Mechanisms of Egg Contamination by Enteritidis: Salmonella

REVIEW ARTICLE
Mechanisms of egg contamination by Salmonella Enteritidis

Inne Gantois1, Richard Ducatelle1, Frank Pasmans1, Freddy Haesebrouck1, Richard Gast2,
Tom J. Humphrey3 & Filip Van Immerseel1
1
Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Research Group Veterinary Public Health and Zoonoses,
Ghent University, Merelbeke, Belgium; 2United States Department of Agriculture, Russell Research Center, Agricultural Research Service, Egg Safety and
Quality Research Unit, Athens, GA, USA; and 3Division of Veterinary Pathology, Infection and Immunity, School of Clinical Veterinary Science, University
of Bristol, Langford, Bristol, UK
Correspondence: Inne Gantois, Department

of Pathology, Bacteriology and Avian
Diseases, Faculty of Veterinary Medicine,
Research Group Veterinary Public Health and
Zoonoses, Ghent University, Salisburylaan
133, B-9820 Merelbeke, Belgium. Tel.: 132
9 264 77 40; fax: 132 9 264 74 94; e-mail:
[email protected]
Received 1 October 2008; revised 11 December
2008; accepted 11 December 2008.
Final version published online 21 January 2009.
DOI:10.1111/j.1574-6976.2008.00161.x
Editor: Simon Cutting
Abstract
Salmonella Enteritidis (SE) has been the major cause of the food-borne salmonellosis pandemic in humans over the last 20 years, during which contaminated hens
eggs were the most important vehicle of the infection. Eggs can be contaminated
on the outer shell surface and internally. Internal contamination can be the result
of penetration through the eggshell or by direct contamination of egg contents
before oviposition, originating from infection of the reproductive organs. Once
inside the egg, the bacteria need to cope with antimicrobial factors in the albumen
and vitelline membrane before migration to the yolk can occur. It would seem that
serotype Enteritidis has intrinsic characteristics that allow an epidemiological
association with hen eggs that are still undefined. There are indications that SE
survives the attacks with the help of antimicrobial molecules during the formation
of the egg in the hens oviduct and inside the egg. This appears to require a unique
combination of genes encoding for improved cell wall protection and repairing
cellular and molecular damage, among others.
Keywords
Salmonella Enteritidis; egg contamination;
eggshell penetration; reproductive tract
colonization; survival in the forming egg;
growth in eggs post-lay.
Eggs as the most important source of

Salmonella Enteritidis (SE) infections in
humans
The epidemiology of SE tells the story of a pathogen that has
found a biological niche in table eggs. SE has caused the
majority of food-borne outbreaks of salmonellosis reported
worldwide since the mid-1980s. In the United States, 298
(80%) of the 371 known-source SE outbreaks from 1985 to
1999 were egg-associated (Patrick et al., 2004). In 2006, a
total of 165 023 confirmed cases of human salmonellosis
were reported in the European Union (EU) via the European
Surveillance System (TESSy) (EFSA, 2007a). SE was identified as the cause of infection in 62.5% of the cases, and
Salmonella Typhimurium in 12.9%. Other serotypes causing
human illness are responsible for o 2% of the human
infections. Other serotypes among the top 10 causes of
human salmonellosis cases in the EU are Infantis, Virchow,
2009 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Newport, Hadar, Stanley, Derby, Agona and Kentucky. Eggs

and egg products were the most often identified food
vehicles in the Salmonella outbreaks (Braden, 2006). These
findings clearly illustrate the link between eggs and human
SE infections. An EU-wide study based on faecal and dust
sampling from layer houses revealed that 30.8% of 5310
commercial large-scale laying hen holdings were Salmonella
positive in 2006 (EFSA, 2007b). SE was the most common
serotype in the laying flock environment (52.3%). Thus,
almost 50% of the isolates were non-SE and the serotype
distribution on layer farms does not match with that found
in table eggs. The overall EU prevalence of Salmonella in
table eggs was 0.8% in 2006, and 4 90% of all egg isolates
were SE (EFSA, 2007a). These data should be interpreted
with caution because the sampling site is not specified. The
remaining 10% of the isolates belonged to different Salmonella serotypes, but these were mostly isolated in only one
EU member state, indicating that the overall importance of
FEMS Microbiol Rev 33 (2009) 718738
719
Egg contamination by Salmonella Enteritidis
non-SE serotypes in eggs is negligible. These data suggest

that SE has some intrinsic characteristics that allow a specific
interaction with either the reproductive organs of laying
hens or the egg components.
Generally, there are two possible routes of egg contamination by Salmonella. Eggs can be contaminated by
penetration through the eggshell from the colonized gut
or from contaminated faeces during or after oviposition
(horizontal transmission) (Messens et al., 2005a; De Reu
et al., 2006). The second possible route is by direct
contamination of the yolk, albumen, eggshell membranes
or eggshells before oviposition, originating from the
infection of reproductive organs with SE (vertical
transmission) (Timoney et al., 1989; Keller et al., 1995;
Miyamoto et al., 1997; Okamura et al., 2001a, b). Figure 1
shows a schematic representation of the egg pathogenesis. It is not yet clear as to which route is most
important for SE to contaminate the egg contents.
Although some authors claim horizontal transmission
to be the most important way to contaminate eggs
(Barrow & Lovell, 1991; Bichler et al., 1996), most authors
claim that vertical transmission is the most important
route (Gast & Beard, 1990; Miyamoto et al., 1997; GuardPetter, 2001).
This review provides an overview of hostpathogen
interactions in the hen reproductive tract and eggs at the
cellular and molecular level. It aims to highlight potential
differences between SE and other Salmonella serotypes that
could allow SE strains to contaminate eggs more successfully
than other serotypes.
(b)
(a)
Penetration of Salmonella
through egg shell and membranes
Salmonella
oral intake
Salmonella in faeces or vagina
Gut colonization
Systemic spread
Ascending infection
(d)
Eggs post lay
Ovaria
(c)
Internal contamination of the eggs

through infection of reproductive organs
Infundibulum
Infundibulum
infection of yolk membranes
Magnum
Magnum
infection of albumen
Oviduct
Isthmus
Isthmus
infection of shell membranes
Shell gland
Shell gland
infection of egg shell
Intestine
Vagina
Right oviduct
Cloca
Fig. 1. Pathogenesis of egg contamination by Salmonella. (a) Salmonella is orally taken up by the hen and enters the intestinal tract. Bacteria colonizing
the intestinal lumen are able to invade the intestinal epithelial cells (gut colonization). As a consequence, immune cells, more specifically macrophages,
are attracted to the site of invasion and enclose the Salmonella bacteria. This allows the bacteria to survive and multiply in the intracellular environment
of the macrophage. These infected macrophages migrate to the internal organs such as the reproductive organs (systemic spread). In addition to
systemic spread, bacteria can also access the oviduct through ascending infection from the cloaca. (b) One possible route of egg contamination is by
Salmonella penetration through the eggshell and shell membranes after outer shell contamination. Surface contamination may be the result of either
infection of the vagina or faecal contamination. (c) The second possible route is by direct contamination of the yolk, yolk membranes, albumen, shell
membranes and egg shell originating from infection of the ovary, infundibulum, magnum, isthmus and shell gland, respectively. (d) Salmonella bacteria
deposited in the albumen and on the vitelline membrane are able to survive and grow in the antibacterial environment. They are also capable of
migrating to and penetrating the vitelline membrane in order to reach the yolk. After reaching this rich environment, they can grow extensively.

720
I. Gantois et al.
Internal egg contamination after

penetration of the eggshell
Outer shell contamination
Following oviposition, any contaminated environment in
the area of the laid egg, such as the nest box, the hatchery
environment or the hatchery truck, can lead to outer shell
contamination. The presence of chicken manure and other
moist organic materials facilitates the survival and growth of
Salmonella by providing the required nutrients and a degree
of physical protection. When eggs are artificially contaminated on the shell with faeces containing Salmonella and
subsequently stored at 25 1C, numbers increase by 12 logs
by day 1 and 45 logs by day 3 (Schoeni et al., 1995). Such a
growth indicates that faeces can serve as a nutritional
reservoir for Salmonella. However, Salmonella can also
survive and grow on the eggshell in the absence of faecal
contamination, especially at lower temperatures and a low
relative humidity (Messens et al., 2006). Salmonella bacteria
probably survive for a longer time at a low temperature due
to the slower metabolism induced by the disadvantageous
conditions on the dry eggshell surface (Radkowski, 2002).
The egg surface can also be contaminated within the hen
reproductive system after formation of the shell, but this will
be discussed further in the text (Humphrey et al., 1991a).
Presuming that no differences exist between different Salmonella serotypes in the interaction with the outer shell,
more prevalent serotypes such as SE are more likely to
contaminate egg surfaces. In order to reduce the risk of
externally contaminated eggs in the food chain, the need to
rapidly remove any faecal contamination should be emphasized. However, intensive control measures in the United
States, such as examining eggs for cracks, and washing and
disinfecting eggs, have not eliminated egg contamination
with SE (Braden, 2006). It is, however, possible that penetration has occurred before examining and washing the eggs.
Factors influencing eggshell and membrane

penetration
Bacteria can easily penetrate through a cracked egg shell
(Fajardo et al., 1995). The intact egg, however, possesses
three physical barriers to bacterial penetration (Fig. 2).
These are the cuticle, which is a hydrophobic proteinaceous
layer covering the eggshell and the pore openings, the
crystalline eggshell and the shell membranes (Ruiz & Lunam, 2002). Shell membranes consist of three different
layers, i.e. the inner and the outer membrane, consisting of
a network of randomly oriented fibres, and a homogenous
third layer of electro-dense material called the limiting
membrane, demarcating the membrane at the interface with
the albumen (Wong-Liong et al., 1997).
Fig. 2. Schematic representation of the egg structure.
In addition to their function as a physical barrier, the

eggshell and shell membranes also act as a chemical barrier.
Although antibacterial proteins have been identified mainly
in the albumen, proteins with well-known antibacterial
properties have also been associated with the eggshell and
shell membranes. Lysozyme is abundant in the limiting
membrane and is also present in the shell membranes, the
matrix and the cuticle of the eggshell (Hincke et al., 2000).
Ovotransferrin has also been identified in the eggshell
membranes and the basal calcified layer, possibly acting as a
bacteriostatic filter (Gautron et al., 2001). Recently, ovocalyxin-36, a novel chicken eggshell and eggshell membrane
protein, has been identified (Gautron et al., 2006). An
antimicrobial role for ovocalyxin-36 was proposed because
its protein sequence is highly similar to lipopolysaccharidebinding proteins, bactericidal/permeability-increasing (BPI)
proteins and Plunc family proteins. These proteins are
involved in antibacterial defence, and therefore it is believed
that ovocalyxin-36 is of particular importance to keep the
eggs free from pathogens. Protein extracts derived from the
cuticle and the outer eggshell matrix indeed possess antimicrobial properties against both Gram-positive and Gramnegative bacteria (Hincke & Wellman-Labadie, 2007). Three
bacterial species, Pseudomonas aeruginosa, Bacillus cereus
and Staphylococcus aureus, were found to be inhibited in the
presence of soluble eggshell matrix proteins, and it was
demonstrated that these proteins might interact and disrupt
the membrane integrity of the bacteria (Mine et al., 2003).
On the other hand, Escherichia coli and SE were weakly
inhibited only at an early stage of incubation time (up to
4 h).
In spite of the protective physical and chemical barriers,
numerous researchers have demonstrated rapid penetration
into the egg by various bacteria, including Salmonella
(Williams et al., 1968; Humphrey et al., 1989, 1991b).
Miyamoto et al. (1998a) observed that after exposing freshly
laid eggs to a Salmonella suspension for 2 h at 25 1C, the
inner eggshell and egg contents were contaminated. Several
721
studies investigated the various factors affecting the probability of bacterial penetration. Both intrinsic and extrinsic
factors are highlighted in a review by Messens et al. (2005a).
The eggshell appears to be more easily penetrated immediately after the egg is laid (Sparks & Board, 1985; Padron,
1990; Miyamoto et al., 1998a). It is suggested that for the
first minutes after oviposition, the cuticle is immature and
some pores may be open. Moreover, when the egg is exposed
to an environment cooler than the chicken body temperature (42 1C), a negative pressure may develop and the
bacteria migrate more easily through the eggshell and
membranes (Board, 1966; Bruce & Drysdale, 1994). In
addition, the cuticle in older eggs becomes dehydrated,
resulting in its shrinkage, and the pores become more
exposed to bacterial penetration (Mayes & Takeballi, 1983).
In recent studies (De Reu et al., 2006; Messens et al., 2007), it
was reported that cuticle deposition is important for the
prevention of penetration, and in the absence of cuticle
deposition, penetration is a frequent event. However, some
research groups (Nascimento et al., 1992; Messens et al.,
2005b) observed no correlation between cuticle deposition
and penetration of Salmonella through the eggshell. Additionally, bacterial penetration was found to be independent
of the pore number (Nascimento et al., 1992; Messens et al.,
2005b; De Reu et al., 2006). As mentioned earlier, temperature is also an important factor affecting the penetration.
Fast penetration is observed when a positive temperature
differential is created between the egg (warm) and the
bacterial suspension (cool) (Mayes & Takeballi, 1983; Bruce
& Drysdale, 1994). It is believed that a positive temperature
differential, combined with the presence of moisture, provides an ideal opportunity for the bacteria to penetrate the
eggshell (Berrang, 1999). The use of different penetration
models, differences in the bacterial strains used, differences
in the number of bacteria inoculated, the temperature and
relative humidity during storage and the egg characteristics
(eggshell quality and egg age) may partly explain the
conflicting results seen in the studies regarding eggshell
penetration, as reviewed by Messens et al. (2005a).
Eggshell penetration by different Salmonella

serotypes and other bacterial species
It has been well demonstrated that penetration of the
eggshell and shell membranes is not a unique characteristic
of SE and that other Salmonella serotypes, and even unrelated bacteria, are capable of passing through these barriers (Sauter & Petersen, 1969, 1974; Mayes & Takeballi,
1983; Jones et al., 2002; De Reu et al., 2006). In a comparative study, the penetration of seven selected bacterial species
originally isolated from egg contents was assessed using two
different egg penetration models (De Reu et al., 2006). The
results indicate that Gram-negative, motile and nonclusterFEMS Microbiol Rev 33 (2009) 718738
ing bacteria penetrate the eggshell most frequently. Using an

agar model, i.e. filling eggs with agar and dipping in a
bacterial suspension, Pseudomonas sp. (60%), Alcaligenes sp.
(58%) and SE (43%) traversed the eggshell most frequently.
However, using intact eggs dipped in a bacterial suspension,
egg contents were most frequently contaminated by SE
(33%), followed by Carnobacterium sp. (17.5%) and Acinetobacter baumannii (14.8%). The results obtained by the two
experimental eggshell penetration assays suggest that shells
can be penetrated by various bacterial species, but that SE
has mechanisms to survive and/or grow in the internal egg
contents, in contrast to the other bacterial species. In a study
of naturally infected flocks, numerous Salmonella serotypes,
such as Enteritidis, Typhimurium and Hadar, were isolated
from eggshells, whereas only Enteritidis was isolated from
egg contents (Humphrey et al., 1991b). Interestingly, only
one egg was positive in both sites, suggesting that internal
egg contamination is more likely to occur during formation
of the egg rather than by penetration through the shell.
Moreover, the relative prevalence of non-Enteritidis serotypes in faecal samples (measured by the overshoe method)
of laying hen flocks (50%) is not consistent with the high
prevalence of SE in table eggs (90%) (EFSA, 2007a). All these
data support the idea that eggshell and egg membrane
penetration are not a specific property of SE and that other
characteristics of this serotype are related to egg contamination. These could include the ability to colonize the hen
reproductive tract and survival and multiplication inside
eggs, both of which could contribute to the epidemiological
association of SE with eggs.
Contamination of eggs during egg

formation
Colonization of the reproductive organs
Several lines of evidence support the view that egg contamination with SE is more likely to take place during the
formation of the egg in the reproductive organs than by
eggshell penetration. In several studies, SE was isolated from
the reproductive tissue of infected birds, in the absence of
intestinal colonization (Lister, 1988). Moreover, SE is capable of persistence in reproductive tissues of naturally and
experimentally infected hens, even though the animals
generate an innate and adaptive immune response to the
infection, indicating that the bacteria can reside intracellularly and escape the host defence mechanisms. The deposition of Salmonella inside eggs is thus most likely a
consequence of reproductive tissue colonization in infected
laying hens (Keller et al., 1995; Methner et al., 1995; Gast &
Holt, 2000a). Very little is known, however, about the exact
site in reproductive tissues where the bacteria reside and the
bacterial and host factors that play a role in the association
722
between the reproductive tissue and Salmonella. The oviduct

can be subdivided into five functional regions. Starting from
the ovary, there are the infundibulum, magnum, isthmus,
uterus and vagina. The infundibulum captures the ovulatory
follicles, the magnum produces the albumen, the isthmus
deposits the eggshell membranes, the uterus forms the
eggshell and the vagina is involved in oviposition. Salmonella
colonizing the oviduct could be incorporated into the
albumen, the eggshell membranes or the eggshell itself,
depending on the site of colonization (magnum, isthmus
and uterus, respectively). Although SE has been isolated
from both the yolk and the albumen, according to most
authors, the albumen is most frequently contaminated,
pointing to the oviduct tissue as the colonization site (Gast
& Beard, 1990; Humphrey et al., 1991b; Keller et al., 1995;
Miyamoto et al., 1997; De Buck et al., 2004c). However,
some studies found the yolk to be primarily contaminated,
suggesting the ovary to be the primary colonization site
(Bichler et al., 1996; Gast & Holt, 2000a; Gast et al., 2002).
One report indicated that several Salmonella strains colonized the ovary significantly more often than the oviduct,
but were deposited at similar frequencies in the yolk and the
albumen (Gast et al., 2007). Because of the very low
incidence of egg contamination in natural infections and
the fact that it is very labour-intensive to examine large
numbers of eggs, not enough studies have been carried out
to definitely establish the principal site of contamination. It
is thus difficult, based on the contamination site in eggs, to
predict the most important colonization site of Salmonella
in the reproductive tract. However, it would be reasonable to
suggest that, given that SE can be isolated from all sites in
the hen reproductive tract, that contamination of any part of
the egg is possible. An overview of all studies on internal egg
contamination through reproductive organ colonization is
presented in Table 1.
It is generally believed that colonization of the reproductive organs is a consequence of systemic spread of Salmonella from the intestine (Vazquez-Torres et al., 1999).
Invasion in the intestinal epithelial cells triggers infiltration
of immune cells, mainly macrophages, resulting in the
uptake of bacteria by these cells. Because of its capability to
survive and replicate in the immune cells, bacteria carried in
the macrophages are spread within the host, resulting in
colonization of the reproductive organs (Keller et al., 1995;
Miyamoto et al., 1997; Okamura et al., 2001a, b; Gast et al.,
2007; Gantois et al., 2008c). Salmonella pathogenicity
island-2 (SPI-2) is essential in the ability to spread within
the host and to cause a systemic infection (Jones et al.,
2001). Using a deletion mutant in the regulator of SPI-2
(ssrA), it was shown that after intravenous infection of
laying hens, the bacterial numbers of the ssrA mutant were
significantly lower in the oviducts and the ovaries as
compared with the wild-type strain. These reduced ssrA
I. Gantois et al.
colony counts in the reproductive organs point to a role for

SPI-2 in the spread or the colonization of the reproductive
tract tissues (Bohez et al., 2008).
Colonization of the reproductive organs has also been
shown to be a consequence of systemic spread after airborne
infections (Baskerville et al., 1992; Leach et al., 1999). It was
even observed that the contamination rate of eggs was much
higher following an aerosol challenge of the laying hens than
following an oral challenge (Leach et al., 1999).
Colonization of the ovary

The extensive permeability of the vascular endothelia observed in the ovary may contribute to the high colonization
rate at this site (Griffin et al., 1984). In the majority of
experimental studies in laying hens, a higher frequency of
ovary colonization is reported, compared with the frequency
of recovery from the oviduct (De Buck et al., 2004b; Gantois
et al., 2006; Gast et al., 2007). Therefore, it is strongly
believed that SE must interact with the cellular components
of the preovulatory follicles. It was indeed shown that SE can
attach to developing and mature follicular granulosa cells
exhibiting different attachment patterns (Thiagarajan et al.,
1994). Higher bacterial numbers in the membranes of the
preovulatory follicles than in the yolk itself suggest that
during transovarian transmission, SE remains attached to
the egg vitelline membranes. A previous study has also
suggested that yolk contamination is more often associated
with the vitelline membrane than with the interior yolk
contents (Gast & Beard, 1990; Gast & Holt, 2000a). It has
been noticed that in vitro attachment of SE to granulosa cells
may involve binding to fibronectin (Thiagarajan et al.,
1996a). Furthermore, a major role of the type 1 fimbriae in
the attachment process was suggested because the in vitro
attachment of SE to granulosa cells was inhibited by
preincubation of the cells with purified fimbrial preparation
(Thiagarajan et al., 1996a). There are also indications that
Salmonella can invade and multiply in granulosa cells
(Thiagarajan et al., 1996a). Howard et al. (2005) compared
the ability of Salmonella to invade ovarian follicles at
different stages of follicular maturity in vitro: the small white
follicles (immature) were more susceptible to Salmonella
invasion than the more mature small and large yellow ones.
These authors believe that the penetration of immature
follicles has practical implications because it can lead to
contamination of eggs after maturation and can cause
continuous transovarian infection of eggs throughout the
reproductive cycle. This statement is, however, questionable
because not all small white follicles will mature and because
the extensive growth of Salmonella in the nutrient-rich
follicles will most likely lead to their degeneration (Kinde
et al., 2000).
723
Table 1. Overview of studies carried out to analyse the internal egg contamination through infection of the reproductive organs
Method
References
Timoney
et al. (1989)
Shivaprasad
et al. (1990)
Gast &
Beard
(1990)
Humphrey
et al.
(1991a)
Thiagarajan
et al. (1994)
Result
Inoculation
route
Strain
Inoculation dose
(log10 CFU mL 1)General result
Oral
SE PT4
The relatively high frequency of internal egg

contamination clearly demonstrates the
potential for egg transmission of SE
Oral,
SE PT8
8.38.6
SE was only cultured from the yolk and egg
intravenous,
white of a small number of eggs until 11 days
cloacal
postinfection
Oral,
SE PT13a
9
Although a high contamination rate of egg
contact
white and yolk was observed, Salmonella
transmission
could not be recovered from any yolk content
sample, suggesting that Salmonella is
contaminating the vitelline membrane
Oral
SE PT4
3, 6, 8
There was no relationship between the
contamination of the egg contents and
antibody status, faecal excretion or the dose
administered
Oral
SE PT8, SE PT28 Not mentioned SE can colonize the preovulatory follicles at
different stages of development. It is
therefore suggested that SE remains attached
to the vitelline membrane instead of
contaminating the yolk content
Keller et al.
(1995)
Oral
SE
Methner
et al. (1995)
Oral
SE
10
Bichler
et al. (1996)
Oral
SE
10
Keller et al.
(1997)
Oral
SE, Salmonella
Typhimurium
Miyamoto
et al. (1997)
Intravenous, SE PT4
intravaginal,
cloacal
Williams
et al. (1998)
Oral
Miyamoto
et al.
(1998b)
Gast & Holt

(1998)
Egg contamination rate
Salmonella
Typhimurium
DT104
Intravaginal, SE PT4
cloacal
Oral
SE PT13
The contamination rate of forming eggs is

much higher than the contamination rate of
laid eggs, indicating that antibacterial factors
within the egg may control the pathogen
before the egg is laid
No correlation was found between the
contamination of the eggshell and that of the
egg content
The hens produced SE-positive eggs at high
frequencies in the first week postinfection.
SE and Salmonella Typhimurium may be equal

in their potential to colonize the tissues of the
reproductive tract and forming eggs, but only
SE was isolated from egg contents after
oviposition
Intravaginal and cloacal inoculation resulted
in the colonization of only the lower oviduct
whereas intravenous infection resulted in
colonization of the entire oviduct
These experiments have demonstrated that
DT104 can contaminate the egg contents
after oral infection
After intravaginal inoculation, SE was
recovered from the uterus and after cloacal
inoculation SE was recovered from the
vagina, indicating that SE only ascends to the
lower parts of the oviduct
This study has shown that infection of 1-dayold chicks can lead to frequent intestinal
colonization and occasional egg
contamination when these birds mature
Yolk: 9.6%
Egg white: 3.6%
Yolk: 0.4%
Egg white: 1.5%
Yolk: 18.5% (first week)
Egg white: 20% (first week)
Yolk contents: 0%
Total egg content:

Inoculum 3: 3.5%
Inoculum 6: 0%
Inoculum 8: 0%
Preovulatory follicle (16 birds):
Membrane: 10 positive samples
Yolk content: four positive samples
Laid eggs:
Yolk: eight positive yolks
Egg white: three positive egg whites
Forming eggs: 27.131.4%
Freshly laid eggs: 00.6%
Yolk: 0%
Egg white: 0.4%
In the first week postinfection:
Eggshell washing: 26.5%
Egg content: 2.9%
Egg white: 43%
Yolk: 41%
Total egg content:
Salmonella Typhimurium: 0%
Total egg content/shell:

Intravenous: 11.5%/7.7%
Intravaginal: 9.6%/12%
Cloacal: 0%/4.6%
Total egg content: 2.1%
Total egg content/shell

Intravaginal: 5%/15%
Cloacal: 0%/0%

724
I. Gantois et al.
Table 1. Continued.
Method
Result
References
Inoculation
route
Strain
Leach et al.
(1999)
Oral,
aerosol
Gast & Holt

(2000a)
Oral
Kinde et al.
(2000)
Oral,
SE PT4
intravenous
9, 6
Okamura &
Holt
(2001a, b)
Intravaginal SE, Salmonella

serotypes
Typhimurium,
Infantis, Hadar,
Heidelberg and
Montevideo
Okamura
et al.
(2001a, b)
Intravenous SE, Salmonella

serotypes
Typhimurium,
Infantis, Hadar,
Heidelberg and
Montevideo
Oral
Salmonella
Pullorum
This study suggests that SE is the predominant

serovar to colonize the reproductive organs of
laying hens among the six serotypes tested
Gast & Holt

(2001)
Oral
Gast et al.
(2002)
Oral,
SE PT13a
aerosol,
intravenous
Oral
SE PT13a wild
type (WT),
passaged SE
PT13a (spleen,
liver)
passaged SE
PT13a
(oviduct and
ovary)
Oral
SE, Salmonella
Heidelberg
9, 9 and 57
One-day-old chicks orally infected with

Salmonella Pullorum produced contaminated
eggs frequently during the period of sexual
maturity as a consequence of reproductive
tract colonization
Deposition of SE within egg yolks appears to
occur infrequently and SE is mostly deposited
on the vitelline membrane
No significant differences were observed in
egg contamination among the three
inoculation routes
Passaged SE strains recovered from ovaries
and oviducts induced a significantly higher
incidence of egg contamination than the WT
SE strain
Intravenous SE PT4
Wigley
et al. (2001)
Gast et al.
(2003)
Gast et al.
(2004)
De Buck
et al.
(2004c)
Gast et al.
(2005b)
Oral
Inoculation dose
Salmonella
7, 24
Typhimurium
DT104
Two SE PT4 and 9
one SE PT13a
SE PT13a
SE, 2 Salmonella 9
Heidelberg strains
and passaged

The egg contamination rate in aerosolinfected birds was much higher compared
with orally infected birds
For all three isolates, the incidence of yolk
contamination was significantly higher than
the incidence of egg white contamination
and no significant difference was observed
between the SE strains
In the orally infected birds, 43% of the
reproductive organs were positive, compared
with 83% in the intravenously infected birds
This study suggests that SE has a specific
advantage over the other Salmonella
serotypes by its capacity to colonize the
vaginal tissues of hens
There was no significant difference in

reproductive tract colonization between the
two serotypes, but Salmonella Heidelberg
was recovered from the eggs at lower
frequencies than SE
The infected birds produced the highest
frequency of contaminated eggs in the first
week postinfection
The Salmonella-passaged strains caused a
significantly higher frequency of egg
contamination than did the WT strains.

Total egg content:
Oral: 1.7%
Aerosol: 1425%
Yolk: 2.5%
Egg white: 0.5%
Egg content/shell
SE: 7.5%/25%
Salmonella Typhimurium: 3.1%/1.6%
Salmonella Infantis: 0%/4%
Salmonella Hadar: 0%/4.9%
Salmonella Heidelberg: 0%/4.5%
Salmonella Montevideo: 0%/1.9%
Yolk/egg white
SE: yolk: 6.9%/2.3%
Other Salmonella serotypes: 0%/0%
Total yolk: 4.3%

Yolk content: 0.5%
Yolk: 47%
Egg white: 02%
Total egg content:
SE PT13a WT: 8.27%
Passaged SE PT13a (spleen, liver):
10.41%
Passaged SE PT13a
(oviduct and ovary): 17%
Total egg content:

SE: 7.0%
Salmonella Heidelberg: 1.14.5%
Shell: 17.4%
Yolk: 20.3%
Egg white: 4.3%
Total egg content:
SE WT: 5%
SE passaged strain: 8.84%
725
Table 1. Continued.
Method
References
Result
Inoculation
route
Strain
Inoculation dose
variants of each
WT strain
Gast et al.
(2007)
Oral
SE PT13a, SE
PT14b,
Salmonella
Heidelberg
Gantois
et al.
(2008c)
Intravenous 2 SE strains,
Salmonella
serotypes
Typhimurium, Heidelberg,
Virchow, Hadar
Furthermore, no correlation was found

between the duration of faecal shedding and
the production of contaminated eggs
The frequency of ovarian colonization was

significantly higher than the frequency of
recovery from the oviduct for all three
Salmonella strains, but no corresponding
difference was observed between the
incidence of deposition in yolk or egg white.
The incidence of egg contamination with SE
was higher than that of Salmonella
Heidelberg
The SE strains showed a higher colonization
of the reproductive organs in comparison
with the Salmonella serotypes Heidelberg,
Virchow and Hadar. No significant difference
was observed between the SE strains and the
Salmonella Typhimurium strain
The fact that Salmonella can interact with the cellular

components of preovulatory follicles raises the question as
to whether serotype Enteritidis harbours some intrinsic
characteristics allowing it to specifically interact with these
cells and, as a consequence, be transmitted to eggs. In a
study by Okamura et al. (2001a, b), it was shown that among
six different Salmonella serotypes, Enteritidis colonized
ovaries and preovulatory follicles at significantly higher
levels than five other serotypes after intravenous inoculation. Because samples in this study were only taken at 4 and
7 days postinfection, and bacteria were still persistent in the
peripheral blood, it cannot be concluded, however, that SE
displays a stronger interaction with follicles than other
serotypes. Similar results were obtained by Gantois et al.
(2008c) in an intravenous infection model, demonstrating a
higher affinity of the serotype Enteritidis for the ovary
compared with other Salmonella serotypes (Hadar, Virchow
and Infantis), except for Typhimurium. The fact that SE and
Salmonella Typhimurium may be equally capable of colonizing the ovary is in accordance with the data obtained by
Keller et al. (1997). Studies comparing invasion of the
serotypes Enteritidis and Typhimurium in ovarian follicles
in vitro yielded conflicting results (Howard et al., 2005;
Mizumoto et al., 2005). Based on the fact that systemic
spread is a characteristic of most Salmonella serotypes, it is
believed that ovarian colonization is not a specific trait
allowing the serotype Enteritidis to contaminate eggs. HowFEMS Microbiol Rev 33 (2009) 718738

Salmonella Heidelberg WT 1: 1.63%
Salmonella Heidelberg passaged
strain 1: 4.95%
Salmonella Heidelberg WT 2: 3.14%
Salmonella Heidelberg passaged
strain 2: 5%
SE:
Yolk: 5.5%
Egg white: 4.1%
Salmonella Heidelberg:
Yolk: 1.5%
Egg white: 1.8%
Total egg content (positive eggs/total

eggs):
SE1: 4/5
SE2: 3/5
Salmonella Typhimurium: 2/5
Salmonella Heidelberg: 0/6
Salmonella Virchow: 1/16
Salmonella Hadar: 0/16
ever, the possibility that SE has a specific ability to interact

and invade the preovulatory follicles cannot be ruled out. A
large-scale study using multiple strains from different
Salmonella serotypes should be carried out in order to
provide more information regarding the serotype specificity
of ovarian colonization and persistence. High levels of
nutrients are available to bacteria invading ovarian follicles.
Therefore, it is to be expected that this should lead to
extensive replication of the bacteria, almost inevitably
resulting in follicular degeneration. Because this is not a
common phenomenon in naturally infected laying hens, as
the laying percentage is usually not reduced, follicle colonization is not believed to be an important source of egg
contamination, although this is under debate.
Colonization of the oviduct

Although several studies reported the vitelline membrane as
the most common site of Salmonella contamination (Bichler
et al., 1996; Gast & Holt, 2000a; Gast et al., 2002), other
reports point to albumen as the principal site of contamination in eggs (Shivaprasad et al., 1990; Humphrey et al.,
1991b; Keller et al., 1995), indicating that SE is colonizing
oviduct tissues. Miyamoto et al. (1997) observed that
developing eggs in a highly contaminated oviduct are likely
to be Salmonella positive. Colonization of the reproductive
tract can be the result of an ascending infection from the
726
cloaca (Reiber et al., 1995; Miyamoto et al., 1997), a

descending infection from the ovary (Keller et al., 1995)
and/or a systemic spread of Salmonella. Depending on the
site of contamination, i.e. the vagina, isthmus and magnum,
Salmonella could be incorporated into the eggshell, the
eggshell membranes or the albumen.
Vaginal colonization
Several authors have focused on the role of the vagina in the
production of SE-contaminated eggs (Barrow & Lovell, 1991;
Keller et al., 1995; Reiber et al., 1995; Miyamoto et al., 1999;
Okamura et al., 2001a, b; Mizumoto et al., 2005). It is
believed that intravaginal infection tends to ascend only to
the lower parts of the oviduct because Salmonella is rarely
recovered from the ovary and the upper oviduct in intravaginally inoculated hens (Miyamoto et al., 1997, 1998b;
Okamura et al., 2001a, b). These studies obtained high egg
contamination rates after intravaginal infection, indicating a
high risk of contamination (primarily eggshell contamination) as the egg passes through a heavily colonized vagina.
When the egg is laid, penetration through the eggshell can
occur, due to suction of the organisms into eggs under the
negative pressure caused by cooling of the egg (Schoeni et al.,
1995; Miyamoto et al., 1998a). In spite of the fact that it is
difficult to distinguish between contamination during formation of the egg or after oviposition, internal egg contamination after vaginal colonization most likely occurs after
penetration of the eggshell and not by internal contamination following ascending infection of the upper oviduct,
although this cannot be ruled out. In a comparative study
with six different Salmonella serotypes, significantly higher
numbers of SE were recovered from the vagina in comparison with strains belonging to other serotypes after intravaginal inoculation (Miyamoto et al., 1998a). The authors
suggested a higher ability of the serotype Enteritidis to attach
to the vaginal epithelium. It was also noticed that the
rank order of the Salmonella invasiveness in vaginal epithelium was dependent on the lipopolysaccharide type, namely
lipopolysaccharide type O9 (SE) 4 lipopolysaccharide type
O4 (Salmonella Typhimurium, Salmonella Heidelberg and
Salmonella Agona) 4 lipopolysaccharide type O7 (Salmonella Montevideo and Salmonella Infantis) and lipopolysaccharide type O8 (Salmonella Hadar) (Mizumoto et al., 2005).
Isthmus and magnum colonization
It is clear that different segments of the oviduct can be
colonized by SE. Using different infection models, the
tubular glands of the isthmus were identified as the predominant colonization site of SE in the oviduct by De Buck
et al. (2004a). Colonization of the isthmus can result in
contaminated eggshell membranes. These observations are
I. Gantois et al.
in accordance with other experimental studies (Bichler et al.,

1996; Miyamoto et al., 1997; Okamura et al., 2001a, b). In
principle, eggshell membrane contamination can also be a
consequence of penetration of Salmonella bacteria after
deposition on the shell during the passage through the
vagina rather than direct contamination of the eggshell
membranes during passage through the isthmus. In addition, when culturing the eggshell and egg contents separately, some albumen sticks to the eggshell, making the
interpretations of the shell membranes as the site of egg
contamination even more complex.
Numerous studies suggest that SE most frequently
migrates into eggs through the upper oviduct in association with the albumen (Gast & Beard, 1990; Hoop &
Pospischil, 1993; Humphrey & Whitehead, 1993; Schoeni
et al., 1995). Detection of SE associated with secretory cells
of the upper and lower magnum by immunohistochemical
staining is in agreement with the hypothesis that the
pathogen may contaminate forming eggs through the albumen (Hoop & Pospischil, 1993; Keller et al., 1995; De Buck
et al., 2004a). Recently, the abilities to invade and proliferate in isthmus and magnum oviduct cells of different
Salmonella serotypes were assessed using a tubular gland
cell primary culture model. All serotypes tested were equally
able to invade and proliferate in the glandular epithelial
cells, suggesting that invasion and proliferation in oviduct
cells is most likely not a unique characteristic of the
serotype Enteritidis (Gantois et al., 2008c). In the study of
Gantois et al. (2008c), it was also shown that a Salmonella
serotype Enteritidis and Typhimurium strain colonized the
oviduct to higher levels than strains belonging to the
serotypes Heidelberg, Virchow and Hadar, even if all
serotypes invaded oviduct cells in vitro. This is in accordance with a previous intravenous infection study by
Okamura et al. (2001a, b), demonstrating that of six serotypes, only Enteritidis and Typhimurium were able to
colonize the reproductive organs at days 4 and 7 postinoculation. One-day-old chicks that were orally infected with
the chicken-adapted Salmonella Pullorum produced a high
amount of contaminated eggs (6.5%) during the period of
sexual maturity as a consequence of reproductive organ
colonization (Wigley et al., 2001). Isolates of SE and
Salmonella Pullorum, together with isolates of Salmonella
Gallinarum and Salmonella Dublin, form a related strain
cluster that share the same lipopolysaccharide-based
O-antigen structure (O-1, 9, 12, characteristic of serogroup
D). Comparative genome analysis of SE and Salmonella
Gallinarum indicated that these serotypes are highly
related and that Salmonella Gallinarum may be a direct
descendant of SE (Thomson et al., 2008). It can be
speculated that these two serotypes harbour the same
characteristics, allowing them to efficiently contaminate
eggs, but this is not clear.
727
Reproductive tract colonization: a matter of

controversy
It is difficult to make comparisons between different experimental studies attempting to determine the preferred site of
colonization or the strongest colonizing and persisting
serotype. Indeed, experimental infection studies use different strains, inoculation methods, infection doses and
laboratory techniques for bacteriological analysis. Individual strains of Salmonella (within and across serotype
boundaries) can differ considerably in their ability to contaminate eggs (Gast & Holt, 2000a, 2001). Four Salmonella
Heidelberg strains colonized the ovaries and oviducts of
inoculated hens at frequencies similar to SE, but were found
significantly less often inside eggs (Gast et al., 2004).
Phenotypic attributes, such as the ability to produce highmolecular-mass lipopolysaccharide and the ability to grow
to a high cell density, have been linked to an enhanced
capability of egg contamination by SE (Guard-Petter et al.,
1997; Guard-Petter, 1998). Recently, a set of small nucleotide
polymorphisms (SNPs), which differ in two SE strains that
vary in egg contamination, were identified (Guard-Bouldin,
2006). In addition, a high-throughput phenotype microarray assaying the growth of bacteria in response to 1920
different culture conditions revealed that these two strains
show dramatic differences in amino acid and nucleic acid
metabolism, which is most likely correlated to the SNPs
(Morales et al., 2005). Furthermore, it was shown that serial
passage through the reproductive organs also enhances egg
contamination (Gast et al., 2003, 2005a). This indicates that
the selective pressure in the reproductive tissues may promote the induction of specific bacterial properties, resulting
in an elevated egg contamination. Numerous studies have
also been performed to study the effect of the inoculation
route on the production of contaminated eggs (Miyamoto
et al., 1997; Gast et al., 2002). While Gast et al. (2002)
reported that oral, aerosol and intravenous inoculations led
to similar frequencies of egg contamination, Miyamoto et al.
(1997) observed a higher contamination rate when birds
were inoculated intravenously and intravaginally. Moreover,
it should be taken into account that using different laboratory techniques to isolate the bacteria from eggs may have an
impact on the outcome of the experiments. In some studies,
the yolk samples are cultured together with the vitelline
membrane and thus some albumen, while other studies
extract yolk contents and thus do not culture the vitelline
membrane. Furthermore, extending the incubation time
from 24 to 48 h can increase the isolation rate of SE from
eggs significantly (Humphrey & Whitehead, 1992), meaning
that studies that do not take account of this may have
underestimated the prevalence of egg contamination. The
use of different pre-enrichment and enrichment media can
also result in different outcomes (Humphrey & Whitehead,
1992). For isolation of Salmonella from whole eggs, it was

found that the Rappaport Vassiliadis broth was superior to
Selenite broth as a selective medium (Humphrey & Whitehead, 1992). Additionally, the outcomes of Salmonella
infections may also be influenced by host susceptibility
characteristics, such as the breed or the line of chickens
(Beaumont et al., 1994, 1999; Keller et al., 1995; Kinde et al.,
2000). Some studies reported that brown-egg layers are
more susceptible than white-egg layers (Keller et al., 1995;
Kinde et al., 2000), and on comparing four different lines of
chickens, it was found that one line was more susceptible to
SE than others (Protais et al., 1996). All these aspects
indicate that care should be taken when interpreting data
obtained from experimental infections.
Virulence factors associated with oviduct

colonization
In order to gain a better understanding of the molecular
mechanisms allowing the serotype Enteritidis to interact with
the hens reproductive tract and to adapt to this particular
ecological niche, a genome-wide screen was carried out by
Gantois et al. (2008b) to identify genes expressed in the
oviduct, using in vivo expression technology. This study
identified the genes involved in cell wall integrity, regulation
of fimbrial operons, amino acid and nucleic acid metabolism,
stress response and motility as being highly induced during
colonization of the reproductive tract. This indicates that the
oviduct is a stressful and damaging environment for Salmonella bacteria, but it also indicates that the bacteria can
counteract this by stress-induced protective and reparative
responses, enabling the bacteria to survive in the hostile
environment and/or escape the host defence reactions.
Other SE factors that play a role in oviduct infections are
fimbriae (De Buck et al., 2003, 2004b; Li et al., 2003). Li et al.
(2003) were the first to identify binding sites for fimbriated
SE in the chicken oviduct. The binding of type 1 fimbriae to
glycosphingolipids and gangliosides from the oviduct mucosa is not uniform along this organ, and is mainly in the
infundibulum. De Buck et al. (2003) clearly demonstrated
that SE isolates are able to adhere to immobilized secretions
of the oviduct. These authors showed that the receptor of
adhesion is also localized inside the tubular gland cells of the
isthmus and adhesion is blocked by the addition of mannose, indicating that the adhesion is mediated by type 1
fimbriae. Conversely, a study using SE isolates differing in
their fimbrial expression found no difference in infection of
the reproductive organs and eggs, making their role equivocal (Thiagarajan et al., 1996b). Nevertheless, a screening for
promoters induced in the albumen showed that SE fimZ is
highly induced during incubation at 42 1C. This may mean
that when Salmonella resides extracellularly in the oviduct
lumen, in the presence of albumen, the transcription of type
728
1 fimbriae will be activated, resulting in bacterial attachment

to the secretory glandular cells (own unpublished data).
There is mounting evidence that lipopolysaccharide is
also of particular importance for SE persistence in the
reproductive tract tissues. Lipopolysaccharide is a major
component of the outer membrane of Gram-negative bacteria and a prime target for recognition by the innate
immune system. At least two different functions have been
attributed to lipopolysaccharides with respect to persistent
reproductive tract infection. It has been suggested that the
composition of lipopolysaccharides is important in determining the survival of SE in avian macrophages and these cells
may be a site where SE resides in the oviduct (He et al., 2006).
Different levels of attachment of different Salmonella serotypes
to chicken vaginal explants possibly also involve a role of the
lipopolysaccharide structure (Mizumoto et al., 2005). Furthermore, it has been shown that high-molecular-weight lipopolysaccharide in SE is correlated with increased egg
contamination (Guard-Petter et al., 1997). Although the exact
role of high-molecular-weight lipopolysaccharide is not yet
known, its presence has been correlated with an unusual
pathology of the reproductive tract, although this was not
reflected in higher egg contamination (Parker et al., 2002).
The type 3 secretion systems-1 and -2 (T3SS-1 and T3SS-2)
may also play a role in egg contamination. T3SS-1 is mainly
associated with bacterial invasion of the intestinal epithelium
via the concerted action of effector proteins (Zhou et al.,
1999), while T3SS-2 is responsible for the establishment of
systemic infection by promoting the intracellular survival of
Salmonella in macrophages, as mentioned earlier. Li et al.
(2008) were the first to confirm the pathogenic role of T3SS-1
and T3SS-2 effectors in SE invasion and intracellular survival
in chicken oviduct epithelial cells. It is believed that invasion
and survival in tubular gland cells of the oviduct is not specific
for serotype Enteritidis (Gantois et al., 2008c). Most likely,
functions exerted by T3SS-1 and T3SS-2 are also required by
Salmonella serotypes other than Enteritidis to invade and
survive inside chicken oviduct epithelial cells (Jones et al.,
2002). Furthermore, a recent study suggested that inactivation
of ssrA, a regulator of T3SS-2, rendered SE unable to colonize
the chicken reproductive tract successfully (Bohez et al., 2008).
Meanwhile, it has become clear that the process of
oviduct colonization is complex and depends on many
factors including fimbriae, flagellae, lipopolysaccharide, cell
wall structure and stress tolerance. Although most, if not all,
bacterial factors, shown to play a role in reproductive tract
colonization, are not specific to the serotype Enteritidis, a
unique regulation of these known virulence factors in the
reproductive tract environment could be one plausible
explanation for the epidemiological association with hens
eggs. This, however, has not been shown yet. It was demonstrated that repeated in vivo passages through the reproductive tissues of chickens increase the ability of an SE strain to
I. Gantois et al.
induce internal egg contamination, whereas serial passage

through the liver and the spleen did not affect the ability of
the strain to cause egg contamination (Gast et al., 2003).
This is an indication that interaction of SE with the
reproductive tissues may either induce or select for the
expression of microbial properties important for egg contamination. The complementarity between phenotypic
traits with relevance for colonization and survival in different tissues may allow SE to traverse the complex series of
events between the introduction of infection and the deposition inside eggs (Gast et al., 2002).
The interaction between Salmonella and

the forming egg
Transfer of Salmonella from the hen to the egg
The survival of SE in forming eggs has been considered
crucial for internal egg contamination (Keller et al., 1995,
1997). Salmonella colonizing reproductive organs can potentially be incorporated into the forming egg, provided the
contamination of the egg contents does not lead to an
abortive egg formation and provided the bacteria are not
killed by the albumen.
Yolk contamination can occur due to ovary colonization by
Salmonella. Degeneration of follicles in the ovary has often
been observed after experimental Salmonella infections, most
likely caused by extensive growth in the nutrient-rich yolk at
chicken body temperature, 42 1C (Kinde et al., 2000). Interestingly, extensive growth in whole eggs when stored at room
temperature (2025 1C) does not lead to changes in the
colour, smell and consistency of the egg contents (Humphrey
& Whitehead, 1993), suggesting that the process of yolk
degeneration is dependent on physiological factors, such as
the temperature. Degeneration of the ovarian follicles would
result in a decline in the production cycle and thus no
production of eggs containing contaminated yolks. The extent
to which the yolk contents become positive after infection of
the ovary is, however, not clear. After intravenous inoculation,
SE cells are confined to the interstitial tissues and not to the
yolk contained in the large follicles (Barrow & Lovell, 1991).
Moreover, experimental studies have suggested that Salmonella
are far more likely to be deposited on the outside of the
vitelline membrane rather than inside the nutrient-rich yolk
during ovary colonization (Gast & Holt, 2001). Inoculation
of Salmonella onto the vitelline membrane in an in vitro egg
contamination model demonstrated that some strains were
capable of penetrating into the yolk contents at a low
frequency during 24 h of incubation at 30 1C (Gast et al.,
2005b), but a similar study reported no positive yolk contents
samples after incubation for 24 h at 42 1C (Guan et al., 2006).
Moreover, SE multiplication on the exterior vitelline membrane both preceded and exceeded multiplication resulting
729
from penetration into the yolk contents during 36 h of

incubation at 30 1C (Gast et al., 2008). This suggests a low
invasion of yolk contents by Salmonella during egg formation.
These data support the possibility of incorporation into the
egg by carriage on the vitelline membrane.
Albumen or shell membrane contamination would occur
when Salmonella colonizes the upper oviduct. According to
Keller et al. (1995), the infection of the forming egg occurs at
this site, before eggshell deposition. Indeed, after oral infection
with SE, about one-third of the forming eggs were positive
compared with 0.6% of the freshly laid eggs (Keller et al.,
1995). This reduction clearly suggests that antibacterial factors
within the albumen can exert a degree of control of SE in
forming eggs. During the c. 26 h required for the formation of
an egg, the ovum spends c. 5 h in the magnum, where it is
surrounded by the albumen, followed by the addition of two
shell membranes in the isthmus. The remaining 21 h are
required for shell deposition in the uterus, after which the
completed egg is moved through the vagina to pass through
the cloaca as it is laid (Solomon, 1997). Survival in the
forming egg could be a possible reason for selective isolation
of the serotype Enteritidis in laid eggs, provided that this
serotype harbours intrinsic or induced factors related to
albumen resistance. Oral infection of laying hens with three
different SE and Typhimurium strains revealed that both
serotypes are equally able to colonize tissues of the reproductive tract and forming eggs in the oviduct before oviposition.
However, only SE, but not Salmonella Typhimurium, was
isolated from egg contents after oviposition (Keller et al.,
1997), suggesting survival strategies of SE inside the forming
eggs. Nevertheless, Salmonella Typhimurium DT104 was
shown to contaminate the egg contents after oral infection of
laying hens (Williams et al., 1998).
Antimicrobial components in the albumen and

vitelline membrane
The reproductive tract produces antimicrobial components
that are incorporated into the albumen, and that are growth
restricting for Salmonella. The most well known are lysozyme
and ovotransferrin. Lysozyme is a muramidase affecting the
cell wall of Gram-positive bacteria (Hughey & Johnson, 1987),
but that has also been shown to form pores in the cell wall of
Gram-negative bacteria (Gast et al., 2005a). Ovotransferrin
possesses two distinct mechanisms of bacteriostatic action
against bacteria. The first is iron chelation, which creates an
iron-deficient environment for bacteria (Mayes & Takeballi,
1983). The second is a direct interaction with the membrane
and induction of damage to biological functions of the
bacterial cytoplasmic membrane (Ibrahim et al., 1998, 2000).
Another group of antimicrobial proteins are those showing proteinase-inhibiting activity. They include ovomucoid,
ovoinhibitors (serine protease inhibitors), cystatin (a cyFEMS Microbiol Rev 33 (2009) 718738
steine protease inhibitor) and ovostatin (Stevens, 1991).

Their function lies in inhibiting tryptic digestion of egg
proteins by bacteria and thus protection of the antimicrobial
activity of albumen proteins.
A recent study identified 11 types of gallinacins (bdefensins) expressed in the segments of the oviduct (Abdel
Mageed et al., 2008). Defensins are antimicrobial peptides
that play significant roles in innate immunity (Sugiarto &
Yu, 2004; Higgs et al., 2005). The greatest expression of
gallinacins was seen in the infundibulum and the vagina
(Ohashi et al., 2005). Recently, Yoshimura et al. (2006)
reported that the expression of gallinacin-1, -2 and -3 was
increased within 24 h in response to SE infection or in
response to purified lipopolysaccharides in cultured vaginal
cells. The study by Abdel Mageed et al. (2008) confirmed that
gallinacin-3 expression was enhanced by lipopolysaccharide
in vivo. Escherichia coli lipopolysaccharide injection in laying
hens also induced gallinacin expression in the ovarian
follicles (Subedi et al., 2007). b-Defensin-11 was identified
to be present in the chicken albumen as well (Mann, 2007).
Remarkably, the albumen contains many proteins that are
connected in some way to lipopolysaccharide binding or
modification. One such albumen component is similar to the
mammalian acyloxyacyl hydrolase, known to cleave acyl
chains from bacterial lipopolysaccharide. Furthermore, proteins containing BPI domains have been identified. These
usually occur in proteins binding and neutralizing lipopolysaccharide and thus eventually mediating the destruction of
bacteria (Elsbach & Weiss, 1998). Such domains also occur in
Tenp, a protein recently identified as an albumen component
(Gue rin-Dubiard et al., 2006), and in the eggshell-specific
protein ovocalyxin-36 (Gautron et al., 2006). Recently,
Silphaduang et al. (2006) were the first to report the presence
of histones H1 and H2B as antimicrobial proteins in the
avian reproductive system, but their functional significance
in the chicken reproductive tract remains obscure. In the
human placenta, histones H2A and H2B show a dosedependent inhibition of the endotoxin activity of lipopolysaccharide by binding to and therefore blocking both the
core and the lipid A moieties (Kim et al., 2002).
It is not known to what degree these antibacterial
components affect different Salmonella serotypes. It is,
however, striking that the function of most albumen proteins is linked to lipopolysaccharide binding. Given that the
O-antigen structure of lipopolysaccharide is a major determinant of serotype specificity, it may be that the lipopolysaccharide structure plays a major role in the SE survival in
forming eggs in vivo and that the lipopolysaccharide chemotype will affect the degree of binding with antimicrobial
components and thus bacterial survival.
Besides the albumen, the vitelline membrane can also
become contaminated (Bichler et al., 1996; Gast & Holt,
2000a; Gast et al., 2002). Recently, a proteomic analysis of
730
the chicken egg vitelline membrane was carried out (Mann,

2008). Most of the components of the vitelline membrane
that were known previously from other egg compartments,
such as lysozyme, ovalbumin, ovotransferrin, ovomucin and
lysozyme, also constitute c. 60% of the dry weight of the
outer vitelline membrane. One outer vitelline membrane
protein was identified as b-defensin 11.
Immunoglobulins are considered to belong to the antimicrobial defence system of avian eggs. Antibodies to
Salmonella have been detected in the egg albumen and yolk
from naturally and experimentally infected chickens (Schiemann & Montgomery, 1991; Desmidt et al., 1996). It has
been suggested that antibodies transferred to the yolk after
hyperimmunization of laying hens have no influence on the
multiplication of Salmonella in the yolk (Takase et al., 1999;
Gurtler & Fehlhaber, 2004). In contrast, Holt et al. (1996)
described a significant difference in the growth behaviour of
SE under the influence of antibodies. These authors, however, performed their experiments by inoculating Salmonella
in a mixture of albumen and yolk, while the two previous
studies were based on inoculations in separated yolk. Thus,
the possibility exists that the antimicrobial components of
the albumen had an additional inhibitory effect on Salmonella. The exact antimicrobial role of immunoglobulins in
avian eggs thus remains to be defined.
Salmonella virulence factors affecting SE

survival in the forming egg
The important role of lipopolysaccharides in conferring
protection against the bactericidal component albumen was
recently confirmed by Gantois et al. (2008a). Applying in
vivo expression technology, the rfbH gene, involved in
lipopolysaccharide O-antigen synthesis, was found to be
transcriptionally induced during growth in whole eggs at
room temperature. After inoculation of a Salmonella DrfbH
strain in albumen at 42 1C, immediate killing was observed
while the wild-type strain was able to survive in albumen
during 24 h. Moreover, the DrfbH mutant was also unable to
grow in whole eggs at room temperature. Lu et al. (2003)
suggested that yafD and xthA play an essential role in the
repair of DNA damage caused by the albumen and hence
confer an advantage to SE to survive in forming chicken
eggs. In a recent paper using transposon mutagenesis, it was
found that the majority of genes associated with SE survival
in albumen at 37 1C are involved in either cell wall structure/
function or nucleic and amino acid metabolism (Clavijo
et al., 2006). Two mutants had insertions in genes unique to
SE. One is homologous to a restriction endonuclease and the
other is the pef operon encoding a fimbrial biosynthesis
gene. Both genes were transformed into a Salmonella
Typhimurium strain, but only the former conferred an
enhanced survival in albumen. The same study also demon2009 Federation of European Microbiological Societies
I. Gantois et al.
strated that survival in the albumen at 37 1C was higher for

serotype Enteritidis compared with Typhimurium and
E. coli. It is striking that many antimicrobial proteins in
albumen bind to lipopolysaccharides while cell wall and
lipopolysaccharide biosynthetic genes of Salmonella seem to
play an important role in albumen survival.
Survival of different Salmonella serotypes in the

forming egg
Some research groups have compared the survival capabilities of strains belonging to different serotypes in albumen at
different incubation temperatures, yielding conflicting results. In one study, a similar survival for SE strains and
Salmonella Typhimurium strains at 37 and 42 1C was shown
(Guan et al., 2006). These findings are in contrast with
earlier studies, demonstrating an enhanced survival in the
albumen at 37 1C for the serotype Enteritidis (Lu et al., 2003;
Clavijo et al., 2006). In the latter studies, strains were
inoculated in the albumen of 1-week-old eggs, while in the
study by Guan et al. (2006), the albumen of freshly laid eggs
was used. It is assumed that fresh albumen (pH = 8.16)
enhances growth compared with stored albumen
(pH = 9.26), and this is most likely caused by the lower pH
of the former (Messens et al., 2004). However, the results
presented in a paper by Humphrey & Whitehead (1993)
suggest that storage has little direct impact on the albumen
with respect to the growth of SE. The lysozyme and
ovotransferrin concentrations in the albumen increased
with the hens age throughout the laying period, which is
reflected in an increased bacteriostatic effect of the albumen
on SE at the mid and the final laying period, possibly
influencing the data (Sellier et al., 2007).
It is striking that studies comparing strains belonging to
different serotypes in their ability to survive in albumen at
chicken body temperature are missing. Recently, the bactericidal effect of albumen at chicken body temperature was
examined for five different Salmonella serotypes (Gantois
et al., 2008c). Remarkably, the strains belonging to the
serotypes Enteritidis, Typhimurium and Heidelberg were
able to survive in the hostile environment of the albumen for
24 h, while the strains belonging to the serotypes Virchow
and Hadar were very susceptible to the albumen, and after
24 h, almost all bacteria were killed. This could explain why
Salmonella Virchow and Salmonella Hadar are almost never
associated with eggs. However, not enough isolates were
compared to draw general conclusions from this study.
Growth of SE in eggs post-lay

Growth patterns of SE in eggs
The risk of human infections following consumption of
Salmonella-contaminated eggs depends on the bacterial
731
numbers present. SE can grow in the contents of naturally

contaminated eggs at room temperature (Humphrey &
Whitehead, 1993). Cogan et al. (2001) observed growth
after 8 days at 20 1C in 7% of whole eggs inoculated in the
albumen near the shell with as few as 2 CFU. It is clear that
this implies a serious threat to human health because
extensive growth in eggs does not lead to changes in the
colour, smell and consistency of the egg contents (Humphrey & Whitehead, 1993). After experimental and natural
infections, some authors point to the albumen as being most
frequently contaminated (Gast & Beard, 1990; Humphrey
et al., 1991b) , while others point to the vitelline membrane
as the most common contamination site (Bichler et al.,
1996; Gast & Holt, 2000a, 2001; Gast et al., 2007). The
albumen is growth restricting for Salmonella because it
contains multiple antimicrobial components, inducing bacterial cell wall and DNA damage (see Growth patterns of SE
in eggs). At temperatures o 10 1C, Salmonella bacteria are
unable to grow in the albumen (Braun & Fehlhaber, 1995;
Schoeni et al., 1995). At room temperature, data are
conflicting and it is difficult to compare the various studies
because the inoculation size, strains, incubation temperatures and period, age of eggs and many other factors vary
(Humphrey & Whitehead, 1993; Braun & Fehlhaber, 1995;
Schoeni et al., 1995; Gast & Holt, 2000b). Recent data
showed that, at 20 1C, upon inoculation with 39 CFU mL 1
albumen, both SE and non-SE strains are able to grow in
separated fresh albumen samples up to 4 106 CFU mL 1
(Clavijo et al., 2006) and, on extending the incubation time,
the number of samples with pronounced growth increased
further . Numerous other studies also observed the growth
of SE in egg albumen at room temperature (Braun &
Fehlhaber, 1995; Schoeni et al., 1995; Duboccage et al.,
2001), indicating that the Salmonella bacteria harbour
intrinsic characteristics to counteract the attacks of the
antimicrobial components present in the egg albumen.
Inoculation of bacteria in the egg albumen of whole eggs
resulted in faster growth than separated egg albumen and
also high numbers of Salmonella bacteria were detected in
the yolk, indicating migration towards the yolk (Cogan
et al., 2001; Messens et al., 2004).
It is believed that Salmonella cells that are deposited in the
albumen are able to migrate to and penetrate through the
vitelline membrane in the egg post-lay, in order to reach the
yolk and thus gain access to a pool of nutrients that are
necessary for its survival and growth. Rapid and extensive
multiplication of SE in the nutrient-rich egg yolks at 25 1C
has been reported (Gast & Holt, 2000b). This was confirmed
in a recent study showing that all strains multiplied rapidly
in yolk contents and reached c. 9.0 log cells mL 1 after 24 h of
incubation at 37 or 42 1C (Guan et al., 2006). Data from
contaminated eggs from either naturally (Humphrey &
Whitehead, 1992) or artificially (Gast & Beard, 1992)
infected hens suggest that there is a delay before yolk

penetration and fast growth occurs in yolk, in eggs stored at
room temperature. This is believed to be because the vitelline membrane in fresh eggs inhibits yolk invasion by
Salmonella. Gradually, the integrity of the vitelline membrane will become lost during storage, resulting in leakage of
nutrients into the albumen. This is considered to allow the
bacteria to migrate to the vitelline membrane and multiply
and invade the yolk (Humphrey & Whitehead, 1993).
Experimentally infected laying hens also often deposit SE
on the vitelline membrane (Bichler et al., 1996; Gast & Holt,
2000a, 2001; Gast et al., 2007). A recent study has suggested
that substantial growth occurred in association with the
vitelline membrane before penetration through the membrane (Gast et al., 2008a). Contamination of the nutrientrich yolk content is uncommon (Gast & Holt, 2001; Gast
et al., 2003), but if it occurs, it leads to rapid bacterial
multiplication (Gast & Holt, 2000b; Guan et al., 2006).
There is considerable controversy on the major deposition
site in eggs. Most likely, the use of different isolation
techniques has a major impact on the outcome of the
experiments. Therefore, the use of imaging such as 3D
localization with bioluminescent or fluorescent Salmonella
bacteria could help to unravel the preferred deposition site
in eggs (Chen et al., 1996).
Studies on the growth of Salmonella in eggs have usually
been performed after direct artificial infection of the eggs
because the production of Salmonella-positive eggs is low
after either natural (Humphrey et al., 1989; Humphrey &
Whitehead, 1992) or experimental (Gast & Beard, 1992)
infection of the hens. The Salmonella growth profiles seen in
naturally contaminated eggs are different from those seen in
eggs contaminated artificially. The latter suggests that
growth is rapid in most eggs and that yolk invasion is
common (Braun & Fehlhaber, 1995; Chen et al., 1996). The
experimental studies, however, used either high contamination levels (104 cells per egg) (Chen et al., 1996), most likely
not representative for naturally contaminated eggs, or
buffered peptone water for the SE solution to be injected,
which enhances bacterial growth in the albumen (Chen
et al., 1996). Data obtained from artificial egg contamination models should thus be interpreted with caution. A
model for artificial egg contamination mimicking the natural situation was developed by Cogan et al. (2001). Using
low numbers of bacterial cells in a low-nutrient, low-iron
suspension as inoculum, a low level of growth was detected
in eggs, comparable to that seen in naturally contaminated
eggs. When few Salmonella bacteria are deposited in the
albumen, very little bacterial multiplication occurs and SE
can persist there at supportive temperatures (Lock & Board,
1992; Hammack et al., 1993; Gast & Holt, 2000b). Humphrey & Whitehead (1993) observed that in artificially
contaminated eggs, the inoculum increased c. 10-fold
732
during the first 24 h postinoculation, as confirmed by Gast &

Holt (2000b). The initial growth phase may involve the
bacterium using its iron reserves, which appear to be
sufficient to support about four generations. When the iron
reserves are exhausted, cells enter a lag phase, where, in the
majority of eggs, there is little or no change in the numbers
of Salmonella organisms. It has been postulated that there
may be leakage of nutrients from the yolk, leading to a
bacterial attraction towards the yolk, some weeks after
storage. Cogan and colleagues (Baron et al., 1997) provide
evidence indicating that the bacteria will then (after weeks of
storage) attach to and penetrate through the vitelline
membrane and gain access to the yolk contents in order to
grow (Baron et al., 1997).
Salmonella virulence factors related to growth

in whole eggs post-lay
It is logical to assume that all Salmonella factors that play a
role in albumen survival or growth in forming eggs will also
play a role in whole egg survival post-lay.
Using in vivo expression technology, it was demonstrated
that expression of SE rfbH, a lipopolysaccharide O-antigen
biosynthesis gene, was strongly induced in eggs at room
temperature (Gantois et al., 2008a). The rfbH gene was
shown to be crucial for growth in eggs at room temperature.
This again demonstrates the importance of lipopolysaccharide in survival in the albumen. This may be important in the
first weeks of egg storage.
During storage, the vitelline membrane gradually deteriorates, resulting in the release of nutrients into the albumen, possibly attracting bacteria that can penetrate the
vitelline membrane and multiply in the nutrient-rich yolk.
It is tempting to speculate that leakage out of the yolk into
the albumen would generate a gradient of amino acids,
sugars or other yolk components, triggering a chemotactic
movement towards the vitelline membrane. This hypothesis
is consistent with the fact that SE grows rapidly in eggs only
after c. 28 days of storage at room temperature (Gantois
et al., 2008a). Flagella are thought to be necessary components for bacterial migration towards the vitelline membrane in whole eggs (Baron et al., 1997). Nonmotile
mutants, such as fliC and motAB mutants, were unable to
move through the albumen towards the yolk; hence, proliferation did not take place (Baron et al., 1997). Moreover,
the nonmotile serotypes Pullorum and Gallinarum were also
not capable of growing in egg contents. Motility is indeed a
significant factor for chemotactic bacteria to move towards
higher concentrations of attractants and to avoid higher
concentrations of repellents by sensing temporal changes in
chemoeffector concentrations (Cogan et al., 2004). Chemotaxis in E. coli is the best-studied signal-transduction network of any living organism. It allows E. coli to sense amino
I. Gantois et al.
acids, sugars, dipeptides and even redox, temperature and

pH changes. In response to these chemical changes, a signal
cascade of methylation/demethylation and phosphorylation/dephosphorylation is switched on. Binding of chemoeffectors to the transmembrane receptors triggers the Che
operon, which transmits the signals to flagellar motors. The
net result of this signal cascade is a change in the direction of
flagellar motor rotation and thus induction of motility
towards or away from a certain trigger. Salmonella harbours
a similar chemotaxis system reacting to chemoeffector
stimuli (Bourret et al., 1989; Sourjik, 2004).
The vitelline membrane comprises a collagenous matrix
overlaid with a layer of glycoproteins (Mariconda et al.,
2006). It is believed that curli fimbriae (Sef17) mediate
bacterial adherence to these glycoproteins such as fibronectin (Bellairs et al., 1963). Yolk invasion and thus multiplication of a curli-deficient strain, an agfA mutant,
occurred significantly less than that in the wild-type SE
strain (Lock & Board, 1992). It is, therefore, suggested that
curli fimbriae are needed to attach to the vitelline membrane, in order to facilitate yolk invasion and multiplication
(Baron et al., 1997). The expression of curli fimbriae has
been investigated under poor (stationary phase) and rich
(exponential phase) nutrient conditions for 15 different
Salmonella strains in a high-pH and iron-restricted medium
at 20 1C (Baron et al., 1997). A correlation has been found
between the expression of curli fimbriae during the late
exponential phase and a high frequency of growth in eggs at
room temperature. This suggests that when bacteria move
closer to the yolk, they will start to grow exponentially, and
thus, strains that show better growth in eggs are those that
are able to express curli fimbriae under growth, rather than
starvation. The fact that genes encoding curli fimbriae
appear to be ubiquitous within the genus Salmonella (Collinson et al., 1991) does not mean that all Salmonella
serotypes are equally effective at multiplying in eggs because
the expression of curli fimbriae can be regulated differently
depending on the environmental triggers and the bacterial
growth phase.
Growth in eggs post-lay by different Salmonella

serotypes
Numerous research reports have compared growth in eggs
between different Salmonella serotypes using various artificial egg contamination models. Messens et al. (2004)
showed that, at 20 1C, after inoculation with 39 CFU mL 1
in the separated albumen, both SE and non-Enteritidis
serotypes were able to grow up to 4 106 CFU mL 1. These
findings were in contrast to those of Lock and Board
(Hammack et al., 1993). In the latter study, the SE strains
showed slow growth in the albumen at 20 and 30 1C, while
the majority of the strains belonging to other serotypes did
733
not multiply, but only survived in the albumen. Data

obtained from growth studies in albumen should be interpreted with caution because most studies report variable
and inconsistent results for independent repeats of the
experiments (Lock & Board, 1992; Hammack et al., 1993;
Messens et al., 2004). Nevertheless, according to most
studies, the capacity to persist and grow in the albumen at
non-hen body temperatures is not a specific trait of serotype
Enteritidis.
Penetration through the vitelline membrane provides an
opportunity for extensive bacterial multiplication inside the
yolk. Penetration of SE through the vitelline membrane has
been reported in several in vitro egg contamination experiments (Humphrey & Whitehead, 1993; Gast & Holt, 2000b).
Recently, a number of studies have compared penetration of
other Salmonella serotypes using the in vitro contamination
model described by Gast and colleagues (Doran et al., 1993;
Gast et al., 2005b; Guan et al., 2006; Murase et al., 2006;
Gantois et al., 2008c). In this model, the yolk and the
albumen are separated and then the yolk is inoculated with
c. 100 CFU of Salmonella onto the exterior surface of the
vitelline membrane, after which the albumen of one single
egg is gently poured onto the yolk. All reports agree that
serotypes, other than Enteritidis, are able to invade the
vitelline membrane and multiply in egg yolk. Furthermore,
the multiplication of different Salmonella serotypes was
assessed by inoculating very small numbers of Salmonella
cells in fresh eggs according to the egg infection model
described by Cogan and colleagues (Cogan et al., 2001;
Gantois et al., 2008c). Except for the serotype Typhimurium,
no significant difference was observed between the serotype
Enteritidis and the strains belonging to the serotypes
Heidelberg, Virchow and Hadar, suggesting that the multiplication strategies inside eggs at room temperature are not
unique for the serotype Enteritidis. The Salmonella Typhimurium strain displayed the lowest frequency of yolk
invasion (Gantois et al., 2008c). However, this finding is in
strong contrast with a finding by Cogan and colleagues
(Baron et al., 1997), showing the highest frequency of yolk
invasion for the serotype Typhimurium. Despite the variability seen within and between experiments and the fact that
even within the serotype Enteritidis different growth patterns have been observed in eggs post-lay (Baron et al.,
1997), the epidemiological association of SE with eggs is
assumed not to be caused by specific multiplication strategies in eggs post-lay because most Salmonella serotypes are
equally effective in multiplying in eggs post-lay.
Conclusion
As opposed to mammals, the chicken embryo does not
develop in the safe environment of the womb, continuously
protected by the dams immune system. Hence, it is not
surprising that the egg has an impressive arsenal of antimicrobial protective mechanisms, including both nonspecific physical barriers and highly efficacious microbiocidal
molecules. Although it is possible to infect eggs with various
bacterial species under the artificial conditions of a laboratory experimental set-up, under natural conditions, this is a
rare event. When it occurs, it usually causes so much damage
that the egg will be easily identified as being infected. SE is
unique in the way that it can pass into the egg and multiply
inside it without inducing noticeable changes. Combining
this exceptional trait with the pathogenicity for the human
intestinal tract allowed this serotype of Salmonella to cause a
pandemic that has lasted for more than a quarter of a
century. Only now are we beginning to understand the
mechanism by which SE contaminates chicken eggs much
more successfully than any other Salmonella serotype.
Evidence is accumulating that contamination of the eggs is
not by penetration through the shell, but by passage from
the hens intestinal tract to the reproductive tract and from
there incorporation into the forming egg on the vitelline
membrane, in the egg white or the shell membranes. It turns
out that many different Salmonella serotypes can pass from
the intestine of the chicken into its blood stream. Even
passage from the blood stream into the hens reproductive
tract is not a unique characteristic of SE. Apparently specific
to SE, however, is its capacity to survive the attacks by
antimicrobial molecules during the formation of the egg in
the hens oviduct. This appears to require a combination of
genes or gene expression patterns encoding for improved
cell wall protection and damage repair, among others. The
exact reason for the epidemiological association of SE with
eggs is, however, still undefined.
Acknowledgements
The authors would like to express their appreciation
to Isabel de Smet, who designed the figures (www.
isabeldesmet.be).
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Mechanisms of egg contamination by Salmonella Enteritidis

Correspondence: Inne Gantois, Department

Eggs as the most important source of

Newport, Hadar, Stanley, Derby, Agona and Kentucky. Eggs

Egg contamination by Salmonella Enteritidis

non-SE serotypes in eggs is negligible. These data suggest

Internal contamination of the eggs

FEMS Microbiol Rev 33 (2009) 718738

2009 Federation of European Microbiological Societies

Internal egg contamination after

Factors influencing eggshell and membrane

Fig. 2. Schematic representation of the egg structure.

In addition to their function as a physical barrier, the

FEMS Microbiol Rev 33 (2009) 718738

Egg contamination by Salmonella Enteritidis

Eggshell penetration by different Salmonella

ing bacteria penetrate the eggshell most frequently. Using an

Contamination of eggs during egg

between the reproductive tissue and Salmonella. The oviduct

colony counts in the reproductive organs point to a role for

Colonization of the ovary

Egg contamination by Salmonella Enteritidis

The relatively high frequency of internal egg

Gast & Holt

Egg contamination rate

FEMS Microbiol Rev 33 (2009) 718738

The contamination rate of forming eggs is

SE and Salmonella Typhimurium may be equal

Total egg content:

Total egg content/shell:

Total egg content/shell

Total egg content: 0.44%

2009 Federation of European Microbiological Societies

Gast & Holt

Intravaginal SE, Salmonella

Intravenous SE, Salmonella

This study suggests that SE is the predominant

Gast & Holt

One-day-old chicks orally infected with

2009 Federation of European Microbiological Societies

There was no significant difference in

Egg contamination rate

Total egg content: 2.6%

Total egg content: 6.5%

Total yolk: 4.3%

Total egg content:

FEMS Microbiol Rev 33 (2009) 718738

Egg contamination by Salmonella Enteritidis

Furthermore, no correlation was found

The frequency of ovarian colonization was

The fact that Salmonella can interact with the cellular

Egg contamination rate

Total egg content (positive eggs/total

ever, the possibility that SE has a specific ability to interact

Colonization of the oviduct

cloaca (Reiber et al., 1995; Miyamoto et al., 1997), a

in accordance with other experimental studies (Bichler et al.,

Egg contamination by Salmonella Enteritidis