Effect of Food Protein Supplements on Salmonella enteritidis Infection and Prevention in Laying Hens

Effect of Food Protein Supplements on Salmonella enteritidis Infection
and Prevention in Laying Hens
Z. G. Kassaify and Y. Mine1
Department of Food Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
ABSTRACT
Reduction of intestinal colonization of Sal-
and a gradual decrease with the other samples. In the
monella enteritidis (SE) during the grow-out period is cru-
prevention study, Salmonella-free chickens were fed the
cial to provide safer eggs, minimize economic losses, and
supplemented feed for 4 wk and then infected orally.
reduce the spread of human salmonellosis. In the search
Fecal samples tested for 4 wk showed that SE was pre-
for novel elimination and prevention methods based on
vented from colonizing the intestinal tract throughout the
feed supplementation, the effects of feed supplemented
test period by nonimmunized egg yolk powder, whereas
the other samples only delayed the colonization. None
with nonimmunized egg yolk powder (did not contain
of the fed supplements disrupted the balance of the intes-
anti-S. enteritidis antibodies), immunized egg yolk pow-
tinal microflora, and the counts in the feces remained
der (with anti-S. enteritidis antibodies), egg yolk proteins,
constant. These results show that the administration of
egg white, and skim milk powder were examined on
only 5.0% (wt/wt) of nonimmunized egg yolk powder
laying hens. In the elimination study, the chickens were
can eliminate and prevent SE colonization in laying hens
orally infected with SE then given a supplemented feed
with no adverse effects. Furthermore, the present results
of 5, 10, or 15% (wt/wt) of each of the test samples. Fecal
indicate that hen egg yolk contains novel anti-adhesive
samples tested weekly showed an absence of SE after the
or immunomodulatory components that may act to pre-
first week of feeding nonimmunized egg yolk powder
vent SE infection.
(Key words: Salmonella enteritidis, laying hen, food supplement, egg yolk, egg yolk antibody)
2004 Poultry Science 83:753–760
INTRODUCTION
into poultry (Moulder, 1985; Finlay and Falkow, 1989).
The adhesion of the pathogen is mediated by bacterial
Salmonella enteritidis is the cause of the foodborne sal-
adhesins, which recognize specific mucosal receptors. In-
monellosis pandemic in humans, in part because it is
hibition of adhesion by blocking the receptors with spe-
the only human pathogen and Salmonella serotype that
cific adhesin analogues or by steric hindrance (Tuomola
contaminates shell eggs. This contamination routinely
et al., 1999) may prevent or eliminate colonization of the
causes human illness without causing illness in the in-
intestine by pathogens and thereby prevent the infection.
fected birds (Petter, 2001). Egg-transmitted salmonellosis
Elimination from or prevention of SE in broilers and
is the primary food poisoning risk in the industrialized
other poultry, especially the table egg-producing layer
world (Levine, 1991; Wachsmuth et al., 1991; Hennessy
flocks (USDA, 1988), before they reach the processing
et al., 1996; USDA, 1996). Salmonella enteritidis is believed
plant will improve the chances of products free from this
to be vertically transmitted from infected ovaries and
organism (Stern et al., 2001). Research has focused on the
oviducts to the eggs of laying hens (Protais, 1989; Suzuki,
development of immunoprophylactic measures, microbi-
1990; Nakamura et al., 1993; Thiagarajan et al., 1994;
ological strategies, and antisalmonella feed additives to
Methner and Meyer, 1995). Another proposed route is
prevent intestinal and tissue colonization of table egg-
that SE penetrates the eggshell from the chicken feces
producing flocks by invasive SE (USDA, 1988; Smith,
deposited on the outside of the egg as it passes through
1989; Schneitz et al., 1990). Passive immunization by oral
the cloaca (Snoeyenbos et al., 1979).
administration of antibodies from external sources is
The ability to adhere to and penetrate intestinal epithe-
thought to be useful for the prevention of gastrointestinal
lial barriers is a common means of entry by Salmonella
infection (Sugita-Konishi et al., 2000). Recently, attention
has been focused on egg yolk immunoglobulin Y (IgY)
obtained from immunized hens as another antibody
?2004 Poultry Science Association, Inc.
Received for publication August 6, 2003.
Accepted for publication December 1, 2003.
1To whom correspondence should be addressed: ymine@
Abbreviation Key: BG = brilliant green; CE = competitive exclusion;
uoguelph.ca.
TS = tryptic soy.
753

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754
KASSAIFY AND MINE
source, because of its high productivity (Bartz et al., 1980;
SE Isolate
Yokoyama et al., 1992; Mine and Kovacs-Nolan, 2002).
Sugita-Konishi et al. (2000) reported that these anti-S.
The isolate used for infection was S. enteritidis PT4
enteritidis antibodies in egg yolk inhibited Salmonella ad-
SA992212 from chickens.2 Challenge inoculum was pre-
hesion and infection of human intestinal epithelial cells
pared from an overnight culture in tryptic soy (TS) broth
in vitro. This inhibition was due to the binding of antibod-
(D4552)3 then serially diluted to the specified viable cell
ies to some specific antigens on the bacterial surface; how-
concentration of 109 cfu/mL. Inoculum was estimated
ever, the specific antibodies were unable to block the
by spectrophotometry, optical density at 660 nm, and
adhesion of SE completely and unable to suppress their
confirmed by colony counts on brilliant green (BG) agar
multiplication inside the cell. Tellez et al. (2001) indicated
(D0702)3 plates containing 20 µg of novobiocin (B231971).3
that a combination of avian-specific probiotic and S. enter-
One milliliter of the challenge inoculum and 1.0 mL of
itidis, S. typhimurium- and S. heidelberg-specific antibodies
sterile TS broth as control were used for the oral infection.
have a beneficial effect in reducing the colonization of SE
Methods of the FDA Bacteriological Analytical Manual
in market-aged broilers; however, this was a combination
(Wallace et al., 1995) were used for the detection and
study with no direct indication if these antibodies alone
identification of SE in the feces of test samples.
have any beneficial effect in reducing SE colonization.
Brady et al. (2002) indicated an antibacterial activity of
Birds
fractionated hen-egg yolk (lipoproteins) against 2 patho-
Forty White Leghorn hens 22 to 24 wk old (elimination
genic streptococcus strains in vitro. Moreover, Sugita-
study), 28 hens (prevention study), and 10 hens (immu-
Konishi et al. (2002) suggested that egg yolk-derived sialy-
nized/nonimmunized egg yolk study) that were specific
loligosaccharides and its derivatives are useful in pre-
pathogen-free were obtained from the university research
venting Salmonella infection when ingested continuously.
station.4 Cloacal swabs and fecal samples were collected
Microbiological strategies employed early exposure of
and tested for SE by the pre-enrichment/enrichment se-
newly hatched chicks to normal adult intestinal flora
lective plating method (Wallace et al., 1995). BG agar
(Lloyd et al., 1977; Snoeyenbos et al., 1978; Barnes et al.,
plates containing 20 µg of novobiocin were used, and
1979; Snoeyenbos et al., 1979; Barnes et al., 1980; Impey
suspected Salmonella colonies were identified biochemi-
and Mead, 1989; Nurmi et al., 1992). Nurmi and Rantala
cally by the lysine iron agar (B211363)3/tryptic sugar iron
(1973) and Snoeyenbos et al. (1978) pioneered the use of
agar (D4402)3 slants biotyping technique. Then SE was
normal gut microflora for colonization control [competi-
positively identified serologically by the somatic O-anti-
tive exclusion (CE) or adhesive probiotics, mainly Lactoba-
serum group D1 (DF 295147)3 agglutination for sero-
cillus strains]. However, to be effective, it is crucial that
grouping and serotyping using flagellar H-antiserum
the CE culture should be given to chicks very early in
single factors m and 7 (DF 2548472, DF 2477475)3 (Wallace
life before they are exposed to SE. Several difficulties are
et al., 1995). Fecal samples were also collected for intesti-
being encountered with long-term preservation of the CE
nal microflora estimation before, during, and after sup-
cultures and with batch-to-batch variability (Ziprin and
plemented feeding and infection. The microorganisms
Deloach, 1993). Furthermore, colonization cannot be pre-
targeted were total aerobic bacteria, total anaerobic bacte-
vented by this method when breeding farms are contami-
ria, Lactobacillus spp., and enterobacteriaceae. The culture
nated (Nurmi et al., 1992; Nakamura et al., 1995), and
media used were TS agar (D4452),3 bacto anaerobic agar
results suggest that such microbiological strategies may
(253610),3 lactobacilli deMan Rogosa Sharpe agar
be ineffective in older hens and layer flocks (Corrier et
(D3052)3 and BG agar, respectively. Serial dilutions were
al., 1993).
prepared in peptone water (PW, D3452),3 and a 0.1-mL
It is important to develop technologies capable of re-
sample of each of the 10?3, 10?5, and 10?7 dilutions was
ducing, eliminating, and preventing the incidence of Sal-
plated on each medium. Plated bacteria were incubated
monella colonization in poultry through a novel
anaerobically in anaerobic chambers with disposable gas
nonantibiotic approach. In this study, we focused on the
generator envelopes for anaerobic environment (B71040)3
eliminating and protective effects of animal-based food
or in CO2-enriched envelopes for Lactobacillus spp.
supplements against SE infection in laying hens. Hence,
(1181440),3 or aerobically, at 30°C for 24 to 48 h. Bacterial
we sought to determine the effects of the food protein
colonies on each medium were then counted.
supplements: egg yolk protein, egg white, and skim milk
Prior to infection, eggs from each bird were cracked,
powder on SE colonization in chickens.
and egg yolks were aseptically separated from the albu-
men. Egg yolk samples were tested for antibodies to SE by
MATERIALS AND METHODS
ELISA using formalin-treated whole SE cells as a coating
All animal experiments were performed with the
antigen and primary and secondary antibodies for the
knowledge and approval of the University of Guelph’s
colorimetric identification as described previously (Mine,
committee on animal use and care.
1997). All birds were negative for SE.
Housing
2Cornelius Poppe, Health Canada, Guelph, ON, Canada.
3BD diagnostic system, Oakville, ON., Canada.
Birds were housed one per cage in separate wire-bot-
4Isolation Unit, Pathobiology Dept. University of Guelph, Canada.
tom, covered metal cage systems in the isolation unit.4

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SALMONELLA ENTERITIDIS INFECTION AND PREVENTION
755
Cages were raised on a metal table, with 3 cages (repre-
Experimental Design
senting 3 replicates of 1 treatment) on each table, keeping
a distance of approximately 60 cm between cages and
Elimination Study. Feed and water were withheld for
100 cm between adjacent tables. Plastic sheets were used
24 h prior to infection. Of the 40 birds, 38 were inoculated
under the wire floors of each cage to catch droppings.
orally with 1.0 mL of 109 cfu/mL of SE and 2 (negative
They were cleaned and replaced daily. Food and water
controls, with 1.0 mL of sterile TS broth, using a syringe
were provided ad libitum.
and a blunt-end catheter. A booster infection of another
1 mL was given 1 wk after the first infection. Three days
Feed Preparation
postinoculation, the infection was confirmed by testing
cloacal swab specimens and fecal samples to monitor SE
level. Once the SE level in the feces reached 104 to105 cfu/
The regular untreated feed5 was soy based and pelleted
g, supplemented feeding commenced. For 4 wk, each 3
and contained no antibiotics (corn 56%; soybean meal,
birds in 3 separate cages (representing 3 replicates of 1
16.2%; wheat shorts, 7.1%; pork meal, 6.0%; limestone,
treatment) were fed, respectively, a supplemented feed
9.5%; dicalcium phosphate, 0.8%; fat, 3.0%; salts, 0.25%;
of nonimmunized egg yolk powder, egg yolk proteins,
and vitamin-premix, 1.0%; wt/wt). Prior to each feeding
egg white powder, or commercial skim milk powder at
study, the feed of each chicken was prepared and stored
concentrations of 5, 10, or 15% (wt/wt). The 2 positive
separately by mixing the regular untreated feed thor-
controls (challenged with SE) and the 2 negative controls
oughly with the designated supplement at the required
(unchallenged) were fed the untreated regular feed [3
concentration. The nonimmunized egg yolk powder with-
birds per concentration per supplement × 3 concentra-
out anti-SE antibodies was prepared from eggs obtained
tions × 4 supplements = 36 birds + 2 positive controls +
from the university research station5 that were cracked
2 negative controls = 40 birds].
after disinfection of the exterior shell surface, and the egg
Prevention Study. For 4 wk, each 3 SE-free birds in 3
yolks were aseptically separated from albumen. Pooled
separate cages (representing 3 replicates of 1 treatment)
egg yolks were freeze-dried and crushed into a fine pow-
were fed respectively a supplemented feed of 5 or 10%
der. All egg yolk samples were tested for antibodies to
(wt/wt) nonimmunized egg yolk powder, 10 or 15% (wt/
SE by ELISA using formalin-killed whole SE cells as a
wt) egg yolk proteins, 10 or 15% (wt/wt) egg white pow-
coating antigen. Samples were negative for the aforemen-
der, or 5 or 10% (wt/wt) commercial skim milk powder.
tioned antibodies. The immunized egg yolk powder (con-
The 2 positive controls (to be challenged with SE) and
taining anti-S. enteritidis antibodies) was prepared as
the 2 negative controls (to remain unchallenged) were
before but from eggs of chickens that were immunized
fed the untreated regular feed [3 birds per concentration
with killed SE cells. In the procedure, 9 White Leghorn
per supplement × 2 concentrations × 4 supplements = 24
hens (20 to 24 wk old) that were specific pathogen free
birds + 2 positive controls + 2 negative controls = 28
were kept in separate cages in the Poultry Research Unit.5
birds]. The singular concentrations in this study were
Hens were intramuscularly vaccinated with 0.4 mL of SE
determined based on results obtained from the elimina-
inoculum prepared from formalin killed cells (109 cfu/
tion study above. At the end of 4 wk postfeeding, feed
mL) in sterile 0.9% NaCl emulsified with an equal volume
and water were withheld for 24 h and all the chickens
of complete Freund’s adjuvant (9007-81-2).6 Hens were
except the 2 negative controls were orally infected with
given a booster 2 wk after the first injection and then
1.0 mL of 109 cfu/mL of SE. Postinfection, the chickens
weekly for 2 wk, with a final booster at 6 wk after the
were fed the untreated regular feed.
first injection. The whole cells of killed Salmonella were
Nonimmunized Vs. Immunized Egg Yolk Powder
also used as a coating antigen in the ELISA method to
Study. To compare the effect of nonimmunized and im-
test for antibodies. Pure IgY7 was used as a standard to
munized egg yolk powder on SE infection, of the 10 birds;
determine the concentration of specific antibodies to SE
8 were inoculated orally with 1.0 mL of 109 cfu/mL of
in the immunized egg yolk powder samples, which was
SE and 2 as the negative control, with 1.0 mL of sterile
estimated to be 8.0 g/kg powder. The other food supple-
TS broth, using a syringe and a blunt-end catheter. A
ments, egg yolk proteins (after lipid fractions were ex-
booster infection of another 1.0 mL was given 1 wk after
tracted by ethanol), and egg white powder were
the first infection. Three days postinoculation, the infec-
commercially obtained from Taiyo Kagaku Ltd.8 Skim
tion was confirmed by testing cloacal swab specimens
milk powder was purchased from a local supermarket.
and fecal samples to monitor SE level. Once the SE level
Neither egg yolk proteins nor skim milk powder con-
in the feces reached 104 to 105 cfu/g, it was monitored
tained specific antibodies to SE.
for 4 wk, at the end of which supplemented feeding com-
menced. For 4 wk, each three birds in 3 separate cages
(representing 3 replicates of 1 treatment) were fed 15%
(wt/wt) nonimmunized or immunized egg yolk powder.
5Arkell Poultry Research Unit, Guelph, Canada.
The 2 positive controls (challenged with SE) and the 2
6Sigma Chemical Co., St. Louis, MO.
7Jackson Immuno Research, West Grove, PA.
negative controls (unchallenged) were fed the untreated
8Taiypo Kaguku Ltd., Yokkaichi, Japan.
regular feed [3 birds per concentration per supplement

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756
KASSAIFY AND MINE
× 1 concentration × 2 supplements = 6 birds + 2 positive
controls + 2 negative controls = 10 birds].
Bacteriologic Examination. Fecal samples were col-
lected once a week starting 4 d postfeeding in the elimina-
tion and immunized vs. nonimmunized studies or 4 d
postinfection in the prevention study for 4 wk. S. enteriti-
dis was detected, identified, and enumerated as outlined
above. Intestinal microflora in fecal samples were also
enumerated weekly. At the end of the experimental pe-
riod, chickens were euthanized. Before and throughout
the feeding and infection period, the weights of hens and
the number and weight of eggs laid per chicken were
monitored weekly for any significant changes in compari-
son with those of the 2 negative control chickens.
Statistical Analysis. Differences in the mean intestinal
bacterial counts between the various diets and that of the
control were analyzed by ANOVA.9 Means were sepa-
rated by Duncan’s multiple range test. Probabilities less
than or equal to P < 0.05 were considered statistically sig-
nificant.
RESULTS AND DISCUSSION
Experiment 1: Effect of Food Supplements
in Preventing and Eliminating SE
Infection in Laying Hens
Elimination Study. The number and incidence of SE
in the feces of hens during this test period with the effect
of the various food supplements are shown in Figure 1.
After infection with 1.0 mL of 109 cfu/mL of SE, the
average log10 level in the fecal samples was 4.9 ± 0.08
cfu/g10 at wk 2. The supplemented feeding commenced
at this level. The incidence of SE in feces decreased rapidly
as shown by the mean log10 levels of the counts, and no
Salmonella were recovered from the feces of those fed the
10 and 15% (wt/wt) nonimmunized egg yolk powder
after 1 wk of feeding. However, the incidence decreased
gradually with the 5% (wt/wt) and was not detected after
2 wk of feeding (Figure 1A), whereas the control mean
log10 levels in the fecal samples remained constant around
5.0 ± 0.27 cfu/g throughout the test period. From the
above results, nonimmunized egg yolk powder signifi-
cantly (P < 0.05) reduced the frequency of colonization
with SE and was able to eliminate the organism at a
concentration as low as 5% (wt/wt) without containing
the SE-specific antibodies. On the other hand, egg yolk
powder is readily available, practical, and economical as
a potential feed supplement. The 10 and 15% (wt/wt)
egg yolk proteins (Figure 1B) showed a similar decreasing
effect in the incidence of SE in the feces, but complete
FIGURE 1. Panel A: effect of feeding nonimmunized egg yolk powder
elimination did not occur until after 2 wk of feeding. Only
on elimination of Salmonella enteritidis from the intestine of laying hens.
a slight decreasing effect on SE was detected with the
Panel B: effect of feeding egg yolk proteins on the elimination of Salmo-
nella enteritidis from the intestine of laying hens. Panel C: effect of feeding
5% (wt/wt) egg yolk proteins when compared with the
egg white on the elimination of Salmonella enteritidis from the intestine
of laying hens. Panel D: effect of feeding skim milk powder on the
elimination of Salmonella enteritidis from the intestine of laying hens. a
Birds were infected with 109 cfu/mL SE. bSupplemented feeding with
9
the various concentrations commenced. Error bar indicates standard
SPSS version 8.0 for Microsoft Windows, SPSS, Chicago, IL.
10
error of the mean. **Significant (P < 0.05) compared with the control.
Log10 colony-forming units per gram ± standard error of the mean.

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SALMONELLA ENTERITIDIS INFECTION AND PREVENTION
757
control mean log10 counts in the feces. These results sug-
Prevention Study. The results of SE detection in the
gest that egg yolk proteins do have an elimination effect,
feces of the hens in this study are shown in Figure 2.
more statistically significant (P < 0.05) at higher concentra-
After the feeding period of 4 wk with the various concen-
tions of the protein. The above observations also indicate
trations of the aforementioned food supplements, the
that the proteins in the egg yolk play a role against the
chickens were infected with 1.0 mL of 109 cfu/mL of SE
colonization of SE, but other components present in the
and then given the untreated control (regular) feed. The
egg yolk also may contribute to this activity.
mean log10 counts in the fecal samples of the chickens
Passive immunization by oral administration of specific
that were fed the nonimmunized egg yolk powder sup-
antibodies has been an attractive approach against gastro-
plement showed that SE was not evident with the 5 and
intestinal pathogens in human and animals. The potential
10% (wt/wt) supplement concentrations (Figure 2A) dur-
application of orally administered egg yolk antibodies for
ing the 4-wk test period when compared with the mean
that purpose has been studied extensively in the past and
log10 counts of the control hens. From the above results,
documented in several reviews (Larsson et al., 1993; Hatta
egg yolk powder treatment was able not only to signifi-
et al., 1997; Mine and Kovacs-Nolan, 2002). However, the
cantly (P < 0.05) reduce the frequency of SE colonization
present work indicated that egg yolk itself contains novel
but also to prevent this organism from colonizing the
anti-infectious factors besides IgY, and they can eliminate
intestinal tract at a concentration as low as 5% (wt/wt)
SE from an infected chicken’s gut. This report is the first
without containing the SE-specific antibodies. These data
to demonstrate such new biological function of egg
are significant because the adhesion of SE in the intestinal
yolk components.
epithelial cells is crucial to the initial phase of infection
The incidence of SE in feces rapidly decreased with the
(Ofek and Doyle, 1994) and so blocking this adhesion in
15% (wt/wt) egg white supplement and was evident after
the intestine may prevent infections if the laying hens
1 wk of feeding (Figure 1C). However, just a slight reduc-
were exposed to the organism during their lifespans. The
tion in the mean log
preventive effect of egg yolk proteins on the incidence of
10 counts was observed with the 5
and 10% (wt/wt) egg white powder. Unsurprisingly, egg
SE in the feces is shown in Figure 2B. The organism was
white proteins at higher concentrations showed an elimi-
not detected for up to 2 wk in the fecal samples of the
nation effect, but it is unclear whether this effect is an
chickens that were fed the 10 and 15% egg yolk proteins,
but then the mean log
anti-adhesive or the result of the various antimicrobial
10 levels of SE increased to 4.7 ±
0.43 cfu/g at 4 wk. These results indicate that the extracted
factors present in egg white (mainly lysozyme and ovo-
egg yolk proteins alone may not be as effective in blocking
transferrin), which are well known as natural antimicro-
SE from colonizing the intestinal tract as the complete
bial agents (Ibrahim, 1997). To our knowledge, there is no
egg yolk supplement.
report on the exclusive effects of egg white components
The results of the egg white protein supplement are
against SE in the animal or human gut. Further studies
shown in Figure 2C. No SE was detected in the feces of
are required to identify the inhibitory mechanism of egg
the chickens that were fed 15% (wt/wt) egg white powder
white and determine whether its activity is antimicrobial
during the 4-wk test period; however, with the 10% egg
or a novel anti-adhesive.
white, the preventive effect was for up to 2 wk. The
Similarly, skim milk also showed a slight elimination
mean log
effect but only with low concentrations at 5 and 10% (wt/
10 levels increased to 4.7 ± 0.20 cfu/g at 4 wk.
Ultimately, similar results were evident with the 5 and
wt). Such concentrations had a gradual decreasing effect
10% (wt/wt) skim milk powder, as shown in Figure 2D.
(as shown in Figure 1D) on the incidence of SE in feces
The nonimmunized egg yolk powder seemed to be the
and no SE was evident after 3 wk of feeding. With the
most effective among all the samples tested in eliminating
15% (wt/wt), there was an initial increase in the mean
and preventing SE from colonizing the intestinal tract of
log10 counts after 1 wk of feeding, and then a steady
laying hens.
slight decrease was observed in the counts afterward.
This initial increase perhaps suggests that, at a high con-
Experiment 2: Effect of Nonimmunized Egg
centration skim milk and its components may act as
Yolk Powder versus Immunized Egg Yolk
growth factors for the organism. The results from skim
Powder in Eliminating SE in Laying Hens
milk can be explained because the nonfat component of
milk has powerful antimicrobial activities, in particular
As documented by many researchers, egg yolk antibod-
lactoferrin, and oligosaccharides in milk have preventive
ies have attracted considerable attention for the preventor
effects against gastrointestinal pathogens as documented
and treatment of viral and bacterial gastrointestinal infec-
by several researchers (Cravioto at al., 1991; Naidu, 2000).
tions. However, one important question arose from our
Finally, commercial skim milk powder contains about 5%
present work. Is the active component in egg yolk, the
(wt/wt) lactose (Holland et al., 1989), and it has been
egg yolk antibodies, or other components? To address
shown that lactose is effective in the prevention of Salmo-
this question, we compared nonimmunized egg yolk and
nella typhimurium infection (Corrier et al., 1990). There-
immunized egg yolk powder in terms of their activity in
fore, in future work we will demonstrate whether the
the elimination of SE in laying hens. The comparative
effect is due to the functional properties of lactose or to
effect of the immunized and nonimmunized egg yolk
the other milk components.
powder on the number and incidence of SE in the feces

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KASSAIFY AND MINE
FIGURE 3. Effect of feeding immunized egg yolk powder with anti-
Salmonella enteritidis-specific antibodies versus nonimmunized egg yolk
powder on the elimination of SE from the intestine of laying hens. aBirds
were infected with 109 cfu/mL SE. bSupplemented feeding with the 2
treatments commenced; the control group was fed untreated feed. Error
bars indicate standard error of the mean. **Significant (P < 0.05) com-
pared with the control.
are shown in Figure 3. A rapid decrease in the mean log10
levels of the counts and an elimination of the organism
were observed when feeding 15% (wt/wt) nonimmu-
nized egg yolk powder (P < 0.05) when compared with
the control group. Incidentally, a similar effect was ob-
served in the mean log10 levels of SE in the feces of those
hens that were fed immunized egg yolk powder supple-
ment that contained anti-S. enteritidis antibodies (specific
IgY, 8 g/kg of yolk powder). These results indicated that
other components in the egg yolk powder may be im-
portant factors in eliminating SE from chicken gut, and
the anti-S. enteritidis antibodies are not primarily the ac-
tive anti-adhesive factors against SE elimination in lay-
ing hens.
This is because egg yolk powder whether containing
the anti-S. enteritidis antibodies or not, significantly (P <
0.05) reduced the frequency of colonization by SE at a
concentration as low as 5% (wt/wt) in the feed. Other
researchers have concentrated on the egg-derived anti-S.
enteritidis antibodies alone as the active components in
preventing SE infection in poultry and did not use nonim-
munized egg yolk powder as a control. In addition, the
preventive effect of the orally administered IgY on gastro-
intestinal infections caused by the enteric pathogens, Sal-
monella spp. in particular, has been studied at length
(Sugita-Konishi et al., 1996; Yokoyama et al., 1998). How-
ever, findings in this study suggest that egg yolk compo-
nents have synergistic effects to prevent Salmonella
FIGURE 2. Panel A: effect of feeding nonimmunized egg yolk powder
on the prevention of Salmonella enteritidis from the intestine of laying
infections in laying hens. It has been reported that, in
hens. Panel B: effect of feeding egg yolk protein on the prevention of
mice, egg yolk derived sialyloligosaccharide, sialylglyco-
SE from the intestine of laying hens. Panel C: effect of feeding egg white
peptide and asialo-yolk derived sialyloligosaccharide
on the prevention of Salmonella enteritidis from the intestine of laying
hens. Panel D: effect of feeding skim milk powder on the prevention
have antibacterial properties and provide protection
of Salmonella enteritidis from the intestine of laying hens. aBirds were
against gastric diseases such as Salmonella infection by
infected with 109 cfu/mL SE after being fed for 4 wk with supplemented
preventing bacteria from binding to the intestine rather
feed containing the designated concentration; the control group was
fed untreated regular feed. Error bar indicates standard error of the
than by activating macrophages (Sugita-Konishi et al.,
mean. **Significant (P < 0.05) compared with the control.
2002). However, the concentration of these sialyloligosac-
charide components in the egg yolk powder is very low
to exhibit an anti-adhesive activity. Therefore, it seems

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SALMONELLA ENTERITIDIS INFECTION AND PREVENTION
759
should determine how this technique could be developed
as a practical intervention method for reducing human
exposure to Salmonella as well as determining the exact
active components in the egg yolk powder that are exhib-
iting such a beneficial effect.
ACKNOWLEDGMENTS
This work was supported by grants from the Poultry
Industry Council (Guelph, ON, Canada), Ontario Egg
Producers’ Marketing Board (Mississauga, ON, Canada),
and the Ontario Ministry of Agriculture and Food
(Guelph).
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