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Avian pathogenic Escherichia coli (APEC)

Maryvonne Dho-Moulin, John Morris Fairbrother

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Maryvonne Dho-Moulin, John Morris Fairbrother. Avian pathogenic Escherichia coli (APEC). Vet-
erinary Research, BioMed Central, 1999, 30 (2-3), pp.299-316. <hal-00902571>

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Review article

Avian pathogenic Escherichia coli (APEC)

Maryvonne Dho-Moulin
a John Morris b
Fairbrother
a
Pathologie aviaire et parasitologie, Inra, centre de Tours, 37380 Nouzilly, France
n
Groupe de recherche sur les maladies du porc (GREMIP), faculté de médecine vétérinaire,
université de Montréal, CP5000, Saint-Hyacinthe, Quebec J2S 7A6, Canada

(Received 4 January 1999; accepted 26 January 1999)

Abstraet- Avian pathogenic E cherichia cn


s i (APEC) cause aerosacculitis, polyserositis, septicemia
l
and other mainly extraintestinal diseases in chickens, turkeys and other avian species. APEC are
found in the intestinal microflora of healthy birds and most of the diseases associated with them are
secondary to environmental and host predisposing factors. APEC isolates commonly belong to cer-
tain scrogroups, Ol, 02 and 078, and to a restricted number of clones. Several experimental mod-
els have been developed, permitting a more reliable evaluation of the pathogenicity of E. coli for chick-
ens and turkeys. Hence, virulence factors identified on APEC are adhesins such as the Fl and P
fimbriae, and curli, the aerobactin iron sequestering system. Kl capsule, temperature-sensitive
hemagglutinin (Tsh), resistance to the bactericidal effects of serum and cytotoxic effects. Experimental
infection studies have shown that the air-exchange regions of the lung and the airsacs are important
sites of entry of E. coli into the bloodstream of birds during the initial stages of infection and that resis-
tance to phagocytosis may be an important mechanism in the development of the disease. They have
also demonstrated that F fimhriae arc expressed in the respiratory tract, whereas P fimbriae are
expressed in the internal organs of infected chickens. The role of these fimbrial adhesins in the
development of disease is not yet, however, fully understood. The more recent use of genetic
approaches for the identification of new virulence factors will greatly enhance our knowledge of
APEC pathogenic mechanisms. Diagnosis of APEC infections is based on the clinical picture, lesions
and isolation of E. coli. This may be strengthened by serotyping and identification of virulence fac-
tors using immunological or molecular methods such as DNA probes and PCR. Approaches for the
prevention and control of APEC infections include the control of environmental contamination and
environmental parameters such as humidity and ventilation. Antibiothcrapy is widely used, although
APEC are frequently resistant to a wide range of antibiotics. Vaccines containing killed or attenuated
virulent bacteria protect against infection with the homologous strain hut are less efficient against het-
ei-ologous strains. Hence, vaccination for colibacillosis is not widely practised because of the large
variety of serogroups involved in field outbreaks. O Inra/Elscvier, Paris.
avian / Escherichia coli / virulence / fimbriae / capsule / aerobactin

*
Correspondence and reprints
Tel.: (33) 2 47 42 77 63; fax: (33) 2 47 42 77 74; e-mail: dhoinouli (-d tours.int-a. li-
Résumé-Escherichia coli pathogènes aviaires (APEC). Les Escherichia coli pathogènes aviaires
(ou APEC) sont responsables d’aérosacculite, de lésions fibrineuses des séreuses, de septicémie et
d’autres pathologies extra-intestinales chez le poulet, la dinde et d’autres espèces de volailles. Les APEC
sont présents dans la flore intestinale des oiseaux sains et la plupart des pathologies qui leur sont asso-
ciées sont secondaires à l’action de facteurs prédisposants. Les APEC appartiennent fréquemment à
trois sérogroupes : O1, 02 et 078, ainsi qu’à un nombre limité de clones. Plusieurs modèles d’infec-
tion expérimentale permettent une évaluation fiable de la virulence pour le poulet et la dinde. Les fac-
teurs de virulence identifiés chez les APEC comprennent des adhésines (fimbriae FI, P et curli), le
système aérobactine d’acquisition du fer, la capsule KI, l’hémagglutinine thermosensible Tsh, la
résistance au pouvoir bactéricide du sérum, ainsi qu’un pouvoir cytotoxique. Des infections expéri-
mentales chez le poulet ont mis en évidence que les zones d’échanges gazeux, sacs aériens et poumons,
constituaient des sites de pénétration des APEC dans la circulation sanguine. La résistance à la pha-
gocytose serait un autre mécanisme important dans le développement de l’infection. Il a été démon-
tré également que les fimbriae FI ne sont exprimés que dans le tractus respiratoire, alors que les
fimbriae P sont exprimés dans les organes internes des poulets infectés. Le rôle de ces adhésines
dans la pathogénie n’est pas complètement élucidé. L’identification de nouveaux facteurs de virulence
par les approches génétiques en cours devrait permettre d’accroître les connaissances sur les APEC.
Le diagnostic de colibacillose repose habituellement sur le tableau clinique, l’observation de lésions
caractéristiques et l’isolement de E.coli. Il peut être confirmé par la détermination du sérotype et la
détection de facteurs de virulence par des méthodes immunologiques ou moléculaires (PCR, sondes
ADN). Dans l’élevage, le contrôle de la contamination de l’environnement ainsi que des paramètres
tels que la température et l’humidité constituent des méthodes de prévention de la colibacillose
aviaire. Les traitements antibiotiques sont largement utilisés bien que les APEC soient fréquem-
ment résistants à plusieurs antibiotiques. Des vaccins à base de bactéries tuées ou à virulence atténuée
peuvent protéger contre la souche homologue d’Ec-oli, mais sont peu efficaces contre des souches hété-
rologues. De fait, la vaccination contre la colibacillose est peu utilisée en raison de la grande diver-
sité des sérotypes d’E. coli qui peuvent être impliqués. &copy; Inra/Elsevier, Paris.

Escherichia coli / aviaire / virulence / fimbriae / capsule / aérobactine


1. INTRODUCTION ease syndrome associated with APEC begins
as a respiratory tract infection and may be
Although Escherichia coli is present in referred to as aerosacculitis or the airsac dis-
the normal microflora of the intestinal tract ease. If unchecked, this infection may evolve
and other host mucosal surfaces and in the into a bacteriemia and a generalized infec-
bird’s environment, only certain of these tion which manifests as a polyserositis. The
strains possessing specific virulence respiratory tract complex is most often
attributes, designated as avian pathogenic observed in birds of 4 to 9 weeks of age and
E. coli (APEC), are able to cause disease. may result in extensive economic losses
APEC are mostly associated with extra- with up to 20 °l° mortality as well as reduced
intestinal infections, principally of the res- growth and feed efficiency and an increased
piratory tract or systemic infections, and condemnation rate at the abattoirs. APEC
result in a variety of diseases which are infection of the respiratory tract is secondary
responsible for severe economic losses 146J. to initial infection with one or more of the
Most of the diseases associated with APEC respiratory tract agents: Newcastle disease
are secondary to environmental and host virus (NDV), infectious bronchitis virus
predisposing factors. Therefore, losses due (IBV) and Mycoplasma gallisepficu
, or
l11
to these diseases may be greatly reduced by with the NDV or IBV vaccine viruses [74,
controlling these factors. In the last several 75, 98]. Susceptibility of birds to APEC
years, the increasing use of more sophisti- infection is increased by deciliation of the
cated molecular approaches for the study of epithelial cells of the upper respiratory tract
bacterial pathogenesis have led to some following exposure to ammonia and dust in
exciting insights into the virulence deter- the immediate environment of the birds.
minants of APEC and the mechanisms by Respiratory tract infection with APEC
which these bacteria are able to develop results in depression, fever and death. Res-
infection and cause disease, although the piratory lesions include aerosacculitis with
pathogenic mechanisms of APEC have not a serous to fibrinous exudate, an initial infil-
yet been fully elucidated. In this chapter, tration with heterophils and a subsequent
we will highlight the recent advances in this
predominance of mononuclear phagocytes.
area, and approaches currently being under- In more generalized infections, lesions of
taken to further our knowledge.
pericarditis and perihepatitis are also
observed. In adult birds, an acute form of
septicemia due to APEC may occur. Lesions
2. DISEASE SYNDROMES may be absent or include pericarditis, peri-
ASSOCIATED WITH APEC tonitis and bile-staining and necrotic foci in
the liver. In laying birds, APEC may infect
APEC are mostly associated with infec- the oviduct via the left abdominal airsac
tion of extraiiitestinal tissues in chickens, leading to salpingitis and loss of egg laying
turkeys, ducks and other avian species with ability. Alternatively, APEC may sporadi-
the exception ofa possihle relationship with cally invade the peritoneal cavity via the
the development of enteritis [44,46],Yolk oviduct leading to peritonitis and death. In
sac infections are most frequently observed broilers, broilcr breeders and layers, APEC
towards the end of the egg incubating period, may cause a specific syndrome called the
usually following fecal contamination of swollen head syndrome, following an ini-
the egg surface. They often result in embry- tial infection with turkey rhinotracheitis
onic mortality or death of the young birds for virus [64] or possibly other viruses such as
up to 3 weeks following hatching. Reten- IBV or NDV. Lesions observed in this syn-
tion of the infected yolk sac and omphalitis drome include cellulitis and oedema of the
are often observed. The most important dis- facial skin and periorbital tissues. In broilers,
APEC are also associated with a cellulitis 4. CHARACTERIZATION OF APEC
or necrotic dermatitis of the lower abdomen
and thighs [33, 41, 71]. This disease has 4.1.
been reported more and more frequently in
Serogroups
recent years and although it does not cause
APEC isolates commonly belong to cer-
clinical signs or mortalities, the associated tain 0 serogroups, particularly 1, 2, 8, 15,
subcutaneous fibrinous lesions result in
18, 35, 78, 88, 109 and 115 [46]. As first
extensive economical losses due to con-
demonstrated by Sojka and Carnaghan [99],
demnation of carcasses [34]. three of these, 0 I, 02 and 078 are the most
APEC are probably not a cause of intesti- frequently recovered from colibacillosis in
nal diseases such as enteritis in poultry, the different countries worldwide, and they
although enterotoxigenic E. coli have occa- represent 15-6l°Io of the total number of
sionally been associated with outbreaks of isolates, depending on the study [ 13, 20, 29,
diarrhea in poultry [4, 59]. 40]. However, many pathogenic isolates do
not belong to these identified pathogenic
serogroups, and they are commonly desig-
3. EPIDEMIOLOGY OF APEC nated as ‘untypable’. This results in diffi-
INFECTIONS culties in identifying APEC strains in vet-

E. coli are normal inhabitants of the lower


erinary laboratories, because at present
diagnosis mainly relies on serogrouping.
digestive tract of many avian species, and
10!-10! colony forming units (cfu) per gram
are usually present in the intestinal contents
4.2. Biochemical properties
of birds. E. coli also colonize the upper res-
piratory tract (pharynx and trachea), and can Several biochemical characteristics have
be isolated from skin and feathers, depend-
been associated with APEC, such as the fer-
ing on the level of environmental contami- mentation of dulcitol or of salicin, but, in
nation [53]. Pathogenic as well as non-
fact, this represents more a correlation with
pathogenic E. coli isolates can be recovered the serogroup of the strains rather than with
at these sites from healthy birds.
their virulence. An example is the positive
The contamination of birds with E. coli correlation between the fermentation of
occurs in the first hours following hatching, adonitol by 035 E. coli strains that were
and E. coli strains rapidly multiply in the responsible for numerous cases of coli-
intestine. Many different strains can be bacillosis in Delaware (USA) between 19811
acquired during the life of a bird. Vertical and 1983 [20].
contamination results from the transmission
of E. coli from breeders, via contaminated
shells during hatching, or in ovo, as a result 4.3. Clonal relationships
of salpingitis [46!.] .
Horizontal contamination with E. coli The molecular characterization of APEC
usually occurs through contact with other strains, which permits the evaluation of
birds, or through feces, contaminated water genetic relatedness, has been widely docu-
and feed. Birds are frequently contaminated mented. Classification of isolates is based
by inhalation of particles present in dust on electrophoretic types (ET), as defined by
which can contain as many as 10!’ cfu of electrophoretic detection of allelic variants
bacteria per gram. Carlson and Whenham of enzyme-coding genes. Certain APEC
[ 17have demonstrated that the risk of col- strains isolated from different countries and
ibacillosis increases with the level of envi- at different times are genetically related and
ronmental contamination. belong to the same clone [114!.Further-
more, it has been demonstrated that some recorded. This permits confirmation of the
clones are specific to APEC: in a compari- pathogenicity of the tested strain.
son of 45 E. coli isolates from poultry, White The direct inoculation of E. coli into the
et al. [115] showed that 83 % of pathogenic airsacs is a ’high performance’ model as it
isolates belong to only five clones, whereas does not require the preliminary action of
each of ten non-pathogenic strains belong
triggering agents, and as it results in typi-
to different clones. Similar results have been cal lesions of colibacillosis with greater
obtained by other methods such as ribotyp-
homogeneity in bird responses, as compared
ing (Coulange et al., unpublished results). to inoculations in the upper respiratory tract
It is noteworthy that some APEC strains
[87,89j.].
belong to the same clones as do pathogenic In all these models, the use of specific
E. coli isolated from extra-intestinal infec-
pathogen-free (SPF) birds is a necessary
tions in humans [ 1 14].
condition to avoid cross contamination with
other pathogens. In some cases, axenic
chickens have been used. This approach has
4.4. Experimental testing of APEC allowed the visualization of the inoculated E.
in animals coli strain in the contaminated tissues with-
out the interference of commensal E. coli

Several experimental models have been strains [30].


developed, allowing the evaluation of the
pathogenicity of E. coli for chickens or 5. VIRULENCE FACTORS OF APEC
turkeys. Pathogenic E. coli isolates are able
to kill embryos or 1-day-old chicks follow-
Several potential virulence factors have
ing subcutaneous inoculation (25, 52]. Both been identified on APEC, mainly from a
of these models give rapid results and permit
the measurement of the virulence of the iso- positive correlation between phenotypic
lates according to their 50 % lethal dose. characteristics and virulence for chickens.
The study of the involvement of these fac-
However, they bypass the natural route of
infection by avoiding the respiratory tract. tors in virulence using experimental mod-

Birds can also be inoculated intravenously els of infection is just beginning. This
involvement includes the adherence ability
[97].
of bacteria to the respiratory tract, mediated
Other experimental models which repro- by fimbriae, the resistance of bacteria to the
duce natural disease in birds at susceptible immunological defences, the multiplication
ages that correspond to those of field dis- of bacteria in the host physiological liquids
ease (2-4 weeks old) have also been used. through the expression of iron siderophores,
Bacteria can be aerosolized [43], inoculated and the ability to produce cytopathic effects.
in the naso-pharynx [98 j, or directly inocu- More recently, genomic methods have been
lated into the trachea [15, 42!, following a used, providing very interesting additional
preliminary challenge with a triggering agent hypotheses.
such as a virus (infectious bronchitis virus,
or Newcastle virus), mycoplasma, or an
increase in ammonia which impairs the nat- 5.1. Fimbriae
ural defences of the respiratory tract. Typi-
cal lesions of colibacillosis are thus repro- 5.1.1. Wae (type 1)

<
!fM
f7y
duced and several criteria, such as weight
gain, presence of fibrin on airsacs, lesions of Evidence that the ability of E. coli to
pericarditis and perihepatitis, and contami- adhere to the epithelium of the respiratory
nation of internal organs and blood can be tract of chickens could be a virulence factor
was first provided by the observation that for isolates of the 02 serogroup [68J. The
a virulent and fimbriated strain was less eas- FimH adhesin is located at the tip of the
ily cleared from the trachea of turkeys than fimbriae, or both at the tip and along the
an avirulent and less fimbriated strain [8]. fimbriae, depending on the APEC strain
These results were strengthened by the [ 18 The significance of these different loca-
demonstration that virulent E. coli strains tions is unknown.
were better colonizers of the chicken tra-
In vivo, type I fimbriae are expressed
chea than avirulent strains, and that these
adhesive properties were mediated by type
mainly in the trachea and in the lungs and
airsacs, but their expression has never been
I fimbriae [24, 26Adhesion of type I fim-
observed in other organs nor in the blood
briae to chicken epithelial cells of the phar-
)30. 9iThis could result from the phase
ynx and trachea was demonstrated both in variation of type I fimbriae, depending on
vivo and in vitro [25, 761. Gyimah and Pan-
the in vivo environmental conditions.
igrahy [49!blocked the specific adherence
of APEC strains to chicken tracheal sections The role of typeI fimbriae in infection is
with specific anti-typeI fimbriae serum and unclear. By using a mutant deleted for the
by D-mannose which is the cellular receptor entire,fim operon, Marc et al. !69] showed
of the adhesin of type 1 fimbriae. that the expression of type I fimbriae is not
necessary for the colonization of the trachea
Typefimbriae consist of a major pro- and airsacs, but that these fimbriae could
tein, FimA, associated with ancillary pro- play a role in the colonization of the lungs.
teins, FimF, FimG and the adhesin FimH.
They areencoded by the fim gene cluster, Type I fimbriae could also play a role in
which is located at 98 mn on the chromo- the interaction of APEC strains with the
some of E. coli, and comprises nine genes,
immune system, although this role is con-
seven of which are present in a single operon
troversial. Orndorff et al. [85suggested
whose expression is controlled by an invert- that type1 fimbriae could protect E. coli
ible element containing the promoter[85]. from phagocytosis. Other studies demon-
strated that the resistance to the bacterici-
The presence of typeI fimbriae is more dal effects of serum was positively corre-
frequent on pathogenic than on non- lated with the presence of typeI fimbriae
pathogenic avian E coli strains, even though [29,I 16]. It was recently demonstrated that
these fimbriae are common among E. co i.
l type1 fimbriae could be mastocyte activa-
Dozois et al. [291 demonstrated the pres- tors via the FimH adhesin, and that this acti-
ence of the,fiwD gene in 74 % of the I 12 2 vation would result in phagocytosis of bac-
APEC isolates but only in 55 % of the E. teria and recruitment of neutrophils at the
coli isolated from healthy birds. Wooley et site of infection [65-67J. Furthennore, Pour-
al. [ 1 16] found that 100 % of the APEC bakhsh et al. [901 showed that highly viru-
strains produced type1 fimbriae as com- lent APEC strains were resistant to the bac-
parcd with 57.5 % of the commensal strains. tericidal effect of macrophages when they
did not express typefimbriae, but were
Several variants of typc1 fimbriae have
been described on APEC and they seem to
sensitive when they did express these fim-
briae.
be related to the scrogroup of the strains
[27. 102]. They differ from classical type Further studies are needed to clarify the
IA fimbriae with respect to the molecular role of type I fimbriae in the virulence of
weight of the major fimbrial subunit and its APEC as a favorable or unfavorable factor.
immunological reactivity. More recently, This topic is also presently controversial for
four variable regions have been identified other extraintestirial pathogenic E. coli, such
in thelin>A gene of an APEC isolate, among as those responsible for urinary tract infec-
which at least two regions could he specific tions.
5.1.2. P fimbriae from chickens with septicemia, or 96 %
(when only isolates of serotypes O1: K1,
P fimbrial adhesins were initially found 02: K1, 035 and 078: K80 were consid-
on E. coli associated with upper urinary tract ered) expressed P fimbrial adhesins of
infections in humans. They mediate bacterial serotype F11, as detected by ELISA.
adherence to uroepithelial cells and are an It is interesting to note that pap-related
important virulence determinant in the DNA sequences were observed in a much
development of pyelonephritis [60!. P fim- higher proportion in E. coli isolates in the
briae are encoded by the pap gene cluster, study by Dozois et al. [29]. It was found
which is chromosomally located and con- that 44 and 31 °lo of 81 and 29 isolates from
sists of 11genes whose structure and func- septicemic chickens and turkeys, respec-
tion have been extensively studied [5 1 ]. P tively, possessed pap-related DNA
fimbriae consist of a major fimbrial subunit, sequences. The presence of pap-related
PapA, and a terminally located fimbrial DNA sequences was significantly more fre-
adhesin, PapG. At least 11serotypes of P quent in isolates from septicemic than from
fimbriae, F7-F16, have been recognized healthy chickens. In isolates from septicemic
based on antigenic differences in the major turkeys, their presence was also associated
fimbrial subunits [51].Receptor specificity with lethality in 1-day-old chicks. Although
of P fimbriae is conferred by the adhesin ap-related
h DNA sequences were present
PapG, for which three variants, classes I, 11 in isolates of serogroups O1, 02 and 078,
and III have been identified [101]. These the in vitro expression of the P fimbrial
variants recognize different isoreceptors of adhesins was only observed in O1and 0188
the globoseries of glycolipids, which contain isolates, even following growth of the bac-
the disaccharide gal-gal and may also be teria in culture conditions optimal for the
distinguished by their mannose-resistant production of these fimbrial adhesins [3 1J.
hemagglutination (MRHA) of different ery- Further examination of these isolates by
throcytes. PCR and Southern blot hybridization [32]
Certain APEC strains also express P fim- demonstrated that only those isolates
brial adhesins [1, 29, 31, 108, 110]. In gen- expressing the P fimbrial adhesin possessed
a complete copy of the.fel gene cluster which
eral, these adhesins have been observed in a
low proportion of the isolates studied. Acht- encodes P fimbrial adhesins of the F1 1I
mann et al. [I] found that 52 % of the 02 serotype. In contrast, the isolates not
isolates from septicemic chickens expressed expressing the P fimbrial adhesin, mainly
P fimbrial adhesins as detected by MRHA. isolates of the 078 serogroup, possessed
Dozois et al. [29, 31 ], however, observed partial or divergent P fimbrial clusters,
the expression of P fimbrial adhesins, as which explained their inability to express
detected by MRHA and immunofluores- these fimbriae.
cence, only in Ol isolates from septicemic The role of P fimbrial adhesins in the
turkeys and in an 018 isolate from a sep- pathogenicity of APEC has not yet been
ticemic chicken, in a study of I 12 E. coli fully elucidated. These adhesins do not
isolates from chickens and turkeys with sep- appear to be involved in bacterial adherence
ticemia. The P fimbrial adhesin from one to chicken tracheal or pharyngeal cells in
of the OI isolates was shown to be closely vitro [108, 110], nor to frozen sections of
related to F11fimbriae associated with E. the chicken trachea[301, suggesting that
coli isolated from upper urinary tract infcc- receptors for these adhesins are not present
tions in humans, by N-terminal amino acid at this site. Pourbakhsh et al. [90, 91 !]
sequencing, immunoblotting, and competi- demonstrated that chickens inoculated with
tive ELISA [88]. Van den Bosch et al. [ 108] an Fl 1 P-Cimbriated APEC strain by the

reported that 78 % of 203 E. coli isolates intratracheal or airsac route mounted a spe-
cific anti-F11antibody response as revealed In addition, Maurer et al. [70] found that
by ELISA, providing evidence that these only half of the examined isolates produced
fimbriae are produced in vivo. No expres- the curli following bacterial growth in cul-
sion of this fimbrial adhesin was observed in ture conditions optimal for curli expression.
bacteria colonizing the trachea of inoculated The role, if any, of curli in the pathogenic
chickens, as detected by immunofluores- process has not yet been elucidated. Nev-
cence, whereas bacteria colonizing the air- ertheless, certain properties of curli, such
sacs, lungs and internal organs of these same as the ability to bind to the major histo-
chickens expressed P fimbriae. These results
compatibility complex class I molecules
provide strong evidence for in vivo phase which are present on most cells of higher
variation of P fimbrial adhesins with respect vertebrates [84], or the ability to bind to the
to their localization in the host and reinforce extracellular matrix and serum proteins [ 81!,
the suggestion that P fimbrial adhesins are
may contribute to bacterial adherence and
not important in the initial colonization of colonization in the initial stages of infec-
the upper respiratory tract but in the later tion.
stages of the infection.

5.1.3. Curli 5.2. Aerobactin iron-sequestering


system
Curli are thin, coiled filamentous struc-
tures of about 2 nm in diameter found on The low concentration of free iron in
the bacterial surface of E. coli and
physiological liquids of animals (about 10-&dquo;
Salmonella spp. [21, 81]. These structures mol.L- is not sufficient to allow bacterial
)
1
mediate bacterial binding to extracellular which requires a concentration of
growth
matrix and serum proteins such as about 10-
6 mol.L-
. Numerous pathogenic
1
fibronectin, laminin, plasminogen and plas- bacteria with invasive abilities have devel-
minogen activator protein [81]. Curli are oped high affinity iron-acquisition systems
optimally expressed in vitro at 26 °C in a which can compete with the host
stationary phase and in a low osmolarity siderophores such as transferrin, and thus
growth medium [82]. favor bacterial growth in low iron environ-
Initially, it was thought that curli were ments.
encoded by the crl gene [81]although it was Dho and Lafont [25] found a positive
subsequently shown that crl plays a role in correlation between the ability of avian
but is not necessary for, the expression of E. coli strains to grow in vitro under iron-
the curli phenotype in an APEC strain [92J.
In fact, crl activates cryptic genes for curli
limiting conditions and the lethality for 1-
day-old chicks. They subsequently demon-
formation [6]. It has more recently been strated that this was due to the expression
shown that the M.!4 (curlin subunit gene) of the aerobactin system [62]. Several stud-
encodes for the major curlin subunit [83]. ies have confirmed that most APEC strains
It appears that most E. coli strains carry (73-98 %) possess and express the aer-
the csgA gene, although the curli are not obactin iron-acquisition system, whereas
always expressed in in vitro growth condi- non-pathogenic strains produce aerobactin
tions 1831. Maurer et al. [70] showed that far less frequently [29, 36, 63]. The high
the c!/4 gene was present in all of the 78 E. correlation observed between the presence of
coli isolates from diseased birds and also in the aerobactin system and the virulence of
all of the 50 commensal E. coli isolates from APEC has recently lead to the use of diag-
healthy chickens. Similarly, Dho-Moulin et nostic tests based on the immunological
al. [28] showed that 298 of the 300 E. coli detection of the IutA protein that is the
from diseased birds possessed the csgA gene. receptor for the ferric aerobactin.
The role of the aerobactin system in the pathogenic E. coli strains were far less lethal
multiplication of APEC in extraintestinal for 1-day-old chicks than the wild-type strain
locations during infection can be highly sus- [23]. More recently, Pourbakhsh et al. [90]
pected. The aerobactin system is expressed showed that three APEC strains possessing
in vivo as shown by the detection of anti- the K1 antigen were more resistant to the
aerobactin antibodies, following intra-tra- bactericidal effects of serum than three other
cheal inoculation of axenic chickens (Bree, APEC strains expressing different K anti-
pers. comm.). gens.
The aerobactin operon is usually carried
by large colV plasmids, which are of at least
80 kb [107, 111In some cases, the pres- 5.4. Temperature-sensitive
ence of large plasmids has been related to
hemagglutinin
APEC virulence [96,109]. Ike et al. [56]
demonstrated that the loss of a large con-
A hemagglutinating activity, preferen-
jugative colV plasmid of an 02 APEC iso-
late resulted in a reduction in virulence, in tially expressed at low temperatures
the loss of resistance to the bactericidal (26-30 °C) and repressed at 42 °C, was
effects of serum, and in the loss of the aer- observed on an APEC strain by Provence
obactin system. The reintroduction of the and Curtiss III [93]. This phenomenon was
called ’temperature sensitive hemaggluti-
plasmid into the parent strain restored these nation’. The tsh gene was cloned from this
three properties. These results support the
idea that the large plasmids of APEC may strain, and it was deduced from its sequence
encode several virulence determinants. The that it encodes a protein of 140 kDa, with
a mature form of 18 kDa. The protein Tsh
presence of the iss gene on the plasmid
colV-1K94, and of the trat gene on drug sequence showed homologies with
resistance plasmids, were shown to be cor- immunoglobulin A proteases from
related with increased survival in serum and Haemophilus influenzae and Neisseria gon-
enhanced virulence of the strains carrying orrhoeae, as well as with the SepA protein
these plasmids [3, 9, 10, 72]. from Shighella flexneri, and with the EspC
].
and EspP proteins which are present on
EPEC and EHEC strains. Neither the pro-
5.3. Capsule teolytic activity of Tsh for A immunoglob-
ulins, nor the activity of SepA, EspB and
Some polysaccharidic capsules of E. coli, EspP are presently known, and the observed
similarities between Tsh and these proteins
especially those containing N-acetyl neu-
raminidic acid, are able to interact with C3 may result from similar secretory systems.
to C3b activators in the classical and alter-
Maurer et al. [70] have demonstrated that
native complement pathways. This induces the tsh gene was present on 46 % of clinical
resistance of the bacteria to the bactericidal E. coli isolates of avian origin, but none
effects of the complement [58]. The KI anti- were found in isolates from healthy animals.
gen is frequently associated with APEC of In another study of 300 avian E. coli origi-
the more pathogenic serogroups such as O11
and 02, and it is also often present on non-
nating from France and Quebec, Dho-
Moulin et al. [28] showed that among tsh
typable APEC strains [46]. positive isolates, the incidence of pathogenic
The Klantigen is poorly immunogenic, isolates (90.6 %) was far higher than that
and could thus be involved in the resistance of non-pathogenic isolates. This suggests
of APEC to the immunological defences of that the Tsh protein could play a role in the
the bird. This hypothesis is strengthened by pathogenic process, although its precise
the observation that K1 mutants of human function has not yet been elucidated.
5.5. Resistance to the bactericidal involved in the pathogenic process. Early
effects of serum studies have suggested that some APEC
might produce exotoxins such as the chick-
Resistance to the bactericidal effects of lethal toxin (CLT) 1105], although produc-
the complement in serum, mediated by bac- tion of this toxin did not seem to be
terial surface structures such as capsule,
widespread in APEC. Subsequently, Emery
lipopolysaccharide, ColV production and et al. [36J demonstrated that in up to 22.5 %
outer membrane proteins, has been associ- of 500 E. coli from colisepticemic chickens
ated with APEC isolates, particularly those or turkeys, two distinct heat-labile toxins
originating from septicemic birds 146, 77, (LT) with cytotoxic activity for Y-and/or
1 17/. For example, Ellis et al. [35) demon- Vero cells were produced. Fantinatti et al.
strated a correlation between serum resis- !37] observed a cytotoxic activity for Vero
tance and virulence for intravenously inoc- cells only in three of the most pathogenic
ulated 3-week-old turkeys, in E. coli isolates isolates in a study of17 isolates from sep-
from turkeys. Dozois et al. [29] observed ticemic chickens. More recently, Blanco et
that serum resistance was associated with al. [ 12J found that only 7 % of 645 E. culi
isolates from septicemic turkeys and with isolates from septicemic or healthy chick-
lethality in isolates from septicemic chick- ens were toxigenic, producing a cytotoxic
ens, in a study of 175 E. coli isolates from response in HeLa but not in Vero cells, an
septicemic and healthy birds. Ike et al. [56] cnterohemolysin, or a new cytotonic product
also found a strong correlation between in HeLa and Vero cells. This toxigenicity
serum sensitivity and lethality for I-day-old did not appear to be related to septicemic
chicks, in 115 E. coli isolates from sep- isolates. No isolates producing enterotox-
ticemic chickens. Wooley [ 1161 found a ins STa or LT, verotoxins VTI, VT2 or
strong association between serum resistance VT2v, or cytotoxic necrotizing factors CNF1I
and isolates from septicemic chickens, in a or CNF2 were detected in this study. Simi-
study of SO E. cnli isolates from septicemic larly, Irwin et al. [57did not find any vero-
and healthy chickens. Nolan et al. [78] toxin-producing E. cnli in cloacal samples
demonstrated a high correlation between from 500 broiler chickens.
serum resistance and lethality for 21-day-
Parreira and Yano [86] demonstrated the
old chickens. In order to investigate the role
production of a cytotoxin active on Vero
of complement resistance in the virulence,
and HeLa cells in 72 % of 50 isolates taken
Nolan et al. [79j produced an avirulent, com-
from chickens with the swollen head syn-
plement-sensitive mutant from a virulent, drome. They have designated this toxin
complement-resistant APEC isolate. This
mutant possessed a 16.2-kDa outer mem- VT2y because of the similarity of its effect
to that of the verotoxins and the neutraliza-
brane protein (OMP) not present in the wild-
tion of its effects by antiserum against VT2.
type strain, suggesting that an as yet uniden- However, in the conditions of stringency
tified OMP may be responsible for the
used in this study, DNA probes for VTI and
complement resistance of this APEC isolate VT2 did not hybridize with VT2y-positive
[80). Further characterization of this mutant isolates. Nevertheless, the production of a
[61suggested that the complement resis- verotoxin, which targets vascular endothc-
tancc of this isolate is due, at least in part, to
its ability to restrict C3 deposition, but not to lium, would he consistent with the lesion
of oedema observed in affected birds.
degrade C3, on the bacterial surfacc.
Enterotoxigenic E. coli have occasion-
5.6. Toxins and cytotoxins ally been isolated from the intestines of
chickens with diarrhea. For example, Akashi
There are few reports demonstrating that et al. [41 detected the genes for STII (STb)
APEC are able to produce toxins that may be in seven of 38 E. coli isolates from fecal
samples of chickens with diarrhea in the [ 113](identifying specific mRNA produced
Philippines. In another study of enterotoxi- by a pathogenic strain) could provide valu-
genic E. coli isolates from chickens with able information in the study of APEC.
diarrhea in the Philippines [106],an LT-like
enterotoxin similar to LTp with respect to
antigenicity and amino acid composition 6. PATHOGENESIS OF APEC
was identified.
INFECTIONS

5.7. Approaches for the identification In the last several years, our understand-
of novel virulence factors ing of how the lesions of diseases due to
APEC develop and of the mechanisms by
In addition to the characterization of puta- which APEC are able to cause these lesions
tive virulence genes and the study of their has greatly increased. This is particularly
role in virulence, novel approaches have the case for the respiratory tract complex,
been recently undertaken to identify and this will be the focus of this review.
genomic regions specific for APEC strains, Natural respiratory tract infection of poultry
which could be included in pathogenicity by APEC is thought to occur via the inhala-
islands. tion of feces-contaminated dust [46]. Clear-
Brown and Curtiss III [16] performed a ance of inhaled particles in the avian lung
seems mainly to be through phagocytosis
genomic subtraction between an APEC iso-
late (serogroup 078) and a K12 E. coli. This by atrial and infundibular epithelial cells of
enabled them to identify and locate 122 the parabronchial region, as there is no
known cellular defence similar to the mam-
unique regions on the chromosome of the
APEC isolate. Five of these unique regions malian aveolar macrophage in the gas-
corresponded to the positions of previously exchange area [100!. Similarly, the avian
airsac has no known resident cellular
reported virulence factors such as the tsh
gene, the group II capsule genes, the rfb
defence mechanisms and must rely on an
gene cluster, and the pathogenicity islands inflammatory influx of heterophils as the
PAI I (LEE) and PAI It. By using a similar first line of cellular defence [38, 39, 103,
approach, Coulange [22] isolated 17 frag- 104]. Hence, the air-exchange regions of
ments specific for a pathogenic strain after the lung and airsacs are rather vulnerable to
a subtractive hybridization between a bacterial colonization and invasion. It has
been shown that the air-capillary region of
pathogenic and a non-pathogenic avian E.
coli strain of serogroup 02. In a collection of the lung is an important site of entry of E.
67 avian E. coli isolates, nine of these frag- coli into the bloodstream of birds [2, 19, 89,
ments were more frequent among 95]. Pourbakhsh et al. [89] also observed
than bacteria adhering to and within the epithelial
pathogenic among non-pathogenic iso-
lates. The construction of mutants in these cells, in the interstitium, and in the lumen
of airsacs and within vascular endothelial
regions will help to understand the role they
cells in chickens following airsac inoculation
play in virulence.
with an APEC isolate. These results indi-
The use of genetic approaches for the
cate that the passage of APEC across the
identification of new virulence factors will
airsac barrier is also a site of entry into the
greatly improve our knowledge of APEC bloodstream early in infection.
pathogenic mechanisms. The application of
methods such as signature-tagged mutage- In order to more fully understand the vir-
nesis [55] (allowing the identification of ulence mechanisms of APEC, Pourbakhsh et
genes which are expressed in vivo by a neg- al. [90] examined the dynamics of infection
ative selection) or arbitrarily primed PCR in chickens following inoculation by the air-
sac route with E. coli isolates of high or low and based on biochemical reactions. The
pathogenicity. At 6 h postinoculation, all indicators used for the identification of E.
isolates had colonized the respiratory tract coli comprise indole production, fermenta-
and internal organs, but bacteria were recov- tion of glucose with gas, presence of a j3-
ered from the pericardial fluid and blood galactosidase, lack of production of hydro-
only in the highly pathogenic isolates. gen sulfide and of urease, and the
Apparently viable bacteria of the highly non-utilization of citrate as a carbon source.
pathogenic isolates, but not the low This diagnosis is strengthened if the iso-
pathogenic isolates, were often observed to lated culture belongs to a known pathogenic
be associated with or within macrophages serogroup (O1, 02, 078) and/or expresses
in the airsacs and lungs of inoculated birds. the aerobactin system. The presence of other
A strong correlation was also observed virulence factors, such as P fimbriae, the
between pathogenicity for chickens in vivo K I capsule and Tsh protein (although these
and the ability to resist the bactericidal factors are not present in a high proportion
effects of chicken macrophages in vitro. in pathogenic strains, they are very infre-
These results suggest that the resistance to quent in non-pathogenic strains), may help
phagocytosis may be an important mecha- to confirm the identification of the isolate
nism in the development of avian colisep- as an APEC.
ticemia.
Serotyping and detection of the aer-
obactin system can be performed using
immunological methods. Other virulence
7. DIAGNOSIS, PREVENTION factors are best detected by molecular meth-
AND CONTROL OF APEC ods such as specific PCR assays or the use of
INFECTIONS
specific DNA probes.

7.1. Diagnosis
7.2. Prevention
A diagnosis of colibacillosis is first sug-
gested by the clinical picture and by the The disease can be prevented by con-
presence of typical macroscopic lesions such trolling environmental contamination in
as airsacculitis, sometimes associated with order to avoid predisposing respiratory infec-
pericarditis and perihepatitis. These lesions tions. The most direct method would be to
can, however, also be caused by other organ- reduce and to control intestinal contamina-
isms. Airsacculitis can be caused by tion by pathogenic serogroups. Weinack et
Mycoplasma and Chlamydia, pericarditis al. !1112] found that pathogenic serotypes
can also be caused by Chlamydia, and per- could be competitively excluded from the
ihepatitis is sometimes caused by Pas- intestinal tract by seeding newly hatched
teurelln, Salmonella or other organisms 1441.] . chicks with the intestinal flora of resistant
Thus, in the presence of lesions evoking col- chickens. Other preventive methods include
ibacillosis, the diagnosis has to be confirmed the reduction of the transmission of E. coli
by the isolation of pathogenic E. coli. Cul- by fumigating the eggs within 2 h after they
tures should be taken from the heart blood have been laid and by discarding eggs that
and affected tissues, such as the liver, spleen, are cracked or those with obvious fecal con-

pericardial sac and marrow, avoiding con- tamination. Infection of the respiratory tract
tamination with the intestinal contents. The of birds can be reduced by maintaining
isolation of E. coli should be verified by mycoplasma-free birds, and by controlling
using appropriate media (McConkey agar, the environmental parameters (humidity,
eosin-methylene blue agar or drigalki agar), ventilation, dust and ammoniac in the air).
7.3. Control 8. CONCLUSION

Presently, the treatment of colibacillosis Recent advances in the study of the vir-
reliesmainly on antibiotherapy. As a high ulence factors of APEC have resulted in a
proportion of pathogenic isolates of E. coli greater understanding of the mechanisms
from poultry are resistant to numerous by which these bacteria are able to develop
antibiotics [5, 11, 94], isolates should be infection and cause disease. Fl fimbriae
tested for antibioresistance before treatment. appear to be involved predominantly in the
The most frequently used antibiotics are initial bacterial colonization of the upper
quinolones, beta-lactamines, tetracyclines respiratory tract. Subsequent multiplication
and sulfamides [46]. Treatment with sub- and persistence of the bacteria following
stances that increase the effectiveness of invasion of the host would be enhanced by
phagocytes, such as ascorbic acid, corti- possession of the aerobactin system and
costerone and deoxycorticosterone, has also resistance to the non-specific immune
been suggested [45, 47, 48]. defences of the host by such bacterial
Various vaccines which employed killed attributes as the presence of the Kl capsule
or attenuated virulent bacteria have been and phase variation of F1 fimbriae. P fim-
brial adhesins may also have a role at this
experimentally tested. They generally con-
fer a good protection against infection with level, since they are expressed exclusively in
the homologous strain, but cross-immunity internal organs. Their role in the infection
against heterologous E. coli strains is not process, as well as that of Tsh and curli, is
as efficacious. Similarly, passive immu- yet to be elucidated. Entry of bacteria into
nization of young birds is satisfactory when the bloodstream from the respiratory tract
birds are further challenged with the homol- appears to occur in the air-exchange regions
of the lungs and the airsacs. However, the
ogous strain. Heller et al. [54] showed that
breeders immunized with vaccines contain- bacterial mechanisms for this entry and for
resistance to the phagocytic defences of the
ing sonicated bacteria harbored detectable
host upon entry, which appears to be an
antibody titers for several months, and that
passive immunity of their chicks against the important attribute of highly pathogenic
homologous E. coli strain was completely strains, remain unknown. Since many APEC
lack the known virulence factors, it is rea-
protective for 2 weeks. Passive immunity
results in increased clearance of bacteria sonable to suppose that additional as yet
from the blood, spleen, liver and lungs [7, uncharacterized factors, possibly expressed
73]. Vaccines against E. coli are not widely only within the host, exist. The current use
of molecular approaches such as subtrac-
employed, probably because of the large
tive hybridization combined with infection
variety of serogroups involved in field out-
breaks. studies in well-defined natural host experi-
mental models using isogenic mutant strains
Experimental assays have attempted to should lead to some exciting insights into
use virulence factors as antigens for vacci-
the pathogenic mechanisms of APEC in the
nation. Highly purified pilus vaccines proved
near future.
to be effective against infection with bacte-
ria possessing the appropriate pili [50]. Bolin
and Jensen [14] passively immunized 18-
ACKNOWLEDGEMENTS
day-old turkeys with a rabbit antiserum pre-
pared against outer membrane preparations The authors wish to thank Clarisse Desautels
of E. coli grown in iron-limiting conditions.
for her invaluable technical assistance in the
When challenged into the airsacs with the
preparation of this manuscript, and Frédériquc
homologous strain, immunized turkeys were Coulangc and Ali Pourbakhsh for their contri-
partially protected against infection. bution to much of our recent experimental work
presented here and to the preparation of the traT of RI00 and iss of CoIV,l-K94, Infect.
Immun. 35 ( 1982) 654-659.
manuscript. Our recent experimental work pre-
sented here was supported by the Minist6re de [I 1]] Blanco J.E., Blanco M., Mora A., Blanco J.,
I’Enseignement Supérieur de la Science, the Prevalence of bacterial resistance to quinolones
Fonds pour la Formation des Chercheurs et à and other antimicrobials among avian
1’Aide a la Recherche du Quebec (FCAR) grant cherichia coli strains isolated from septicemic
s
E
and healthy chickens in Spain, J. Clin. Micro-
0214, and Natural Sciences and Engineering biol. 35 (1997) 2184-2185.
Research Council of Canada (NSERC) grant
2294. [12! Blanco J.E., Blanco M., Mora A., Blanco J.,
Production of toxins (enterotoxins, verotoxins,
and necrotoxins) and colicins by VT strains iso-
lated from septicemic and healthy chickens:
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