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179
Received for publication August 13, 1995.
Accepted for publication August 27, 1996.
1
Mention of trade names in this publication does not imply
endorsement by North Carolina State University or the North Carolina
Agricultural Research Service, Raleigh, NC 27695 of the product
named nor criticism of similar products not mentioned.
Principles of
Ex Ovo
Competitive Exclusion and
In Ovo
Administration of
Lactobacillus reuteri
1
F. W. EDENS,* C. R. PARKHURST,* I. A. CASAS,
‡
and W. J. DOBROGOSZ
†
*Department of Poultry Science and
†
Department of Microbiology, North Carolina State University, Raleigh,
North Carolina 27695-7635, and
‡
BioGaia Biologics, Inc., 6213D Angus Drive, Raleigh, North Carolina 27613
ABSTRACT The data that have been presented
indicate that the in ovo use of competitive exclusion (CE)
agents is feasible for both chickens and turkeys.
However, there are many pitfalls that await the use of in
ovo application of CE agents, including the use of
nonspecies-specific intestinal microbes and the use of
harmful proteolytic, gas-producing and toxin-producing
intestinal microbes. Of the potential CE agents that have
posthatch application, only Lactobacillus reuteri has been
shown to be safe and effective in terms of not affecting
hatchability and in having a prolonged effect in the
hatched chick or poult. Lactobacillus reuteri administra-
tion in ovo increases its rate of intestinal colonization and
decreases the colonization of Salmonella and Escherichia
coli in both chicks and poults. Additionally, mortality
due to in-hatcher exposure to E. coli or Salmonella is
reduced with in ovo L. reuteri. Use of antibiotics in ovo
may preclude the use of co-administered CE agents, but
Gentamicin
and L. reuteri are a compatible mixture
when administered in ovo in separate compartments.
Nevertheless, the intestinal morphology can be affected
by both the CE agent and by antibiotics. Lactobacillus
reuteri both in ovo and ex ovo will increase villus height
and crypt depth, and Gentamicin
in ovo causes a
shortening and blunting of the villus. Both Gentamicin
and L. reuteri in ovo suppress potentially pathogenic
enteric microbes, but with diminished antibiotic effects
shortening and blunting of the intestinal villi does not
correct itself. Goblet cell numbers increase significantly
on the ileum villus of chicks treated with Gentamicin
in
ovo, and this is presumably due to the increase in
potentially pathogenic bacteria in the intestinal tract.
Diminishing antibiotic effects posthatch would then
negatively affect the absorption of nutrients and reduce
growth at least in a transitory manner. Thus, L. reuteri
administration in ovo singly or in combination with
Gentamicin
followed by L. reuteri via drinking water or
feed appears to have potential to control many enteric
pathogens in poultry.
Additional work in the use of in ovo CE cultures is
mandated because there is a world-wide movement to
reduce antibiotic use in poultry due to increased
microbial resistance to antibiotics. Use of naturally
occurring intestinal bacterial cultures, either in mixed
culture or as single well-defined cultures, has potential
for immediate use in the poultry industry.
(Key words: competitive exclusion, Lactobacillus reuteri, in ovo, hatchability, livability)
1997 Poultry Science 76:179–196
INTRODUCTION
The advent of competitive exclusion (CE) technolo-
gies for the control of pathogenic enteric bacteria in
poultry as well as humans has received considerable
interest during the past two decades. Intensive efforts by
groups of USDA scientists (Bailey, 1987, 1988; Bailey et
al., 1988; Ziprin et al., 1989, 1990, 1991; Cox et al., 1990,
1992a,b; Corrier et al., 1990a,b, 1991a,b, 1992a,b,c, 1994,
1995a,b; Hinton et al., 1990, 1991a,b; Hume et al., 1993;
Hollister et al., 1994a,b,c, 1995; Nisbet et al., 1994) signal
the importance of this technology and its application to
the poultry industry.
The CE concept was applied first to the domestic fowl
by Nurmi and Rantala (1973) and Rantala and Nurmi
(1973) when they attempted to control a severe outbreak
of Salmonella infantis in Finnish broiler flocks. In their
studies, it was determined that very low challenge doses
of Salmonella (1 to 10 cells into the crop) were sufficient
to initiate salmonellosis in chickens. Additionally, they
determined that it was during the 1st wk posthatch that
the chick was most susceptible to Salmonella infections.
Use of a Lactobacillus strain did not produce protection,
and this forced them to evaluate an unmanipulated
population of intestinal bacteria from adult chickens that
were resistant to the S. infantis. On oral administration
of this undefined mixed culture, adult-type resistance to
EDENS ET AL.
180
Salmonella was achieved. This procedure later became
known as the Nurmi or CE concept. The term “competi-
tive exclusion” was applied first in poultry by Lloyd et
al. (1974).
In traditional terms, CE in poultry has implied the
use of naturally occurring intestinal microorganisms in
chicks and poults that were ready to be placed in
brooder houses. Competitive exclusion products ex-
ploded onto the market after the initial report by Nurmi
and Rantala (1973); however, many of the products were
simply not effective in the control of potential enteric
pathogens. Therefore, for a short period of time,
development of probiotics for CE of potential pathogens
in chickens slowed. Nevertheless, research is now
widespread in this area, and potential new products are
being introduced regularly.
Many of the CE products can be applied to the
surface of embryonated chicken and turkey eggs;
however, little has been done to extend the concept of
CE to in ovo administration. Work published by Cox et
al. (1992b) was the first to show that the Nurmi concept
of CE could be extended to administration to hatching
embryos. Edens et al. (1991) also had demonstrated the
potential for the use of Lactobacillus reuteri for in ovo
administration for induction of CE in hatching chickens
and turkey poults.
It is known that many commercial hatcheries are
contaminated with Salmonella and many other enteric
pathogens that may limit CE activity in chickens and
turkeys (Cox et al., 1990, 1991; Goren et al., 1988). The in
ovo route of administration is an attractive method of CE
application because chicks and poults will hatch with an
intestinal tract colonized with beneficial bacteria.
CONCEPT OF COMPETITIVE EXCLUSION
The idea that intestinal bacteria played a role in
maintenance of health was originated by Metchnikoff
(1908) when he studied “lactic acid bacteria” in fer-
mented milk products and their use to increase lon-
gevity and maintenance of youthful vigor in humans.
His landmark publication sparked research efforts
around the world, and by the 1930s, evidence was
accumulating to show that normal intestinal microflora
inhibited the growth of intestinal pathogens.
However, Greenberg (1969) first used the term CE for
the situation in which one species of bacterium more
vigorously competed for receptor sites in the intestinal
tract than did another species in a report dealing with
total exclusion of Salmonella typhimurium from maggots
of blow flies. He observed that S. typhimurium would
only survive if there was a reduction or elimination of
the normal intestinal microflora. In 1971, van der Waaij
and associates used a synonymous term “colonization
resistance” when studying pathogen colonization in
mice.
Competitive exclusion against Salmonella can be
induced by exposure of day-old chicks to fecal and cecal
bacterial flora from adults. Indeed, in some European
countries, fecal and cecal contents are used to induce CE
in growing poultry (Wierup et al., 1988). Several
attempts have been made to isolate pure cultures of
protective microflora (Impey et al., 1982, 1984) and
mixed cultures (Impey et al., 1982, 1984; Stavric et al.,
1985; Stavric, 1987; Gleeson et al., 1989) to induce
competitive exclusion of Salmonella, but Mead and
Impey (1986) have shown that there are many problems
associated with this method because there is a decided
lack of adequate selective isolation media that would
permit the detailed analysis of the cecal microflora, a
lack of clear means to determine in vitro whether a
particular strain is protective, and the need to identify
all component cecal microorganisms.
MECHANISM OF
COMPETITIVE EXCLUSION
The mechanisms used by one species of bacteria to
exclude or reduce the growth of another species are
varied, but Rolfe (1991) determined that there are at
least four major mechanisms of CE. These mechanisms
are: 1) creation of microecology that is hostile to other
bacterial species, 2) elimination of available bacterial
receptor sites, 3) production and secretion of an-
timicrobial metabolites, and 4) selective and competitive
depletion of essential nutrients.
The microecology of the intestinal tract is the
determining factor in specific microorganismal viability.
The production of volatile fatty acids at a pH below 6.0
is known to decrease the populations of Salmonella and
Enterobacteriacea (Meynell, 1963). Disruption of the
normal intestinal microbial populations with antibiotics
will abolish this mechanism of CE because the concen-
trations of volatile fatty acids produced by the intestinal
bacteria will decrease and gut pH will increase toward a
more alkaline range. In newly hatched chicks, the
volatile fatty acid concentration and pH are not
sufficient to chemically exclude pathogens (Barnes et al.,
1979, 1980a,b).
In order for pathogens to attach to the intestinal
epithelium, there must be available sites for adhesion to
occur. The adhesion is mediated through the
polysaccharide-containing components attached to the
cell wall (Soerjadi et al., 1982). This cell wall component
mediates adherence of common bacteria to each other
and to the intestinal epithelium, preventing other
bacteria from attaching to the epithelium and effectively
blocking all receptor sites.
Antimicrobial substances produced and secreted by
natural inhabitants of the intestinal tract can either kill
or inhibit growth of pathogens (Rolfe, 1991). In this
capacity, research with the acid-loving Lactobacillus has
shown that as a group, they produce significant
amounts of bacterial growth inhibitory substances. Of
these, reuterin, a product of glycerol metabolism that is
secreted by L. reuteri, has broad-spectrum killing abilities
in the intestinal tract of chickens (Talarico et al., 1988).
SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION
181
The importance of L. reuteri as a CE agent is being
investigated by the authors of this review.
Competition for available nutrients as a means to
control intestinal bacterial populations is probably not
the most effective means for CE. Rolfe (1991) indicated
that there were many environmental factors that came
into play that either enhanced availability of nutrient
from the diet of the host or, through manipulation of
dietary ingredients, enhanced the growth of certain
microbial populations that may result in exclusion of
other bacterial species. However, we have shown that by
manipulating the lactose concentration in the diets of
chicks and poults, we can selectively provide an
advantage for the enhancement of L. reuteri (Casas et al.,
1993).
LACTOBACILLUS
AND GROWTH IN
DOMESTIC FOWL
Tortuero (1973) determined that Lactobacillus acidophi-
lus inoculation to day-old broiler chicks improved
weight gain, feed conversion, fat digestibility, and
nitrogen retention. Additionally, cecal weights were
reduced and the production of fecal material (the prime
source of carcass microbiological contaminants) was
reduced significantly. Along with these determinations
was the observation that intestinal microflora was
altered significantly in that there was a significant
increase in Lactobacillus colonies and a total decrease in
Enterococci colonies. The conclusion from this work was
that Lactobacillus intestinal flora could increase the
weight of the growing bird through a correction of fat
malabsorption. The observations by Tortuero (1973) that
L. acidophilus improved these growth performance
characteristics seemed to be even better when the birds
were raised on built-up litter. This observation sug-
gested that there was something in the litter that
interacted with the L. acidophilus to further improve
performance. The litter constituent is now known to be
related to the kinds of microbial flora that were shed
from birds previously on that litter and is now referred
to as CE.
Earlier work by Larousse (1970) indicated that L.
acidophilus inoculation resulted in equal or superior
growth performance in chicks given antibiotics as
growth promotants (Kol’cova, 1962; Tortuero, 1970). In
fact, the L. acidophilus colonies were increased even
further with antibiotic treatment.
Krueger et al. (1977) found that the addition of a
complex of Lactobacillus to the diets of laying hens
improved egg production and hatchability. Growth of
broiler chickens was improved when Lactobacillus cul-
tures were added to diets, and when suboptimal amino
acid composition of the diet was a confounding factor,
birds fed Lactobacillus cultures grew at a rate that was
equal to the growth of control broilers (Dilworth and
Day, 1978). In contrast, Watkins and Kratzer (1984) did
not find any advantage in the continuous use of a
Lactobacillus culture in the production of broiler chick-
ens, as there were no differences in body weight or feed
conversion.
Francis et al. (1978) and Potter et al. (1986) observed
increased growth in turkeys fed L. acidophilus and other
Lactobacillus cultures and zinc bacitracin, but the effect of
the two dietary supplements on the growth of the
turkeys was not additive. Nevertheless, both Lactobacil-
lus and zinc bacitracin decreased the coliform and total
aerobic counts in the feed and in the intestinal tract of
the poults, and zinc bacitracin also decreased intestinal
Lactobacillus colonies. Damron et al. (1981) did not find
any beneficial effects of L. acidophilus and other Lac-
tobacillus cultures in turkey breeder hens in terms of egg
production, body weight change, fertility, hatch of fertile
eggs, hatchability of all eggs set, or egg specific gravity.
LACTOBACILLUS
INTERACTION
WITH ENTERIC PATHOGENS
Watkins et al. (1979, 1982) reported that broiler chicks
inoculated with L. acidophilus were more resistant to the
pathogenic effects of E. coli. The addition of L. acidophilus
acidified the crop, cecum, and colon of the inoculated
chicks, and this acidification appeared to increase the
competitiveness of the L. acidophilus against the other
intestinal microflora. Watkins et al. (1982) found that
shedding of L. acidophilus increased from 34 to 76% but
did not increase further with subsequent dosing.
Nevertheless, prophylactic treatment with L. acidophilus
decreased the shedding of pathogenic E. coli 100 to 47%,
but this did not occur with therapeutic dosing with L.
acidophilus. Watkins and Miller (1983a) further suggested
that L. acidophilus increases competitive gut exclusion
against harmful organisms (S. typhimurium, Staphylococ-
cus aureus, and E. coli) in the intestinal tract, especially in
the crop but not to the same degree in the ceca and
colon, of the chicken, as suggested earlier by Fuller
(1973, 1977, 1978). Shahani et al. (1976) suggested that L.
acidophilus competed against other bacteria through the
production of a bacteriocin much like many other lactic
acid bacteria that produce bacteriocins for bacterial
exclusion (Klaenhammer, 1988). Watkins and Miller
(1983b) also demonstrated that L. acidophilus colonized
the epithelial cells of the gastrointestinal tract through a
close relationship and through actual physical attach-
ment.
Therefore, the colonization of lactic acid bacteria in
the chicken and turkey intestinal tract appears to have a
beneficial effect in these species. The benefit appears to
be associated with the production of bacteriocins of
some species and by reuterin (a metabolic product
secreted by L. reuteri), which aid CE of harmful and
pathogenic microorganisms (such as Salmonella, En-
terococci, and Escherichia).
Competitive exclusion against Salmonella can be
induced by exposure of day-old chicks to fecal and cecal
bacterial flora from adults. In Finland and Sweden, adult
fecal and cecal contents are used to induce CE in
growing poultry (Wierup et al., 1988), and in the last 2
EDENS ET AL.
182
yr, several European countries have allowed fecal and
cecal cultures for the CE of Salmonella. Several attempts
have been made to isolate pure cultures of protective
microflora (Impey et al., 1982, 1984) and mixed cultures
(Impey et al., 1982, 1984; Stavric et al., 1985; Stavric, 1987;
Gleeson et al., 1989) to induce CE of Salmonella, but
Mead and Impey (1986) have shown that there are many
problems associated with this method, as there is a
decided lack of adequate selective isolation media that
would permit the detailed analysis of the cecal
microflora, a lack of clear means to determine in vitro
whether a particular strain is protective, and the need to
identify all component cecal microorganisms. In 1995, an
effective continuous flow intestinal culture of 29 differ-
ent microbes was licensed by USDA for use as a CE
culture in the U.S. (Corrier et al., 1995a,b).
LACTOBACILLUS REUTERI
IN THE
NORMAL INTESTINAL MICROFLORA
Of all of the intestinal microflora of the avian
intestinal tract, the Lactobacillus genus predominates
similar to the condition found in the mammalian
gastrointestinal tract (Fuller, 1973, 1977, 1978; Kandler et
al., 1980; Soerjadi et al., 1981b; Sarra et al., 1985; Axelsson
and Lindgrin, 1987; Axelsson et al., 1989). In the chicken,
there are three predominant species of Lactobacillus (L.
reuteri, Lactobacillus salivarius, and Lactobacillus animalis),
but only L. reuteri has the potential to produce reuterin,
a clearly defined antibacterial substance that is an
intermediary metabolite of glycerol (Talarico et al., 1988,
1990; Axelsson et al., 1989; Chung et al., 1989; Talarico
and Dobrogosz, 1989, 1990). This observation is signifi-
cant as one considers the importance of Lactobacillus in
the process of CE of organisms such as the Salmonella,
Campylobacter, Listeria, Enterococci, and E. coli from the
intestine of the domestic fowl.
Reuterin, secreted by L. reuteri, has broad-spectrum
antimicrobial activity extending to the inhibition of at
least 25 different genera of prokaryotic microbial
pathogens (both Gram-negative and Gram-positive) and
to at least 10 different eukaryotic protozoan pathogens
frequently found in intestines of most mammalian and
avian species (Chung et al., 1989). It was proposed by
Dobrogosz et al. (1989) that L. reuteri, through its in vivo
ability to produce reuterin, confers an ecological advan-
tage allowing L. reuteri to play a modulating role in the
growth of all enteric microflora (Axelsson et al., 1989;
Chung et al., 1989; Talarico and Dobrogosz, 1989;
Talarico et al., 1988). It is known that: 1) L. reuteri cells
normally reside in the gastrointestinal tract of healthy
chickens, with the highest numbers found in the crop
and ceca (Casas, unpublished data). Inclusion of lactose
(whey) in the diet increases the number of L. reuteri
found in the ceca (Edens et al., 1991; Parkhurst et al.,
1991), and dietary lactose supplements have been
reported to reduce the numbers of Salmonella found in
the gastrointestinal tract of chickens and turkeys (Cor-
rier et al., 1990a,b; Hinton et al., 1990; Edens et al., 1991;
Parkhurst et al., 1991); 2) Lactobacillus reuteri cells are able
to convert the natural substrate glycerol into reuterin,
which is secreted by L. reuteri and has potent an-
timicrobial activity; 3) Reuterin in concentrations as low
as 10 to 30 mg/mL can kill Salmonella, Escherichia, and
Campylobacter (and other bacteria) within 30 to 90 min;
and 4) The number of chickens and turkeys that test
positive for Salmonella increases significantly when they
are harvested and delivered for processing.
THE
LACTOBACILLUS REUTERI
—
REUTERIN SYSTEM
The L. reuteri—reuterin system is a natural system of
broad-spectrum antimicrobial activity associated with
anaerobic metabolism of glycerol by L. reuteri (Axelsson
et al., 1989; Chung et al., 1989). A considerable amount of
background information on this system is now available.
Lactobacillus reuteri was first isolated by Lerche and
Reuter (cited by Kandler et al., 1980), but until 1983 it
was classified as Lactobacillus fermentum (Kandler and
Weiss, 1986). It is now a recognized species of obligately
heterofermentative Lactobacillus residing in the gastroin-
testinal tract of healthy humans, cattle, swine, poultry,
and other animals (Kandler and Weiss, 1986). It,
perhaps, is the dominant heterofermenter in this
ecosystem (Sarra et al., 1985). It has been shown that L.
reuteri is in all regions of the proximal gastrointestinal
tract (stomach to ileum) in nursed piglets within 1 to 2 d
after birth. In comparable colostrum-deprived piglets it
took approximately 2 wk for a similar colonization to
occur (Dobrogosz et al., 1989).
Reuterin has-broad spectrum antimicrobial activity.
Concentrations of 15 to 30 mg/mL inhibit growth of
Gram-negative and most Gram-positive bacteria, yeast,
fungi, and protozoa. Concentrations of reuterin 60 to 150
mg/mL are required to kill lactic acid bacteria, including
L. reuteri itself (Chung et al., 1989). It is unknown why
the latter group of microorganisms is more resistant in
this regard. It is suspected that this differential sensitiv-
ity may have ecosystem (i.e., the gastrointestinal tract)
implications.
Lactobacillus reuteri is the only bacterial species able to
produce and secrete reuterin. Reuterin has been isolated,
purified, and identified as an equilibrium mixture of
monomeric hydrated monomeric and cyclic dimeric
forms of 3-hydroxypropionaldehyde (3-HPA) (Talarico
and Dobrogosz, 1989). A purified and characterized
enzyme, B
12
-dependent glycerol dehydratase, is respon-
sible for conversion of glycerol into reuterin (Talarico
and Dobrogosz, 1990). Additionally, a nicotinamide
adenine dinucleotide (NAD
)
+
-dependent oxidoreductase
is responsible for reducing reuterin to 1,3-propanediol
(Talarico et al., 1990). These two enzymes constitute a
pathway by which L. reuteri cells can use glycerol as an
alternate hydrogen acceptor during carbohydrate fer-
mentation. Use of this pathway increases available
cellular adenosine triphosphosphate yields and growth
SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION
183
rates significantly above those observed in the absence
of glycerol (Talarico et al., 1990).
A few other bacterial species (e.g., Enterobacter) are
able to use glycerol in a similar manner. These species,
however, produce 3-HPA only as a transient metabolic
intermediate that is immediately reduced to 1,3-
propanediol. Lactobacillus reuteri is unique in its ability to
1) produce more 3-HPA than is reduced, and 2) secrete
the excess 3-HPA, thereby imparting antimicrobial
activity to the surrounding environment (Talarico et al.,
1990). Reuterin production is believed to occur in vivo in
the gastrointestinal (and perhaps genito-urinary) tract,
providing an ecological competitive advantage to L.
reuteri cells among other benefits to the host animals.
The L. reuteri—reuterin system is believed to play a role
in colonization resistance against potential pathogens.
Also, significantly deeper crypts and higher villi are
observed in the ileal regions of chickens supplemented
with 10
5
cfu L. reuteri per gram of feed than in
corresponding control birds (Casas, unpublished data).
Villus damage during weaning in piglets is ameliorated
by L. reuteri 1063 (a swine-specific strain) supplementa-
tion to sows and piglets during the weaning process
(Lindgren, 1990).
Reuterin is a natural metabolite, and it is nonmuta-
genic based on the Ames test. It exerts rapid killing and
appears to “self destruct” (most likely through cell
uptake followed by derivatization). Resistance to reute-
rin’s antimicrobial activity has not been detected
(Dobrogosz et al., 1989).
Smith and Tucker (1980) and Ziprin et al. (1989) have
reported that the newly hatched chick is very suscepti-
ble to Salmonella infections, but as the chick ages through
5 d posthatch its resistance increases. Ziprin et al. (1989)
concluded from their study that increasing resistance to
Salmonella infections could be attributed to development
of a competent T cell-dependent immune system;
however, this did not rule out the contributing effects of
acquired resistance through colonization of the intestinal
tract of beneficial microflora, such as L. reuteri, as well.
In fact, the increasing resistance to Salmonella infections
after hatch may well be dependent upon both the
development of the T cell-mediated immune response
and the acquisition of beneficial microflora, such as L.
reuteri, in the gut. The timing of these two independent
events would appear to be more than a simple
coincidence.
Because the newly hatched chick is very susceptible
to Salmonella infections and colonizations, many at-
tempts have been made to make the chick more resistant
to these potentially harmful non-host-specific Salmonella
(Impey et al., 1982, 1984; Stavric et al., 1985; Mead and
Impey, 1986; Stavric, 1987; Gleeson et al., 1989; Impey
and Mead, 1989). In addition to attempts to make the
bird more resistant to colonization, attempts are being
made to define the age- and environment-related factors
that influence the persistence and pathogenicity of many
organisms, especially the Salmonella (Gast and Beard,
1989).
Gast and Beard (1989) found that chicks inoculated
orally between 1 and 8 d posthatch with S. typhimurium
showed decreasing mortality with increasing age, but
the pathogenic organism persisted in the ceca of the
7-wk-old broiler. They also noted that the birds
inoculated at 1 d posthatch had greater numbers of S.
typhimurium cfu at 7 wk than did the birds inoculated at
8 d posthatch. Furthermore, S. typhimurium adhered to
the epithelium of the ceca in the birds inoculated at 1 d
more readily than in the birds inoculated at 3, 5, or 7 d
posthatch, but age did not affect the recovery of the
organism from the spleen or the liver. These observa-
tions by Gast and Beard (1989) strongly suggest that a
symbiotic relationship between the avian host and
protective microflora in the intestinal tract develops
after hatch so that S. typhimurium inoculation after 1 d
posthatch was less severe. Additionally, the age-related
decrease in mortality further indicated that some kind of
adaptation or developed resistance had occurred.
Soerjadi et al. (1981a,b) and Stavric et al. (1987) have
shown that resistance to Salmonella is developed only
when there is colonization of the intestinal tract with
beneficial microflora. Based upon the work by Gast and
Beard (1989) and others (Snoeyenbos et al., 1978, 1985;
Rigby and Pettit, 1980a), it is readily apparent that
beneficial organisms can induce CE of Salmonella.
Because there is a predominance of Lactobacillus in the
intestinal tracts of chickens that are capable of resistance
to Salmonella colonization, it was believed that the
Lactobacillus population was responsible (Fuller, 1977,
1978; Kandler et al., 1980; Soerjadi et al., 1981b; Sarra et
al., 1985; Axelsson et al., 1987).
However, an interesting observation by Soerjadi et al.
(1981b) indicated that a single application of a mixture
of two crop and cecal isolates of Lactobacillus ssp. into
the crop of newly hatched chicks did not prevent
Salmonella colonization in the cecum and only played a
minor role in CE of Salmonella in the crop. Barnes et al.
(1980b) also reported that Lactobacillus ssp. obtained
from sources other than the chicken host did not
establish colonies in the intestinal tract of chickens. More
importantly, Barnes et al. (1980b) reported that a mixture
of L. acidophilus, L. fermentum, and Lactobacillus salivarius
did not prevent Salmonella colonization, whereas a
complex mixture of cultured facultative anaerobes plus
anaerobes from adult chickens did prevent Salmonella
colonization. Additionally, Barnes et al. (1980b) made
another significant observation when they reported that
E. coli isolated from the same habitat as that of the
Lactobacillus ssp. survive when co-administered with the
Lactobacillus ssp. Barnes and Impey (1980) further
implied that organisms that are inhibitory for Salmonella
may also be inhibitory for other bacteria, thus disturbing
the ecological balance of gut microflora. Barnes and
Impey (1980) also suggested that Lactobacillus ssp. may
help to confer intestinal protection when applied
together with other organisms from a cecal suspension,
but can make the situation worse when added alone.
EDENS ET AL.
184
Because in the chicken there are three predominant
species of Lactobacillus (L. reuteri, L. salivarius, and L.
animalis), it seems reasonable to make the assumption
that it is these beneficial microorganisms that impart the
advantage against Salmonella that we term CE. Impey
and Mead (1989) are very clear as to their conviction
that it is Lactobacillus that provide the means of CE, as
they were able to show that administration of the adult
cecal cultures rich in Lactobacillus immediately arrested
the growth of Salmonella organisms.
With the discovery that L. reuteri is a major compo-
nent of the intestinal microflora of chickens and other
animals (Dellaglio et al., 1981; Sarra et al., 1985;
Dobrogosz et al., 1991; Edens et al., 1993), it appeared
that the population dynamics of L. reuteri might be
involved in establishing a favorable microecology in the
intestinal tract via its ability to secrete lactic acid, acetic
acid, and reuterin, which act as modulators of intestinal
tract microbial populations (Dobrogosz et al., 1991). The
earlier that L. reuteri is given to chicks and poults, the
more capable they were in developing CE potential
against Salmonella (Casas et al., 1993).
SITE(S) OF ACTION OF
LACTOBACILLUS
IN THE PROCESS OF CE
The site of Lactobacillus-induced inhibition of further
growth of the Salmonella organisms would appear to be
at the level of the mucosal region in the intestinal tract.
It is our hypothesis that it is the reuterin (Talarico et al.,
1988; Axelsson et al., 1989; Chung et al., 1989; Talarico
and Dobrogosz, 1989) produced by the L. reuteri, the
predominant Lactobacillus in the chicken (Talarico et al.,
1988), that would interact with the pathogens at this
level to inhibit further growth. Because it is the
adherence of Salmonella organisms to the mucosa that
predisposes the chick to colonization and infection, it
appears that the precolonization of the mucosa by
Lactobacillus is the initial step in establishing CE of the
pathogens. Lactobacillus must colonize the entire gas-
trointestinal tract in order for them to be effective in CE.
Gast and Beard (1989) and Impey and Mead (1989) have
shown that timing of Salmonella challenge to newly
hatched chicks is critical in the development of
salmonellosis. Although Gast and Beard (1989) did not
find a significant reduction in the frequency of recovery
of S. typhimurium from the liver and spleen of chicks
inoculated at different times posthatch, their data do
suggest that penetration may be affected by prior
exposure of the gastrointestinal tract to Lactobacillus.
This hypothesis would be in agreement with the
conclusion of Turnbull and Snoeyenbos (1974), who
found decreased penetration of Salmonella through the
intestinal mucosa of chicks of increasing age. Presuma-
bly, this phenomenon could be associated with the
increased numbers of Lactobacillus in the intestinal tract
and in association with the mucosa.
LACTOBACILLUS
INVOLVEMENT IN
REDUCED PATHOGENICITY OF
SALMONELLA
Factors that would violate the integrity of the mucosa
allow the invasion of pathogenic organisms such as S.
typhimurium more readily than if the mucosa were not
damaged. Chicks reared in cages and subjected to either
a coccidial challenge (Eimeria tenella), a S. typhimurium
challenge, or both were more susceptible to salmonello-
sis in the presence of E. tenella (Kosugi et al., 1986) There
is a greater recovery of S. typhimurium from the cecal
epithelium of gnotobiotic chicks with E. tenella infection
24 h after inoculation with S. typhimurium than in
control chicks (Fukata et al., 1987).
However, coccidiosis infection is not the only exacer-
bating factor that allows pathogenic organisms, such as
the Salmonella, to penetrate the mucosa and infect the
chicken. Low environmental temperature is a strong
inducer of salmonellosis in the chicken (Thaxton et al.,
1974). In fact, any factor that alters the body temperature
control of the chicken or turkey tends to make it more
susceptible to bacterial infections of all sorts (Pasteur et
al., 1878; Hess, 1887; Wagoner, 1890; Keyes, 1916; Fenn,
1922; Scholes and Hutt, 1942; Ram and Hutt, 1955; Hutt,
1958; Edens et al., 1984).
Rigby and Pettit (1980b) and Rigby et al. (1980)
demonstrated that the potential to increase the shed rate
of Salmonella was greatly increased as the result of
preparation for transport to market, catching and
loading in shipping crates, and transport to the slaugh-
ter house. In fact, the crates in which the birds were
transported became infected with Salmonella and re-
mained infected even after washing. Not only did the
rate of shed increase during transport, but chickens
found to be clean of Salmonella also showed infection
after arrival at the slaughter house. In this case, the
normally commensal relationship between the colonized
organism and the host had been severely interrupted,
allowing the organism to be shed to infect other,
formerly clean, animals. If Salmonella are to be persistent
in the environments in which poultry are to be grown, it
is important to make sure that the birds are exposed to
the beneficial bacteria, ostensibly Lactobacillus, that
would potentiate CE of the Salmonella, as soon as
possible.
The fact that Salmonella are persistent in the environ-
ment (Rigby and Pettit, 1980b; Rigby et al. (1980) points
out the importance of existing Salmonella and their role
in the infection of new flocks. Lahellec et al. (1986)
studied the role of resident Salmonella in broiler houses
at the time of placement of chicks and determined that
they were the most important in the developing
salmonellosis than those Salmonella isolated during the
rearing period. Soerjadi-Liem and Cumming (1984)
surveyed 20 broiler flocks in Australia and found that
the level of Salmonella-positive birds could range from 0
to 30%, but in flocks that had been reared on built-up
litter the incidence of Salmonella-positives was signifi-
cantly lower than from flocks reared on new litter. This
SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION
185
finding, once again, points out the necessity of early
seeding of the chicks with beneficial microflora such as
L. reuteri.
Another factor of consideration for the use of L.
reuteri to induce CE of Salmonella is that L. reuteri and
other members of the Lactobacillus family utilize lactose
readily in their metabolism. A study by Ofek et al. (1978)
indicated that residues of various sugars on the gut
epithelium may serve as receptors for the binding of
certain human and animal pathogens to the intestinal
mucosa. Ofek and Beachey (1980) and Ofek and Sharon
(1990) reported later that the interaction between the pili
and the carbohydrate is specific and that the interaction
could be blocked by a soluble form of the sugar. McHan
et al. (1989) studied the effect of selected sugars on the
attachment of S. typhimurium to the cecum of the chick.
Attachment of the organism was reduced by the
presence of N-acetyl-D-galactose, L-fucose, D-galactose,
L+ arabinose, and D+ mannose. Oyofo et al. (1989) also
found mannose to significantly reduce (73%) S. typhimu-
rium adherence to the ceca of chicks, and in addition
they determined lactose had a highly significant effect
(47% reduction) on adherence as well. Oyofo et al. (1989)
presented these sugars in the drinking water of S.
typhimurium-challenged chicks. Oyofo et al. (1989)
pointed out that mannose and lactose may act to inhibit
S. typhimurium attachment via different mechanisms.
Mannose may interact with mannose-sensitive type-1
fimbrae on the bacterium. Lactose on the other hand,
known to inhibit in vivo the growth of pathogens
(Schaible, 1970), may act by the enhancement of the
growth of Lactobacillus, which, in turn, inhibit the
growth of pathogens such as S. typhimurium (Oyofo et
al., 1989). This hypothesis would be in line with the
conclusion of Soerjadi et al. (1981a,b), who proposed that
members of the Lactobacillus family are responsible for
CE of pathogens from the gut of chickens. This
hypothesis would also be in concert with our hypothesis
that L. reuteri enhances CE in avian species, as it is the
predominant Lactobacillus in the chicken and turkey
intestinal tract.
Bailey (1988) pointed out that there was a need to
refine the CE techniques so that there would be a
realistic and practical chance to reduce, if not eliminate,
Salmonella from poultry production. Although he dis-
cussed CE at length and found many practical advan-
tages to the implementation of the procedure, there may
be serious drawbacks such as the lack of USDA and
FDA approval to use some of the existing techniques in
the U.S. He pointed out that it is not currently realistic
to think about imminent development of an anti-
colonization vaccine. Additionally, he suggested that
genetic resistance to Salmonella be bred into the commer-
cial poultry strains. The need to develop further CE
methodology centers on reports by Poppe (1984) and
Poppe et al. (1986), who determined that even with
chlorination of drinking water, the incidence of infection
from Salmonella of chicks in a contaminated environment
was not affected. Regardless of the chlorination treat-
ment of the drinking water, poults automatically
decreased their enteric populations of Salmonella be-
tween 14 and 21 d of age. The results from Poppe’s
study were in agreement with those of Al-Chalaby et al.
(1985), who found that drinking water sanitation failed
to inhibit the incidence of natural Salmonella in broiler
chickens. These results again suggest the development
of naturally occurring beneficial microflora of the gut,
presumably L. reuteri and other Lactobacillus species,
which predominate in the avian species.
Thus, it is apparent that new methodologies as-
sociated with the development of a workable CE
program are needed and are of potential value to the
poultry industry. Therefore, the use of the most
prevalent Lactobacillus, L. reuteri, in the chicken as a
model organism to promote CE in the broiler chicken
and turkey poult appears to have merit.
CURRENT
IN OVO
CE TECHNOLOGY
Incubation, hatching, and hatchery processing can be
a primary source of pathogen exposure for the chick or
poult, and this exposure can lead to early mortality.
Pathogens reside on the eggshell and in its membranes,
and these pathogens are widely disseminated during
hatching. Because there is a need to protect the chick
from eggborne pathogens, the industry has resorted to
the use of shell decontaminants and antibiotics given
either in ovo or at hatch. With the introduction of the
Nurmi Concept of CE (Nurmi and Rantala, 1973),
extensive research efforts are being made to develop CE
cultures for general use ex ovo as an application to
hatched birds ready to be placed in the brooder house.
However, little has been done to extend the concept
of CE to in ovo administration even though many
products can be sprayed in hatcher to achieve early
posthatch intestinal colonization in chicks and poults.
From the multitude of papers that have been published
on the concept of CE of avian intestinal bacterial
pathogens, only one paper, to our knowledge, has
extended the CE concept to in ovo application.
Cox and colleagues (1992) administered in ovo to
embryonated chicken eggs an undefined cecal culture of
bacteria. At hatch, the chicks were given S. typhimurium
in challenge doses ranging from 1,000 to 10 million cfu
per chick (Figure 1). Administration of a 1,000-fold
dilution of the cecal culture reduced the Salmonella
colonization in chicks given the 1,000 or 100,000 cfu
challenge doses but not at the 10 million cfu challenge
dose. Chicks given the 1,000,000-fold dilution of the
cecal culture showed a small suppression in Salmonella
colonization when challenged with 1,000 Salmonella cfu
but not at higher challenge doses.
In a second experiment, Cox et al. (1992) demon-
strated dose and delivery site effects of in ovo (air cell
and amnionic fluid) on chick hatchability. Dose of the
CE culture, ranging from undiluted to 1 million-fold
dilution, had a significant negative effect on hatchability
EDENS ET AL.
186
FIGURE 1. Inhibition of ex ovo intestinal colonization by Salmonella
typhimurium given as oral challenge doses of 10
3
,10
5
,or10
7
cfu per
newly hatched chick inoculated in ovo (air cell) with an undefined
bacterial culture (Untreated control vs 1,000-fold or
1,000,000-fold dilutions of the culture) from chicken ceca (from Cox et
al., 1992). The competitive exclusion treatments within a S. typhimurium
challenge dose with no common lower case letter differ significantly (P
≤ 0.05).
FIGURE 2. Effect on hatchability of 18 d chick embryos inoculated
with an undefined bacterial culture from chicken ceca administered as
undiluted (AC 0, AM 0), 1,000-fold diluted (AC 3, AM 3), or
1,000,000-fold diluted (AC 6, AM 6) culture material delivered into the
air cell (AC) or into the amnionic fluid (AM) (from Cox et al., 1992).
The different lower case letters associated with hatchability percen-
tages for the various competitive treatments indicate significant
treatment differences (P ≤ 0.05).
(Figure 2). The site of CE administration affected
significantly the hatchability rate. Air cell administration
reduced hatching rate but allowed Salmonella-resistant
chicks to hatch. However, when the CE mixture was
delivered below the air cell into the amnionic fluid, the
undiluted and the 1,000-fold dilution prevented any
hatching, and at 1 million-fold dilution there was less
than 50% hatch.
LACTOBACILLUS REUTERI IN OVO
STUDIES WITH CHICKEN AND
TURKEY EMBRYOS
Over the past 5 yr, we have been working with the
naturally occurring intestinal microorganism L. reuteri.A
considerable body of knowledge on L. reuteri has now
been amassed for publication from our research efforts.
In the arena of intestinal bacteriology, we have deter-
mined the following: 1) L. reuteri is the predominant
heterofermentative species of Lactobacillus that resides
symbiotically in the gastrointestinal tract of healthy
species of poultry and other vertebrates, including
humans; 2) L. reuteri confers protection against potential
pathogens, provides nutrients, affects intestinal morpho-
genesis, interacts with the host’s gut-associated lym-
phoid tissue and immune system, and is an essential
component of normal, beneficial microbiota in the
gastrointestinal tract; 3) L. reuteri produces reuterin (3-
HA), which has broad-spectrum antimicrobial activity;
and 4) L. reuteri in posthatch feed application improves
performance and decreases Salmonella presence in the
gastrointestinal tract.
In early 1990, we obtained hatching eggs severely
contaminated with an E. coli. Hatchability was consis-
tently around 40% and livability of the chicks was only
about 20%. We decided to test L. reuteri in ovo in these
contaminated eggs. Not knowing the best time of
administration, dose of administration, or site of ad-
ministration, we chose to administer 10
7
L. reuteri cells
per live embryo at incubation Day 18 (E18) and E19,
giving injections into the air cell, the amnionic fluid, and
directly into the embryo itself. Control embryos were
manipulated the same way, being swabbed in 70%
ethanol and being given a sham injection into the air
cell, amnionic fluid, or into the embryo itself. After the
in ovo inoculations, the L. reuteri-inoculated and control
embryos were placed into separate hatchers and were
allowed to hatch without additional interventions. As
soon as we determined that maximum hatch had
occurred on E21, hatchabilities of the chicks from the
four treatments were determined, and randomly from
each of the four treatments, 10 chicks were collected for
determination of cecal colonization of L. reuteri using a
patented methodology. A total of three separate experi-
ments were conducted for this early in ovo study with L.
reuteri and involved a total of 300 viable embryos per
treatment.
Lactobacillus reuteri administration allowed signifi-
cantly more chicks inoculated in ovo at both E18 and E19
to hatch than control uninoculated groups (Table 1). The
hatch rate of L. reuteri-inoculated embryos was approxi-
mately 60%, whereas uninoculated embryos hatched at a
rate less than 50%. There was a high rate of L. reuteri
colonization in the ceca of chicks inoculated at both E18
and E19, but none of the control chicks were found to
contain L. reuteri (Table 1). Although these chicks were
not retained for livability and growth studies, the data
indicated that for the first time it may be possible to
apply the Nurmi Principle to embryos in ovo as well as
to chicks ex ovo. These observations suggested that a
systematic study of in ovo application of L. reuteri was
SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION
187
TABLE 1. Effect of in ovo Lactobacillus reuteri on hatchability and cecal
colonization in broiler chickens exposed in ovo to Escherichia coli
A,B
Mean percentages in a row for a particular variable with no common superscript differ significantly (P ≤
0.001).
Hatchability Cecal L. reuteri positives at hatch
Day of incubation L. reuteri-inoculated Control L. reuteri-inoculated Control
(%)
18 60.8
A
49.1
B
75.0
A
0
B
19 60.0
A
46.2
B
87.5
A
0
B
TABLE 2. Dose and site (air cell or amnionic fluid) of in ovo
administration of Lactobacillus reuteri on percentage hatchability
of viable broiler chick embryos at 18 d of incubation
1
Means are from four hatchability trials with 100 viable embryos
per treatment per trial. (n = 400 embryos per treatment). No significant
difference among treatments (P ≥ 0.05).
Treatment Hatchability
1
(%)
Control 92.5
Air cell 10
4
cfu per embryo 96.2
Air cell 10
6
cfu per embryo 93.7
Air cell 10
8
cfu per embryo 91.8
Amniotic fluid
10
4
cfu per embryo 94.2
10
6
cfu per embryo 92.3
10
8
cfu per embryo 89.9
warranted. Nevertheless, based upon this observation
and the observations made by Cox et al. (1992), one must
weigh the effects of an undefined culture and the effects
of a well-defined single organism for use in ovo.
In an effort to further define the potential for use of L.
reuteri as an in ovo CE agent for chickens, a series of
experiments were conducted in which we used the same
protocol as described above. We used both air cell as
well as amnionic fluid administration of L. reuteri in
various dosages ranging from 10
4
to 10
6
cfu per embryo.
For the sake of convenience, we chose E18 or transfer
day for the administration of L. reuteri in ovo. Data from
these studies are summarized in Table 2. Hatching rate
of our control embryos was 92.5% over four trials. This
mean hatch rate is compared to air cell and amnionic
administration and to doses of L. reuteri in both sites. In
the air cell, hatch rate varied from 92 to 96% in the L.
reuteri chicks, and these hatch rates were not different
from the control hatch rate of 92.5%. Lactobacillus reuteri
in the amnionic fluid allowed hatch rates between 90
and 94%, and this was overall slightly less than that
noted with air cell application of L. reuteri. However,
these hatch rates from amnionic fluid administration of
L. reuteri were not significantly different from the
control hatch rate of 92.5%. These data indicated that L.
reuteri was a safe organism that could be administered in
ovo to chicken eggs without sacrificing hatchability
(Table 2).
The chicken responses to in ovo application of L.
reuteri were very encouraging, and a decision was made
to test turkey embryos as well. In each of eight trials
with E24 turkey embryos obtained from a commercial
hatchery, we included a vehicle control (phosphate-
buffered saline at pH 6.5) to test the vehicle effect on
hatchability. The treatment groups were control with no
violation of the eggshell, vehicle control, and L. reuteri
administered into the air cell in phosphate-buffered
saline in doses of 10
4
to 10
8
cfu per E24 turkey embryo.
Hatchability and livability are significant concerns
when one considers in ovo application of CE agents. In
Table 3, we show that hatchability is not reduced when
as many as 10
8
cfu L. reuteri are given in ovo to E24
turkey embryos, but 7-d livability was reduced when the
two highest L. reuteri doses were compared to controls.
Many of the poults that had received the L. reuteri in ovo
at the doses rate of 10
7
or 10
8
cfu showed some signs of
mild diarrhea and some dehydration prior to death,
indicating that there is an effective in ovo dose of L.
reuteri that will promote excellent hatchability and
livability (Table 3). Therefore, 10
6
L. reuteri cells was
chosen as a standard in ovo dose for subsequent studies.
The turkey embryo is also tolerant to L. reuteri given
in ovo in doses ranging from 10
6
to 10
8
cfu per embryo
(Table 4). These dose ranges were selected randomly to
test the colonization rates of the ceca from turkey poults
at hatch after being inoculated in ovo on E24. In ovo
inoculation on E24 promoted a significant increase in
total Lactobacillus in the cecum at hatch, and more than
33% of the treated embryos were L. reuteri positive at
hatch, whereas none of the controls were positive (Table
4).
There are two questions that must be answered to
make conclusions about a CE agent: 1) Will the CE agent
reduce the colonization rate of intestinal pathogens? and
2) Will there be improvement in the performance of the
flock after a CE agent has been applied? Shown in
Figure 3 are the results of a study in which L. reuteri was
given in ovo (10
6
cfu per embryo) to 400 broiler embryos
at E18 and hatched in a hatcher contaminated by
embryos seeded with S. typhimurium (ST-10, a naladixic
acid- novobiocin-resistant strain). In a separate hatcher,
another 400 embryos were exposed only to ST-10. To
accomplish the Salmonella exposure, four embryos were
given 10
3
cells ST-10 at the time of external pipping and
placed back into each of the two hatchers. At the time of
hatching, all hatchlings had a cecal ST-10 colonization
EDENS ET AL.
188
TABLE 3. Effect of Lactobacillus reuteri in ovo inoculation of turkey embryos at
24 d of incubation on percentage hatchability and percentage 7-d livability
a.b
In a column, mean percentages with no common superscript differ significantly (P ≤ 0.05). Each mean
represents percentage hatch of 800 embryos, and the percentage livability represents responses of the total
number poults hatched from the in ovo inoculated embryos.
Treatment Hatchability
7-d
Livability
(%)
Untreated control 98
a
97
a
PBS vehicle 89
b
95
a
L. reuteri,10
4
cfu 97
a
98
a
L. reuteri,10
5
cfu 94
a
98
a
L. reuteri,10
6
cfu 94
a
92
a
L. reuteri,10
7
cfu 92
ab
74
b
L. reuteri,10
8
cfu 95
a
77
b
TABLE 4. Effect of Lactobacillus reuteri given in ovo into the air cell of turkey embryos at 24 d of
incubation on cecal colonization of total Lactobacillus and L. reuteri in
hatching turkey poults removed directly from the hatcher
a–c
In a column, means with no common superscript differ significantly (P ≤ 0.05).
L. reuteri
Treatment Total Lactobacillus Positives
(Log
10
cfu per cecum) (%)
Control <1.0 × 10
2c
0
b
PBS vehicle <5.0 × 10
1c
0
b
L. reuteri,10
6
cfu 3.3 × 10
5b
>33
a
L. reuteri,10
7
cfu 1.2 × 10
6a
>33
a
L. reuteri,10
8
cfu 4.7 × 10
5b
>33
a
level of about 10
2
cfu/g. At 5 d after hatching, the chicks
exposed only to ST-10 had a cecal ST-10 population of
more than 10
7
cfu/g of cecum, whereas those given L.
reuteri plus ST-10 had cecal ST-10 populations of 10
6
cfu/g. This represented a one log difference or a 99%
reduction in the cecal population of ST-10 in the L.
reuteri-treated chicks. By 15 d the cecal ST-10 population
in those exposed only to ST-10 had decreased to a level
that was only slightly higher than the levels found in the
L. reuteri-treated chickens. The cecal level of ST-10
continued to decrease in both groups through 21 d,
when the cecal populations of ST-10 in both groups was
about 10
3
cfu/g cecum. These observations indicated
that L. reuteri does have the potential to decrease cecal
Salmonella loads but that it does not completely
eliminate the intestinal pathogen with a single in ovo
treatment.
A similar experiment has been conducted in which
turkey embryos were given L. reuteri (10
6
cfu per
embryo at E24) as an in ovo CE treatment along with in-
hatcher Salmonella (ST-10) exposure. At 1 and 5 d
posthatch, those poults given L. reuteri had fewer ST-10
cfu/g cecum than did the poults exposed only to ST-10
without L. reuteri treatment (data not shown). The
differences in these results were due to the presence of
L. reuteri, which, we believe, provided some advantage
to the microflora in the intestinal tract and reduced the
ability of S. typhimurium to find attachment sites or were
killed out right by the action of reuterin produced by the
L. reuteri.
The influence of time in the hatcher has to be taken
into account also. It has been reported from the field
that the longer a new hatchling remains in the hatcher,
the more likely that it can develop debilitating, often
times terminal, enteric diseases. In an effort to determine
whether L. reuteri would affect this phenomenon, a
study was conducted in which L. reuteri (10
6
cfu per
embryo) was given in ovo into the air cell of 400 viable
E24 turkey embryos in each of two hatchers. At the time
of external pipping, four embryos were given 10
3
cfu ST-
10 at the time of external pipping and placed back into
each of two hatchers, and the hatching process was
allowed to continue. As the poults hatched, they were
segregated into early-hatch (before 27 d) vs late-hatch
(27 to 28 d) poults. At E28, the poults were placed into
batteries according to the time of hatch and by
treatment. The data presented in Table 5 indicate that
early-hatching poults, which spent the longest time in
the hatcher before being placed in brooders, had the
greatest ST-10 load in the ceca, and this load was
significantly greater than that found in later-hatching
poults, which spent the least time in the hatcher.
However, at 5 d after hatching the cecal ST-10 levels had
increased in both groups and there was no difference
between early- and late-hatching poults exposed only to
ST-10.
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189
TABLE 5. Effect of Lactobacillus reuteri given in ovo (air cell) to viable turkey poult embryos at 24 d incubation on Salmonella
typhimurium (ST-10 via oral challenge to four externally pipped embryos per each of two hatchers), total Lactobacillus,
and L. reuteri cecal colonization at 1 and 5 d after hatching
a–c
Means in a column with no common superscript differ significantly (P ≤ 0.05).
S. typhimurium L. reuteri Total Lactobacillus
Treatment Day 1 Day 5 Day 1 Day 5 Day 1 Day 5
(Log
10
cfu/g cecum)
Control (ST-10 only)
Early hatch 7.1
a
9.0
a
0
b
0
c
4.6
a
8.1
a
Late hatch 5.3
b
8.6
a
0
b
<1.5
c
5.7
a
8.1
a
L. reuteri + ST-10
Early hatch <1.7
c
7.1
b
5.6
a
7.1
a
5.6
a
7.3
a
Late hatch <1.7
c
7.6
b
4.4
a
5.7
b
4.4
a
7.5
a
FIGURE 3. Effect of a single Lactobacillus reuteri dose given in ovo to
broiler embryos at 18 d of incubation and subsequently challenged at
external pipping via oral inoculation with 10
3
cfu Salmonella typhimu-
rium (ST-10, a naladixic acid-, novobiocin-resistant strain) on cecal ST-
10 levels from hatch through 21 d of age. Within an age, treatment
means with no common lower case letter differ significantly (P ≤ 0.05).
Poults given L. reuteri at E24 had significantly fewer
ST-10 cfu/g of cecum at hatch than did the poults
exposed only to ST-10. However, there was no differ-
ence between ST-10 cfu/g cecum in L. reuteri-treated
early- and late-hatching poults (Table 5). At 5 d after
hatching the ST-10 loads in the ceca of both the early-
and late-hatching L. reuteri-treated poults had increased
significantly, but the ST-10 cecal loads in these poults
were significantly less than that found in the poults
exposed to ST-10 alone.
In poults exposed to ST-10 alone, the cecal levels of L.
reuteri in both early- and late-hatching poults could not
be determined at 1 d after hatch (Table 5). At 5 d after
hatch, there was less than 1.5 cfu/g cecum of L. reuteri in
only the late-hatching poults, whereas none was found
in the early-hatching poults. Conversely, poults given L.
reuteri in ovo had L. reuteri in the ceca at levels of 5.6 and
4.4 cfu/g cecum in the early- and late-hatching poults,
respectively (Table 5). At 5 d after hatching, cecal L.
reuteri levels had risen to 7.1 and 5.7 cfu/g cecum in the
early- and late-hatching poults, respectively.
When the total Lactobacillus populations were deter-
mined, it appeared that neither early- nor late-hatching
had affected their total numbers in either the ST-10
exposed alone or in the L. reuteri plus ST-10 exposure
regimen (Table 5). However, in those poults given L.
reuteri at E24, the L. reuteri accounted for all Lactobacillus
at 1 d, and the majority of Lactobacillus at 5 d after hatch.
On the other hand, in those poults exposed only to ST-
10, Lactobacillus other than L. reuteri made up the total
cecal populations at both 1 and 5 d after hatching. These
data show that a single in ovo exposure can stimulate the
production of L. reuteri in the cecum of turkey poults,
but if L. reuteri is not made available early in the life of
the poult, a long period of time is required before L.
reuteri is able to colonize the cecum of the turkey. The
consequences of delayed L. reuteri intestinal colonization
could be early mortality from enteric pathogens or
morbidity and reduced performance.
The second criterion concerning the influence of a CE
agent is the performance of the flock. In this experiment,
we gave L. reuteri in ovo (10
6
cfu per embryo at E18) and
challenged the chicks at hatch with S. typhimurium (ST--
10, 10
3
cfu per chick). The broiler chickens were placed
on litter floors in isolation rooms and were provided
with starter, grower, and finisher diets from the North
Carolina Agricultural Research Service over a
40-d growing period. In this experiment, L. reuteri in the
form of GaiaFeed
was supplemented to the Salmonella
+ L. reuteri group at the feed application rate of 2% of
the diet from the time of placement in the isolation
rooms until the end of the experiment at 40 d of age.
The results of this experiment are presented in Table 6.
Within 6 d after Salmonella challenge, the mortality
rate in the Salmonella-challenged broilers had reached
34%, whereas in the L. reuteri-treated broilers, mortality
was only 6%. Mortality induced by the ST-10 used in
this experiment and others seemed to peak within the
first 5 to 7 d after challenge regardless of the poultry
EDENS ET AL.
190
TABLE 6. Effect of Lactobacillus reuteri (given in ovo to embryos at 18 d of incubation) plus
Salmonella typhimurium challenge at day of hatch on body weights and mortality
of broiler chickens at 6 and 40 d of age
a,b
In a row, means with no common superscript differ significantly (P ≤ 0.05).
1
Numerical differences between Salmonella only and L. reuteri + Salmonella treatments.
2
Number of chicks weighed in parentheses.
Treatment
Parameter Salmonella only L. reuteri + Salmonella Difference
1
6-d mortality, % 34
a
6
b
26
40-d mortality, % 41
a
9
b
32
6-d body weight, g 72
b
(297)
2
107
a
(423) 35
40-d body weight, g 1,728
b
(266) 1,934
a
(410) 206
TABLE 7. Protective effect at 22 d of age of air cell application
of Lactobacillus reuteri in broiler chicken embryos
co-administered Gentamicin
into the amnionic fluid at Day 18
of incubation followed by in-hatcher exposure to an
enteropathogenic Escherichia coli given to four embryos
at the time of external pipping
AB
Means in a column, with no common superscript differ
significantly (P ≤ 0.01).
Treatment Body weight Mortality
(g) (%)
Absolute control 842
b
1.42
b
E. coli-challenged
Control 803
b
9.52
a
Gentamicin
(0.2 mg per embryo) 819
b
4.26
b
L. reuteri (10
6
cfu per embryo) 874
a
3.56
b
L. reuteri + Gentamicin
882
a
0.00
b
species being studied. By 40 d after ST-10 challenge,
mortality reached 41% in the Salmonella controls and
was only 9% in the Salmonella + L. reuteri group. These
data are indicative that L. reuteri, used as a CE agent in
ovo and in combination with L. reuteri feed supplementa-
tion after hatching, can provide significant protective
benefits against a strong enteric pathogen such as this S.
typhimurium strain.
At 40 d of age, L. reuteri treatment allowed for an
increase in body weight of 206 g (Table 6). These data
suggest strongly that there is an advantage to providing
L. reuteri as a CE agent via the in ovo route coupled with
posthatch application in the feed, and the sooner that
this organism is given to the chick or poult, the better its
posthatch performance will be.
In development of L. reuteri as an in ovo CE agent, we
wanted to be as practical as possible. Therefore, studies
were conducted in which Gentamicin
, an antibiotic
used in ovo and ex ovo in both chickens and turkeys, and
L. reuteri were co-administered in embryos that were
exposed in-hatcher to an enteropathogenic E. coli (Table
7). There were five treatment groups in this experiment
and these consisted of 1) Absolute Control with no L.
reuteri, E. coli, or Gentamicin
; 2) Control: E. coli-
challenged; 3) Gentamicin
(0.2 mg per embryo) + E.
coli-challenged; 4) L. reuteri (10
6
cfu per embryo) + E. coli
challenge; and 5) L. reuteri (10
6
cfu per embryo) +
Gentamicin
(0.2 mg per embryo) + E. coli challenge.
The results (Table 7) of this study indicate that 1)
Gentamicin
and L. reuteri reduce E. coli-associated chick
mortality, 2) the combination of L. reuteri and Gen-
tamicin
had an additive effect by further decreasing E.
coli-associated mortality, and 3) body weights more
uniform and were increased significantly by L. reuteri +
E. coli and the L. reuteri + Gentamicin
+ E. coli
combination in comparison to the Absolute Control, E.
coli Control, and the Gentamicin
+ E. coli group.
The in ovo administration of L. reuteri or Gentamicin
or both to chicks and poults had significant positive
effects. Oftentimes, we wonder how these effects are
exerted. Do antibiotics simply kill off populations of
bacteria? Do the CE agents simply cover all attachment
sites on the intestinal epithelium? Do the CE agents
produce enormous volumes of bacteriocins or other
antimicrobials which either inhibit bacterial growth or
act as bactericidals? In each case, I believe the question is
answered with an enthusiastic “Yes”.
Casas et al. (1993; unpublished data) have demon-
strated that L. reuteri has a positive effect on the
morphology of the intestinal tract. They have demon-
strated that the ileum villus height was increased
significantly by L. reuteri and that crypt depth was
increased as well. We have seen this posthatch event on
many occasions, and it is consistent in broiler chickens
and in turkey poults (unpublished observations).
However, at no time have we looked at the effect of in
ovo application of L. reuteri on the general morphology
of the ileum villus. In another study with co-
administration of L. reuteri and Gentamicin
, we did
observe with the use of scanning electron microscopy
that at the time of external pipping, before S. typhimu-
rium (ST-10 at 10
3
cfu per chick at hatch), L. reuteri had
no negative effects on the tall, cylindrical-shaped
morphology of the ileum villus (Figure 4). However, in
ovo administration of Gentamicin
into the amnionic
fluid appeared to shorten and blunt the ileum villus
morphology at the time of hatching. The co-
administration of L. reuteri with Gentamicin
appeared
to reverse the shortening and blunting effect of Gen-
SYMPOSIUM: CURRENT ADVANCES IN AVIAN EMBRYOLOGY AND INCUBATION
191
FIGURE 4. Scanning electron micrographs of villi in the ileum of externally pipping chicks at 21 d of incubation afterin ovo treatment at 18 d of
incubation with L. reuteri (10
6
cfu per embryo, in the air cell), Gentamicin
(0.2 mg per embryo, in the amnionic fluid), the combination of
Lactobacillus reuteri into the air cell plus Gentamicin
into the amnionic fluid, or untreated Controls.
tamicin
and returned the hatching chick ileum villus
morphology to a normal tall, cylindrical shape (Figure
4).
After hatching, we challenged the chicks with S.
typhimurium (ST-10 at 10
3
cfu per chick at hatch) and
determined the effects of L. reuteri, Gentamicin
, and
posthatch Salmonella infection on growth of the villi and
the number of goblet cells per villus at 1, 3, and 6 d
posthatch by staining the ileum sections with periodic
acid Schiff to visualize the goblet cells, which stain a
bright red under this histochemical protocol (Figure 5).
There was an approach toward a plateau number of
goblet cells per villus as the chicks reached 3 to 6 d of
age in control conditions without ST-10 challenge. The
exception to this assessment was that the Gentamicin
-
treated chicks showed a small increase in goblet cell
numbers at 6 d of age. This increase was attributed to
the diminished effect of Gentamicin
and to the
possibility that some pathogenic organisms may have
been colonizing the intestinal tract at this time.
However, with Salmonella infection, there is an
indication that in the Gentamicin
group, there was an
induction of a goblet cell proliferation by 3 d, and that at
6 d, with development of maximal Salmonella numbers
in the ileum, Control, and Gentamicin
-treated chicks
had significantly larger goblet cell numbers than were
found in L. reuteri or the L. reuteri + Gentamicin
treatments. These data show that there was a significant
effect of microbial populations (good and bad organ-
isms) on the morphology of intestinal villi, and that
there was an antibiotic effect, although it was expressed
differently.
The shortened and blunted appearance of the ileum
villus in the Gentamicin
-treated groups did not correct
itself over the 6 d after hatch. In fact, with ST-10
challenge at hatch, the Control ileum villi also became
shortened and blunted. In those chicks that had been
EDENS ET AL.
192
FIGURE 5. Effect of in ovo Lactobacillus reuteri, alone and in combination with Gentamicin
, on the expression of goblet cells per ileum villus as
influenced by Salmonella typhimurium challenge to the broiler chicks after hatching.
treated with L. reuteri, and in the combination of L.
reuteri + Gentamicin
with ST-10 challenge at hatch, the
ileum villus retained its tall, cylindrical morphology
through the 6 d posthatch experimental period (micro-
graphs are not presented). We believe that the L. reuteri
in ovo (air cell) treatment had some prolonged beneficial
effects and that the posthatch ST-10 infection was
depressed by the presence of L. reuteri and Gentamicin
.
With the diminished effects of the single Gentamicin
in
ovo treatment around 3 d posthatch, ST-10 colonization
increased and, as a result, goblet cell numbers per villus
increased and the shortened, blunted appearance of the
ileum villus had no opportunity to return to normal.
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