Received: 2 September 2021 Revised: 19 January 2022 Accepted: 24 January 2022

DOI: 10.1111/1541-4337.12929

COMPREH ENSIVE REVIEWS IN FOOD SCIENCE AND FOOD SAFETY

A systematized review and qualitative synthesis of potential

risk factors associated with the occurrence of non-O157
Shiga toxin-producing Escherichia coli (STEC) in the
primary production of cattle

Susan M. Withenshaw1 Richard P. Smith1 Rob Davies2 Alice E. O. Smith1

Elizabeth Gray1 John Rodgers2

1 Departmentof Epidemiological Sciences,

Animal and Plant Health Agency –
Weybridge, New Haw, UK
2 Department of Bacteriology, Animal and
Plant Health Agency – Weybridge, New
Haw, UK
Correspondence
Susan Withenshaw, Department of
Epidemiological Sciences, Animal and
Plant Health Agency – Weybridge,
Woodham Lane, New Haw, KT15 3NB,
UK.
Email: susan.withenshaw@apha.gov.uk
Funding information
Department for Environment, Food and
Rural Affairs, Grant/Award Numbers:
CR2000E, CR2008.
Abstract
Human infection with Shiga toxin-producing Escherichia coli (STEC) causes an
estimated 2.8 million cases of acute illness worldwide each year. Serogroup O157
is the most commonly diagnosed STEC in humans, but cases linked to non-O157
STEC serogroups have increased recently due to increased surveillance and
improvements to detection methods. Cattle are an important reservoir for STEC
O157 and the same may be true for non-O157 STEC; therefore, reducing the
occurrence of these pathogens in cattle could mitigate human infection risk. A
systematized literature review of articles published within the Scopus database
since 2010 (employing a partially systematic approach) was therefore conducted
followed by qualitative synthesis of evidence to provide a structured overview of
potential risk factors for non-O157 STEC in primary cattle production. Overall,
few relevant studies were identified (n = 22), highlighting that more studies are
needed. Consistently significant associations were only identified with respect
to cattle age (broadly higher rate of isolation from young animals compared
to adults) and season of sampling (generally increased isolation of non-O157
STEC in summer). However, wide variation in study designs, including notable
differences in laboratory detection methods, means drawing more general
conclusions is currently not possible based on the results of this review. How-
ever, it is likely that the development of more sensitive methods for non-O157
STEC detection in potential livestock reservoirs and increased standardization
across statistically sound epidemiological investigations are required to identify
pertinent risk factors.

KEYWORDS
bovine, epidemiology, Escherichia coli, risk factors, STEC

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the
original work is properly cited and is not used for commercial purposes.
© 2022 The Authors. Comprehensive Reviews in Food Science and Food Safety published by Wiley Periodicals LLC on behalf of Institute of Food Technologists

Compr Rev Food Sci Food Saf. 2022;1–28. wileyonlinelibrary.com/journal/crf3 1

2 RISK FACTORS FOR NON-O157 STEC IN CATTLE
1 INTRODUCTION
Escherichia coli are common bacteria and commensal con-
stituents of the mammalian gut flora (Conway et al., 2004;
Kaper et al., 2004), but some strains have acquired viru-
lence factors and can cause disease in humans and ani-
mals (Fremaux et al., 2008). Among these pathogenic E.
coli are Shiga toxin-producing E. coli (STEC) strains, which
are characterized by the production of Shiga toxins (Stx1
and Stx2) encoded by the genes stx1 and stx2 (Scheutz
et al., 2012; Shridhar et al., 2017). Human STEC infec-
tion usually leads to sporadic cases or larger outbreaks
of uncomplicated diarrhea, but can also progress to life-
threatening disease in infants and the immunocompro-
mised (e.g., hemolytic-uremic syndrome) (Byrne et al.,
2014).
Human infection with STEC is estimated to cause 2.8
million cases of acute illness worldwide each year (Majow-
icz et al., 2014). In 2019, it was the third most frequent bac-
terial agent detected in food-borne outbreaks in the Euro-
pean Union, with a total of 7775 laboratory confirmed cases
in humans and a case-fatality rate of 0.21% (EFSA & ECDC,
2019). Under-reporting of cases is likely to mean that the
true burden of infection is still greater. For example, in the
United Kingdom, a national study of gastrointestinal dis-
ease estimated that the rate of STEC E. coli incidence in the
community was 7.4 times greater than the rate reported to
national surveillance (0.3 vs. 0.042 cases per 1000 person-
years) (Tam et al., 2012).
There are over 100 different serogroups of STEC, dis-
tinguished by their somatic (O) antigens (Byrne et al.,
2014). Serogroup O157 is currently the most commonly
diagnosed in humans in the United Kingdom and most
other countries in Europe. However, it has been demon-
strated in many parts of the world that non-O157 STEC
strains collectively account for a higher proportion of lab-
oratory confirmed human STEC cases than O157 (e.g., in
the United States; Hadler et al., 2011), and recent microbio-
logical risk assessments by the World Health Organization
(WHO) and the European Food Safety Authority (EFSA)
concluded that any Stx-producing E. coli pose a risk to pub-
lic health, regardless of serotype (EFSA, 2013; WHO, 2018).
In addition, until recently most public health laboratories
have used testing methods that are specific for STEC O157;
therefore, it is likely that the human incidence of non-O157
STEC has been underestimated, especially in milder cases
(Cooley et al., 2013).
Six further STEC serogroups (O26, O45, O103, O111,
O121, and O145) have been regularly reported as posing
significant public health risks, with the severity of illness
often similar to STEC O157 (Gould et al., 2013; Shridhar
et al., 2017). The number of human cases linked to non-
O157 STEC serogroups has increased over recent years; in
England there was a 20-fold increase in reports of non-
O157 STEC serogroup isolations between 2011 and 2016
(Byrne et al., 2014; PHE, 2017). This trend is underpinned
by increased surveillance and improvements in detection
methods (Byrne et al., 2014), which have revealed a higher
prevalence of non-O157 STEC and more frequent involve-
ment in human illness than was once appreciated. There
is therefore a need to understand more about pathways for
human infection, and to identify control measures.
Current evidence suggests that approximately half of
all human STEC (including O157) illnesses worldwide are
foodborne, and a recent synthesis of outbreak data and
expert opinion indicated that a range of food types may be
implicated as sources of infection globally, including fresh
produce (i.e., fruit and vegetables) as well as livestock prod-
ucts (WHO, 2018). Source attribution is challenging, how-
ever, due to the potential complexity of the risk pathways
involved (e.g., contaminated vegetables may result from
the use of irrigation waters contaminated by infected live-
stock), and continual evolution of E. coli colonies (includ-
ing the potential loss and gain of virulence genes over time)
making it difficult to trace the origin of contamination.
Such was highlighted in a large outbreak of STEC O104:H4
linked to beansprouts in Europe in 2011 (King et al., 2012;
Karch et al., 2012). However, beef has been identified as
the most common food type to be associated with human
STEC outbreaks in a number of global regions (WHO,
2018). Cattle are also widely considered to be an important
reservoir for STEC O157, which they can harbor asymp-
tomatically in their gastrointestinal tract and shed inter-
mittently in their feces (Chapman et al., 1997; Paiba et al.,
2003), and this may also be true for non-O157 STECs. Iden-
tifying risk factors for the occurrence of non-O157 STEC in
cattle during primary production may therefore be key to
developing control measures that could reduce the risk of
both direct and indirect zoonotic transmission to humans
(Karmali et al., 2010).
Much of the existing research into the occurrence
of STEC in primary cattle production focuses only on
serogroup O157 (e.g., Ellis-Iversen et al., 2007; Gunn et al.,
2007; Paiba et al., 2002, 2003; Pritchard et al., 2009; Synge et
al., 2003; Schouten et al., 2004; Smith et al., 2016). Research
into the occurrence and risk factors of non-O157 STEC
serogroups is sparse but is increasing due to the greater
recognition of these STEC serogroups as important human
pathogens and increased reporting of hybrid pathotypes.
This is especially true following the large outbreak of STEC
O104:H4 in Europe in 2011 (King et al., 2012; Karch et al.,
2012). A review of the existing literature is therefore timely
RISK FACTORS FOR NON-O157 STEC IN CATTLE
to consolidate current knowledge on the patterns of occur-
rence and potential risk factors for non-O157 STEC in pri-
mary cattle production, and to identify knowledge gaps
that should be prioritized for focusing future research. The
specific objective of this review was to identify existing
published research studies that report the isolation of non-
O157 STEC within primary cattle production, along with
epidemiologically relevant detail of the circumstances of
isolation, so that potential risk factors may be determined.
2 MATERIALS AND METHODS
Existing literature was searched for in an online database,
and relevant epidemiological data relating to the occur-
rence of non-O157 STEC in primary cattle production were
retrieved and synthesized. Resource constraints meant
that a full systematic review was not possible. However,
a systematized review process was employed, whereby
several systematic steps for literature searching and data
retrieval were followed (Grant & Booth, 2009). A flow
chart depicting the different stages of the review process
is shown in Figure 1.
2.1 Literature search
A search for relevant original research publications was
carried out on the online abstract and citation database
Scopus on March 1, 2021. Titles, abstracts, and keywords
were searched against the following criteria: (“Escherichia
coli” OR “E. coli”) AND (“verocyt*” OR “shigat*” OR
“Shiga-toxin” OR “vtec” OR “stec” OR “O111” OR “O26”
OR “O145” OR “O103”) AND (“bovine” OR “cattle”).
The titles and abstracts of articles matching these crite-
ria were retrieved and subjected to screening. The search
was restricted to publications written in English because
the time and budget needed to acquire translations of non-
English publications was not available, though the search
was not otherwise restricted by country of research. For the
same reasons, the search was also restricted to articles pub-
lished after the January 1, 2010. The development of meth-
ods for detecting non-O157 STEC has progressed, particu-
larly during the last 10 years, with increasing sensitivity,
confirmation of virulence and serogroup via polymerase
chain reaction (PCR) methods, and work toward validated
protocols (Ludwig et al., 2020). It was therefore felt that a
cut-off date of 2010 was a suitable compromise given the
resource constraints of the project, as a large proportion of
relevant research publications were likely to be identified
despite these restricted search methods.
3
2.2 Literature screening
Each retrieved publication was first assessed for rele-
vance by reviewing the title and abstract. Publications were
excluded at this stage if at least one of the following exclu-
sion criteria were met: (1) the data presented were not
empirical, (2) data related only to the isolation of serogroup
O157, (3) data were not presented on the isolation of STEC
strains of E. coli, and (4) isolations were not directly or indi-
rectly from cattle and were not collected on cattle farms or
at slaughterhouses. Publications that did not meet any of
these criteria, or were ambiguous with respect to these cri-
teria according to title and/or abstract alone, were carried
through to the next stage of screening.
Each remaining publication was then read in full and
excluded if at least one of exclusion criteria 1–4 were
met according to the full text. All publications were also
assessed against a fifth criteria at this stage and excluded
from the review if this was met: (5) the reporting of the iso-
lation of non-O157 STEC was not stratified by at least one
epidemiological characteristic. The process of screening
publications was carried out by two reviewers (Reviewer
A and Reviewer B), who each screened approximately half
of the publications that were retrieved via the database
search.
In addition to the systematized process for identifying
eligible articles described above, the reference lists of suit-
able publications identified via this initial identification
and screening exercise were also interrogated for suitable
eligible articles using the same exclusion criteria. This
additional stage of article identification was performed on
an ad hoc basis and was not carried out systematically, and
so may have introduced bias into the identification and
selection of articles included in this review. During this ad
hoc phase, publications were included regardless of pub-
lication date, but non-English language publications were
still excluded.
2.3 Data collection
Each publication included in this review was then inter-
rogated to extract data relating to study design, labora-
tory methods, and reported epidemiological patterns of
non-O157 STEC occurrence. The specific data items sought
from each study are listed in Table S1. Initial review
and data collection was performed by a single individual
(Reviewer A). Each publication was then reviewed by a
second individual (Reviewer B), who cross-checked the
data extracted by the first individual, amended if necessary,
and sought to fill any data gaps if possible.
4 RISK FACTORS FOR NON-O157 STEC IN CATTLE

FIGURE 1 Flow chart depicting the number of publications included in this review and reasons for exclusion at each stage of screening

2.4 Data synthesis
The collected data were synthesized by Reviewer B using
a qualitative approach. Details of general study design
were first summarized to provide a broad overview of the
countries and types of premises (farms or slaughterhouses)
where empirical data relating to the epidemiology of non-
O157 STEC in cattle have been collected, and the types of
studies reported (e.g., longitudinal vs. cross-sectional stud-
ies). The different STEC serogroups that were targeted for
isolation across all publications was then determined.
To provide a structured overview of potential risk factors
for non-O157 STEC in primary cattle production, trends
in non-O157 STEC occurrence were synthesized according
to different epidemiological variables (e.g., cattle age, cat-
tle type, sampling season). For each variable, publications
that reported relevant data were identified. The non-O157
STEC prevalence reported in each publication was then
tabulated against the reported categories of the variable
(along with results of formal statistical tests if available)
to enable trends in pathogen occurrence to be compared
and contrasted across studies. The epidemiological vari-
ables investigated were not defined prior to the literature
review. Rather, synthesis is presented for an epidemiologi-
cal variable if relevant data were obtained from at least one
publication during the review process itself.
Where possible, data were collected from each publi-
cation relating to defined serotypes of known stx status
RISK FACTORS FOR NON-O157 STEC IN CATTLE
(i.e., specific pathotypes). However, some publications pre-
sented data on multiple non-O157 serogroups in combina-
tion. Some publications also presented data on the occur-
rence of STEC and non-STEC isolates combined even if
the serogroup was defined. As such, potential risk factors
could not always be attributed to specific pathotypes. These
uncertainties are highlighted where relevant throughout
this review. It should also be noted that studies were
included in this review only if they reported data on strains
of E. coli that produce Shiga toxins and, by definition, this
may also include enterohaemorrhagic E. coli (EHEC) as
well as STEC. To simplify the reporting of results, only the
term STEC is used in this review.
During the data collection stage of the review pro-
cess, a high degree of variability between publications
became evident with respect to the laboratory methods
for pathogen isolation and characterization. Since differ-
ent methods may vary in sensitivity and so confound
comparisons of study results, differences in the methods
reported by each publication were taken into considera-
tion when synthesizing the epidemiological results. The
laboratory methods themselves were therefore first syn-
thesized. Specifically, an attempt was made to categorize
the laboratory methods reported in each publication into
categories to enable simplified comparisons between pub-
lications. The categories were defined according to six
different methodological details that were considered the
most likely to introduce bias into the interpretation of epi-
demiological results, and included the approach used for
the characterization of serogroups and virulence genes,
and the order in which serogroup and virulence profiles
were determined. A full list of criteria used to categorize
the methodological details are listed in Table S2. These
details were extracted from each publication by Reviewer
B. Unique combinations of these methodological details
were then identified and classified into unique categories
(hereafter referred to as methods A–R).
3 RESULTS AND DISCUSSION
3.1 Overview of articles identified
The literature search retrieved 985 publications in total.
After an initial sift based on title and abstract, a more in-
depth appraisal of the remaining publications yielded a
final list of 22 papers of relevance to this review, which are
summarized in Table S3.
Publications related to studies conducted in the United
States (×9 publications), Canada (×3), Belgium (×2), New
Zealand (×2), Australia (×1), Japan (×1), Portugal (×1),
Republic of Ireland (×1), Scotland (×1), and South Africa
(×1). The majority of studies involved sampling of animals
5
on one or more farms (15 studies), with the remainder
involving sampling at slaughterhouses or from slaughter-
house transport vehicles (seven studies). Of the studies car-
ried out on farms, six took place at feedlots; these are dif-
ferentiated in this review due to feedlots being an intensive
farming system which, although common in North Amer-
ica, differ from the less intensive systems generally
employed in Europe in terms of management and poten-
tially also risk pathways for pathogens such as STEC. Most
studies were carried out at commercial farms or slaughter-
houses; however, six farm studies (including five carried
out at feedlots) were conducted on herds of cattle that were
managed by a research facility. Thirteen of the studies were
cross-sectional in nature, whereby samples were collected
from farms or slaughterhouses on a single occasion only
or, if visited multiple times, different animals were sam-
pled on each sampling occasion. In contrast, nine studies
reported the results of longitudinal sampling whereby the
same group or groups of animals were sampled on multi-
ple occasions over the duration of the study, which lasted
anywhere between 1 and 9 months.
All except one study reported targeted screening of sam-
ples for predetermined non-O157 STEC serogroups. Fifteen
studies targeted multiple different serogroups, while six
studies targeted a single serogroup each. The remaining
study did not target specific serogroups and instead per-
formed post hoc characterization of the O and H antigens
of E. coli colonies that were positive for virulence genes
(stx1, stx2, and eae) (Ballem et al., 2020). In total, eight
different serogroups were targeted across all studies, but
there was variation in the number of serogroups targeted
per study, and the number of studies targeting each
serogroup overall. The most common target serogroup
was O26, which was tested for in 20/22 studies, and was
the sole non-O157 serogroup targeted in five studies (Enge-
len et al., 2020a; Jaros et al., 2016; McCabe et al., 2019;
Paddock et al., 2014; Sasaki et al., 2013). Other commonly
targeted serogroups were O111 (15 studies), O103 (14
studies), O145 (14 studies), O45 (11 studies), and O121 (11
studies). Serogroups O113 and O177 were also tested for in
one study each. Prevalence of each E. coli serogroup and
pathotype was variable between studies, and sometimes
not reported; details are given in Table S3 to provide
context for interpreting the epidemiological patterns
outlined in the remainder of this review.
3.2 Variation in STEC detection
methods
An in-depth comparison and appraisal of laboratory meth-
ods used for the detection and characterization of non-
O157 STEC are beyond the scope of this review. However,
6 RISK FACTORS FOR NON-O157 STEC IN CATTLE
the laboratory methods described by the studies discussed
further in this review were variable in several respects, and
this should be borne in mind when attempting to com-
pare epidemiological outcomes. In total, 18 different varia-
tions on STEC detection and characterization were identi-
fied across the 22 publications and this variation is broadly
summarized in Table 1.
All reported methods involved the enrichment of raw
samples (though the specifics of enrichment conditions
were variable) prior to testing for the presence of STEC
by determining serogroup (O-types) and virulence profile
(including the presence of stx1/stx2). However, while the
majority of studies incorporated methods to isolate bacte-
rial colonies prior to pathogen characterization, six stud-
ies used PCR to test DNA extracted directly from enriched
samples (methods Q and R in Table 1; Cernicchiaro et al.,
2014; Hallewell et al., 2016; Paddock et al., 2014; Schnei-
der, Klopfenstein, et al., 2018; Schneider, Lewis, et al.,
2018; Stanford et al., 2017). Furthermore, even if bacte-
rial colonies were first isolated, there was between-study
variability with respect to the order that isolates were
serotyped and virulence profiled. For example, nine stud-
ies employed a method whereby bacterial isolates were
first tested for the presence of prespecified target serotypes
and then only virulence profiled if positive for the target
serotype of interest (methods A–H in Table 1; Baines &
Erb, 2013; Engelen et al., 2020a; Irshad et al., 2017; Jaros
et al., 2016; McCabe et al., 2019; Montso et al., 2019; Sasaki
et al., 2013; Pearce et al., 2004; Stanford et al., 2016). In con-
trast, testing methods in five studies involved determining
the virulence profiles of isolated colonies first before then
determining the serotypes only of those colonies that tested
positive for stx genes (and sometimes other virulence fac-
tors) (methods I–M in Table 1; Ballem et al., 2020; Engelen
et al., 2020b; Ekiri et al., 2014; Mellor et al., 2016; Pearce et
al., 2004). A third variation of this process, reported in four
studies, was the simultaneous characterization of isolates’
serotype and virulence profiles, whereby the virulence pro-
file of an isolate was determined regardless of its serotype
(or vice versa) (methods N–P in Table 1; Cull et al., 2017;
Cernicchiaro et al., 2020; Dewsbury et al., 2015; Paddock et
al., 2014).
In addition to variation in the sequence of pathogen
characterization, the methods for E. coli isolation when
used were not standard across studies. Some always sub-
jected enriched samples to immunomagnetic separation
(IMS) prior to plating on growth media (methods A–C,
J–K, and O–R in Table 1; e.g., Cernicchiaro et al., 2014,
2020; Dewsbury et al., 2015; Ekiri et al., 2014; Irshad et
al., 2017; Hallewell et al., 2016; Pearce et al., 2004; Pad-
dock et al., 2014; Sasaki et al., 2013; Schneider, Klopfen-
stein, et al., 2018; Stanford et al., 2017; Schneider, Lewis, et
al., 2018; Stanford et al., 2016) while others did not or, in the
case of Jaros et al. (2016), only when target serotypes were
not isolated when sample enrichments were first incu-
bated without the use of IMS. When used, the types and
manufacturers of the IMS beads themselves were variable
between studies, and a variety of growth media and spe-
cific incubation conditions were reported across all stud-
ies that attempted isolation of bacterial colonies. Further-
more, if bacterial colonies were isolated, then subsequent
characterization was then carried out either on individual
colonies or on pools of multiple colonies which were com-
bined according to one of several different criteria (e.g.,
methods H and K–O in Table 1).
Methods to characterize the serotypes of isolated bac-
teria included either serological techniques, PCR-based
techniques (singleplex or multiplex), or a combination of
both, and while the majority of studies reported using mul-
tiplex PCR to determine the presence of specific virulence
genes (with template DNA extracted either directly from
sample enrichments [methods Q and R in Table 1] or from
isolated bacterial colonies), Pearce et al. (2004) used DNA
hybridization in addition to PCR, and the use of a “rapid
flow test” was reported by Baines and Erb (2013).
Current laboratory methods used for STEC detection in
carrier animals, especially for non-O157 serotypes, gener-
ally have low sensitivity, meaning that only high levels of
organisms would have been detected in any of the studies
that are discussed further below, and prevalence of non-
O157 STEC is likely to have been under-estimated in all
cases. The different laboratory testing methods reported
will undoubtedly have also varied in their sensitivity. Addi-
tional variation in the types of sample collected (e.g., recto-
anal mucosal swab samples, rectal feces, and feces col-
lected from the ground), whether or not samples were
pooled prior to testing, and the transport/storage condi-
tions implemented following collection (e.g., refrigeration
during transport compared to ambient temperature) may
have compounded this variation further by differentially
affecting the number/concentration and/or viability of any
organisms present before testing even began. As a result,
some studies may have had more power than others to
detect significant epidemiological patterns in the occur-
rence of non-O157 STEC by virtue of employing more
sensitive sampling and laboratory processing methods.
Disentangling true between-study differences in the epi-
demiological patterns of non-O157 STEC occurrence from
differences arising due to study methodology (including
differences in study type and scale in addition to laboratory
testing methods used) is therefore problematic and makes
it difficult to identify general trends and to draw clear con-
clusions. Such differences are highlighted where necessary
throughout the remaining sections of this review.
TA B L E 1 Overview of laboratory methods used for the detection and characterization of E. coli in the studies reported in the 22 research publications that were included in this review
Serogroup and
Sequence of IMS used on enriched Serogroup virulence genes Bacterial
Method pathogen sample to separate characterization Virulence gene characterized for colonies
reference characterizationa target serogroups method(s) characterization method(s) same isolate pooled
RISK FACTORS FOR NON-O157 STEC IN CATTLE

Ab S-V Yes Singleplex PCR Multiplex PCR Yes No

B S-V Yes Multiplex PCR Multiplex PCR Yes No
C S-V Yes Serology Multiplex PCR Yes No
Db S-V No Singleplex PCR and serology Multiplex PCR Yes No
b c
E S-V Sometimes Singleplex PCR and serology Multiplex PCR Yes No
F S-V No Singleplex and multiplex Multiplex PCRo Yes No
PCR
G S-V No Polyvalent serologyk Rapid-flow test No (different isolates) No
H S-V No Singleplex PCR Multiplex PCR Yes Yesl
Id V-S Yes Multiplex PCR and serology Multiplex PCR Yes No
f g
J V-S Yes Serology or multiplex PCR Multiplex PCR Yes No
K V-S No Serology DNA hybridization and multiplex Yes Yesh
PCR
Lm V-S No Serology Multiplex PCR Yes Yesn
M V-S No Singleplex PCR Multiplex PCR Yes Yesn
N Simultaneous Yes Multiplex PCR Multiplex PCR Yes Yese
O Simultaneous Yes Multiplex PCR Multiplex PCR Yes Yesl
P Simultaneous Yes Multiplex PCR Multiplex PCR Yes (but isolates Yes
pooled)
(Continues)
7
 8

TA B L E 1 (Continued)
Serogroup and
Sequence of IMS used on enriched Serogroup virulence genes Bacterial
Method pathogen sample to separate characterization Virulence gene characterized for colonies
reference characterizationa target serogroups method(s) characterization method(s) same isolate pooled
Q Simultaneous No Multiplex PCR Multiplex PCR No (E. Coli not n.a.
isolated)i
Rb Simultaneous Yesj Multiplex PCR Multiplex PCR No (E. Coli not No
isolated)i
Abbreviations: IMS, immunomagnetic separation.
a
S-V = characterization of virulence genes for serogroup-positive samples or isolates only; V-S = characterization of serogroup for virulence gene-positive samples or isolates only; simultaneous = characterization of
serogroup and virulence genes performed simultaneously for all samples or isolates.
b
Initial PCR screening for target serogroups directly on sample enrichment. Only positive enrichments characterized further.
c
IMS not used on all enriched samples but was used for samples where isolates of any target serogroups were not retrieved from direct culture plates.
d
Initial PCR screening for target serogroups and virulence genes (simultaneously) on sample enrichment. Only enrichments positive for virulence genes and at least one target serogroup characterized further.
e
Suspect non-O157 colonies were pooled and tested via PCR for serogroups and virulence genes (simultaneously). Individual isolates were then only further characterized if a pool was positive for both a target serogroup
and virulence gene.
f
IMS beads did not include all target serogroups so selection for some serogroups may have been under-estimated.
g
Isolates that did not react with antisera via slide agglutination were subject to multiplex PCR for serotyping instead.
h
Initial sweep of mixed colonies from culture plate tested for presence of virulence genes by PCR. Only colonies from PCR-positive plates were then subjected to further characterization.
i
Multiplex PCR directly on sample enrichment rather than isolated bacterial colonies.
j
IMS used prior to serogroup characterization but not prior to virulence gene characterization.
k
Suspect non-O157 colonies subjected to polyvalent testing for three different combinations of non-O157 serogroups: Group 1 = O26, O55, O111, O119, O126; Group 2 = O83, O114, O125, O127, O128; Group 3 = O44, O112,
O124, O142.
l
Suspect non-O157 colonies were pooled and tested via PCR for target serogroups. Individual isolates were then only tested if a pool was positive for at least one target serogroup.
m
Initial PCR screening for virulence genes directly on sample enrichment. Only positive enrichments were further characterized.
n
Pooled colonies tested for virulence genes; individual colonies tested if pools were positive.
o
Only O177 colonies positive for bfp gene were further screened for the presence of other virulence genes, including stx.
RISK FACTORS FOR NON-O157 STEC IN CATTLE
RISK FACTORS FOR NON-O157 STEC IN CATTLE
3.3 Risk factors associated with cattle
characteristics
3.3.1 Age
Eight studies reported the occurrence of non-O157 STEC by
cattle age, but results were variable and not all identified
significant associations (Table 2).
A cross-sectional study of 21 dairy farms in Portugal
found that overall sample prevalence of non-O157 STEC
(comprising multiple different serotypes) was significantly
higher (Fisher’s exact test p < .05) in the feces of Holstein-
Friesian heifers aged 6–18 months (70/115 animals were
positive; 45% prevalence) than in lactating cows of the
same breed aged more than 24 months old (42/253 animals
were positive; 16% prevalence) (Ballem et al., 2020).
Elsewhere in Europe, cross-sectional sampling of 19 Bel-
gian dairy farms using overshoe swabs of occupied pens
resulted in a higher overall percentage of non-O157 STEC
positive samples (serotypes O26, O103 and O145 combined)
being collected from pens housing young animals aged 1–
24 months (11/51 samples positive; 22%) compared to adult
cattle (5/28 samples positive; 18%), though the percentage
of positive samples from new born calves aged less than 1
month was lower than both older age categories (1/9 sam-
ples positive; 11%) (Engelen et al., 2020b). However, the
total number of samples collected in this study was rela-
tively small (n = 88), fewer than half the farms involved
(9/19) tested positive for non-O157 STEC, and the statis-
tical significance of differences between age groups was
not reported since this was not the primary goal of the
research. In a subsequent study by the same authors which
involved three of the same farms, Engelen et al. (2020a)
reported further variation in the presence of E. coli O26 by
age. The three farms were each visited on three separate
occasions, and groups of young calves (aged less than 6
months old) were sampled via recto-anal mucosal swabs
(RAMS) during each visit. Most of the sampled groups
contained different animals at each visit. The highest per-
centage of E. coli O26-positive animals (results from all
three farms combined) were in the 15–30 days age cate-
gory (44%), while very few animals younger than 14 days
old were positive for E. coli O26 (7%). However, the total
number of samples collected and that were positive per age
category was not disclosed. Furthermore, most E. coli O26
isolates were positive for the virulence genes stx1 and/or
stx2, though 25 isolates from one farm were stx-negative.
Therefore, the reported results represent a combination of
STEC and non-STEC O26.
Outside of Europe, two longitudinal farm studies
described variation in fecal shedding of non-O157 STEC
in cohorts of cattle studied over time. Fecal shedding of
several E. coli serogroups (STEC and non-STEC combined)
9
and virulence-associated genes (stx1, stx2) was found to be
highly variable over time in a longitudinal study of beef
cattle steers in Canada carried out at a research facility
feedlot (Hallewell et al., 2016). Thirty steer calves, sourced
from a closed commercial herd, were each sampled (rec-
tal feces) on four occasions: at weaning (age of 196 ± 21
days), after arrival at the feedlot (1 day later), after 30 days
on the feedlot, and after 135 days on the feedlot. In general,
the prevalence of STEC increased over time, with preva-
lence of O26, O111, and O121 being significantly higher on
the final sampling occasion compared to at weaning. How-
ever, there was no significant difference in the prevalence
of O45 and O103, and the prevalence of serogroup O121
decreased over the same time period. The study also inves-
tigated changes over time in the prevalence of virulence
genes (all serogroups combined) and found a significantly
higher prevalence of at least one stx gene in samples col-
lected from animals aged 53 or 70 weeks compared to when
weaned at 49 weeks of age. However, this study took place
over the 5 months immediately following movement of
cattle from a ranch to a feedlot, and as they were transi-
tioned onto a different feeder diet. As such, the temporal
change in the occurrence of serogroups and virulence fac-
tors identified in this study may not solely be attributed to
age and may be confounded by other factors including cat-
tle movement, contact with other animals or a new con-
taminated environment, and diet. In contrast, a study car-
ried out on a different research farm in the United States
(Ekiri et al., 2014) found that the prevalence of non-O157
STEC (serogroups combined) in rectal fecal samples from
48 beef steers was highest during the postweaning period
(16%) and lower during finishing (6%) and slaughter (2%)
(i.e., decreased over time). However, this was based on a
descriptive analysis only, and the results are also likely
to be confounded by changes to diet and movement of
animals from a rearing to finishing site that took place
throughout the course of the study.
In addition to the farm studies described above,
three cross-sectional slaughterhouse studies reported age-
associated differences in the occurrence of non-O157 STEC
in cattle according to the collection of samples postslaugh-
ter. In New Zealand, Jaros et al. (2016) collected RAMS
sample from a total of 695 calves and 895 adults (represent-
ing 655 dairy farms and 354 beef farms) across four different
slaughterhouses and found a significantly higher preva-
lence of STEC O157 and O26 (combined) in calves aged 4–7
days old (42/695 positive samples; 6%) compared to adult
cattle (16/895 positive samples; 1.8%) (χ2 test p < .001). Simi-
larly, Mellor et al. (2016) tested 1500 fecal samples collected
from across 31 slaughterhouses in Australia and found
a higher prevalence of STEC (multiple serogroups com-
bined) in samples collected from the carcasses of young
cattle (504/744 positive samples; 67.8%) compared to adult
10 RISK FACTORS FOR NON-O157 STEC IN CATTLE

T A B L E 2 Details of cattle age-associated patterns in the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) according
to the 22 research publications included in this review. Eight of the 22 studies reported occurrence by cattle age category
Reported
prevalence (%) (No. Statistical significance
Non-O157 E. coli of positive/No. of of difference between
Reference pathotype Age category samples) categories
(Ballem et al., 2020) Multiple STEC 1. 6–18 mo 45.2 (70/155) Category 1 > Category 2
(serogroups 2. ≥24 mo 16.5 (42/254) (p < .05); Fisher exact
combined) test
(Engelen et al., 2020b) STEC O26, O103, O145 1. <1 mo 11.1 (1/9) Descriptive only
(serogroups 2. 1–24 mo 21.6 (11/51)
combined) 3. adults 17.9 (5/28)
(Engelen et al., 2020a) E. coli O26 1. <14 d 7a Descriptive only
(STEC + non-STEC) 2. 15–30 d 44a
(Jaros et al., 2016) STEC O157 and O26 1. 4–7 d 6.0 (42/695) Category 1 > Category 2
(serogroups 2. adults 1.8 (16/895) (p < .001); χ2 test
combined)
(Mellor et al., 2016) Multiple STEC 1. young (carcass weight 67.8 (504/744) Category 1 > Category 2
(serogroups ≤250 kg) (p < .001); Fisher
combined) 2. adult (carcass 32.2 (243/756) exact test
weight >250 kg)
(Hallewell et al., 2016) E. coli O145 1. 196 ± 21 d (weaning) 26.7 (8/30) Category 4 > Category 1
(STEC + non-STEC) 2. 197 ± 21 d (feedlot arrival) 10.0 (3/30) (p < .05); GLMM
3. 227 ± 21 d (30 days on 26.7 (8/30)
feed)b
4. 332 ± 21 d (135 days on 66.7 (20/30)
feed)c
E. coli O26 1. 196 ± 21 d (weaning) 23.3 (7/30) Category 3 and
(STEC + non-STEC) 2. 197 ± 21 d (feedlot arrival) 56.7 (17/30) 4 > Category 1 and 2
3. 227 ± 21 d (30 days on 83.3 (25/30) (p < .05); GLMM
feed)b
4. 332 ± 21 d (135 days on 80.0 (24/30)
feed)c
E. coli O111 1. 196 ± 21 d (weaning) 36.7 (11/30) Category 4 > Category 1
(STEC + non-STEC) 2. 197 ± 21 d (feedlot arrival) 10.0 (3/30) (p < .05); GLMM
3. 227 ± 21 d (30 days on 43.3 (13/30)
feed)b
4. 332 ± 21 d (135 days on 86.7 (26/30)
feed)c
E. coli O121 1. 196 ± 21 d (weaning) 56.7 (17/30) Category 3 > Category 1;
(STEC + non-STEC) 2. 197 ± 21 d (feedlot arrival) 23.3 (7/30) Category 4 < Category
3. 227 ± 21 d (30 days on 83.3 (25/30) 1 (p < .05); GLMM
feed)b
4. 332 ± 21 d (135 days on 6.7 (2/30)
feed)c
E. coli O45 1. 196 ± 21 d (weaning) 33.3 (10/30) No difference between
(STEC + non-STEC) 2. 197 ± 21 d (feedlot arrival) 53.3 (16/30) categories (p > .05);
3. 227 ± 21 d (30 days on 50.0 (15/30) GLMM
feed)b
4. 332 ± 21 d (135 days on 46.7 (14/30)
feed)c
E. coli O103 1. 196 ± 21 d (weaning) 70.0 (21/30) No difference between
(STEC + non-STEC) 2. 197 ± 21 d (feedlot arrival) 86.7 (26/30) categories (p > .05);
3. 227 ± 21 d (30 days on 73.3 (22/30) GLMM
feed)b
4. 332 ± 21 d (135 days on 73.3 (22/30)
feed)c
(Continues)
RISK FACTORS FOR NON-O157 STEC IN CATTLE
TA B L E 2 (Continued)
Non-O157 E. coli
Reference pathotype
(Ekiri et al., 2014) Multiple STEC
(serogroups
combined)
(McCabe et al., 2019) STEC O26
Abbreviations: d, days; GLMM, generalized linear mixed model; mo, months.
a
Number of samples collected and number positive per age category was not disclosed.
b
Fed a silage-based diet (70% barley silage, 25% barley grain, and 5% mineral and vitamin supplement; dry matter basis).
c
Fed a barley grain-based finishing diet (85% barley, 10% barley silage, and 5% supplement; dry matter basis).
animals (243/756; 32.2%; Fisher’s exact test p < .001). The
young cattle age category included all veal, young beef,
and young dairy animals with a carcass weight ≤250 kg,
and adults were beef and dairy animals with a carcass
weight >250 kg. In contrast, a study investigating STEC
O26 shedding in beef cattle at the time of slaughter at
three large commercial abattoirs in the Republic of Ire-
land found no significant difference in the rate of shedding
between different age groups (McCabe et al., 2019). How-
ever, only 10/1317 RAMS samples (0.76%) tested positive
for STEC O26 in this study, and this low prevalence is
likely to have affected analytical power (though the statis-
tical methods used were not reported in detail). It is worth
noting, however, that no positive samples were identified
in cattle older than 30 months at the time of sampling
(Table 2).
Age-related patterns of non-O157 STEC occurrence var-
ied across the studies identified here. However, most stud-
ies reported a broadly higher rate of isolation from young
animals compared to adults, suggesting that young cattle
may be an important on-farm reservoir of pathogenic E.
coli. This is consistent with trends seen in several stud-
ies of O157 STEC which have identified increased shed-
ding in calves compared with adult cattle (Cho et al.,
2009; Nielsen et al., 2002; Paiba et al., 2003). More fre-
quent occurrence of STEC in younger cattle may be the
result of a less well-developed immune system, under-
developed rumen function, or lower diversity of protec-
tive gut microflora compared to adult cattle (Mir et al.,
2016; Zhao et al., 2013), which may result in increased
susceptibility of younger animals to STEC infection (Lew-
erin et al., 2019). Super-shedding individuals may also be
more common in younger age groups compared to adults
(McCabe et al., 2019), leading to higher levels of contam-
ination in the environment of younger animals and con-
tributing to increased rate of STEC transmission within
11
Reported
prevalence (%) (No. Statistical significance
of positive/No. of of difference between
Age category samples) categories
1. Postweaning (1–3 mo) 15.6 (15/96) Descriptive only
2. Finishing (5 mo) 6.4 (3/47)
3. Slaughter (8 mo) 2.1 (1/47)
1. 11−18 mo 1.4 (2/142) No difference between
2. 19–24 mo 0.9 (4/459) categories; Details of
3. 25–30 mo 0.8 (4/495) statistical test not
31–36 mo 0.0 (0/143) reported
37+ mo 0.0 (0/78)
these age groups. However, the studies differed in the age
categories and E. coli pathotypes investigated, number of
samples collected, and statistical methods used; therefore,
comparisons across studies should be made with caution.
3.3.2 Weight
Two slaughterhouse studies carried out in New Zealand
reported the detection of non-O157 STEC according to
cattle weight (live weight or carcass weight; Table 3).
Multivariable logistic regression models were constructed
by Jaros et al. (2016) using data collected in the same
study of four slaughterhouses that was described ear-
lier. Carcass weight was investigated as one of several
variables to identify risk factors for the presence of E.
coli O26 (STEC and non-STEC combined) in RAMS sam-
ples. Occurrence of E. coli O26 in calf samples (age 4–
7 days old) and adult samples was investigated sepa-
rately. Mean carcass weight of calves and adult cattle
was reported at 15.7 kg (95% confidence interval [CI]
9.7–21.7) and 271.2 kg (95% CI 133.9–408.4), respectively,
and was not a significant predictor of positivity in either
age group (Table 3). Interestingly, the same conclusion
was drawn for STEC O157, which was investigated in the
same study. In contrast, Irshad et al. (2017) did iden-
tify a significant effect of carcass weight on the odds of
RAMS samples from calves aged less than 7 days old
at slaughter being positive for E. coli O26 (STEC and
non-STEC combined), whereby the odds of being posi-
tive decreased by 0.05 with every 1 kg increase in the
weight of calves (p = .05 in a multivariable generalized
linear mixed model [GLMM]). No such effect was seen
for E. coli O103 (p = .86) nor O145 (p = .86), although
these serogroups had an overall lower prevalence (23%
and 16%, respectively) compared to serogroup O26 (45%
12
T A B L E 3 Details of cattle weight-associated patterns in the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC)
according to the 22 research publications included in this review. Two of the 22 studies reported occurrence by weight category
Reference Non-O157 E. coli pathotype
(Jaros et al., 2016) E. coli O26
(STEC and non-STEC)
(Irshad et al., 2017) E. coli O26
(STEC and non-STEC)
E. coli O103
(STEC and non-STEC)
E. coli O145
(STEC and non-STEC)
Abbreviation: GLMM, generalized linear mixed model.
prevalence overall; Table S3), which may have affected
statistical power. This study only included samples col-
lected from very young calves; therefore, any confounding
effect of animal age is likely to have been minimal. How-
ever, the range of carcass weight is also likely to have been
limited, and so it is difficult to envisage how such a finding
would be broadly applicable to on-farm control. In addi-
tion, STEC and non-STEC positivity was combined in the
analysis.
3.3.3 Gender
Three different slaughterhouse studies presented data on
the occurrence of STEC stratified by the gender of the ani-
mals sampled, and none found evidence of a statistically
significant difference (Table 4). In the study by Jaros et
al. (2016) involving four slaughterhouses in New Zealand,
there was no significant effect of gender on PCR detection
of E. coli O26 (STEC and non-STEC combined) in RAMS
samples from calves (male vs. females; p = .48) or adults
(male vs. castrated male vs. female; p = .875) according to
univariable logistic regression models (Table 4). Likewise,
Irshad et al. (2017) found that gender (male vs. female)
was not a significant predictor of RAMS samples from veal
calves being PCR-positive for neither E. coli O26, O103
nor O145 (STEC and non-STEC combined) according to
univariable and multivariable logistic regression models
(p > .05 in all case; Table 4). Finally, McCabe et al. (2019)
stated that there was no significant difference in the detec-
tion of STEC O26 in RAMS samples collected from beef cat-
tle of different gender in their abattoir study in the Repub-
lic of Ireland. The authors did not indicate the number of
samples collected or proportion positive from males versus
females, nor provide further detail of the statistical results
obtained in relation to a comparison of positivity between
genders. However, as noted earlier, the study suffered from
RISK FACTORS FOR NON-O157 STEC IN CATTLE
Statistical significance
of difference between
Relationship identified categories
Calves: No relationship identified p > .05; GLMM
Adults: No relationship identified p > .05; GLMM
Negative relationship; odds of being p = .05; GLMM
positive decreased by 0.05 with every
1 kg increase in calf carcass weight
No relationship identified p = .86; GLMM
No relationship identified p = .86; GLMM
low analytical power due to very low overall prevalence of
the target pathogen (0.76%).
In contrast to the results from abattoir sampling, one
farm study involving eight feedlot farms in the United
States did identify a significant association between the
occurrence of non-O157 STEC and the gender of the cat-
tle sampled, though this was based on indirect sampling of
floor feces rather than samples taken from individual cat-
tle (Cull et al., 2017). Considering all farms together, a total
of 1886 pen-floor fecal samples were collected, and 3.3%
(63/1886) tested positive for at least one of the non-O157
STEC serogroups investigated (O26, O45, O103, O111, O121,
O145, or O157; Table S3). A multivariable GLMM found
that within-pen prevalence of non-O157 STEC in pen-floor
fecal samples (serogroups combined) varied significantly
by the sex composition of the pen, with higher preva-
lence in steer-only pens (model-adjusted mean within-pen
prevalence estimate 3.1% with confidence interval 1.3%–
7.1%) compared to heifer-only (1.1%, CI 0.3–3.8) or mixed
pens (0.5%, CI 0.2–2.8) (p = .02; Table 4). Such differ-
ences may not just reflect an effect of gender per se, how-
ever, which is likely to be confounded by differences in
behavioural dynamics, stress, and diet experienced by ani-
mals depending on the composition of the pen in which
they are housed. Physiological state of individuals may also
be a confounding factor. For example, in a longitudinal
farm study involving 12 cattle herds in England, Smith et al.
(2016) found a significantly higher prevalence of STEC
O157 in the feces of males compared to females, but only
if the males were castrated; the same may be true of non-
O157 STEC serogroups. In addition, the farms in this study
varied in terms of the type of cattle raised (beef, dairy, or
both); therefore, sex composition of pens may have been
confounded by the types of farms involved if, for example,
heifer-only pens were primarily associated with farms rais-
ing dairy cattle.
T A B L E 4 Details of cattle gender-associated patterns in the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) according to the 22 research publications included in
this review. Four of the 22 studies reported occurrence by gender category
Reported prevalence (%) Statistical significance of
(No. of positive/No. of difference between
Reference Non-O157 E. coli pathotype Gender category samples) categories
(Jaros et al., 2016) E. coli O26 1. Male (calves) 32.6% (167/512) No difference between
(STEC and non-STEC 2. Female (calves) 55.1% (65/183) categories 1 and 2 (p = .48);
RISK FACTORS FOR NON-O157 STEC IN CATTLE

combined) 3. Male (adults) 6.9% (12/173) univariable GLMM

4. Castrated male (adults) 7.3% (26/355) No difference between
5. Female (adults) 8.2% (30/367) categories 3, 4 and 5
(p = .85); univariable
GLMM
(Irshad et al., 2017) E. coli O26 1. Male 47.3% (100/211) No difference between males
(STEC and non-STEC 2. Female 42.0% (37/88) and females (p = .53)
combined) univariable GLMM; (p =
.59) multivariable GLMM
E. coli O103 1. Male 21.8% (46/211) No difference between
(STEC and non-STEC 2. Female 25.0% (22/88) categories 1 and 2 (p = .54)
combined) univariable GLMM; (p =
.72) multivariable GLMM
E. coli O145 1. Male 15.6% (33/211) No difference between
(STEC and non-STEC 2. Female 15.0% (14/88) categories 1 and 2 (p = .95)
combined) univariable GLMM; (p =
.09) multivariable GLMM
(McCabe et al., 2019) STEC O26 1. Male Not reported per gender No difference between males
2. Female Not reported per gender and females; Full statistical
results not reported
(Cull et al., 2017) Multiple STEC 1. Steer-only pens 3.1% (CI 1.3–7.1) Category 1 > Categories 2 and
(serogroups combined) 2. Heifer-only pens 1.1% (CI 0.3–3.8) 3 (p = .02); GLMM
3. Mixed steer and heifer pens 0.5% (CI 0.2–2.8)
Abbreviation: CI, confidence interval; GLMM, generalized linear mixed model.
13
14
3.3.4 Breed and herd type
Five studies reported occurrence of non-O157 STEC
according to cattle breed or herd type, and the majority
reported no difference (Table 5). All three of the slaugh-
terhouse studies already described in this review recorded
the breed of cattle from which RAMS samples were col-
lected (Irshad et al., 2017; Jaros et al., 2016; McCabe et, al.,
2019). However, the number of samples collected per breed
was very uneven in all cases, which may have resulted in
a lack of power to identify significant differences between
some breeds. Of the RAMS samples collected from calves
across four New Zealand slaughterhouses by Jaros et al.
(2016), 35.8% of Friesian calves (168/469) were positive for
E. coli O26 (STEC and non-STEC combined), while 28.2%
Jersey calves (62/220) were positive. The remaining sam-
ples were collected from three different breeds of beef cat-
tle, and one third (2/6) were positive. While a univariable
logistic regression model identified a lower odds ratio of
detecting E. coli O26 in Jersey calves compared to Friesian
calves (odds ratio [OR] 0.70, CI 0.50–1.00, p = .05), the
effect was not significant when incorporated into a multi-
variable model (p = .40; Table 5). Irshad et al. (2017) also
found no significant effect of breed (Friesian, Hereford,
or Jersey) on the detection of E. coli O26, O103, or O145
(STEC and non-STEC combined) in RAMS samples col-
lected from young veal calves. The number of samples col-
lected and proportion positive per breed was not reported,
but p > .05 for the breed variable in all univariable and mul-
tivariable models (Table 5).
With respect to adult cattle, Jaros et al. (2016) found sim-
ilar proportions of E. coli O26-positive RAMS samples from
Hereford cattle (14/159; 8.8%), Friesian cattle (20/276; 7.2%),
Angus cattle (22/314; 7.0%), and Jersey cattle (5/75; 6.7%)
from the four New Zealand slaughterhouses, and breed
was not a significant variable in either univariable or multi-
variable models (p = .86 and p = .22, respectively). McCabe
et al. (2019) also reported that there was no significant
effect of breed on detection of STEC O26 in their slaughter-
house study in the Republic of Ireland. However, the differ-
ent breeds sampled were not described, and, as previously
mentioned, prevalence of STEC O26 was very low (0.76%),
which is likely to have resulted in low analytical power.
No studies of live animals within a farm setting were
identified as reporting comparisons of non-O157 STEC
occurrence according to cattle breed, though two studies
made broader comparisons between herd types. In Japan,
Sasaki et al. (2013) compared the prevalence of STEC O26
in beef and dairy cattle by collecting rectal content sam-
ples from 10 healthy adult cattle on each of 25 beef farms
RISK FACTORS FOR NON-O157 STEC IN CATTLE
and 25 dairy farms. All of the dairy cattle sampled were the
Holstein-Friesian breed (250/250), whereas the beef cattle
were a combination of Japanese Black (70/250), Holstein-
Friesian (20/250), or first-generation hybrids of these two
breeds (160/250). Just one animal each from beef and dairy
cattle herds tested positive for STEC O26, and with such
a low prevalence it was not possible to make a statisti-
cally robust comparison of prevalence across herd types
(Table 5). Prevalence of the target STEC serogroups was
similarly low in the study of eight feedlots in the United
States by Cull et al. (2017), with just 3.3% of pen floor fecal
samples (63/1886) testing positive for at least one of the
non-O157 STEC serogroups investigated (O26, O45, O103,
O111, O121, O145 or O157), and just 1.5% of samples testing
positive for STEC O103, which was the most prevalent non-
O157 STEC serogroup and detected on all eight farms. The
authors reported that cattle type (beef or dairy) was not a
significant predictor of pen-level prevalence of non-O157
STEC (all serogroups combined), or STEC O103 or STEC
O145. The proportion of positive samples per cattle type
was not reported. However, representation of cattle types
was unbalanced in this study (five feed lots fed beef cat-
tle only, one fed dairy cattle-only, and two fed both beef
and dairy cattle), making statistically robust comparisons
between cattle types problematic.
Identifying robust patterns of STEC occurrence accord-
ing to cattle breed or herd type may be complicated by
potential confounding factors that make it difficult to con-
fidently determine the primary drivers of any difference.
For example, while different cattle breeds may vary in sus-
ceptibility to infection as a result of differences in underly-
ing genetics, which has indeed been demonstrated under
controlled experimental conditions for STEC O157 (Jeon
et al., 2013), breed is often also a predetermining factor of
herd type (e.g., beef or dairy). Any breed-associated dif-
ferences in STEC occurrence may therefore instead reflect
differences in pathogen exposure as a result of differences
in herd management between beef and dairy herds. Fur-
thermore, variation in the way that herds are managed
and resulting risk pathways for infection within broad herd
type categories (e.g., closed dairy herds vs. flying dairy
herds; suckling beef herds vs. fattening beef herds) means
that an investigation of risk factors within broad herd type
categories may be more informative than a wider compar-
ison of beef and dairy herds. Indeed, a study of risk fac-
tors for shedding STEC O157 in cattle herds in England
found no difference in risk of shedding between beef fat-
tener and dairy herds, but significantly lower risk of shed-
ding in suckling beef herds compared to both beef fattener
and dairy herds (Smith et al., 2016).
RISK FACTORS FOR NON-O157 STEC IN CATTLE 15

T A B L E 5 Details of patterns in the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) associated with cattle breed or
herd type according to the 22 research publications included in this review. Five of the 22 studies reported occurrence by cattle breed or herd
type category
Reported prevalence (%) Statistical significance
Non-O157 E. coli Breed or herd type (No. of positive/No. of of difference between
Reference pathotype category samples) categories
(Jaros et al., 2016) E. coli O26 1. Friesian (calves) 35.8% (168/469) Category 1 > Category 2 (p
(STEC and 2. Jersey (calves) 28.2% (62/220) = .05); univariable
non-STEC 3. Other breeds (calves) 33.3% (2/6) GLMM
combined) 4. Friesian (adults) 7.2% (20/276) No difference between
5. Jersey (adults) 6.7% (5/75) categories 1, 2 or 3 (p =
6. Angus (adults) 7.0% (22/314) .40); multivariable
7. Hereford (adults) 8.8% (14/159) GLMM
8. Other breeds (adults) 9.6% (7/71) No difference between
Categories 4 - 8 (p =
.86) univariable GLMM;
(p = .22) multivariable
GLMM
(Irshad et al., 2017) E. coli O26 1. Friesian See footnotea No difference between
(STEC and 2. Hereford See footnotea categories (p > .05);
non-STEC 3. Jersery See footnotea univariable and
combined) multivariable GLMM
E. coli O103 1. Friesian See footnotea No difference between
(STEC and 2. Hereford See footnotea categories (p > .05);
non-STEC 3. Jersery 3. Jersery univariable and
combined) multivariable GLMM
E. coli O145 1. Friesian See footnotea No difference between
(STEC and 2. Hereford See footnotea categories (p > .05);
non-STEC See footnotea See footnotea univariable and
combined) multivariable GLMM
(McCabe et al., 2019) STEC O26 Different breeds sampled See footnotea Full results not reported
not reported but authors state no
significant effect of
breed.
(Sasaki et al., 2013) STEC O26 1. Dairy 0.4% (1/250) Statistical comparison not
(Holstein-Friesian) possible due to low
2. Beef (Japanese Black, 0.4% (1/250) prevalence
Holstein-Friesian, F1
cross)
(Cull et al., 2017) Multiple STEC 1. Beef See footnoteb No difference between
(serogroups 2. Dairy See footnoteb categories (p > .20);
combined) univariable GLMM
STEC O103 1. Beef See footnoteb No difference between
2. Dairy See footnoteb categories (p > .20);
univariable GLMM
STEC O145 1. Beef See footnoteb No difference between
2. Dairy See footnoteb categories (p > .20);
univariable GLMM
Abbreviation: GLMM, generalized linear mixed model.
a
Number of samples collected and proportion positive per breed was not reported.
b
Proportion of positive samples per herd type was not reported.
16
3.4 Risk factors associated with
features of herd management
3.4.1 Housing
Two longitudinal cohort studies assessed whether the
shedding of non-O157 STEC varied depending on whether
cattle were housed indoors or were outside (Table 6).
Little evidence was found in support of an effect. The
first study involved the sampling of 49 calves on a mixed
beef and sheep farm in northern Scotland (Pearce et al.,
2004). Rectal fecal samples were collected from individ-
ual calves at birth and then weekly for up to 21 weeks.
The study animals were allowed to run together in
the same field on pasture for the first 11 weeks of the
study (August–November), and were then housed together
indoors on straw bedding for the remainder of the study
period (November–January). The most common non-O157
serogroups detected were O26 and O103, with overall sam-
ple prevalence of E. coli O26 being 17.3% (115/664 samples;
STEC and non-STEC combined) and E. coli O103 being 5.1%
(34/664 samples; STEC and non-STEC combined). Accord-
ing to the results of two-tailed Fisher’s exact tests, there
was no significant change in the rate of shedding of either
E. coli O26 or E. coli O103 either 1 or 2 weeks after hous-
ing compared with the results seen the week before hous-
ing (p = 1.00 in all cases), although shedding of E. coli
O103 was not detected at all in the second week after hous-
ing. However, the time elapsed between sampling animals
at pasture and indoors was short and may not have been
sufficient for resulting changes to animals’ susceptibility
and/or exposure to infection, and subsequent fecal shed-
ding, to become apparent.
In contrast, a longitudinal study of 48 beef cows and
their calves on a research farm in the United States found
that prevalence of non-O157 STEC (multiple serogroups
combined) in rectal fecal samples was higher when housed
in a winter dry lot (29.0% of 124 samples were posi-
tive) compared to when they were at pasture (8.3% of
48 samples were positive) (Ekiri et al., 2014). However,
this was a descriptive analysis only. Furthermore, sam-
ples were collected on three different occasions across 6
months when housed in the dry lot but on just a sin-
gle occasion when at pasture. This unbalanced sampling
design may have resulted in STEC prevalence at pas-
ture being underestimated relative to prevalence when
housed, especially if shedding is intermittent over time
(as has been demonstrated for STEC O157; Smith et al.,
2010).
Further investigation of the effect of housing conditions
and periodic changes to herds’ housing situation (e.g.,
rehousing in the winter after being at pasture in the sum-
mer) on occurrence of non-O157 STEC in cattle is war-
RISK FACTORS FOR NON-O157 STEC IN CATTLE
ranted. Certain housing conditions may favor survival of
non-O157 STEC in the environment and lead to increased
exposure of cattle to infection when housed, as has been
found for STEC O157 (Ellis-Iversen et al., 2008). House-
flies have also been identified as a potential vector for non-
O157 serogroups according to a study at feedlots and dairy
farms in the United States (Puri-Giri et al., 2016); there-
fore, the presence of these and other pests inside housing
could contribute to infection risk and dynamics of within-
farm transmission. Equally, risk of exposure while at pas-
ture may vary depending on local conditions and should
be explored. For example, long-term presence of several
STEC serogroups on grazing pasture following contami-
nation with cattle manure has been demonstrated experi-
mentally in a study in the Netherlands (van Overbeek et al.,
2020), and a study in the United States detected carriage of
multiple STEC serogroups by wild birds sampled near agri-
cultural land (Navarro-Gonzalez et al., 2020). Time inter-
vals between manure spreading and cattle grazing on pas-
ture and local abundance of wild birds may therefore have
consequences for cattle infection and shedding of STEC.
Furthermore, a study of 32 beef herds in Scotland also
demonstrated a complex interaction between the effect of
being at pasture versus housed and the presence of wild
geese on the shedding of STEC O157 (Synge et al., 2003).
Such complex interactions between herd management and
contact with wildlife may also be relevant for non-O157
STEC. Physiological changes to individuals’ susceptibility
to infection as a result of stress from re-housing and associ-
ated group mixing, and concurrent dietary changes, could
also influence risk of infection and patterns of fecal shed-
ding (Ellis-Iversen et al., 2008).
3.4.2 Herd size and pen size
Only one study reported non-O157 STEC occurrence in
relation to the size of the herd. This was the study by Cull
et al. (2017) carried out across eight feedlots in the United
States. Total herd size of all farms in this study was very
large, but also varied within the study on an order of mag-
nitude with between 8000 and 85,000 cattle per feedlot.
Despite this variation, a univariable GLMM found that
within-feedlot prevalence of non-O157 STEC (serogroups
combined) did not vary significantly by feedlot capacity
when those with < 25,000 were compared to those with
≥25,000 animals (p > .20; Table 6). In the same study,
the number of cattle/pen ranged from 30 to 582, but pen
size (categorized as < 100, 100–200, 200–300, or > 300
animals/pen) was not a significant predictor of within-
pen prevalence when investigated in separate multivari-
able models for non-O157 STEC combined, STEC O103 or
STEC O145 (p > .20 in all cases; Table 6).
T A B L E 6 Details of cattle herd management factors and associated occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) according to the 22 research publications
included in this review. Four of the 22 studies reported occurrence by different categories of herd management
Statistical significance
Herd management Non-O157 E. coli of difference between
Referencea feature pathotype Categories compared Reported prevalence categories
(Pearce et al., 2004) Housing E. coli O26 1. 1 week prior to housing See footnoteb No difference between
RISK FACTORS FOR NON-O157 STEC IN CATTLE

(STEC and non-STEC) 2. 1 week after housing See footnoteb categories (p = 1.00);
3. 2 weeks after housing See footnoteb Fisher’s exact test
Housing E. coli O103 1. 1 week prior to housing See footnoteb No difference between
(STEC and non-STEC) 2. 1 week after housing See footnoteb categories (p = 1.00);
3. 2 weeks after housing See footnoteb Fisher’s exact test
(Ekiri et al., 2014) Housing/diet Multiple STEC 1. Winter dry lot (fed on 29.0% (36/124; collected across 3 Descriptive only
(serogroups dry alfalfa-grass mixed sampling visits)
combined) diet) 8.3% (4/48; collected during 1
2. Pasture (grazing native sampling visit)
pasture)
(Cull et al., 2017) Herd size Multiple STEC 1. <25,000 See footnoteb No difference between
(serogroups 2. ≥25,000 See footnoteb categories (p > .20);
combined) univariable GLMM
Pen size Multiple STEC 1. <100 animals/pen See footnoteb No difference between
(serogroups 2. 100–200 animals/pen See footnoteb categories (p > .20);
combined) 3. 200–300 animals/pen See footnoteb univariable GLMM
4. >300 animals/pen See footnoteb
(Montso et al., 2019) Management system E. coli O177 1. Intensive 33.3% ± 14.43c No difference between
(STEC and non-STEC) 2. Semi-intensive 33.3% ± 14.43c categories (p > .05);
3. Extensive 50.0% ± 14.18c GLM
Abbreviations: GLMM, generalized linear mixed model; GLM, general linear model.
a
See Table 2 for details of (Hallewell et al., 2016).
b
Prevalence per category was not reported.
c
Number of samples collected per farm and per farming system was not reported.
17
18
Herd and pen size may be expected to influence both
individuals’ susceptibility and exposure to STEC infection.
For example, group size may affect stress levels and subse-
quently immune function and also contact rates between
susceptible and infectious individuals. In addition, over-
all herd size might determine how a herd and the farm
operation as a whole is managed, and so potentially influ-
ence multiple risk pathways for animals’ exposure to STEC
while on farm (e.g., risk of incursion as a result of num-
ber of staff members or deliveries by external contractors
or vets required, or increased risk of contact with environ-
mental contamination if larger herds are dispersed over
a larger area while at pasture). Although the single study
identified here did not report an effect of herd or pen size
on occurrence of non-O157 STEC, the pen and group sizes
involved were much larger than those generally seen on
commercial cattle farms outside of North America. Fur-
ther studies involving a broader range of herd types and
sizes are therefore needed to robustly assess whether herd
and/or pen size has broad relevance to risk of non-O157
STEC infection in cattle. Future studies should also con-
sider the influence of potential confounding factors when
investigating the effect of herd size. For example, while
Cho et al. (2013) found that small herds with < 100 cat-
tle were associated with increased rate of STEC O157 shed-
ding, many of the smaller herds were organic whereas all
of the larger herds were managed conventionally. Disen-
tangling the effects of herd size and other management
characteristics may therefore be difficult and depend on
the nature of farms that can be recruited for study.
3.4.3 Type of farming system
A study of beef and dairy cattle in South Africa found
that the occurrence of E. coli O177 (STEC and non-STEC
combined) did not vary significantly between intensive,
semi-intensive, and extensive animal production systems
(Montso et al., 2019), with prevalence in rectal fecal sam-
ples being 33.3 ± 14.43% in both intensive and semi-
intensive farms, and 50.0 ± 14.18% in extensively man-
aged farms (Table 5). A total of 780 fecal samples were
collected from eight farms over a period of 6 months, and
376 isolates were identified as serogroup O177. However,
the number of samples collected per farm, or per farm-
ing system, was not reported, and it is assumed (though
not clear) that the results provided relate to the percent-
age (and 95% CI) of O177-positive fecal samples per farming
system. The primary aim of this study was an assessment
of the genetic virulence and antimicrobial resistance pro-
files of an atypical enteropathogenic E. coli serogroup, and
few epidemiological details were provided. The broader
relevance of this result is therefore unclear. Further inves-
RISK FACTORS FOR NON-O157 STEC IN CATTLE
tigation of the impact of farming system on the occur-
rence of non-O157 STEC in cattle may be warranted, given
that increased shedding of STEC O157 has been identi-
fied in organic farms compared to conventional herds (Cho
et al., 2013). However, as previously discussed, awareness
is needed of potential confounders such as herd size.
3.4.4 Diet, feed supplements, and
vaccination
Two farm studies reported data on non-O157 STEC occur-
rence in relation to characteristics of animals’ feed. How-
ever, potential confounders were present in both cases,
making it difficult to draw firm conclusions about the role
of diet in the risk of shedding. Hallewell et al. (2016) col-
lected rectal fecal samples from 30 beef steers on a feed-
lot in Canada over a period of 20 weeks and reported how
the prevalence of several STEC serogroups changed over
time and in relation to changes in diet. Serogroups O145
and O111 were found to increase in prevalence at the time
when cattle were fed a concentrate-based finishing diet
compared to when sampled at weaning, though prevalence
of serogroup O121 decreased over the same time period
(6.7% compared to 56.7%), and prevalence of O45 and O103
did not change significantly over time (Table 5). Changes
in diet were confounded in this study by changes in ani-
mal age, especially since the animals were relatively young
throughout the study (aged 11–12 months old at the final
sampling occasion) and with an immune system that was
likely to still be developing. The difference in combined
prevalence of several non-O157 STEC serogroups in beef
cattle when moved from pasture to a dry feedlot on a beef
farm in the United States (Ekiri et al., 2014; described ear-
lier) may also have been related to a change in diet. Feed
type changed from grazing of native pasture while kept
outdoors to feeding of a dry alfalfa-grass mixed diet while
housed in a winter dry lot. It is therefore possible that the
higher prevalence detected while located in the dry feedlot
(29.0% vs. 8.3%; Table 5) was a result of changes to diet, and
associated changes to animals’ microflora and immune sta-
tus. Investigations of risk factors for shedding STEC O157
in young cattle herds in England and Wales found that a
number of feed types offered to cattle were associated with
shedding (Smith et al., 2016); further investigation into the
effect of feed composition on the shedding of non-O157
STECs may identify similar trends.
Significant associations between feed composition and
occurrence of non-O157 STEC in RAMS samples collected
from individual animals were, however, identified in an
experimental randomized control trial on beef cattle at
a farm research facility in the United States (Schneider,
Klopfenstein, et al., 2018). The study concluded that the
RISK FACTORS FOR NON-O157 STEC IN CATTLE
probability of detecting STEC O45 in feces was positively
associated with increased fiber content in the animals’ diet,
with odds of detection being 9.1 times greater in steers
fed 22% neutral detergent fiber (NDF) compared to those
fed 17% NDF (p < .001 in multivariable logistic regression
model), although no such effect was found for STEC O103,
O111, or O121 (nor indeed for O157). The study also found
that the source of dietary fiber influenced the probability of
fecal shedding for some STEC serogroups, but not in a con-
sistent manner: cattle were more likely to shed STEC O103
if fed a modified distiller’s grain diet compared to a corn
bran diet (p = .02 for the interaction between fiber level and
dietary source), but the opposite was true for STEC O111
(p = .03 for the interaction term). For comparison, Synge
et al. (2003) found that the feeding of distillers’ grains was
a risk factor for shedding STEC O157 in beef suckler cows.
In addition to feed composition, the consequences of
using prebiotics as feed supplements in domestic cattle on
the shedding of non-O157 STECs were reported in three
studies (two of which analyzed the same samples using dif-
ferent methods); however, just one of these studies iden-
tified a significant effect. Experimental treatment of beef
heifers at a farm research facility in the United States found
that treatment with a prebiotic significantly reduced the
proportion of animals shedding non-O157 STEC in feces
(serogroups combined; Baines & Erb, 2013). The prebi-
otic treatment consisted of a dose of 14 g per day of a
commercial yeast-based product (Celmanax), R which the
authors had previously shown to be effective at reducing
O157 and non-O157 STEC colonization of cell culture in
vitro (Baines et al., 2011b, 2011a). However, the effect iden-
tified in the experimental in vivo experiment was only evi-
dent in spring, with no such effect being apparent in sum-
mer.
In contrast, a randomized control trial carried out at
a feedlot in the United States involving 17,148 beef steers
found no significant effect of administering a direct-fed
microbial (DFM) product on the fecal shedding of non-
O157 E. coli serogroups in cattle, though it is unclear if the
analysis conducted in this study was based on the presence
of only E. coli serogroups with virulence genes, or if non-
STEC E. coli were also included within the outcome vari-
able (Cernicchiaro et al., 2014). Cattle were divided across
40 pens (mean 430 steers per pen), and allocated to receive
the DFM or not. The DFM product consisted of Lactobacil-
lus acidophilus strain NP51 and Propionibacterium freuden-
reichii NP24 which was dosed at ≥106 CFU per animal per
day throughout the study period, which lasted for a mean
of 87 days. Thirty floor fecal samples per pen were collected
weekly during the last 4 weeks of the trial, and the feed
treatment was found to have no significant effect on fecal
prevalence of E. coli serogroups O26, O45, O103, or O121
according to GLMMs (p > .05 in all cases). Statistical com-
19
parisons of serogroups O111 and O145 were not possible due
to very low prevalence. In the same study, some experi-
mental groups were treated with an E. coli O157:H7 vac-
cine, and this too was found to have no effect on fecal shed-
ding of any of the non-O157 serogroups tested for, when
either given as the only treatment or in combination with
the DFM (though it did significantly reduce the fecal shed-
ding of serogroup O157). In an extension to the study by
Cernicchiaro et al. (2014), Paddock et al. (2014) used an
alternative method to test the same samples for the pres-
ence of STEC O26 only, but also concluded that neither the
feed supplement nor vaccine treatment had any impact on
fecal shedding.
3.5 Risk factors associated with
environmental factors
3.5.1 Season or month of sampling
Studies carried out in four different countries (USA,
Canada, Republic of Ireland and New Zealand) reported
non-O157 STEC occurrence in cattle with respect to the sea-
son or month of sampling (Table 7). However, in general,
low prevalence of the STEC serogroups investigated meant
that studies often lacked the statistical power needed to
identify robust seasonal patterns. Furthermore, the stud-
ies identified here were variable in their sampling design
and season definitions, which makes between-study com-
parisons of seasonal trends problematic.
Most of the studies that reported seasonal trends were
carried out in the central states of the United States and
in general identified an increase in the occurrence of non-
O157 STEC in summer months (comprising June, July,
and/or August) compared to other times of year (Table 7).
A study on a single large commercial feedlot in central
United States (a more precise location was not reported)
found an overall higher prevalence of non-O157 STEC in
the feces of finisher cattle in summer compared to win-
ter (Dewsbury et al., 2015). Floor feces were collected
weekly for 12 weeks in summer (June–August 2013) and
10 weeks in winter (January–March 2014), totaling 576
samples collected from across 24 pens (121–627 cattle per
pen) in each season. The seasonal difference in preva-
lence was statistically significant for STEC O103 accord-
ing to a GLMM (prevalence in summer was 1.6% vs.
0.0% in winter; p < .05), but prevalence of other non-
O157 STEC serogroups was too low for a seasonal com-
parison. However, the authors reported that no non-O157
STEC serogroups detected were ever detected during win-
ter months.
In contrast, a different feedlot study of cow-calf herds
in Mississippi and Nebraska, USA, did detect a significant
20 RISK FACTORS FOR NON-O157 STEC IN CATTLE

T A B L E 7 Details of season or month of sampling and associated occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC)
according to the 22 research publications included in this review. Six of the 22 studies reported occurrence by season or month of sampling
Statistical significance
of difference between
Reference Non-O157 E. coli pathotype Season Reported prevalence categories
(Dewsbury et al., 2015) STEC O103 1. Summer (June–Aug.) 1.6% (95% CI 0.0–4.4) Summer > Winter
2. Winter (Jan.–Mar.) 0.0% (p < .05); GLMM
(Schneider et al., 2018b) STEC O45 1. Winter (Feb.) 1.2% (4/340) Summer > all other
2. Spring (Apr.) 0.3% (1/340) categories (p = .02);
3. Summer (July) 10.0% (34/340) GLMM; OR = 4.2, 95%
4. Autumn (Sep.) 1.5% (5/340) CI: 1.3–14.0
STEC O111 1. Winter (Feb.) 0.0% (0/340) Summer > all other
2. Spring (Apr.) 1.8% (6/340) categories (p < .001);
3. Summer (July) 13.5% (46/340) GLMM; OR = 23.0,
4. Autumn (Sep.) 5.6% (19/340 95% CI: 5.6–90.9
STEC O145 1. Winter (Feb.) 0.6% (2/340) Summer > all other
2. Spring (Apr.) 0.6% (2/340) categories (p = .007);
3. Summer (July) 10.9% (37/340) GLMM; OR = 8.3, 95%
4. Autumn (Sep.) 0.6% (2/340) CI: 1.9–37.0
STEC O26 1. Winter (Feb.) 0.6% (2/340) No difference between
2. Spring (Apr.) 0.0% (0/337) categories (p > .05);
3. Summer (July) 16.2% (55/340) GLMM
4. Autumn (Sep.) 0.6% (2/340)
STEC O121 1. Winter (Feb.) 0.0% (0/340) No difference between
2. Spring (Apr.) 0.0% (0/340) categories (p > .05);
3. Summer (July) 4.7% (16/340) GLMM
4. Autumn (Sep.) 0.6% (2/340)
(Cull et al., 2017) Multiple STEC (serogroups 1. June 2.8%, 95% CI 1.1–7.1 June > July (p = .03);
combined) 2. July 1.5%, 95% CI 0.6–3.7 June > August (p <
3. August 0.6%, 95% CI 0.1–1.8 .001); July > August (p
= .04); GLMM
STEC O145 1. June 1.5%, 95% CI 0.0–4.7 June > July(p < .01);
2. July 0.2%, 95% CI 0.0–1.0 GLMM
3. August 0.2%, 95% CI 0.0–1.6
STEC O103 1. June 0.1%, 95% CI 0.0–1.7 August > June (p = .01);
2. July 2.1%, 95% CI 1.0–4.4 August > July (p =
3. August 2.4%, 95% CI 1.1–5.4 .02); GLMM
(McCabe et al., 2019) STEC O26 1. Winter 0.0% (0/327) Descriptive only
2. Spring 1.02% (4/391)
3. Summer 1.21% (3/248)
4. Autumn 0.85% (3/351)
(Jaros et al., 2016) E. coli O26 (STEC and 1. July - Calves only 43.2% (57/132) July > other months
non-STEC) 2. August - Calves only 33.3% (138/415) (p = .006); univariable
3. September - Calves only 25.0% (37/148) GLMM
July > other months (p
= .004); multivariable
GLMM
E. coli O26 (STEC and 1. Winter - Adults only 7.2% (14/195) No difference between
non-STEC) 2. Spring - Adults only 9.0% (14/155) categories (p = .51);
3. Summer - Adults only 8.8% (24/272) univariable GLMM
4. Autumn - Adults only 5.9% (16/273)
(Continues)
RISK FACTORS FOR NON-O157 STEC IN CATTLE 21

TA B L E 7 (Continued)
Reference Non-O157 E. coli pathotype Season
(Stanford et al., 2016) E. coli O26 (STEC and 1. Winter (Dec.–Feb.)
non-STEC) 2. Spring (Mar.–May)
3. Summer (June–Aug.)
4. Autumn (Sept.–Nov.)
E. coli O45 (STEC and Winter (Dec.–Feb.)
non-STEC) Spring (March–May)
Summer (June–Aug.)
Autumn (Sept.–Nov.)
E. coli O103 (STEC and Winter (Dec.–Feb.)
non-STEC) Spring (March–May)
Summer (June–Aug.)
Autumn (Sept.–Nov.)
E. coli O121 (STEC and Winter (Dec.–Feb.)
non-STEC) Spring (March–May)
Summer (June–Aug.)
Autumn (Sept.–Nov.)
E. coli O111 (STEC and Winter (Dec.–Feb.)
non-STEC) Spring (March–May)
Summer (June–Aug.)
Autumn (Sept.–Nov.)
E. coli O145 (STEC and Winter (Dec.–Feb.)
non-STEC) Spring (March–May)
Summer (June–Aug.)
Autumn (Sept.–Nov.)
Abbreviation: CI, confidence interval; GLMM, generalized linear mixed model; OR, odds ratio.
a
Study was carried out across 2 years and proportion positive per year is shown; total number of samples collected per season was not reported.
Statistical significance
of difference between
Reported prevalence categories
Yr1: 37.5%; Yr2: 65.9%a Winter < other seasons
Yr1: 76.0%; Yr2: 78.8%a (p < .001); GLMM
Yr1: 92.7%; Yr2: 98.0%a
Yr1: 71.7%; Yr2: 86.2%a
Yr1: 42.5%; Yr2: 80.9%a Winter < other seasons
Yr1: 93.3%; Yr2: 95.5%a (p < .001); GLMM
Yr1: 92.7%; Yr2: 98.8%a
Yr1: 92.5%; Yr2: 98.6%a
Yr1: 57.5%; Yr2: 92.3%a Winter < other seasons
Yr1: 93.8%; Yr2: 96.3%a (p < .001); GLMM
Yr1: 88.8%; Yr2: 99.7%a
Yr1: 96.7%; Yr2: 98.6%a
Yr1: 12.5%; Yr2: 48.5%a Winter < other seasons
Yr1: 55.6%; Yr2: 62.9%a (p < .001; GLMM)
Yr1: 74.3%; Yr2: 85.4%a
Yr1: 70.4%; Yr2: 79.5%a
Yr1: 45.0%; Yr2: 18.8%a Winter > other seasons
Yr1: 2.2%; Yr2: 9.8%a (p < .001; GLMM)
Yr1: 4.7%; Yr2: 12.4%a
Yr1: 5.4%; Yr2: 14.8%a
Yr1: 22.5%; Yr2: 12.2%a Winter > other seasons
Yr1: 4.4%; Yr2: 11.0%a (p < .001; GLMM)
Yr1: 8.9%; Yr2: 3.2%a
Yr1: 2.3%; Yr2: 4.8%a

seasonal trend and determined that the odds of detecting
three different STEC serogroups was greater in summer
compared to all other seasons combined (Schneider, Lewis,
et al., 2018). In contrast to the study by Dewsbury et al.
(2015), this study involved four different herds and used a
slightly different seasonal definition. Pen floor fecal sam-
ples were collected from each herd on four occasions dur-
ing a single year, representing winter (February), spring
(April), summer (July), and autumn (September). Eighty-
five samples were collected per farm in each season, result-
ing in a total of 1360 samples, of which 14.4% overall were
positive for at least one of the STEC serogroups tested
for (O26, O45, O103, O121, O145, or O157). Sample preva-
lence of all serogroups was highest on the summer sam-
pling occasion compared to all others, and the difference
was statistically significant for O45 (OR = 4.2, p = .02),
O111 (OR = 23.0, p < .001), and O145 (OR = 8.3, p = .007)
(Table 7). For other non-O157 serogroups, there was a
lack of power to make seasonal comparisons due to very
small numbers of positive samples being identified at other
times of year other than summer. Furthermore, there was
between-farm variability in the occurrence of all STEC
serogroups investigated and seasonal patterns were not
consistent.
The study of eight commercial feedlots in the United
States by Cull et al. (2017), carried out across Texas and
Nebraska, reported that within-pen prevalence of non-
O157 STEC (serogroups combined) varied significantly by
summer month, with a higher prevalence in June (model-
adjusted prevalence estimate 2.8%) compared to July (1.5%,
p = .03) and August (0.6%, p < .001; Table 7). A simi-
lar seasonal pattern was identified for STEC O145 when
modeled separately, with a significantly higher prevalence
in June (1.5%,) compared to July (0.2%, p < .01). How-
ever, the pattern for STEC O103 was different, with preva-
lence being significantly higher in August (2.4%) compared
to June (0.1%, p = .01) and July (2.1%, p = .02). Preva-
lence of other STEC serogroups was too low to be mod-
eled separately, and data were only collected from June
to August in a single year; therefore, more general sea-
sonal comparisons outside of summer months were not
discernible.
22
A similar seasonal pattern was identified in a study
of fecal samples collected from slaughterhouse lorries in
Alberta, Canada (Stanford et al., 2016). This study ana-
lyzed the presence of STEC and non-STEC combined in a
total of 1749 samples, and found that serogroups O26, O45,
O103, and O121 were significantly less prevalent in cooler
winter months (December–February) compared to other
seasons (p < .001; Table 7). However, in contrast, preva-
lence of the more uncommon serogroups O111 and O145
was significantly higher during winter compared to other
seasons (p < .001), although they were still not common
in winter (<10% of samples overall were positive for these
serogroups; Table 7).
Two studies performed outside of North America
reported occurrence of STEC in relation to season, but due
to low prevalence of non-O157 STEC identified in these
studies the patterns identified were not conclusive. In the
Republic of Ireland, McCabe et al. (2019) found that the
highest levels of STEC O26 shedding in beef and dairy cat-
tle at slaughter occurred in the summer, and the lowest lev-
els occurred in the winter. However, this difference was not
statistically significant due to the overall low prevalence of
STEC O26 (0.76% of 1317 cattle sampled were positive). In
their cross-sectional study of slaughter cattle at four abat-
toirs in New Zealand, Jaros et al. (2016) collected samples
from adult cattle each month across 2 years, and fortnightly
from calves between July and October each year. No sig-
nificant seasonal variation in the PCR detection of E. coli
O26 (STEC and non-STEC combined) was identified for
adult cattle when comparing spring, summer, autumn, and
winter months (p = .51 in a univariable GLMM), though
the months included in each seasonal category was not
reported. In contrast, prevalence in calves was significantly
higher in July (43.2%) compared August (33.3%) or Septem-
ber (25.0%) according to both univariable and multivari-
able GLMMs (p < .05; Table 7).
Robust conclusions about seasonality of non-O157 STEC
detection are difficult to draw from the studies identi-
fied due to the generally very low prevalence of the target
pathogens. Where significant seasonal trends were identi-
fied, higher prevalence during summer months appeared
to be the most common trend, which is similar to the pat-
terns often identified for STEC O157 (e.g., Gunn et al.,
2007; Schouten et al., 2004; Sheng et al., 2016). Compara-
tively lower shedding of STEC during winter months might
reflect a combination of decreased STEC survival in the
environment during colder weather and lower rates of
infection or shedding of by individuals (e.g., due to sea-
sonal changes to feed). However, seasonal trends are likely
to be the result of a number of interacting factors and
therefore highly context-dependent and variable accord-
ing to local conditions and herd management practices.
Furthermore, seasonal shifts in the composition of micro-
RISK FACTORS FOR NON-O157 STEC IN CATTLE
bial communities in the environment and in the cattle gut
have been demonstrated (Dowd et al., 2008; Franz et al.,
2005), and it is therefore highly plausible that seasonal-
ity of environmental occurrence and cattle infection risk
is variable across STEC serotypes. Discerning such trends
and making comparisons between serotypes with confi-
dence would require higher temporal resolution investi-
gations with greater analytical power (e.g., involving more
farms and collection of more samples).
3.5.2 Weather
In addition to seasonality, two studies highlighted asso-
ciations between STEC occurrence and localized weather
events. In their study of beef cattle on four farms in the
United States, Schneider, Lewis, et al. (2018) found that
the odds of detecting STEC O45 in feces was significantly
lower if a precipitation event had occurred within the week
prior to sampling (OR = 0.07, 95% CI: 0.006–0.8), but this
association was not identified for other non-O157 STEC
serogroups investigated (O26, O103, O111, O121, and O145)
nor for O157 STEC. In contrast, a different study involving
fecal sampling of transport trailers at two Canadian slaugh-
terhouses reported that the prevalence of E. coli O103 was
significantly higher (p < .05) when rainfall was 3.3 mm/day
above the seasonal average of 3.7 mm/day, though this
analysis was based on serogroup-only information (i.e.,
STEC and non-STEC combined) and the authors did not
report the raw prevalence data (Stanford et al., 2017).
The same two studies also identified significant associ-
ations with temperature, but patterns were not consistent
across serogroups. In the first study (Schneider, Lewis, et
al., 2018), there was an increase in the odds of detecting
STEC O103 for every 5◦ C increase in ambient temperature
on the day of sampling (OR = 4.5, 95% CI 1.5–13.6), but no
similar effect was found for other serogroups. In the second
study (Stanford et al., 2017), the prevalence of E. coli O45
and O121 in winter was significantly lower if temperatures
were lower than the seasonal average (daily mean temper-
ature 12.3◦ C lower than seasonal average of −10.4◦ C), but
prevalence of E. coli O111 was significantly lower if winter
temperatures were higher than average (daily mean tem-
perature 10.5◦ C warmer than seasonal average of −10.4◦ C).
In the summer, the prevalence of E. coli O26 was signif-
icantly lower if temperatures were higher than average,
but no effect was found for other non-O157 serogroups.
Interestingly, this study found that serogroup O157 was
more responsive to severe weather events than non-O157
serogroups.
The fact that the atypical weather events described in
these two studies had varying effects on the occurrence
of different E. coli serogroups highlights the potential
RISK FACTORS FOR NON-O157 STEC IN CATTLE 23

complexity of disentangling seasonal and weather- cattle production were identified by this review, highlight-
associated effects. This complexity is compounded by ing that further empirical studies investigating potential
the often low prevalence of the target serogroups under risk factors are needed. Furthermore, a large proportion
investigation, and the combined use of STEC and non- of the studies reported results from North America (nine
STEC pathogens in analyses in some cases, which make it studies from the United States and three studies from
difficult to draw broad conclusions. Canada), where intensive cattle farming involving very
large herds and feedlot rearing is commonplace. The rel-
evance of the epidemiological patterns identified to other
3.5.3 Geographical variation cattle-rearing regions such as Europe, where herd sizes
are generally much smaller and herd management is less
The occurrence of non-O157 STEC was found to vary sig-
intensive, may therefore be limited, especially with respect
nificantly by geographical location in three different stud-
to aspects of herd management and seasonality.
ies where formally investigated. The presence of STEC
Seven of the 22 studies involved sampling of animals
O26 in young calves (4–7 days old) at slaughter varied
or vehicles at slaughterhouses rather than on farm. While
between four slaughter plants located in different geo-
risk factors identified through such studies are still rele-
graphical regions of New Zealand (Jaros et al., 2016), and
vant to the overall control of non-O157 STEC in the food
geographical differences were identified in the odds ratio
chain, they may not be applicable to the on-farm situation
of detecting STEC O26 in adult beef cattle on four farms in
and control within the farm environment since findings
the United States, with samples more likely to be positive
are likely to be strongly influenced by the experiences of
from the two farms in Nebraska compared to the two farms
the animals after leaving the farm (e.g., transport-related
in Mississippi (Schneider, Lewis, et al., 2018). In addition,
stress). In addition, of the 15 studies carried out on farms,
a study of slaughterhouse cattle in Canada also found that
six were carried out at research facilities. While such facil-
the presence of Shiga-toxin encoding genes varied signifi-
ities are likely to emulate conditions on commercial farms
cantly by the geographic origin of cattle for all of the non-
to some extent, they are nevertheless ‘‘artificial’’ farm envi-
O157 serogroups investigated; (O103, O45, O26, O121, O111,
ronments and therefore the results of such studies may not
and O145; Stanford et al., 2016).
be directly applicable to commercial farms. Several of these
Broad geographical variation in the occurrence of non-
studies applied experimental treatments to different ani-
O157 STEC may reflect differences in regional environ-
mal groups and measured how this affected STEC occur-
mental conditions and prevalence of infection. However,
rence. Such studies in controlled environments are valu-
reports of farm level variation in STEC occurrence and
able for identifying how specific management practices
prevalence are not uncommon, even within the same
may influence the risk of STEC occurrence in cattle, but
region. For example, a survey of herd-level STEC O157
may not replicate the complexity of factors within the com-
prevalence involving 952 beef cattle farms in Scotland esti-
mercial setting of primary cattle production.
mated that only 22.8% of farms had at least one animal
The reported prevalence of non-O157 STEC varied con-
shedding the pathogen in feces (Gunn et al., 2007). Simi-
siderably between studies and overall was very low. Iden-
larly, for non-O157 STEC, estimated prevalence on farms
tification of significant epidemiological patterns via for-
in Northern Portugal ranged from 5% to 65.4% accord-
mal statistical analyses was often therefore not possible,
ing to Ballem et al. (2020). Drawing conclusions about
or subject to a large degree of uncertainty, due to lack of
broad geographical differences in STEC occurrence based
power within datasets. For some studies, this meant that
on a relatively small number of farms or slaughterhouses
patterns of occurrence were reported via descriptive statis-
may instead reflect farm-level differences that are driven
tics only, or that statistical comparisons of occurrence were
by a multitude of different interacting factors rather than
performed without accounting for confounders. Epidemi-
true geographical differences in pathogen distribution.
ological patterns for the same STEC serotypes were some-
The high degree of farm-level variation in STEC occur-
times not consistent between studies, and differences in
rence should be taken into account when planning future
study size and design, and resulting analytical power, may
surveillance and investigations of risk factors for occur-
have contributed to this variation. Epidemiological pat-
rence of non-O157 STEC on farms.
terns also varied between STEC serogroups when investi-
gated within the same study. Variation in prevalence and
resulting analytical power may also have contributed to
4 GENERAL DISCUSSION different patterns being identified. Any true differences in
the epidemiology of different non-O157 STEC serogroups
Overall, very few studies reporting epidemiological infor- (e.g., due to their filling different ecological or biological
mation on the occurrence of non-O157 STEC in primary niches) could have consequences for control but require
24
corroboration via further investigation that would benefit
from the use of more sensitive and standardized detection
methods.
The most common STEC serogroup targeted for inves-
tigation in the studies identified here was O26. This is not
surprising given that after O157, STEC O26 is the serogroup
that has most commonly been associated with human ill-
ness worldwide so far (Valilis et al., 2018). Investigations of
other non-O157 STEC serogroups are relatively rarer, and
this may partly result from laboratory methods for detec-
tion being less well developed for these serotypes.
Variation in methodology, and resulting differences in
selectivity of STEC detection, makes it difficult to draw
comparisons between studies in terms of the epidemiolog-
ical patterns identified. This is compounded by the fact
that investigators often performed data analysis on test
results relating to multiple STEC serotypes in combination,
or relating to the presence of both STEC and non-STEC
pathotypes even if serotype was specified. The definition of
STEC also differed between studies, with some only includ-
ing samples or isolates that were positive for the virulence
factor eae in addition to stx genes, while others included all
stx-positive samples regardless of eae presence; therefore,
like-for-like comparisons between these investigations are
not possible. Furthermore, sporadic shedding of STEC by
infected cattle, which is well documented (e.g., Matthews
et al., 2006; Smith et al., 2010) may have exacerbated differ-
ences in the epidemiological patterns identified between
studies, especially where the number of samples collected
was small relative to the total cattle population size, which
was often the case.
Although this review included a comprehensive descrip-
tive synthesis of data collected from the selected publi-
cations, the review process was not fully systematic and
was subject to some limitations that ought to be high-
lighted. Due to time and resource constraints, only a sin-
gle electronic database was used to retrieve articles and
relevance screening was not cross-checked (i.e., the deci-
sion to include or exclude each article based on title and
abstract was taken by a single individual); this may have
resulted in some bias to which publications were included
in the review and it is possible that additional relevant
publications may have been retrieved by searching mul-
tiple databases and if all relevance screening was con-
ducted in parallel by a second reviewer. Further relevant
publications may have been omitted as a result of restrict-
ing to the English language and limiting the initial litera-
ture search to articles published from 2010 onwards. It is
also possible that the inclusion criteria used for identify-
ing suitable studies for this review resulted in some poten-
tially relevant publications being omitted, as publications
were only included if they specifically reported screen-
ing of samples for at least one non-O157 STEC serotype.
RISK FACTORS FOR NON-O157 STEC IN CATTLE
Studies that report the occurrence of STEC more gener-
ally without defining the serotype or testing only for O157
(e.g., Venegas-Vargas et al., 2016) have therefore not been
included in this review and some relevant epidemiological
information may therefore have been missed. Further clar-
ity on the risk factors for non-O157 STEC in cattle may be
gained from a fully systematic or scoping review. However,
despite these limitations, to the authors’ knowledge, this is
the first time that epidemiological data on the occurrence
of non-O157 STEC in primary cattle production have been
reviewed in a systematized manner and the data collated
here provide a useful foundation for further synthesis and
identification of knowledge gaps.
5 CONCLUSION
Drawing broad conclusions about risk factors for the occur-
rence of non-O157 STEC in cattle during primary produc-
tion from published literature is currently difficult. Occur-
rence of non-O157 STEC has been reported with respect to
several intrinsic animal-level characteristics (age, weight,
gender, breed, and herd type), herd management prac-
tices (e.g., housing type, herd and group size, and diet),
and season. However, consistently significant associations
were only identified with respect to cattle age and sea-
son of sampling according to the evidence gathered in
this review. These variables, at least, should be further
investigated as potential risk factors for infection and fecal
shedding of non-O157 STEC in primary cattle production;
however, other pertinent risk factors, as well as potential
mitigation measures are likely to be identified with the
development of more sensitive and specific methods
of detection for non-O157 STEC (e.g., the development
of enhanced selective and indicator culture media and
PCR primers with increased selectivity for certain STEC
serogroups) and increased standardization of sampling,
sample processing and detection methods across statis-
tically sound epidemiological investigations. In addition,
this review focused on occurrence of non-O157 STEC in
cattle only, but the role of other livestock species as reser-
voirs cannot be ruled out (e.g., contact with lambs at
petting zoos; Byrne et al., 2014) and should also be con-
sidered in future investigations. Finally, this review specif-
ically focused on identifying epidemiological trends for
non-O157 serogroups of STEC and not O157 STEC. How-
ever, as evidenced by many of the publications that were
reviewed, and as shown by recent work by Hoyle et al.
(2021), STEC serogroups often do not occur in isolation
within the same farm. A review of epidemiological pat-
terns of STEC in general (including both non-O157 and
O157 STEC) may therefore be a useful extension of the work
presented here.
RISK FACTORS FOR NON-O157 STEC IN CATTLE
AC K N OW L E D G M E N T S
This work was supported by the Department for Environ-
ment, Food and Rural Affairs under Project CR2000E and
Project CR2008.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AU T H O R CO N T R I B U T I O N S
Data curation, investigation, methodology, and writing—
original draft: Susan M. Withenshaw. Conceptualiza-
tion and Writing—review and editing: Richard P. Smith.
Conceptualization and Writing—review and editing: Rob
Davies. Data curation, investigation, methodology, and
writing—original draft: Alice E. O. Smith. Conceptual-
ization, funding acquisition, project administration, and
writing—review and editing: John Rodgers.
ORCID
Susan M. Withenshaw https://orcid.org/0000-0003-
0125-024X
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S U P P O RT I N G I N F O R M AT I O N
Additional supporting information may be found in the
online version of the article at the publisher’s website.
How to cite this article: Withenshaw, S. M.,
Smith, R. P., Davies, R., Smith, A. E. O., Gray, E., &
Rodgers, J. (2022). A systematized review and
qualitative synthesis of potential risk factors
associated with the occurrence of non-O157 Shiga
toxin-producing Escherichia coli (STEC) in the
primary production of cattle. Compr Rev Food Sci
Food Saf, 1–28.
https://doi.org/10.1111/1541-4337.12929