PERSPECTIVE ARTICLE

VETERINARY SCIENCE
published: 14 October 2014
doi: 10.3389/fvets.2014.00005

Is the concept of avian pathogenic Escherichia coli as a

single pathotype fundamentally flawed?
Charlotte Collingwood 1 , Kirsty Kemmett 1 , Nicola Williams 2 and Paul Wigley 1 *
1
Department of Infection Biology, Institute of Infection and Global Health, School of Veterinary Science, University of Liverpool, Neston, UK
2
Department Epidemiology and Population Health, Institute of Infection and Global Health, School of Veterinary Science, University of Liverpool, Neston, UK

Edited by: Avian Pathogenic Escherichia coli (APEC) is a major pathogen within the poultry indus-
Guillermo Tellez, University of
try. However disease, especially in broiler chickens, may be caused by range of E. coli
Arkansas, USA
genotypes that carry few, if any, virulence factors associated with APEC. Furthermore,
Reviewed by:
Raul G. Barletta, University of commensal E. coli in the intestines of healthy birds may carry an array of APEC virulence
Nebraska Lincoln, USA factors suggesting they have potential to cause disease when opportunity arises. Given
Pierre Germon, National Institute for the diseases caused by APEC, namely colibacillosis and salpingitis peritonitis syndrome,
Agricultural Research, France
are syndromic in nature and the great diversity of the strains causing disease we suggest
*Correspondence:
it is wrong to consider disease as the result of a single APEC pathotype. Whilst it is clear
Paul Wigley , University of Liverpool,
Leahurst Campus, CH64 7TE Neston, certain pathogenic E. coli can be considered as APEC, much of the disease-associated
UK with E. coli in domestic poultry is as much a consequence of increased host susceptibil-
e-mail: paul.wigley@liv.ac.uk ity due to stress, immunosuppression, co-infection, or poor welfare. This leads to more
“opportunistic” infections rather than the result of infection with a specific pathotype. As
such the current use of the term APEC for all cases of E. coli infection in the chicken is
fundamentally flawed.
Keywords: chicken, Escherichia coli, APEC, broiler chicken, eggs

Avian Pathogenic Escherichia coli (APEC) is both a primary and of their virulence determinants and are more variable between
secondary pathogen of the chicken and other avian species (1). It isolates. These virulence determinants include bacterial capsule
is considered to be a member of the extra-intestinal pathogenic that helps avoid host innate immunity, adhesins such as fimbriae
E. coli (ExPEC) along with human Uropathogenic (UPEC) and involved in attachment to host cells and tissues, hemolysins that
neonatal meningitis-associated E. coli (NMEC) that cause disease lyse host blood cells and multiple systems for acquisition of iron
outside the intestine. APEC infection may occur in broiler (meat) needed by the bacterium. In APEC, no single common virulence
chicken, turkey, and egg-laying sectors. In all sectors, infection factor has been identified in all strains. Although certain genes
is syndromic in nature. In the broiler chicken, APEC infections associated with pathogenicity are common in APEC, including:
are considered to typically lead to colibacillosis; a syndrome that iss associated with serum resistance (15), ibeA associated with
includes respiratory tract infection, air sacculitis, pericarditis, per- invasion (16), and sitA associated with iron acquisition, they are
ihepatitis, splenomegaly, and swollen head syndrome. In mature not found in all isolates. Virulence genes including iss, iroN, and
laying hens, reproductive tract infection leading to salpingitis or the iuc and cva operons are often associated with large plas-
salpingo-peritonitis syndrome (SPS) is common. mids including the Colicin V (ColV) plasmid (11). Certain E. coli
Avian pathogenic Escherichia coli is amongst the greatest health serotypes such as O1 and O78 are more frequently associated with
threats to the developed poultry industries and its emergence per- colibacillosis. Potentially these lineages harbor the genetic back-
haps reflects the decrease in the prophylactic use of antimicrobials bone required to acquire virulence mechanisms. Representatives
or their use as growth promoting agents. Furthermore, the close of these serotypes have been genome sequenced and character-
genetic relationship between APEC and other ExPEC associated ized (17, 18) and whilst there is little doubt these represent highly
with human disease along with evidence from experimental ani- pathogenic variants of APEC, the reality is that in commercial
mal models have lead to suggestions that APEC may represent a broiler chickens colibacillosis is caused by a wide range of E. coli
zoonotic risk (2–8). serotypes. Recently in our laboratory we have, for the first time,
Whilst in recent years, APEC has become accepted as a primary compared E. coli isolates causing colibacillosis within a broiler
pathogen rather than a consequence of respiratory or immuno- flock with those carried as intestinal commensals (19). These data
suppressive viral infections, our understanding of APEC and its showed that many colibacillosis – associated isolates carry few,
pathogenesis has remained relatively limited, due, at least in part, if any, of the genes most commonly ascribed as APEC virulence
to its great diversity and genomic plasticity (3, 4, 9–13). This vari- factors. In essence, they have the genotype of “intestinal com-
ation is true of other ExPECs too (14). E. coli associated with mensals.” Equally, virulence genes may be found in “commensal”
human intestinal disease harbor certain defining virulence fac- E. coli residing in the intestines of presumed healthy birds. Fur-
tors such as Type III secretion systems, Shiga-like toxins, and thermore, isolates from cases of colibacillosis are not associated
enterotoxins. ExPEC are, in general, less well defined in terms with any specific phylogenetic group. It appears that soon after

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Collingwood et al. Is the concept of an APEC pathotype is fundamentally flawed?

hatch chicks acquire a diverse range of E. coli as part of their (over 70%) in both SPS isolates and in systemically isolated E. coli
microbiota, likely to be sourced from the hatchery environment. in colibacillosis, iss which was found in 83% SPS isolates was only
Within this population are isolates carrying virulence-associated found in 25% of broiler colibacillosis isolates whilst ibe, was found
genes (1), though the frequency of such genes decreases as birds in more than 60% of broiler isolates but was found in <25% of
age (19). E. coli found in the intestinal tract are likely to form a layer SPS isolates. This may be coincidence, but does suggest that
reservoir of potential infection, we have previously termed such different virulence factors may play roles causing what are very
isolates as potential APEC or pAPEC (19), and may be associated different diseases. The use of the subcutaneous infection model
with early infection and mortality. Such isolates could be consid- for cellulitis in day old chicks has recently identified pic, a serine
ered opportunists in a compromised avian host as a consequence protease, as a putative a virulence factor (22). However as yet, this
of production-related stress, immunosuppression, or prior infec- is only an association with reduced virulence, has no mechanism
tion (1, 19, 20). Whilst opportunistic infections are likely to reduce ascribed and has not been identified in other models. A problem
in likelihood in older birds, they represent a clear risk in broiler is that phenotypes of virulence factors in the chicken are poorly
production. understood, partly as a consequence of the difficulty in reproduc-
In laying hens, the situation is a little clearer. We have previously ing experimental infections, in particular of the reproductive tract,
shown that APEC isolates from an outbreak of SPS in a commercial so making our understanding of the mechanisms of APEC disease
layer flock was caused by a single isolate that displayed features of rather rudimentary. Recently developed infection models for SPS
both APEC and UPEC (2). Full genomic analysis of this isolate is may clarify some of the virulence factors and mechanisms that
ongoing. Recently, we have looked at the distribution of key viru- underlie this disease syndrome (23, 24). However, such models rely
lence in genes in 188 SPS isolates from the UK through PCR. These on direct delivery to the reproductive tract and, in common with
findings suggest the majority possess iss, hlyF (a hemolysin), and colibacillosis models, where delivery is direct to the air sacs (18,
the iron acquisition genes iucC and iroN but other virulence genes 25), may fail to detect important factors involved in the initiation
are less frequently found (Figure 1). Whilst within the 188 isolates of infection and colonization of tissues.
there is variation, there does at least seem to be at least greater So one may pose the question should APEC be defined as any E.
commonality of virulence factors than in colibacillosis, although coli isolated from a diseased or sick bird or should the term be more
seven of these isolates do not posses any of the genes screened for. narrowly defined? It is clear that certain well-characterized isolates
This closer relationship between certain genotypes and disease and can be defined as APEC. They can cause disease in experimental
layers has also been described in Denmark where it appears certain models and possess a range of virulence factors. However disease,
lineages are common throughout the country, though there is still and in particular colibacillosis, may result from infection with an
considerable diversity (21). Intriguingly in our UK-based studies isolate which bears few or none of the hallmarks by which we
although genes of the iuc aerobactin operon are found commonly would define APEC, other than the fact it has caused disease. The

FIGURE 1 | Distribution of APEC associated virulence determinants in (increased serum survival), hlyF (a hemolysin), iucC (aerobactin), iroN (iron
188 E. coli isolates from cases of salpingitis peritonitis syndrome in UK acquisition), cva (colicin operon), iutA (iron transport), tsh (a hemagglutinin),
laying hens. Virulence determinants detected by PCR using previously and ibeA (invasion). Data previously presented at American Society for
described methods (13, 19). The genes tested and their function were iss Microbiology, Annual General Meeting, Denver, June 2013.

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Collingwood et al. Is the concept of an APEC pathotype is fundamentally flawed?

use of the term APEC for E. coli that cause what in all likelihood is Escherichia coli isolates of human and avian origin. J Clin Microbiol (2006)
opportunistic infection in the chicken is fundamentally flawed. It is 44:3484–92. doi:10.1128/JCM.00548-06
4. Moulin-Schouleur M, Reperant M, Laurent S, Bree A, Mignon-Grasteau S, Ger-
perhaps more appropriate to consider both colibacillosis and SPS
mon P, et al. Extraintestinal pathogenic Escherichia coli strains of avian and
as disease syndromes caused by an array of variable genotypes, that human origin: link between phylogenetic relationships and common virulence
are as dependent on host susceptibility as any virulence factor pos- patterns. J Clin Microbiol (2007) 45:3366–76. doi:10.1128/JCM.00037-07
sessed by the pathogen. It is probably fair to say APEC is successful 5. Johnson TJ, Wannemuehler Y, Johnson SJ, Stell AL, Doetkott C, Johnson
pathogens that are far more likely to lead to disease in poultry than JR, et al. Comparison of extraintestinal pathogenic Escherichia coli strains
from human and avian sources reveals a mixed subset representing potential
commensal E. coli. However, disease is not just restricted to those
zoonotic pathogens. Appl Environ Microbiol (2008) 74:7043–50. doi:10.1128/
isolates we can define as having an APEC pathotype, and trying to AEM.01395-08
pin all incidences of colibacillosis or SPS on an APEC pathotype 6. Tivendale KA, Logue CM, Kariyawasam S, Jordan D, Hussein A, Li GW, et al.
is flawed. Of course such concepts are more challenging both to Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E.
scientists and producers when identifying specific causative agents coli strains and are able to cause meningitis in the rat model of human disease.
Infect Immun (2010) 78:3412–9. doi:10.1128/IAI.00347-10
and their mechanisms are the norm. 7. Vincent C, Boerlin P, Daignault D, Dozois CM, Dutil L, Galanakis C, et al. Food
There are also implications in disease control. It is difficult to reservoir for Escherichia coli causing urinary tract infections. Emerg Infect Dis
control bacteria that are normally a commensal and although vac- (2010) 16:88–95. doi:10.3201/eid1601.091118
cination against APEC (26), especially in laying hens, has value 8. Mora A, Viso S, Lopez C, Alonso MP, Garcia-Garrote F, Dabhi G, et al. Poul-
it is not feasible to achieve either economically or immunologi- try as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-
ST95 in humans. Vet Microbiol (2013) 167:506–12. doi:10.1016/j.vetmic.2013.
cally in young broiler chicks. As such, good hatchery hygiene and 08.007
management are important in controlling early mortalities, with 9. La Ragione RM, Woodward MJ. Virulence factors of Escherichia coli serotypes
good management and welfare likely to reduce the risk of col- associated with avian colisepticaemia. Res Vet Sci (2002) 73:27–35. doi:10.1016/
ibacillosis in growing broilers. This also includes effective control S0034-5288(02)00075-9
10. Ewers C, Janssen T, Kiessling S, Philipp HC, Wieler LH. Molecular epide-
of other pathogens including respiratory viruses where E. coli is
miology of avian pathogenic Escherichia coli (APEC) isolated from colisep-
a common secondary pathogen. Vaccination may not be effective ticemia in poultry. Vet Microbiol (2004) 104:91–101. doi:10.1016/j.vetmic.2004.
against such a diverse microbial population and removal of E. coli 09.008
as part of the microbiota may have other implications we cannot 11. Johnson TJ, Siek KE, Johnson SJ, Nolan LK. DNA sequence of a ColV plasmid and
foresee. That said, approaches to sequence multiple genomes of prevalence of selected plasmid-encoded virulence genes among avian Escherichia
coli strains. J Bacteriol (2006) 188:745–58. doi:10.1128/JB.188.2.745-758.2006
E. coli from the chicken could reveal a common “core” genome
12. Dziva F, Stevens MP. Colibacillosis in poultry: unravelling the molecular basis
in disease-associated E. coli, identifying a genetic relationship that of virulence of avian pathogenic Escherichia coli in their natural hosts. Avian
cannot be found when considering virulence factors alone. Such Pathol (2008) 37:355–66. doi:10.1080/03079450802216652
an approach may also lead to the identification of novel targets for 13. Timothy S, Shafi K, Leatherbarrow AH, Jordan FTW, Wigley P. Molecular epi-
future vaccines. demiology of a reproductive tract-associated colibacillosis outbreak in a layer
breeder flock associated with atypical avian pathogenic Escherichia coli. Avian
In conclusion, we believe that E. coli disease in the chicken can- Pathol (2008) 37:375–8. doi:10.1080/03079450802216579
not be simply defined as being caused by a single pathotype of E. 14. Manges AR, Tabor H, Tellis P, Vincent C, Tellier PP. Endemic and epidemic
coli. In particular, colibacillosis is perhaps better defined as disease lineages of Escherichia coli that cause urinary tract infections. Emerg Infect Dis
caused by E. coli rather than by Avian Pathogenic E. coli, and that (2008) 14:1575–83. doi:10.3201/eid1410.080102
the term APEC be reserved for the smaller number of well-defined 15. Foley SL, Horne SM, Giddings CW, Robinson M, Nolan LK. Iss from a virulent
avian Escherichia coli. Avian Dis (2000) 44:185–91. doi:10.2307/1592523
“bona fide” pathogenic isolates with a range of defined virulence 16. Germon P, Chen YH, He L, Blanco JE, Bree A, Schouler C, et al. ibeA, a virulence
determinants that can reproduce disease in animal models. There factor of avian pathogenic Escherichia coli. Microbiology (2005) 151:1179–86.
are APEC, but not all disease-associated with E. coli in the chicken doi:10.1099/mic.0.27809-0
is caused by APEC. 17. Johnson TJ, Kariyawasam S, Wannemuehler Y, Mangiamele P, Johnson SJ,
Doetkott C, et al. The genome sequence of avian pathogenic Escherichia coli
strain O1: K1: H7 shares strong similarities with human extraintestinal patho-
ACKNOWLEDGMENTS genic E. coli genomes. J Bacteriol (2007) 189:3228–36. doi:10.1128/JB.00537-07
We wish to thank Dr. Tim Wallis and Ridgeway Biologicals for the 18. Dziva F, Hauser H, Connor TR, van Diemen PM, Prescott G, Langridge GC,
panel of SPS-associated isolates. Charlotte Collingwood is sup- et al. Sequencing and functional annotation of avian pathogenic Escherichia
ported by a BEMB Educational Trust Ph.D. award. Kirsty Kemmett coli serogroup O78 strains reveal the evolution of E. coli lineages patho-
genic for poultry via distinct mechanisms. Infect Immun (2013) 81:838–49.
was supported by a BBSRC DTG Ph.D. award. doi:10.1128/IAI.00585-12
19. Kemmett K, Humphrey T, Rushton S, Close A, Wigley P, Williams NJ. A lon-
REFERENCES gitudinal study simultaneously exploring the carriage of APEC virulence asso-
1. Antao EM, Glodde S, Li G, Sharifi R, Homeier T, Laturnus C, et al. The chicken ciated genes and the molecular epidemiology of faecal and systemic E. coli in
as a natural model for extraintestinal infections caused by avian pathogenic commercial broiler chickens. PLoS ONE (2013) 8:e67749. doi:10.1371/journal.
Escherichia coli (APEC). Microb Pathog (2008) 45:361–9. doi:10.1016/j.micpath. pone.0067749
2008.08.005 20. Kemmett K, Williams NJ, Chaloner G, Humphrey S, Wigley P, Humphrey T.
2. Rodriguez-Siek KE, Giddings CW, Doetkott C, Johnson TJ, Fakhr MK, Nolan The contribution of systemic Escherichia coli infection to the early mortali-
LK. Comparison of Escherichia coli isolates implicated in human urinary ties of commercial broiler chickens. Avian Pathol (2014) 43:37–42. doi:10.1080/
tract infection and avian colibacillosis. Microbiology (2005) 151:2097–110. 03079457.2013.866213
doi:10.1099/mic.0.27499-0 21. Pires-dos-Santos T, Bisgaard M, Christensen H. Genetic diversity and virulence
3. Moulin-Schouleur M, Schouler C, Tailliez P, Kao MR, Bree A, Germon P, profiles of Escherichia coli causing salpingitis and peritonitis in broiler breeders.
et al. Common virulence factors and genetic relationships between O18:K1:H7 Vet Microbiol (2013) 162:873–80. doi:10.1016/j.vetmic.2012.11.008

www.frontiersin.org October 2014 | Volume 1 | Article 5 | 3

Collingwood et al. Is the concept of an APEC pathotype is fundamentally flawed?

22. Barbieri NL, de Oliveira AL, Tejkowski TM, Pavanelo DB, Rocha DA, Conflict of Interest Statement: The authors declare that the research was conducted
Matter LB, et al. Genotypes and pathogenicity of cellulitis isolates reveal traits in the absence of any commercial or financial relationships that could be construed
that modulate APEC virulence. PLoS ONE (2013) 8:e72322. doi:10.1371/ as a potential conflict of interest.
journal.pone.0072322
23. Chaudhari AA, Kariyawasam S. An experimental infection model for Escherichia
coli egg peritonitis in layer chickens. Avian Dis (2014) 58:25–33. doi:10.1637/ Received: 30 May 2014; accepted: 28 June 2014; published online: 14 October 2014.
10536-032213-Reg.1 Citation: Collingwood C, Kemmett K, Williams N and Wigley P (2014) Is the concept
24. Pors SE, Olsen RH, Christensen JP. Variations in virulence of avian pathogenic of avian pathogenic Escherichia coli as a single pathotype fundamentally flawed? Front.
Escherichia coli demonstrated by the use of a new in vivo infection model. Vet Vet. Sci. 1:5. doi: 10.3389/fvets.2014.00005
Microbiol (2014) 170:368–74. doi:10.1016/j.vetmic.2014.02.043 This article was submitted to Veterinary Infectious Diseases, a section of the journal
25. Sadeyen JR, Kaiser P, Stevens MP, Dziva F. Analysis of immune responses Frontiers in Veterinary Science.
induced by avian pathogenic Escherichia coli infection in turkeys and their Copyright © 2014 Collingwood, Kemmett , Williams and Wigley. This is an open-access
association with resistance to homologous re-challenge. Vet Res (2014) 45:19. article distributed under the terms of the Creative Commons Attribution License (CC
doi:10.1186/1297-9716-45-19 BY). The use, distribution or reproduction in other forums is permitted, provided the
26. La Ragione RM, Woodward MJ, Kumar M, Rodenberg J, Fan H, Wales AD, et al. original author(s) or licensor are credited and that the original publication in this
Efficacy of a live attenuated Escherichia coli O78:K80 vaccine in chickens and journal is cited, in accordance with accepted academic practice. No use, distribution or
turkeys. Avian Dis (2013) 57:273–9. doi:10.1637/10326-081512-Reg.1 reproduction is permitted which does not comply with these terms.

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