirements, as well as on the actual incidence of
infection. In the United States, where most health care is private, the incidence of culture-
confirmed infections reported to the active surveillance system FoodNet is 16.4/100,000
population in 2012
1
. In the United Kingdom, where most health care is paid for publicly, the
incidence is 13/100,000
2
. For the European Union, it was 21/100,000 in 2010
3
.
The actual number of infections that occur is greater than the number that are diagnosed and
reported, because some ill people never consult a clinic, or are not cultured. The number of
infections occurring can be estimated in several ways. The pyramidal method uses estimates of
the likelihood of each step, from illness to consultation to culture to isolation to reporting. Using
this method, CDC has estimated that 29 Salmonella illnesses occur for every one reported, or
approximately 475/100,000
4
. Another method is to culture a cohort of the population whenever
they develop diarrhea. In this way it has been estimated that in England and Wales, the actual
number of infections was 4.7 times the nationally reported rate, or 60/100,000
5
(p112) . A third
method, developed in Denmark and the Netherlands, uses a serosurvey to include even
inapparent infections. This method suggested that in 1999 the true rate of immunizing exposures
in Denmark was 21%, 570 times higher than the reported rate of infection
6
. Applying this method
to 8 European countries, the annual seroconversion rate varied from 6 to 547 per 1000, and did
not correlate with reported infections
7
. The ratio of serologically-determined infections to
reported infections varied from 100 2000. These efforts show how difficult it is to make useful
comparisons using reported incidences.
Attributing infections to sources
Determining food and other sources of Salmonella infection is important to target control
measures at those sources, and to evaluate whether they are effective. One source of data for
this source attribution comes from a series of reported foodborne outbreaks where specific foods
were implicated
8
. It is important to note that this represents the foods that were actually
consumed, rather than the ultimate reservoirs of infection. A food is often a mlange of different
ingredients, and so an outbreak investigation sometimes may implicate one food commodity or
ingredient (a so-called simple food), or investigation may implicate a compound of multiple
commodities or ingredients (a so-called complex food). Either can be used for attribution. For
example, in the European Community, a simple outbreak attribution is regularly reported by
EFSA. In 2010, EFSA summarized the distribution of food vehicle types for 341 foodborne
outbreaks of salmonellosis: eggs accounted for 44%, bakery items for 14%, buffet items for 12%,
and broiler, pork and beef for 5% apiece
9
(p334). Some bakery items and buffet items may be
complex, and some contain eggs, but that contribution is not easily captured in this analysis.
CDC recently published an analysis of 4,589 foodborne outbreaks reported in the US from 1998
2008, that systematically allocated the illness due to complex foods across the spectrum of food
types
10
. This analysis included data from 877 outbreaks due to Salmonella, nearly half of which
were due to complex foods. This analysis also used the number of illnesses in each outbreak, so
that the estimate could be applied to the previous estimate of the overall number of infections. In
384
this analysis the top five sources for salmonellosis were: Vegetables growing on vines or stalks
20.7%, poultry 19%, eggs 14.8%, fruits and nuts, 13%; and beef, 7.3% (online table 4)
A different approach to source attribution depends on subtyping libraries of bacterial strains from
humans, foods and animal reservoirs, and comparing the distribution of subtypes to estimate the
contribution of each reservoir. This approach, first developed in Denmark, is used there to
generate the annual Salmonella account of attribution to specific food animal species
11, 12
. This
method is strongest when the libraries represent an array of different food sources, when the
subtyping is robust, and when it can be easily repeated. The statistical approaches to source
attribution continue to evolve, and more advanced estimates blending several data sources are
likely to be helpful.
Trends over time
Our purpose ultimately is to understand the sources well enough to reduce the likelihood of
transmission from them. Following time trends in the surveillance can detect new or growing
problems, and can document the effectiveness of the control measures that are instituted. In the
1980s and early 1990s, many countries experienced dramatic increases in salmonellosis as a
global pandemic of SE swept around the world, possibly through layer breeder flock
contamination, and SE became the dominant serotype in many countries
13, 14
. More recently,
some countries have documented profound decreases in the incidence of salmonellosis. In the
United Kingdom, a sharp decline in salmonellosis began in 1998, as a voluntary Salmonella control
program was instituted by the egg laying and boiler industry, that included systematic
vaccination
15
. Bolstered by promotion that encouraged consumers to look for those eggs in the
market, the incidence of salmonellosis dropped by 80%. In Denmark, a comprehensive control
program focused on screening broiler flocks, and swine, and egg laying flocks for Salmonella, and
taking control measures at the pre-harvest level has led to a reduction from the peak of 90
infections /100,000 to 20/100,000 in 2011 many of which are travel related
12, 16
. In the EU as a
whole, though Enteritidis remains the most frequently reported serotype, the collective incidence
of salmonellosis has dropped from 31/100/000 in 2007, the year that new control measures were
applied to breeding flocks across the Union, to 21/100,000 in 2010, a decrease of 32%
3, 9
.
In the United States, there has been no change in the reported incidence of human salmonellosis
since active surveillance began in sentinel sites in 1996
1
. This summary reporting masks progress
made through the enactment in 1996 of HACCP based food safety systems for red meat and
poultry and extensive voluntary efforts by the egg industry to control SE which appear to be
having some effect
17, 18
. In fact, the incidence of both Enteritidis and Typhimurium infections has
declined substantially since the 1990s. In 2009, these two serotypes, which are relatively common
in food animal reservoirs, together accounted for only 37% of serotyped Salmonella, (compared to
50% in 1995) while other serotypes, particularly I 4,5,12:i:-, Newport and Javiana have increased in
frequency. Nonetheless, Enteritidis remains the most frequently reported serotype, and remains
in egg-laying and broiler flocks, as well as other food sources.
As noted above, much salmonellosis is directly attributable to animal reservoirs, particularly to
poultry and eggs. In the US, while much regulatory and industry attention has focused on
reducing the contamination of whole poultry carcasses at slaughter, the public is increasingly
consuming poultry in the form of parts or ground products. Continued focus on reducing
contamination at slaughter, as well as pre-slaughter measures on the ranch, farm and lairage pen
may help make further progress.
Antimicrobial resistance in Salmonella is complex, and also varies widely from one region of the
world to the other. The public health impact of resistance to drugs that are important in human
385
medicine has been well documented, and include greater rates of hospitalization and death
19
. The
selective pressure of antimicrobials used in agriculture have contributed to the resistance in non-
typhoid salmonellosis
20
. It is critical to consider the critical needs of human medicine in selecting
antibiotics for prudent use in agriculture, to limit those uses to those that treat illness in animals,
and to monitor the trends in incidence and antimicrobial use over time, so that policies can be
adjusted.
In the developing world, resistance is emerging in association with nosocomial salmonellosis
reminiscent of the outbreaks more common in the developed world in the 1950s, and some may
be more related to selective pressure in medical settings than to agriculture. Multiresistant
Typhimurium and Enteritidis strains have caused an increase in community-acquired bacteremic
infections in East Africa with case fatality ratios above 20%
21, 22
.
Emerging sources of salmonellosis
Outbreak investigations have heralded emerging and unsuspected sources of salmonellosis. Just as
incidence varies depending on the nature of the healthcare system, so the types of outbreaks
detected also depend on the capacity of the public health surveillance systems in place. In 1996,
the US launched PulseNet, a national molecular subtyping network that targets Salmonella, STEC,
and Listeria monocytogenes, based on uniform strain subtyping in all 50 states and the food
regulatory agencies using pulsed field gel electrophoresis. While the method is simple, the
network is powerful. PulseNet increased the capacity to detect outbreaks by approximately an
order of magnitude, and has been particularly helpful in detecting dispersed clusters
23
. Whether
they are small and localized within one state, or large multi-state outbreaks, most of these
outbreaks would simply be missed without PulseNet. Investigating these dispersed outbreaks has
been particularly important in identifying unsuspected and unusual hazards. Since 2006, outbreak
investigations have identified at least nine food vehicles of salmonellosis that had not been noted
in the United States before, each of which presents an opportunity to improve our food safety
system*. These dispersed outbreaks are often due to foods that are contaminated at low levels,
and are distributed to many states.
Many of the recent outbreaks are due to produce items: fruits, vegetables, nuts and seeds. Fresh
produce has accounted for an increasing proportion of all foodborne outbreaks and outbreak
associated cases over the last several decades in the United States, as tracked by outbreaks
24
, and
in the most recent source attribution, produce accounted for 44% of salmonellosis
10
. Some of this
is imported produce, and some is domestically grown in the US. In the summer of 2012, a large
outbreak of serotypes Typhimurium and Newport infections linked to whole cantaloupes from
one US farm led to 261 illnesses, 94 hospitalizations and 3 deaths
25
. Contamination in the field
and uncleanable equipment in the packing shed contributed to the outbreak. Cantaloupes and
tomatoes also have been recurrent vehicles
26, 27
. Seed sprouts continue to be a challenge, partly
controlled by voluntary seed treatment with up to 20,000 ppm chlorine to inactivate the
pathogens in the seeds
28
. As a result of these produce related outbreaks, the USFDA has recently
proposed regulations to reduce contamination of produce during production and packing
29
.
Peanut butter has been the vehicle of three large outbreaks, each related to poor in-plant
practices. As a result of these and other outbreaks due to processed low moisture foods, the
USFDA has proposed regulations requiring process controls in a variety of food processing
venues
30
.
Recent observations on Salmonella in the plants we eat indicate that the bacteria may not just be
passive contaminants, but exhibit active behavior, and can internalize in plants and suppress their
immune reactions
31
. For example, Salmonella on the surface of a leaf are attracted to the
products of respiration coming from open stomata, and enter them
32
. On the flower of a tomato
plant, Salmonella placed with pollen on a stamen travel with the pollen tube to the ovule, and can
386
subsequently be recovered inside the tomato that forms, and Salmonella placed on the leaf of a
tomato plant can subsequently be detected in the tomato itself
33, 34
. It is possible that the
frequent observation of outbreaks in association with plant-derived foods may reflect
contamination that is arising from a two-host life cycle that allows some types of Salmonella to
pass from one herbivore to another via the plants they eat
35
.
Salmonella can have complex transmission pathways that go beyond the intestinal tract, as
illustrated by the adaptation of serotypes like Enteritidisto transovarian vertical transmission in
hens, and perhaps by cycles that involve colonization and invasion of plants. Recent work on
cattle suggests that peripheral lymph nodes may be an important source of Salmonella for ground
beef suggesting a transdermal source of infection
36
, such as biting flies. There may well be more
yet to be determined.
Salmonella can be transmitted without involving foods at all. Outbreaks of waterborne
salmonellosis can occur when the integrity of a drinking water system is breached. Novel pets, pet
foods and urban agriculture have been an increasing challenge. In the US, the growing popularity
of small backyard chicken flocks means that chicks from small breeders can be ordered over the
internet, and introduced into homes in many cities. In 2012, 3 outbreaks were related to at least 3
chicken breeders where hatchery hygiene was wanting, and leding to 264 culture confirmed
illnesses, 94 hospitalizations, and 3 deaths, that,
25
. There were also eight outbreaks with 347
cases and 55 hospitalizations of several serotypes linked to small turtles sold illegally in the US
(where they are banned by the USFDA since 1975 because of the risk of salmonellosis), and one
outbreak of Typhimurium infections with 30 cases, 4 hospitalizations and a death linked to pygmy
hedgehogs
25
. A large three year outbreak of Typhimurium infections with 376 cases, and 56
hospitalizations was traced to one breeder of dwarf aquatic frogs; the outbreak was controlled by
changing conditions in the breeding tanks
37
. A recurrent problem with serotypes I 4,5,12:i:-
/Typhimurium in frozen feeder rodents from one source that snake fanciers use as food for their
pets made approximately 500 ill between 2009 and 2012 in both the UK and the US
25, 38, 39
.
The globalization of the food supply, including many perishable foods, means that some outbreaks
are multinational and even global in scope. Indeed, of the 9 new food vehicles identified since
2006, 6 were imported foods. A recent investigation linked an outbreak of 425 Bareilly infections
linked to raw tuna used in spicy tuna sushi rolls
25
. The tuna had been scraped from the bones of
tuna at a facility in South India, frozen, and shipped to the US. Genuinely global outbreaks have
been detected on several occasions, affecting persons on multiple continents
40
. This is likely to be
an extremely underreported event, as detecting such outbreaks depends on rapid international
exchange of subtype information before outbreaks can be detected, for which there is no formal
mechanism. The fact that it happens at all is due to the universal use of serotyping. We have
trained public health microbiologists more than 80 countries in PulseNet methodology; when this
or some other universal method is applied routinely and the results shared, many more global
outbreaks are likely to be detected.
The Challenge of Culture Independent Diagnostic Testing (CIDT)
A new trend is of deep concern. New diagnostic platforms are being implemented now that could
obviate the need to isolate bacteria pathogens in clinical diagnostic laboratory medicine, replacing
culture with tests that detect DNA or antigens from the pathogens of interest. Lack of isolates
threatens public health surveillance based on laboratory-confirmed cases; outbreak detection
networks based on molecular subtyping of the isolate in public health labs, and regulators ability
to confirm a food has the strain that caused the outbreak
41, 42
. At a collaborative workshop
Culture-Independent Diagnostics Forum: Charting a Path for Public Health hosted by CDC in April
2012, short and long term strategies were proposed. For the short term, strategies are needed
that promote reflex culture in the event that a CIDT is positive for Salmonella, so that a strain is
387
isolated for public health purposes, as well as to determine the antimicrobial resistance. A longer
term approach depends on the ability to sequence part or all of the whole genome, and then to
extract from that sequence the data needed by public health to characterize and subtype the
organism. Such a solution depends on the research and development of many partners. Without
some strategy, surveillance for Salmonella and other enteric bacterial pathogens may lose much
sensitivity in detecting outbreaks and tracking trends..
In the 21st century, better control of Salmonella infection will depend on better understanding of
the pathways it takes to infect humans, so that better, more focused interventions can be devised.
This will require:
* Making the transition from traditional diagnostic and public health microbiology to DNA
sequence based microbiology.
* Giving even greater attention to reducing preharvest contamination of animals and plants, as
well as better sanitation during slaughter.
* Providing better information about the range of foods and other sources of Salmonella, and how
to intervene to prevent contamination of those foods.
* Forming international surveillance and investigation networks, so that global outbreaks can be
detected and controlled.
Footnote page 4: * peanut butter, broccoli powder, dry dog food, frozen pot pies, hot peppers,
white and black pepper, papayas, pine nuts, raw scraped tuna.
The findings and conclusions in this presentation are those of the author and do not necessarily
represent the views of the Centers for Disease Control and Prevention
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May 27-28-29, 2013
Saint-Malo France
Session 7
Chairpersons:
Communications
391
An outbreak of Salmonella Newport associated with mung bean sprouts,
Germany, October/November 2011
Bettina ROSNER
1
, Christophe BAYER
1,2
, Helen BERNARD
1
, Christina FRANK
1
, Rita PRAGER
3
,
Wolfgang RABSCH
3
, Petra HILLER
4
, Burkhard MALORNY
4
, Beatrice PFEFFERKORN
5

and Klaus STARK
1

1
Robert Koch-Institute, BERLIN, Germany;
2
Postgraduate Training for Applied Epidemiology, Berlin, Germany and European
Programme for Intervention Epidemiology Training, STOCKHOLM, Sweden;
3
Robert Koch Institute, National Reference Centre for Salmonella and Other Bacterial
Pathogens, WERNIGERODE, Germany;

4
Federal Institute for Risk Assessment, BERLIN, Germany;
5
Federal Office of Consumer Protection and Food Safety, BERLIN, Germany
ABSTRACT
The number of reported Salmonella Newport infections in Germany increased from an average of two to three cases per
week to 39 in week 44 in October 2011. Mung bean sprouts were suspected as the infection vehicle in this outbreak
because mung bean sprouts contaminated with S. Newport had been detected at a distributor in Germany. In a case-
control study, we compared notified adult S. Newport case-patients with notified S. Enteritidis patients of the same age
group regarding their food consumption, in particular sprouts, in the 3 days before onset of illness. S. Newport isolates
from case-patients and food samples were subtyped. We conducted trace-back investigations for sprouts starting from
locations where case-patients had eaten. Median age of case-patients (n=106) was 38 years (range 0-91 years), 52%
were female. Sprouts showed the strongest association with disease among identified risk food items in univariate
analysis. In multivariable logistic regression analysis only sprout consumption was associated with S. Newport infection.
Molecular subtyping patterns of human isolates and the mung bean sprouts isolate were indistinguishable. Sprouts were
traced back to a sprout producer in the Netherlands. Epidemiological, laboratory and trace-back evidence point to
sprouts as the vehicle of infection. Since sprouts are frequently contaminated with microorganisms, consumption of raw
or insufficiently cooked sprouts may impose a health risk.
INTRODUCTION
Salmonella enterica serovar Newport has been an uncommon cause of acute gastroenteritis in
Germany. In 2010, a total of 25,310 cases of Salmonella infection were notified, of which only 83
(0.3%) were caused by S. Newport [1]. Outbreak-related cases have occurred only rarely in
previous years. S. Newport outbreaks in other European countries and the USA were associated
with the consumption of various food items, for example, ground beef, lettuce, and alfalfa sprouts
[2-5]. In November 2011, an increase of S. Newport isolates was observed at the National
Reference Centre for Salmonella and other bacterial enteric pathogens (NRC) at the Robert Koch
Institute (RKI) and other diagnostic laboratories. Furthermore, routine outbreak algorithms for the
national database of notifiable infectious diseases at the RKI revealed a substantial increase of
notified S. Newport cases from an annual average of 2-3 cases per week (2001-2010) to 39 in week
44 of 2011. Mung bean sprouts were suspected as the vehicle in the outbreak because two lots of
mung bean sprouts contaminated with an unspecified serovar of Salmonella had been found at
sprout producer A in the Netherlands. Furthermore, S. Newport had been detected in a mung
bean sprouts sample taken in October 2011 during routine sampling at a sprout distributor in
northern Germany. These mung bean sprouts originated from one of the contaminated lots
produced in the Netherlands by producer A. Here we describe the outbreak investigation launched
to identify the source of the outbreak, including an analytical epidemiological study,
microbiological analyses, and trace-back investigations.
A case was defined as a laboratory confirmed S. Newport infection in a person with at least one
symptom of an acute gastroenteritis (diarrhoea, abdominal pain, vomiting, fever), and onset of
392
symptoms between 20 October and 08 November 2011. To test the hypothesis that consumption
of mung bean sprouts was associated with illness, we conducted a case-control study. S. Newport
case-patients 18 years of age were compared with a control group of S. Enteritidis case-patients
notifed in October-November 2011 regarding frequency of exposure to suspected risk factors.
Cases and controls were frequency matched by age groups (18-31 years, 32-48 years, 49-88 years).
All case-patients and controls were interviewed using a standardised questionnaire. Questions
referred to the 3 days before disease onset and were focussed on the consumption of sprouts and
dishes frequently containing sprouts. Univariate and multivariable logistic regression analyses
were performed, and exposure-specific odds ratios (OR) and 95% confidence intervals (CI) were
calculated. Isolates from S. Newport case-patients and various food items were characterised by
pulsed-field gel electrophoresis (PFGE) and multiple-locus variable number tandem repeat
analyses (MLVA). Starting from Asian restaurants and other locations where case-patients
reported to have eaten in the 3 days before disease onset, mung bean sprouts were traced back to
the distributors and producers of the sprouts.
RESULTS
In total, the outbreak in Germany comprised 106 cases (Figure 1). Median age was 38 years (range
0-91 years). Fifty-two per cent of case-patients were female. Hospitalisation due to S. Newport
infection was reported for 28% of the cases. No deaths were reported. Cases were notified in 15 of
the 16 German federal states. Fifty cases and 45 controls were included in the case-control study.
Sprouts had been consumed by 14/43 (33%) of S. Newport and 1/45 (2%) of S. Enteritidis cases
and showed the strongest association with disease among identified risk food items in univariate
analysis (OR: 21.2; 95% CI: 2.9-917.9). In multivariable analysis, controlled for age group and
gender, only sprout consumption was associated with S. Newport infection (OR: 18.4; 95% CI: 2.3-
150.1). From the 106 S. Newport case-patients attributed to the outbreak 32 isolates were
available for PFGE analysis. All human isolates showed an identical PFGE pattern. The PFGE pattern
was indistinguishable from the pattern of the mung bean sprout isolate, which originated from the
sample taken in October during routine food sampling at the distributor in northern Germany.
Sprouts served at six Asian restaurants and other locations where case-patients had eaten before
falling ill could be traced back via one or more distributors to producer A in the Netherlands.
DISCUSSION
We describe the largest S. Newport outbreak in Germany reported to date, involving 106 cases.
Combined efforts of epidemiologists, microbiologists and food safety authorities identified
contaminated mung bean sprouts as the source of the outbreak. The case-control study revealed a
strong and significant association between sprout consumption and S. Newport infection. The
epidemic curve showed a distinct peak from 21 October-5 November 2011, which is consistent
with an infection vehicle that was in circulation only for a limited time period due to a short shelf-
life. Self-reported sprout consumption could explain only one third of the cases. This is in line with
other epidemiological outbreak investigations where sprouts have been identified as the vehicle of
infection [5, 6]. Sprouts are often used as garnish or side dishes, or are served mixed with other
food items, which makes consumption difficult to remember by patients. The long time lag
between exposure period and interview may be another reason why the proportion of cases who
recalled sprout consumption was small. Contaminated fresh produce has increasingly been
recognized as an important source of foodborne outbreaks [7]. Shortly after the large STEC
O104:H4 outbreak in Germany caused by fenugreek sprouts [6], sprouts, once again, were the
vehicle of infection in the outbreak described here.
393
CONCLUSIONS
Since sprouts are known to be frequently contaminated with microorganisms, inlcuding
Salmonella, consumer advice clearly stating the health risks associated with sprout consumption
and the safe preparation of sprouts before consumption is essential for prevention of illnesses.
REFERENCES
1. Robert Koch Institute. Infektionsepidemiologisches Jahrbuch meldepflichtiger Krankheiten fr 2010.
Berlin: Robert Koch Institute; 2011.
2. Schneider JL, White PL, Weiss J, Norton D, Lidgard J, Gould LH, et al. Multistate outbreak of multidrug-
resistant Salmonella Newport infections associated with ground beef, October to December 2007. J Food
Prot. 2011; 74(8): 1315-9.
3. Lienemann T, Niskanen T, Guedes S, Siitonen A, Kuusi M, Rimhanen-Finne R. Iceberg lettuce as
suggested source of a nationwide outbreak caused by two Salmonella serotypes, Newport and Reading, in
Finland in 2008. J Food Prot. 2011; 74(6): 1035-40.
4. Irvine WN, Gillespie IA, Smyth FB, Rooney PJ, McClenaghan A, Devine MJ, et al. Investigation of an
outbreak of Salmonella enterica serovar Newport infection. Epidemiol Infect. 2009; 137(10): 1449-56.
5. Van Beneden CA, Keene WE, Strang RA, Werker DH, King AS, Mahon B, et al. Multinational outbreak of
Salmonella enterica serotype Newport infections due to contaminated alfalfa sprouts. JAMA. 1999; 281(2):
158-62.
6. Buchholz U, Bernard H, Werber D, Bhmer MM, Remschmidt C, Wilking H, et al. German outbreak of
Escherichia coli O104:H4 associated with sprouts. N Engl J Med 2011; 365(19): 1763-70.
7. Lynch MF, Tauxe RV, Hedberg CW. The growing burden of foodborne outbreaks due to contaminated
fresh produce: risks and opportunities. Epidemiol Infect. 2009;137(3):307-15.
TABLES AND FIGURES
Figure 1. Notified Salmonella Newport cases in Germany by date of disease onset, 01 October 15
December 2011 (n=138). 106 S. Newport cases were notified with disease onset from 20 October 08
November 2011 (outbreak period).
Figure 2. Pulsed-field gel electrophoresis (PFGE) of XbaI restriction enzyme digested genomic DNA from the
Salmonella Newport outbreak strain. Lanes 1-3: human isolates from case-patients; lane 4: mung bean
394
sprout isolate; lane S: the PulseNet universal standard Salmonella enterica serovar Braenderup H9812
strain.
S 1 2 3 4
395
Changing trends in antimicrobial resistance patterns
in Salmonella enterica serovar Typhimurim DT193
in England and Wales, 1981-2011
Oluwaseun ESAN
Health Protection Agency, 61 Colindale Avenue, NW9 5EQ LONDON UK
ABSTRACT
Over the past 30 years, trends in human salmonellosis in England and Wales have undergone significant changes.
Between 1981 and 1987, 44% of reported isolates were Salmonella enterica subsp. enterica serovar Typhimurium (S.
Tm). Between 1987 and 1997 the epidemic of S. Enteritidis PT 4 overshadowed a national increase in S. Tm DT 104.
The change in prevailing salmonella serovars in humans in UK was also accompanied by dramatic changes in
antimicrobial resistance.
Between 1981 and 1987, 20% (2198/11592) of all resistant indigenous S. Tm cases exhibited resistance to
sulphonamides and tetracyclines (SuT) with S. Tm DT204 accounting for majority of this R-type (60%, 1345/2198). The
dominance between 1992 and 2008 of the S.Tm DT 104 clone led to ampicillin, chloramphenicol, streptomycin,
sulphonamides, and tetracyclines (ACSSuT) becoming the prevalent R-type. Since 2009, the rise in multiresistant clones
of monophasic S. Tm in Europe has led to resistance to ampicillin, streptomycin, sulphonamides and tetracyclines
(ASSuT) becoming the prevalent R-type. In 2011 over 70% (1307/1786) of all indigenously acquired S. Tm
demonstrated resistance to at least one antimicrobial, with 44% resistant to A,S,Su,T.
INTRODUCTION
Salmonella enterica subsp. enterica serovar Typhimurium (S. Tm) has undergone considerable
changes in the number of human isolates with antimicrobial drug resistance and particularly
multiple resistance (to four or more drugs) in the UK and other parts of Europe and North
America. Historically, multiple resistance in S. Tm was commonly associated with the international
spread of a series of multiresistant clones, with S. Tm DT 104 isolates resistant to ampicillin,
cholramphenicol, streptomycin, sulphonamides and tetracyclines (ACSSuT) dominating from the
late 1980s to the early 2000s [9, 10, and 11]. Since the decline of human cases of S. Tm DT 104,
other phage types, in particular DT 193 have increased, with isolates exhibiting resistance to at
least four antimicrobials. S. Typhimurium DT 193 is highly heterogeneous due to several phage
types converting to DT 193 through the acquisition of plasmids, some of which encode genes for
resistance to a range of antimicrobials [4, 7, 9].
METHODS AND MATERIALS
The details of specimen testing, typing and reporting in England and Wales have been reported
elsewhere [1, 2]
The common R-type was selected based on the frequency of reporting over the thirty year period.
The analytical approach adopted was to review the incidence of S. Tm DT 193, and R-type against
the serovar and all salmonella incidence.
RESULTS
In 1981, cases of S. Tm reported to the PHLS accounted for 39% (3946/10040) of all human
salmonellosis, and 42% of indigenously acquired salmonellosis. By 1983, a twofold increase in
indigenously acquired S. Tm infections was recorded (7083 cases), representing the highest
number of human infections with S. Tm.
Following on from this period, reported numbers of indigenous infections fluctuated between 18%
and 43% of Salmonella reports, with S. Tm DT 104 having the greatest impact representing over
60% of all S. Tm human infections at the peak of its incidence in 1995. Reported cases of S. Tm
infections represented a 70% increase in the 1981 levels, but only accounted for 24% of all human
salmonellosis due to the concomitant elevated levels of the epidemic S. Enteritidis PT 4 strain.
396
Subsequently there was a steady decline in the number of S. Tm cases up until 2006, following
which reports have increased as a result of the spread of monophasic S. Tm DT 193. Nevertheless
numbers and overall proportions have remained 50% below levels seen in 1981 (Figure 1).
The changes in dominance of certain phage types of S. Tm have had an impact on circulating
resistant strains. In the first decade of electronic reporting, over 60% of isolates were sensitive to
the panel of antimicrobials tested; by the next decade (1991-2000) there was a reversal in the
proportion of resistant isolates, with 76% of all isolates resistant to at least one of the
antimicrobials used for testing. Resistance to A, C, S, Su and T (ACSSuT) was by far the most
common pattern accounting for 43% (13802/32425) of all R-types. Following on from this period,
ACSSuT and ASSuT became the dominant resistance patterns with 16% and 13% of isolates
reported with these patterns respectively (Table 1).
In 1981, over half (53%, 36/67) of S. Tm DT 193 were resistant to at least one antimicrobial,
representing only 3% (67/1268) of all resistant S. Tm isolates. By 1991, the numbers of drug-resistant
isolates had increased to 94% of DT 193 (1169/1242), and accounted for 39% of all S. Tm resistant
isolates. This increase was a result of the emergence of multiple resistant (ASSuT) strains accounting
for 40% of all resistant isolates with a further 30% resistant to tetracyclines alone. Prior to 1991, a
multiple resistant pattern with resistance to ampicillin, streptomycin, sulphonamides, tetracyclines,
ciprofloxacin and ceftazidime (ASSuTCpCz) emerged in S. Tm DT 193 between 1987 and 1989, where
this pattern represented 55% of all S. Tm DT 193 and 22% of all S. Tm resistant isolates. Other
derivations of ASSuT have also been observed; in particular resistance to A, Su, T and trimethoprim
(Tm) (ASuTTm) emerged in 1995, representing over half of all drug-resistant isolates of DT 193
(386/704). Following the dramatic changes in resistance patterns in 1991 and 1995, all patterns have
been on the decline except ASSuT. This pattern has been on the increase since 2006, with levels
observed in 2011 only 7% lower than the 1991, the year it emerged (Figure 2).
DISCUSSION
The most dramatic change in the past thirty years is the shift from S.Tm DT 104 as the dominating
phagetype to S.Tm DT 193. At the time of S. Tm DT 104 dominance, resistant to ACSSuT was the
most common R-type in circulation, with over half of cases having this pattern at the peak of the
outbreak in 1996. The R-type was also seen in other parts of the world [10, 11, and 12]. Studies
have shown ACSSuT in DT 104 is chromosomally encoded, with plasmid- derived sequences coding
for resistance to these antimicrobials [11]. While S. Tm DT 104 is a clonal strain, S. Tm DT 193 is
heterogenous in nature [4, 9, and 10]. Further, there is evidence for the conversion of both DT 104
and DT 204 to DT 193 following acquisition of plasmids [4, 9]. This may explain the dominance of
ASSuT and T R-types and more recently only ASSuT in S. Tm DT 193. This mechanism to acquire
plasmids may be responsible for the different variations of ASSuT seen in resistant isolates, most
especially the sudden emergence of ASuTTm in 1996. There is evidence to show different clones of
S.T m DT 193 can display different R-types, and that specific R-type patterns can be successfully
used to define and investigate outbreaks. In an outbreak of S. Tm DT 193 linked to cooked ham in
North West England and North Wales in 1991, all outbreak strains were resistant to Su, Tm and
furazolidone (SuTmFu) [14]. This highlights the potential for non-dominant R-types to cause
outbreaks of public health concern.
Where S. Tm DT104 outbreaks were predominantly linked to bovine products, other food sources
such as poultry and porcine meats have also been implicated. In contrast monophasic S. Tm DT
193 of R-type ASSuT is predominantly porcine-related [7, 11].
The European Food Safety Authority (EFSA) baseline studies on prevalence of Salmonella in EU
holdings with breeding pigs show one in three were positive for Salmonella, with S.Tm the second
most frequently isolated serovar [3]. This has necessitated the need for national control
programmes (NCP) for salmonella in pigs [8]. Currently there is a voluntary scheme (Zoonosis
397
Action Plan ZAP) in the UK, which has been running for nine years. Hopefully information from
this scheme will guide the implementation for the overall National Control Plan (NCP), which is
proposed for 2013 [7, 8].
Furthermore, there has been a widespread increase of S. Tm DT193 strains lacking the second
phase flagella [5, 6]. These monophasic strains also exhibit multiple resistance, with ASSuT as the
most common R-type, and are becoming the most frequently reported strains of S. Tm in the UK
ACKNOWLEDGMENTS
The authors wish to acknowledge the input of staff in the HPA Gastrointestinal Bacteria Reference Unit for
identification and typing over the past three decades.
REFERENCES
1. Anderson ES. The phage typing of salmonellae other than S. Typhi. In: van Oye E, editor. The World
Problem of Salmonellosis. Dr W. Junk, The Hague: pp 89-110, 1964.
2. Bale JA, De Pinna E, Threlfall EJ, Ward LR. Salmonella identification: serotypes and antigenic formula.
Kauffmann-White Scheme 2007. Health Protection Agency 2007. ISBN: 978-0-901144-91-1.362.
3. European Food Safety Authority. Analysis of the baseline survey on the prevalence of Salmonella in
holdings with breeding pigs in the EU, 2008 - Part A: Salmonella prevalence estimates. EFSA Journal 2009;
7(12):1377
4. Hampton MD, Threlfall EJ, Frost JA, Ward LR, Rowe B. Salmonella Typhimurium DT 193: differentiation
of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid
virulence (spv) gene probe. J Appl Bacteriol. 1995 Apr; 78(4): 402-8.
5. Hopkins KL, de Pinna E, Wain J. Prevalence of Salmonella enterica serovar 4,[5],12:i:- in England and
Wales, 2010. Euro Surveill. 2012 Sep 13;17(37). pii: 20275
6. Hopkins KL, Kirchner M, Guerra B, Granier SA, Lucarelli C, Porrero MC, Jakubczak A, Threlfall EJ, Mevius
DJ Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new pandemic strain? Euro Surveill.
2010 Jun 3; 15(22): 19580
7. Miller AJ, Twomey DF, Davies RH, Teale CJ, Williamson SM, Reichel R, Featherstone CA, Cook AJ, Snow
LC, Armstrong JD. Salmonella serovars and antimicrobial resistance patterns on a sample of high
seroprevalence pig farms in England and Wales (2003-2008). Zoonoses Public Health. 2011 Dec; 58(8): 549-
59
8. Paiba G, Armstrong D, and Wight A. National control programme for Salmonella in pigs. Veterinary
Record 2011 168: 569
9. Threlfall EJ, Ward LR, Ashley AS, Rowe B. Plasmid-encoded trimethoprim resistance in multiresistant
epidemic Salmonella Typhimurium phage types 204 and 193 in Britain. Br Med J. 1980 May 17; 280(6225):
1210-1.
10. Threfall EJ. Epidemic Salmonella Typhimurium DT 104--a truly international multiresistant clone. J
Antimicrob Chemother. 2000 Jul; 46(1): 7-10.
11. Threfall EJ. Antimicrobial drug resistance in Salmonella: problems and perspectives in food- and water-
borne infections. FEMS Microbiol Rev. 2002 Jun;26(2):141-8.
12. Threfall EJ, Lawson AJ, Walker RA and Ward LR. Salmonella Typhimurium DT 104: rise and fall of a
multiresistant epizoonotic clone. SCIEH Weekly Report. 2001 Jun 5; 35(2001/22): 142-145
13. Thornton L, Gray S, Bingham P, Salmon RL, Hutchinson DN, Rowe B, Newton D, Syed QU. The problems
of tracing a geographically widespread outbreak of salmonellosis from a commonly eaten food: Salmonella
Typhimurium DT193 in north west England and north Wales in 1991. Epidemiol Infect. 1993
Dec;111(3):465-71.
398
TABLES AND FIGURES
Figure 1. Reported Salmonella Typhimurium incidence by common phage types, England and Wales, 1981-
2011
Figure 2. Common resistance patterns of Salmonella Typhimurium DT 193, 1981-2011
0
1000
2000
3000
4000
5000
6000
7000
8000
1
9
8
1
1
9
8
3
1
9
8
5
1
9
8
7
1
9
8
9
1
9
9
1
1
9
9
3
1
9
9
5
1
9
9
7
1
9
9
9
2
0
0
1
2
0
0
3
2
0
0
5
2
0
0
7
2
0
0
9
2
0
1
1
N
u
m
b
e
r

o
f

S
.
T
m

r
e
p
o
r
t
s

Year
S.Typhimurium S.Tm DT193 S.Tm DT104
0
100
200
300
400
500
600
1
9
8
1
1
9
8
3
1
9
8
5
1
9
8
7
1
9
8
9
1
9
9
1
1
9
9
3
1
9
9
5
1
9
9
7
1
9
9
9
2
0
0
1
2
0
0
3
2
0
0
5
2
0
0
7
2
0
0
9
N
u
m
b
e
r

o
f

r
e
s
i
s
t
a
n
t

i
s
o
l
a
t
e
s

Year
ASSuT T
ASSuTCpCz ASuTTm
399
Table 1. Common resistance pattern of all
S. Typhimurium, 1981-2011
1981-1990 (N = 58089) N (%)
T 2714 (5)
SuT 2679 (5)
SuTTm 2230 (4)
SuTm 1994 (3)
Ssu 1875 (3)
Other Patterns 8072 (14)
Fully sensitive 38525 (66)
1991-2000 (N= 42706) N (%)
ACSSuT 13802 (32)
ACSSuTTm 4101 (10)
T 2813 (7)
ASSuT 1847 (4)
SuTTm 1772 (4)
2001-2011 (N=17048) N (%)
ACSSuT 2730 (16)
ASSuT 2285 (13)
T 1292 (8)
ACSSuTTm 1225 (7)
ACSSuTNxCpl 634 (4)
ASuTTm 446 (3)
A-Ampicillin, C- Chloramphenicol, Nx-Nalixidic acid, Cpl reduced susceptibility to Ciprofloxacin S- Streptomycin, Su-
Sulphanamide, T Tetracycline, Tm-Trimethoprim
400
The benefit of Salmonella control in Sweden who benefits and who pays the cost?
Susanna STERNBERG LEWERIN
1
, Helene WAHLSTRM
2
, Sofie IVARSSON
3

and Kristian SUNDSTRM
4

1
Department of Biomedical Sciences and Veterinary Public Health, Swedish University
of Agricultural Sciences, UPPSALA, Sweden;
2
Zoonosis Centre, National Veterinary Institute, UPPSALA, Sweden;
3
Swedish Institute for Communicable Disease Control, SOLNA, Sweden;
4
Institute for Food and Agricultural Economics, Lund University, Sweden
ABSTRACT
Recently an assessment of the costs and the economic benefits of the Swedish salmonella control programme was made.
The results indicated an economic benefit of the current programme. Most of the benefits stem from avoidance of human
salmonellosis. This is a political incentive for investing money in salmonella control in the feed-to-food chain. However, the
farming industry may be less convinced, unless a return of their costs for salmonella control can be seen. The current study
is an attempt to assess who pays the different costs and who benefits in what way from the salmonella control. The cost
and the benefits of the current Swedish salmonella control are unevenly distributed between the farming industry, the food
industry, the government and the taxpayers. At present, there is support for the current control programme among both the
farming industry and the taxpayers. This may stem from an appreciation of the actual economic benefits by both parties but
more likely has other reasons.
BACKGROUND
The national salmonella control programme in Sweden was initiated more than 50 years ago and
covers the entire chain from feed to food (3). All subspecies of Salmonella enterica are included and
any finding of salmonella in animals, food or feed is notifiable according to the Swedish law on
zoonoses (8). Measures to eliminate/eradicate salmonella are taken upon any positive finding.
Restrictions are put on infected holdings until they can be declared free from salmonella.
The costs for on-farm eradication are shared by the government and the farmers. The level of co-
funding varies with animal species and whether the farm is affiliated to a voluntary control
programme. No funding is given for eradication on broiler holdings or beef herds buying animals
from more than five sources. For other food-producing animals, 50% of documented costs are
covered by the government, unless the farm is affiliated to a voluntary control programme, in which
case up to 70% of documented costs may be covered by the government. The cost of HACCP
controls in feed mills is paid by the feed producers. The surveillance in slaughterhouses and cutting
plants is paid for by tax money while the food industry pays for control in food items.
A consortium was recently commissioned to perform cost-benefit analyses of reducing the
salmonella prevalence in pigs, on EU level (4, 5). The results indicate large differences between
countries in terms of the profitability of salmonella control. While it is important to estimate the
benefits and costs of reducing the salmonella prevalence in high-prevalence countries, analysing a
potential change in the current control measures may be more relevant in low-prevalence countries
with a functioning salmonella control already in place.
A general estimation of the costs and the benefits of the Swedish salmonella control was attempted
in 1993 (2) but since then no analysis of the costs and benefits of the programme was performed
until 2011 when the Swedish salmonella committee (an advisory committee with representatives
from authorities and the animal industry) commissioned an evaluation of the costs and benefits of
the control programme as compared to other possible options. The potential increase in reported
domestic human cases in Sweden, in case of cessation of the current control, was estimated by
evaluating two different alternative salmonella control strategies (7). Furthermore, the
underdetection and underreporting of salmonella cases and possible economic effects of
introducing these alternative salmonella control strategies were assessed (6).
401
Estimated costs and benefits of the current programme
The cost for the current control programme in the feed-to-food chain was 11.5 million euro/year
(M). The results of the evaluation indicated an economic benefit of the current programme (as
compared to the alternative control strategies) ranging between 9 and 105 M, depending on the
scenario and cost estimates that were used. Implementing alternative strategies was estimated to
increase the costs for human illness by between 12 and 104 M while the costs for increased disease
due to Salmonella Dublin in cattle ranged between 3.9 and 7.9 M. The estimated benefits from
decreased control costs varied between 3.9 and 7.1 M.
The mean estimated costs and benefits in the 8 different scenarios assessed in the recent studies are
illustrated in figure 1.
Figure 1. Estimated benefits and costs of the Swedish salmonella control programme as compared to 8
different alternative scenarios
Distribution of the costs and benefits
The main stakeholders, bearing the costs and receiving the benefits of the current Swedish
salmonella control, are the farming industry, the food industry, the government and the taxpayers.
Most of the benefits stem from avoidance of human salmonellosis. This is a political incentive for
investing money in salmonella control in the feed-to-food chain. However, the farming industry may
be less convinced, unless a return of their costs for salmonella control can be seen. This paper is an
attempt to assess who pays the different costs and who benefits in what way from the salmonella
control. Based on figures from the previous studies, the following estimates were made.
The total government cost for the current control (including surveillance from-feed-to-food and on-
farm eradication of all detected salmonella infections) amounts to 2.8 M while the cost for the
farming industry amounts to 8.7 M.
Benefits from avoiding human salmonellosis represent up to approximately 100 M (mean 58 M)
saved from taxpayers money, and up to approximately 8 M (mean 5.9 M) for the farming
industrys avoidance of production losses due to S. Dublin. Benefits for the food industry represent
1.1 M in avoided surveillance costs.
If the current control ceased, the government would save about 1.5 M and the farmers about 0.4
M in on-farm eradication costs for cattle and pigs. The cost for surveillance in food items, including
surveillance in slaughterhouses and cutting plants would be reduced by less than 0.1 M. However,
if the current control ceased, this cost would most likely have to be borne by the food industry and
the government would thus add an extra 1.2 M to the benefits from saved surveillance costs.
The industrys costs for feed control and voluntary surveillance in cattle and pigs would be reduced
by 2-5 M, depending on what alternative surveillance would be introduced.
Thus, the taxpayers would save about 2.7 M if the current control programme ceased, but the
increased costs for human illness would far outweigh those benefits.
In contrast, the farmers would save about 0.4 M if the current control ceased but the dairy sector
may eventually suffer much higher costs due to increased S. Dublin infections. Moreover, the food
402
industry would have to pay more for surveillance, according to the microbiological criteria in the EU
legislation. Thus, the benefits of lower control costs are far outweighed by the increased costs for
the food-producers as well.
The distribution of the mean costs and benefits is illustrated in figure 2a and 2b, respectively. The
distribution of the current costs for the programme is shown in figure 3.
Figure 2a. Distribution of benefits from the Swedish salmonella control programme (mean estimates from
comparison with alternative strategies)
Figure 2b. Distribution of costs of the Swedish salmonella control programme (mean estimates from
comparison with alternative strategies)
Figure 3. Distribution of the current costs of the Swedish salmonella control programme (total 11.5million
/year)
DISCUSSION
The perceived added value of the current salmonella control for the consumers is hard to assess. The
Swedish consumers assume that food items on the Swedish market are free from salmonella. There
is a perceived benefit from the production of domestic food of animal origin. This may be due to the
view that Swedish animal production maintains a high standard as regards animal welfare and
antimicrobial usage, as well as salmonella status. A recent study by the Swedish Board of Agriculture
(1) indicates that Swedish consumers pay between 0.2 and 0.4 M more for Swedish meat than they
403
would for the same amount of imported meat. However, the prices paid to the farmers are similar,
or lower, than the EU average.
The Swedish farmers pay a large part of the current control costs. The poultry farmers would be little
affected if the programme were abandoned, due to harmonised EU legislation on salmonella in
poultry. The cattle farmers would be most affected, as an increase in salmonella prevalence (S.
Dublin in particular) would lead to production losses in the dairy sector, and this would not be
outbalanced by reduced control costs.
Salmonella-free feed is a prerequisite for salmonella-free animal production. In the current
programme, costs for the control in feed amount to 4.7 M. This is paid by the feed industry and
added onto feed prices (and thus ultimately paid by the farmers).
There is support for the current control programme among the farming industry and the taxpayers.
This may stem from an appreciation of the actual economic benefits by both parties but more likely
has other reasons.
The Swedish farmers have a long tradition of preventive disease control programmes and their
organisations are trusted to run these programmes for the benefit of the farming industry. The
salmonella programme is run by the government, but the farmers organisations participate to such
an extent that it enjoys a general acceptance among farmers. Moreover, the direct risk of zoonotic
infection from salmonella infected livestock would be highest for people in direct contact with the
animals so the farmers may see an immediate benefit.
The government control costs are paid from the budget of the Ministry of Agriculture while the
expected benefits mainly accrue to the Ministry of Health and Social Affairs. There is a current
political consensus around the Swedish salmonella control but this may be disrupted. The global
financial crisis as well as the intent to place more of the responsibilities for safe products on the
producers themselves may lead to questioning the value of the control programme.
CONCLUSIONS
The cost and the benefits of the current Swedish salmonella control are unevenly distributed
between the farming industry, the food industry, the government and the taxpayers. The main
benefit is avoided human illness, while the farmers bear the majority of the costs. Nevertheless,
there is still a general support for the national control of salmonella and each stakeholder appears to
have their own reasons for this.
REFERENCES
1. Anonymous. Added value for Swedish meat. (in Swedish) SJV report 2008:5 available at
www.jordbruksverket.se
2. Engvall A, Andersson Y, Cerenius F. The economics of the Swedish Salmonella control- A cost-benefit
analysis. Proceedings: International course on Salmonella control in animal production and products , by SVA
and WHO August 1993.
3. European Food Safety Authority, European Centre for Disease Prevention and Control; The European
Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in
2010. Available at www.efsa.europa.eu
4. SANCO. FCC Consortium analysis of the costs and benefits of setting a target for the reduction of
Salmonella in slaughter pigs; 2010. Available at www.ec.europa.eu
5. SANCO. FCC Consortium analysis of the costs and benefits of setting a target for the reduction of
Salmonella in breeding pigs; 2010. Available at www.ec.europa.eu
6. Sundstrm K, Wahlstrm H, Ivarsson S, Sternberg Lewerin S, 2013. Economic effects of introducing
alternative salmonella control strategies in Sweden. Submitted.
7. Wahlstrm H, Sternberg Lewerin S, Sundstrm K, Ivarsson S, 2013. Estimation of the expected change in
domestic human salmonella cases in Sweden in 2010, given a hypothetical relaxation of the current
salmonella control programme. Submitted
8. Zoonoslagen, SFS 2006:1039 available at www.jordbruksverket.se
404
Multistate outbreak of Salmonella Stanley infection
in the European Union, 2011-2012:
investigations from fork to farm
Cline GOSSNER
1
, Sophie BERTRAND
2
, Elisabeth KANITZ
3
, Jaime MARTINEZ-URTAZA
1
,
Pete KINROSS
1
, Johanna TAKKINEN
1
, Marc STRUELENS
1
, Ernesto LIBANA
4
, Frank BOELAERT
4
,
Marta HUGAS
4
, Pia MAKELA
4
, Wesley MATTHEUS
2
, Christina FRANK
5
,
Katalin KRISZTALOVICS
6
, Kirsten MOOIJMAN
7
, Lieke VAN ALPHEN
1,8
, Denis COULOMBIER
1

and Daniela SCHMID
3

1
European Centre for Disease Prevention and Control (ECDC), STOCKHOLM, Sweden;
2
Belgian NRC for Salmonella, Scientific Institute of Public Health (WIV-ISP),
BRUSSELS, Belgium;
3
Austrian Agency for Health and Food Safety (AGES), VIENNA, Austria;
4
European Food Safety Authority (EFSA), PARMA, Italy;
5
Robert Koch Institute (RKI), BERLIN, Germany;
6
National Centre for Epidemiology, BUDAPEST, Hungary;
7
National Institute for Public Health and the Environment (RIVM), EURL-Salmonella,
BILTHOVEN, the Netherlands;
8
Staten Serum Institute (SSI), COPENHAGEN, Danmark
ABSTRACT
In July 2012, EU-wide investigations of a multistate outbreak of Salmonella Stanley infection were initiated. Following the
report from Belgium (29 June 2012) of an outbreak of S. Stanley infection through the Epidemic Intelligence Information
System for Food and Waterborne Diseases, Austria, Czech Republic, Germany, Greece, Hungary, Italy, Slovak
Republic, Sweden and the United-Kingdom reported cases sharing indistinguishable XbaI-PFGE patterns.
Affected Member States gathered available epidemiological and microbiological information with the support of the
European Centre for Disease Prevention and Control, the European Food Safety Authority and the European Reference
Laboratory for Salmonella. Comparative analyses of human, food, animal and environmental isolates fingerprints were
performed. A common hypothesis-generating trawling questionnaire was developed.
Retrospective investigation showed that the outbreak had already been on-going since August 2011. Between 01 August
2011 and 23 January 2013, 679 non-travel related cases were identified. Case interviews did not show any common
exposure suggesting the source of the outbreak. Results of investigations by food and veterinary authorities as well as
results of the PFGE comparison analysis strongly suggested that the turkey production chain was the primary source of
the outbreak. However, contribution of other food and animal sources, such as beef, pork and broiler meat could not be
excluded.
EU-wide trace-back and trace-forward of the turkey production chain was recommended to understand the distribution of
the infected animals and contaminated meat. Ecological studies testing spatial correlation between the cases and the
animal and food distribution would contribute to the epidemiological investigations. This outbreak highlights the
importance of sharing of information at an early stage between the public health and veterinary sectors at national and
international level during outbreak investigation. This is a prerequisite to rapidly investigate events from fork to farm.
INTRODUCTION
Initial alert and launch of the investigations
On 29 June, the National Reference Centre for Salmonella in Belgium reported through the
Epidemic Intelligence Information System for Food and Waterborne Diseases (EPIS-FWD) platform
a significant increase of non travel-related Salmonella Stanley infections (S. Stanley) in 2012 with
26 cases since the beginning of the year and more recently, 10 cases between 11 and 25 June [1,
2]. The baseline observed during previous years was 3 to 6 S. Stanley cases annually on average.
The 20 strains typed showed indistinguishable XbaI-PFGE profile and resistance to nalidixic acid.
Between 2007 and 2011, 2 611 cases of S. Stanley infection were reported through the European
Surveillance System (TESSy). Most of these S. Stanley cases are travel related outside the EU.
The XbaI-PFGE pattern of the outbreak strain was shared through EPIS-FWD and compared with
historical molecular typing data of human S. Stanley strains at the EU level (previously known as
405
PulseNet Europe data, up to 2006) and with national human typing data in the EU Member States.
Preliminary results from the comparison with historical datasets showed a high diversity of S.
Stanley among human isolates in Europe and underlined the absence of this specific pattern in the
available databases.
Respectively on 03 and 11 July, Germany and Hungary replied to the urgent inquiry reporting an
increase in number of non-travel related cases in the first semester of 2012. By the end of July, 64
S. Stanley cases (36 cases in Hungary, 20 cases in Belgium and eight cases in Germany) shared the
outbreak XbaI-PFGE profile. Retrospective investigations in Hungary showed that cases with the
outbreak XbaI-PFGE profile were identified since August 2011.
METHOD
EPIS-FWD is an information exchange platform for public health risk assessment of food and
waterborne events. EPIS-FWD gathers all 27 EU Member states, the three EEA countries (Norway,
Iceland and Lichtenstein) plus Australia, Canada, Japan, New-Zealand, South-Africa, Switzerland,
Turkey and the United States. The objective of the communication platform is to timely inform all
participating countries about an event of potential EU-wide dimension and to facilitate the
coordination of the response to food and waterborne outbreaks. The platform was used
successfully during this outbreak.
In July 2012, ECDC launched an EU-wide investigation of this multistate outbreak of S. Stanley. The
first step was to establish an EU epidemic case definition for S. Stanley infections, to prepare a
data collection for an EU line listing, and to agree with the affected countries on the coordination
of investigation. A confirmed case was defined as a laboratory confirmed case of S. Stanley with
XbaI-PFGE pattern matching the Belgium outbreak strain, with onset of symptoms after August
2011 and without travel history outside the EU in 1-7 days prior the onset of symptoms. A
probable case was defined as a laboratory confirmed case of S. Stanley with no XbaI-PFGE
available and with the onset of symptoms after August 2011 and without travel history outside the
EU in 1-7 days prior the onset of symptoms. Any case of S. Stanley infection with an XbaI-PFGE
profile not matching the outbreak profile was excluded.
Hypothesis generating interviews of the cases were managed independently at the national level
with a locally developed questionnaire or with specific hypothesis generating questionnaire
developed by ECDC that was put available for the countries in EPIS-FWD.
To raise awareness about this event and communicate about the preliminary findings, ECDC
published successively two rapid risk assessments on 27 July and on 29 August [3, 4].
Between 01 August 2011 and 27 August 2012, Austria, Belgium, Czech Republic, Germany and
Hungary reported a cumulative number of 257 cases, 117 of which were PFGE-confirmed. For the
following of the investigation, a Member State was considered as affected when at least one case
was microbiologically confirmed.
To identify the possible sources of this outbreak, multi-sectorial investigations were initiated. Both
the public health and the food and veterinary sectors at national and EU levels were engaged in
the investigations with the objective 1) to pull together data on isolation of S. Stanley in humans,
feed, food, animal and environment to perform a comparative analysis of these isolates, and 2)
eventually to identify an epidemiological link between the cases and the distribution of any
suspected vehicle.
In coordination with EFSA, the European Union Reference Laboratory (EU-RL) for Salmonella and
DG SANCO, ECDC set up a database for PFGE profiles of human, food, feed, animal and
environmental isolates of S. Stanley. Based on a recommended data collection protocol, S. Stanley
PFGE typing data from humans, as well as from animal, food, feed and environmental sources
were collated at ECDC for comparison of baseline genetic diversity and distribution of the
406
outbreak strain over time, country and potential food sources before and during the outbreak
period.
In order to reach countries that do not have access to the EPIS-FWD platform, the International
Food Safety Authorities Network (INFOSAN) of the World Health Organisation issued an alert on
20 July 2012 asking for any information that would contribute to the investigation.
In addition, the Rapid Alert System for Food and Feed (RASFF) platform of DG SANCO was daily
scanned to learn about any S. Stanley notifications in food and feed.
RESULTS
From fork
As of 23 January 2013, Austria, Belgium, Czech Republic, Germany, Greece, Hungary, Italy, Slovak
Republic, Sweden and the United-Kingdom reported cases sharing indistinguishable XbaI-PFGE
patterns. The cumulative number of probable and confirmed human cases was 684 cases (figure
1).
Figure 1: Distribution of cases of non-travel-related Salmonella Stanley infections (probable and confirmed
cases) by Member States and month of report, August 2011 January 2013, as of 23 January. (n=684)
Considering the high diversity of S. Stanley among human historical isolates, the finding of one
single XbaI profile in isolates from different countries suggested a multinational outbreak related
to a persisting source of infection or to multiple sources in the EU that were contaminated with a
single clone of S. Stanley.
Several local outbreaks were investigated by the local and national health authorities. In October
2011, the Austrian health authorities investigated an outbreak of 32 S. Stanley cases that ate at a
turkey kebab stand in the region of Carinthia. While there were no leftover of turkey meat, a sauce
sample and a dishcloth from the stand tested positive for S. Stanley. PFGE analysis of the human
strains and the food and environmental strains were indistinguishable with the outbreak strain. In
August 2012, the Austrian health authorities investigated another local outbreak of S. Stanley
infections that occurred in Upper Austria. Cases attended a community event where they reported
having eaten potato salad which was later discovered to having been prepared by an
asymptomatic S. Stanley case.
In Hungary, the health authorities investigated an outbreak that occurred in June-July 2012 in a
summer camp. Cases reported the consumption of meat balls which contained turkey meat. No
meat balls were available for microbiological analysis. However, some frozen turkey meat from the
same batch that was used for the preparation of the meat balls was positive for S. Stanley with the
outbreak PFGE profile.
These three local investigations provided strong microbiological evidence for turkey meat as a
possible vehicle of infection although this hypothesis could not be verified with analytical
epidemiological investigations.
407
Apart from these local outbreak investigations, interviews of the cases with the national and EU
trawling questionnaires did not generate any hypotheses that could undoubtedly link the cases
epidemiologically.
to farm.
Between 2004 and 2010, S. Stanley serovar has been relatively rarely reported from food and
animals by EU Member States, Norway and Switzerland, with only 55 isolations reported. However
this increased to 311 in 2011. About 96% of the S. Stanley isolations in 2011 were from turkey
fattening flocks, turkey breeding flocks and turkey meat. In addition some isolates reported in
2011 came from: broiler flocks, Gallus gallus breeding flocks, broiler meat, pigs, and other poultry
and hedgehogs [5]. These results swiftly directed the investigation towards turkey as a possible
vehicle of infection.
The S. Stanley isolations from turkeys and turkey meat in 2011 were made by five Member States:
Austria, Czech Republic, Germany, Hungary and Slovenia. Hungary also provided preliminary data
from 2012 (up to the end of June): no S. Stanley isolations had been reported from turkey
breeding flocks, whereas 258 fattening flocks from 24 holdings yielded S. Stanley isolates [5].
In the years 20042010, no S. Stanley isolations were reported related to turkeys or meat thereof
in the EU/EEA countries. Similarly no S. Stanley findings were made in the EU-wide baseline survey
on Salmonella in turkey flocks in the years 20062007.
Food and veterinary investigations conducted in 2012 in Austria, Belgium, Germany, Czech
Republic, Hungary and Poland identified the presence of S. Stanley isolates with an
indistinguishable XbaI-PFGE fingerprint and a common resistance to nalidixic acid among majority
of isolates originating from the turkey production chain (turkeys and turkey meat). Isolates with
indistinguishable PFGE patterns were also detected in several EU Member States (France,
Germany, Hungary, Czech Republic, Poland) from broiler flocks (breeding and fattening chicken
flocks), meat from other animal species (broiler meat, beef and pork) and from animal feed.
Comparison animal/food/human isolates
In Austria, 14 human isolates from 2011 and 2012 with indistinguishable XbaI-PFGE patterns from
the outbreak profile were analysed with a second enzyme (BlnI). The results showed
indistinguishable patterns among them and indistinguishable to the Belgium outbreak profile
digested with BlnI. This pattern using BlnI was also identified in 14 veterinary isolates (from turkey
fattening farms, turkey hatchery, parental flock farm) and three food isolates.
From September to December 2012, EU/EEA countries provided over 500 PFGE profiles to the
joint human, food, feed, animal and environmental isolates database. The majority (about 70%) of
these PFGE profiles from humans and from animal, food, feed and environmental sources were
indistinguishable from the outbreak strain PFGE profile. Ninety-two percent of the food, animal
and environmental isolates with indistinguishable XbaI-PFGE outbreak profile originated from
turkey production.
CONCLUSION
The concomitant emergence of S. Stanley serovar across the EU in humans (non travel-related
infections), turkey and turkey meat suggested a relationship between the upsurge of S. Stanley
cases and either the consumption of or the indirect exposure to contaminated turkey or turkey
meat.
408
Comparative molecular epidemiological analyses of human, food, animal and environmental
isolates PFGE fingerprints provided further evidence strongly supporting the hypothesis of the
turkey production chain as the primary source of the outbreak. However, contribution of other
food and animal sources, such as beef, pork and broiler meat could not be excluded.
Subsequently to the finding of a S. Stanley isolate with the outbreak strain PFGE profile in turkey
stick from Austria, the Austrian food authorities issued on 10 September a Rapid Alert System for
Food and Feed (RASFF) notification [6].
EU-wide trace-back and trace-forward investigations of the turkey production chain were
recommended to understand the distribution of the infected animals and contaminated meat
across countries. Ecological studies testing spatial correlation between the cases and the animal
and food distribution could have contributed to the epidemiological investigations [5].
No related cases were identified outside the EU. The number of cases reported in all EU affected
countries was decreasing consecutively between September and December 2012 (figure 1),
indicating that the spread of contaminated food has ceased and fewer people have been exposed
to contaminated food products [7].
Multi-sectorial collaboration
Fork to farm investigations rely on the timely sharing of information between the public health
and veterinary sectors at national and international level during an outbreak investigation. It was
the pull-in and crossing of epidemiological and microbiological data both from the public health
side and the food and veterinary side that led to rapid hypothesis generation and eventually
identified the most likely source of the EU-wide outbreak. Despite the relative small number of
infections in most of the affected Member States, there was active sharing of information among
countries.
At the EU level, the different mechanisms to collect and disseminate information were activated.
ECDC, EFSA, EU-RL Salmonella and DG SANCO coordinated to gather, combine and analyse
available epidemiological and microbiological information from farm-to-fork.
The EPIS-FWD platform has showed its usefulness in detecting at an early stage the multinational
dimension of an outbreak in humans assuming that the event was initially reported in a timely
manner by a member country. In order to facilitate the exchange of information between public
health and food and veterinary experts during foodborne outbreak investigation, the development
of a joint communication platform could be foreseen.
DISCLAIMER
The authors Ernesto Libana, Frank Boelaert, Marta Hugas, Pia Makela are employed with the
European Food Safety Authority (EFSA) in its BIOHAZ Unit that provides scientific and
administrative support to the BIOHAZ Panel, and the BIOMO Units that supports EFSAs scientific
activities in the area of monitoring of biological hazards The present article is published under the
sole responsibility of the author and may not be considered as an EFSA scientific output. The
positions and opinions presented in this article are those of the authors alone and are not
intended to represent the views or scientific works of EFSA. To know about the views or scientific
outputs of EFSA, please consult its website under http://www.efsa.europa.eu.
409
REFERENCES
1. Yde M, Naranjo M, Mattheus W, Stragier P, Pochet B, Beulens K, et al. Usefulness of the European
Epidemic Intelligence Information System in the management of an outbreak of listeriosis, Belgium, 2011.
Euro Surveill. 2012;17(38).
2. European Centre for Disease Prevention and Control (ECDC). Epidemic Intelligence tools: EPIS. 2010 [10
January 2013]; Available from:
http://www.ecdc.europa.eu/en/activities/epidemicintelligence/Pages/EpidemicIntelligence_Tools.aspx.
3. European Centre for Disease Prevention and Control (ECDC). Rapid risk assessment - update: Multi-
country outbreak of Salmonella Stanley infections Published in the Early Warning and Response System
(EWRS) 29 August 2012.
4. European Centre for Disease Prevention and Control (ECDC). Epidemiological update: Multistate
outbreak of Salmonella Stanley infections, 12 December 2012. [20 January 2013]; Available from:
http://ecdc.europa.eu/en/press/news/Lists/News/ECDC_DispForm.aspx?List=32e43ee8-e230-4424-a783-
85742124029a&ID=807&RootFolder=%2Fen%2Fpress%2Fnews%2FLists%2FNews.
5. European Centre for Disease Prevention and Control (ECDC), European Food Safety Authority (EFSA).
Joint ECDC/EFSA rapid risk assessment: Multi-country outbreak of Salmonella Stanley infections, 20
September 2012. Available from:
http://ecdc.europa.eu/en/publications/Publications/Forms/ECDC_DispForm.aspx?ID=962
6. Rapid Alert System for Food and Feed (RASFF). Salmonella Stanley (presence /25g) in turkey sticks from
Austria, with raw material from Hungary, Notification 2012.1296, Notified on 10 September 2012. [20
January 2013]; Available from: https://webgate.ec.europa.eu/rasff-
window/portal/index.cfm?event=notificationDetail&NOTIF_REFERENCE=2012.1296.
7. European Centre for Disease Prevention and Control (ECDC). Epidemiological update: Multistate
outbreak of Salmonella Stanley infections, 30 January 2013. [07 February 2013]; Available from:
http://ecdc.europa.eu/en/press/news/Lists/News/ECDC_DispForm.aspx?List=32e43ee8%2De230%2D4424
%2Da783%2D85742124029a&ID=838&RootFolder=%2Fen%2Fpress%2Fnews%2FLists%2FNews.
410
Salmonella Brandenburg resistant to ciprofloxacin
in patients hospitalized in the same clinic, France, 2000-2012
Damien MOULY
1*
, Olivier BAUD
2*
, Jean Michel THIOLET
1**
, Claude BERNET
2**
,
Martine BESSON
2*
, Martine DENIS
3
, Pierre-Hugues GUILLERM
4
, Vronique VAILLANT
1**
,
F. MAILLARD
5
, Nathalie JOURDAN-DA SILVA
1
and Simon LE HELLO
5

1
French Institute for Public Health Surveillance,
*
InVS-Cire Auvergne, 60 avenue de l'union sovitique, 63057 CLERMONT FERRAND, France
**
InVS, 12 rue du val d'osne, 94415 SAINT MAURICE, France
2*
Cclin Sud Est, Arlin Auvergne, 58 Rue Montalembert, 63000 CLERMONT FERRAND, France
**
Cclin Sud Est, 20 route de Vourles, 69230 SAINT GENIE LAVAL, France
3
Anses-Ploufragan laboratory, National Reference laboratory for Salmonella,
BP 53, 22440 PLOUFRAGAN, France
4
CLIN Clinic A, 75 Alle des Ailes, 32000 VICHY, France
5
Institut Pasteur, French National Reference Center for E. coli, Shigella and Salmonella,
25-28 rue du Docteur Roux, 75724 PARIS, France
ABSTRACT
Between September 2010 and January 2012, six patients infected with Salmonella enterica serotype Brandenburg with
high-level resistance to ciprofloxacin, were reported. All six patients were hospitalized in the same clinic before the S.
Brandenburg strain was isolated. An investigation was launched in order to identify a potential common source in the
clinic and implement appropriate control measures. Confirmed cases were individuals with isolated ciprofloxacin
resistant S. enterica serotype Brandenburg reported to the French National Reference Center (FNRC) E. coli, Shigella
and Salmonella. From January 2000 to July 2012 the FNRC identified a total of 20 cases including the 6 cases
mentioned above. Among these isolates 13 corresponded to samples taken from patients during their stay at the clinic, 7
being taken from other patients after hospitalization in the clinic. The investigations performed took into account the 15
most recent cases and kitchen staff of the clinic as potential source of contamination. Control measures have since been
implemented by the clinic and standard precautions have been reinforced. The hypothesis of a chronic carriage and
intermittent excretion among staff members is particularly taking in consideration.
INTRODUCTION
Between September and October 2010 five patients with identified multi-drug resistant
Salmonella enterica serotype Brandenburg (resistant even to the key antibiotic ciprofloxacin
(Brandenburg CIP-R)) were hospitalized in a clinic in the Auvergne region in France and were
reported to the local health authority. Patients were hospitalized in different medical services. At
the end of 2011, a sixth patient testing positive for Brandenburg CIP-R was also reported.
Brandenburg is a rare serotype causing salmonellosis in humans in France. On average the French
National Reference Center (FNRC) for E. coli, Shigella and Salmonella has received 50 clinical
strains in each of the previous 3 years compared to 100-150 before. Several outbreaks of S.
enterica serotype Brandenburg have been reported in the literature (1-5). High-level resistance to
fluoroquinolones in Salmonella is rarely described and mainly associated with specific serotypes,
e.g. Typhimurium and Choleraesuis in Asia, and more recently Kentucky in Africa (6).
Between 2010 and 2011, the occurrence of six patients with Brandenburg CIP-R strains isolated in
a health care facility is notable for two reasons: the repetition of one infrequent strain and its
persistence within the same health facility (over a long period).
The aim of this paper is to present the investigations conducted to (i) describe a hospital-acquired
outbreak of Brandenburg CIP-R strain (ii) confirm the existence of an epidemic strain and
characterize it and (iii) find a common source and contributing factors in the transmission facility.
411
Description of the care facility
The health care facility is a private clinic for profit with a capacity of 147 beds divided in 4 medical
services (medicine, surgery, rehabilitation unit, ambulatory unit). Previously the hospital meals
were cooked and prepared on-site by kitchen staff. Today, however, the central kitchen of the
clinic is managed by an external provider which follows (as does the institution) the HACCP
approach. The patients meals are cooked off-site but prepared on site by the kitchen staff.
Epidemiological and staff investigation
A case was defined as any person with or without digestive symptoms with identified (from a
biological sample) S. enterica serotype Brandenburg highly resistant to various antibiotics,
including fluoroquinolones (ciprofloxacin). Information about the health status of individuals, the
context of the microbiological analysis, and history of hospitalization was gathered for each case.
A food history questionnaire was conducted for the cases reported from late 2010 onwards.
As no common exposure to a single health care worker was found, investigations focused on the
kitchen and its staff for potential sources of contamination.
The kitchen was visited by the investigation team first time in January 2012. Kitchen staff were
invited to answer a questionnaire and be screened. Information about daily professional activity,
working schedule, health status, lifestyle and hand hygiene were collected. Screening consisted in
examining six stool samples per person at 24h to 48h intervals. A further series of 3 stool samples
per person was recommended in the event of new occurrences of Salmonella.
Microbiological investigations
All human Salmonella strains received by the FNRC are serotyped using the White-Kauffmann-Le
Minor scheme.
A total of 407 samples of serotype Brandenburg (antigenic formula 4,512:l,v:e,n,z15), one per
patient, were tested for a panel of 32 antibiotics, including those used to treat severe
salmonellosis (i.e. imipenem, ceftriaxone, ciprofloxacin and co-trimoxazole). The Brandenburg
samples strains were chosen as follows: all those collected by the FNRC since 2009 (n=187), all
those isolated in the Auvergne region between 1992 and 2008 (n=44) and a subset of
representative samples isolated in 1997 (n=40), 2000 (n=45), 2002 (n=41), and 2008 (n=50).
All strains resistant to nalidixic acid and/or ciprofloxacin were extensively characterized for their
resistance mechanisms: the presence of beta-lactam resistance genes, plasmid-mediated
quinolone resistance genes, class 1 integron gene cassettes, and Salmonella genomic island 1
(SGI1) was detected by PCR using previously published methods. PCR and sequencing were also
used to screen for quinolone-associated mutations in subunits of the DNA gyrase (gyrA and gyrB)
and the topoisomerase IV (parC and parE) (6).
PulseNet standard pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA was
performed on a subset of 52 representative Brandenburg strains isolated from 1992 to 2012,
including all those resistant to quinolones.
Investigation of the clinics kitchen
In July 2012, a total of 22 samples were collected from the clinics kitchen by swabbing
environmental surfaces: 12 from the cold room (wall (6), floor (2), air conditioning (4)), 4 in the
central kitchen (worktop (2), floor (1), air conditioning grill (1)), and 6 in the two refrigerators.
After enrichment in BPW at 37C during 182H, a second enrichment was performed on MKTTn,
RVS and MSRV at 41.5C during 243H. Each enrichment was then streaked on XLD and
RAPIDSalmonella plates. The presence of typical colonies was checked for after 24h at 37C.
Ethic and deontology aspects
Recording, processing and preservation of epidemiological data was conducted in accordance with
authorization N 341 194 v 42 of the Commission nationale de linformatique et des liberts
(CNIL).
412
RESULTS
Occurrence of Brandenburg CIP-R
Twenty cases of S. enterica serotype Brandenburg with extensive phenotypic resistance to several
drugs, in particular to ciprofloxacin, were identified by the FNRC E. coli, Shigella and Salmonella
on 407 samples tested between 1992 and 2012. Four of these occurred prior to 2010, the oldest
case dating back to 2000 (Figure 1).
Brandenburg CIP-R was identified for 50% of cases in urine, 42% in feces and for one case in blood.
For three cases with urinary carriage, the strain was aslo identified in their stools. Two cases
tested respectively 6 and 10 months after initial positive identification, were still contaminated.
Median age of cases was 80.0 years (mean 78.6 years, min: 60, max: 100 years), women were
predominantly affected (female to male ratio was 9), 44% of cases had symptoms of
gastrointestinal problems at the time of diagnosis. All cases had been hospitalized in the clinic in
different medicals services (surgery, rehabilitation unit and ambulatory unit) prior to the
identification of Brandenburg CIP-R Salmonella. Seventy percent of cases were hospitalized in the
clinic at the time of diagnosis and for all other cases, samples were taken outside of the clinic but.
Median duration of hospitalization for all cases was 29 days (Average: 32 days Min: 3 days max: 87
days). Cases had between 1 and 6 stays at the clinic (1.3 on average) in different services for
different (medical) reasons.
Clonality of the Brandenburg CIP-R population
Genetic relatedness of 52 Brandenburg isolates was studied using PFGE (Figure 2). All Brandenburg
CIP-Rs were clustered into a homogeneous group (based on >80% similarity).
Ciprofloxacin resistance in the 20 Brandenburg CIP-R isolates had for the majority a high level of
MIC (>32 mg/L) and presented gyrA and parC mutations. The majority contained a double
mutation in gyrA (Ser83 and Asp87) and a single parC substitution (Ser80 encoding an isoleucine
residue). One also had either gyrB or other parC mutation. In gyrA, all isolates contained a change
at codon Ser83 to phenylalanine, whereas mutations in the Asp87 codon resulted in substitutions
to asparagine, tyrosine or glycine residues. No plasmid-mediated quinolone resistance genes, such
as qnr, aac(6)-Ib-cr, and qepA, was found in Brandenburg CIP-R isolates. Apart from quinolone
resistance, other resistance was also seen in some Brandenburg CIP-R isolates (Figure 2). The most
prevalent patterns included resistance to amoxicillin (due to the TEM gene), streptomycin,
spectinomycin, kanamicin, tobramycin, gentamicin, sulfamethoxazole, trimethoprim,
chloramphenicol and tetracycline. Different complex integrons were found (900 to 1500 bp).
Interestingly, 4 additional strains tested were found also in patients hospitalized in the same clinic
between 1992 and 1994 and displayed the same resistance pattern but limited for quinolones to
nalidixic acid, suggesting a precursor strain of the epidemic Brandenburg CIP-R.
Epidemiological investigation
A food history questionnaire completed by reported cases from late 2010 to July 2012 did not
allow us to identify any particular food or dietary supplement as the source of contamination.
All members of kitchen staff responded to the questionnaire (n = 12). Although each person was
assigned a main activity, during weekends, holiday periods and holidays meal preparation,
distribution and dishwashing responsibilities were shared by all members of kitchen staff. The
preparation of certain dishes (powdered custard, cooking rice, pasta, etc.) was performed the day
before the consummation. The median age of employees was 51 years (mean 49 years, Min: 38
years Max: 58 years) and the staff was predominantly female (female to male ratio = 5).
Employees had been working in the clinic for an average of 19.3 years (min 4 years max: 30). All
said that they washed their hands regularly (between 10 and 15 times per day) with or without
alcohol-based gel. Four employees reported trips abroad in recent years: United Kingdom, Italy,
Spain, Caribbean islands. One person reported regular visits to Turkey over the previous twenty
years. This person also reported having gastrointestinal problems since 2001. During the interview
413
no other employee reported digestive problems over the previous few years. Finally, the dates of
hospitalization of cases occurred between September 2009 and April 2012 and staff rosters were
concordant for 2 people, including the one with digestive symptoms.
The rate of kitchen staff screening participation was 92% (11/12). Ten participants fully completed
screening of the first series of six stools, one participant partially completing this step (3/6 stools).
The average time between the first and sixth stool sample was 18 days, i.e. 3 days on average
between 2 samples for each participant. Eight percent of the samples were provided less than 24
hours apart. All analyzes (69 stool samples) were negative. Further screening (a series of 3 stools)
of the same participants was performed following the occurrence of new cases in April 2012. The
participation rate for this second campaign was also 92%. All analyzes (n = 32) returned negative.
No Salmonella was detected from the environmental samples collected in the clinic kitchen.
Moreover, there were no domestic pets in the clinic or in its environment.
DISCUSSION
The identification of 20 cases of Salmonella enterica serotype Brandenburg with extensive
phenotypic resistance to several drugs, identified for the first time in France, all hospitalized in the
same care facility between 2000 and 2012, suggests the existence of a common and persistent
source in the health facility for over 10 years. The low severity of symptoms in the cases and the
high proportion of asymptomatic carriers (56%) suggest low level contamination and probably
significant underdiagnosis.
To date, the hypothesis of transmission through contaminated food transfer (contact with
contaminated hands or surfaces) from the intermittent excretion of a chronic carrier working in
the kitchen intermittent excretion is particularly taking in consideration. The arguments in favor of
this hypothesis are several: structural and material non-conformities identified in kitchens by
health authorities (results not shown), the consumption of meals by all cases, no new cases
reported since the outsourcing of kitchen and food services, the presence of regular
gastrointestinal symptoms among the kitchen staff and concordance between the dates of
admission and scheduling of employees. The negative results of stool screening cannot exclude
this hypothesis. Indeed excretion is intermittent and stool testing were made away from the
occurrence of the event.
Control measures related to food safety have been implemented by the clinic. Standard
precautions have been reinforced. Epidemiological investigations are still ongoing. No new cases
have been reported since the outsourcing of the kitchen facilities to an external provider.
REFERENCES
1. Burns C, Mair NS, Hooper WL. An outbreak of Salmonella Brandenburg infection caused by infected pork
products. Mon Bull Minist Health Public Health Lab Serv 1965 Jan;24:9-14.
2. Baker MG, Thornley CN, Lopez LD, Garrett NK, Nicol CM. A recurring salmonellosis epidemic in New
Zealand linked to contact with sheep. Epidemiol Infect 2007 Jan; 135(1): 76-83.
3. Hamada K, Tsuji H. Salmonella Brandenburg and S. Corvallis involved in a food poisoning outbreak in a
hospital in Hyogo Prefecture. Jpn J Infect Dis 2001 Oct; 54(5): 195-6.
4. Mammina C, Aleo A, Romanelli G, Marconi P, Di Noto AM, Donato R, et al. A food borne outbreak of
Salmonella enterica serotype Brandenburg as a hint to compare human, animal and food isolates identified
in the years 2005-2009 in Italy. J Prev Med Hyg 2011 Mar; 52(1): 9-11.
5. Wong TL, Nicol C, Cook R, MacDiarmid S. Salmonella in uncooked retail meats in New Zealand. J Food
Prot 2007 Jun; 70(6): 1360-5.
6. Le Hello S, Hendriksen RS, Doublet B, Fisher I, Nielsen EM, Whichard JM, et al. International spread of an
epidemic population of Salmonella enterica serotype Kentucky ST198 resistant to ciprofloxacin. J Infect Dis
2011 Sep 1 ; 204(5) : 675-84.
414
TABLES AND FIGURES
0
1
2
3
4
5
6
7
8
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012
Year of isolation
blood sample stool sample urine sample
Figure 1 : epidemic curve of cases of Salmonella enterica serotype Brandenburg with extensive phenotypic
resistance to several drugs, identified between 2000 and 2012.
Figure 2 : Analysis of Xba-I PFGE profiles obtained among 52 susceptible or resistant Salmonella enterica
serotype Brandenburg isolates from humans during the period 1992 to 2012. Strain code, Xba-I and
antimicrobial susceptibility patterns and "departement" of acquisition are indicated to the right of the PFGE
profiles. Panel used for susceptibility testing: A, amoxicillin; C, chloramphenicol; Cip, ciprofloxacin; G,
gentamicin; K, kanamycin; Nal, nalidixic acid; Sul,sulfamethoxazole; Sp, spectinomycin; S, streptomycin; Te,
tetracycline; Tmp, trimethoprim; T, tobramycin.
415
Seventeen years of extra-intestinal nontyphoidal human Salmonella infections
in the United States, 1995 to 2011
Kathleen E FULLERTON, Kelly A JACKSON, Richard BISHOP, Patricia I FIELDS
and Barbara E MAHON
Centers for Disease Control and Prevention (CDC), 1600 Clifton Rd NE MS C-09, ATLANTA, GEORGIA, USA
30333
Abstract:
Salmonella causes an estimated 1.2 million human infections, 23,000 hospitalizations, and 450 deaths in the United
States annually. Almost all Salmonella serotypes are classified as nontyphoidal, indicating that infection typically
presents as gastroenteritis, not typhoid fever. However, nontyphoidal serotypes can cause extra-intestinal infections.
We analyzed data on the clinical source (including stool, blood, or urine only) of laboratory-confirmed nontyphoidal
human Salmonella infections reported to the Centers for Disease Control and Prevention (CDC) from 1995 to 2011,
excluding the highly invasive serotypes Choleraesuis and Dublin. During 1995 to 2011, 575,908 nontyphoidal
Salmonella infections were reported. Of these, 472,860 (82%) isolates were from stool, 26,634 (4.76%) were from
blood, and 25,973 (4.5%) were from urine; the others were from other or unknown sites. Among the 525,467 stool,
blood, and urine isolates, the most frequently reported serotypes were Typhimurium (including Typhimurium var. 5-)
(22%), Enteritidis (21%), Newport (10%), Heidelberg (5.1%), and Javiana (4.5%). The percent isolated from blood
varied among the top five serotypes (mean 5.5%, range 1.8% [serotype Newport] to 11.9% [serotype Heidelberg]). The
percent isolated from urine was similar among the top five serotypes (mean 4.1%, range 2.7% to 5.5%) Among
serotypes with more than 100 isolates reported, those with the highest percentage of blood isolations were IIIa
18:z4,z23:- (42.4%), Lomalinda (22.3%), Urbana (17.1%), Sandiego (15.4%), and Telelkebir (15.4%). By contrast, the
serotypes with the highest percentage of urine isolations were IIIb 61:l,[v],[z13]:1,5,[7] (39.0%), Cubana (33.0%),
Tennessee (28.0%), Rissen (25.4%) and IIIa 18:z4,z23:- (22.4%). Human nontyphoidal Salmonella infections in the
United States show marked variation in distribution of sites of infection. Although all serotypes are most commonly
isolated from stool, some are more commonly isolated from blood and others from urine. Characterization of
microbiological and epidemiological features of serotypes may contribute to understanding their virulence.
416
A European outbreak of S. Newport associated
with melons from South America
Ian FISHER on behalf of the International Outbreak Control Team*
Health Protection Agency, 61 Colindale Ave, NW9 5EQ,London, UK
ABSTRACT
An isolate of Salmonella Newport was isolated from a ready-to-eat watermelon fan in England in November 2011. In
December 2011an increase in cases of S. Newport were identified in Scotland and England and Wales. Information that
was disseminated via the Rapid Alert for Food and Feed system and the ECDC Epidemic Intelligence Information
System on the epidemiology and microbiology of the cases subsequently identified additional cases in Germany and
Ireland. In total 63 cases across six countries were identified with indistinguishable Pulsed-Field Gel Electrophoresis
patterns between them and the watermelon fan from England.
Of the 63 cases, 46 (73%) were interviewed and 59% of these reported watermelon consumption.
Whole genome sequencing was preformed on a subset of the isolates from the UK and the watermelon fan, which
confirmed that they were all from one outbreak strain.
The multi-disciplinary investigation involving epidemiological, microbiological and food tracing investigations led to the
source of these watermelons being identified as one region in Brazil.
INTRODUCTION
In November 2011 the Health Protection Agency (HPA) Food Water and Environment (FWE)
Laboratory in Preston, England, confirmed the presence of Salmonella in a ready to eat sliced
watermelon fan purchased from a major supermarket retailer. The isolate was sent to the
Gastrointestinal Bacteria Reference Unit (GBRU) at Colindale, London who reported it as
Salmonella enterica subspecies enterica serovar Newport in December 2011. On 13 December
2011, the result was communicated through the Rapid Alert System for Food and Feed (RASFF) of
the European Commission. [1]
Health Protection Scotland (HPS) reported four cases of S. Newport all with the same pulsed-field
gel electrophoresis (PFGE) profile which had not previously been seen. Concurrently in England,
Wales and Northern Ireland, reporting of S. Newport infections exceeded expected levels.
Molecular analysis of isolates from the human cases from all four countries indicated a PFGE
profile indistinguishable from the sliced watermelon isolate.
Four cases were reported in Ireland in January 2012. Also in January, Germany reported through
the Epidemic Intelligence Information System (EPIS) at the European Centre of Disease Prevention
and Control (ECDC), 14 S. Newport isolates that were indistinguishable from the sliced watermelon
isolate PFGE profile. This differed to that of isolates from cases in an outbreak of S. Newport linked
to the consumption of mung bean sprouts which the Robert Koch Institute (RKI) investigated at
the same time [2].
A multi-agency outbreak control team (OCT) was convened comprising membership from HPA,
Public Health Wales (PHW), HPS and the UK Food Standards Agency (FSA). The HPA also
communicated with the RKI regarding the German cases and both the Health Protection
Surveillance Centre (HPSC) and the National Salmonella, Shigella and Listeria Reference Laboratory
(NSSLRL) regarding cases from Ireland. They and colleagues from their food safety authorities
subsequently joined the OCT.
Isolates from patients and food samples were characterized and compared. In England, Wales and
Northern Ireland, all isolates were sent to GBRU; isolates in Scotland to the Scottish Salmonella,
Shigella, and Clostridium Difficile Reference Laboratory; in Ireland to the NSSLRL and in Germany
to the National Reference Centre for Salmonella and other Bacterial Enteric Pathogens at the RKI,
Wernigerode.
417
PFGE using the PulseNet-Europe/Salm-gene protocol [3] was performed on all S. Newport isolates
from patients with an onset of illness date reported during the outbreak period. The results of
PFGE analysis together with antimicrobial resistance profiles were used to inform case definitions
in epidemiological investigations.
A subset of isolates from UK cases (24 of 37 outbreak isolates, the watermelon fan and 11 non-
outbreak isolates) were selected for whole genome sequencing (WGS). Genomic DNA was
extracted using the Wizard Genomic DNA Purification Kit, and samples were sequenced using
multiplex libraries on the Illumina HiSeq platform using 100bp paired-end reads.
Cases were defined as persons with laboratory confirmed infection with fully antimicrobial
sensitive S. Newport exhibiting the outbreak PFGE profile (as per the watermelon isolate) with
onset of illness between 31
st
October 2011 and 31st January 2012.
A questionnaire was designed to collect information on fruit consumption and was shared with
each member country of the OCT. The questionnaire gathered detailed information on
consumption of different types of fruit, including melon, and addressed whether products were
pre-packaged, sliced, cubed, mixed with other fruit and where they were purchased from and
consumed. All cases were interviewed using this questionnaire (translated into the local language
where necessary), although some countries added some further questions as well. The frequency
of watermelon consumption in the general population was analysed in Germany and the UK.
Trace-back investigations were undertaken by all countries.
RESULTS
A single strain of S. Newport was found to be involved in this outbreak, with the PFGE profile
SNWPXB.0110. All isolates were fully susceptible to the antimicrobials tested. WGS revealed near-
identical sequences between the watermelon isolate and 24 outbreak isolates from the UK. Five
isolates had one single nucleotide polymorphism (SNP). The non-outbreak strains differed by
several thousand SNPs. There were a combined total of 63 confirmed cases reported with onset
dates during the outbreak period.
Onset dates ranged from 5
th
November 2011 to 19
th
January 2012, with almost half (47.5%) of
these between the 28
th
November and 11
th
December 2011.
Cases were predominantly female (71%) and ranged in age from 6 months to 95 years of age, with
the greatest number (22%) in children aged 5 years or less. Cases were distributed across several
regions within countries.
In total, 46 (73%) of confirmed cases from six countries were successfully interviewed using the
outbreak case exposure questionnaire.
Consumption of pre-packed, pre-sliced and whole watermelon was reported by 27 (59%) of
interviewed cases. An additional two cases reported possible consumption of watermelon. While
the remaining 17 cases reported no consumption of watermelon, two reported consumption of
other types of melon (Galia and Cantaloupe). Data on watermelon consumption extracted from
the UK National Diet and Nutrition Survey dataset [4] indicated that the level of between 5-10% of
the UK population report watermelon consumption at that time Consumption reported by cases
(64%) was therefore considerably higher than would be expected in the general population.
The RKI study showed that of the persons who could remember whether they had consumed
watermelon (n=87), 7 (8%) affirmed consumption. Both of these data indicate that consumption of
melons among cases was higher than the expected background levels.
The Food Safety Agency of Ireland performed a trace-back on the watermelon consumed by the
family cluster. It had arrived in a shipment from Brazil and further work traced it back to the same
source as the watermelon fan found positive for the outbreak strain in November 2011. German
authorities traced watermelons from a chain of supermarkets where cases had reported
purchasing watermelon, to a distributor in the Netherlands. From there, watermelons were traced
418
back to the same grower in Brazil associated with the contaminated watermelon fan and the Irish
cases and to an affiliated grower working in the same geographical region in Brazil. The
watermelon market has been developed by Brazil in recent years and Brazilian imports to the UK
have been around 6,000 tonnes per annum in recent years. Brazil also exports similar quantities to
Germany and the Netherlands. [UK Department for Environment, Food and Rural Affairs:
unpublished data]
DISCUSSION
Molecular, epidemiological, and trace-back investigations identified a multi-country outbreak of S.
Newport gastroenteritis linked to consumption of watermelon imported from Brazil. Molecular
analysis determined the contaminated watermelon strain and human isolates from confirmed
cases in the UK, Ireland and Germany indistinguishable from one another by PFGE and, for a
subset, by WGS.
Trace-back investigations identified Brazilian watermelons as a common factor in cases associated
with the outbreak. Consumption of watermelon was reported by a high proportion of cases across
all six countries as compared to the general population (UK National Diet and Nutrition Survey,
German RKI study). Cases declined in February, coinciding with the end of the Brazilian
watermelon importation season.
The same melon grower was associated with the contaminated watermelon fan from England and
human cases in Ireland and Germany. The melons associated with the Scottish cases were traced-
back to a different grower in the same region. However, the isolates from Scottish cases shared a
common sequence with the other cases, therefore it is likely Brazilian watermelons from a
particular area were the vehicle, but these were not confined to one specific grower.
Cases reported consumption of watermelons prepared in a variety of ways indicating the melons
themselves were contaminated and contamination did not occur during processing. Furthermore,
a range of different retailers, with different importers, processors and distributors were reported.
It is not possible to confirm at what point contamination occurred but there is likely to have been
a common source of S. Newport which contaminated the produce at some unidentified point
between growing and distribution.
CONCLUSION
The use of standardised microbiological techniques facilitates the identification of international
outbreaks of foodborne pathogens. Their investigation depends on the rapid dissemination of
epidemiological information and the use of common epidemiological tools to assist in the
detection of common vehicles and sources.
ACKNOWLEDGEMENTS
The International Outbreak Control Team consisted of; Lisa Byrne, Ian Fisher, Tansy Peters, Alison
Mather, Nick Thomson,

Bettina Rosner, Helen Bernard, Paul McKeown,

Martin Cormican, John
Cowden, Victor Aiyedun,

Chris Lane, Joanne Aish, Gautam Adak, Victor Aiyedun, Derek Brown,
Lynda Browning, Paul Cook, Cecilia Ellis, Andrew Fox, Elizabeth de Pinna, Patricia Garvey, Kathie
Grant, Dorothy Guina-Dornan, Petra Hiller, Colin Houston, Naomi Launders, Burkhard Malorny,
Brendan OBrien, Beatrice Pfefferkorn, Rita Prager, Wolfgang Rabsch, Roland Salmon, Robert
Smith, Brian Smyth, Kirsten Stone and Heidi Wichmann-Schauer.
They were ably assisted by public health practitioners and others within each country to ensure
the full investigation could be completed successfully.
419
REFERENCES
1. European Commission. Food and Feed Safety. Rapid Alert System for Food and Feed. [Accessed 5
th
March 2013]. Available at: http://ec.europa.eu/food/food/rapidalert/index_en.htm
2. Robert Koch Institute. Salmonella Newport outbreak in Germany and The Netherlands, 2011 - Mung
bean sprouts likely vehicle of infection [in German]. Epid Bull. Berlin (Germany): Robert Koch Institute;
2012. p. 177-84. Available at :
http://www.rki.de/DE/Content/Infekt/EpidBull/Archiv/2012/Ausgaben/20_12.pdf
3. Peters TM, Berghold C, Brown D, Coia J, Dionisi AM, Echeita A et al. Relationship of pulsed-field profiles
with key phage types of Salmonella enterica serotype Enteritidis in Europe: results of an international multi-
centre study. Epidemiol Infect. 2007 Nov;135(8):1274-81
4. Whitton, C.; Nicholson, S.K.; Roberts, C.; Prynne, C.J.; Pot, G.K, Olson A et al. National Diet and Nutrition
Survey: UK food consumption and nutrient intakes from the first year of the rolling programme and
comparisons with previous surveys. Br J Nutr. 2011 Dec:106 (12):1899-914
420
Evidence for Reptile-Exotic-Pet-Associated-Salmonellosis
(REPAS) in Germany
Wolfgang RABSCH
1
, Michael PEES
2
, Bastian PLENZ
2
, Angelika FRUTH
1
, Rita PRAGER
1
,
Sandra SIMON
1
, Volker SCHMIDT
2
, Sebastian MNCH
1
and Peggy G BRAUN
3

1
National Reference Centre for Salmonella and other bacterial Enterics, Robert Koch Institute,
Wernigerode, Germany;
2
Clinic for Birds and Reptiles, University of Leipzig, Germany;
3
Institute of Food Hygiene, Leipzig, Germany
ABSTRACT
Reptiles are suspected to be a source for salmonellosis in humans, and numerous case reports exist on salmonellosis in
infants related to reptiles. It was therefore the aim of this study to obtain data on reptiles kept in households with children
diseased from an exotic Salmonella serovar, including the Salmonella status and possible transmission paths, and to
compare the isolates with those found in the respective child. 79 affected families were contacted, and in 19 households
and a total of 36 reptiles, samples of the oral cavity, the cloacae, the skin and the stomach (lizards) were collected.
Salmonella isolation was conducted using a strict protocol with repeated enrichment and serotyping which was followed
by pulsed-field gel electrophoresis (PFGE) identification in cases of identical serovars found.
Almost 50% of the households answered that they kept at least one reptile. 68% of the examined reptiles were bearded
dragons (Pogona vitticeps). Altogether 319 isolates were investigated and 44 different serovars identified. In 79% of the
households, in at least one reptile the identical serovar was found and confirmed by PFGE. In 84% of all bearded
dragons examined, the identical serovar was confirmed. In most reptiles several serovars were found.
The results demonstrate that reptiles and especially bearded dragons shed different serovars of Salmonellae including
those serovars that were isolated from diseased children in the respective households. Therefore, from the
epidemiological point of view, we propose to call this special kind of epidemic as Reptile-Exotic-Pet-Associated-
Salmonellosis (REPAS). Hygiene protocols and parents education is therefore highly necessary to reduce the risk of
transmission.
INTRODUCTION
Numerous reports exist on the prevalence of Salmonella (S.) enterica in captive reptiles. In
contrast to early studies, more recent publications demonstrate a higher prevalence in lizards (up
to 76%) in comparison to tortoises and turtles [1]. Geue and Lschner [2] demonstrated a
significantly higher prevalence for Salmonella in collections with only purchased reptiles and
reptiles bought in pet shops were affected most often (89%).
Most reports of reptile-associated salmonellosis are in babies and children younger than 5 years.
Fatal outcomes following reptile-associated salmonellosis in babies have been reported [3, 4].
Newer studies and surveys indicate that other reptile species, especially lizards, may also play a
more important role [5, 6]. These reports demonstrate that today reptile-associated Salmonella
infections in humans are a world-wide problem. Thomas et al. [7] concluded that the potential of
captive and pet wildlife to transmit Salmonellae to humans should not be underestimated, and
that epidemiological studies on sources for human salmonellosis should simultaneously
investigate both the human cases and the wild and domestic animals in contact with them. It was
therefore the aim of this study to obtain data on possible links between captive reptiles and
salmonellosis in children by examining both the children with salmonellosis and all reptiles kept in
the respective households.
The study was conducted from July 2010 to October 2011. Within this period, the National
Reference Centre (NRC) for Salmonella and other Bacterial Enterics at the Robert Koch Institute
(Wernigerode, Germany) examined 206 cases of salmonellosis in children not older than three
years. 65% of the Salmonella isolates did not belong to S. Typhimurium and S. Enteritidis and were
therefore of interest for this study. From these cases, 79 parents were contacted on a randomly
421
basis and asked about the presence of reptiles in the respective households. 35 (44.3%) answered
positive. As inclusion criteria, parents had to agree that all reptiles in the respective household
could be sampled and their health status assessed, and the time period between detection of
clinical salmonellosis in the child and the sampling of the reptiles was not more than three weeks.
Swabs were taken from the oral cavity, the cloaca, and the skin on the ventral region of the
reptile. In lizards, an additional swab sample was taken from the stomach. Bacterial isolation and
identification was conducted with repeated enrichment and examination of several colonies in
each sample, in order to find as many different Salmonella serovars as possible: All samples were
immediately placed into a tube containing Rappaport-Vassiliadis (RV) medium (Oxoid, Wesel,
Germany). If Salmonella-suspicious colonies were observed on the agar, samples were taken from
at least five different colonies and subcultured on Brilliant Green agar (SIFIN, Berlin, Germany) for
confirmatory testing with biochemical methods. Additionally all strains were typed at the NRC
according to the White-Kauffmann-Le Minor scheme by slide agglutination with O- and H-antigen
specific sera (SIFIN, Berlin, Germany and SSI Copenhagen, Denmark) to define the serovar.
The isolates of identical serovars confirmed in the diseased child as well as the reptile were
compared using pulsed-field gel electrophoresis (PFGE). PFGE was carried out according to the
standardized protocol for subtyping Salmonella [8]. 40 strains (human and animal) were
investigated by PFGE. Cases from households with minor pattern differences were repeated.
Identical PFGE patterns were considered to represent the same epidemiological type. Up to four
differences in PFGE restriction fragment pattern were considered to be the result of a single
genetic event, and isolates were designated as subpatterns or related patterns. More than four
differences were considered to represent an epidemiologically-significant difference.
RESULTS
Nineteen households met the criteria and were included in the study. Salmonella serovars of these
cases belonged mainly to subspecies I (12 cases), but also Sub-species II (1), Subspecies IIIa (1),
Sub-species IIIb (2) and Subspecies IV (3), see Table 1. One S. Newport strain (6,8:z10:1,2:z67; d-
tartrate-, malonate+) was investigated in Paris (Institut Pasteur) because of its unusual
biochemical properties. The strain exhibited two atypical characteristics: D-tartrate negative and
malonate positive. The e,h and the 1,2 flagellar phases have been confirmed by fliC and fljB
sequencing. The MLST profile (ST118) was identical to those of serovar Newport lineage II [9].
Sampling areas were spread well over Germany, including eight federal states. All children except
one were less than 15 months of age, most were less than six months. All children showed clinical
symptoms of gastroenteritis including fever, with some being critically ill. Altogether, 36 reptiles
were kept in the households included in this study. Details are listed in Table 1.
A total of 319 Salmonella isolates were investigated and 44 different serovars were identified. All
serovars except one (S. Eastbourne; 9,12:e,h:1,5) were found in only one household in this study.
Between one and four different serovars were identified within individual reptiles. Only from
three reptiles (two snakes, one chameleon) no Salmonella was isolated. In nine reptiles in which
identical Salmonella serovars were detected, only this serovars but no other Salmonella were
isolated (details see Table 1).
In 15 of the examined 19 households, an identical human-pathogenic serovar was found in at least
one reptile. In six cases, the respective Salmonella serovar was isolated from the oral cavity, in two
cases (lizards) it was found in the stomach. In 16 cases Salmonella were detected in the cloaca,
and in seven cases the skin swab sample was positive. The identical serovars found in infected
children and reptiles belonged to the following subspecies: I (10), II (1), IIIa (1), IV(3) (details see
Table 1). PFGE demonstrated identical patterns in nine cases (PFGE data not shown) and related
patterns in six cases (repeated PFGE, see also Figure 1). Details are given in Table 1.
422
DISCUSSION
In most published case reports on reptile-associated salmonellosis, only fecal samples or fecal
swabs were used for Salmonella detection. In contrast, this study was designed to obtain as much
information as possible on Salmonella serovars shed by the reptiles. Sampling of the oral cavity
(and stomach) as well as the cloaca should provide information on shedding via both orifices and if
Salmonella are present within the whole digestive system, whereas samples from the skin should
demonstrate whether Salmonella may also be trans-mitted via direct contact with the animal.
Repeated enrichment and culture was necessary to provide reliable information on the Salmonella
status in the reptiles examined. Combining the molecular typing using PFGE with other available
data, such as serological typing as in this study, is highly recommended for accurate analysis and
comparison of samples [20].
Up to four different serovars were found within one reptile, and serovars within a collection were
usually identified in several animals. This indicates that Salmonella as a part of the normal flora are
spread amongst individuals within captive reptile collections and therefore probably shed over
long periods of time. These results are in accordance with observations that if one reptile carries
Salmonella, nearly all other reptiles of the respective owner are also affected [3].
Most isolates were found in cloacal swabs. However, since in some cases the identical serovar was
only found on the skin or the stomach, it can be assumed that shedding via the cloaca is
intermittent and a negative cloacal or fecal sample will not prove that the animal does not harbor
Salmonella. Given the anatomy and behavior of most reptiles, the presence of Salmonella on skin
samples was not surprising but underlines the general problem when handling reptiles. Therefore,
to increase the detection rate of Salmonella in reptiles, several sampling points should be used.
In 79% of the examined households, the identical serovar was found in the diseased child and at
least one reptile. The epidemiological association between these isolates was first confirmed using
biochemical typing. PFGE then confirmed all cases with either completely identical patterns (9
households) or only differences in a few fragments indicating single genetic variations (6
household, PFGE data shown in Fig. 1), as they have been described in disease outbreaks [8].
All publications cited above indicate that although infections attributed to exposure to reptiles and
other exotic pets represent only a small proportion of all human salmonellosis cases, it is likely an
under-estimated and growing problem in Europe [11] and in the USA [12] that deserves closer
attention. In 2007 more than 500,000 reptiles were imported to Germany only via the
Frankfurt/Main airport [13].
From the epidemiological point of view, and in addition to a recommendation already made
before (Reptile-Associated Salmonellosis, RAS [14]) we propose to call this special kind of
epidemic Reptile-Exotic-Pet-Associated-Salmonellosis (REPAS). The main argument for this
proposal is that over the last years, the way of trading reptiles has changed considerably and this
will probably continue in the future. The main risk of Salmonella transmission from reptiles to
humans is not due to European wild species, but as the results of this study also demonstrate
today mainly due to exotic imported reptile species. Furthermore, following recent
examinations Salmonella shedding is higher in reptiles kept in captivity in comparison to wild
reptiles [2,10] and pet reptiles are obviously in closer contact to humans. These arguments
justify the inclusion of exotic pet into the term describing the problem. The risk to human health
connected to the reptile pet market has been highlighted recently [15] and the accurate
description of the problem using REPAS might be important to convey the problem in education
and assist the European Commission to give recommendations to harmonise animal welfare and
public health.
Concluding, it needs to be emphasized that educational measures will be the key to reduce the
risk of Salmonella transmission to children. It is the responsibility of all professionals dealing with
reptiles (pet shop owners, veterinarians, breeders) or human health to increase the awareness of
423
this problem. In the authors opinion the risk of REPAS can easily be minimized, using a reasonable
management protocol without the need to remove reptiles from households with young children.
ACKNOWLEDGEMENT
The authors thank Franois-Xavier Weill, Centre for Reference and Research on Salmonella, Enteric
Bacterial Pathogens Unit, Institut Pasteur, Paris, for the confirmation of the unusual S. Newport.
We thank the Member States in the FWD network for submitting detailed serovar data.
Furthermore we thank Marita Wahnfried, Susanne Kulbe, Dagmar Busse for skillful technical
assistance, and Rachel Marschang, University of Hohenheim, for linguistic support.
REFERENCES
1. Pasmans F, Martel A, Boyen F, Vandekerchove D, Wybo I, Van Immerseel F, et al. Characterization of
Salmonella isolates from captive lizards. Vet. Microbiol. 2005; 110: 28591.
2. Geue L, Lschner U. Salmonella enterica in reptiles of German and Austrian origin. Vet. Microbiol. 2002;
84: 79-91.
3. Woodward DL, Khakhria R, Johnson WM. Human salmonellosis associated with exotic pets. J. Clin.
Microbiol. 1997; 35(11): 2786-90.
4. Mermin J, Hutwagner L, Vugia D, Shallow S, Daily P, Bender J, et al. Reptiles, amphibians, and human
Salmonella infection: a population-based, case-control study. Clin. Infect. Dis. 2004; 38 Suppl 3: S253-S261.
5. Willis C, Wilson T, Greenwood M, Ward L. Pet reptiles associated with a case of salmonellosis in an
infant were carrying multiple strains of Salmonella. J. Clin. Microbiol. 2002; 40(12): 48023.
6. Weiss B, Rabsch W, Prager R, Tietze E, Koch J, Mutschmann F, et al. Babies and bearded dragons: sudden
increase in reptile-associated Salmonella enterica serovar Tennessee infections, Germany 2008. Vector
Borne Zoonotic Dis. 2011; 11(9): 12991301.
7. Thomas AD, Forbes-Faulkner JC, Speare R, Murray C. Salmonellosis in wildlife from Queensland. J. Wildl.
Dis. 2001; 37(2): 22938.
8. Goering RV. Pulsed field gel electrophoresis: A review of application and interpretation in the molecular
epidemiology of infectious disease. Inf. Gen. Evol. 2010; 10(7): 86675.
9. Sangal V, Harbottle H., Mazzoni CJ, Helmuth R, Guerra B, Didelot X, et al. Evolution and population
structure of Salmonella enterica serovar Newport. J. Bacteriol. 2010; 92(24): 6465-76.
10. Scheelings TF, Lightfoot D, Holz P. Prevalence of Salmonella in Australian reptiles. J. Wildlife Dis.
2011;47(1):111.
11. Bertrand S, Rimhanen-Finne R, Weill FX, Rabsch W, Thornton L, Perevoscikovs J, et al. Salmonella
infections associated with reptiles: The current situation in Europe. Euro Surveill. 2008; 13(24): pii=18902.
12. Center for Disease Control and Prevention. Outbreak of salmonellosis associated with pet turtle
exposures - United States, 2011. Morb. Mort. Wkly. Rep. 2012; 61(3): 79.
13. Hatt, JM, Fruth A, Rabsch W. Reptilienassoziierte Salmonellosen - aktuelle Informationen fr
Tierrztinnen und Tierrzte. Tieraerztl. Pra. 2009; 37: 188-193.
14. De Young B, Andersson Y, Ekdahl K. Effect of Regulation and Education on Reptile-associated
Salmonellosis. Emerg Infect Dis. 2005; 11: 398403.
15. Arena PC, Steedman C. and Warwick C. Amphibian and reptile pet markets in the EU: An investigation
and Assessment, http://animal-public.de/2012/05/wissenschaftler-fordern-verbot-von-terraristikborse,
accessed December 15, 2012.
424
Table 1: Reptile-Exotic-Pet-Associated-Salmonellosis (REPAS) in different households and confirmation of
the identity using Pulse-field Gel Electrophoresis.
House-
hold
Child Reptile species No. of
serovars
identified
Localization
of identical
serovar
PGFE
result
*
Age
(months)
Serovar
1 11 S. Eastbourne 9,12 : e,h : 1,5 Pogona vitticeps 3 Oral cavity,
Cloaca, Skin s (2)
Pogona vitticeps 1 Cloaca
2 3 S. Ealing 35:g,m,s,:- Pogona vitticeps 1 - -
3 8 S. Cotham 28 : i : 1,5 Pogona vitticeps 3 Skin s (2)
4 2 S. subspez. IV 44:z4,z23:- Pogona vitticeps 3 Stomach i
5 6 S. Tennessee 6,7 : z29 : - Pogona vitticeps 3 Skin (see
above)
i
6 35 S. Monschaui 35 : m,t : - Pogona vitticeps 1 Cloaca
s(1)
7 2 S. subspec. IIIa 41 : z4,z23 : - Pantherophis guttatus 4
a
Cloaca
i Pantherophis guttatus 1 -
Chameleo calyptratus 1 -
8 3 S. subspec.IIIb 14,24;z10:z Pantherophis guttatus 1 - -
9 7 S. Newport 6,8:z10:1,2:z67 (d-
tartrate-,malonate+)
Pantherophis guttatus 2 Cloaca
s (2)
Pantherophis guttatus 1 Cloaca
10 13 S. Kandla 17:z29:- Pogona vitticeps 2 Cloaca
i
11 5 S. Monschaui Pogona vitticeps 2 -
-
Pogona vitticeps 2 -
12 5 S. subspec. IIIb 48:r:z Chameleo calyptratus 1 -
-
Chameleo calyptratus 1 -
Python sp. 0 -
Python sp. 1 -
Python sp. 2 -
Python sp. 1 -
Python sp. 1 -
13 3 S. subspec. II 42:r:- Pogona vitticeps 2 Oral cavity,
stomach,
cloaca, skin
i
14 11 S. Potsdam 6,7:l,v:e,n,z15 Testudo hermanni 1 Cloaca i (2)
15 7 S. Jangwani 17:a:1,5 Pogona vitticeps 2 Cloaca
i Pogona vitticeps 1 Oral cavity,
cloaca
16 5 S. Kumasi 30:z10:enz15 Physignathus cocincinus 1 Oral cavity
i
Physignathus cocincinus 1 Cloaca
17 5 S. subspec. IV 48:g,z51:- Pogona vitticeps 2 Oral cavity
s (1)
Pogona vitticeps 2 Skin
18 3 S. subspec. IV 44:z4,z23:- Pogona vitticeps 1 Cloaca, skin
i
Mauremys reevesii 1 Cloaca, skin
19 3 S. Eastbourne 9,12:e,h:1,5 Pogona vitticeps 1 Oral cavity,
cloaca
i
i = identical pattern; s = similar (number of minor pattern differences); in brackets: number of different fragment pattern
a
S. Subspec. IIIa 41:z4,z23:- S. Subspec. IIIb 42:k:z35; S. Paratyphi B Var.dT+ Java 4,5,12:b:1,2 phage type: Worksop; S. Florida
1,6,14,25:d:1,7 were detected in the snake
425
Figure 1. Pulsed-field gel electrophoresis (PFGE) patterns of Reptile-Exotic-Pet-Associated-Salmonellosis
(REPAS) cases from children and reptiles in different households. Shown are cases with minor pattern
differences. H - human isolate; R - reptile isolate.
426
May 27-28-29, 2013
Saint-Malo France
Session 7
Chairpersons:
Posters
427
DT7a: a very uncommon Salmonella phage type related to a human outbreak
A.A. LETTINI, E. RAMON. A. LONGO, C. SACCARDIN, M.C. DALLA POZZA,
,
P. ZAVAGNIN, E. CORTINI, K. ANTONELLO, E. MARAFIN, L. BARCO and A. RICCI
Istituto Zooprofilattico Sperimentale delle Venezie, National Reference Laboratory
for Salmonellosis, LEGNARO (PD), Italy
INTRODUCTION
A marked increase in the prevalence of a monophasic variant of Salmonella Typhimurium (S.
4,[5],12,i:-) has been noted in several European countries in the last ten years. On June 2011 an
outbreak of Salmonella 4,[5],12,i:- was reported among attendees of a wedding reception. The
source of this outbreak was identified as a cooked pork product served during the wedding party.
All Salmonella strains isolated from humans and the contaminated pork products were phage
typed as (DT) 7a, a rare phage-type, similar to DT 7, but differentiable based on a strong reaction
with phage 29. Afterwards, the farm of origin of pigs slaughtered for the production of the
contaminated product was identified, and faecal samples resulted positive for Salmonella
Typhimurium. Despite the difference in the serovar, also the Typhimurium strains isolated from
pigs were phage-typed as DT7a. All the strains isolated within the outbreak from animals, humans
and pork products were further subtyped by PFGE, MLVA, plasmid profile and resistance patterns,
in order to investigate the genetic relationship among these strains belonging to the two different
serovars. In addition, a collection of epidemiologically unrelated S. Typhimurium and S. 4,[5],12:i:-
isolates phage-typed as DT 7a, were characterized through the same subtyping methods.
MATERIALS & METHODS
In this study, 24 Salmonella strains linked to a food-borne outbreak, and 53 Salmonella strains
epidemiologically unrelated and belonging to the same phage type DT7a, were characterized.
Phage-typing was performed following the protocol of the HPA, Colindale, London (1). Serotyping
was performed according to the White-Kauffmann-Le Minor scheme by slide agglutination and
confirmed by PCR as previously described (2). PFGE was carried out with XbaI as restriction
enzyme according to the Pulse-Net protocol (3). Antimicrobial susceptibility of strains was tested
by Sensititre System (TREK Diagnostic Systems), then PCR was used to amplify the resistance genes
(4). MLVA typing was performed using the protocol described by Lindstedt et al. (5). MLVA profiles
were assigned according to the nomenclature suggested by Larsson et al. (6). Finally, plasmid
profile was analysed by using a previously described protocol (7).
RESULTS
Salmonella outbreak strains: Twenty-one isolates were serotyped as S. 4,[5],12:i:- and 3 strains as
S. Typhimurium. All the strains were phage typed as DT7a, and showed the same PFGE profile
(STYMXB.0079). Twenty isolates (83%) had R-type ASSuT, 1 isolate expressed an additional
resistance to colistin (ASSuTCol), 1 isolate was fully susceptible and 2 isolates had R-type ASSu.
Overall the phenotypic resistance patterns identified were confirmed by the PCR assays for the
specific resistance genes. MLVA identified 2 profiles. The most prevalent profile (3-13-11-n.a.-
0211) was detected for 23 (96%) out of the 24 isolates investigated. Only one strain expressed
another profile (3-12-7-n.a.-0211), which differed at loci STTR5 and STTR6
Salmonella DT7a strains epidemiologically unrelated: All Salmonella strains (53) were phage
typed as DT7a Twenty-nine out of them (55%) were serotyped as S. 4,[5], 12,i,-, and the remaining
24 strains (45%) as S. Typhimurium. 85% (45/53) of the strains were MDR and the most prevalent
R-type was ASSuT showed by 25 (47%) strains. STYMXB.0079 was the most prevalent PFGE profile
showed by 37 out of the 53 strains. 13 isolates presented profiles strictly correlated to the
dominant one, and 3 strains presented a profile clearly different from the dominant one. MLVA
428
identified 20 profiles differing at loci STTR9, STTR5 and STTR6. The 3 most common MLVA profiles
accounted for 36% of strains. In table 1 the results obtained for all strains investigated are
summarized.
DISCUSSION & CONCLUSION
The results of this study suggest that Salmonella phage type DT7a is a peculiar clone, common to
both S. Typhimurium and its monophasic variant. Data generated by using different sub-typing
methods reveal that DT7a is a very clonal phage type. It was not possible to correlate specific PFGE
or MLVA profiles to a source, and very similar subtypes were shared among different sources.
Salmonella strains analyzed in this study were highly conserved, thus it is foreesable that the
phylogenetic position of the two serovars in the context of this particular phage-type is very close,
suggesting that it is still one unique clone.
REFERENCES
1. Anderson, E.S., et al. 1977. Bacteriophage-typing designations of Salmonella Typhimurium. J. Hyg.
(Lond.) 78: 297-300.
2. Barco et al. A rapid and sensitive method to identify and differentiate Salmonella enterica serotype
Typhimurium and Salmonella enterica serotype 4,[5],12:i:- by combining traditional serotyping and
multiplex polymerase chain reaction. Foodborne Pathog Dis. 2011 Jun; 8(6): 741-
3. Ribot EM, et al. Standardization of pulsed-field gel electrophoresis protocols for the subtyping of
Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis. 2006 Spring; 3(1):
59-67.
4. Beutlich J, et al. Med-Vet-Net WP21 Project Group. Antimicrobial resistance and virulence determinants
in European Salmonella genomic island 1-positive Salmonella enterica isolates from different origins. Appl
Environ Microbiol. 2011 Aug 15; 77(16): 5655-64.
5. Lindstedt et al. Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp.
enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis. J Microbiol
Methods. 2004 Nov; 59(2): 163-72.
6. Larsson et al.Development of a new nomenclature for Salmonella Typhimurium multilocus variable
number of tandem repeats analysis (MLVA). Euro Surveill. 2009 Apr 16; 14(15).
7. Kado CI and Liu ST. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol.
1981 Mar; 145(3): 1365-73.
Table 1
Salmonella DT7a isolates used in this study
(*) PFGE profiles with or without one additional band respect to STYMXB.0079
429
Phenotypic and Molecular Characterisation
of multiresistant monophasic Salmonella Typhimurium (1,4,[5],12:i:-)
in Greece, 2006-2011
Georgia MANDILARA
1
, Maria LAMBIRI
1
, Michalis POLEMIS
2
, Maria PASSIOTOU
3

and Alkiviadis VATOPOULOS
1

1
National Reference Centre for Salmonella, National School of Public Health & Central Public
Health Laboratory, Centre of Disease Control and Prevention, VARI, Attica ,Greece;
2
Hellenic Centre of Disease Control and Prevention, ATHENS, Greece;
3
Veterinary Reference Centre for Salmonella, CHALKIS, Greece
INTRODUCTION
Recently, multiresistant S. enterica serovar 1,4,[5],12:i:-, a monophasic variant of S. Typhimurium
(1,4,[5],12:i:1,2) emerged, and is now among the most common serovars isolated from humans in
many countries [1]. In Greece, monophasic Typhimurium was recorded for the first time in human
isolates in 2007 (0.3% of total isolates), increased sharply thereafter, and since 2009 is the 3rd most
frequent serovar (Table 1). In this study, we present the results of the phenotypic and molecular
characterisation of 119 S. serovar 1,4,[5],12:i:- strains, of human, food and animal origin isolated in
Greece since 2006.
Serotyping of Salmonella spp. isolates was performed by the slide agglutination method according
to the White-KaufmannLe Minor Scheme [7]. To confirm that strains serotyped as Salmonella
serovar 1,4,[5]:i:- were Salmonella Typhimurium monophasic variants, one multiplex PCR assay was
applied to detect the presence of a specific for S. Typhimurium IS200 fragment, and the phase 2
(fljB) flagellar antigen gene [6]. Susceptibility testing was performed by the agar disk diffusion
method (Kirby-Bauer) according to CLSI [2] The following antibiotics (Biorad) were tested: ampicillin
(A), amoxicillin-clavulanic, ceftazidime, ciprofloxacin, chloramphenicol (C), ceftriaxone, kanamycin,
tobramycin, netilmicin, nalidixic acid (Na), streptomycin (S), spectinomycin (Sp), sulphonamides
(Su), tetracycline (T), trimethoprim (Tm), sulphamethoxazole-trimethoprim. Phage typing was
performed on 50 isolates, according to Health Protection Agency protocol. Pulsed Field Gel
Electrophoresis (PFGE-XbaI) was performed according to the Pulse-Net protocol [5]. Fingerprints
were analyzed using GelCompar II v.4.1 software.
RESULTS
From the 119 isolates serotyped as monophasic S. 1,4,[5],12:i:-, 97 (82%) were confirmed by the
PCR assays as Typhimurium monophasic variants. Several multiresistant patterns were observed
(Table 2), the ASSuTSpTm pattern predominated (56%), in all sources. PFGE analysis identified 17
unique profiles; 79 out of 97 isolates (81%), represented by the three predominant profiles
(STYMXB.0010, STYMXB.0079 and STYMXB.0131). The most frequent PFGE profile was
STYMXB.0010 (47%). The most frequently identified PTs were DT120 (62%) and DT193 (24%),
represented in all sources. Combining PFGE profile and R-type of all isolates, the STYMXB.0010 and
ASSuTSpTm cluster was predominant (40%); most of the isolates of this cluster belonged to DT120.
This clone, represented in humans and pigs is the most frequently occurring clone in Greece.
430
CONCLUSIONS
The main clone of monophasic Typhimurium (1,4,5,[12]:i:-) circulating in humans in Greece is that
of phage type DT120, R-type ASSuTSpTm and PFGE profile STYMXB.0010. This clone predominates
among pig isolates as well, indicating that pigs may be a reservoir of this clone in Greece. Clone of
DT193, R-type ASSuT and PFGE profile STYMXB.0131, is the predominant within several European
countries [3,4], but of low frequency in Greece. This is an ongoing study, given that NRCS 2012
data, reveal a further increase of S. monophasic Typhimurium isolates in Greece (>15% of total
isolates)
Table 1: Salmonella Enteritidis, Typhimurium and monophasic Typhimurium isolates, 2006-2011, in Greece
(n=total number of Salmonella isolates in humans).
2006
(n=697)
2007
(n=633)
2008
(n=599)
2009
(n=409)
2010
(n=241)
2011
(n=416)
Enteritidis(1)
a
407 (58%) 353 (56% ) 378 (63% ) 208 (51% ) 73 (30% ) 130 (31% )
Typhimurium(2)
a,b
87 (13% ) 68 (11% ) 40 (7%) 39 (10%) 55 (23% ) 86 (21% )
monophasic
Typhimurium
c
-
2 (0%)
(16)
a
5(1%)
(10)
a
18 (4%)
(3)
a
11 (5%)
(3)
a
17 (4%)
(3)
a
All others 203 (29%) 210 (33%) 176 (29%) 144 (35%) 102 (42%) 183 (44%)
a
serovar rank,
b
excluding monophasic variants,
c
after confirmation by PCR.
Table 2: Combination of R-type & PFGE profile (n=97) and Phage Type (n=50) for S. monophasic
Typhimurium.
R-type PFGE STYMXB. PT
ASSuTSpTm (54/97, 56%) 0010(39)
b
23H,11PG, 1 PL,4F DT120(24)
c
,DT193(2),DT97(1)
0079 (9) 3H,5PL,1F -
0131 (3) 2H,1PL DT120(2)
Other (3) 2H,1C UT
d
(2)
ASSuT( 22/97, 23%) 0010 (4) 2PG,2F DT 193(3)
0079 (3) 1H,1PL,1F -
0131 (7) 5H,1PG, 1PL DT193(3)
Other (8) 4H,4F DT193(2)
Other resistance patterns
a
(21/97, 21%) 0010 (3) 1H,1PG,1F DT120(2), DT193(1)
0079 (6) 1H,1PG,4F DT120(1), DT195(1), UT
d
(1)
0131 (5) 5H DT193(1)
Other (7) 6H,1F DT120(2), DT7(2)
TOTAL (%) 0010 (46/97,47%)
0079 (18/97,19%)
0131 (15/97,15%)
Other (18/97,19%)
DT120 (31/50,62%)
DT193 (12/50,24%)
Other (7/50,14%)
a
T (n=6), ACSSuSpTTm (n=4), SuSSpTm (n=4), ASSuSpTm (n=3), AT (n=1), S (n=1), AS (n=1), ASSuTTm (n=1),
b
H:human, F: food, PG: pig, PL:poultry,
C:cattle origin,
c
13 human, 10 animal (all of pig origin), 1 food origin,
d
Untypable:,isolates that do not react with any of the typing phages
431
REFERENCES
1. EFSA-Scientific Opinion on monitoring and assessment of the public health risk of Salmonella
Typhimurium-like strains. EFSA J. 2010; 8(10): 1826.
2. CLSI 2010. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Tenth
Edition. M02-A10.
3. Hopkins KL, Kirchner M, Guerra B, Granier SA, Lucarelli C, Porrero MC, et al. Multiresistant Salmonella
enterica serovar 4,5,[12]:i:- in Europe: a new pandemic strain? Euro Surveill. 2010;15(22):pii=19580.
Available from: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19580
4. Hopkins KL, de Pinna E, Wain J. Prevalence of Salmonella enterica serovar 4.[5],12:i:- in England and
Wales, 2010. Euro Surveill. 2012;17(37): pii=20275. Available from:
http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=20275
5. Pulsenet International 2009: One-Day Standardized Laboratory protocol for molecular subtyping of
Esherichia coli O157:H7, Salmonella serotypes, Shigella sonnei and Shigella flexneri by Pulsed Field Gel
Electrophoresis (PFGE) Protocol.
6. Tennant S, Diallo S, Levy H, Livio S, Sow SO, Tapia M, et al. Identification by PCR of non-typhoidal
Salmonella enteric serovars associated with invasive infections among febrile patients in Mali. PLOS Negl
Trop Dis.2010; 4(3): 1-9.
7. White-Kauffmann-Le Minor Scheme Antigenic Formulae of the Salmonella serovars (9
th
edition) 2007.
WHO Collaborating Centre for Reference and Research on Salmonella.
432
Molecular Typing of Monophasic Salmonella 4,[5],12:i:- Strains
Isolated in Belgium, 2008-2011
Ccile BOLAND
1
, Sophie BERTRAND
2
, Wesley MATTHEUS
2
, Katelijne DIERICK
2

and Pierre WATTIAU
1

1
Veterinary and Agrochemical Research Centre, BRUSSELS, Belgium;
2
Scientific Institute of Public Health, BRUSSELS, Belgium
INTRODUCTION
Monophasic variants of Salmonella Typhimurium have emerged in the mid-1990s in Europe.
During the last years, these strains have increasingly been reported in the context of outbreaks or
surveillance programs such as the 3 foodborne outbreaks that occurred in neighboring France in
2010 and 2011 (1-3, 8, 9). To assess the distribution of Salmonella 4,[5],12:i:- subtypes in the
Belgian food chain and compare it to the subtypes associated with human infections, a molecular
assessment was initiated. The study investigated all isolates serotyped as 4,[5],12:i:- and isolated
during the period 2008-2011 at the Belgian Salmonella National Reference Laboratories for Public
Health, Animal Health and Food. This serotype accounts for about 4% of all Salmonellae isolated
yearly in Belgium.
PCR
A duplex PCR method was performed to differentiate S. Typhimurium monophasic variants from
authentic S. Typhimurium and from other variants as recommended by the European Food Safety
Authority (1, 10)
Multilocus variable number of tandem repeats analysis (MLVA)
MLVA analyses were performed as described (7) and following the nomenclature proposed by
Larsson et al. (6).
RESULTS
Two hundred ninety-eight isolates, 114 of human, 165 of animal or food origin and 19 of
unreported origin, were first analyzed by a duplex PCR test able to differentiate S. Typhimurium
monophasic variants (STMV) from authentic S. Typhimurium and from other variants (1, 10). Of
these, 197 isolates (66.1%) displayed the STMV PCR profile. MLVA subtyping identified 56 different
profiles, representing a Simpsons Diversity index of 0.942. Four profiles were predominant and
accounted for 45% of all Belgian STMV, namely 3-13-10-NA-0211 (30 isolates), 3-12-10-NA-0211
(23 isolates), 3-12-11-NA-0211(18 isolates) and 3-12-9-NA-0211 (17 isolates). These profiles have
been previously reported in other European countries (4, 5). Eight isolates displayed the 3-13-9-
NA-0211 profile, a MLVA profile circulating in several European countries during the period 2006-
2011 and linked to the 2011 food outbreak in France(3-5). In total, 58% of all tested STMV isolates
displayed a MLVA profile already reported in Europe while the other profiles were so far
unreported.
DISCUSSION
STMV displaying MLVA profiles encountered in other European countries were observed in
Belgium. Other isolates displaying MLVA profiles unreported previously were also observed. MLVA
failed to point out a preferred contamination source or dominant MLVA subtype associated with
human infections.
433
REFERENCES
1. Anonymous. 2010. Scientific Opinion on monitoring and assessment of the public health risk of
Salmonella Typhimurium-like strains. EFSA Journal 2010; 8(10): 1826. [48 pp.].
2. Bone, A., H. Noel, S. Le Hello, N. Pihier, C. Danan, M. E. Raguenaud, S. Salah, H. Bellali, V. Vaillant, F. X.
Weill, and N. Jourdan-da Silva. 2010. Nationwide outbreak of Salmonella enterica serotype 4,12:i:-
infections in France, linked to dried pork sausage, March-May 2010. Euro Surveill 15: pii=19592.
3. Gossner, C. M., D. van Cauteren, S. Le Hello, F. X. Weill, E. Terrien, S. Tessier, C. Janin, A. Brisabois, V.
Dusch, V. Vaillant, and N. Jourdan-da Silva. 2012. Nationwide outbreak of Salmonella enterica serotype
4,[5],12:i:- infection associated with consumption of dried pork sausage, France, November to December
2011. Euro Surveill 17: pii=20071.
4. Hopkins, K. L., E. de Pinna, and J. Wain. 2012. Prevalence of Salmonella enterica serovar 4,[5],12:i:- in
England and Wales, 2010. Euro Surveill 17: pii=20275.
5. Hopkins, K. L., M. Kirchner, B. Guerra, S. A. Granier, C. Lucarelli, M. C. Porrero, A. Jakubczak, E. J.
Threlfall, and D. J. Mevius. 2010. Multiresistant Salmonella enterica serovar 4,[5],12:i:- in Europe: a new
pandemic strain? Euro Surveill 15: pii=19580.
6. Larsson, J. T., M. Torpdahl, R. F. Petersen, G. Sorensen, B. A. Lindstedt, and E. M. Nielsen. 2009.
Development of a new nomenclature for Salmonella Typhimurium multilocus variable number of tandem
repeats analysis (MLVA). Euro Surveill 14: pii=19174.
7. Lindstedt, B. A., T. Vardund, L. Aas, and G. Kapperud. 2004. Multiple-locus variable-number tandem-
repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and
multicolor capillary electrophoresis. J Microbiol Methods 59: 163-72.
8. Raguenaud, M., S. Le Hello, S. Salah, F. Weill, A. Brisabois, G. Delmas, and P. Germonneau. 2012.
Epidemiological and microbiological investigation of a large outbreak of monophasic Salmonella
Typhimurium 4,5,12:i:- in schools associated with imported beef in Poitiers, France, October 2010. Euro
Surveill 17: pii=20289.
9. Switt, A. I., Y. Soyer, L. D. Warnick, and M. Wiedmann. 2009. Emergence, distribution, and molecular and
phenotypic characteristics of Salmonella enterica serotype 4,5,12:i. Foodborne Pathog Dis 6: 407-15.
10. Tennant, S. M., S. Diallo, H. Levy, S. Livio, S. O. Sow, M. Tapia, P. I. Fields, M. Mikoleit, B. Tamboura, K. L.
Kotloff, J. P. Nataro, J. E. Galen, and M. M. Levine. 2010. Identification by PCR of non-typhoidal Salmonella
enterica serovars associated with invasive infections among febrile patients in Mali. PLoS Negl Trop Dis 4:
e621.
434
Diversity of human Salmonella Typhimurium
and Salmonella enterica 4,[5],12:i- strains
isolated in Emilia Romagna Region, Italy
Elena CARRA
1
, Marina MORGANTI
2
, Francesco CORPUS
1
, Erika SCALTRITI
2
,
Federica BERGAMINI
1
and Stefano PONGOLINI
2

1
Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia-Romagna (IZSLER),
MODENA, Italy
2
Istituto Zooprofilattico Sperimentale della Lombardia e dellEmilia-Romagna (IZSLER),
PARMA, Italy
INTRODUCTION
According to national surveillance data, in Italy Salmonella Typhimurium (ST) and Salmonella
enterica 4,[5],12:i- (STM) constitute more than fifty per cent of all Salmonella enterica isolates
from human enteric infections over the period 2009-2011
(1)
. The aim of this study was to evaluate
the genetic variability of ST and STM strains isolated from human infections in Emilia-Romagna, a
Region in the North of Italy with a population of five millions.
From September 2011 to September 2012 926 human Salmonella enterica strains were isolated
and serotyped resulting in 320 ST (34.6%) and 252 (27.2%) STM. The serotype was confirmed by
PCR for the deletion of fljB flagellar gene in monophasic variant isolates
(2)
. Genotyping was
performed by Pulsed-field gel electrophoresis (PFGE) using XbaI restriction enzyme following the
PulseNet standard protocol
(3)
and by the multiple-locus variable-number tandem-repeat analysis
(MLVA) based on the amplification of five loci: STTR9-STTR5-STTR6-STTR10pl-STTR3 according to a
previously described protocol
(4)
. To analyse the clonal relationships among the isolates, based on
the PFGE profiles, a dendrogram was generated using the unweighted paired group method with
arithmetic average (UPGMA) based on the Dice Similarity Index by BioNumerics 6.6 Software. The
MLVA profiles were imported as character data into BioNumerics, used for categorical clustering
and a Minimum Spanning Tree (MST) was constructed. Simpsons Index of Diversity (SID) and
Shannons Index of Diversity (H) were calculated to compare the discriminatory ability of two
techniques
(5)
.

RESULTS
PFGE showed 143 different profiles, each profile included 1-65 isolates, and 71 were unique to one
single isolate. PFGE profiles sharing by more than 5 isolates were 26 including 363 isolates. MLVA
showed 130 different profiles, each profile included 1-50 isolates, most variability was due to the
STTR5 and the STTR6 (20 alleles each) with a SID value of 0.796 (95% CI: 0.779-0.813) and 0.864
(95% CI: 0.850-0.878) respectively. At STTR10pl locus, located on the pSLT plasmid, only 68 isolates
had PCR product showing 15 different alleles. Twenty MLVA profiles were observed in more than 5
isolates, including 381 isolates, and 66 profiles in one single isolate. A SID value of 0.973 (95% CI:
0.967-0.978) and of 0.967 (95% CI: 0.962- 0.972) was obtained for PFGE and MLVA respectively.
The p-value calculated according to the jackknife pseudo-values resampling method resulted 0.11.
The Shannons index of diversity values resulted 6.105 (95% CI: 6.092-6.118) and 5.769 (95% CI:
5.755-5.783) for PFGE and MLVA respectively. The p-value calculated according to Hutcheson 't'
test
(6)
for the Shannon index between techniques resulted 0.0027.
The dendrogram obtained from the PFGE profiles identified twenty distinct clusters (I XX) with
greater than 74% similarity (Figure 1). Cluster analysis using a MST based on MLVA profiles (Figure
1) detected one major (A) and eight minor (B-I) clusters. In the MLVA cluster A most isolates of the
major PFGE clonal groups (IV-V-VI) were grouped.
435
DISCUSSION
In order to determine the diversity of human ST and STM strains isolated in Emilia Romagna
Region the MST based on the MLVA profiles proved to be useful to investigate relatedness among
strains dividing 572 isolates into one major and eight minor clusters. More than one PFGE profiles,
represented by different colours, could correspond to one MLVA profile, represented as node in
the MST. On the contrary in some minor MLVA clusters (C, G, E) different nodes (different MLVA
profiles) belonged to the same PFGE cluster.
In this study SID values calculated for PFGE and MLVA were high and no significant difference was
observed between them, indicating that both methods were similarly appropriate to discriminate
different genotype. Shannons index of diversity gave a significantly higher value for PFGE in
comparison with MLVA, indicating a greater discriminatory ability of PFGE than MLVA in
consistence with the MST results. Different results obtained with SID and H can be ascribed to the
nature of these indices: SID is a dominance index that gives more weight to common profiles,
while H is able to better highlight rare subtypes.
CONCLUSION
The combination of MLVA and PFGE results allowed to improve subtype discrimination and to
establish clear clonal relations among isolates.
REFERENCES
1. Luzzi I et al. (2012), ISTISAN Congressi 12/C2, pag.4.
2. Tennant SM et al. (2010), PLoS Negl Trop Dis Mar 9;4(3):e621.doi: 10.1371/journal.pntd.0000621
3. http://www.pulsenetinternational.org/ SiteCollectionDocuments/pfge/
4. Lindstedt et al. (2004), J. Microbiol. Methods 59, 163-172.
5. http://darwin.phyloviz.net/ComparingPartitions/
6. Hutcheson, K. (1970) A test for comparing diversities based on the Shannon formula. J. Theor. Biol., 29,
151-4
TABLES AND FIGURES
Figure 1: Minimum-spanning tree based on MLVA
allelic profiles of 320 Salmonella serotype
Typhimurium and 252 serotype 4,[5],12:i- isolates.
Each node represents a unique MLVA profile, and
nodal size is proportional to the number of isolates.
The different lines indicate the distance between the
circles, where a thick line represents a closer
distance than a thin line. The colors of the circles
indicate different PFGE clusters. Nine MLVA clusters,
differing in no more than 1 of the five VNTR loci, are
regarded as a group and are highlighted (MLVA
cluster A, pink; cluster B, violet; cluster C, orange;
cluster D, yellow; cluster E, light blue; cluster F,
brilliant green; cluster G, gray; cluster H, green;
cluster I, dark violet).
A
B
C
D
E
F
G
I
N isolates; PFGE
clusters
436
Salmonella serovars and phage types associated with specific animal
reservoirs and their epidemiology situation in the United Kingdom
Doris MUELLER-DOBLIES and Robert H. DAVIES
Bacteriology and Food Safety Department, AHVLA Weybridge, Woodham Lane, New Haw,
ADDLESTONE, KT15 3NB, United Kingdom
INTRODUCTION
In 2011, 9,015 cases of human salmonellosis were confirmed in the UK, of which 28.5% were
caused by S. Enteritidis, and 23.8 % were caused by S. Typhimurium. S. Enteritidis has traditionally
has been mainly attributed to eggs, whereas S. Typhimurium is often linked to pork and pork
products; however, travel-related infections and infections caused by imported food also play an
important role. Several Salmonella outbreaks have been linked to non-food animal related
sources, such as vegetable produce, fruits, chocolate and contact with reptiles. We analysed the
distribution of Salmonella serovars reported from livestock species and the contribution they
might make to human cases.
Data on the number of reported human salmonellosis cases were obtained from the Health
Protection Agency (HPA). These were compared to data from livestock compiled by the Animal
Health and Veterinary Laboratories Agency (AHVLA). Under the UK Zoonoses Order 1989,
laboratories isolating Salmonella from any species listed in the Order have to send the isolate to
the Salmonella reference laboratory at AHVLA Weybridge for further typing.
Salmonella reports from livestock are reported as incidents in this publication. An incident is
defined as the first isolation (and all subsequent isolations) of the same serovar or
serovar/phagetype combination from an animal or group of animals and their environment within
30 days. This definition was chosen, as there are often several isolations of the same serovar from
a group of animals which represent one epidemiological unit.
RESULTS
The number of reported human salmonellosis cases in the UK has dropped significantly over the
past decade, from 15,434 in 2000 to 9,015 in 2011. This reduction was mainly due to a reduction
in cases caused by S. Enteritidis (from 8,618 in 2000 to 2,566 in 2011), but also, to a lesser extent,
to a reduction of S. Typhimurium cases (from 2,688 in 2000 to 2,141 in 2011, including
monophasic and biphasic strains). This decline in human cases of S. Enteritidis coincides with a
decline in S. Enteritidis reports from chickens, believed to be mainly the result of the introduction
of the National Control Programmes for the control of Salmonella. While there were 37 S.
Enteritidis incidents reported from chickens in 2007, only eight were reported in 2011. The likely
correlation between the reduction in S. Enteritidis cases in humans and in chickens is also
supported by the fact that phagetype 4 (PT4), which used to be the predominant phagetype in
laying hens in the 1990s now only represents 10.6% of S. Enteritidis reports in humans, compared
for example with 57.2% in 2000 or 53.4% in 1993.
While the number of S. Typhimurium cases in people has decreased slightly over the past decade,
the proportion of monophasic strains of S. Typhimurium has increased from 247 in 2000 to 953 in
2011. Human S. Typhimurium isolates and monophasic strains of S. Typhimurium now mainly
belong to phagetypes 193 and 120 which are also commonly seen in pigs. The emergence of
monophasic strains of S. Typhimurium has been seen across many European countries with two
clonal lineages described. S. Typhimurium and monophasic strains of S. Typhimurium are the most
commonly reported serovars in British pigs, together representing more than 75% of incidents. S.
437
Typhimurium phagetypes from pigs were mainly U288 (37.7%) and DT193 (22%), while
monophasic strains belonged to phagetypes 193 (80%) and 120 (10%).
The predominant serovars in turkeys, S. Derby, S. Kottbus and S. Kedougou represented only
0.32%, 0.33% and 0.16% of human cases, respectively, and are therefore not considered major
sources of human salmonellosis.
In cattle, S. Dublin represented the majority of incidents, which is rarely seen in people (0.24% of
cases); in sheep, the situation is more complex, with the most common Salmonella being the
diarizonae serovars, S. 61:k:1,5,7 and S. 61:-:1,5,7 which are rarely seen in people; however, S.
Montevideo accounted for 22.7% of sheep incidents in 2011 and it is unclear if there is a common
link between S. Montevideo infection of sheep and of people. An increase in S. Montevideo
reports can also be seen in broiler chickens, but it has not yet been established if there is a
common link between human and animal cases.
In 2010, an increase in the number of S. Typhimurium DT8 cases in people was seen with a strong
correlation between infection and the consumption of duck eggs.
The number of S. Montevideo cases in people has almost tripled within a year, from 58 in 2010 to
155 in 2011, even though only representing 1.7% of human cases in 2011.
DISCUSSION
While it seems to be obvious that the reduction in human salmonellosis cases caused by S.
Enteritidis was mainly due to the reduction of prevalence in the chicken industry, and particularly
in the egg laying sector, the impact the emergence of monophasic strains of S. Typhimurium has
on human salmonellosis cases is not clear. The contemporaneous correlation between an increase
in human and pig incidents suggests a link which would need further studies to be evaluated in
detail.
While it was estimated that 2.6% of human salmonellosis cases across the EU may be attributable
to turkeys (Efsa Panel on Biological Hazards, 2012), it seems likely that consumption of turkey
meat in the UK has a much lesser impact on human salmonellosis cases, based on the prevalence
of serovars such as S. Derby and S. Kedougou found in UK turkeys and the prevalence of those
serovars in the human population.
The recent increase in human salmonellosis cases caused by infection with S. Montevideo may be
linked to the rise in cases among sheep, chickens and turkeys; however, it could also be due to a
confounding factor, like contaminated grain or other feed ingredients being used both for human
consumption and as an ingredient in animal feed. To date, no epidemiological investigation has
been preformed to investigate possible links or confounding factors.
CONCLUSION
Salmonella serovars seen in livestock species other than chickens and pigs in the UK only represent
a small minority of Salmonella cases in people and therefore do not seem to have a major impact
on human case numbers. However, emerging serovars such as multi-resistant S. Kentucky, S.
Infantis, S. Heidelberg or S. Stanley found in poultry in some other EU countries or S. Typhimurium
DT8 in duck egg production need to be closely monitored.
REFERENCES
Efsa Panel on Biological Hazards, 2012. Scientific opinion on an estimation of the public health impact of
setting a new target for the reduction of Salmonella in turkeys. EFSA Journal 10, 2616-2616.
438
About three cases of intestinal carriage
of Salmonella enterica serovar Toulon in NICU
S. Marcello VITALITI
1
, Francesca DI BERNARDO
2
, Aurora ALEO
3
and Caterina MAMMINA
3

1
Neonatal Intensive Care Unit, ARNAS Ospedale Civico, Di Cristina & Benfratelli,
PALERMO, Italy;
2
Laboratory of Microbiology, ARNAS Ospedale Civico, Di Cristina & Benfratelli,
PALERMO, Italy;
3
Department of Sciences for Health Promotion G. DAlessandro, University of Palermo,
PALERMO, Italy
INTRODUCTION
Non-typhoidal salmonellae are major etiologic agents of enteric infection. These organisms can be
problematic even in otherwise healthy hosts, because of possible bacteremic spread and focal
infection. However, infections by non-typhoidal salmonellae are especially challenging in poorly
immunocompetent individuals, including newborns and infants, who may experience prolonged
intestinal carriage and are at risk of developing serious and potentially lethal invasive infection.
We report the asymptomatic intestinal carriage of S. enterica serovar Toulon by three infants who
were staying in the neonatal intensive care unit (NICU) of the ARNAS General Hospital Civico,
Benfratelli & Di Cristina, Palermo, Italy. The epidemiological investigation, which has been carried
out after the accidental detection of the first case, is also described.
Patients
1
st
case: he was an extremely preterm newborn with congenital heart disease born on October 6,
2011. He was admitted soon after the birth and discharged to a pediatric cardiac surgery unit on
January 16, 2012. During his NICU stay, he was fed with hydrolysed cow's milk proteins and
powdered infant formula (PIF). S. Toulon was isolated from a rectal swab following the transfer of
the infant to the surgery unit. The rectal swab was performed as a screening at admission the
cardiac surgery unit. No symptoms or signs of enteritis were present neither at admission nor
through the entire clinical course.
2
nd
case: he was a preterm newborn with respiratory distress born on October 13, 2011. He was
admitted soon after the birth and discharged home on January 31, 2012. His was fed with sterile
liquid infant formula. S. Toulon was recovered from a rectal swab during the active surveillance
program, which was started in the NICU soon after the alert about the first isolation.
3
th
case: she was a premature twin girl with cerebral cortical malformation. She was born on
November 2, 2011, admitted to the NICU on November 25 and discharged home on February 3,
2012. She was fed with sterile liquid infant formula. S. Toulon was isolated from the nasogastric
tube and rectal swab.
The latter two infants were asymptomatic for gastrointestinal events during their NICU stay.
No antibacterial drug treatment was administred in any of the three cases.
All samples were processed by standard techniques. The three Salmonella isolates underwent
serotyping and antibacterial drug susceptibility testing against 16 agents using the Kirby-Baer
method on Meller-Hinton agar. The three isolated were fully susceptible to the panel of tested
antibiotics.
Epidemiological investigation and infection control intervention
According with the infection control policy of It was not possible to reliably identify the time of
acquisition of S. Toulon by the three cases, because the investigation started when the first case
was discovered after his discharge. But, as the figure shows, their NICU stay overlapped for six
weeks. Therefore, a cross-transmission was possible.
439
All healthcare workers of the NICU were interviewed to assess their health status, but were not
screened. Parents of cases 2 and 3 were also interviewed about possible risk factors of exposure to
Salmonella, such as housing type and pet owning, but nothing of relevance was reported.
The epidemiological investigation included also environmental and PIF sampling. Environmental
specimens were collected from multiple surfaces and equipment in the NICU using Dacron swabs
pre-moistened with sterile water. Samples of infant foods (opened or unopened) were also
collected and cultured for Salmonella using 100 g samples, when available.
Contact precautions were adopted in NICU after the detection of the two carriers. Hand washing
and general control infection measures were reinforced.
The investigation failed to detect the source of Salmonella, but proved that spread of this
organism was restricted and eventually stopped by the infection control measures.
No further cases were detected in the following month by the active surveillance cultures.
Figure. Pattern of spread of S. Toulon in NICU
case 1
case 2
case 3
O
c
t
o
b
e
r

2
0
1
1

N
o
v
e
m
b
e
r

2
0
1
1
D
e
c
e
m
b
e
r

2
0
1
1
J
a
n
u
a
r
y

2
0
1
2
DISCUSSION
S. Toulon is an exceedingly rare serovar. In the Enternet-Italy database only one isolate has been
recorded in 2010 from a pediatric patient. More recently, two isolates have been reported from a
survey on 499 European wild boars hunted in the Latium region, Italy. This suggests a possible
misunderstood circulation of this serovar. Of interest, this serovar is lactose fermenting, that can
jeopardize its detection in the routine diagnostic microbiologic analysis. Indeed, less than 1% of
nontyphoidal Salmonella are lactose positive and, generally, this biochemical character is a
predictor of non-Salmonella isolates.
Our observation shows a colonization in three NICU infants by an atypical Salmonella serovar. It
is well known, on the other hand, that infants may have prolonged intestinal carriage of
Salmonella species.
Infection control measures were effective in interrupting transmission, even if the source and the
route of entry remained obscure. The more reliable hypothesis was the entry of S. Toulon in NICU
via an healthcare workers or, alternatively, via the infant (case 3) who required admission after
living at home for about three weeks. Cross-transmission via the hands of caregivers was likely
responsible for the additional cases.
PIF analysis yielded no results. However, even if the poor sensitivity of cultural methods on these
food products is widely agreed, in our case a possible role for PIF appears to be unlikely because
only one of the three infants was fed with PIF. Moreover, the large use of PIF in the NICU would
have reasonably caused a larger number of colonization cases.
Our experience suggests also that the NICU is a healthcare setting especially prone to importation
of potentially pathogenic organisms from the community.
440
REFERENCES
Galanis E, Lo Fo Wong DMA, Patrick ME, et al. Web-based surveillance and global Salmonella distribution,
20002002. Emerg Infect Dis 2006; 12: 3818.
Hohmann E. L. Non typhoidal salmonellosis. Clinical Infectious Diseases 2001; 32: 2639.
McDonough PL, Shin SJ, Lein DH. Diagnostic and public health dilemma of lactose-fermenting Salmonella
enterica serotype Typhimurium in cattle in the Northeastern United States. J Clin Microbiol. 2000; 38: 1221-
6.
Zottola T, Montagnaro S, Magnapera C, Sasso S, De Martino L, Bragagnolo A, D'Amici L, Condoleo R,
Pisanelli G, Iovane G, Pagnini U. Prevalence and antimicrobial susceptibility of Salmonella in European wild
boar (Sus scrofa); Latium Region - Italy. Comp Immunol Microbiol Infect Dis. 2013; 36: 161-8.
441
A Multi-Country Outbreak of Salmonella Newport
Associated with Watermelon
Elizabeth DE PINNA
1
, Tansy PETERS
1
, Clare MAGUIRE
1
, Alison MATHER
2
, Nick THOMSON
2
,
Chris LANE
3
and Ian FISHER
3
1
Public Health England, Microbiology Services Division, Gastrointestinal Bacteria
Reference Unit, Colindale, LONDON, UK;
2
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton,
CAMBRIDGE, UK;
3
Public Health England, Health Protection Services, Colindale, LONDON, UK
INTRODUCTION
Salmonella enterica subsp. enterica Newport (S. Newport) is one of the more common Salmonella
serovars and has been implicated in several food poisoning outbreaks.
In November 2011, a Food, Water and Environment (FWE) Laboratory of the Health Protection
Agency isolated S. Newport from a ready-to-eat sliced watermelon fan.
In December 2011, Health Protection Scotland (HPS) reported four cases of S. Newport that were
indistinguishable by Pulsed-field Gel Electrophoresis (PFGE). This PFGE profile had not previously
been seen and was shown to be indistinguishable from the PFGE profile of the watermelon isolate.
The reporting of S. Newport cases in England, Wales and Northern Ireland also exceeded expected
levels in December 2011. In January 2012, Germany and Ireland also reported an increase in the
number of S. Newport cases.
On 16
th
January 2012, a multi-agency outbreak control team (OCT) was convened to investigate
the increased incidence of cases of S. Newport. Food safety authorities from the United Kingdom
(UK), Germany and Ireland joined the OCT.
METHODS
Microbiological investigations:
The isolates of Salmonella from patients and food were serotyped according to the Kauffmann-
White scheme [1]. PFGE was performed on all of the isolates using the Pulse Net-Europe/Salm-
gene protocol [2].
A sub-set of isolates was selected for whole genome sequencing: 24 of the patients confirmed as
part of the outbreak in the UK; eleven isolates of S. Newport from patients not part of the
outbreak; and the isolate from the watermelon. Samples of the genomic DNA were sequenced
using multiplex libraries on the Illumina HiSeq platform using 100bp paired-end reads. The
sequence data were aligned to the reference strain S. Newport SL254.
Epidemiological investigations:
Food histories were obtained from the patients infected with the outbreak strain of S. Newport.
Food safety authorities carried out trace-back investigations of watermelons.
RESULTS
Microbiological:
PFGE analysis found the S. Newport isolate from the watermelon had a unique profile that had not
previously been seen. This profile was designated SNWPXB.0110. During the outbreak period 1
st

December 2011 to 3
rd
February 2012 a total of 63 human cases of S. Newport from the six
countries all with the SNWPXB.0110 PFGE profile were reported.
Whole genome sequencing showed a high divergence between the outbreak isolates and the
reference strain SL254, with ~50,600 single nucleotide polymorphisms (SNPs) separating the
outbreak isolates from the reference strain SL254. The sequencing data revealed near-identical
sequences between the watermelon and 24 outbreak isolates. Five isolates had a single SNP
442
compared to the watermelon isolate, of which two were within genes of known function - an
acetyltransferase and a sensory histidine kinase.The eleven non-outbreak isolates did not cluster
with the watermelon isolate.
Epidemiological:
In total, food histories were obtained from 42 of the confirmed cases. Of these, 27 of the cases
reported consumption of watermelon.
In England, trace-back of the contaminated watermelon fan indicated the product originated from
a batch of watermelons imported from Brazil. Trace-back in Scotland found watermelons had also
been imported from Brazil but from a different grower. Trace-back on watermelons consumed by
cases in Ireland found them to be from the same Brazilian source as the watermelon fan.
Watermelons purchased in Germany were found to have been from a distributor in the
Netherlands who imported them from Brazil.
DISCUSSION
Molecular, epidemiological and trace-back methods were used to link the consumption of
watermelons imported to six European countries from Brazil to an outbreak of S. Newport.
The contamination of a ready-to-eat watermelon fan with S. Newport was detected during a local
food survey by a FWE laboratory of the HPA in November 2011. Monitoring of the incidence of
Salmonella infection in the human population across the six countries involved in this outbreak
highlighted an increase of cases of S. Newport reported during the period 1
st
December 2011 to 3
rd

February 2012.
Molecular typing by PFGE of the isolates using the same protocol in each country enabled the
profiles to be compared. Analysis of the PFGE profiles showed the isolate from the watermelon
had a unique profile that had not previously been seen SNWPXB.0110. Analysis of the PFGE
profiles from the human cases showed that 63 had the SNWPXB.0110 profile and were
indistinguishable from the watermelon isolate.
Whole genome sequencing showed a sub-set of the human isolates and the watermelon isolate
had near identical sequences. Whole genome sequencing did not cluster non-outbreak S. Newport
isolates with the watermelon isolate. Both PFGE analysis and whole genome sequencing show the
human isolates to be indistinguishable from the watermelon isolate. This suggested that
watermelon was the source of the outbreak.
The food histories obtained from the human cases showed 64% of the cases reported consuming
watermelon and this is considerably higher than would be expected in the general population in
these countries at this time of year. This also suggests watermelon was the source of this
outbreak.
Trace-back of watermelons in all six countries showed them to have been imported from Brazil,
with the growers all being located in the same geographical region. Salmonellosis outbreaks
associated with a variety of melons have been reported previously in the United States and
Canada. In August 2006, there was an outbreak of S. Newport linked to watermelon in New York
State [3]. To our knowledge, this is the first outbreak of Salmonella associated with melons in
Europe and highlights the importance of this food as a potential source of infection.
ACKNOWLEDGEMENTS
The investigation of this outbreak could not have been carried out without the help of:
The staff of the Salmonella Reference Laboratory, HPA, Colindale, London.
Members of the International Outbreak Control Team.
The whole genome sequencing was supported by Wellcome Trust grant WT076964.
443
REFERENCES
1. Grimont, P.A.D. and Weill, F.X. 2007. Antigen formulas of the Salmonella serovars. WHO Collaborating
Centre for Reference and Research on Salmonella, Institut Pasteur, Paris, France.
http://www.pasteur.fr/ip/portal/action/WebdriveAction/oid/01s-000036-089
2. Peters T. et al. Relationships of pulsed-field profiles with key phages types of Salmonella Enteritidis in
Europe: results of an international multi-centre study. Epidemiol Infect. 2007 Nov; 135(8): 1274-81
3. Centres for Disease Control and Prevention (CDC). Foodborne Outbreak Online Database (FOOD).
Available at http://wwwn.cdc.gov/foodborneoutbreaks/Default.aspx
444
Foodborne disease outbreaks associated
with the consumption of raw milk cheese in France, August September 2012
Sandra GIRON
1
, Simon LE HELLO
2
, Siham SALAH
3
, Mariola MAZUR
4
, Renaud LAILLER
5
,
Delphine BARATAUD
1
, Damien MOULY
1*
and Nathalie JOURDAN-DA SILVA
1
1
French Institute for Public Health Surveillance
2
Institut Pasteur, French National Reference Center for E. coli, Shigella and Salmonella
3
General Directorate for Food Sanitary Emergency Unit
4
DDCSPP63, Departmental Social Cohesion and the Protection of Populations, PUY-DE-DME
5
ANSES, French Agency for Food, Environmental and Occupational Health & Safety,
Maisons-Alfort Laboratory for Food Safety, Unit Bacterial Characterization and Epidemiology
* Corresponding author : Damien Mouly, InVS regional office Auvergne, 60 avenue de
l'union sovitique, 63200 Clermont Ferrand, France
INTRODUCTION
August 30th, 2012: Notification of foodborne outbreak in France (Auvergne) involving 10 patients
who all consumed raw milk cheese (St Nectaire). Salmonella serotype Dublin (S. Dublin) was
identified in one hospitalized case. At the same time, two other foodborne outbreaks associated
with the consumption of the same type of cheese were notified in France (Auvergne and
Bretagne). These foodborne outbreaks had many characteristics in common (consumption of St
Nectaire, incubation period and symptoms, dates of onset of symptoms closely grouped in time,
high attack rate, i.e. 86%). Considering all these factors, the most likely hypothesis was a common
source of contamination through consumption of St Nectaire cheese distributed nationally. A
national alert was launched on August 31
st
and an outbreak control team, comprising the national
institute for public health surveillance (InVS), the French National Reference Center (FNRC) for E.
coli, Shigella and Salmonella and various veterinary services, was formed to describe the health
impact of the foodborne outbreaks, identify the producer(s) of the contaminated cheese and
propose appropriate control measures.
Cases definition
- A confirmed case was defined as someone with isolated S. Dublin, identified by the FNRC
between August 22
nd
and September 11
th
, 2012;
- A probable case was defined as someone with gastroenteritis and with an epidemiological link to
a confirmed case.
Notifications of foodborne outbreaks occurring during the same period with symptoms and
incubation consistent with Salmonella contamination were also examined. Cases and other
participants who shared the same meals were interviewed by phone about symptoms and their
food intake during the three days before the onset of symptoms.
Veterinary investigation
Traceback investigations of cheese consumed by the cases were conducted by the veterinary local
offices and the General Directorate for Food.
Microbiological investigation
Identification of Salmonella isolates was performed by using conventional methods, and
serotyping performed on the basis of the White-Kauffmann-Le Minor scheme.
Comparison of profiles of strains found from human (stools) and non-human (uneaten pieces of
the consumed St Nectaire) origin was performed using PulseNet standard pulsed-field gel
electrophoresis (PFGE) of XbaI-digested chromosomal DNA.
445
RESULTS
Active case finding identified 15 foodborne outbreaks in France between August 12
th
and 31
st
,
2012. This included 9 clusters in Auvergne and three isolated cases associated with consumption
of St. Nectaire cheese (Figure 1).
Finally, 103 cases (16 confirmed and 87 probable) of 123 exposed persons developed symptoms
after eating raw milk cheese (attack rate = 84%) between August 12
th
and September 2
nd
. Ages
ranged between 5 and 78 years. Ten non-immunocompromised cases were hospitalized (median
age = 39 years; median length of stay = 3 days). No death was reported.
Traceback investigations identified 2 different producers of St Nectaire living in Auvergne (farm A
and B) and 1 refiner C receiving cheese from farm A. In one of these two producers, S. Dublin was
isolated from one cow in each of the 2 producers herds (1 premature calf from a symptomatic
cow and 1 asymptomatic cow after microbiological research in milk).
In one month, 17 strains of S. Dublin were identified by the FNRC in 16 different individuals
samples. The biochemical profile of these strains corresponded to the usual profile found in this
serotype including the absence of gamma-glutamyltransferase activity.
In addition, 4 uneaten pieces of consumed St Nectaire remaining in private homes tested positive
for S. Dublin with the same antigenic formula 9,12:g,p:. These strains were isolated from two
different producers of cheese.
According to the comparison results carried out by the French Agency for Food, Environmental
and Occupational Health & Safety (ANSES), the PFGE patterns of these strains of human (stools)
non-human origin (St Nectaire) were identical (SDUBX0003). This profile is the dominant one
found in the PFGE database of ANSES (142 of 168, i.e. 85%), suggesting the weak discriminatory
power of PFGE for this serotype.
September 4th, 2012: withdrawal / recall of contaminated lots of cheese was implemented by the
producers at the request of the General Directorate for Food (regional office). Among the 7580 Kg
of cheese produced by the two farms, 17% (1335 pounds) was immediately recalled from the
market.
DISCUSSION
Epidemiological, microbiological and veterinary investigations helped identify two different
producers of St Nectaire as the sources of the outbreak.
The hypothesis of a common source for the two farms strains cannot be confirmed (molecular
typing methods are not discriminating enough for this serotype).
While S. Dublin preferentially affects immunocompromised persons, morbidity and virulence
being higher than in other serotypes [1-3]. This foodborne outbreak of S. Dublin is characterized
by its limited impact on health (people of all ages were affected, patients showed no individual risk
factor). The effectiveness of epidemiological, microbiological and veterinarian investigations
enabled actors to implement control measures very quickly after the launch of the alert.
REFERENCES
1. LR Nielsen. Overview of pathogenesis, epidemiology and diagnostic tools Necessary for successful
monitoring and eradication of Salmonella Dublin from the Danish cattle population prize assignment
"Professor Dr.med.hc CO Mindefond Jensens." Department of Large Animal Sciences, University of
Copenhagen, 2009. 70 p.
2. Centers for Disease Control (CDC). Salmonella dublin and Raw Milk Consumption - California, MMWR.
Morbidity and mortality weekly report 33:14 1984 Apr 13 pg 196-8.
3. V. Vaillant, S. Haeghebaert, J.C. Desenclos, P. Bouvet, F. Grimont, P.A. Grimont 3, A.P. Burnens. Outbreak
of Salmonella Dublin infection in France, November-December 1995. Eurosurveillance Vol 1 n2 august
1996
446
TABLES AND FIGURES
Figure 1: Epidemic curve representing the foodborne disease outbreaks (blue squares) and isolated
case with respect to the date of the onset of symptoms and the place of origin of the contaminated
St Nectaire cheese, August-September 2012.
447
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
Koen DE REU
1*
, Marc HEYNDRICKX
1
, Geertrui RASSCHAERT
1
, Sophie BERTRAND
2
,
Christa WILDEMAUWE
2
, Pierre WATTIAU
3
, Hein IMBERECHTS
3
, Lieve HERMAN
1
,
Richard DUCATELLE
4
, Stephanie VAN WEYENBERG
1
and Isabelle DEWAELE
5

1
Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit,
Brusselsesteenweg 370, 9090 MELLE, Belgium;
2
National Reference Centre for Salmonella and Shigella, Bacterial Diseases Division, Scientific
Institute of Public Health (IPH), Juliette Wytsmanstraat 14, 1050 BRUSSELS, Belgium;
3
Veterinary and Agrochemical Research Centre (CODA-CERVA), Groeselenberg 99,
1180 BRUSSELS, Belgium;
4
Ghent University, Faculty of Veterinary Medicine, Department of Pathology, Bacteriology
and Poultry Diseases, Salisburylaan 133, 9820 MERELBEKE, Belgium;
5
Formerly at ILVO; now Poulpharm, Prins Albertlaan 111, 8870 IZEGEM, Belgium
* Corresponding author: Koen.Dereu@ilvo.vlaanderen.be
ABSTRACT
The aim of the study was to characterize human and layer farm related Salmonella Enteritidis (SE) isolates collected
from 2000-2010, to determine whether the types were comparable for layer and human isolates (a) before
implementation of vaccination (Period 1; 2000-2004), (b) during voluntary vaccination (Period 2; 2005-2006) and (c)
during implementation of the national control program (NCP) for Salmonella including mandatory vaccination against SE
(Period 3;2007-2010) as well as to investigate whether a different type distribution has arisen in either of the populations
since the implementation of the NCP. Therefore, phage typing and multiple-locus variable number tandem-repeat assay
(MLVA) typing were performed.
While PT4 and PT21 were predominantly isolated in layers and humans before 2007, a significant reduction of those
PTs was observed in both populations in the period 2007-2010. The relative proportion of PT4b, PT21c and PT6c was
found to have increased considerably in the layer population and to a lesser extent in the human population since 2007.
In the human population, PT8, PT1 and other PTs were more frequently isolated compared to the previous periods. The
proportion of most PTs (e.g., PT4, PT21, PT8, PT1) was not found to be significantly different between both populations
in Period 1, while a significant difference was found in Period 3.
When comparing the proportion of the predominant MLVA types Q2 and U2, no significant difference was found between
the layer and human population in the three periods and between periods within each category (layer and human). A
significant difference in isolate distribution among MLVA clusters I and II was found between human and layer isolates
recovered during Period 3 and in the human population between Period 1 and 3.
Results confirm the link between SE in layers and the occurrence of the pathogen in humans, although the correlation
seems to be reduced in Belgium since the implementation of the NCP.
INTRODUCTION
The aim of this comparison was twofold. First, the PT and MLVA distribution between layer farm
and human isolates was investigated and compared for the following periods: (a) before the
implementation of vaccination (Period 1; 2000-2004), (b) during periods to determine whether a
different PT and/or MLVA type distribution has arisen in either of the populations since the
implementation of the NCP. In relation to the layer farm isolates, the aim was to investigate
whether vaccination has led to a shift in the SE population colonizing the laying hens.
The isolate collection, serotyping, phage typing, MLVA typing (Dewaele et al. 2012b) and statistical
analysis is described in detail in Dewaele et al. (2013) or can be asked in detail to the
corresponding author of this proceeding.
448
RESULTS
Results are outlined in detail in Dewaele et al. (2013) or can be asked in detail to the
corresponding author of this proceeding.
DISCUSSION
In conclusion, the present study identified interesting features about the trends in PT and MLVA
types in SE from human and layer populations within different epidemiological contexts. Although
prevalence figures already indicated that the vaccination of laying hen flocks has contributed to a
significant reduction of Belgian human SE cases since 2004. In the present study, a significant
reduction of the most prevalent phage types PT21 and PT4 was noticed in both populations since
the implementation of a NCP with mandatory vaccination against SE. The shift towards PT4b,
PT21c and PT6c in the layer population since 2007 indicates that probably some persisting SE
isolates remain on layer farms, which was also observed by Dewaele et al. (2012b). However, the
limitations of phage typing should also be considered. Because the relation between observed
human and layer types has reduced since the implementation of a NCP with mandatory
vaccination indicates a relative reduction of Belgian eggs related to Belgian human SE cases.
However, SE isolates present on layer farms still seem to have an important share in the reported
human SE cases which was suggested by indistinguishable PT and MLVA types observed in both
isolatevoluntary vaccination (Period 2; 2005-2006) and (c) during the implementation of the NCP
including mandatory vaccination (Period 3; 2007-2010). Secondly, the present study aimed to
investigate the PT and MLVA distribution of human SE isolates and layer farm SE isolates between
these collections during the period 2007-2010. Still, further investigation is necessary to estimate
the most important sources responsible for human SE infections and the route of transmission.
Therefore, it is important to include SE isolates from other animal sources and from food sources
(e.g., eggs and egg products) from importing countries in connection to Belgium. Unfortunately, SE
isolate collections from other animal and food sources are very rare. Alternatively, comparison of
available PT data from humans and layer farms from other European countries can also be
informative.
ACKNOWLEDGEMENTS
This research was funded by the Belgian Federal Public Service for Health, Food Chain Safety and
Environment (RF 6195).
REFERENCES
1. Dewaele I, Rasschaert G, Bertrand S, Wildemauwe C, Wattiau P, Imberechts H, Herman L, Ducatelle R, De
Reu K and Heyndrickx M (2012b). Molecular characterization of Salmonella Enteritidis: Comparison of an
optimized Multi-Locus Variable-Number of Tandem Repeat Analysis (MLVA) and Pulsed-Field Gel
Electrophoresis. Foodborne Pathog. Dis. 9(10): 885-95.
2. Dewaele I, Rasschaert G, Wildemauwe C, Van Meirhaeghe H, Vanrobaeys M, De Graef E, Herman L,
Ducatelle R, Heyndrickx M and De Reu K (2012a). 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. Poult. Sci. 91(11): 2727-35.
3. Dewaele I, Heyndrickx M, Rasschaert G, Bertrand S, Wildemauwe C, Wattiau P, Imberechts H, Herman L,
Ducatelle R, Van Weyenberg S and De Reu K. (2013). 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. Zoonoses and Public Health, accepted.
449
Epidemiology and antimicrobial resistance
of human and avian Salmonella isolated
in N'Djamena, Chad
Djim-Adjim TABO
1,2,3
, Colette D. Diguimbaye
3
, Sophie A. GRANIER
4
, Frdrique MOURY
4
,
Anne BRISABOIS
4
, Rachid ELGROUD
5
and Yves MILLEMANN
2

1
Facult des Sciences Exactes et Appliques (FSEA), Universit de NDjamena, Chad
2
ENVA-UPSP MASQ (Microbiologie des Aliments, Scurit et Qualit),
7, avenue du Gnral de Gaulle, 94704 MAISONS ALFORT, France
3
Laboratoire de Recherches Vtrinaire et Zootechnique de Farcha (LRVZ/F),
BP : 433 NDJAMENA, Tchad
4
Universit Paris-Est, ANSES, Laboratoire de Scurit des Aliments de Maisons-Alfort,
Unit Caractrisation et Epidmiologie Bactrienne, 23, avenue du Gnral de Gaulle,
94706 MAISONS-ALFORT cedex, France
5
Laboratoire dhygine et dinspection des denres alimentaires dorigine animale,
Dpartement des sciences vtrinaires, Universit de Mentouri, CONSTANTINE, Algeria
INTRODUCTION
Salmonella infection is a major health concern and continues to have a serious economic impact
worldwide (Flor et al., 2011). Nontyphoidal salmonellosis is usually acquired by ingestion of
contaminated water or food, particularly foods of animal origin such as beef, poultry, eggs, and
dairy products. Poultry meat and derived products are a major source in many developed
countries (Hald et al., 2004). In Chad, a country located in the middle of Africa, Salmonella strains
and other major zoonotic bacterial pathogens are not often isolated and identified, and the
resistance of these pathogens including Salmonella to commonly used antimicrobial is rarely or in
no way assessed. This study represents the first Chadian investigations to gather knowledge on
Salmonella infections and resistance in humans, in laying hen and broiler chicken in NDjamena.
Study design, sample collection, Salmonella identification and antimicrobial resistance
Our study was carried out during two seasons, from August 2010 to January 2011 and from
September 2011 to February 2012, and involved five hospitals and sixteen out of the 30 functional
laying hen and broiler chicken farms identified in NDjamena, Chad.
The samples collected were analysed according to French Norm for Salmonella Spp. NF ISO
6579/2002. Serotyping and antimicrobial susceptibility was carried out respectively according to
the Scheme of White-Kauffmann-Le Minor (Guibourdenche et al., 2010) and the recommendations
of Clinical and Laboratory Standards Institute (CLSI, 2006).
RESULTS
Prevalence, distribution and antibiotic susceptibility of the Salmonella serotypes
A total of 128 pooled samples collected from 16 NDjamenas poultry farms, were analysed for the
presence of Salmonella and of these, 56 (44%) pooled samples were positive for Salmonella. In the
hospitals, over a total of 420 human faeces sampled and analysed, 13.1% were positive for
Salmonella. At the end of our study, 139 Salmonella strains, belonging to 41 different serotypes
were isolated. The majority of these serotypes were pan-susceptible to all antibiotics tested,
except some S. Colindale isolates that exhibited a decreased susceptibility to fluoroquinolones, S.
Limete isolates resistant to three antimicrobial, S. Entiritidis isolates that were resistant to
fluoroquinolones and S. Minnesota isolates resistant to five different antimicrobial classes (Table
1).
450
DISCUSSION AND CONCLUSION
Before our research, there was no recent information available on Salmonella prevalence and
antibiotic resistance on Chadian hospitals and poultry farms. The present results of our study
provide better information concerning prevalence rate and antimicrobial resistance on Salmonella
contamination in hospitals, broiler and laying hen farms of NDjamena. The different serotypes
and antibiotic resistance profiles that were evidenced highlight importance of Salmonella diversity
in Chad, the contribution of avian isolates to human cases of salmonellosis and its capacity to
colonize all types of environment throughout the world (Majowicz et al., 2010). This study also
provides the first baseline survey to build an educational program about food safety for producers,
retailers and consumers of poultry and derived products.
AKNOWLEDGEMENTS
This study was carried out with a grant of the French cooperation. We wish to acknowledge the
MASQ-ENVA staff for their entire availability. We are also grateful for the technical support of the
CEB unit staff (ANSES, Maisons-Alfort Laboratory for Food Safety). We also do not want to forget
to express our gratitude to the LRVZ and the University of NDjamena for their financial
contribution.
REFERENCES
1. Flor, M., Sanchas-Vargas, Maisam, A., Abu-El-Haija, Oscar, G., Gomez-Duarte, 2011. Salmonella
infections: An update on epidemiology, management, and prevention. Travel Medicine and Infectious
Disease. 9:263-277.
2. Guibourdenche, M., Roggentin, P., Mikoleit, M., Fields, P.I., Bockemuhl, J., Grimont, P.A., and Weill FX.,
2010. Supplement 2003-2007 (No. 47) to the White-Kauffmann-Le Minor scheme. Res. Microbiol. 161:26-
29.
3. Hald, T., Vose, D., Wegner, H.C., Koupeev, T., 2004. A Bayesian approach to quantify the contribution of
animal-food sources to human salmonellosis. Risk Anal. 24: 255269.
4. Majowicz, S.E., Musto, J., Scallan, E., Angulo, F.J., Kirk, M., OBrien, S.J., Jones, T.F., Frazil, A., Hoekstra,
R.M., 2010. The global burden of non-typhoidal Salmonella gastroenteritis. Clin. Infect. Dis. 50: 882-889.
451
TABLES AND FIGURES
Table 1: Characterisation of Salmonella isolated from hospitals and poultry farms in NDjamena.
Serovars
Origin of
isolates
Number of
isolates
Prevalence
rate (%)
Antimicrobial phenotype
Resistance profiles
Nb of Isolates
(+) / tested
S. Colindale
Avian 16
(13.6 %)
NA
1
, OFX
1
, ENR
1
12/16
Human 3 Pan-susceptible
2
3/3
S. Minnesota Avian 15 (10.8 %)
AMP, CHL, SSS, SXT,
STR, TET
15/15
S. Stanleyville Human 8 (5.8 %) Pan-susceptible 8/8
S. Havana
Avian 5
(5 %)
Pan-susceptible 5/5
Human 2 Pan-susceptible 5/5
S. Kottbus
Avian 4
(5 %)
Pan-susceptible 4/4
S. Idikan
Avian 3
(4.3 %)
Pan-susceptible 3/3
S. Riggil Avian 5 (3.6 %) Pan-susceptible 5/5
S. Anatum Human 5 (3.6 %) Pan-susceptible 5/5
S. Muenchen
Avian 3
(2.9 %)
Pan-susceptible 3/3
S. Enteritidis
Avian 1
(2.2 %)
Pan-susceptible 1/1
Human 2 NA, OFX, CIP 2/2
S. Limete Avian 1 (0.7 %) SXT, SSS, TET 1/1
S. Typhimurium Avian 1 (0.7 %) Pan-susceptible 1/1
Others Salmonella Avian & Human 58 (41.7 %) Pan-susceptible 58/58
1= decreased susceptibility, 2= susceptible to all antimicrobials tested, Nb= number
AMP: ampicillin, CHL: chloramphenicol, ENR: enrofloxacin, NA: nalidixic acid, OFX: ofloxacin, CIP: ciprofloxacin, SSS:
sulphonamids, STR: streptomycin, SXT: trimethoprim-sulfamethoxazole (= cotrimoxazole), TET: tetracyclin.
452
Ad hoc study on Salmonella Stanley outbreak strains
Dariusz WASYL
1
, Magdalena ZAJC
1
, Katarzyna DOMASKA-BLICHARZ
2

and Andrzej HOSZOWSKI
1

1
National Veterinary Research Institute, Department of Microbiology, PUAWY, Poland;
2
National Veterinary Research Institute, Department of Poultry Diseases, PUAWY, Poland
INTRODUCTION
An increasing number of Salmonella enterica subsp. enterica serovar (S.) Stanley noted recently in
humans in several EU countries has led to joint ECDC/EFSA action aimed at the detection of
infection source [1]. The outbreak onset was set after August 2011 and the outbreak strain was
defined as S. Stanley with PFGE pattern matching the Belgium strain without outside the EU
travel history within a week before the onset. The strain shows microbiological resistance to
nalidixic acid and ciprofloxacin.
In response to the ECDC/EFSA request ad hoc characteristics of the isolates obtained in Poland
was performed to confirm their epidemiological relations.
Nine S. Stanley isolated from September 2011 to February 2012 from official sampling in turkey
farms within national Salmonella control program or hygienic checks at slaughter plants were used
for analysis. Antimicrobial resistance was tested with microbroth ditulion reference method and
interpreted according to EUCAST epidemiological values. XbaI and BcuI-PFGE was run according to
PulseNet protocol, as well as ERIC-PCR and plasmid proffiling were used to increase discrimination.
Quinolone Resistance Determining Regions (QRDR) of the genes encoding for gyrase and
topoisomerase IV subunits and Plasmid Mediated Quinolone Resistance (PMQR) determinants
were amplified and relevant amplicons were sequenced and deposited in GenBank.
RESULTS
The analysed S. Stanley were isolated from three turkey farm environments (boot swab samples)
and turkey meat (N=5) on different processing steps, including neck skin samples and minced
meat. One isolate was obtained at fox farm, from a sample of turkey by-product intended for
feeding. All isolates showed microbiological resistance to nalidixic acid (MIC >64 mg/L) and
ciprofloxacin (MIC = 0.25 0.5 mg/L). Single isolate was also resistant to tetracycline (MIC >64
mg/L), and three others to ampicillin (MIC >32 mg/L), gentamycin (MIC > 16 >32 mg/L) and
sulfamethoxazole (MIC >1024 mg/L). All isolates were gyrA (Asp87Tyr), parC (Thr57Ser) mutants.
Three plasmid profiles were observed: all isolates contained plasmid of 4,7 kb, but one had an
additional plasmid of 3,9 kb and three others carried a plasmid >53.1 kb. All isolates were tested
negative for plasmid mediated determinants encoded by qnrA, qnrB, qnrC, qnrD, and qnrS.
DISCUSSION
S. Stanley is common serovar in Asia and most of human cases in Europe have been related to
travel [2] or imported food [3]. An increase of infections with this rare serovar acquired in the EU
have been recently noted [1]. Herewith ad hoc study run in responce to ECDC and EFSA request
confirmed the presence of ciprofloxacin resistant S. Stanley in turkey primary production and its
spread into the food chain, including slaughter plant hygiene checks, turkey meat and by-products.
The described isolates were the only representatives of that serovar amongst over 20.000
Salmonella isolates refered to NVRI for reference tesing since 2005. Molecular typing has shown
genetic homogeneity of the isolates that might be related to the current pan-European outbreak
[1].
453
The application of traditional typing methods such as plasmid profiling and resistance patterns
enabled identification of four isolate groups. Being aware of the limitations of the method due to
plasmid instability in bacterial host, in a short term, they might serve as valuable epidemiological
marker in outbreak investigations.
The observed MIC values corresponded to nucleotide substitutions found in QRDR [4] and no
previously described in S. Stanley PMQR mechanisms were found [5].
In conclusion, ad hoc study confirmed that few available S. Stanley isolates fit into the
description of outbreak strain and might compromise public health.
REFERENCES
1. ECDC Joint ECDC/EFSA rapid risk assessment: Multi-country outbreak of Salmonella Stanley infections.
Update. Stockholm: 2012. Available from: http://ecdc.europa.eu
2. Hendriksen RS, Le Hello S, Bortolaia V, Pulsrikarn C, Nielsen EM, Pornruangmong S, et al.
Characterization of isolates of Salmonella enterica serovar Stanley, a serovar endemic to Asia and
associated with travel. J Clin Microbiol. 2012; 50(3): 709-20.
3. Kirk MD, Little CL, Lem M, Fyfe M, Genobile D, Tan A, et al. An outbreak due to peanuts in their shell
caused by Salmonella enterica serotypes Stanley and Newport-sharing molecular information to solve
international outbreaks. Epidemiol Infect. 2004 ;132(4): 571-7.
4. Hopkins KL, Davies RH, Threlfall EJ. Mechanisms of quinolone resistance in Escherichia coli and
Salmonella: recent developments. Int J Antimicrob Agents. 2005; 25(5): 358-73.
5. Hopkins KL, Wootton L, Day MR, Threlfall EJ. Plasmid-mediated quinolone resistance determinant qnrS1
found in Salmonella enterica strains isolated in the UK. J Antimicrob Chemother. 2007;59(6):1071-5.
454
TABLES AND FIGURES
Figure 1. Summary characteristics of tested S. Stanley isolates
R-type: Nal nalidixic acid, Cip ciprofloxacin, Tcy tetracycline, Amp ampicillin, Gen gentamycin; Str
streptomycin, Smx sulfamethoxazole
QRDR: substitutions in quinolone resistance regions of qyrA and parC (GenBank Accession JX970947JX970964), no
substitutions found in gyrB and parE
Plasmid profile: plasmid molecular weight was assessed against Escherichia coli V517 plasmids (53,1 kb, 7,2 kb, 5,1 kb,
4,7 kb, 3,9 kb, 3,0 kb, 2,7 kb, 2,1 kb)
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455
A perfect storm : Misdiagnosed typhoid fever in a laboratory worker
in a context of possible vaccine failure
Karen H. KEDDY*
1,2
, Arvinda SOOKA
1
, Husna ISMAIL,
1,2
Nomsa TAU
1,2
, M. Alfred MTHANTI
1

and Anthony M. SMITH
1,2

1
Centre for Enteric Diseases (CED), National Institute for Communicable Diseases,
National Health laboratory Service, Johannesburg, South Africa and Faculty of Health
Sciences, University of the Witwatersrand, Johannesburg, South Africa
2
Faculty of Health Sciences, University of the Witwatersrand, JOHANNESBURG,
South Africa
Abstract:
Typhoid fever remains an important disease in developing countries, with an estimated
burden of 21,65 million cases and over 200 000 deaths annually world-wide. In recent years,
the numbers of typhoid fever cases notified in South Africa have decreased. Typhoid fever is
a well-recognised hazard for microbiology laboratory workers, and those who are frequently
exposed to Salmonella enterica serotype Typhi should receive vaccine. In September 2012, a
27 year-old laboratory worker presented with signs of malaise, fever and mild diarrhoea.
Blood cultures were done. The diagnostic laboratory identified Salmonella on MALDI
Biotyper, and reported non-typhoidal Salmonella (NTS), susceptible to fluoroquinolones. The
patient was treated with oral ciprofloxacin for seven days. The isolate was referred to the
Centre for Enteric Diseases (CED) for confirmation where Salmonella Typhi, where
intermediately resistant to fluoroquinolones, was confirmed. The patient was still excreting
Salmonella Typhi in her stool and was retreated with azithromycin and ciprofloxacin, for 14
days. Subsequent stools were negative for Salmonella Typhi. The patient had recently
received typhoid vaccine. This vaccine was found to be part of a batch recalled by the
manufacturer, because of potentially low (below specification) antigen content. Pulsed-Field
Gel Electrophoresis (PulseNet protocol) and multilocus variable-number tandem-repeat
analysis confirmed that the isolate was 100% identical to a cluster of selected Salmonella
Typhi isolates (H58 haplotype), received as part of national surveillance for typhoid fever, on
which the patient had worked. This case report highlights critical issues: misreporting of the
isolate as NTS, with associated public health implications: typhoid fever is a notifiable
disease in South Africa and follow up of both the patient and the patients contacts are
integral to disease management. Failure to update antimicrobial reporting according to new
CLSI guidelines by the diagnostic laboratory resulted in inappropriate treatment being used
initially and the organisms was not cleared from the patients gut. No vaccine is 100%
protective, in this case, the vaccine may have been faulty; and failure to adhere to good
laboratory practice puts that individual at risk for laboratory-acquired infection.
456

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Salmonella and Salmonellosis

We can thus speculate that these different entry processes may

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Salmonella and Salmonellosis 2013

Salmonella and Salmonellosis 2013

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