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Methods in
Molecular Biology 2291
Stephanie Schüller
Martina Bielaszewska Editors
Shiga Toxin-
Producing
E. coli
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
Shiga Toxin-Producing E. coli
Methods and Protocols
Edited by
Stephanie Schüller
Norwich Medical School, University of East Anglia, Norwich, UK
Martina Bielaszewska
National Reference Laboratory for E. coli and Shigellae, National Institute of Public Health,
Prague, Czech Republic
Editors
Stephanie Schüller Martina Bielaszewska
Norwich Medical School National Reference Laboratory for E. coli and Shigellae
University of East Anglia National Institute of Public Health
Norwich, UK Prague, Czech Republic
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1338-2 ISBN 978-1-0716-1339-9 (eBook)
https://doi.org/10.1007/978-1-0716-1339-9
© Springer Science+Business Media, LLC, part of Springer Nature 2021

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
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Preface
Since its discovery in 1982, Shiga toxin-producing E. coli (STEC) has gained notoriety for
causing severe renal and neurological disease due to the production of potent Shiga toxins.
The importance of this pathogen has been underlined by the publication of the Methods in
Molecular Medicine book E. coli: Shiga Toxin Methods and Protocols edited by Dana Philpott
and Frank Ebel in 2003. Novel methodologies have been developed since which have
contributed to the detection, clinical diagnosis, and treatment of STEC infections as well
as a better understanding of its epidemiology and pathogenesis. In particular, the accessibil-
ity of whole genome sequencing and bioinformatic tools has demonstrated the fluidity of
the STEC genome and blurring of boundaries with other E. coli pathotypes which has
become relevant in the large 2011 outbreak in Germany caused by a Shiga toxin-producing
enteroaggregative E. coli hybrid strain. Similar to the 2006 “Spinach outbreak” in the USA,
the source of the German epidemic was traced to contaminated fresh produce, thus empha-
sizing the need to understand STEC persistence in the extraintestinal environment. In
addition, it has become evident that STEC virulence factors including Shiga toxins are
released within outer membrane vesicles and extracellular vesicles and can thus travel within
the host over long distances and contribute to pathogenesis. Another shift in perspective has
been evoked by the discovery that the expression of STEC virulence genes in the gut is
largely governed by environmental cues including physicochemical factors and signals from
the host cells and resident microbiota. This knowledge is reflected in the sophistication of
biologically relevant STEC infection models including fermenters, stem cell-derived orga-
noids, and advanced in vivo models. In this book, we have aimed to include these new
technologies for the detection, characterization, and investigation of STEC and Shiga
toxins. We hope this book will become a valuable resource for clinicians, epidemiologists,
members of the food and farming industry, and researchers interested in STEC
pathogenesis.
We would like to thank the series editor John Walker for the invitation to edit this book
and guiding us through every step of the process. Most importantly, we are grateful to all
authors who shared their methodology and expertise and dedicated time to writing their
chapters despite the ongoing COVID pandemic.
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Integrated Approach for the Diagnosis of Shiga Toxin-Producing

Escherichia coli Infections in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Stefano Morabito, Fabio Minelli, and Rosangela Tozzoli
2 Shiga Toxin-Producing E. coli in Animals: Detection, Characterization,
and Virulence Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Stefanie A. Barth, Rolf Bauerfeind, Christian Berens,
and Christian Menge
3 Identification of Shiga Toxin-Producing Escherichia coli
Outbreaks Using Whole Genome Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Stefan Bletz, Alexander Mellmann, and Barbara Middendorf-Bauchart
4 Predicting Host Association for Shiga Toxin-Producing E. coli
Serogroups by Machine Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Nadejda Lupolova, Antonia Chalka, and David L. Gally
5 Isolation and Characterization of Shiga Toxin Bacteriophages . . . . . . . . . . . . . . . . 119
Lorena Rodrı́guez-Rubio and Maite Muniesa
6 Lambda Red–Mediated Recombination in Shiga
Toxin-Producing Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Kenneth G. Campellone and Alyssa M. Coulter
7 Functional Analysis of Shiga Toxin-Producing Escherichia coli
Biofilm Components in Plant Leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Nicola J. Holden, Kathryn M. Wright, Jacqueline Marshall,
and Ashleigh Holmes
8 Virulence Factor Cargo and Host Cell Interactions of Shiga
Toxin-Producing Escherichia coli Outer Membrane Vesicles . . . . . . . . . . . . . . . . . . 177
Martina Bielaszewska, Lilo Greune, Andreas Bauwens, Petra Dersch,
Alexander Mellmann, and Christian Rüter
9 Isolation and Characterization of Shiga Toxin-Associated
Microvesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Annie Willysson, Anne-lie Ståhl, and Diana Karpman
10 Thin-Layer Chromatography in Structure and Recognition Studies
of Shiga Toxin Glycosphingolipid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Johanna Detzner, Gottfried Pohlentz, and Johannes Müthing
11 Identification of Nanobodies Blocking Intimate Adherence
of Shiga Toxin-Producing Escherichia coli to Epithelial Cells . . . . . . . . . . . . . . . . . 253
David Ruano-Gallego and Luis Ángel Fernández
vii
viii Contents
12 Determining Shiga Toxin-Producing Escherichia coli Interactions

with Human Intestinal Epithelium in a Microaerobic Vertical
Diffusion Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Conor J. McGrath and Stephanie Schüller
13 Human Epithelial Stem Cell-Derived Colonoid Monolayers
as a Model to Study Shiga Toxin-Producing Escherichia coli–Host
Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Karol Dokladny, Julie G. In, James Kaper, and Olga Kovbasnjuk
14 Use of the Dynamic TIM-1 Model for an In-Depth Understanding
of the Survival and Virulence Gene Expression of Shiga Toxin-Producing
Escherichia coli in the Human Stomach and Small Intestine . . . . . . . . . . . . . . . . . . 297
Ophélie Uriot, Sandrine Chalancon, Carine Mazal,
Lucie Etienne-Mesmin, Sylvain Denis, and Stéphanie Blanquet-Diot
15 Measuring Effector-Mediated Modulation of Inflammatory
Responses to Infection with Enteropathogenic and Shiga
Toxin-Producing E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Georgina L. Pollock, Cristina Giogha, and Elizabeth L. Hartland
16 Interaction of Bovine Lymphocytes with Products of Shiga
Toxin-Producing Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Andrew G. Bease, Robin L. Cassady-Cain, and Mark P. Stevens
17 Infection of Immunocompetent Conventional Mice with Shiga
Toxin-Producing E. coli: The DSS + STEC Model . . . . . . . . . . . . . . . . . . . . . . . . . . 353
Gregory Hall, Shinichiro Kurosawa, and D. J. Stearns-Kurosawa
18 Infant Rabbit Model for Studying Shiga Toxin-Producing
Escherichia coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
Jennifer M. Ritchie
19 Citrobacter rodentium Lysogenized with a Shiga Toxin-Producing
Phage: A Murine Model for Shiga Toxin-Producing E. coli Infection . . . . . . . . . . 381
Laurice J. Flowers, Shenglan Hu, Anishma Shrestha,
Amanda J. Martinot, John M. Leong, and Marcia S. Osburne
20 Overview of the Effect of Citrobacter rodentium Infection
on Host Metabolism and the Microbiota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Eve G. D. Hopkins and Gad Frankel
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Contributors
STEFANIE A. BARTH • Friedrich-Loeffler-Institut/Federal Research Institute for Animal

Health, Institute of Molecular Pathogenesis, Jena, Germany
ROLF BAUERFEIND • Institute for Hygiene and Infectious Diseases of Animals, Justus Liebig
University Gießen, Gießen, Germany
ANDREAS BAUWENS • Institute for Hygiene, University of Münster, Münster, Germany
ANDREW G. BEASE • The Roslin Institute and Royal (Dick) School of Veterinary Studies,
University of Edinburgh, Midlothian, UK
CHRISTIAN BERENS • Friedrich-Loeffler-Institut/Federal Research Institute for Animal
MARTINA BIELASZEWSKA • National Reference Laboratory for E. coli and Shigellae, National
Institute of Public Health, Prague, Czech Republic; Institute for Hygiene, University of
Münster, Münster, Germany
STÉPHANIE BLANQUET-DIOT • Microbiology Digestive Environment and Health, UMR
UCA-INRA 454 MEDIS, Université Clermont Auvergne, Clermont-Ferrand, France
STEFAN BLETZ • Institute of Hygiene and National Consulting Laboratory for Hemolytic
Uremic Syndrome (HUS), University Hospital Münster, Münster, Germany
KENNETH G. CAMPELLONE • Department of Molecular & Cell Biology, Institute for Systems
Genomics, University of Connecticut, Storrs, CT, USA
ROBIN L. CASSADY-CAIN • The Roslin Institute and Royal (Dick) School of Veterinary Studies,
SANDRINE CHALANCON • Microbiology Digestive Environment and Health, UMR
ANTONIA CHALKA • Division of Infection and Immunity, The Roslin Institute and Royal
(Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK
ALYSSA M. COULTER • Department of Molecular & Cell Biology, Institute for Systems
Genomics, University of Connecticut, Storrs, CT, USA
SYLVAIN DENIS • Microbiology Digestive Environment and Health, UMR UCA-INRA
454 MEDIS, Université Clermont Auvergne, Clermont-Ferrand, France
PETRA DERSCH • Institute for Infectiology, Center for Molecular Biology of Inflammation
(ZMBE), University of Münster, Münster, Germany
JOHANNA DETZNER • Institute for Hygiene, University of Münster, Münster, Germany
KAROL DOKLADNY • Division of Gastroenterology, Department of Internal Medicine,
University of New Mexico Health Sciences Center, University of New Mexico School of
Medicine, Albuquerque, NM, USA
LUCIE ETIENNE-MESMIN • Microbiology Digestive Environment and Health, UMR
LUIS ÁNGEL FERNÁNDEZ • Department of Microbial Biotechnology, Centro Nacional de
Biotecnologı́a, Consejo Superior de Investigaciones Cientı́ficas (CNB-CSIC), Madrid,
Spain
LAURICE J. FLOWERS • Department of Molecular Biology and Microbiology, Tufts University
School of Medicine, Boston, MA, USA; Tufts University Graduate School in Biomedical
Sciences, Boston, MA, USA; Department of Dermatology, University of Pennsylvania,
Philadelphia, PA, USA
ix
x Contributors
GAD FRANKEL • MRC Centre for Molecular Bacteriology and Infection, Department of Life
Sciences, Imperial College London, London, UK
DAVID L. GALLY • Division of Infection and Immunity, The Roslin Institute and Royal
CRISTINA GIOGHA • Centre for Innate Immunity and Infectious Diseases, Hudson Institute of
Medical Research, Clayton, VIC, Australia; Department of Molecular and Translational
Science, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton,
VIC, Australia
LILO GREUNE • Institute for Infectiology, Center for Molecular Biology of Inflammation
GREGORY HALL • Department of Pathology and Laboratory Medicine, Boston University
School of Medicine, Boston, MA, USA; Toxikon Corporation, Bedford, MA, USA
ELIZABETH L. HARTLAND • Centre for Innate Immunity and Infectious Diseases, Hudson
Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and
Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash
University, Clayton, VIC, Australia
NICOLA J. HOLDEN • Cell & Molecular Sciences, The James Hutton Institute, Dundee, UK
ASHLEIGH HOLMES • Cell & Molecular Sciences, The James Hutton Institute, Dundee, UK
EVE G. D. HOPKINS • MRC Centre for Molecular Bacteriology and Infection, Department of
Life Sciences, Imperial College London, London, UK
SHENGLAN HU • Department of Molecular Biology and Microbiology, Tufts University School
of Medicine, Boston, MA, USA; Institute of Animal Science, Guangdong Academy of
Agricultural Sciences, State Key Laboratory of Livestock and Poultry Breeding, Key
Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture
and Rural Affairs, Guangdong Key Laboratory of Animal Breeding, Guangzhou, China
JULIE G. IN • Division of Gastroenterology, Department of Internal Medicine, University of
New Mexico Health Sciences Center, University of New Mexico School of Medicine,
Albuquerque, NM, USA
JAMES KAPER • Department of Microbiology & Immunology, University of Maryland School of
Medicine, Baltimore, MD, USA
DIANA KARPMAN • Department of Pediatrics, Clinical Sciences Lund, Lund University,
Lund, Sweden
OLGA KOVBASNJUK • Division of Gastroenterology, Department of Internal Medicine,
University of New Mexico Health Sciences Center, University of New Mexico School of
Medicine, Albuquerque, NM, USA
SHINICHIRO KUROSAWA • Department of Pathology and Laboratory Medicine, Boston
University School of Medicine, Boston, MA, USA
JOHN M. LEONG • Department of Molecular Biology and Microbiology, Tufts University
School of Medicine, Boston, MA, USA
NADEJDA LUPOLOVA • Division of Infection and Immunity, The Roslin Institute and Royal
JACQUELINE MARSHALL • Cell & Molecular Sciences, The James Hutton Institute, Dundee,
UK
AMANDA J. MARTINOT • Department of Infectious Diseases and Global Health, Tufts
Cummings School of Veterinary Medicine, North Grafton, MA, USA
CARINE MAZAL • Microbiology Digestive Environment and Health, UMR UCA-INRA
CONOR J. MCGRATH • Norwich Medical School, University of East Anglia, Norwich, UK
Contributors xi
ALEXANDER MELLMANN • Institute of Hygiene and National Consulting Laboratory for

Hemolytic Uremic Syndrome (HUS), University Hospital Münster, Münster, Germany;
Institute for Hygiene, University of Münster, Münster, Germany
CHRISTIAN MENGE • Friedrich-Loeffler-Institut/Federal Research Institute for Animal
BARBARA MIDDENDORF-BAUCHART • Institute of Hygiene and National Consulting
Laboratory for Hemolytic Uremic Syndrome (HUS), University Hospital Münster,
Münster, Germany
FABIO MINELLI • Istituto Superiore di Sanità, Rome, Italy
STEFANO MORABITO • Istituto Superiore di Sanità, Rome, Italy
MAITE MUNIESA • Department of Genetics, Microbiology and Statistics, University of
Barcelona, Barcelona, Spain
JOHANNES MÜTHING • Institute for Hygiene, University of Münster, Münster, Germany
MARCIA S. OSBURNE • Department of Molecular Biology and Microbiology, Tufts University
GOTTFRIED POHLENTZ • Institute for Hygiene, University of Münster, Münster, Germany
GEORGINA L. POLLOCK • Centre for Innate Immunity and Infectious Diseases, Hudson
Institute of Medical Research, Clayton, VIC, Australia; Department of Molecular and
Translational Science, Faculty of Medicine, Nursing and Health Sciences, Monash
University, Clayton, VIC, Australia
JENNIFER M. RITCHIE • University of Surrey, Guildford, UK
LORENA RODRÍGUEZ-RUBIO • Department of Genetics, Microbiology and Statistics, University
of Barcelona, Barcelona, Spain
DAVID RUANO-GALLEGO • Department of Microbial Biotechnology, Centro Nacional de
Biotecnologı́a, Consejo Superior de Investigaciones Cientı́ficas (CNB-CSIC), Madrid,
Spain
CHRISTIAN RÜTER • Institute for Infectiology, Center for Molecular Biology of Inflammation
STEPHANIE SCHÜLLER • Norwich Medical School, University of East Anglia, Norwich, UK
ANISHMA SHRESTHA • Department of Molecular Biology and Microbiology, Tufts University
ANNE-LIE STÅHL • Department of Pediatrics, Clinical Sciences Lund, Lund University,
Lund, Sweden
D. J. STEARNS-KUROSAWA • Department of Pathology and Laboratory Medicine, Boston
University School of Medicine, Boston, MA, USA
MARK P. STEVENS • The Roslin Institute and Royal (Dick) School of Veterinary Studies,
ROSANGELA TOZZOLI • Istituto Superiore di Sanità, Rome, Italy
OPHÉLIE URIOT • Microbiology Digestive Environment and Health, UMR UCA-INRA
ANNIE WILLYSSON • Department of Pediatrics, Clinical Sciences Lund, Lund University,
Lund, Sweden
KATHRYN M. WRIGHT • Cell & Molecular Sciences, The James Hutton Institute, Dundee, UK
Chapter 1
Integrated Approach for the Diagnosis of Shiga

Toxin-Producing Escherichia coli Infections in Humans
Stefano Morabito, Fabio Minelli, and Rosangela Tozzoli
Abstract
Shiga toxin-producing Escherichia coli (STEC) are human pathogens causing severe diseases, such as
hemorrhagic colitis and the hemolytic uremic syndrome. The prompt diagnosis of STEC infection is of
primary importance to drive the most appropriate patient’s management procedures. The methods to
diagnose STEC infections include both direct isolation of the STEC from stool samples and the identifica-
tion of indirect evidences based on molecular, phenotypic, and serological applications. Here, the proce-
dures in use at the Italian Reference Laboratory for E. coli infections are described.
Key words Shiga toxin, Real time PCR, STEC isolation, Vero cell assay, ELISA, Anti-lipopolysacchar-
ide antibodies, Serology
1 Introduction
Shiga toxin-producing Escherichia coli (STEC) cause a spectrum of

diseases in humans, including either enteric or systemic syndromes.
In fact, following infection different outcomes may occur. These
include asymptomatic carriage of the microorganism, mild diar-
rhea, or hemorrhagic colitis, and the life-threatening hemolytic
uremic syndrome (HUS). The latter represents the most severe
manifestation of STEC infection, usually occurring in children,
the elderly, and immuno-compromised patients [1]. HUS is char-
acterized by hemolytic anemia, thrombocytopenia, and acute renal
failure [2] and is the major cause of kidney impairment in children,
resulting in fatality in 2–7% of cases [3]. HUS can also cause long-
term sequelae, such as renal impairment, hypertension, or neuro-
logical injury [2, 4].
The use of antimicrobials to treat STEC infections is contro-
versial [5], as it has been observed that it may provoke an increase in
the production or release of Shiga toxins (Stx) [6]. Antimicrobials
Stephanie Schüller and Martina Bielaszewska (eds.), Shiga Toxin-Producing E. coli:

Methods and Protocols, Methods in Molecular Biology, vol. 2291, https://doi.org/10.1007/978-1-0716-1339-9_1,
1
2 Stefano Morabito et al.
administration may thus represent a significant risk factor for the

progression of the infection towards the most severe forms of
STEC-induced disease, HUS [7].
The management of STEC infection is mainly supportive, and
in severe cases it includes hemodialysis [8]. It has been recently
described that patients with STEC-induced HUS had great benefit
from volume expansion by generous intravenous fluids administra-
tion upon early diagnosis of STEC infection, and it was proposed
that this practice may lead to positive effects on both short- and
long-term disease outcomes, by reducing organ damage [9]. There-
fore, a prompt etiological identification is pivotal for the clinical
management of the patients.
Stx is the major virulence factor of STEC and belongs to a
heterogenous family of AB5 toxins, including two major antigeni-
cally distinct types, Stx1 and Stx2. STEC may possess Stx1 or Stx2
gene or a combination of the genes encoding the two types. Large
variability in stx gene sequences has been described, and they can be
divided in several subtypes of both stx1 and stx2, with some of them
being more associated to the severe disease [10]. In fact, although
Stx1 has been also linked to human illness, STEC that produce
Stx2, and particularly subtypes Stx2a, Stx2c, and Stx2d, are more
often associated with the development of the most severe forms of
infection [11, 12].
Apart from the production of Shiga toxins, STEC are pheno-
typically indistinguishable from the commensal E. coli, commonly
present in the human intestine. Therefore, the detection of STEC
in complex samples and the confirmation of the E. coli isolates as
STEC are based on the identification of the presence of the
Stx-coding genes and/or the Stx themselves. This is carried out
by means of molecular biology methods, such as the Real-Time or
conventional PCR, and assays aiming at identifying the cytopathic
effect induced by the Stx onto monolayers of cultured cells, respec-
tively. Finally, the indirect evidence of the presence of STEC infec-
tion may be revealed by detecting circulating antibodies against the
lipopolysaccharide (LPS) of E. coli in serum samples of patients.
In the present chapter, the integrated approach for the diagno-
sis of STEC infections carried out at the Istituto Superiore di Sanità
(ISS) in Rome is described. ISS is the National Institute for Public
Health in Italy and acts as National Reference Laboratory for STEC
infections and as European Union Reference Laboratory for E. coli
including STEC according to the 625/2017 EU Regulation. ISS
coordinates the National Registry of HUS in Italy and the Labora-
tory receives clinical samples from the Italian network of pediatric
nephrology units for the diagnosis of STEC infections in cases of
HUS and, to a lesser extent, bacterial cultures or isolated strains for
their confirmation as STEC. Hereafter, the procedures in place in
the ISS are reported, and the different approaches used are critically
addressed.
Laboratory Diagnosis of STEC Infections 3
2 Materials
Prepare all solutions using ultrapure water (resistivity 18.2 MΩ/cm

at 25 C) and analytical grade reagents.
Dissolve the oligonucleotides and probes used for Real-Time
PCR (RT-PCR) in nuclease-free water.
Adjust the pH of the media and solutions at room temperature
(25 C) using NaOH or HCl solutions, if needed.
2.1 Reagents 1. Fecal sample collected into a dedicated sterile container (1–2 g
and Media of feces are sufficient).
for the Detection 2. Mixed or pure bacterial cultures.
and Isolation of STEC
3. Tryptone soy broth (TSB): 17 g/L casein peptone (pancre-
atic), 2.5 g/L dipotassium hydrogen phosphate, 2.5 g/L glu-
cose, 5 g/L sodium chloride, 3 g/L soy peptone (papain
digest.), pH 7.3.
4. MacConkey agar: 17 g/L peptone, 3 g/L proteose peptone,
10 g/L lactose, 1.5 g/L bile salts, 5 g/L sodium chloride,
0.03 g/L neutral red, 0.001 g/L crystal violet, 13.5 g/L agar,
pH 7.1.
5. Cefixime-tellurite sorbitol MacConkey agar (CT-SMAC):
20 g/L peptone, 10 g/L sorbitol, 5 g/L sodium chloride,
1.5 g/L bile salts, 0.03 g/L neutral red, 0.001 g/L crystal
violet, 13.5 g/L agar, 0.0025 g/L potassium tellurite,
0.05 mg/L cefixime, pH 7.1.
6. Rhamnose MacConkey agar (RMAC): 17 g/L peptone, 3 g/L
proteose peptone, 10 g/L rhamnose, 1.5 g/L bile salts, 5 g/L
sodium chloride, 0.03 g/L neutral red, 0.001 g/L crystal
violet, 13.5 g/L agar, pH 7.1.
7. TBX agar: 20 g/L peptone, 1.5 g/L bile salts, 0.075 g/L
X-ß-D-glucuronide, 15 g/L agar, pH 7.2.
8. Kit for DNA purification.
9. Primers and probes for RT-PCR assays (see Table 1): Prepare
stock solutions by dissolving oligonucleotides to the final con-
centration of 100 μM and probes to the final concentration of
50 μM; store at 20 C in 100 and 50 μL aliquots, respectively.
Prepare working solutions by diluting the stock solutions in the
ratio of 1:5 with nuclease-free water to obtain the concentra-
tion of 20 μM for the primers and 10 μM for the probes.
10. RT-PCR kit: It contains the Mastermix for amplification. It
may or may not include the Internal Amplification Control
(IAC). In case the IAC is present, follow the instructions
supplied with the kit. Store at 20 C. Dilute the Mastermix
to obtain a 1 concentration in each reaction.
Table 1
Genes detected with RT-PCR and oligonucleotides used
Gene target Oligonucleotides forward, reverse, and probes (50 –30 ) Reference
stx1 stx fwd: TTTGTYACTGTSACAGCWGAAGCYTTACG [17]
stx rev: CCCCAGTTCARWGTRAGRTCMACRTC
Probe-CTGGATGATCTCAGTGGGCGTTCTTATGTAA
stx2 stx fwd: TTTGTYACTGTSACAGCWGAAGCYTTACG [17]
stx rev: CCCCAGTTCARWGTRAGRTCMACRTC
Probe-TCGTCAGGCACTGTCTGAAACTGCTCC
eae eae fwd: CATTGATCAGGATTTTTCTGGTGATA [18]
eae rev: CTCATGCGGAAATAGCCGTTA
Probe-ATAGTCTCGCCAGTATTCGCCACCAATACC
rfbEO157 O157 fwd: TTTCACACTTATTGGATGGTCTCAA [17]
O157 rev: CGATGAGTTTATCTGCAAGGTGAT
Probe-AGGACCGCAGAGGAAAGAGAGGAATTAAGG
wbdIO111 O111 fwd: CGAGGCAACACATTATATAGTGCTTT [17]
O111 rev: TTTTTGAATAGTTATGAACATCTTGTTTAGC
Probe-TTGAATCTCCCAGATGATCAACATCGTGAA
wzxO26 O26 fwd: CGCGACGGCAGAGAAAATT [17]
O26 rev: AGCAGGCTTTTATATTCTCCAACTTT
Probe-CCCGTTAAATCAATACTATTTCACGAGGTTGA
ihp1O145 O145 fwd: CGATAATATTTACCCCACCAGTACAG [17]
O145 rev: GCCGCCGCAATGCTT
Probe-CCGCCATTCAGAATGCACACAATATCG
wzxO103 O103 fwd: CAAGGTGATTACGAAAATGCATGT [19]
O103 rev: GAAAAAAGCACCCCCGTACTTAT
Probe-CATAGCCTGTTGTTTTAT
11. Sterile loops for bacteriology (1 and 10 μL).

12. Microcentrifuge tubes (1.5/2 mL).
13. RT-PCR tubes (0.2 or 0.1 mL).
14. Micropipettes and sterile tips.
15. Incubator (37 C).
2.2 Reagents, Media, 1. Fecal sample collected into a dedicated sterile container (1–2 g
and Type of Samples of feces are sufficient).
for the Detection 2. Saline solution: Dissolve 8.5 g NaCl in 1 L of ultrapure water,
of Free Shiga Toxin sterilize by autoclaving.
3. 10 trypsin/EDTA solution: 2 g/L EDTA, 5 g/L trypsin in
saline solution. Store at 20 C.
4. 200 mM L-glutamine (100 stock). Store at 4 C.
5. 100 penicillin/streptomycin stock solution: 10,000 U/mL

penicillin G, 10 mg/mL streptomycin sulfate in saline solution.
Store at 20 C.
6. Gentamicin solution (50 mg/mL). Store at 4 C.
7. Trypan blue staining solution (use according to the producer’s
instruction).
8. Vero cells (cell line from the kidney of Cercopithecus aethiops,
ATCC CCL-81).
9. Medium 199 with Earle’s salts, supplemented with 5% fetal calf
serum, 2 mM glutamine, and a 1 mix of antibiotics (penicil-
lin/streptomycin). Alternatively, Minimal Essential Medium
supplemented as above can be used for Vero cells growth.
Store at 4 C.
10. 25 cm2 tissue culture–treated flasks.
11. 96-well plates with lids, flat bottom, tissue culture treated.
12. Cell culture incubator (37 C, 5% CO2).
13. Anti-Stx1 and anti-Stx2 neutralizing polyclonal antibodies
(produced in rabbits at ISS).
14. Sterile syringes.
15. 0.22 μm syringe filters.
17. Sterile loops for bacteriology (10 μL).
18. Sterile disposable microcentrifuge tubes (1.5/2 mL).
2.3 Reagents 1. Serum sample in a sterile vial (50 μL is sufficient).

for the Detection 2. Carbonate buffer: Solution A: 0.86 g/10 mL NaHCO3; solu-
of Anti-LPS Antibodies tion B: 1.06 g/10 mL Na2CO3. Add 4.53 mL of solution A to
in Serum Samples distilled water (about 1 L), adjust pH to 9.6 with solution B
by ELISA and make up to 1 L with distilled water.
3. Phosphate-buffered saline (PBS): 8.0 g/L NaCl, 0.2 g/L KCl,
1.44 g/L Na2HPO4∙2H2O, 0.24 g/L KH2PO4, pH 7.4.
4. ELISA buffer: PBS with 3% skim milk (w/v) and 0.1% Tween
20 (v/v). Store at 4 C.
5. Detection substrate (diethanolamine/p-nitrophenyl phos-
phate). Use according to the manufacturer’s instructions.
6. Anti-human total IgG (secondary antibody) conjugated with
alkaline phosphatase. Use according to the manufacturer’s
instructions.
7. Tris-buffered saline (TBS): 1.21 g/L Tris–HCl, 7.9 g/L NaCl,
pH 7.2. Store at 4 C.
8. Phenol crystalline.
9. 3 M sodium acetate.
10. Absolute ethanol.

11. Polystyrene Multiwell plates for immunoassay recommended
for ELISA.
12. Beckmann Optima XPN-90 Ultracentrifuge with the SW41
rotor (or equivalent ultracentrifuge).
13. Sorvall centrifuge with the SS34 rotor (or equivalent
centrifuge).
14. Corex glass tubes.
15. Nutrient agar (e.g., TSA, TSB added with agar 15 g/L).
16. 50 mL disposable tubes.
17. 1.5 mL sterile disposable tubes.
19. Sterile distilled water.
20. E. coli LPS reference strains.
21. Water bath at 65 C.
22. Incubator (37 C).
3 Methods
3.1 Molecular Historically, the most common serogroups of STEC strains isolated
and Microbiological from HUS cases were the so-called “top five,” namely O157, O26,
Approach O111, O103, and O145 [13] and thus most of the diagnostic
to the Diagnosis methods focused on the detection of these features. Nowadays,
of STEC Infections many more STEC serogroups are reported as causes of human
severe disease, and the serogroup (e.g., O157) is no longer consid-
ered as a hallmark of pathogenicity [14]. The common feature of all
STEC is the presence of genes encoding Shiga toxin(s). The aim of
the method presented here is in line with this paradigm, being
centered on the detection of the Stx1- and Stx2-coding genes
(Stx2a to Stx2g with the exception of Stx2f, see Note 1) [15] and
the intimin-coding gene eae, whose product is involved in the
peculiar “attaching and effacing” colonization mechanism. The
detection of genes targeting to the top-five serogroups is still part
of the methodology described but is only carried out in presence of
a positive result in the eae PCR, and the procedure is followed by
the attempt of isolation anyway (see Note 2).
The present method is used to test clinical samples (fecal speci-
mens, mixed and pure bacterial cultures), sent to the Italian Refer-
ence Laboratory for the diagnosis of STEC infections. DNA
samples may also be sent to the Laboratory and are also processed
with this method (screening step only).
The procedure for the complex matrices (fecal samples and
mixed bacterial cultures) is divided in two sections, consisting of
the screening followed by culture or subculture aimed at isolating
the STEC strain identified at the screening step. The detection of

the STEC virulence genes is carried out by RT-PCR using DNA
extracted from bacterial cultures obtained following the enrich-
ment of the clinical samples or the growth of the bacterial culture
or isolates. When a positive result for the stx genes is obtained,
single colonies are screened for the presence of the same genes, to
isolate the STEC strain. This latter part of the procedure is not
applied to the confirmation of pure STEC cultures.
The procedure as a whole is sequential: in case of the detection
of stx1 and/or stx2, the isolation of the STEC is attempted. The
detection of the eae gene is performed on the stx-positive cultures
or may be carried out simultaneously to the Stx-coding genes. In
case of positivity to both the stx and the eae genes, the detection of
top-five serogroup-specific genes may be carried out to aid the
isolation. As a matter of fact, when some of the top-five serogroups
are suspected (following the detection of one or more top-five
serogroup associated genes in the screening of fecal samples),
solid media such as CT-SMAC or RMAC, developed to facilitate
the isolation of STEC O157 and O26 respectively, may be used.
STEC O157 are resistant to cefixime and potassium tellurite and do
not ferment sorbitol, whereas some STEC O26 do not ferment
rhamnose and can be thus identified as colorless colonies onto
CT-SMAC and RMAC, respectively (see Note 2). An exception
are sorbitol-fermenting STEC O157:NM (nonmotile) strains,
which are susceptible to tellurite and cannot be thus isolated on
CT-SMAC.
The procedure consists of the following steps: Sample prepara-
tion, template DNA purification, setting up and running the
RT-PCR assays, and solid media plating for isolation.
3.1.1 Preparation Keep fecal specimens refrigerated at 4 C if they were shipped at

of Fecal Samples room temperature or refrigerated, or at 20 C if they were sent
frozen in dry ice. Let the sample equilibrate at room temperature
before starting the analysis. Inoculate aseptically 1 g of fecal speci-
men (1 g approximately corresponds to a 10 μL loopful) into
10 mL of TSB and incubate at 37 C for 18–24 h without shaking.
Proceed with the DNA extraction step.
3.1.2 Preparation Store bacterial cultures frozen, refrigerated, or at room tempera-

of Bacterial Cultures ture, depending on their nature (liquid broths, agar plates, cultures
with cryo-preservatives, cultures streaked on sloped agar in tubes).
Inoculate the bacterial culture samples, either mixed or pure cul-
tures, in 10 mL of TSB using a sterile loop (1 μL) and incubate at
37 C for 18–24 h without shaking. Proceed with the DNA
extraction step.
3.1.3 Preparation Transfer the rectal swab into 10 mL of TSB and incubate at 37 C
of Rectal Swabs for 18–24 h without shaking. Proceed with the DNA extraction.
3.1.4 DNA Extraction Extract DNA from 1 mL of the enrichment culture using any
commercial non-immobilized resin according to the manufac-
turer’s protocol. Use the purified DNA as a template for RT-PCR
analyses described below. Dilute the DNA 1:10 before use. In case
of isolated strains, dilute 1:100. Purify DNA from control strains
(i.e., strains possessing the genes targeted by the RT-PCR) using
the same kit and dilute 1:100.
3.1.5 Setting Up The amplification reactions for the identification of stx1 and stx2
and Running of the RT-PCR genes are conducted as multiplex, along with Internal Amplification
Control (IAC). As for the other targets (eae or serogroup-
associated genes), they may be amplified as separate reactions
along with the internal amplification control.
According to the ISS protocol, set up the PCR reactions in total
of 20 μL for each sample as described in Table 2, and analyze each
sample in duplicate.
Run the PCR for all targets, except for wzxO103, using the
following thermal profiles:
1. 5 min at 95 C; 2. 15 s to 95 C; 3. 60 s to 60 C. Repeat steps
2 and 3, 40 times.
Run the amplification of the wzxO103 gene as follows:
1. 5 min at 95 C; 2. 15 s to 95 C; 3. 60 s to 55 C. Repeat steps
2 and 3, 40 times.
3.1.6 Interpretation The positive test sample has an increase in fluorescence as the
of PCR Results amplification cycles increase, relative to the target gene channel
(Fig. 1) (see Note 3). The correct evaluation of the results is
primarily based on the observation of controls, which shall produce
amplification in positive controls and will not yield any curve in
negative controls. In the case one of controls do not produce the
expected result, the assay should be repeated. Additional to the
observation of the controls, the IAC should also be considered
when evaluating PCR results (see Note 4). Check that there is no
inhibition in the tested samples by examining the amplifications
related to IAC. The IAC in use at ISS is the pUC19 [16] (Table 2).
A good amplification of the IAC has the Ct (threshold cycle) values
of 25–33 (see Note 5). In some cases, when analyzing complex
samples, positivity may be detected in the lower regions of the
graph (around or beyond the 35 amplification cycles). If such late
positivity is detected, the result is assessed on a case-by-case basis. If
the positivity affects only one of the two replicas, the test is not
repeated and is considered positive for signals that rise within the
35th amplification cycle. In contrast, the samples are considered
negative, on a case-by-case basis, if the signal rises beyond the 35th
amplification cycle.
Table 2
Schemes for the preparation of the RT-PCR reactions
Reagent Final concentration or amount

Mix per sample (stx1/stx2)
Real-time PCR Mastermix 1
Primer stx FWD 1 μM
Primer stx REV 1 μM
stx1 Probe FAM 0.2 μM
stx2 Probe ROX 0.2 μM
Internal Amplification Control primer FWD 0.5 μM
Internal Amplification Control primer REV 0.5 μM
Internal Amplification Control probe HEX 0.2 μM
DNA sample 2 μL
Internal Control DNA pUC19 Ten copies
Water to the final volume of 20 μL
Mix per sample (eae and serogroup-associated genes)
Real-time PCR Mastermix 1
Primer FWD 0.5 μM
Primer REV 0.5 μM
Probe FAM 0.2 μM
Internal Amplification Control Assay Same as above
DNA sample 2 μL
Internal Control DNA pUC19 Ten copies
Water to the final volume of 20 μL
3.1.7 Isolation When the presence of stx genes in the RT-PCR screening is
of the STEC from detected, the isolation is attempted by streaking the enrichment
the Positive Cultures culture onto a solid media such as the TBX agar or MacConkey
agar, followed by screening of isolated typical E. coli colonies using
the same RT-PCR procedure used in the screening of stool samples
and bacterial cultures. In case of the positivity for stx genes, the eae
gene and one of the serogroup-specific genes, alternative media
known to provide selectivity or specificity towards certain E. coli
serogroups are used to select the colonies. In particular, the
CT-SMAC is selective and differential for E. coli O157:H7, which
does not ferment sorbitol and is resistant to cefixime and tellurite,
whereas RMAC allows to visualize some E. coli O26 colonies that
Fig. 1 RT-PCR amplification curves. Panel a: The positive controls-related curves raise approx. at 18–20 cycle.
Panel b: The curves related to positive samples raise typically later than those generated by the amplification
of pure cultures (as the positive controls in a)
do not ferment rhamnose. It has to be noted that sorbitol-

fermenting STEC O157:NM (non-motile) strains are indeed sus-
ceptible to tellurite and cannot be thus isolated on CT-SMAC.
Media capable of identifying all STEC strains are not available,
and the isolation of STEC from enrichment cultures still relies
mostly on the use of the general media as the TBX agar or Mac-
Conkey agar. Several commercial chromogenic media (e.g.,
CHROMagar™) are proposed for this purpose but they may not
work properly with all STEC serogroups and their suitability should
be verified by the laboratory.
3.2 Detection of Free Free fecal Shiga toxin is detected in order to identify the STEC
Shiga Toxin in Fecal infection in the absence of STEC isolation or other microbiologi-
Samples cal, molecular, or serological evidence (see below). The assay is very
sensitive and can detect the presence of the free fecal toxin after the
bacterium has been cleared following, e.g., an antibiotic treatment.
The use of the Vero cell assay (VCA) requires the expertise in
cellular biology, the morphology of the Stx-mediated cytotoxic
effect, and neutralization experiments with anti-Stx1 and/or anti-
Stx2 antibodies to confirm the specificity of the observed cytotox-
icity (see Note 6). For these reasons, this assay is carried out by
reference laboratories only.
1. Detach confluent Vero cell monolayers from the flask’s wall by
digestion with trypsin/EDTA (final concentration of 1) for
10 min at 37 C.
2. Resuspend the cells in cell culture medium M199 and perform
vital cells count in a Burker chamber through trypan blue
staining.
3. Use a part of the cells to propagate the cell culture by seeding
approximately 1010 cells into a 25 cm2 fresh cell culture flask
containing M199 supplemented with penicillin/streptomycin
(about 10 mL of medium) (see Note 7). Incubate at 37 C and
5% CO2 until the cells are confluent (this takes approx. 5 days).
4. Seed the other part of the cell suspension into a 96-well micro-
plate. Seed approximately 5 104 cells/well in a final volume
of 180 μL of M199 medium supplemented with penicillin/
streptomycin (as described in Subheading 2) and 100 μg/mL
of gentamicin. Incubate at 37 C with 5% CO2 until the cells
reach a semiconfluent layer, which may take approx. 24–48 h.
5. Prepare the fecal extract by suspending approximately 1 g or
1 mL (if liquid) of the fecal sample in 1 mL of sterile saline
solution in a 1.5 mL tube and mix thoroughly by inverting the
tube several times.
6. Centrifuge the suspension at a high speed (13,000 g for
10 min at 4 C), transfer the supernatant to a new tube, and
sterilize by filtration through a 0.22 μm filter.
7. Prepare two-fold dilutions (1:2 and 1:4) of the sterile fecal

extract, and add 20 μL of each dilution and the undiluted
extract to the cell monolayers in the microplate wells. This
results in final filtrate dilutions 1:10, 1:20, and 1:40.
8. Incubate at 37 C and 5% CO2 for 72 h. Observe the appear-
ance of a cytopathic effect (CPE) after 24, 48, and 72 h to
monitor the progression of the CPE, which includes the
detachment of the rounded cells from the well surface followed
by the cell death.
9. If needed, confirm the specificity of the CPE by applying serum
neutralization test (see Note 6).
3.3 Detection This method is used in patients with HUS to get an indirect
of Antibodies Against evidence of STEC infection when the results of the direct methods
STEC LPS in Patients’ (STEC virulence gene PCRs, STEC isolation, and free fecal Shiga
Sera Using ELISA toxin detection) are negative. The approach is based on the detec-
tion of antibodies against E. coli LPS circulating in the patient’s
serum using an ELISA assay. The test described here is a colorimet-
ric assay, based on the use of p-nitrophenyl phosphate (pNPP)
which turns yellow (λmax ¼ 405 nm) when dephosphorylated by
the alkaline phosphatase coupled to the secondary antibody used to
detect the circulating antibodies which are captured by the LPS
used to sensitize the ELISA plates.
The test is used to screen for a limited number of LPSs repre-
senting the STEC strains which are most frequently associated with
HUS (O157, O26, O103, O111, O145) (see Note 8).
3.3.1 LPS Preparation Use a reference strain for each serogroup you wish to probe for the
presence of antibodies in the sera in order to prepare the reference
LPS.
1. Inoculate the reference strain in 1 mL of TSB and incubate in a
static flask overnight at 37 C.
2. Streak each overnight bacterial culture onto seven plates of
nutrient agar (e.g., TSA) and incubate overnight at 37 C.
3. Resuspend the bacteria from the seven plates in 12 mL of TBS
in a 50 mL tube.
4. Heat the bacterial suspension to 65 C in a water bath.
Pre-warm 25 mL of liquid phenol to 65 C.
5. Add 25 mL of the pre-warmed phenol to the bacterial suspen-
sion under a hood and incubate at 65 C for 1.5 h in
water bath.
6. Cool to room temperature and centrifuge at 800 g for
45 min at 4 C.
7. Recover the aqueous phase and add 20 mL of distilled water
pre-warmed at 65 C to the tube and incubate again at 65 C
for 90 min.
8. Cool at room temperature and centrifuge at 800 g for 90 min

at 4 C.
9. Collect the supernatant in a clean flask and add 3 M Na-acetate
(one-tenth of the recovered volume, to get a final concentra-
tion of 0.3 M Na-acetate) and 5 volumes of absolute ethanol
(kept at 20 C). Store at 4 C overnight.
10. Centrifuge the solution at 10,000 g and at 4 C in Corex
glass tubes for 45 min. Discard the supernatant and resuspend
the pellet in deionized water.
11. Centrifuge at 250,000 g (38,000 rpm with a SW41 Ti
rotor) for 4 h at 4 C; resuspend the pellet (i.e., isolated LPS)
in 200 μL deionized water and store at 20 C.
Identify the working dilution of the LPS by using a serum
sample known to be positive for the specific anti-LPS antibody
(see Note 9).
3.3.2 ELISA Protocol 1. Prepare the working dilution of the reference LPS needed for
the assay (see Note 9) in carbonate buffer and add 100 μL to
the wells of an ELISA plate; use two wells per serum sample.
Perform this step on day 1. Incubate overnight at 37 C.
Perform steps 2–6 on day 2.
2. Wash the microplate(s) with carbonate buffer three times
(200 μL per well).
3. Add ELISA buffer (200 μL per well) and incubate at 37 C for
30 min.
4. Remove the ELISA buffer and add the patient’s serum (diluted
to 1:500 and 1:1000 in ELISA buffer) (100 μL per well). Add
positive and negative controls for LPS antigens and serum
samples, respectively (see Note 10). Incubate at 37 C for
90 min.
5. Remove the serum and the controls and wash the wells three
time with ELISA buffer (200 μL per well).
6. Add secondary antibody (anti-human total IgG) conjugated
with alkaline phosphatase diluted 1:4000 in ELISA buffer
(100 μL per well). Incubate at 37 C for 90 min.
7. Remove the secondary antibody and wash with ELISA buffer
(200 μL per well).
8. Add the diethanolamine/p-nitrophenyl phosphate substrate
and incubate under the conditions indicated by the supplier.
9. Read the plate with a spectrophotometer at λ ¼ 405 nm after
15 min and 30 min of incubation at the dark, e.g., wrapped in
an aluminum foil.
3.3.3 Interpretation A serum is considered positive for a specific anti-LPS antibody, if

of the Results the OD405 value is at least one-third of the positive control value or
higher (e.g., the positive control serum OD405 ¼ 2.350 and the test
sample OD405 ¼ 0.783 or higher). The negative controls’ OD405
values shall not exceed one-tenth of those yielded by the
corresponding positive control.
4 Notes
1. The lack of the determination of stx2f gene is due to the fact that
until recently this subtype had been reported mainly in the animal
reservoir. Nonetheless, a RT-PCR targeting stx2f is available at
the ISS website (https://www.iss.it/about-eu-rl-vtec).
This RT-PCR can be applied when an stx2 and stx1 RT-PCR
negative result is obtained in the presence of free Shiga Toxin
detected in the Vero cell assay.
2. If one of the top five serogroups is suspected, an immuno-
magnetic serogroup-specific enrichment may be attempted to
augment the chance to isolate the STEC, which may be present
in stools of HUS patients in low amounts, for the following
purpose of characterization. This piece of the protocol in our
laboratory works best only with STEC O157 and O26 among
the top five and sometimes its application may also be detri-
mental as there can be the simultaneous presence of an E. coli
belonging to one of the target serogroups and the STEC: this
could lead in losing the STEC present in the sample when
following the serogroup rather than the Stx-coding genes.
Therefore, the immuno-magnetic serogroup-specific enrich-
ment is not going to be discussed here.
3. Different probe labels and quenchers can be used for the
RT-PCR assays, but they should be selected according to the
compatibility with the RT-PCR apparatus in use (i.e., availabil-
ity of different detection channels).
4. If no fluorescence related to IAC is observed, the reaction shall
be repeated diluting the sample 1:10 or following re-extraction
of nucleic acid where possible if the problem persists.
5. The Ct value corresponds to the time point at which the
amplification curve starts to grow, corresponding to the begin-
ning of the exponential amplification.
6. Verification of the specificity of the CPE by a neutralization
assay with anti-Stx1 and/or anti-Stx2 antibody is carried out if
the CPE observed is not morphologically clear. Neutralization
is performed by incubating the fecal extracts with a preparation
of antibodies against the Stx1 and/or Stx2 for one hour at
37 C followed by inoculation onto the Vero cells monolayers
in the same conditions as those described for the VCA. At ISS,

in-house produced polyclonal antibodies raised in rabbits are
used diluted 1:2000, but any poly- or monoclonal neutralizing
antibody anti-Stx1 or Stx2 can be used, after determining the
best conditions for the assay. The titer of the antibody to be
used has been determined empirically at ISS by neutralizing the
supernatant of an overnight culture of an STEC strain with
serial dilutions of the polyclonal antibody and registering the
highest dilution, which is still active to neutralize the CPE
induced onto Vero cells monolayers.
7. The number of cells transferred to the flask is not a crucial
parameter. The indicated number of seeded cells is meant to be
a kind of a guidance but the growth of the cells in flask may
develop differently in different laboratories, and the right cou-
ple number of cells/days to the monolayer should be deter-
mined in each laboratory.
8. There are more than 180 different serogroups identified for
E. coli, and it would not be feasible to test for each and every of
them into a single ELISA assay. To restrict the test to the most
reported STEC serogroups in human cases of disease is a
valuable strategy. At ISS, we prepare LPS of any new STEC
serogroup we identify and add it to the panel for period of six
months to monitor the circulation of the STEC serogroup
concerned.
9. The working dilution of the LPS is determined empirically at
ISS by running the ELISA assay after sensitizing the ELISA
plate with dilutions of the newly prepared LPS and using for
the detection a previously assayed serum sample positive for the
presence of antibodies against the concerned LPS. The work-
ing dilution to be used is the one providing the best signal-to-
noise ratio.
10. Human serum samples from patients with HUS that have been
previously characterized are used as positive controls, in wells
sensitized with the corresponding LPS antigens. The negative
controls of the antigens are carried out by sensitizing the wells
with the specific LPS and conducting the entire ELISA test
without adding the serum sample. Finally, the highest serum
concentration (1:500) will be placed in a well that has not been
added with the LPS antigen and subjected to all the following
ELISA test steps to exclude non-specific reactions. Such reac-
tions may occur, due to cross-reactions between the serum
samples and the microwell’s plastic. In case these non-specific
reactions are detected, it is necessary to use further dilutions of
the serum sample or eliminate the complement from the serum
by incubation at 56 C for 30 min.
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MNH.0b013e328354a62e 10. Scheutz F, Teel LD, Beutin L, Piérard D,
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and characterization of verocytotoxin-
Chapter 2
Shiga Toxin-Producing E. coli in Animals: Detection,

Characterization, and Virulence Assessment
Stefanie A. Barth, Rolf Bauerfeind, Christian Berens, and Christian Menge
Abstract
Cattle and other ruminants are primary reservoirs for Shiga toxin-producing Escherichia coli (STEC) strains
which have a highly variable, but unpredictable, pathogenic potential for humans. Domestic swine can carry
and shed STEC, but only STEC strains producing the Shiga toxin (Stx) 2e variant and causing edema
disease in piglets are considered pathogens of veterinary medical interest. In this chapter, we present general
diagnostic workflows for sampling livestock animals to assess STEC prevalence, magnitude, and duration of
host colonization. This is followed by detailed method protocols for STEC detection and typing at genetic
and phenotypic levels to assess the relative virulence exerted by the strains.
Key words Shiga toxin, Shiga toxin-producing Escherichia coli, STEC, Virulence genes, Host–cell
interactions, Prevalence, PCR, Pulsed-field gel electrophoresis, Multilocus sequence typing, Vero cell
cytotoxicity assay, ELISA, Adhesion assay, Invasion assay
1 Introduction
Shiga toxin-producing E. coli (STEC) are food-borne pathogens

that can evoke diarrhea, hemorrhagic colitis (HC), and hemolytic
uremic syndrome (HUS) in humans [1]. Cattle and other rumi-
nants are primary reservoirs for STEC serotypes frequently asso-
ciated with human disease, e.g., O157:H7 [2] and non-O157
STEC serogroups including O26, O45, O103, O111, O121, and
O145 [3–5]. Humans principally acquire the infection via the oral
route by consumption of food items or water contaminated with
fecal matter from livestock and wild ruminants [2]. Domestic swine
can also carry and shed STEC but the role of swine in the epidemi-
ology of STEC-related human disease is likely minor [6]. STEC
strains producing the Shiga toxin (Stx) 2e variant cause edema
disease (ED) in piglets and are referred to as the edema disease
E. coli (EDEC) subtype of STEC [7]. In this chapter, all
Stephanie Schüller and Martina Bielaszewska (eds.), Shiga Toxin-Producing E. coli:

Methods and Protocols, Methods in Molecular Biology, vol. 2291, https://doi.org/10.1007/978-1-0716-1339-9_2,
19
20 Stefanie A. Barth et al.
Stx-producing E. coli are referred to as STEC, with the exception of

porcine pathogenic STEC-encoding Stx2e for which the acronym
EDEC is used.
1.1 Edema Disease ED (syn. E. coli enterotoxaemia) is an acute, severe, and often fatal
of Swine (ED) swine disease that primarily affects piglets during the first 2 weeks
after weaning. ED is common in most countries with intensive
swine production and, due to high morbidity and mortality, can
have a significant economic impact on farms. ED is caused by
certain STEC strains that are able to colonize the porcine small
intestine and produce Stx2e (EDEC). Bacterial colonization of the
porcine intestine is mediated by the ability of these bacteria to
adhere to villous epithelial cells via their cytoadhesive F18 fimbriae.
Two major subtypes of F18 fimbriae are distinguished by serologic
and molecular methods and both have been detected in EDEC
strains: F18ab fimbriae (previously called F107 fimbriae) and
F18ac (formerly termed 2134P, 8813, or Av24 fimbriae) [8]. The
expression of receptors for these fimbriae on the apical enterocyte
surface is inherited as a dominant trait among pigs and determines
susceptibility to diseases caused by F18-fimbriated pathogenic
E. coli [9]. Most EDEC strains belong to E. coli serogroups
O138, O139, and O141 and also to others such as O8, O147,
and O149. Some strains are O-non-typeable [10]. Similar to
enterotoxigenic E. coli (ETEC), many EDEC strains produce
heat-labile E. coli enterotoxin I (LT-I), or heat-stabile E. coli enter-
otoxins I or II (ST-I, ST-II), and/or F4 or F5 fimbriae in addition
to Stx2e and F18 fimbriae [11]. These strains may be regarded as
EDEC/ETEC hybrids.
Clinical manifestation in weaned piglets is a hallmark of
ED. Piglets usually acquire EDEC during the suckling period or
early after weaning via the fecal–oral route. The sudden increase in
nutrient protein due to the change of feed and the concomitant
withholding of protective milk antibodies during weaning are
assumed to initiate massive growth of the pathogen in the small
intestine and to trigger the pathogenesis of ED [12]. Symptoms
and lesions are extensively elicited by Stx2e after its translocation
from the pathogen colonization site across the intestinal epithelium
into the bloodstream. Stx2e causes a systemic microangiopathy
characterized by fibrinoid necrosis of endothelial and smooth mus-
cle cells in small arteries and arterioles. Subsequently, perivascular
edema, hemorrhage, and ischemic necrosis occur in several loca-
tions, conspicuously in the subcutis of the forehead, eyelids and
submandibular region, in the submucosa of the larynx and stom-
ach, and in the brain. Focal perivascular damage of the brain tissue
is accompanied by a progredient neurologic dysfunction, e.g.,
ataxia, paralysis, convulsions, and lateral recumbency. Infarction
and malacia in the brain stem are the main causes of death in
affected pigs [12, 13]. However, in cases where EDEC/ETEC
STEC in Animals 21
hybrid strains are implicated, diarrhea and subsequent dehydration

can predominate the clinical appearance [14, 15]. Treatment of ED
rarely proves successful and economically reasonable, particularly in
severely affected pigs. Measures taken during an outbreak rather
focus on the protection of healthy mates from infection or from the
onset of disease. Several meta- and prophylactic measures have been
suggested for control and prevention of ED [16, 17]. The discov-
ery that Stx2e and F18 fimbriae are not only crucial virulence
factors in the pathogenesis of ED but also are protective antigens
has stimulated efforts to develop specific protocols for active and
passive immunization [18–20]. The European Commission has
granted a marketing authorization valid throughout the EU for
two Stx2e-based toxoid vaccines and an F18-based live E. coli
vaccine for swine [21]. On most farms, the most successful strategy
to prevent outbreaks of ED is a combination of several measures
instead of any single activity [16].
1.2 STEC Carriage Since a targeted treatment for STEC-induced human diseases is still
and Shedding by not in sight, prevention of human infection with STEC from animal
Ruminants and environmental sources has the highest priority. Prophylactic
measures to prevent exposure are challenging as up to 86% of cattle
shed STEC with their feces [2, 22]. Bovines can already become
infected at calves’ age by minute infectious doses [23]. After initial
replication in the ileum, cecum, and colon, persistent infection is
established and followed by prolonged shedding of the bacteria for
several months [24, 25]. While strains considered to be particularly
virulent to humans, like those of serovar O157:H7, preferably
colonize epithelia covering lymphoid follicles [26] and the squa-
mous epithelium [27] at the recto-anal junction, STEC of other
serovars evenly colonize the large bowel mucosa in numerous
microcolonies [26]. In principle, experimental and natural STEC
infections of cattle remain asymptomatic [28, 29]. STEC are able to
induce bloody diarrhea in calves [30, 31], but infections of adult
cattle establish in the absence of intestinal inflammation. STEC
have adopted a commensal-like lifestyle in the intestinal milieu of
bovines [32]. In periods of low pathogen exposure (on pasture),
STEC shedding rates may temporarily drop below the detection
limit for the bacteria [33], but the same STEC clone can be main-
tained in a single herd for several months and years [25, 34]. Even
though STEC are shed for longer periods by calves than by adult
cattle [35] and the latter harbor Stx-specific antibodies [36], a
previous STEC infection does not protect from reinfection even
with the same strain [35, 37]. Shedding of STEC by cattle at high
numbers (commonly defined as >104 colony-forming units [cfu]/
g of feces), so-called super-shedding, seems to be the major source
of deposition into the environment and has an important role in
augmenting cattle-to-cattle transmission [38]. Such shedding is
not confined to certain animals [39] but viewed as an occasional
phenomenon with the potential to occur in any individual bovine.

These relatively rare super-shedding events contribute significantly
to human risk [40].
1.3 STEC Virulence Identification of the STEC virulence gene profiles is the basis of the
Assessment and Its modern approach to an impact assessment of STEC on public
Challenges health [41]. Numerous attempts have been undertaken to subtype
the many different STEC strains that are shed by animals in order to
predict a given strain’s degree of threat to human health. Various
levels of host adaptation have been traced back to certain patterns
of virulence genes and their expression levels. E.g., STEC O157:
H7 strains express iha, espA, rfbE, and ehxA to different extents
according to their origin from natural infections of humans or cattle
[42]. Spontaneous Stx production is higher in HUS-associated
STEC clones than in bovine STEC isolates, and Stx1 production
is induced more strongly by iron deprivation in vitro in the former
[43]. A reduced capacity to produce Stx2 in bovine STEC corre-
lates with the presence of the Q21 allele of the late antiterminator
protein Q upstream of stx in the genome of stx-converting pro-
phages, whereas strongly inducible Stx production seems to be
linked to the Q933 allele [44]. Indeed, a support vector machine
analysis of bovine and human E. coli O157 isolate sequences iden-
tified cattle strains more likely to be a serious threat to human
health by comparing them with sequences from human isolates
[45]. This distinction was possible despite the fact that the majority
of the isolates involved were members of previously defined patho-
genic lineages and encoded key virulence factors. The major differ-
ences between human and bovine E. coli O157 isolates were the
relative abundances of predicted prophage proteins. However, the
confidence in relying on such analyses to predict human pathoge-
nicity of STEC isolates was severely shattered by the appearance of
unexpected novel and unusual STEC strains possessing a blended
virulence profile combining genetic patterns of STEC and human
adapted enteroaggregative E. coli (EAEC), rarely detected in animal
hosts before [46, 47]. Although the O104:H4 STEC/EAEC
hybrid strain that caused the 2011 German outbreak appears to
be preferably adapted to humans, the strain’s potential to colonize
intestinal epithelial cells of humans and cattle [48] indicates that
even STEC strains with an unusual genotype can colonize hosts of
various species. Indeed, the outbreak strain colonizes calves under
experimental conditions [49], its genetic markers are present in the
cattle population [50], and the strain has been grouped in the midst
of bovine commensal strains in a recent comprehensive genome
analysis unveiling the evolutionary sources [51].
Colonization of the mucosal site is a complex process but a
highly conserved feature of intestinal E. coli strains [52]. Different
outcomes of bacterial infection in various hosts may result inter alia
from differential abilities to interact with the respective epithelial
STEC in Animals 23
cells [53], specific differences in cellular receptor distribution

[54, 55], and altered expression of bacterial factors [42]. In the
natural reservoir, virulence factors of bacterial pathogens counter-
act elements of the immune control generating a balance between
pathogen and host [56]. In order to appraise the relative level of
host adaption and virulence of an outbreak strain to possible reser-
voir hosts, comparing its interaction with host-specific cells in vitro
and linking its reaction profile to that of defined bovine- and
human-associated E. coli strains may be instrumental to raise the
level of preparedness against future outbreaks implicating unusual
STEC strains.
Several methods have been deployed to support risk assessment
in the public health context, including:
l Detection of stx genes in conjunction with the presence of the
LEE locus (namely eae) and genes encoding for EHEC hemoly-
sin (ehxA).
l Whole genome sequencing of strains and analysis of the viru-
lence associated gene profile and the resistance profile.
l Vector machine analysis of isolate sequences to predict human
adaptation [45].
l Phenotypic confirmation of Stx production by Vero cell assay
[48, 57, 58] or by ELISA [59] (see Subheading 4.4).
l Antimicrobial susceptibility testing using antimicrobial disc dif-
fusion or broth dilution assays according to the CLSI
protocol [60].
l Assessment of adherence and invasion capabilities for epithelial
cells [46, 48, 58, 61] (see Subheading 4.5).
l Assessment of virulence gene transcription and its regulation in
cultured mammalian cells [48] (see Subheading 4.6).
2 Diagnostic Workflows for STEC Detection in Animals
The pathogenesis of STEC-associated diseases originates from col-

onization and multiplication of the pathogens at intestinal mucosal
surfaces. STEC strains, including the highly virulent O104:H4
strain, which caused the large outbreak of HUS and HC in Ger-
many in 2011, are noninvasive [46, 48, 49]. Despite the fact that
viable bacteria were occasionally found at necropsy in mesenteric
lymph nodes in natural hosts [62], STEC have not been detected in
extraintestinal tissues during the course of systemic disease
[63, 64]. Consequently, fecal matter is the principal diagnostic
sample for detecting STEC and EDEC in animals.
Shiga toxins (Stxs), potent bacterial exotoxins produced and

released by STEC [65], represent the principal virulence factors
implicated in pathogenesis [66]. Many human pathogenic STEC
strains inherit the ability to settle on the enteric mucosa by inducing
attaching and effacing (AE) lesions leading to a tight association of
single bacteria or small-size colonies with intestinal epithelial cells.
The genes involved are encoded by the locus of enterocyte efface-
ment (LEE) in the STEC chromosome [67, 68]. While the LEE is a
key determinant in pathogenesis, not all STEC possess it, indicating
that some strains deploy alternative virulence and colonization
factors [69]. The occurrence of an outbreak caused by the unusual
yet highly virulent O104:H4 hybrid strain, which lacked the LEE
locus [46], stresses the fact that Stx is the only common denomina-
tor of STEC strains posing a threat to susceptible hosts. This notion
can be extended to O80:H2 STEC/ExPEC (extraintestinal patho-
genic E. coli) hybrid strains [70, 71] and also to EDEC strains
deploying different subtypes of F18 fimbriae to colonize the por-
cine intestine. An important diagnostic tool to identify STEC,
specifically those of serotype O157:H7, in clinical samples, food
samples, and feces is sorbitol MacConkey agar. STEC strains of
O157:H7 serotype are not capable of fermenting sorbitol, which
allows for an easy identification of suspicious colonies on solid agar
plates. Immunomagnetic separation applying magnetic beads
coated with anti-O antibodies, e.g., anti-O157 coated beads,
helps to enrich STEC of the respective O serogroup for a
subsequent cultural detection. The increasing clinical importance
of sorbitol-fermenting O157 strains (SF-O157) in Central Europe
[72], the recognition of STEC strains of other serogroups as
human health threat [73], and the SF capabilities of porcine patho-
genic EDEC [74] strictly limit the value of this simple biochemical
property for diagnostic use when it comes to analyzing samples
from animal sources. Similarly, a hemolysin produced by STEC
(designated EHEC hemolysin) causes hemolytic zones on specific
blood agar plates [75] and is present in many but not all STEC
isolates of human and animal origin [76–78]. Taken together,
methods to detect STEC in animals are essentially based on the
detection of a limited number of virulence factors by molecular
biological methods with the gene-encoding Stx being the primary
target [57, 67].
A strict application of molecular methods for clinical diagnosis
of ED in pigs or for the assessment of STEC prevalence in animal
populations has the advantage that the respective laboratory pro-
cedures can be performed under conditions of comparably low
biosafety precautions. By contrast, directed work with STEC iso-
lates must be conducted under biosafety level 3 conditions in many
countries. Since certain STEC strains are considered potential
biological warfare agents, restrictive measures for accessing the
laboratories and the material therein apply. The reader is strongly
STEC in Animals 25
advised to seek approval by the competent authorities of the coun-

try in which the work is to be conducted to ensure strict compliance
with the pertinent regulations before work is taken up. If a
biological material confirmed to contain STEC strains—with cul-
tures of isolated STEC in particular—is forwarded to other labora-
tories, shipping may also have to meet specific requirements. Such
are laid down in the “UN Recommendations on the Transport of
Dangerous Goods” and regulations based thereupon like the
“Accord relatif au transport international des marchandises Dan-
gereuses par Route” (ADR) and the International Air Transport
Association (IATA) global standards.
2.1 Detection Implementation of effective control and prevention measures

of EDEC in Piglets against postweaning syndromes in swine generally requires that
their causes are accurately identified. Definitive diagnosis of ED is
essentially based on (a) appearance of typical clinical signs and/or
lesions in pigs in combination with (b) presence of viable EDEC or
EDEC/ETEC bacteria in these pigs. Consequently, diagnosis of
ED includes systematic application of different diagnostic proce-
dures usually according to the following order:
l Recording the medical history.
l Clinical examination of affected pigs,
l Necropsy of representative perished or euthanized pigs, includ-
ing gross and histopathological examinations (facultative).
l Bacteriological analysis of intestinal contents or feces from
representative pigs.
ED must be suspected if sudden death, neurological signs,
subcutaneous edema, dyspnea, and unusual sound of squeals are
observed in piglets during the first weeks after weaning [79]. Gross
and microscopic lesions such as gelatinous subcutaneous edema,
submucosal edema in the stomach and mesocolon, pulmonary
edema, hemorrhage, arteriopathy, perivascular edema, and ence-
phalomalacia corroborate the suspicion [16, 17]. A presumptive
diagnosis may be easier in an ED outbreak when affected animals
display the full range of signs [17]. However, laboratory-based
diagnosis is indispensable since ED must be differentiated from a
number of diseases that may occur with similar manifestations, e.g.,
water deprivation, vitamin E-selenium deficiency, poisoning
(sodium chloride, selenium, lead, organic arsenic), pseudorabies,
Gl€asser’s disease, classical swine fever, Teschen/Talfan disease, sep-
ticemia, and meningoencephalitis caused by Streptococcus suis or
Salmonella enterica serovar Choleraesuis [16]. The presence of
EDEC or EDEC/ETEC in affected piglets is considered proven
as soon as suspect E. coli bacteria are cultured from intestinal
contents or feces and typical virulence factors or genes are subse-
quently confirmed in these bacteria (virotyping).
2.1.1 Specimen EDEC are noninvasive bacteria shed by infected pigs with their
Collection feces. Thus, intestinal content and intestinal mucosal swabs are
the most appropriate specimens for laboratory examination as
they usually yield high EDEC counts in culture when collected
from the distal jejunum, ileum, or colon of piglets as long as the
animals have not yet received antibiotic treatment [80]. Samples
should be obtained from perished or euthanized piglets immedi-
ately after their death to avoid artifacts by overgrowth of
non-causative microorganisms. Rectal swabs and fecal samples
from affected piglets are also highly suitable for bacterial culture
of EDEC and EDEC/ETEC. Sometimes it is more convenient for
the collector and causes less discomfort to the animals when fecal
samples are collected from freshly voided feces on the pen floor.
This procedure is acceptable if the material really originates from
piglets with suspected ED. Bacterial counts of EDEC and EDEC/
ETEC can decrease rapidly after the onset of symptoms in infected
piglets and can be low in samples associated with chronic or mild
forms of ED. Therefore, samples provide the best chance to isolate
the causative bacterial pathogen when taken from acutely and
severely affected animals during the first days of sickness. Analo-
gous to porcine E. coli diarrhea, we recommend to take samples
from at least 3–5 representative piglets in an outbreak [81].
Group and environmental sampling strategies have become
increasingly important for the efficient surveillance of infectious
diseases in modern pig production systems. Boot swab (syn. sock
swab) techniques have been developed to collect fecal samples from
pen floors and have proven valuable for detecting diarrheagenic
E. coli at nurseries [82]. Boot swabs are obtained by walking
through the pen of concern while wearing boots covered with a
sterile disposable plastic sock and a polyethylene overboot on the
outside. Another method of sampling pigs at pen-level utilizes
pieces of cotton ropes that are transiently exposed as a toy to the
animals of interest. Pigs chew on these ropes due to their natural
exploratory behavior, thereby soaking the cotton with “oral fluids.”
These oral fluids can be recovered and represent an appropriate test
matrix potentially containing not only antibodies against various
pathogens but also the viral and bacterial pathogens themselves
[83]. We have used boot swabs and oral fluids successfully to detect
EDEC and EDEC/ETEC strains in batches of weaned piglets (data
not published).
All samples should be collected in clean, sterile containers and
stored on ice or refrigerated until they are further processed.
2.1.2 Sample Shipment To obtain accurate and reliable test results, all samples must be
processed and submitted to the bacteriological examination as soon
as possible, optimally within 3 h. Keep samples on ice or refrigerated
(4–6 C) during shipment to the laboratory. Consider the use of a
bacteriological transport medium such as Stuart medium, if labora-
tory processing cannot be started within 24 h [16].
STEC in Animals 27
2.1.3 Sample Analysis Under Subheading 4.1, we describe detailed protocols that have
proved valuable in our hands for isolation and subsequent confir-
mation of EDEC and EDEC/ETEC from clinical or environmental
samples by PCR. Parts of that section represent updates from an
earlier version [84]. Please note that boot swabs and cotton ropes
require specific processing in the laboratory to harvest the speci-
mens that can then be cultured for bacteria (see Subheadings 4.1.1
and 4.1.2). Multiplex PCR techniques make it possible to rapidly
screen large numbers of bacterial isolates for a variety of discrimina-
tive virulence genes, thus facilitating detection and classification of
pathogenic E. coli with great sensitivity and specificity. The porcine
STEC/ETEC multiplex PCR was originally introduced by Casey
and Bosworth [85]. We complemented the original protocol to
additionally facilitate detection of the intimin gene (eae) which is
a distinguishing virulence marker of enteropathogenic E. coli
(EPEC) and some STEC [86]. The porcine EDEC duplex PCR
combines the F18-specific primers of the aforementioned multiplex
PCR and Stx2e-specific primers published by Scheutz et al. [87].
2.2 Detection In the past four decades, several hundred articles have reported the
of STEC in Ruminants detection of STEC in farmed ruminants, in animals displayed at
farm fairs, and kept in petting zoos. Reported estimates of the
STEC prevalence on cattle farms in Europe vary widely between
0% and 86% [88–93]. Multiple factors are known to influence
STEC shedding including diet [94, 95], precipitation, ambient
temperature, region, and season [96–98]. Carriage of STEC by
cattle and sheep can range from low to very high, i.e., super-
shedding [40, 99]. Individual STEC shedding by cattle may vary
significantly from birth to slaughter in terms of numbers of bacteria
shed and also with regard to the strains detectable [22] as calves
become exposed to a plethora of different STEC strains in the first
month after birth [29, 58].
According to Zoonoses Directive 2003/99/EC, European
member states are obliged to collect relevant and comparable data
on zoonoses, zoonotic agents, and food-borne outbreaks but noti-
fication requirements differ significantly from country to country
[41]. In 2018, testing of 1690 sample from animal units (animals
or herds or flocks) was reported by only six member states. Overall,
the presence of STEC was reported in 7.6% of them. There is still
large use of methods that only detect E. coli O157 in samples from
animals. Other animal samples were tested using the ISO TS
13136:2012 method, implemented for food and animal feeding
stuffs rather than samples taken from livestock intra vitam [100].
Only limited attempts have been made to precisely estimate the
STEC prevalence in ruminants at a nationwide scale. Two national
cross-sectional surveys in Scotland [101, 102] demonstrated the
presence of E. coli O157 on approximately 20% of farms producing
cattle for human consumption. A structured survey in England and
Wales during 1999 estimated herd-level STEC O157 prevalence to

be 38.7% [103], while a 2003 convenience survey in England and
Wales identified STEC O157 on 32.2% of 255 farms [104]. The
British E. coli O157 in Cattle Study (BECS) between September
2014 and November 2015 on 270 farms across Scotland and
England and Wales [105] revealed herd-level prevalence estimates
for E. coli O157, with the majority of strains being stx positive, of
23.6% for Scotland and of 21.3% for England and Wales. Other
STEC serotypes were not monitored in these studies.
2.2.1 Study Designs In lieu of continuous and comprehensive monitoring and reporting
by competent authorities for STEC carriage and shedding by live-
stock in many countries, timely and tailored prevalence studies are
required to provide a sound basis for implementing pre- and post-
harvest food safety measures to prevent human disease. Studies
must be designed according to good epidemiologic practices and
may be manifold depending on the hypothesis to be proven. Two
examples are provided hereafter. For a cross-sectional study,
authors of the BCES study systematically identified to-be-sampled
farms taking into account estimated prevalences and geographic
information as well as participation in a previous STEC prevalence
study [105]. Sampling teams were sent once to each farm. The
sample group was the group of nonbreeding cattle closest to
slaughter on the day of the visit. At the sampling visit, a question-
naire was completed through a face-to-face interview. Another
study aimed at assessing STEC shedding by cattle between birth
and slaughter and only considered a small number of farms
[22]. On each farm, groups of 25 heads of cattle were monitored
on average. Each animal among the beef groups was sampled at
intervals of approximately 2 4 weeks from birth to slaughter.
Sampling was interrupted for the periods when the cattle were kept
on pasture and were thus not accessible for regular examinations.
Some animals were also sampled on the day of slaughter before
transport to the abattoir, immediately upon arrival at the abattoir,
and just before slaughter.
2.2.2 Specimen Even though fecal matter is the prime sample to be taken, other
Collection sampling matrices are also feasible:
l Natural fecal pats (freshly voided) [105, 106]:
This matrix is appropriate when sampling in the pens aims to
avoid approaching and handling of single animals. Samplers
should ensure that they do not sample from the same pat twice
nor from old, dried, or desiccated pats. The number of pats
taken from each group depends on group size [102, 107,
108]. For each sample, a 30 mL universal container is filled to
just below the threaded portion with feces taken from several
locations on a fresh pat. Samplers may preferentially target areas
STEC in Animals 29
on the surface of the pat where mucus is apparent to appreciate

the recto-anal junction as principal colonization site of O157:
H7 strains, which is believed to primarily result in surface con-
tamination of the pat [26].
l Rectal swabs [22]:
This is the matrix of choice when shedding of single animals
is to be assessed. The assessment may be quantitative (i.e.,
enumeration of STEC bacteria) or qualitative over time (i.e.,
typing of isolated STEC strains) or combinations thereof. All
animals are identified by their ear tag numbers. Rectal swabs are
immediately transferred into sterile tubes to prevent environ-
mental contamination.
l Grab samples [109]:
Rectal content can be obtained from animals via palpation
with a gloved hand. Fresh gloves have to be used for each
individual sampling to protect from cross-contamination
between animals. Such samples combine the advantage of
being traceable to single animals and being of significant vol-
ume. However, the rectal ampulla may be empty at the time of
sampling which particularly hampers sampling of younger ani-
mals. Grab samples may be obtained from more cranial areas of
the rectum in adult animals, but this sampling method requires
enhanced efforts and manipulation of animals with the accom-
panied risk of the sampler getting harmed.
l Perineal or hide swab samples [106, 110]:
Bovine manure can harbor STEC at typical environmental
temperatures for >49 days [111]. Dirt and feces that collect on
the hides of cattle can therefore be contaminated with E. coli
O157:H7 for long periods of time [112]. Hide swab samples
can be collected from a 500-cm2 area of the hide around the
anus or of the rump using a sterile sponge stick (e.g., 3M,
St. Paul, MN, U.S.A.) moistened with 25 mL of PBS, with a
new sponge stick used for each animal.
l Manila rope [113]:
Ropes (1–1.5 m long) are fastened to bunk rails within each
pen to be available overnight for cattle to rub or chew. The
following morning, ropes are collected aseptically and returned
to the laboratory for bacterial culture. For each pen, several
ropes can be deployed in order to obtain data representative of
the entire group of pen-mates. To classify pens as high or low
prevalence in longitudinal studies conducted during the summer
and winter feeding periods, regular sampling over a period of
two full years may be necessary [113]. This sampling procedure
allows for a simple handling, avoidance of animal contact but
monitoring of groups of animals rather than individuals.
l Boot swabs [114, 115]:

This procedure allows for a quick sampling of animal groups
but is advantageous over pat or rope sampling as walking the pen
by the sampler results in the collection of a composite sample
likely best reflecting the shedding pattern of the entire animal
group (also see Subheading 4.1.1). The boot swab sampling
technique is established for environmental sampling of myco-
bacteria in dairy herds, an approach overcoming the issue of
sampling location and further reducing effort and cost
[116]. The detection limit of this approach in terms of within-
herd prevalence is low [117], but depends on the laboratory
methods used to detect the pathogen of interest. The technique
has not yet been applied for the detection of STEC on cattle
farms, but is being used routinely for monitoring the presence of
antimicrobial resistant Enterobacteriaceae in pig pens [115].
A study by Stanford et al. [118] showed that collection of rectal
fecal samples from all animals per pen provided superior isolation of
E. coli O157:H7 compared with oral swabs, pooled fecal pats, and
manila ropes, although labor and animal restraint requirements for
fecal sample collection were high. Depending on the setting in the
animal holding and the research question to be answered, investi-
gators have to carefully select the sampling method if application of
several methods in parallel is considered not feasible.
2.2.3 Sample Shipment Samples are labeled and have to be kept cool during immediate
transport to the laboratory. Microbiological examination ought to
start within 3 h after sampling. If it takes longer, samples should be
transported on dry ice and transferred to 80 C upon arrival at the
laboratory for later processing. However, different bacterial strains
are affected by this procedure to different extents, resulting in a
shifted population of cultivable strains.
2.2.4 Sample Analysis The laboratory workflow to be applied for prevalence estimation
has to be adjusted to the principal question to be answered. For
determining the intra- or interherd prevalence, simple grading of
samples as STEC positive or negative may be sufficient. For a better
understanding of the STEC population and its dynamics in an
animal population, isolation and characterization of strains is indis-
pensable. Thereby, the analysis may be restricted to STEC O157:
H7 strains which can easily be selected by their sorbitol-fermenting
inability if this is deemed suitable in the given setting and the lack of
information on STEC strains of other serotypes is acceptable. In
these instances, qualitative methods may be used to analyze indi-
vidual (e.g., fecal swab, grab sample) or composite samples (e.g.,
boot swab). If the quantity of shedding is of interest, e.g., to
determine the number of super-shedders in a herd or the average
number of STEC bacteria shed related to diet or season, quantita-
tive culture- or PCR-based methods have to be used.
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“That’s no good here and you know it’s no good,” said the officer.
“Wainboro! And a year old too. Why didn’t you come and get your
permit when you got to town? You’ve been in this game long enough
to know you’ve got to do that. All these concessions have permits,
except those under carnival management.”
“Some towns—” began McDennison.
“Never mind about some towns. You know you’ve got to get a
permit in this town. Why didn’t you do it?”
The harassed performer began again, “You guys⸺”
“Never mind about that now,” said the officer. “I was sent here to
see your permit and to bring you down to the office if you didn’t have
it. You know all about it; you were at the Elks’ Fair three years ago.
You better come along and get your permit, Charlie. You’ll have to
take care of a fine, too.”
“You don’t mean now?” the diving wonder asked. “Ain’t you going
to leave me do my trick? I go on in about five minutes. You fellers
sure got the knife in us. If I belonged in this here town⸺”
“Come on, McDennison,” said the officer in a way of quiet finality.
“You know the game as well as I do. We’re not interested in your
trick, only your permit. Come on, get your duds on. I guess you’ve
been through all this before. Come on, speed up.”
Diving Denniver cast his cigarette from him, bestowing a look of
unutterable contempt on the officer. In that sneering scorn he
seemed to include the whole of Farrelton and all constituted
authorities the world over. And Hervey joined him in his contempt
and loathing. Diving Denniver had been through all that before. He
knew the permit towns and the non-permit towns and the towns
where a “tip” would save him the expense of a permit. Hervey had
not dreamed that this enchanted creature ever had to do anything
but dive, he did not know that the wonder of two continents had hit
Farrelton penniless.
I will not recount the language used by Diving Denniver as he
pulled on a shabby suit of clothes and threw a funny little derby hat
on the back of his head. How prosaic and odd he looked! But his
language was not prosaic; it was quite as spectacular as his famous
exploit—his trick, as he called it. Poor McDennison, it was all he had
to sell—his trick. And sometimes he had so much trouble about it.
A funny little figure he made trotting excitedly along with the
officer, his derby hat on the back of his head bespeaking haste and
anger. He smoked a cigarette and talked volubly and swore as he
hurried away, leaving Hervey staring aghast.
Such a troublesome and distracting thing it is to be a wonder of
two continents.
CHAPTER XXIX
THE WHITE LIGHT
Well at all events, Hervey might now inspect freely the sanctum of
the diving wonder. His enthusiasm for the hero was not dimmed.
Even the derby hat had not entirely covered up Diving Denniver.
Here was just another exhibition authority. That a cop should make
so free with Diving Denniver, even calling him Charlie!
Hervey went into the tent, and stood looking about. Muffled by the
distance he could hear the frightful monotonous music of the merry-
go-round playing Little Annie Rooney for the millionth time. On the
red board were strewn the leavings of Diving Denniver’s supper. The
smutty little oil-stove reeked of kerosene. A long, up-ended box did
duty as a washstand and on this, beside a tin basin, was the
photograph of a girl. A couple of candles burned and sputtered. On
the tent pole hung a broken mirror.
Diving Denniver’s bathrobe and his white bathing suit trimmed
with gold braid lay on the converted couch just as he had thrown
them in his hurry and anger. The very bathrobe, half off and half on
the couch, seemed eloquent of his high disgust at the tyrannical
interruption of his work. Hervey surmised that he would speak with
the management of the carnival on his way out; he wondered why
the two had not gone in that direction. But in truth the diving wonder
did not love his public enough to consider it in his sudden dilemma.
He never went up when the wind was strong. And he was not
thinking of the expectant throng now.
Hervey longed to don that gorgeous exhibition suit. Could he slip it
on in a hurry? With him it was but one step from impulse to action
and in a few seconds he had thrown off his suit and was gazing at
himself in the dirty old mirror, clad in the white and gold habiliments
of the international wonder. How tightly it fitted! How thrillingly
professional it made him feel! What a moment in his young life!
Suddenly, something very extraordinary happened. The trodden
grass at his feet shimmered with a pale brightness. Clearly he saw a
couple of cigarette butts in the grass. It was as if some one had
spilled this brightness on the ground. Then it was gone. And there
was only a dim light where the candles sputtered on the makeshift
table. That was a strange occurrence.
He stepped out of the tent and there was the patch of brightness
near the Ford sedan. How plainly he could read the flaunting words
on the spare tire, THREE HUNDRED FOOT DIVE. Then suddenly,
the square tank and the foot of the dizzy ladder were bathed in light.
A long, dusty column was poking around as if it had lost something.
The sedan was again illuminated. The bright patch moved under the
tent and painted an area of the canvas golden. Was it looking for
Diving Denniver, the wonder of two continents, to come forth and
make his three hundred foot dive?
It found the tank and the ladder again and made them glowing
and resplendent. Then there was wafted on the air the robust sound
of the band playing real music. It drowned the tin-pan whining of the
merry-go-round and sent its rousing strains over the fence which
bore the forbidding sign. What a martial tumult! It made the cane
ringers pause, sent the carriers of kewpie dolls to a point of vantage,
and left the five-legged calf forlorn and alone. Louder and louder it
sent forth its rousing melody.
Come take a ride o’er the clouds with me
Up in the air mid the stars.
Hervey Willetts stood petrified. He was in the hands of the gods—
or the devils. I have sometimes wondered if he ever, ever thought.
Behind every act, good or bad, there is some kind of intention. And I
have told you about boys whose intentions were not of the best. But
what of this boy? There was just never anything behind his acts. No
boy could catch him. Yet the band and the waiting light caught him.
And what did they do to him? The light seemed to be waiting for him,
there at the foot of the ladder. All else was darkness. Only the area
of brightness bathing the ladder and the big tank with its metal
corners. It seemed to say, “Come, I am going up with you.” And, God
help him, he went to it as a moth flies to a flame.
When he had ascended a few feet, he remembered that Diving
Denniver went up very slowly seeming to test each rung. He knew
now that this had been for effect and to make the climb seem long.
For the rungs were sound and strong. Also the performer had
occasionally extended his arm. The substitute realized that there had
been good reason for that, for the breeze was more brisk as he
ascended and he knew that the diver had thus held out his hand by
way of keeping tabs on the breeze.
The small tank permitted no divergence from the straight descent.
To land outside it⸺
He went up slowly, but did not pause at each rung. He could be
reckless, but not theatrical. But he did hold out his hand every few
feet and the gay breeze cooled his sweaty palm. Was the wind too
strong? What would Diving Denniver do? Go back? But in any case
Hervey could not do that. He never turned back.
He continued ascending, up, up, up. He could feel the ladder
sway a little. When he was about half-way up, the breeze made a
little murmur where it was cut by one of the wires extending off
slantingways, far off down to the earth somewhere. It was funny how
he could see these wires in the circle of light that had accompanied
him in his long climb, but could not follow them with his eyes to their
distant anchorages. Each wire disappeared in the darkness, and he
had an odd fear that they did not go anywhere. He saw the lights of
the carnival, but no human beings. Were they gazing at him—
hundreds of upturned faces?
Up, up, up he went. Was there no end to it? Now he did really feel
the force of the breeze. Was it too strong? How could he decide
that? He could hear the band, but he knew it would cease playing
when he reached the top. In that one brief moment of suspense it
would cease playing. His companion light moved with him like a
good pal. And beyond and below all was darkness except for the
lights of the carnival.
Up, up, up he climbed. And he came at last to the little platform at
the top, as big as the top of a stepladder. It was just a little shelf fixed
to the fifth or sixth rung from the top. But the part of the ladder above
that would serve as a back and he could lean against it. By fancying
the ground was right below him, by eliminating the distance from his
mind, he was able to squirm around and get onto this tiny shelf. He
did not know how Diving Denniver did this, but he managed it.
Standing on the little shelf and leaning back, he could feel the
ladder shake under him. Of course, there were several ladders
clamped together and the extending wires could not hold the
towering structure absolutely taut. But it was steady at the top.
Far below him was a square frame of lights marking the sides of
the tank which had been illuminated during his ascent. Within it the
water shimmered. His senses swam and he closed his eyes, then
opened them and got control of himself. A straight down dive would
do it. Would it? Yes, he was sure. He let go the ladder and laid his
two hands palm to palm above his head.
There was no music now.
HERVEY MADE THE GREAT DIVE.
CHAPTER XXX
STUNT OR SERVICE
The next thing he knew he was lying propped up against a tree
and people were crowding about him. He knew this was not in tribute
to him for he heard a voice say, “Some crazy little fool, all right.”
“Did you ’phone?” he heard some one ask.
“Yes, he’ll be here soon.”
“He isn’t the regular one, is he?” another asked.
“Don’t ask me,” another answered; “I just followed the crowd.”
All the while a boy in a scout suit was moving his hand around
near Hervey’s foot. Emerging from his stunned condition, Hervey
had an odd impression that this boy was stirring something in a bowl.
Far off was the monotonous, incessant music of the merry-go-round.
Then, as Hervey blinked his eyes and brushed his soaking hair back
with a wet hand it seemed as if this boy were playing the music, for
his hand moved in time with that muffled clamor. Hervey lapsed off
into unconcern again and closed his eyes. It was only giddiness.
When he opened them again, he watched the boy with a kind of
detached curiosity. He felt a tightening sensation in his leg. Then he
realized that the boy had been drawing a bandage tighter and tighter
around his calf by revolving a stick. Still Hervey was only vaguely
interested.
“Stopped?” some one asked.
“Yep,” said the boy. He sat at Hervey’s feet with hands clasped
around his drawn-up knees. Soon he arose and stood looking as if to
ascertain on his own account if some one were coming.
“Who are you looking for?” Hervey asked weakly.
“The doctor,” answered the boy. He was a tall boy. As he stood
looking, he kicked something with his foot.
“What’s that?” Hervey asked.
The boy picked it up and dangled it in front of him, laughing. It was
just about recognizable as the body of a kewpie doll, and it was a
ghastly sight, for the head hung loose and the body was mangled
and out of shape. “Glad you’re not as bad off as that, hey?” said the
scout. “I won that blamed thing ringing canes and I got—I bet I got
three yards of cloth off it; there goes.” And twirling it cruelly by one
leg, he hurled it gayly over the heads of the throng.
“You people get away from here, go on,” said the robust voice of a
policeman. “Go on, all of yer, get away from here; he ain’t hurt much.
Go on, chase yourselves, you kids.”
“He can’t chase me anyway,” said Hervey.
“That’s a good one,” laughed the boy. “Nor me either; I’m the
surgeon general or whatever you call it.”
“You can’t chase me,” said Hervey to the policeman. “That’s
where I’ve got the laugh on you.”
“If I was your father, I’d chase you to the padded cell,” the
policeman commented, then busied himself clearing away the
loiterers.
The scout examined his twisted bandage and gave it one more
twist. Then he sat down on the ground beside Hervey. Two or three
men and the policeman lingered about, but did not bother these two.
“That was some crazy stunt all right,” said the scout.
“Did I—where did I fall?” Hervey asked.
“You went in the tank, but only just, I guess. Your foot must have
knocked the edge; four of the electric bulbs were broken. I don’t think
there’s any glass in your foot; anyway, I stopped it bleeding. Gee,
boy, I did murder that kewpie doll! How the dickens did you happen
to do that, anyway?” Hervey told him briefly.
“Good night, some daredevil! I dived to-day, but I had the whole
river to dive in. Me for that tank stuff—not.”
“Are you a scout in this town?” Hervey asked. “Yep, South
Farrelton. I was here last night and I had my fortune told and the old
woman told me I’d be lucky. I was all right. And believe me, so were
you.”
“How were you lucky?” Hervey asked.
“Oh, things came my way. I’m here with my patrol to-night; I guess
the cop chased them—good thing. They’d have trampled all over
you.”
“They’re always chasing people,” Hervey said. “They came and
got that diving wonder even, they’re so blamed fresh. And he’s a
wonder of two continents. Anyway, I’m always lucky.”
“I’ll say you are!”
“I’m going out to Montana, maybe to South America. I bet you can
do what you want down there. They weave Panama hats under the
water; gee, I bet I could do that. I always land right side up, that’s
one thing about me.”
“It’s a darned good thing,” said the scout.
Hervey did not bother to ask him his name, but the boy told him; it
was Wyne Corson. “That’s a good first name, hey?” he said. “Wyne?
It’s better than lose. There’s a scout in our troop named Luze—they
call us Win and Lose. He’s a Hungarian on his great granddaughter’s
side, I guess. Here comes the crowd back; I guess the doctor’s
coming.”
The doctor came and kneeled down, brisk, smiling and efficient.
He seemed not to take any interest in the spectacular exploit, only in
the injured foot. “Well, I guess you’re all right,” he said after treating
and bandaging the foot. “You won’t be able to run any marathon
races to-morrow.”
“Can I the next day?” Hervey asked.
“No, you can’t the next day,” the doctor laughed. “Who’s going to
take you home?”
Then he offered to do it himself and Wyne Corson got the hero’s
brown shirt and knickerbockers from the tent and maneuvered him
into them. He even placed the treasured hat on his head at an
unconventional angle. He seemed to have an inspired appreciation
of Hervey’s bizarre character. Then they helped him to the waiting
car. Gaping stragglers watched the self-appointed understudy of the
diving wonder as he limped between the doctor and the scout, past
the enclosure of the five-legged calf, and around the festooned
platform where the merry dance was on. Whirling couples paused to
stare at him and one girl ran out and boldly inspected the celebrity
from head to foot. “Oh, he has the brightest eyes,” she confided to
her waiting partner, “and the funniest little hat with all sorts of buttons
on it. Do you know who he reminds me of? Peter Pan.”
At the doctor’s car half a dozen scouts stood about gazing at
Hervey. They hardly knew what to make of him, but they had a kind
of instinctive respect for him and showed it. I am not sure that this
was just on account of his daredevil exploit. There was something
about him and that’s all there is to it. Good or bad, he was different.
“Did I do the right thing?” Wyne Corson ventured to ask the
doctor. He had hoped he might be asked to accompany Hervey, but
apparently this was not to be.
“Oh yes indeed—the only thing,” said the doctor. “You were on the
job and efficient and clever. That’s the kind of thing I like to see.”
“You ought to have seen what he did,” Wyne ventured. Was he
falling for this cracked-brained youngster too?
“I don’t believe I’d care to see that,” said the doctor with brisk
good-humor.
And there stood Wyne Corson with his scout comrades about him.
They did not comment upon his efficiency nor the doctor’s ready
compliment.
“Did he talk to you? What did he say?” asked one.
“Where does he live?” asked another.
“Is he friendly, sort of?” asked a third.
“For the love of Christopher, why didn’t you talk to him
yourselves?” laughed Wyne. “He wouldn’t eat you up. Come on, I’m
going to treat to ice cream again, then let’s go home.”
CHAPTER XXXI
HOPELESS
He sat in a big old-fashioned chair in the living room with his
injured foot upon a stool, in deference to the powers that be. There
was a knock on the front door and presently young Mr. Ebin Talbot,
scoutmaster, poked his head around the casing of the living room in
a way of mock temerity.
“May I come in and have a look at the wonder of wonders?” he
asked. “How are we; getting better?”
“It hurts a little when I stand on it.”
“Then the best thing is not to stand on it, hey? Like the advice to a
young man about to stand on his head on a steeple—Don’t. Good
advice, huh? Well Herve, old boy, I’ve got you where I want you at
last; your foot’s hurt and you can’t get away from me. Did you ever
hear the story about the donkey that kicked the lion? Only the lion
was dead. Well, I’m the donkey and you’re the lion; I’ve got you
where you can’t jump down my neck. Do you know that was a crazy
thing you did, Herve? You just put yourself in my power. Maybe you
did it so you wouldn’t have to go to school, huh? Where’s your dad?”
“He’s at the store.”
“Have you heard about this conspiracy to send you to military
school?” Poor man, he was trying to reach Hervey by the good pal
method. He drew his chair close and spoke most confidentially. “I
think we can beat it,” he said.
“Leave it to me,” said Hervey.
“You’re not worrying?”
“I’d be there about three days,” said Hervey.
“I think you’d be there about three years, my boy.”
“What do you bet? Everybody’s calling me a crazy daredevil. Do
you think I wouldn’t be enough of a daredevil to get away from a
military school? Bimbo, that’s a cinch.”
It seemed to be something that Hervey was quite looking forward
to; a lure to new adventure. Mr. Talbot went on another tack.
“Well, I thought if we could slip you into the Scouts in time, we
could beat your dad to it.”
“I’ll beat them all to it, all right,” said Hervey vaguely. “They
arrested that wonder—even of two continents he’s a wonder—but I
gave them a good run. I nearly bit that feller’s hand off when he
grabbed me. Do you dare me that I won’t get away from military
school?”
“Oh goodness no, but listen, Herve.” He became soft and serious.
“You can listen, can’t you? You haven’t got anything else to do—now.
You know that boy who put the jigamerig around your leg?”
“Carter—something like that?”
“You don’t remember his name, Herve? Wyne Corson. That fellow
is in the troop they’ve got down in the south end; they’ve got quite an
outfit. One of them—he’s just a kid—wants to have a hat like yours.
When you jumped, you jumped right into the hearts of the Raccoon
Patrol; you didn’t hit the tank at all. Well, that fellow was—now listen,
here’s a knockout for you. Do you know how those fellows happened
to be at the carnival last night?”
“Do you think I bother ringing canes?” said Hervey.
“Well, it’s good he won a kewpie doll, now isn’t it? But that’s not
the knockout. He won a prize yesterday and he was giving his patrol
a kind of a blowout last night at the carnival. I think there’s going to
be a shortage of pop-corn for the next forty-’leven years.”
“Well, yesterday morning he was up the river with that scout—that
little stocky fellow; did you notice him?”
“No.”
“Well, he noticed you. They were up on Blackberry Cliff; as near
as I can make out they’re always out for eats. There was a girl in a
canoe down below; she belongs in that white house right across
from the cliff. What I’m telling you is in this afternoon’s paper—you
can see it. Well sir, the canoe upset, and this Wyne, he dived from
the Cliff—that’s pretty high, you know, Herve, and he got her and
swam to shore with her—now wait. Here’s the punch. He gets the
Ellen C. Bentley reward for this year. You remember nobody got it
last year. He goes on a trip to California next summer—six weeks.
Naturally he was feeling pretty good last night. And he never told you
a word about it! Think of those two things that scout did yesterday!
Dived from a cliff and saved a life, won a trip across the continent,
then put a what-d’ye-call-it around your leg when you might have
bled to death after making a crazy dive that didn’t get you anything—
not one blessed thing.”
“Do you think I didn’t have any fun?”
“Hervey, boy, why did you do it? Why—why did you do it? A crazy
fool thing like that!” Hervey was silent, a trifle abashed by the
seriousness and vehemence of his visitor.
“Why did you do it?”
“I—I couldn’t help it.”
Young Ebin Talbot just looked at him as a wrestler might look,
trying to decide where to take his adversary. “I guess so,” he said
low and resignedly.
But he was not to be beaten so easily. “Hervey, there are only two
boys in this town who could do what Wyne Corson did, and he is one
of them and you’re the other one. Why are you never in the right
place at the right time?”
Hervey flared up, “Do you mean to tell me I don’t know any one
who could do that—what Wyne Corson did? Do you bet me I don’t?”
“Oh, for goodness’ sakes, Hervey! You did a hair-brained thing, a
big stunt if you will; and Corson did a heroic act. And here you are
making bets with me about something of no importance. What’s the
matter with you? Why I was paying you a compliment!”
“You said I don’t know anybody who could swim out like that. Do
you say I can’t—do you dare me⸺”
Young Mr. Talbot held up his hand impatiently. Hervey not only
never did the right thing, but he even couldn’t talk about the right
thing. Like many men who are genial in hope, he was impatient in
failure. He had not Mr. Walton’s tolerant squint.
“Please don’t dare me, Hervey. Dares and stunts never get a boy
anywhere.”
“How do you know how many fellers can do a thing?” Hervey
demanded.
“Well, all right then, Hervey, I don’t,” said Mr. Talbot rising. “But let
me just say this to you. I know you could do what Corson did
yesterday and it was a glorious thing, and brings him high reward.
Also, if it’s any satisfaction for you to know it, I believe you could find
a way of escaping from a military school. You see, I give you full
credit. I think there is hardly a single thing that you could not do—
except to do something with a fine purpose. Just to stand on your
head isn’t enough; do you see? The first time you do a brave,
reckless thing for service you’ll be the finest scout that ever lived.
None of them can touch you on action, but action means nothing
without motive. It’s just like a car jacked up and the wheels going
round; it never gets anywhere.”
“Didn’t I do a service to Diving Denniver?” Hervey demanded.
“Well, did you? Honor bright; did you? Did you want to help him?
Was that the idea? Come on now, Hervey. Fair and square, was it?”
“No, it wasn’t.”
“You did it because⸺”
“Didn’t I tell you it was because I couldn’t help it?” said Hervey
angrily.
CHAPTER XXXII
UPS AND DOWNS
Young Mr. Talbot gave Hervey up. I think he lost patience too
readily. As for Mr. Walton, he was past the stage of quiet argument
with his stepson. He was as firm in resolve as he was patient in
discussion. And never was Hervey more bent on action that was his
harassed guardian from the moment he was apprised of the carnival
escapade. Even gentle Mrs. Walton, who had pled after the satchel
episode, thought now that it was better for Hervey to go to military
school than to break his neck.
“Well, he won’t even break rules there,” said Mrs. Walton.
As for Hervey, he was not worrying about military school. He
never thought or worried about anything. He would meet every
situation as it came. He was not staggered by Wyne Corson’s
opportunity to go west. To give him credit, he was not selfish or
envious. He forgot all about Wyne Corson.
One matter he did bear in mind and it was the very essence of
absurdity. With his own narrow escape to ponder on, and Wyne
Corson’s splendid deed to thrill him (if he was capable of a thrill) he
must set off as soon as he was able to prove his all-important claim
that there was another individual capable of doing what Mr. Talbot
had said that only he and Corson could do. He accepted the young
scoutmaster’s declaration not as a compliment, but as a kind of dare.
That is how his mind worked and I am giving you just the plain facts.
I told you in the beginning that no one understood him.
But now he was to receive something as near to a shock as he
had ever received. He sought out Diving Denniver in his sanctum
and approached him rather sheepishly (for him) for he knew not how
his feat had impressed the wonder of two continents. It was the last
day of the carnival, the matter of the permit had been adjusted, and
Diving Denniver was that evening to dive for the last time in
Farrelton. On this occasion he wore his regular clothes and his little
derby hat was on the back of his head as he packed his trunk in
anticipation of departure.
“Hello,” said Hervey.
“Hello, yer gol blamed little fool.”
“Well, I did it, didn’t I?” said Hervey defensively.
“Sure you did it, but you were just lucky. You’re just a crazy kid,
that’s all. That there kid that’s got his name in the papers fer savin’ a
girl’s life, now he’s a regular guy, he is. If you want to jump why don’t
you get in the big parade, kid?” He folded some clothing and did not
pay much attention to Hervey as he talked. “If yer want ter pull the
big stuff why don’t yer get in with them guys. This here ain’t narthin’.”
“Do you know what a scoutmaster told me?” demanded Hervey,
somewhat aroused. “He said that only two fellers—me and that other
feller—could dive off that cliff and swim to shore with a girl. So as
long as you’re a friend of mine will you come and show him that you
can do it? Just to show him he’s not so smart. Then he’ll see you’re
a friend of mine, and he’ll see you can do it. Hey? So I can put it all
over him. Hey?”
“Naah, cut that stuff, kid. Why wuz yer thinkin’ I can swim and
save lives? I ain’t much on swimmin’, kid.” He reached over to where
Hervey sat dangling his legs from the makeshift table and good-
naturedly ruffled his hair. “Yer got me wrong, kid. What’s bitin’ yer
anyways? This here is a trick, that’s all it is. I know me little trick.
Why wouldn’ I? I been doin’ it fer seven years. There ain’t narthin’ to
it when yer once get it right. Did yer think this here wuz a kind of an
adventure like? Hand me them two saucers, will yer. Listen here, kid.
Here’s how it is. When yer know how ter do it there’ ain’t narthin’ to
it; see? An’ if yer try it when yer don’t know how, yer a blame fool. I
bet yer kin swim better’n what I can, at that. I jus’ do me turn, kid.
See?”
Hervey was staggered. “Ain’t you the wonder of two continents?”
he asked. “Don’t you say it yourself?”
“Sure thing, and I’m sorry I didn’t make it five continents when I
wuz printin’ it. What’s a couple of continents more or less? Pull that
there broken glass down and let’s have it, will yer? Yer don’t think yer
done narthin’ big do yer?” He paused and faced Hervey for just a
moment. “Dis here is just a trick, kid. Go on and join them kids
what’s doin’ the divin’. Come out o’ yer trance, little brother. You’ze
got the makin’s of a regular hop, skip and jumper, yer has. Wuz yer
old man sore at yer?”
Hervey felt as if the bottom had fallen out of the earth. Not that he
wanted praise and recognition; he never craved those. But what he
had done was just nothing at all. He was no more a hero than a man
who tried to commit suicide is a hero. And the wonder of two
continents was just a good-humored, tough little young man who
knew a trick! How brave and splendid seemed the exploit of Wyne
Corson now! That was not a trick.
“You beat it home now,” said McDennison, “and don’t go inter no
business what yer ain’t got the dope on. A kid like you oughter had
that trip ter the coast. Look at me, I ain’t got the carfare ter open up
in Bridgeburgh Fair.”
Hervey went away, not exactly heavy-hearted, for he was never
that. And not exactly thoughtful, for he certainly was never that. But
disgruntled. And even that was unusual with him. He might have had
that trip to the coast. Or at least on a dozen different occasions, he
might have won such a reward. But for all his fine bizarre deeds he
got just nothing; not even honor. And the pity of it was he could not
figure this out. He never remembered what anybody told him; he
never pondered. Yet I think that poor Diving Denniver did some
good; I think he almost reached him.
On the way home, he was saved from any of the perils of thought
by the allurements of action. Near the entrance to the carnival was a
basket full of booklets about Farrelton the Home Town. There was a
sign above this basket which read. Free—Take One. Hervey did not
take a booklet, but he took the sign. It was an oblong wooden sign
and had a hole in it to hang it up by. By inserting a stick in this hole,
he could twirl the sign around as he ambled homeward. He became
greatly preoccupied with this pastime and his concentration
continued till he reached the Aunt Maria Sweet Shoppe. Here were
bottles of honey and tempting jars of preserves standing on a display
shelf outside, and he coyly set the Free—Take One sign on these,
proceeding homeward with that air of innocence that he knew how to
affect.
Crossing the deserted Madden farm, he discovered a garter
snake. It was a harmless little snake, but it filled its destiny in
Hervey’s life. It was necessary for him to lift it on the end of his stick
and, before it wriggled off, send it flying through the broken window
of the Madden barn. This was not easy to do, because the snake
would not hold still. With each cast, however, it seemed to become
more drowsy, until finally it hung over the stick long enough for
Hervey to get a good aim and send the elongated missile flying
through the broken, cobweb-filled window.
The shot was so successful that Hervey could not refrain from
giving an encore. One good sling deserved another. So up he
vaulted to the sill of the old window, brushing ancient cobwebs out of
his eyes and hair, and down he went inside. But he went down
further than he had expected to, for the flooring was quite gone from
the old barn and he alighted all in a heap on a pile of dank straw in
the cellar.
Four unbroken walls of heavy masonry arose to a height of ten or
twelve feet. Far above him, through the shrunken, rotted shingles,
little glints of sunlight penetrated. A few punky boards strewn in this
stenchy dungeon gave evidence that the flooring above had rotted
away before being entirely removed. Perhaps there had been an
intention to lay a new flooring. But it was many years since the
Maddens had gone away and now there were rumors that the
extensive farm land was to become a bungalow colony.
As Hervey lifted one of the punky boards it broke in the middle
and fell almost in shreds at his feet. A number of little flat bugs,
uncovered in their damp abode, went scooting this way and that after
similar shelter. The snake too, recovered from the shock of being a
missile, wriggled off to some agreeable refuge amid the rotting litter
of that dank prison.
CHAPTER XXXIII
STORM AND CALM
Hervey’s fortunes were never at a lower ebb than when he stood
in that damp cellar as the night came on and tried to reconcile
himself to sleeping on the straw. Even the morrow held only the hope
that by chance some one would discover him in his dreadful
dungeon. It was not until a rotten board, laid diagonally against the
foundation, had collapsed with him that he gave up and threw
himself down with a feeling as near to despair as his buoyant nature
had ever experienced.
Through the cracks and crevices of the shingles high overhead,
he watched the light die away. A ray from the declining sun streamed
through the window from which he had fallen, lingered for a few
moments, then withdrew leaving the place almost in darkness. Such
a price to pay for a merry little game with a snake!
Meanwhile, events occurred which were destined to have a
bearing on Hervey’s life. At about half past nine that night, young Mr.
Talbot emerged from the Walton house and encountered Wyne
Corson coming in through the gateway. They both laughed at the
encounter.
“Missionary work?” Mr. Talbot inquired.
“You beat me to it?” laughed Wyne.
“No, I’m through,” Mr. Talbot said. “He isn’t even home; nobody
knows where he is. No, I’m through working on that prospect, and I
wouldn’t waste my time if I were you, Corson. He’s going to military
school and I guess that’s the best place for him.”
“The fellows in my troop are crazy about him,” said Wyne.
“They might better be crazy about you,” Mr. Talbot answered. “If
they’re as crazy as all that, they’re better off without another crazy
fellow in their troop. Come on, walk along with me; there’s no one

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1 Integrated Approach for the Diagnosis of Shiga Toxin-Producing

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Shiga toxin-producing Escherichia coli (STEC) cause a spectrum of

Stephanie Schüller and Martina Bielaszewska (eds.), Shiga Toxin-Producing E. coli:

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Prepare all solutions using ultrapure water (resistivity 18.2 MΩ/cm

11. Sterile loops for bacteriology (1 and 10 μL).

5. 100 penicillin/streptomycin stock solution: 10,000 U/mL

2.3 Reagents 1. Serum sample in a sterile vial (50 μL is sufficient).

10. Absolute ethanol.

the STEC strain identified at the screening step. The detection of

3.1.1 Preparation Keep fecal specimens refrigerated at 4 C if they were shipped at

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Microbiol 73(6):1892–1898. https://doi. producing Escherichia coli by automated 50

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Shiga toxin-producing E. coli (STEC) are food-borne pathogens

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