01999 Applied Poultry Science, Inc

BACTERIAL
PENETRATION OF THE
EGGSHELLAND SHELL
MEMBRANES OF
HATCHING
THE CHICKEN EGG:A REVIEW
M. E. BEFUNGI
USDA, ARS,PPMQRU, Russell Research Center, Athens, GA 306045677
Phone: (706) 546-3551
FAX;.(706) 546-3633
E-mail: m berrang@ars.usda.gov
N . A. COX
USDA, ARS, PMSRU, Russell Research Center, Athens, GA 30604-5677
J. E FRANK
Universityof Georgia, Food Science and Technology,Athens, GA 30602
R. J. BUHR
USDA, ARS, PPMQRU, Russell Research Center,Athens, GA 306045677

Primary Audience: Researchers, Hatchery Managers

hatching cabinet. Such contamination can be

DESCRIPTION
OF PROBLEMcarried onto the grow-out farm and pose a
Bacteria including the human pathogen significant food safety hazard. Understanding
salmonellae can readily penetrate the shell the manner in which bacteria can penetrate
and membranes of an intact hatching egg. The the shell is important if producers hope to
result of this penetration is contamination not intervene. This information may be of use to
only of the embryo within but also of many hatchery managers, breeder managers, and
other chicks during hatch in the commercial producers in their efforts to control the

1 To whom correspondence should be addressed

500
Salmonella problem in the commercial hatch-
ery and its product, the chick.
How BACTERIA
PENETRATE
EGGSHELLS
AND MEMBRANES
Bacteria can penetrate the outer struc-
tures of an egg. Once within the egg, bacteria
can cause depression of hatchability and/or
contamination of the chick. Contamination of
the chick with a human enteropathogen has
important food safety implications.
MECHANISM FOR TRANS-SHELL
PENETRATION
Eggshells can be penetrated by bacteria
when water or some other Liquid is present,
especially if there is a temperature differential
between the egg and the liquid. When an egg
is laid, it is warmer than the environment, since
the body temperature of the hen is 42°C. The
warm egg cools to environmental tempera-
ture, and this cooling causes the contents of
the egg to contract. Such contraction tends to
form a negative pressure within the egg.
Bacteria present in the environment or on the
egg surface can then be pulled into and
through the eggshell and its membranes [l,21.
This method of trans-shell contamination
has been confirmed through years of research.
Early research showed that application of a
mechanical vacuum to simulate a negative
pressure could cause penetration of a partial
eggshell [3]. Later experiments proved that
immersion of an egg into a cool cell suspen-
sion led to penetration of the intact egg [41.
W-s et al. [5] found that eggs are pene-
trated almost immediately when challenged
with moist Salmonella-contaminated chicken
feces. They [5] further noted that shell thick-
ness did not have a significant effect on bac-
terial penetration, but the presence of cuticle
plugging the shell pores is more important.
Also of interest was their observation that
bacterial motility is not related to ability to
penetrate.
AREA O F EGG MOST LIKELY TO BE
PENETRATED
By covering areas of the egg with molten
wax, Vadehra et al. [6] tested which area is
most likely to be penetrated by Pseudomonas
aeruginosa, a common egg spoilage organism.
BACTERIAL PENETRATION OF EGGS
They found that the blunt or air cell end is most
prone to penetration when challenged by a
temperature differential immersion. Interest-
ingly, this is the only area where the inner and
outer shell membranes do not remain in close
contact, forming the air cell which may re-
spond more rapidly to a change in tempera-
ture than the rest of the egg contents.
PRODUCTION STAGES WHERE AN
EGG MAY BE PENETRATED
Since moisture and presence of bacteria
are needed to allow penetration, any stage of
production where these two factors may be
present provides an opportunity for bacterial
invasion.When the factor of a positive temper-
ature differential (egg warmer than the envi-
ronment) is added, there is an increased
possibility of bacterial attack. Padron [i'l found
that when eggs were placed on Satmonella-
contaminated nest box shavings for 10 min,
the eggshell and membranes were penetrated
by that Salmonella organism in 59% of the
samples. In a field survey, Smeltzeret al. found
that eggs laid on the dirty chicken house floor
were more likely to exhibit internal bacterial
contamination than were eggs laid in a nest
box [8]. Eggs are most vulnerable to bacterial
penetration in the first 30 to 60 sec after lay
before the cuticle hardens and effectivelycaps
the pores.
Other stages of egg production and
handling can be implicated in bacterial pene-
tration. After collection, eggs are usually
placed in a cold room at the farm until hatch-
ery personnel transport them to the hatchery.
When cool eggs are removed from the farm on
a warm day, they may "sweat"due to conden-
sation of moisture from the air. Despite the
lack of a positive temperature differential, the
presence of moisture on the egg surface may
provide opportunity for contamination and
causes concern among many researchers as
reviewed by Bruce and Drysdale [2].
METHODS
USED TO
MEASURE
BACTERIAL
PENETRATION
Many articles have been published on the
measurement of bacterial invasion of eggs.
However, several stand out as real contribu-
tions in the area of how best to assess the
penetration of intact eggs or membranes.
 Review Article
BERRANG et al.
EGGSHELL AND MEMBRANES
Williams and Whittemore [9] reported an
excellent test for bacterial invasion under sim-
ulated fecal contamination conditions. This
method essentially involves gluing a short
aluminum tube to the area of interest and
fllinp it with sterile chicken feces. The feces
can then be seeded with a known bacteria.
Penetration is demonstrated by culturally
retrieving the microorganism from within the
egg. The egg is emptied and the inside of the
shell (or shell and membranes, depending on
the interest of the researcher) is sampled with
a swab. This method is excellent for examina-
tion of specific regions of the shell. However,
if one is interested in the entire egg or large
numbers of samples, this method becomes
cumbersome.
Board and Board [lo] developed a
method whereby the entire egg surface can
be quickIy and easily tested for bacteria1 pen-
etration. The intact egg is first subjected to a
positive temperature differential challenge by
immersion in a cool bacterial cell suspension.
After drying, the egg is emptied of contents
and fdled with microbiological growth me-
dium with the addition of tetrazolium. Upon
reduction by bacterial growth, tetrazolium is
chemically changed to formazon, which is a
deep red color. After the growth media has
hardened, the egg is sealed with paraffin and
incubated to allow for bacterial growth. The
resulting red spots appear through the shell
using a standard portable candler. This same
method has been adapted for use to show that
Campylobacterjejuni can penetrate the outer
structures of an egg [ll].
Another method developed to test in-
tact eggs for penetration uses a luminescent
strain of Salmonella enleritkiis (121. In this
method the egg is inoculated by immersion in
a culture of luminescent S. enteritidis. The
egg is then sealed in a plastic bag with the
luciferase substrate decanal. Luminescence
could then be detected through the shell with
an imaging system.This method is exciting and
shows promise because the egg does not
need to be opened or cracked during the
penetration test. However, it does require
some specialized expertise in the manufac-
ture, handling, and detection of luminescent
cultures.
AS an alternative to using bacterial cul-
tures, Kim and Slavik [13] developed an indi-
501
cator test whereby blue lake dye was applied
to eggs. Penetration was assessed by detection
of blue spots on the membranes below the
surface of the shell. The dye penetration
method correlated well with eggshell penetra-
tion by Salmonella.
MEMBRANE ALONE
The literature does not contain many
references to measurement of bacterial
penetration of membranes alone. Lifshitzetal.
[14] were able to test penetration of these
structures by emptying the egg, removing the
shell from the wide end of the egg, and leaving
the membrane intact. The egg was then set
into a bacterial cell suspension and the inte-
rior was filled with sterile broth. Periodic
culture of the broth from inside the shell
was used to determine the extent of bacterial
penetration.
A more practical and repeatable method
was originally developed to test the membrane
as a barrier to radioactive amino acids. The
membrane can be placed across the opening
in a ball and socket ground glass connection.
The radioactive amino acid is placed in the
upper tube with the ball part of the connection
and assayed in the lower socket section [15].
This type of method was modified by Berrang
et ul. [16]. An apparatus was fashioned with
PVC pipe and a union, whereby eggshell
membrane was used to cover the only opening
between an upper and lower chamber. When
a cell suspension of Salmonella typhimurium
was placed in the upper chamber, penetration
could be demonstrated by using a syringe
and needle to aseptically draw samples from
the bottom chamber through a sealed rubber
septum.
Wong et ul. [17] developed a method to
examine eggshell membrane microscopically
following positive temperature differential
immersion of an egg in a Salmonella cell sus-
pension. A confocal laser scanning micro-
scope was used to visualize Salmonella within
the meshwork of the outer membrane. With a
Salmonella-specific antibody conjugated to a
color-producing chemical, this method could
allow detection of penetration without the
need for culturing.
502
THEEGGS
DEFENSE
AGAINST
BACTERIAL
INVASION
Despite the fact that bacteria can pene-
trate the shell and membrane, the egg is not
without defense against these invaders.
PHYSICAL DEFENSES
The outermost physical defense that a
bacterial cell encounters on an egg is the cuti-
cle. The cuticle is a very effective barrier to
water and carbon black particles [MI. Despite
the fact that the cuticle allows gas passage, it
seems to effectively fill the pores of the egg-
shell [Z]. However, this defense is not perfect.
A small percentage of eggs are laid without
cuticle; these eggs may easily be contaminated
by water and carbon black [MI. Even when
cuticle is present, for the first few minutes
after lay it is an ineffective barrier to bacterial
invasion until it hardens [19]. Nevertheless,
the hardened cuticle is the major barrier to
liquid and therefore is of utmost importance
in bacterial exclusion [19].
However, the eggshell is an ineffective
barrier to bacterial penetration. The pores
are wide enough to allow entry. Eggshells
characterized to be of excellent quality (from
eggs with specificgravityabove 1.090 and pre-
sumably thicker shell than poor quality eggs)
were found to be more resistant to penetration
by salmonellae [20], possibly because of the
longer time needed for bacteria to move
through a thick shell.
Eggshell membranes do not have an in-
herent anti-bacterial property and can be
penetrated by bacteria. However, the mem-
branes do add some protective value to the
shell alone, improving the ability to hold out
bacteria over the short term [21]. The time
needed for bacteria to penetrate the combined
inner and outer eggshell membranes is not
clearly related to the amount of open space
between fibers in the outer surface of the outer
membrane [16]. When comparing the shell,
inner, and outer membranes for ability to pre-
vent bacterial entry, the inner membrane is the
most effective [14] because of the tighter
meshwork of the inner membrane relative to
the outer membrane.
BACTERIAL, PENETRATION OF EGGS
CHEMICAL DEFENSES
The egg’s albumen is a substance unin-
viting to most bacteria. The pH of egg albumen
is about 7.6 when laid. This pH rises to about
9.5 during storage, unfavorable for microbial
growth [22,23]. Conalbumin, an iron-binding
agent is also present in the albumen. Unless
this chemicalcan be quenched with iron, there
will not be enough free iron available to allow
microbial growth in the egg [22]. Further
protection is afforded the egg by the action
of lysozyme which can lead to the rupture
of procaryotic cell walls [22]. The typical in-
cubator temperature also seems to increase
the antibacterial effect of egg albumen [24].
Nevertheless, in an in-vitro test Pseudomonas
putida cells could move through the albumen;
those reaching the surface of the yolk were
able to reproduce quickly, leading to a gener-
alized infection of the egg [l].
While bacterial cells that contact the al-
bumen early may be killed, the area within
the shell and membranes can provide a safe
niche. Bacteria can remain in the membranes,
which have been shown to select for gram
negative over gram positive bacteria [19].
When the chick hatches, these bacteria are
ingested or can cause cross contamination.
Thus, in the hatching egg industry the chemical
defenses of the egg are not totally effective.
Also, in a fertile egg, the chemistry changes at
about the 7th day of embryonic development
and is no longer as hostile to bacteria such as
Salmonella [25].
RESULT
OF
INVASION
BACTERIAL OF
HATCHING
EGGS
In the past much of the concern regarding
bacterial infection of hatching eggs resulted
from the possibility of decreased hatch or
rotting eggs in the incubators. Current con-
cern has shifted to the food safety aspects of
bacterial invasion. Human enteropathogen
invasion of hatching eggs is a real problem.
Salmonellae have been isolated from hatching
eggs in the field [26, 27, 28, 291. The trans-
mission of Salmonella through an integrated
poultry company has been documented. Bains
et al. [30] details the chain of Salmonella from
breeder feed to the nest box, the hatchery,
onto the grow-out farm, and to dressed car-
casses. Broiler hatcheries have been shown to
 Review irticle
BERRANG et al. 503

be reservoirs for Salmonella. In one study71%
of eggshell fragment samples were contami-
nated [27]. In another study involving six
commercial breeder hatcheries, 15.2% of
the eggshell fragments were contaminated
with salmonellae [a]. Salmonella contamina-
tion of eggs can lead to extensive cross con-
tamination of chicks in the hatching cabinet
[31, 32, 331. In a 1996 study [34] Salmonella
were introduced into the day-of-hatch chick
through an assortment of body openings
(mouth, cloaca, eye, nasal passage, and navel).
All of these routes produced birds contami-
nated with Salmonella. These data emphasize
the need to control salmonellae in breeder
flocks, hatching cabinets, hatchery environ-
ments, and broiler houses to minimize the
production of seeder chicks. Salmonella iso-
lates from the hatchery have been found to
occasionally make their way not only to the
grow-out farm but also to the processing plant
and the final product [35].
In order to produce chicken and chicken
products free of human bacterial pathogens,
the source of the pathogens needs to be inter-
rupted. The hatching egg and the hatchery are
very important control points for introduction
of some human pathogens such as Salmonella
into the growing chicken and ultimately onto
the final product [36,37,38].

CONCLUSIONS
AND APPLICATIONS
1. Bacteria, including salmonellae, can penetrate eggshells and associated membranes.
Penetration can be detected and measured and may lead to cross contamination of many
chicks in the hatchery. This situation can cause a food safety problem when the contami-
nation is carried onto the farm and the processing plant.
2. The natural defenses of the egg are not entirely adequate to prevent penetration and
suMval of salmonellae.
3. Likelihood of penetration can be lessened by avoiding contact between the egg and
contaminated surfaces or substancessuch as feces or dirty nest pads. Also, excess moisture
(other than properly applied disinfectant solutions) on the eggs must be avoided, especially
at times of positive temperature differential (egg warmer than the environment).

AND NOTES
REFERENCES
1. Lock, J.L,J. Dolman, and R.G. Board, 1992. Ob-
senations on the mode of bacterial infection of hen’s
eggs. FEMS Microbiol. Letters 100:71-74.
2. Bruce, J. and E M . Drysdale, 1994. Trans-shell
transmission. Pages 63-91 in: Microbiology of the Avian
Egg. 1st Edition. RG. Board and R. Fuller, ed. Chapman
and Hall, London, England.
3. Haines, RB. and T. Moran, 1940. Porosity of, and
bacterial invasion through, the shell of the hen’s egg.
J. Hyg. 4 0 4 5 H 6 1 .
4. Stokes, J.L,W.W.Osborne, andH.G. Bayne, 1956.
Penetration and growth of Salmonella in shell eggs. Food
Res.21:510-518.
9. Williams, J.E. and AD. Whittemore, 1967. A
method for studying microbial penetration throu
outer structures of the avian egg. Avian Dis. ll:46f%%
10. Board, P A and R.G. Board, 1967. A method of
studying bacterial penetration of the shell of the hen’s egg.
Lab. Prac. 16:472473,482.
11. Neill, S.D., J.N.Campbeband JJ.O’Brien, 1985.
Egg penetration by
14:313-320.
m.
Avian Path.
12. Chen, J., R.C. Clarke, and M.W. Grimths,
. . 1996.
.
Use of luminescent strains of Salmonella ententldls to
monitor contamination and survival in eggs. J. Food
Prot. 59:915-921.

--
5. WUliams, J.E,LH. Dillard, and G.O. Hall, 1968.
The penetration patterns of Salmonella ‘ ’
the outer structures of chicken-=e
6. Vadehra, D.V., RC. Baker, and H.B. Naylor, 1970.
Infection routes of bacteria into chicken eggs. J. Food Sci.
3561-62.
7. Padron, M.N., 1990. pen-
etration through the eggshell of hatching eggs. Avian Dis.
34A63465.
8. Smeltzer, T.I., K. Orange, B. Peel, and 6. Runge,
1979. Bacterial penetration in floor and nest box eggs
from meat and layer birds. A u t . Vet. J. 55592-593.
13. Kim, J.W. and M.F. Slavik, 1996. Use of blue lake
asan indicator of bacterial penetration into eggs. J. Rapid
Methods Automat. Microbiol. 418S190.
14. Lifshitz A, R.C. Baker, and H.B. Naylor, 1964.
The relative importance of chicken e exteriorstructures
in resisting bacterial penetration. J.Food Sci. 29:94-99.
15. Wedral, EM., D.V. Vadehra, and RC. Baker,
1971. Mechanism of bacterial penetration through the
eggs of -&. 2. Effect of penetration and growth
on permeability of inner shell membrane. J. Food Sci.
36520-522.
16. Berrang, M E , J.F. Frank, RJ. Buhr, J.S. Bailey,
and N . h Cox, 1999. Eggshell membrane structure and
504
penetration by ella twhimurium. J. Food Prot.
6273-76.
17. Wong, J.W., J.F. Frank, and S. Bailey, 1997. Visu-
alization of eggshell membranes and their interaction
. . using confocal
with-ententldls scanning laser
microscopy. J. Food Prot. 601022-1028.
18. Bo+ RG. and N.A. Halls, 1973. The cuticle: A
bamer to liquid and particle penetration of the shell of
the hen’s egg. Br. Poultry Sci. 14:69-97.
19. Sparks, N.H.C., 1987. The hen’s eggshell: A resis-
tance network. Aslib Index Theses 36:294.
20. Sauter, EA.and C.F. Petersen, 1974.The effect of
egg shell quali on penetration by various salmonellae.
Poultry Sa. 537159-2162.
21. Garibaldi, J.A. and J.L Stokes, 1958. Protective
role of shell membranes in bacterial spoilage of eggs.
Food Res. 23283-290.
22. Board, RG., 1974. Non-specific antimicrobial de-
fenses of the avian egg, embryo, and neonate. Biol. Rev.
49:15-49.
23. Sharp, P.F. and C.K. Powell, 1931. Increase in the
H of the white and yolk of hens’ eggs. Ind. Eng. Chem.
53:196-199.
24. Trader, H.S. and RG. Board, 1984.The influence
of incubation temperature and pH on the antimicrobial
P :s
ro erties of hen egg albumen. J. Appl. Bacteriol.
6 3-61.
25. Romanoff, kL a n d k Romanoff, 1949.The Avian
Egg.John Wiley and Sons, New York, NY.
26. Greenfield J., C.H. Bizland, and H.D. McCaus-
land, 1971. Detection of --contaminated eggs.
Poultry Sci. 50652-653.
27. Cox, N.A., J.S. Bailey, J.M. Mauldin, and LC.
Blankenship, 1990. Research note: Presence and impact
of &@&la contamination in commercial broiler
hatchenes. Poultry Sci. 69:1606-1609.
28. Cox, N.A.,J.S. Bailey, J.S. Mauldin, LC. Blanken-
ship, and RL Wilson, 1991. Research note: Extent of
salmonellae contamination in breeder hatcheries. Poultrj
Sci. 70416418.
BACTERIAL PENETRATION OF EGGS
29. Blankenship. LC., J.S. Bailey, N.A. Cox, N.J.
Stern, R. Brewer, and 0. Williams, 1993. Two-step mu-
cosal competitive exclusion flora treatment to diminish
salmonellae in commercial broiler chickens. Poultry Sci.
72: 1667-1672.
30. Bains, B.S. and M.A. MacKenzie, 1974.Transmis-
sion of Salmonellathrough an integrated poultry organi-
zation. Poultry Sci. 53:1114-1118.
31. Bailey, J.S., N.A. Cox, and M.E Berrang, 1994.
Hatchery acquired salmonellae in broiler chicks. Poultry
Sci. 73:1153-1157.
32. Cason, J.A., N . k C0q.andJ.S. Bailey, 1994.Trans-
mission of Salnlpnella during hatching of
broiler chicks. Avlan D% i %??!%.
33. Bailey, J.S., RJ. Buhr, N.A. Cox, a n d M.E
Berrang, 1996. Effect of hatcher cabinet sanitization
treatments on -cross contamination and hatch-
ability of broiler eggs. Poultry Sci. 75191-196.
34. Cox, N.A., J.S. Bailey, and M . E Berrang, 1996.
Alternative routes for Salmonellaintestinal tract coloni-
zation of chick. J. Appl. Poultry Res. 5:282-288.
35. Lahellec, C. and P. Colin, 1985. Relationship be-
tween serotypes of salmonellae from hatcheries and rear-
ing farms and those from processed poultry carcases.
Br. Poultry Sci. 26:179-186.
36. Cox, N.A., J.S. Bailey, and M.E Berrang, 1994.
Attempts to break the salmonellae in oult
chemically treating the freshly laid egg. A g e 3 7 6 % k 2
84th General Meeting, American Society of Microbiol-
ogy, Las Vegas, NV.
37. Cox, SA.and J.S. Bailey, 1995. Reducin bacteria
from farm through processing. Pages 28-31 in: #roc. 30th
Natl. Meeting on Poultly Health and Processing,Ocean
City. MD.
38. Cox, N.A., J.S. Bailey, and NJ. Stern, 1995. Re-
search efforts to intervene in the Salmonellacolonization
of broiler chicks. Pages 193-199 in: Proc. XI1 European
Symposium on the Quality of Poultry Meat,Edinburgh,
Scotaland.