Download In Vitro Studies of Chicken Egg Yolk Antibody (IgY

IMMUNOLOGY AND MOLECULAR BIOLOGY
In Vitro Studies of Chicken Egg Yolk Antibody (IgY) Against
Salmonella enteritidis and Salmonella typhimurium
E. N. Lee,* H. H. Sunwoo,* K. Menninen,† and J. S. Sim*1
*Department of Agricultural, Food and Nutritional Science, University of Alberta;
and †AAFRD, Animal Health Lab Branch, Edmonton, Alberta, Canada, T6G 2P5
medium. The growth rate of S. enteritidis incubated with
S. enteritidis-specific IgY was fourfold less than that of the
control group during a 4-to-6-h incubation. Cell counts of
S. typhimurium incubated with S. typhimurium-specific IgY
were reduced by 1.6 log cfu/mL in comparison to that
of the control group after 6 h of incubation. The specific
binding activity of IgY was further evaluated by using
immunofluorescence and immunoelectron microscopy. It
was found that Salmonella-specific IgY could bind to the
antigens expressed on the Salmonella surface, resulting in
structural alterations of the bacterial surface.
ABSTRACT Chicken egg yolk antibody (IgY) raised
against Salmonella enteritidis or Salmonella typhimurium
was found in highly specific activity levels by ELISA. S.
enteritidis- and S. typhimurium-specific IgY powder, prepared by freeze-drying the egg yolk water-soluble fraction, contained 15.5 and 10.0% of specific IgY, respectively. Anti-S. enteritidis IgY cross-reacted 55.3% with S.
typhimurium. The cross-reactivity of anti-S. typhimurium
IgY with S. enteritidis was 42.4%. Salmonella-specific IgY
was demonstrated to inhibit Salmonella growth in liquid
(Key words: immunoglobulin Y, Salmonella enteritidis, Salmonella typhimurium, antibacterial activity,
immunofluorescence and immunoelectron microscopy)
2002 Poultry Science 81:632–641
binding to bacteria may be essential to promote their
antibacterial properties. In this regard, it is necessary that
IgY binding activity and its effect on bacteria be investigated in more detail to provide basic information on the
antibacterial properties of IgY.
Two Salmonella serovars, Salmonella enteritidis and Salmonella typhimurium, were selected as bacterial antigens
to raise IgY because they have been the main cause of
salmonellosis outbreaks in humans and infections in
chickens (O’Brien, 1988; CDC, 1990; Roberts et al., 1996;
Khakhria et al., 1997). In vivo studies have shown the
preventive effect of IgY on Salmonella infection (Peralta
et al., 1994; Yokoyama et al., 1998a,b). However, the immunological properties of IgY on the binding activity
against Salmonella have not been reported previously.
Therefore, the objective of this study was to investigate
the antibacterial properties of IgY against S. enteritidis
and S. typhimurium for its specificity and activity by using
various in vitro methods, such as ELISA, growth inhibition assay, and microscopy.
INTRODUCTION
Chicken egg yolk antibody, referred to as Immunoglobulin Y (Leslie and Clem, 1969), differs from mammalian
IgG in terms of structural and immunological properties
(Higgins, 1975; Kobayashi and Hirai, 1980). It also has
advantageous characteristics over mammalian IgG, including stability under various physico-chemical conditions and its suitability as an immunological tool (BarJoseph and Malkinson, 1980; Vieira et al., 1984; Losch et
al., 1986; Shimizu et al., 1988, 1992, 1994; Yolken et al.,
1988; Otani et al., 1991). IgY can be produced in large
quantities from the yolk of chicken eggs by simple methods, and without distress to the animals (Kwan et al.,
1991; Akita and Nakai, 1993). Therefore, IgY has received
much attention due to its potential as an alternative to
conventional mammalian antibodies.
Antibacterial properties of IgY have been one of the
main interests in IgY studies. Many reports have shown
that IgY demonstrated an immune function in preventing
bacterial shedding or infection in vivo (Ikemori et al.,
1992; Peralta et al., 1994; Imberechts et al., 1997; Yokoyama et al., 1998 a,b; Marquardt et al., 1999). Antibody
MATERIALS AND METHODS
Bacteria and Culture Conditions
Bacteria were cultured to use for the immunization
of chickens. Salmonella enteritidis (ESO 9325-92) and S.
2002 Poultry Science Association, Inc.
Received for publication December 1, 2000.
Accepted for publication November 30, 2001.
1
To whom correspondence should be addressed: jsim@afns.
ualberta.ca.
Abbreviation Key: TSB = tryptic soy broth; WSF = water-soluble
fraction.
632
BINDING ACTIVITY OF IGY AGAINST SALMONELLA
typhimurium (ATCC 14028) were obtained from the Animal Health Laboratories Branch, Alberta Agriculture,
Food and Rural Development, Edmonton, Alberta, Canada. Bacteria were cultured in tryptic soy broth (TSB)2
and harvested by centrifugation. The formalin-inactivated cells were lyophilized and stored at −20 C until
used.
Immunization of Chickens
Chickens were immunized to obtain Salmonella-specific
IgY loaded eggs. All chickens were cared for in accordance with the Canadian Council on Animal Care guidelines of animal welfare. Immunization of hens was carried
out as described (Sunwoo et al., 1996). Lyophilized S.
enteritidis (500 µg of cell/mL; 3 µg of protein/mL) and
S. typhimurium (500 µg of cell/mL; 4.5 µg of protein/mL)
whole cells were emulsified with an equal volume of
Freund’s incomplete adjuvant3 to immunize eight 23-wkold Single Comb White Leghorn chickens intramuscularly. Booster immunizations were given at 2 wk and 4
wk after the initial immunization in the same manner.
Eggs were collected daily and stored at 4 C until used.
Isolation of Water-Soluble Fraction
Containing IgY from Egg Yolk
The water-soluble fraction (WSF) was isolated from egg
yolk to utilize for the analysis of IgY properties. The WSF
was prepared using the water dilution method developed
by Akita and Nakai (1992). The egg yolk separated from
egg white was first mixed gently with eight volumes of
cold distilled water (acidified with 0.1 N HCl to a pH of
4.0) and then with cold, acidified, distilled water (pH 2.0)
to make a final dilution of 1:10. After mixing well, the
mixture was adjusted to pH 5.0∼5.2 and incubated at 4
C for 12 h. The WSF was obtained by centrifugation at
3,125 × g at 4 C for 20 min and then stored at −20 C
until analyzed. The WSF containing specific IgY with high
activity levels, determined by the following ELISA or
non-specific IgY, was neutralized with 0.1 N NaOH and
lyophilized. The IgY powder was analyzed for concentrations of protein, total IgY, and specific IgY.
ELISA
The prepared WSF and IgY powder were assayed by
an ELISA procedure as described by Sunwoo et al. (1996,
2000) with modifications.
Specific Activity of IgY. The specific binding activity
of IgY against whole bacterial cells was tested as follows.
A microtiter plate4 was coated with 150 µL of lyophilized
S. enteritidis (1.67 mg of cells/mL; 10 µg of protein/mL)
2
Difco Laboratories, Detroit, MI 48232.
Sigma Chemical Co., St. Louis, MO 63103.
4
COSTAR威, Corning Incorporated, Corning, NY 14831.
5
Vmax威, Molecular Devices Co., Sunnyvale, CA 94089-1136.
3
633
or S. typhimurium (1.11 mg of cells/mL; 10 µg of protein/
mL) whole cells in carbonate-bicarbonate buffer (0.05 M,
pH 9.6). The WSF (diluted 1:1,000 in PBS, 150 µL per well)
containing specific egg yolk antibodies (IgY) or nonspecific IgY as a control was reacted with coated antigens.
The same volume (150 µL) of rabbit anti-chicken IgG
conjugated with horseradish peroxidase3 (diluted 1:1,000
in PBS) and freshly prepared substrate solution, 2-2′azino-bis (3-ethylbenzthiazoline-6-sulfonic acid)3 in 0.05
M phosphate citrate buffer (pH 5.0) containing 30% hydrogen peroxide were used for secondary antibody and
substrate, respectively. Absorbance of the mixture was
read at 405 nm by a kinetic microplate reader.5
Cross-Reactivity of IgY. The cross-reactivity of IgY
was determined by using the above ELISA and the following bacterial cells: S. enteritidis, S. typhimurium, Escherichia
coli O157:H7 and E. coli 987P. Wells of the microtiter plate
were coated with 150 µL of lyophilized whole cells in
carbonate-bicarbonate buffer at the following concentrations: S. enteritidis, 1.67 mg/mL; S. typhimurium, 1.67 mg/
mL; E. coli O157:H7, 0.31 mg/mL; and E. coli 987P, 0.5
mg/mL. IgY powder specific for S. enteritidis (40 to 10
µg/mL) or S. typhimurium (64 to 16 µg/mL) was serially
diluted with PBS and added (150 µL per well) to react
with coated antigens. The cross-reactivity of anti-S. enteritidis or anti-S. typhimurium IgY against selected bacteria
was determined by comparing activities against those
bacteria with activity against S. enteritidis or S. typhimurium, respectively.
Total IgY Concentration. To measure the total IgY
concentration of the WSF or IgY powder, ELISA was
performed as described above, except the microtiter plate
was coated with 150 µL of rabbit anti-chicken IgG3 at a
final concentration of 3.75 µg/mL. Samples of the WSF
were diluted 1:90,000 with PBS. Specific or nonspecific
IgY powder was reconstituted and serially diluted with
PBS (2 to 0.125 µg/mL). Twofold serial dilutions of purified chicken IgG3 (1 mg/mL) in PBS (0.5 to 0.031 µg/
mL) were used as the reference antibodies to prepare a
standard curve. The standard curves were then compared
to provide a relative measurement of total IgY concentration.
Specific IgY Concentration. The concentrations of S.
enteritidis- or S. typhimurium-specific IgY were measured
by ELISA as described by Sunwoo et al. (2000). Wells of
a microtiter plate were coated with 150 µL of rabbit antichicken IgG3 (3.75 µg/mL) and lyophilized S. enteritidis
(1.67 mg/mL) or S. typhimurium (1.67 mg/mL) whole
cells in carbonate-bicarbonate buffer (0.05 M, pH 9.6).
Twofold serial dilutions of reconstituted specific (12.5 to
1.56 µg/mL) and nonspecific (4.5 to 0.28 mg/mL) IgY
powder in PBS were added to wells (150 µL per well)
coated with S. enteritidis or S. typhimurium whole cells.
Wells coated with rabbit anti-chicken IgG were filled with
two-fold serial dilutions of purified chicken IgG3 (1 mg/
mL) in PBS (0.5 to 0.008 µg/mL). Secondary antibody,
substrate and the measurement of absorbance, were identical as described in a previous section, Specific Activity
of IgY. The optical density at 405 nm was converted to
634
LEE ET AL.
micrograms of specific IgY per milligram of IgY powder
by using a quantitative standard curve determined by
the titration between rabbit anti-chicken IgG and purified
chicken IgG.
Protein Assay
The Bio-Rad protein assay6 (Microtiter Plate Protocol),
based on the method of Bradford (1976), was performed
using purified chicken IgG3 (1 mg of protein/mL) as the
reference protein.
Growth Inhibition Assay
This assay was conducted to investigate whether the
binding activity of anti-Salmonella IgY could inhibit Salmonella growth in a liquid medium. The same strain of S.
enteritidis or S. typhimurium used as an antigen for immunizing chickens was subcultured on blood agar plates
(5% defibrinated sheep blood in Columbia Agar)7 and
suspended in TSB. The suspension was adjusted to an
optical density of 0.05 at 600 nm, corresponding to a cell
density of approximately 2.7 × 107 cfu/mL. The same
volume of 20% (vol/vol) glycerol in TSB was added to
the suspension, which was then stored at −70 C until
used. Two milliliters of prepared bacterial culture were
mixed with 2 mL of TSB and incubated at 37 C with
shaking. The turbidity of the culture (optical density at
600 nm) was measured by a spectrophotometer8 at 1-h
intervals. The growth curve was plotted until the stationary phase was reached.
Specific or nonspecific IgY powder was reconstituted
to 90, 180, and 360 mg/mL with TSB. IgY solution was
then centrifuged at 1,500 × g at 4 C for 20 min. The supernatant was taken and sterilized by using a 0.22-µm membrane filter.9 Two milliliters of specific or nonspecific IgY
solution were then added to the same volume of prepared
S. enteritidis or S. typhimurium culture. The bacteria and
IgY mixtures were incubated at 37 C with shaking. Aliquots of samples (100 µL) were taken at 0, 2, 4, and 6 h
of incubation. Plate counts were performed by the spread
plate method on TSB agar plates2 in duplicate. The inoculated plates were incubated at 37 C overnight. The number
of colony-forming units per plate was counted to determine the total number of bacteria colony-forming units
per mL of sample.
Microscopic Analyses
The specific binding activity of Salmonella-specific IgY
against Salmonella was further evaluated by microscopic
observation. Immunofluorescence and immunoelectron
microscopy were carried out to visualize Salmonella
bound by IgY.
Immunofluorescence Microscopy. One hundred microliters of S. enteritidis (3.8 × 102 cfu/mL) or S. typhimurium (1.5 × 104 cfu/mL) cells suspended in PBS were
incubated with the same volume of specific IgY or nonspecific IgY (100 µg of IgY powder/mL PBS) or without
IgY at 37 C for 1 h. After washing with PBS two times,
fluorescein isothiocyanate-conjugated rabbit anti-chicken
IgG3 diluted 1:250 in PBS was added and then incubated
at 37 C for 1 h. The samples were washed as before and
resuspended in 50 µL of PBS. Cell suspension (10 µL)
was smeared on the microscope slide, which was then
air-dried and a coverslip was mounted by using a drop
of mounting buffer (Glycerol-PBS, pH 7.2). Immunofluorescent staining of specimens was detected using a 2001
confocal laser scanning microscope.10
Immunoelectron Microscopy. One milliliter of S. enteritidis (3.8 × 102 cfu/mL) or S. typhimurium (1.5 × 104
cfu/mL) cells suspended in PBS was centrifuged at 12,800
× g for 10 min. One milliliter of specific IgY or nonspecific
IgY (100 µg of IgY powder/mL of 1% BSA in PBS) was
added to the cell pellets. After incubation at 37 C for 1
h, samples were washed with 1% BSA in PBS, two times,
before adding 100 µL of rabbit anti-chicken IgG3 (diluted
1:14 in 1% BSA in PBS), followed by incubation at 37 C
for 1 h. After being washed, samples were incubated with
300 µl of goat anti-rabbit IgG gold conjugate3 (diluted
1:25 with 1% BSA in PBS). The suspended cells were used
for negative staining and ultrathin sectioning.
For negative staining, bacterial cells were washed with
distilled water two times and subsequently mounted on
a 300 mesh copper grid. Grid-mounted samples were
stained with 2% uranyl acetate. After being washed and
dried, specimens were observed with a transmission electron microscope.11
For the ultrathin section method, bacteria treated with
antibodies were washed with 1% BSA in PBS two times,
fixed with 2.5% glutaraldehyde for 1 h, and postfixed
with 1% osmium tetroxide for 1 h. The fixed samples were
dehydrated in a graded series of ethanol and embedded
in Spurr’s medium (Spurr, 1969). After infiltration with
Spurr’s medium, polymerization was accomplished at 70
C for 12 h. The specimens were then thin sectioned with
an ultramicrotome.12 Ultrathin sections were mounted on
a 200-mesh copper grid and stained with 2% uranyl acetate and then with lead citrate. The specimens were examined with a transmission electron microscope.11
Statistical Analyses
6
Bio-Rad Laboratories, Hercules, CA 94547.
Oxoid, Basingstoke, Hampshire, RG248PW UK.
8
Spectronic 20, Bausch and Lomb, Rochester, NY 14604-2701.
9
Millipore Co., Bedford, MA 01730.
10
Molecular Dynamics, Sunnyvale, CA 94085-4520.
11
Hitachi H-7,000 TEM, Tokyo, 105-8717Japan.
12
Ultracut E model, Reichert-Jung, Austria.
7
The results were analyzed by analysis of variance.
When testing the significance of the differences between
the experimental and control group, Student’s t-test was
used. Data are presented as means ± standard deviation.
A probability level of P < 0.05 was considered statistically significant.
635
BINDING ACTIVITY OF IGY AGAINST SALMONELLA
FIGURE 2. The change of specific activity of IgY in the egg yolk from
chickens immunized with Salmonella enteritidis whole cells or Salmonella
typhimurium whole cells during the immunization period. The level of
specific activity in a 1,000-fold dilution of the water-soluble fraction
was measured by ELISA using bacterial whole cells as an antigen and
expressed as an ELISA value [optical density (OD) at 405 nm]. Values
are the mean of quadruple samples. Vertical bars indicate the standard
deviation. Arrows indicate the week of immunization.
In this study, the WSF containing anti-S. enteritidis IgY
or anti-S. typhimurium IgY was obtained with a purity
(total IgY in protein) of 21.5 or 21.4%, respectively. The
recovery of IgY might exist in the range of 92 to 96%,
based on results of Akita and Nakai (1993), who used the
same conditions (10-fold dilution to a final pH of 5.0 to
5.2) to obtain the WSF. Consequently, IgY present in the
WSF could be produced in large quantities by using the
water dilution method characterized as simple and economical.
FIGURE 1. The concentrations of protein and total IgY in the watersoluble fraction obtained from chickens immunized with (a) Salmonella
enteritidis; (b) Salmonella typhimurium whole cells during the immunization period. Values are the mean of quadruple samples. Vertical bars
indicate the standard deviation.
RESULTS AND DISCUSSION
Concentrations of Protein
and Total IgY in the WSF
The total IgY concentration (Figure 1) was relatively
constant (P > 0.05) during the immunization period, and
the total average IgY concentration was 7.08 ± 1.24 and
7.09 ± 1.40 mg/mL for anti-S. enteritidis IgY and anti-S.
typhimurium IgY, respectively. There was no difference
between those two values (P > 0.05), which indicates that
the total IgY concentration in the egg yolk is independent
of the type of antigens used to raise antibodies as shown
by Sunwoo et al. (1996).
Specific Activities of IgY Against
Salmonella Antigens
As shown in Figure 2, the level of specific activities of
IgY increased 1 wk after the initial immunization and
then rose constantly. The lag time of 1 wk can be explained
by the time it takes for specific antibodies produced in
chicken serum to be transferred and accumulated in egg
yolk as reported by Li et al. (1998). The activity of anti-S.
enteritidis IgY against S. enteritidis reached a peak (optical
TABLE 1. Concentrations of protein, total IgY and specific IgY
in IgY powder prepared from the water-soluble fraction
containing Salmonella-specific or nonspecific IgY.
Values are the mean of quadruple samples ± SD
Concentration (mg/g)
IgY powder
Anti-S. enteritidis
Specific IgY
Anti-S. typhimurium
Specific IgY
Nonspecific IgY
Protein
Total
IgY
590 ± 38
129 ± 10
605 ± 41
468 ± 62
140 ± 35
93 ± 21
Specific
IgY
20 ± 7
14 ± 6
0.14 ± 0.037
636
LEE ET AL.
FIGURE 3. The cross-reactivity of (a) anti-S. enteritidis IgY; (b) antiS. typhimurium IgY with other members of enterobacteriaceae, including
Escherichia coli O157:H7 and E. coli 987P. Values are the mean of triplicate
samples. Vertical bars indicate the standard deviation. Asterisks denote
values that differ (P < 0.05) from E. coli O157:H7 and E. coli 987P.
density of 1.10) at 8 wk and declined thereafter. AntiS. typhimurium IgY activity against S. typhimurium also
showed the same pattern as that of anti-S. enteritidis IgY.
The level of activity increased to a maximum optical density of 0.62 at 7 wk and then decreased.
The overall activity of anti-S. typhimurium IgY was
lower (P < 0.05) than that of anti-S. enteritidis IgY. This
difference may be attributed to the prior immune status
of chickens or inactivated S. typhimurium as a weak immunogen. Chickens immunized in this study were not tested
for previous exposure to S. typhimurium or to bacteria
carrying cross-reacting antigens with S. typhimurium. Another reason for comparatively low activity of anti-S. typhimurium IgY could possibly be suggested from findings
that inactivated S. typhimurium is less immunogenic than
live S. typhimurium and thus inactivated S. typhimurium
immunogens do not stimulate a sufficient immune response to eliminate those bacteria in chickens (Germanier,
1972; Barrow et al., 1990; Hassan et al., 1991). However,
the value of anti-S. typhimurium IgY activity may not
be low, considering S. typhimurium whole cells as crude
antigens in comparison to the degree of activity of IgY
against purified S. typhimurium lipopolysaccharide (LPS)
shown in Sunwoo et al. (1996).
FIGURE 4. The effect of IgY on the growth of Salmonella in a liquid
medium. Bacteria (approximately 1 × 107 cfu/mL) were grown in tryptic
soy broth mixed with Salmonella-specific or nonspecific IgY powder at
37 C with shaking: (a) growth curves of S. enteritidis or S. typhimurium;
(b) growth of S. enteritidis incubated with 0.9 mg of specific IgY powder/
mL; (c) growth of S. typhimurium incubated with 0.63 mg of specific
IgY powder/mL. Viable cells were counted by the plate count method.
Values are the mean of triplicate samples. Vertical bars indicate the
standard deviation. Asterisks denote values that differ (P < 0.05) from
nonspecific control IgY.
It is thus likely that both Salmonella-antigens used in the
form of inactivated bacterial whole cells are immunogenic
enough to induce an immune response and produce Salmonella-specific antibodies in chickens. The immunogenicity of an antigen is influenced by several factors, including the species or strain being immunized, antigen
properties and dosage, or the route of administration and
BINDING ACTIVITY OF IGY AGAINST SALMONELLA
637
adjuvant (Kuby, 1997). In this study, the production of
Salmonella-specific antibody could be efficiently elicited
in Single Comb White Leghorn chickens immunized intramuscularly with S. enteritidis or S. typhimurium whole
cells emulsified with Freund’s incomplete adjuvant. As
a consequence, IgY in the WSF was assessed to possess
specific binding activity against Salmonella whole cells
and could be used for further study.
Properties of IgY Powder
The specific IgY concentration of total IgY was 16%
and 10% for S. enteritidis-specific IgY powder and S. typhimurium-specific IgY powder, respectively (Table 1). The
specific IgY concentration in nonspecific IgY powder,
which was prepared as a control from nonimmunized
chicken egg yolk, was significantly lower (P < 0.05) than
that of specific-IgY powder as expected. The purity (total
IgY in protein) of S. enteritidis-specific IgY powder and
S. typhimurium-specific IgY powder was 21.9 and
23.1%, respectively.
Cross-Reactivity of IgY
The anti-S. enteritidis IgY cross-reacted 55.3% with S.
typhimurium in contrast to significantly low cross-reactivity (P < 0.05) of anti-S. enteritidis IgY with E. coli O157:H7
or E. coli 987P as shown in Figure 3a. Anti-S. typhimurium
IgY also showed low cross-reactivity with both E. coli
strains (P < 0.05), although it did cross-react 42.4% with
S. enteritidis (Figure 3b).
The high cross-reactivity of anti-Salmonella IgY between
S. enteritidis and S. typhimurium compared to E. coli strains
can be explained by the fact that both Salmonella spp. share
somatic antigens (O:1 and O:12) and common epitopes
on the different flagellin (Le Minor, 1984; van Zijderveld
et al., 1992). Anti-Salmonella IgY used in this study was
a polyclonal antibody against bacterial whole cells, and,
thus, it may be possible to raise antibodies against crossreacting antigens. The cross-reactivity of IgY can add
more value to antibacterial properties of IgY in that IgY
might have an antibacterial effect on bacteria with crossreacting antigen as well as target bacteria.
Growth Inhibitory Effect
of Anti-Salmonella IgY
The growth curves of S. enteritidis and S. typhimurium
showed similar patterns that consisted of lag (0 to 2 h of
incubation time), exponential (2 to 6 h of incubation time),
and stationary phase (Figure 4a). Accordingly, S. enteritidis or S. typhimurium was incubated with IgY for 6 h
during which samples were taken at 2-h intervals to perform the growth inhibition assay. In this assay, different
concentrations of specific or nonspecific IgY were used
while considering the importance of antibody quantities
to effectively interact with antigens.
The growth of S. enteritidis incubated with specific IgY
showed a significant reduction in bacterial growth after
FIGURE 5. Immunofluorescence micrographs of S. enteritidis incubated with (a) specific IgY; (b) nonspecific IgY; and (c) without IgY
(magnification 400 ×).
638
LEE ET AL.
FIGURE 6. Immunofluorescence micrographs of S. typhimurium incubated with (a) specific IgY; (b) nonspecific IgY; and (c) without IgY
(magnification 400 ×).
4 h incubation (P < 0.05). However, control IgY had no
effect on bacterial growth, which maintained a lag phase
and exponential phase from 0 to 2 h and 2 to 6 h of
incubation, respectively (Figure 4b). Cell counts of the
specific and control groups increased by 0.3 log cfu/mL
and 1.2 log cfu/mL, respectively, during 4 to 6 h of incubation, indicating that bacteria in the specific treatment
group proliferated four times less than the control group.
The difference in bacterial growth between the two
groups implied that specific IgY has an inhibitory effect
on the growth of S. enteritidis. The effective concentration
of S. enteritidis-specific IgY in the inhibition of bacterial
growth was 0.9 mg/mL, which was determined by specific IgY concentration of IgY powder (20 mg/g) without
considering loss of specific IgY during the preparation of
IgY solution.
The growth patterns of S. typhimurium incubated with
S. typhimurium-specific or nonspecific IgY (control) were
similar to those observed in the experiment of S. enteritidis
with IgY as shown in Figure 4c. There was no difference
in bacterial growth between the two groups during 4 h
of incubation (P > 0.05). However, the degree of bacterial
growth when incubated with specific IgY decreased 16fold compared to that of the control group. Consequently,
cell counts of the specific group were reduced by 1.6 log
cfu/mL in comparison to those of the control group at 6
h of incubation time. The concentration of specific IgY
that showed a growth-inhibitory effect was 0.63 mg/mL.
As a result, both Salmonella-specific IgY were found to
inhibit the growth of homologous Salmonella in a liquid
medium. The mechanism by which antibodies can suppress bacterial growth is not clearly understood. The agglutination of bacterial cells cross-linked by antibodies,
which lead to bacterial cells with less motility and opportunity to take nutrients and proliferate than a free-motile
single bacterial cell, may not be one of the causes of
bacterial growth inhibition. There have been reports that
bacterial growth is inhibited by antibodies in solid medium where bacteria cannot aggregate, demonstrating
that the reduction of bacterial colony counts mediated by
antibodies is not attributed to the agglutination reaction
(Feldmann et al., 1992; Sadziene et al., 1992). In addition,
Kubo et al. (1973) reported that the agglutinating property
of IgY can be displayed only at raised salt concentrations
or low pH conditions due to the steric hindrance caused
by so closely aligned Fab arms of IgY.
Therefore, there may be other reactions between IgY
and bacteria to cause the growth inhibition of bacteria
than the agglutination reaction. The specific binding of
IgY to bacteria appears to be involved in bacterial growth
inhibition. Particular components expressed on the bacterial surface, which are crucial factors for the bacterial
growth, may be recognized and bound by related polyclonal antibody, IgY. This binding may block or impair
the function of growth-related bacterial components and
lead to bacterial growth inhibition. Outer membrane protein, lipopolysaccharide, fimbriae (or pili), and flagella
may be included in these bacterial surface components
(Sim et al., 2000). Therefore, the specific binding activity
BINDING ACTIVITY OF IGY AGAINST SALMONELLA
639
FIGURE 7. Immunoelectron micrographs of S. enteritidis incubated with specific IgY (specific group) or nonspecific IgY (control group): negatively
stained specimen of (a) specific group (magnification 6,000 ×), (b) control group (6,300 ×); ultrathin sectioned specimen of (c) specific group (9,000
×), (d) control group (5,100 ×).
of IgY, as a potential candidate for the major antibacterial
property, requires more intensive studies to uncover the
mechanism of inhibition.
Microscopic Observation
of IgY Binding to Salmonella
Figure 5 shows immunofluorescence micrographs, in
which fluorescence was observed in S. enteritidis incubated with S. enteritidis-specific IgY; however, there was
no fluorescence in the control groups. The presence of
fluorescence implies that IgY binds specifically to bacteria, demonstrating the specific binding activity of IgY
against the bacteria. A positive result was also obtained
from the experiment of S. typhimurium with IgY. S. typhimurium-specific IgY was shown to possess specific binding activity against S. typhimurium as indicated by fluorescence in Figure 6a. The immunofluorescence microscopy performed could be a preliminary test to confirm
the specific binding activity of IgY and to further evaluate
its activity through immunoelectron microscopy.
Figure 7 shows immunoelectron micrographs of S. enteritidis incubated with S. enteritidis-specific IgY or nonspecific IgY. Immunogold particles were observed around
bacteria incubated with specific IgY (Figures 7a and 7c),
in contrast to the observation of bacteria incubated with
nonspecific IgY (Figures 7b and 7d). Bacteria labeled with
immunogold indicated that S. enteritidis were bound by
specific IgY, which substantiates the specific binding
property of IgY against S. enteritidis. The experiment of
S. typhimurium with IgY also resulted in the presence of
immunogold particles around bacteria incubated with S.
typhimurium-specific IgY as shown in Figures 8a and 8c.
S. typhimurium-specific IgY was verified to have specific
binding activity against S. typhimurium as well.
Furthermore, immunoelectron microscopic observation revealed the distribution of immunogold particles
around the bacterial surface. It was also found that the
640
LEE ET AL.
FIGURE 8. Immunoelectron micrographs of S. typhimurium incubated with specific IgY (specific group) or non-specific IgY (control group):
Negatively stained specimen of (a) specific group (magnification 9,000 ×), (b) control group (9,000 ×); Ultrathin sectioned specimen of (c) specific
group (6,000 ×), (d) control group (6,000 ×).
bacterial surface was structurally altered as presented in
Figures 7c and 8c. Bacterial surface bound by IgY was
rough in contrast to the smooth surface of control bacteria.
These findings can be indicative that specific IgY is
attached to components exposed on the bacterial surface,
resulting in the structural alterations of the bacterial surface. One of the possible causes leading to the inhibition of
bacterial growth is the reaction between bacterial surface
components and IgY raised against those components as
explained previously. This assumption may be corroborated on the basis of this morphological change of bacteria
with bound IgY. Further studies remain to be carried out
to define which bacterial surface components are bound
by specific IgY and how those binding activities of IgY
result in the growth inhibition of bacteria.
In conclusion, the binding activity of chicken egg yolk
antibody (IgY) against S. enteritidis or S. typhimurium resulted in inhibiting bacterial growth in vitro. Microscopic
observation revealed the structural alterations of Salmonella surface bound by IgY. In vitro IgY studies may suggest that IgY binds to Salmonella surface molecules, which
are critical for bacterial growth, and leads to the functional
impairment of those components.
ACKNOWLEDGMENTS
Support for this research was provided in part by the
Canada-Alberta Hog Industry Development Fund, the
Poultry Industry Council of Canada and NLRI international cooperative research fund from Korea. The authors
thank all staff in the Poultry Research Center, University
of Alberta, for the technical services and assistance in the
care of animal used in this research.
REFERENCES
Akita, E. M., and S. Nakai. 1992. Immunoglobulins from egg
yolk: Isolation and purification. J. Food Sci. 57:629–634.
BINDING ACTIVITY OF IGY AGAINST SALMONELLA
Akita, E. M., and S. Nakai. 1993. Comparison of four purification
methods for the production of immunoglobulins from eggs
laid by hens immunized with an enterotoxigenic E. coli strain.
J. Immunol. Methods 160:207–214.
Bar-Joseph, M., and M. Malkinson. 1980. Hen egg yolk as a
source of antiviral antibodies in the enzyme linked immunosorbent assay (ELISA): A comparison of two plant viruses.
J. Virol. Methods 1:179–183.
Barrow, P. A., T. O. Hassan, and A. Berchieri. 1990. Reduction
in fecal excretion of Salmonella typhimurium strain F98 in
chickens vaccinated with live and killed S. typhimurium organisms. Epidemiol. Infect. 194:413–426.
Bradford, M. M. 1976. A rapid and sensitive method for the
quantification of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal. Biochem.
72:248–254.
Centers for Disease Control 1990. Salmonella surveillance. Annual Summary 1988. CDC, Atlanta, GA.
Feldmann, R. C., B. Henrich, V. Kolb-Bachofen, and U. Hadding.
1992. Decreased metabolism and viability of Mycoplasma hominis induced by monoclonal antibody-mediated agglutination. Infect. Immun. 60:166-174.
Germanier, R. 1972. Immunity in experimental salmonellosis. III.
Comparative immunization with viable and heat-inactivated
cells of Salmonella typhimurium. Infect. Immun. 5:792–797.
Hassan, J. O., A. P. A Mockett, D. Catty, and P. A. Barrow.
1991. Infection and reinfection of chickens with Salmonella
typhimurium: Bacteriology and immune responses. Avian
Dis. 35:809–819.
Higgins, D. A. 1975. Physical and chemical properties of fowl
immunoglobulins. Vet. Bull. 45:139–154.
Ikemori,Y., M. Kuroki, R. C. Peralta, H. Yokoyama, and Y. Kodama. 1992. Protection of neonatal calves against fatal enteric
colibacillosis by administration of egg yolk powder from
hens immunized with K99-piliated enterotoxigenic Escherichia coli. Am. J. Vet. Res. 53: 2005–2008.
Imberechts, H., P. Deprez, E. Van Driessche, and P. Pohl. 1997.
Chicken egg yolk antibodies against F18ab fimbriae of Escherichia coli inhibit shedding of F18 positive E. coli by experimentally infected pigs. Vet. Microbiol. 54:329–341.
Khakhria, R., D. W. Woodward, M. Johnson, and C. Poppe.
1997. Salmonella isolated from humans, animals and other
sources in Canada. 1983–92. Epidemiol. Infect. 119:15–23.
Kobayashi, K., and H. Hirai. 1980. Studies on subunit components of chicken polymeric immunoglobulins. J. Immunol.
124:1695–1704.
Kubo, R. T, B. Zimmerman, and H. M. Grey. 1973. Phylogeny
of immunoglobulins. Pages 417–477 in The Antigens. Vol.1
M. Sela, ed. Academic Press, San Diego, CA.
Kuby, J. 1997. Immunology. 3rd ed. J. Kuby, ed. W. H. Freeman
and Company, New York.
Kwan, L., E. Li-Chan, N. Helbig, and S. Nakai. 1991. Fractionation of water-soluble and -insoluble components from egg
yolk with minimum use of organic solvents. J. Food Sci.
56:1537–1541.
Le Minor, L. 1984. Bergery’s Manual of Systematic Bacteriology.
1st ed. Williams and Wilkins, Baltimore, MD.
Lesile, G.A., and L. W. Clem. 1969. Phylogeny of immunoglobulin structure and function III. Immunoglobulins of the
chicken. J. Exp. Med. 130:1337–1352.
Li, X., T. Nakano, H. H. Sunwoo, B. H. Paek, H. S. Chae, and
J. S. Sim. 1998. Effects of egg and yolk weights on yolk
antibody (IgY) production in laying chickens. Poult. Sci.
77:266–270.
Losch, Y., I. Schranner, R. Wanke, and L. Jurgens. 1986. The
chicken egg, an antibody source. J. Vet. Med. B33:609–619.
Marquardt R. R., L. Z. Jin, J. W. Kim, L. Fang, A. A. Frohlich,
and S. K. Baidoo. 1999. Passive protective effect of egg-yolk
antibodies against enterotoxigenic Escherichia coli K88+ infec-
641
tion in neonatal and early-weaned piglets. FEMS Immunol.
Med. Microbiol. 23:283–288.
O’Brien, J. D. P. 1988. Salmonella enteritidis infection in broiler
chickens. Vet. Rec. 122:214.
Otani, H., K. Matsumoto, A. Saeki, and A. Hosono. 1991. Comparative studies on properties of hen egg yolk IgY and rabbit
serum IgG antibodies. Lebensm. Wiss. Techonol. 24:152–158.
Peralta, R.C., H. Yokoyama, Y. Ikemori, M. Kuroki, and Y. Kodama. 1994. Passive immunization against experimental salmonellosis in mice by orally administered hen egg yolk antibodies specific for 14-kDa fimbriae of Salmonella enteritidis.
J. Med. Microbiol. 41:29–35.
Roberts, T. A., A. C. Baird-Parker, and R. B. Tompkin. 1996.
Salmonellae. Pages 217–264 in Microorganisms in Foods 5.
Characteristics of microbial pathogens. Blackie Academic
and Professional, London.
Sadziene, A., P. A. Rosa, P. A. Thompson, D. M. Hogan, and
A. G. Barbour. 1992. Antibody-resistant mutants of Borrelia
burgdorferi: In vitro selection and characterization. J. Exp.
Med. 176:799–809.
SAS Institute. 1991. SAS威 User’s Guide: Statistics. Version 6.04
Edition. SAS Institute, Cary, NC.
Shimizu, M., H. Nagashima, K. Hashimoto, and T. Suzuki. 1994.
Egg yolk antibody (IgY) stability in aqueous solution with
high sugar concentrations. J. Food Sci. 59:763–765.
Shimizu, M., H. Nagashima, K. Sano, K. Hashimoto, M. Ozeki,
K. Tsuda, and H. Hatta. 1992. Molecular stability of chicken
and rabbit immunoglobulin G. Biosci. Biotech. Biochem.
56:270–274.
Shimizu, M., R. C. Fitzsimmons, and S. Nakai. 1988. Anti-E. coli
immunoglobulin Y isolated from egg yolk of immunized
chickens as a potential food ingredient. J. Food Sci.
53:1360–1366.
Sim, J. S., H. H. Sunwoo, and E. N. Lee. 2000. Ovoglobulin IgY.
Pages 227–252 in Natural Food Antimicrobial Systems. A. S.
Naidu, ed. CRC Press, Boca Raton, FL.
Spurr, A. R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastructure Res. 26:31–43.
Sunwoo, H. H., X. Li, E. N. Lee, Y. K. Kim, and J. S. Sim.
2000. Preparation of antigen-specific IgY for food application.
Pages 311–322 in Egg Nutrition and Biotechnology. J. S. Sim,
S. Nakai., and W. Guenter, ed. CAB International, New York.
Sunwoo, H.H., T. Nakano, W. T. Dixon, and J. S. Sim. 1996.
Immune responses in chickens against lipopolysaccharide of
Escherichia coli and Salmonella typhimurium. Poult. Sci.
75:342–345.
van Zijderveld, F. G., A. M. van Zijderveld, and J. Anakotta.
1992. Comparison of four different enzyme-linked immunosorbent assays for the serological diagnosis of Salmonella enteritidis infections in chickens. J. Clin. Microbiol. 30:2560–
2566.
Vieira, J. G. H., M. A. D. Oliveira, E. M. K. Russo, R. M. B.
Maciel, and A. B. Pereira. 1984. Egg yolk as a source of
antibodies for human parathyroid hormone (hPTH) radioimmunoassay. J. Immunoassay 5:121–129.
Yokoyama, H., K. Umeda., R. C. Peralta., T. Hashi., F. C. Icatlo.,
M. Kuroki., Y. Ikemori., and Y. Kodama. 1998a. Oral passive
immunization against experimental salmonellosis in mice
using chicken egg yolk antibodies specific for Salmonella enteritidis and S. typhimurium. Vaccine16:388–393.
Yokoyama, H., R. C. Peralta, K. Umeda, T. Hashi, F. C. Icatlo,
M. Kuroki, Y. Ikemori, and Y. Kodama. 1998b. Prevention
of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk Salmonella-specific antibodies. Am.
J. Vet. Res. 59:416–420.
Yolken, R.H., F. Leister, S. E. Wee, R. Miskuff, and S. Vonderfecht. 1988. Antibodies to rotaviruses in chickens’eggs: A
potential source of antiviral immunoglobulins suitable for
human consumption. Pediatrics 81:291–295.