Tubular Glands of the Isthmus Are the Predominant Colonization Site
of Salmonella Enteritidis in the Upper Oviduct of Laying Hens
J. De Buck,1 F. Pasmans, F. Van Immerseel, F. Haesebrouck, and R. Ducatelle
Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine,
Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium
tion of the invasiveness of S. enteritidis in the oviduct of
laying hens. Loops in the isthmus and magnum of laying
hens were made under anaesthesia. S. enteritidis was
inoculated into each loop. After 1 h, tubular gland cells
were isolated from the infected tissue under gentamicin.
S. enteritidis invasiveness was measured as the ratio of
isolated bacteria per isolated tubular gland cell. This ratio
was higher (P < 0.01) in the isthmus (1.3 × 10−3) than in
the magnum (5.3 × 10−5). In a third experiment, laying
hens were intravenously infected with 5 × 107 cfu S. enteritidis bacteria. The number of intracellular bacteria was
counted in the tubular gland cells of the isthmus and
magnum after isolation under gentamicin. The ratio of
isolated bacteria per isolated tubular gland cell was again
significantly higher in the isthmus as compared with in
the magnum. In all 3 assays, the tubular gland cells of
the isthmus were more heavily invaded than those of
the magnum.
ABSTRACT Salmonella enterica serovar Enteritidis is the
serovar most frequently isolated from chicken eggs. Colonization of the upper oviduct of hens is believed to play
an important role in egg contamination. The interaction
of S. enteritidis with gland epithelial cells of the isthmus
and the magnum was, therefore, studied in vitro and in
vivo. In the first experiment, S. enteritidis bacteria were
added to confluent monolayers of primary cultures of
chicken tubular epithelial cells of the isthmus (ICTEC) or
magnum (MCTEC). Intracellular bacteria in ICTEC and
MCTEC were confirmed by a gentamicin protection
assay. Internalization in the glandular cells was corroborated by confocal scanning microscopy. Although S. enteritidis was able to invade and proliferate intracellularly
during 24 h in the cell culture of both segments, this was
significantly more so in the ICTEC. In a second experiment, an in vivo loop model was developed for investiga-
(Key words: cell culture, isthmus, magnum, proliferation, Salmonella enteritidis)
2004 Poultry Science 83:352–358
chicken eggs through the upper oviduct (Gast and Beard,
1990b; Shivaprasad et al., 1990; Hoop and Pospischil,
1993; Humphrey, 1994; Keller et al., 1995).
S. enteritidis can be isolated from the reproductive tissues of artificially and naturally infected hens (Humphrey
et al., 1989; Hoop and Pospischil, 1993; Withanage et al.,
1999; Kinde et al., 2000; Okamura et al., 2001b). To date,
little attention has been paid to the localization of Salmonella bacteria in infected oviducts. The infection of the
oviducts in several infection models has been described
in terms of colony-forming units per gram of oviductal
tissue (Hassan and Curtiss, 1997; Miyamoto et al., 1997;
Okamura et al., 2001a,b) or merely as percentage of positive samples (Timoney et al., 1989; Gast and Beard, 1990a;
Barrow and Lovell, 1991; Baskerville et al., 1992; Barnhart
et al., 1993; Hoop and Pospischill, 1993; Keller et al., 1995,
1997; Reiber et al., 1995; Withanage et al., 1998; Withanage
et al., 1999; Kinde et al., 2000). However, there are some
INTRODUCTION
Table eggs are the most important source of Salmonella
enterica serovar Enteritidis (S. enteritidis) infection in humans (Hogue et al., 1997; Hald et al., 1998; Humphrey,
1999; Meyer, 1999; Palmer et al., 2000; Van Loock et al.,
2000). Understanding the mechanism that leads to S. enteritidis colonization of eggs is essential to reduce the
public health risk associated with consumption of infected eggs. However, the pathogenesis of egg contamination is still not completely understood. The current concept of vertical transmission of S. enteritidis in chickens
considers contamination of the shell surface as the egg
passes through the vagina and cloaca, contamination of
the yolk in the ovary, or contamination during passage
through a colonized oviduct. Several studies have suggested that S. enteritidis most frequently migrates into
2004 Poultry Science Association, Inc.
Received for publication July 16, 2003.
Accepted for publication October 20, 2003.
1
Correspondence should be addressed: [email protected].
Abbreviation Key: BGA = brilliant green agar; HBSS = Hanks’ buffered salt solution; ICTEC = chicken tubular epithelial cells of the isthmus; MCTEC = chicken tubular epithelial cells of the magnum; MEM =
minimum essential medium; TBS = Tris-buffered saline.
352
SALMONELLA INVASION IN OVIDUCTAL GLAND CELLS
reports in the literature that mention an association of
serovar Enteritidis with tubular gland cells of the oviduct,
both after natural infection (Hoop and Pospischil, 1993)
and after experimental infection (Keller et al., 1995).
In a previous study we showed that serovar Enteritidis
isolates can adhere to isthmus gland secretions (De Buck
et al., 2003), which led us to the hypothesis that serovar
Enteritidis may selectively colonize the isthmus glands
of the oviduct. The bacteria could then be transported to
the lumen of the oviduct with the isthmus secretions and
thus become incorporated into the eggshell membranes,
which could explain the bacteriological finding of shell
association of the serotype.
In this study, we aimed to find the predominant site
in the upper oviduct where the invasion and proliferation
of S. enteritidis takes place to elucidate the colonization
of the oviduct, which possibly leads to egg contamination.
MATERIALS AND METHODS
Bacteria
Experiments were carried out with the Salmonella enteritidis phage type 4 (PT4) strain NIDO 76Sa88 (Desmidt
et al., 1998). The strain was cultured overnight at 37°C
on Isosensitest agar2 plates. Ten colonies were transferred
into brain heart infusion broth3 and cultured for 20 h at
37°C while shaking. The suspensions were checked for
purity and the number of colony-forming units was determined by plating 10-fold dilutions on Isosensitest agar.
The suspensions were stored overnight at 4°C and were
then used in the experiments. The minimal inhibitory
concentration for gentamicin was lower than 0.5 µg/mL.
Isthmus and Magnum Tubular
Gland Cell Isolation and Culture
Isthmus and magnum tubular gland cells were obtained following the method of Jung-Testas et al. (1986)
with minor modifications. Seven-day-old chicks were
given daily subcutaneous injections of 1 mg of estradiol
benzoate in 0.5 mL of sesame oil for 10 d. After 4 wk the
chicks received a second stimulation by reinjection for 7
consecutive d with the same daily amount of estradiol
benzoate. The oviducts of the estrogen-treated chicks
were aseptically removed. The magnum and isthmus
were separated, slit open longitudinally, and washed 3
times in Hanks’ buffered salt solution (HBSS). Both segments of the oviduct were minced into very small pieces.
The tissue was then dissociated for 1 h at 37°C in minimum essential medium (MEM4) containing collagenase
1 mg/mL (180 U/mg solid) and penicillin-streptomycin.
2
Oxoid, Basingstoke, UK.
Biolife, Milan, Italy.
4
Life Technologies, Paisley, Scotland, UK.
5
BD Falcon, San Jose, CA.
6
Sigma, St. Louis, MO.
3
353
Tissue pieces were allowed to sediment, and then the
supernatant was removed, and 10 mL of HBSS without
Ca2+ and Mg2+, containing trypsin (0.25%) and EDTA (3
mM), was added. The tissue was allowed to dissociate
for 10 min. The supernatant was removed and pooled
with the supernatant of a second trypsin/EDTA dissociation. The resulting cell suspension was filtered, using a
cell strainer5 (∅ 70 µm) and centrifuged at 300 × g. The
cell pellets were rinsed twice with MEM containing 10%
fetal calf serum (FCS). The cells were planted at 1 × 106
cells/mL in Petri dishes in MEM supplemented with 15%
FCS, insulin (0.12 IU/mL), penicillin-streptomycin (50
µg/mL), and estradiol (50 nM). The dishes were placed
in the cell incubator at 37°C and 5% CO2. After 1.5 to 2 h,
fibroblasts were attached to the Petri dishes. The epithelial
cells in the supernatant were seeded into tissue culture
96-well plates.
Invasion Assay
At d 2 postisolation, the cells were rinsed 3 times with
HBSS and fresh medium without antibiotics was added
to the wells. At d 3 postisolation the wells were checked
for confluence and the culture medium was replaced with
medium containing 5 × 106 cfu/mL Salmonella bacteria.
The plates were then centrifuged at 1,500 × rpm for 10
min at 37°C to sediment the bacteria onto the confluent
cell layer. The plates were incubated for 1 h at 37°C, and
the wells were then rinsed 3 times with MEM medium.
MEM medium with 50 µg gentamicin was added, and
the plates were incubated for 1, 4, or 24 h. The wells were
rinsed 5 times with HBSS, and the cells were lysed by
adding 100 µL of 1% Triton X-100 in aqua dest to the
wells. After 10 min, 100 µL of HBSS was added to the
wells. Ten-fold dilutions were made of the resulting suspension and plated onto brilliant green agar (BGA2)
plates. Intracellular bacteria were measured by calculating the colony-forming units per milliliter of culture medium after an overnight incubation at 37°C. Percentage
of invasion was calculated as the number of intracellular
bacteria compared with the number of bacteria brought
in contact with the cultured cells. The reported values
are the means of 3 independent experiments with 3 replicates per experiment.
Immunofluorescent Staining
of Intracellular Bacteria
An invasion assay was performed on isthmus glandular cells, grown in 50-mm Petri dishes as described above.
The cells, however, were not lysed but fixed in ice-cold
methanol for 10 min. A rabbit anti-S. enteritidis polyclonal antiserum was diluted 1/1,000 in PBS with 2.2%
skim-milk powder, added to the fixed cells, and incubated
for 1 h at 37°C. The cells were washed 4 times with PBS.
A tetramethyl rhodamine isothiocyanate (TRITC)-labeled
goat anti-rabbit IgG6 was diluted 1/100 in PBS with 2.2%
skim-milk powder and incubated in the dark for 1 h at
37°C. The cells were again washed 4 times. For fluorescent
354
DE BUCK ET AL.
staining of the secretion granules, fluorescein isothiocyanate (FITC)-labeled Lens culinaris lectins6 at 25 µg/mL in
PBS were added, and the cells were incubated in the dark
for 1 h at 37°C (De Buck et al., 2003). The cells were
washed 4 times and dried. The cells were examined under
a confocal laser scanning microscope.7
Anesthesia and Loop Construction
Commercial Salmonella-free chickens (checked by serology and bacteriology) of 20 wk old were premedicated
intramuscularly with 0.05 mg/kg buprenorphine hydrochloride (Temgesic8) and 0.05 mg/kg atropine. Anesthesia was induced by administration of isoflurane8 through
a mask. Following intubation with a 3.0 uncuffed tracheal
tube,9 a continuous oxygen flow of 1.5 to 2.0 L/min was
administered carrying 1.5 to 3% isoflurane, depending on
the depth of the anesthesia desired. The hens were kept
under anesthesia until they were euthanized at the end
of the experiment. The birds were covered with a sterile
surgical blanket and defeathered at the abdominal surface. After disinfection of the incision area with a polyvidone iodine solution,10 the abdomen was opened through
a midline incision avoiding perforation of the ventral air
sacs, and the oviduct segments of interest were carefully
exposed. Loops of the oviductal segments were ligated
using surgical suture (Vicryl 3.011) in the magnum and
isthmus. A control loop was constructed between the
magnal and isthmal loops. The loops were 1.5 to 2.0 cm
long. Sufficient blood supply was ensured to all separate loops.
Oviduct Loop Assay
An overnight culture of S. enteritidis PT4 76Sa88 was
washed 2 times in HBSS, and a 5 × 107 cfu/mL bacterial
suspension was prepared in HBSS. The loops were inoculated with 1 mL of the bacterial suspension or with pure
HBSS (for the control loop) using a 27-ga needle. After
dosing, 2 mL of HBSS containing 400 µg/mL of gentamicin was sprayed over the loops, and the loops were reintroduced into the abdomen, and the abdominal wall was
sutured. After 1 h, 1 mL of HBSS containing gentamicin
(400 µg/mL) was injected into each loop to obtain a final
concentration in the loop of approximately 200 µg/mL.
After 5 min the hen was euthanized by intravenous injection with a solution of mebenzoniumiodide (50 g/mL),
embutramide (200 mg/mL), and tetracaine hydrochloride
(5 mg/mL) (T6112). This loop assay was performed on
3 hens.
Cell Isolation and Bacteriological Counts
from Oviduct Loops
The loops were immediately aseptically removed from
the abdominal cavity and slit open longitudinally. The
tissue pieces were rinsed 3 times in HBSS containing 100
µg/mL gentamicin. Tissue samples from the loops were
frozen in Tissue-Tek13 for immunohistochemistry or fixed
in 10% buffered formalin for hematoxylin-eosin staining.
The control loops were rinsed 5 additional times in fresh
HBSS to remove the gentamicin. The control loop was
bacteriologically examined for the presence of Salmonella
by a preenrichment-enrichment procedure in buffered
peptone water (BPW) and tetrathionate brilliant green
broth.2 Tubular gland cells were isolated from the infected
loops by a modified protocol of Jung-Testas et al. (1986),
as described above, but with an additional 50 µg/mL
of gentamicin in all dissociating solutions and without
penicillin and streptomycin. The cells were counted and
then lysed in 5 mL 1% triton X-100 aqua dest solution.
After 5 min of lysis, 5 mL HBSS was added. The number
of Salmonella bacteria in the resulting lysate was counted
by plating 10-fold dilutions in HBSS onto BGA. The bacterial load is given as the ratio of isolated bacteria per
isolated gland cell, because it cannot be excluded that a
cell is occupied by more than one bacterium.
Immunohistochemistry
Thin sections (6 µm) of the frozen loop samples were
mounted onto glass slides coated with 3-aminopropyltriethoxysilane. They were rinsed with Tris-buffered saline
(TBS) (pH 7.5). The sections were incubated at 37°C for
30 min in a 1/4,000 (TBS pH 7.2) dilution of rabbit antiS. enteritidis antiserum and again rinsed with TBS. Peroxidase-labeled goat anti-rabbit IgG, diluted 1/400 (TBS pH
7.2) was added. After an incubation of 30 min at 37°C,
the sections were rinsed with PBS, stained with 3,3′-diaminobenzidine, and counterstained with hematoxylin.
Isolation of Intracellular Salmonella
Bacteria After Intravenous Infection
Three commercial Salmonella-free laying hens (checked
by serology and bacteriology), 20 wk of age, were intravenously infected with 5 × 107 cfu of S. enteritidis 76Sa88
bacteria in 0.5 mL of PBS. Tubular gland cells from the
magnum and the isthmus of all 3 hens were isolated at
4 d postinfection in the same way as was done for the
ligated oviductal loops. Intracellular Salmonella bacteria
were counted by plating 10-fold dilutions of the lysed
cells in HBSS onto BGA. The infection ratio was calculated
as described above.
7
Leica TCS SP2, Wetzlar, Germany.
Schering-Plough, Kenilworth, NJ.
Hudson RCI, Temecula, CA.
10
B. Braunol Medical, Prague, Czech Republic.
11
Johnson-Johnson, New Brunswick, NJ.
12
Intervet, Bedrijvenlaan, Belgium.
13
Sakura finetek Europe, Zoeterwoude, The Netherlands.
8
9
Statistics
The infection ratios in the loop model and after intravenous infection between isthmus and magnum were analyzed with the paired Student t-test after log10 transforma-
SALMONELLA INVASION IN OVIDUCTAL GLAND CELLS
FIGURE 1. Invasion and proliferation of Salmonella enterica serovar
Enteritidis in primary cell cultures of tubular gland cells of the isthmus
(white bar) and magnum (grey bar), expressed as percentage of invaded
bacteria relative to the inoculum after 1, 4, or 24 h of incubation. All
bars represent the mean value of 3 independent experiments in triplicate.
Stars indicate significant differences (P < 0.05) in the percentage of
invasion between the isthmus and magnum for each separate time point.
The error bars indicate the standard error of the mean.
tion (Neter et al., 1996).14 Differences in intracellular bacteria in the cell cultures between the isthmus and the
magnum were analyzed with the Student t-test.
RESULTS
Invasion, Proliferation, and Replication
of S. Enteritidis in Cultured Isthmus
and Magnum Glandular Cells
S. enteritidis bacteria were isolated from isthmus and
magnum glandular cells (Figure 1). The amount of intracellular bacteria in the isthmus and magnum glandular
cells was significantly increased after 4 and 24 h compared
with the first time point. Differences (P < 0.05) in the
percentage of intracellular bacteria occurred between
magnum and isthmus after 1, 4, and 24 h of incubation.
355
FIGURE 2. Confocal scanning microscopic image of invaded Salmonella enterica serovar Enteritidis 76Sa88 bacteria in the tubular gland
cells of the isthmus and magnum. The bacteria were immunochemically
labeled with tetramethyl rhodamine isothiocyanate (TRITC) (red); granules of the isthmal tubular glands were labeled by fluorescein isothiocyanate (FITC)-labeled Lens culinaris lectines (green). Z-Y and Z-X sections
through a 3-dimensional stack of confocal images show the bacteria
between the secretion granules.
hematoxylin-eosin of formol-fixed thin section of all
loops. The control loops were negative for Salmonella. The
S. enteritidis bacteria were able to invade the oviductal
tissue of the loops of all 3 hens (Table 1). The mean count
(log10 cfu) of intracellular bacteria in the isthmal (4.48 ±
0.20) and the magnal (2.97 ± 0.65) loops differed (P <
0.01). The ratio of isolated Salmonella bacteria per isolated
tubular gland cell of the isthmus (1.3 ± 0.37 × 10−3) was
Immunofluorescent Staining
of S. Enteritidis in Isthmal
Tubular Gland Cells
The presence of the bacteria inside the cells was confirmed by confocal laser scanning microscopy. After 1 h
of incubation with the cells, S. enteritidis 76Sa88 was
detected inside the chicken tubular ICTEC by immunofluorescent labelling (Figure 2). The Z-X and Z-Y sections
through a stack of confocal laser scanning microscope
images showed that the S. enteritidis bacteria were localized intracellularly between the secretion granules, which
were stained by FITC-labeled Lens culinaris lectins.
Invasion of S. Enteritidis in Loops
in the Isthmus and the Magnum
The tissue of the loops remained intact during incubation with S. enteritidis as demonstrated by staining with
14
SPSS 9.0, SPSS Inc., Chicago, IL.
FIGURE 3. Immunohistochemical staining of Salmonella enterica serovar Enteritidis in an infected isthmal loop. The Salmonella enteritidis
bacteria are indicated by arrows. Counterstaining was done with hematoxylin. Bar = 10 µm.
356
DE BUCK ET AL.
TABLE 1. Isolation of intracellular Salmonella enterica serovar Enteritidis bacteria from the tubulary
gland cells of inoculated loops of the isthmus and magnum of anesthetized laying hens
Segment
Experiment
Isthmus
1
2
3
Mean
Magnum
Cells1
(n)
Infection
ratio2
×
×
×
×
×
×
×
×
1.7 × 10−3
1.3 × 10−3
9.9 × 10−4
1.3 ± 0.4 × 10−3*
2.4
3.1
1.8
2.4
2.7
3.1
4.9
3.6
1
2
3
Mean
107
107
107
107
107
107
107
107
8.2 × 10−5
7.3 × 10−5
3.4 × 10−6
5.3 ± 4.3 × 10−5*
1
Total number of isolated gland cells.
Ratio of the number of isolated intracellular Salmonella bacteria per isolated tubular gland cell.
*Statistically significant difference (P < 0.05) between the isthmal and magnal loop.
2
significantly greater from that of the magnum (5.28 ± 4.30
× 10−5). In Table 1 the total number of isolated cells and
the infection ratio are given. The S. enteritidis bacteria
were demonstrated inside the tubular glands by immunohistochemistry (Figure 3). Few bacteria were observed in
association with the surface epithelium.
Isolation of S. Enteritidis from the Isthmal
and Magnal Tubular Gland Cells
After an Intravenous Infection
The load of intracellular bacteria in the isthmus and in
the magnum, measured as the ratio of isolated S. bacteria
per isolated tubular gland cell, called the infection ratio,
is shown in Table 2. The mean infection ratio of isthmus
(2.1 × 10−4) was significantly higher than that of the magnum (4.8 × 10−5).
DISCUSSION
In the present study, invasion and replication of S.
enteritidis was demonstrated in the primary cell cultures
of tubular gland cells of the isthmus and magnum of
laying hens. Furthermore, an in vivo loop model was
developed for studying the invasion of oviductal tissue
in laying hens. This in vivo model confirmed the observations in the in vitro cell cultures. Also in the intravenous
inoculation model the invasion of oviductal tissues was
confirmed. Depending on the model, invasiveness was 3
to 25 times higher in the isthmus glandular epithelial cells
as compared within magnum gland epithelial cells.
In the loop model, most of the S. enteritidis bacteria
were associated with tubular gland cells. Few bacteria
were observed attached to the surface epithelium, which
predominantly consists of ciliated cells (Bakst and Howarth, 1975). Previously, we have been unable to demonstrate adhesion to the surface epithelium of explants of the
magnum and isthmus by scanning electron microscopy
(unpublished results).
When the tubular gland cells were isolated from the
intravenously infected laying hens, in an identical manner
as for the preparation of primary cultures, S. enteritidis
bacteria were detected intracellularly. Therefore, the primary cultures of tubular gland cells of the oviduct are a
useful model to further investigate the intracellular colonization of oviduct glandular epithelial cells.
In the present study, we showed that the isthmus has
higher numbers of intracellular S. enteritidis after an intravenous infection and after inoculation into ligated oviductal loops and that the tubular epithelial cells of the
isthmus are more readily invaded in vitro. These observations are in accordance with observations of others after
experimental infections (Keller et al., 1995; Miyamoto et
al., 1997; Okamura et al., 2001b), where the isthmus is
TABLE 2. Isolation of intracellular Salmonella enterica serovar Enteritidis bacteria from
the magnum and isthmus of intravenously inoculated laying hens
Segment
Experiment
Isthmus
1
2
3
Mean
Magnum
Mean
1
1
2
3
Cells1
(n)
7.2
5.3
9.9
7.4
1.2
6.0
2.1
6.7
×
×
×
×
×
×
×
×
106
106
106
106
108
107
107
107
Number of isolated tubular gland cells.
Ratio of the number of isolated intracellular Salmonella bacteria per isolated tubular gland cell.
*Statistically significant difference (P < 0.05) between the isthmus and magnum.
2
Infection
ratio2
1.4
4.2
1.8
2.1
4.6
7.3
6.7
4.8
×
×
×
×
×
×
×
×
10−5
10−4
10−4
10−4*
10−6
10−5
10−5
10−5*
SALMONELLA INVASION IN OVIDUCTAL GLAND CELLS
more frequently or heavily contaminated. Analysis of
eggs laid by infected hens has shown that the shell, containing the eggshell membranes and produced by the
isthmus, is often the most heavily infected site of surface
decontaminated eggs (Bichler et al., 1996; Miyamoto et
al., 1997; Okamura et al., 2001b). Thus, from the results
of bacterial culturing of oviduct segments and eggs after
experimental infections and from the results of this study,
S. enteritidis is suggested to have adapted best to the
isthmus segment of the chicken oviduct.
In the present study we showed that S. enteritidis could
be located intracellularly in a colonized oviduct. After 24
h of incubation in the primary cell cultures, replication
of the bacteria inside the cells was observed. Further studies are needed to show if indeed a persistent infection
can be established in the tubular glands of the upper
oviduct. The clustered and intermittent production of infected eggs (Humphrey et al., 1989) could be explained
by the capacity of Salmonella bacteria to proliferate intracellularly in the oviduct during long periods, wait for an
undefined stimulus to come out of the cells, and colonize
the forming egg, as previously suggested by Keller et al.
(1995). Following such stimulus, the bacteria may egress
from the glandular cells, together with the glandular secretions. The high affinity of S. enteritidis isolates for the
isthmal secretions, which contributes to the production
of the eggshell membranes (De Buck et al., 2003), could
enable the bacteria to become incorporated into the forming egg.
In conclusion, evidence from 3 independent assays indicated that S. enteritidis preferably invaded the tubular
glandular cells of the isthmal segment in comparison with
the magnum.
REFERENCES
Bakst, M., and B. Howarth. 1975. SEM preparation and observation of the hen’s oviduct. Anat. Rec. 181:211–225.
Barnhart H., D. W. Dreesen, and J. L. Burke. 1993. Isolation of
Salmonella from ovaries and oviducts from whole carcasses
of spent hens. Avian Dis. 37:977–980.
Barrow P. A., and M. A. Lovell. 1991. Experimental infection
of egg-laying hens with Salmonella enteritidis phage type 4.
Avian Pathol. 20:335–348.
Baskerville A., T. J. Humphrey, R. B. Fitzgeorge, R. W. Cook,
H. Chart, B. Rowe, and A. Whitehead. 1992. Airborne infection of laying hens with Salmonella enteritidis phage type
4. Vet. Rec. 130:395–398.
Bichler, L. A., K. V. Nagaraja, and D. A. Halvorson. 1996. Salmonella enteritidis in eggs, cloacal swab specimens, and internal
organs of experimentally infected White Leghorn chickens.
Am. J. Vet. Res. 57:489–495.
De Buck, J., F. Van Immerseel, G. Meulemans, F. Haesebrouck,
and R. Ducatelle. 2003. Adhesion of Salmonella enterica serovar Enteritidis isolates to chicken isthmal glandular secretions. Vet. Microbiol. 93:223–233.
Desmidt, M., R. Ducatelle, J. Mast, B. M. Goddeeris, B. Kaspers,
and F. Haesebrouck. 1998. Role of the humoral immune system in Salmonella enteritidis phage type four infection in
chickens. Vet. Immunol. Immunopathol. 63:355–367.
Gast, R. K., and C. W. Beard. 1990a. Isolation of Salmonella
enteritidis from internal organs of experimentally infected
hens. Avian Dis. 34:991–993.
357
Gast R. K., and C. W. Beard. 1990b. Production of Salmonella
enteritidis contaminated eggs by experimentally infected
hens. Avian Dis. 34:438–446.
Hald, T., H. C. Wegener, and B. B. Jorgensen. Annual report on
zoonoses in Denmark 1998. Pages 18–20 in Danish Zoonoses
Centre and Danish Veterinary and Food Administration,
Ministry of Food, Agriculture and Fisheries, Copenhagen.
Hassan J. O., and R. Curtiss, III. 1997. Efficacy of a live avirulent
Salmonella Typhimurium vaccine in preventing colonization
and invasion of laying hens by Salmonella Typhimurium and
Salmonella enteritidis. Avian Dis. 41:783–791.
Hogue, A., P. White, J. Guard-Petter, W. Schlosser, R. Gast, E.
Ebel, J. Farrar, T. Gomez, J. Madden, M. Madison, A. M.
McNamara, R. Morales, D. Parham, P. Sparling, W. Sutherlin,
and D. Swerdlow. 1997. Epidemiology and control of eggassociated Salmonella enteritidis in the United States of
America. Rev. Sci. Tech. Off. Int. Epizoot. 16:542–553.
Hoop, R. K., and A. Pospischil. 1993. Bacteriological, serological,
histological and immunohistochemical findings in laying
hens with naturally acquired Salmonella enteritidis phage
type 4 infection. Vet. Rec. 133:391–393.
Humphrey, T. J., A. Baskerville, S. Mawer, B. Rowe, and S.
Hopper. 1989. Salmonella enteritidis phage type 4 from the
contents of intact eggs: A study involving naturally infected
hens. Epidemiol. Infect. 103:415–423.
Humphrey, T. J. 1994. Contamination of eggshell and contents
with Salmonella enteritidis: A review. Int. J. Food Microbiol.
21:31–40.
Humphrey, T. J. 1999. Contamination of eggs and poultry meat
with Salmonella enterica serovar Enteritidis. Pages 183–192
in Salmonella enterica serovar Enteritidis in humans and
animals. A. M. Saeed, T. K. Gast, M. Potter, P. G. Wall, ed.
Iowa State University Press, Ames, IA.
Jung-Testas, I., T. Garcia, and E. E. Baulieu. 1986. Steroid hormones induce cell proliferation and specific protein synthesis
in primary chick oviduct cultures. J. Steroid Biochem.
24:273–279.
Keller, L. H., C. E. Benson, K. Krotec, and R. J. Eckroade. 1995.
Salmonella enteritidis colonization of the reproductive tract
and forming and freshly laid eggs. Infect. Immun.
63:2443–2449.
Keller, L. H., D. M. Schifferli, C. E. Benson, S. Aslam, and R. J.
Eckroade. 1997. Invasion of chicken reproductive tissues and
forming eggs is not unique to Salmonella enteritidis. Avian
Dis. 41:535–539.
Kinde, H., H. L. Shivaprasad, B. M. Daft, D. H. Read, A. Ardans,
R. Breitmeyer, G. Rajashera, K. V. Nagaraja, and I. A. Gardner. 2000. Pathologic and bacteriologic findings in 27-weekold commercial laying hens experimentally infected with
Salmonella enteritidis, phage type 4. Avian Dis. 44:239–248.
Meyer, H. 1999. Animals as sources of infections in humans—
Salmonelloses. Dtsch. Tierarztl. Wochenschr. 106:344–351.
Miyamoto, T., E. Baba, T. Tanaka, K. Sasai, T. Fukata, and A.
Arakawa. 1997. Salmonella enteritidis contamination of eggs
from hens inoculated by vaginal, cloacal and intravenous
routes. Avian Dis. 41:296–303.
Neter, J., M. H. Kutner, C. J. Nachtsheim, and W. Wasserman.
1996. Applied Linear Statistical Models. Irwin Press, New
York.
Okamura, M., Y. Kamijima, T. Miyamoto, H. Tani, K. Sasai, and
E. Baba. 2001a. Differences among six Salmonella serovars in
abilities to colonize reproductive organs and to contaminate
eggs in laying hens. Avian Dis. 45:61–69.
Okamura, M., T. Miyamoto, Y. Kamijima, H. Tani, K. Sasai, and
E. Baba. 2001b. Differences in abilities to colonize reproductive organs and to contaminate eggs in intravaginally inoculated hens and in vitro adherences to vaginal explants between Salmonella enteritidis and other Salmonella serovars.
Avian Dis. 45:962–971.
Palmer, S., S. Parry, D. Perry, R. Smith, M. Evans, L. Nehaul,
R. Roberts, M. Walapu, and D. Wright. 2000. The role of
358
DE BUCK ET AL.
outbreaks in developing food safety policy: Population based
surveillance of Salmonella outbreaks in Wales 1986–98. Epidemiol. Infect. 125:467–472.
Reiber, M. A., D. E. Conner, and S. F. Bilgili. 1995. Salmonella
colonization and shedding patterns of hens inoculated via
semen. Avian Dis. 39:317–322.
Shivaprasad, H. L., J. F. Timoney, S. Morales, B. Lucio, and R.
C. Baker. 1990. Pathogenesis of Salmonella enteritidis infection
in laying chickens. I. Studies on egg transmission, clinical
signs, fecal shedding, and serologic responses. Avian Dis.
34:548–557.
Timoney, J. F., H. L. Shivaprasad, R. C. Baker, and B. Rowe.
1989. Egg transmission after infection of hens with Salmonella enteritidis phage type 4. Vet. Rec. 125:600–601.
Van Loock, F., G. Ducoffre, J. M. Dumont, M. L. Libotte-Chasseur, H. Imberechts, M. Gouffaux, J. Houins-Roulet, G. Lamsens, K. De Schrijver, N. Bin, A. Moreaus, L. De Zutter, and
G. Daube. 2000. Analysis of foodborne disease in Belgium
in 1997. Acta Clin. Belg. 55:300–306.
Withanage, G. S., K. Sasai, T. Fukata, T. Miyamoto, and E. Baba.
1999. Secretion of monoclonal antibodies against a fimbrial
structure of Salmonella enteritidis and certain other serogroup
D salmonellae and their application as serotyping reagents.
Vet. Immunol. Immunopathol. 67:185–193.
Withanage, G. S. K., K. Sasai, T. Fukata, T. Miyamoto, E. Baba,
and H. S. Lillehoh. 1998. T lymphocytes, B lymphocytes,
and macrophages in the ovaries and oviducts of laying hens
experimentally infected with Salmonella enteritidis. Vet. Immunol. Immunopathol. 66:173–184.