Note: Descriptions are shown in the official language in which they were submitted.
WO 96/04364 21961 0Q PCT/US95/09656
SACCI3AROMYCES TREATMENT TO DIMINISH CAMPYLOBACTER
AND SALMONELLA POPULATIONS IN POULTRY
BACKGROUND OF THE INVENTION
u
Field of the Invention
This invention relates to a method for reducing the presence
of human enteropathogenic bacteria in poultry.
Description of the Related Art
The consumption of improperly prepared poultry products has
resulted in numerous cases of human intestinal diseases. It has
long been recognized that Salmonella spp. are causative agents of
such diseases, and more recently Campylobacter spp., especially
Campylobacter jejuni, has also -been implicated. As many as two
million cases of salmonellosis occur annually in the United States
(Stavric et al., Journal of Food Protection, Volume 56, No. 2, 173-
180, February 1993); twice as many cases of campylobacteriosis are
thought to occur (Krienberg et al., Food Technology, pages 77, 80,
81, and 96, July 1987). Both microorganisms may colonize poultry
gastrointestinal tracts without any deleterious effects on the
birds and, although some colonized birds can be detected,
asymptomatic carriers can freely spread the microorganisms during
production and processing, resulting in further contamination of
both live birds and carcasses. Poultry serves as the primary
reservoir for Salmonella and Campylobacter in the food supply (Jones
et al., Journal of Food Protection, Volume 54, No. 4, 259-262,
April 1991; Jones et al., Journal of Food Protection, Volume 54,
No. 7, 502-507, July 1991) . The intestinal contents of chickens
may harbor up to 10' Campylobacter and/or Salmonella per gram, and
cross contamination during processing is frequent (Oosterom et al.,
Journal of Food Protection, Volume 46, No. 4, 339-344, April 1983)
Studies have demonstrated that fecal material constitutes the major
source from which edible parts of chickens are contaminated in
processing plants. Therefore, to significantly reduce the level
of contamination on processed poultry, pathogen-free or nearly
pathogen-free- birds must be delivered to the processing plant,
(Bailey, Poultry Science, Volume 72, 1169-1173, 1993).
Better control measures -are needed to minimize the spread of
these and other human enteropathogenic bacteria; and the most
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promising approach to achieve this end has been to decrease the
incidence and level of colonization by these microorganisms in
poultry gastrointestinal tracts. To date, the most effective means
for controlling Salmonella colonization is competitive exclusion
(CE). Although theexact mechanism of CE protection is unclear,
it is likely to be in-fluenced by factors such as pH, Eh, production
of inhibitory substances such as H~S, bacteriocins, fatty acids,
and conjugated bile acids; competition for nutrients and receptor
sites; and local immunity (Mead et al., Letters in Applied
Microbiology, Volume_10, 221-227, 1990). Comp"etitive exclusion
treatment involves introduction of intestinal flora from pathogen-
free adult birds into newly hatched chicks. A study by Nurmi et
al. (Nature, Volume 241, 210-211, January 19, 1973), first reported
the use of the competitive exclusion technique. The reference
discloses inoculation.of 1, to. 2 day old chicks, by oral gavage with
a 1:10 dilution of normal intestinal contents from healthy adult
birds. One day lates, the chicks were challenged with Salmonella.
After 8-22 days, the birds were examined for the presence of
Salmonella. It was found that only 33% of the treated birds were
colonized with Salmonella whereas 100% of the untreated birds were
colonized with Salmonella. Originally, a suspension of crop and
intestinal tract materials obtained from healthy, adult birds was
used. _In later studies, cecal content was cultured anaerobically
in a liquid medium. It was found that preparations of subcultured
intestinal contents,from healthy, adult birds conferred protection
to young chicks whose intestinal or qut microflora had not yet been
established. Administrationof undefined CE preparations to chicks
speeds up the maturation of the gut flora in the newly-hatched
birds and also provides a substitute for the natural process of
transmission of micro.flora from the adult hen to its offspring.
Snoeyenbos et al., U.S. Patent No. 4,335,107 (1982) developed a
technique designed to reduce salmonellae in poultry where the
source of CE microfI,ora was lyophilized fecal. droppings which were
propagated by anaerobic culture. Mikkola et al., U.S. Patent No.
4,657,762 (1987) discloses the nse of_intestinal, fecal and cecal
contents as a sourGe of,,,CE microflora. Treatment with their
culture required _media to.be anaerobic and,pH balanced. Neither
of these CE treatments addressed.Campylobacter.
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Since CE was known to be effective against Salmonella, a
similar method for the control of Campylobacter was investigated by
Stern et al. (Avian Diseases, Volume 32, 330-334, 1988). It was
found, however, that treatment with CE preparations such as
described by Nurmi et al. (1973), Snoeyenbos et al. (1982) and
Mikkola et al (1987), did not affect Campylobacter colonization.
After treatment with five different CE cultures, colonization was
observed after challenge by Campylobacter in 81 of 84 chicks, and
45 of 46 control chicks. Shanker et al. confirmed these
observations (Epidemiol. Infect., Volume 104 101-110, 1990). Stern
and Stern et al. (Poultry Science, Vol. 73, 402-407, 1994)
disclose a mucosal competitive exclusion
(MCE) method and preparation effective against Campylobacter and
'Salmonella, using an anaerobic culture of the mucin layer scraped
'from cecal epithelia and an anaerobic culture of a cut piece of the
washed ceca. This undefined MCE culture contains a diversity of
flora that successfully competes with Salmonella and diminishes
levels of Campylobacter in the chick.
The treatments discussed above all relate to the use of
undefined mixtures of organisms obtained from cecal contents or
cecal wall scrapings which are subcultured. While these undefined
cultures have generally proven to be effective in reducing
colonization of chickens with foodborne pathogens, there are
concerns regarding their safety since there is the possibility of
transmission of etiological agents associated with human foodborne
disease and/or the transmission of avian disease. Because of
the safety concerns and difficulties in standardizing the bacterial
cdmposition and/or efficacy of undefined CE cultures, there is a
need to develop defined compositions which exhibit the potency of
undefined culture in order to diminish the presence of human
enteropathogenic bacteria in poultry. Stavric et al. (Journal of
Food Protection, Volume 56, No. 2, 173-180, February 1993) disclose
that the formulation of effective defined cultures is difficult
because of insufficient knowledge of the underlying protective
mechanism(s) and interactions between gut microflora. Furthermore,
the reference discloses, there is a lack of a sound scientific
basis :for the selection of potentially protective strains. To
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date, defined cultures of single isolates of Clostridium spp.,
Streptococcus faecalis, Bifidobacterium spp., and Bacteroides
hypermegas have been examined. Furthermore, preparations
containing several strains of single species, such as
Bacteroides spp., Bifidobacterium spp., and Escherichia spp. have
also been evaluated. None of these CE preparations consistently
protect chicks against Salmonella challenge. The reference states
that there has been one report claiming mixtures of lactobacilli
protect poultry against Salmonella colonization.
Stavric further reports that the use of probiotics containing
one to eight bacterial strains of different genera failed to
protect poultry against Salmonella, although the data is limited.
Studies with larger numbers of bacterial strains from different
genera have shown limited success. Therefore, it was surprising
=to find that the present invention, which is a defined CE
preparation of yeast, reduced the populations of Gram-negative
enteropathogenic Campylobacter and Salmonella in poultry.
Summary of the Invention
An embodiment of the present invention is to provide a
defined competitive exclusion composition to reduce pathogen
colonization in poultry.
An embodiment of the present invention is to provide a
defined competitive exclusion composition which diminishes the
presence of Campylobacter and Salmonella in poultry.
An embodiment of the present invention is to provide a
method for reducing pathogen colonization in poultry.
An embodiment of the present invention is to provide a
method for diminishing the presence of Campylobacter and
Salmonella in poultry.
An embodiment of the present invention is to provide a
method for reducing levels of colonization of human
enteropathogenic bacteria in poultry comprising administering
a defined competitive exclusion preparation consisting of
Saccharomyces boulardii ATCC 74352 in amounts effective to
reduce levels of colonization of human enteropathogenic
bacteria in poultry.
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An embodiment of the present invention is to provide a
method for reducing levels of colonization of human
enteropathogenic bacteria in poultry comprising administering
by oral gavage a defined competitive exclusion preparation
consisting of Saccharomyces boulardii ATCC 74352 in amounts
effective to reduce levels of colonization of human
enteropathogenic bacteria in poultry.
Further aspects, embodiments and advantages of the
invention will become apparent from the following description.
Detailed Descrintion of the Invention
The importance of enteric infections in humans has been
increasingly well recognized over the last dozen years. The
relationship of poultry contamination and human infection has,
likewise, become well documented. The ability to. diminish this
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=
health hazard by interventions at poultry processing plants is also
well known. During broiler production and processing, fecal
materials containing pathogens are _transferred onto meat and
persist into the food processing kitchens.
The application of yeasts as competitive-exclusion microflora - --
for the reduction of pathogen colonization in poultry has been
discovered. _It was surprising that different species and strains
of Saccharomyces diminish the populations of gram-negative
enteropathogenic bacteria such as Campylobacter and Salmonella. S.
boulardii is a non-pathogenic yeast originally isolated growing on
lychee fruit in Indochina in the 1920s (Surawicz et al.,
Gastroenterology, Volume 96, 981-988, 1989). Since 1962, it has
been used in several countries to treat antibiotic-associated
diarrhea in humans. It has been used widely in Europe and is under
study in the United States for treatment of patients whose
intestinal microflora has been compromised by intensive antibiotic
therapy (Surawicz et al., American Journal of Gastroenterology,
Volume 84, 1285-1287, 1989; Gastroenterology, Volume 104, A7.86,
1993). Often in these patients, antibiotic resistant pathogens
take advantage of the lack of competing organisms and colonize the
intestines causing severe_-_and sometimes fatal diarrhea.
Administration of S. boulardii prevents toxin formation by Gram-
positive Clostridium difficile (Buts et al., Journal of Pediatric
Gastroenterology and Nutrition, Volume 16, 419-425, 1993) and
reduces the concentrations of several aetiological agents of diarrhea
(McFarland et al., Microbial Ecology in Health and
Disease, Volume 6, 157-171, 1993).
Conditions are quite different in the ceca of a chicken than
in the intestines of a human since the presence of Salmonella and
Campylobacter species does not often result in a diseased bird.
There are several attributes of S. boulardii which indicate it has
potential as a competitive inhibition composition in poultry.
First S. boulardii is rather thermophilic with an unusual optimum
growth temperature of 37 C. It therefore is able to withstand the
higher body temperature of birds which is about 41.5 C for
chickens. Second, the yeast has been shown to survive gastric acid
in the stomachs of mammals to reach the intestines (Bluehaut et --
al., Biopharmaceutics and Drug Disposition, Volume 10, 353-364,
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WO 96104364 219'610O PCT/US95109656
i
1989), which indicates that it might survi,ve passage through the
crop, proventriculus, and gizzard of chickens to reach the
intestines and ceca. Third, it has demonstrated antagonistic
activity in vitro and in vivo against various bacterial pathogens
(Elmer et al., Antimicrobial Agents and Chemotherapy, January 1987,
pp. 129-131); and last, S. boulardii can survive either aerobically
or anaerobically, potentially making the culture and administration
of the organism easier and more reliable than anaerobic cultures.
The method of this invention is applicable to any avian animal
whether domestic oF: wild and particularly to poultry that are
raised for human consumption which could serve as carriers for the
target pathogens. Poultry includes all domestic fowl raised for
eggs or meat andõ includes chickens, turkeys, geese, ducks,
pheasants, and the like.
The target pathogens include all human enteropathogenic
bacteria capable of colonizing poultry. Of particular interest are
Salmonella and Campylobacter species
Yeast includes any species and strains ofõ Saccharomyces such
as S. boulardi.i, S. cerevisiae, S. carlsbergensis, S. elZipsoideus, S.
intermedius, for example, and of particular interest are
Saccharomyces boulardfi and S. cerevi,siae. _
Poultry is treated by administering an gffective amount of
yeast. The yeast can be administered by oral ,gavag,e, in drinking
water, in feed, by spraying newly hatched chicks with an aqueous
suspension, or a combination of the above. Yeast treatment is most
effective if administ:ered as.early and as frequently as possible.
For example, an effective amount of yeast in an aqueous suspension,
is sprayed on birds when they are 50-75% hatched, followed by
completion of the incubation period. In chickens, for example,
hatching trays can be removed from the hatching cabinet after the
eggs have been incubated in a setter for 16 days and in a hatching
cabinet for about Z.5 days, and each tray is sprayed so that each
hatching chick and/or unhatched egg rece,iveS the yeastõpreparation.
The hatching trays are then returned to the hatching cabinet to
complete incubation.._
~
After the bird-s hatch, an effective amount of yeast can be
added to the birds'_ first drinking water and is left in place until
all has been consumed. In chickens, for example, an approximately
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about 1:200 dilution of yeast is placed in 1-gallon drinker jars
which are placed in a broiler house at a ratio of approximately 1
jar per 200 chickens. The jars are left in place until all the
water has been consumed (approximately 4 hours), resulting in
consumption of approximately about lOml of diluted yeast solution
per chick.
Alternatively, the preparation may be effectively administered
by adding approximately about 5% by weight of a freeze-dried or
encapsulated yeast preparation to feed, injecting in ovo, spraying
directly on chicks after all the chicks are pipped, or by
administering through the farm water system.
The following examples are intended only to further illustrate
the invention and are not intended to limit the scope of the
invention as defined by the claims.
Exam lg e 1
Preparation of the Yeast Competitive Exclusion Culture
Saccharomyces boulardii (s.b.), accession number ATCC 74012,
was obtained from the American Type Culture Collection (12301
Parklawn Drive, Rockville, Maryland 20852-1776)
Saccharomyces cerevisiae (s.c.) is used as purified baker's
yeast from Fleishmann, Fleishmann's RAPIDRISET"yeast.
YEAST IN DRINKERS
One colony of yeast is inoculated per lOml of sabouraud
dextrose broth (SDB, Difco, Detroit, MI) and cultured at 30"C for
24 hours. Each lOml culture was poured into bottles containing SDB
(500m1 each) and allowed to grow at 30 C for 48 hours. The
cultures were then pelleted by centrifugation at 1000xg for 10
minutes. Each culture is separately suspended in 2000ml of water
for drinker bottles.
YEAST GAVAGING SOLUTIONS
. Eight petri dishes containing sabouraud dextrose agar (SDA,
Difco, Detroit, MI) are surface swabbed with a cotton-tipped swab
inoculated with either S. boulardii or S. cerevisiae. The cultures
are incubated at 30 C for 24 hours and each harvested with a cotton
tipped swab into 6m1 of phosphate buffered saline (PBS) 0.2m1 of
the solution were administered perorally to each chick with a
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+. , . , .
rubber-tipped needle. The yeast solutions were enumerated by
plating serial 10-fold dilutions onto SDA and colony forming units_
(CFU) determined.
YEAST IN FEED
Dried baker's yeast is mixed in the feed at 5% by weight.
Baker's yeast is also mixed in the drinking water using 10
grams/2000m1 of water.
Examole 2
Test for Yeast Efficacy Aaainst Camgvlobacter
50 day-of-hatch chicks were purchased from a local hatchery
and placed in groups of 10 into 5 separate isolation units equipped
,with nipple drinkers and a filtered air supply. One group was
:given 0.2 ml of a S. boulardii preparation, as described above in
Example 1, by oral gavage daily for seven days. A second group
received Saccharomyces cerevisiae in the same manner. A third group
received a freeze-dried preparation of S. cerevisiae mixed in feed
to make a preparation of approximately about 5- yeast in feed. The
fourth group received no treatment (positive control). The fifth
group received no treatment (negative control). On day 5, each
chick, except those in the negative control group, received, by
oral gavage, approximately 3.2 x 105 Campylobacter cells which was
a mixture of three strains of Campylobacter originally isolated from
chickens. In addition to daily gavage yeast treatment or feed
supplement, the yeast was also added to the drinking water as
described in Example 1. Stir plates were used to keep the yeast
suspended as it was delivered through nipple drinkers to the
chicks. On day 8, the chicks were killed by cervical dislocation
and individually weighed. Their ceca were aseptically removed and
placed in small stomacher bags. The ceca and contents were diluted
1:4 in phosphate-buffered saline and blended for 30 seconds. The
/ suspensions were serially diluted and plated on CEFEX agar for
recovery of Campylobacter spp. (Stern et al., Journal of Food Protection,
Volume 55, 663-666, 1992).
The plates were incubated under a microaerobic
environment at 42 C for 24 hours prior to enumerating Campylobacter
colonies.
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The results are presented in Table 1 below.
TABLE 1
D MrN GH,D - M v OBA TER OTONT ATTON O Hr K NG DUE TO
~
YEAST TREATMENT
Group Description Mean Log Difference, # Colonized
Campylobacter/g Positive Birds/10
Ctrl
1. S.b. gavage 2.58 4.57 4
2. S.c. gavage 5.29 1.86 8
3. S.c. 5% in feed 4.82 2.33 7
4. Positive control 7.15 0 9
5. Negative control <10 ---- 0
A reduction in Campylobacter levels of 4.6 log was noted in
chicks receiving S. boulardii by oral gavage as compared to chicks
receiving no yeast treatment. Chicks receiving S. cerevisiae by
oral gavage or in the feed showed reduced Campylobacter levels of
1.9 and 2.3 log respectively. Not only were the Campylobacter
populations reduced, but, perhaps more importantly, the incidence
of colonization was also reduced. Nine out -of ten of the positive
control birds were found to have Campylobacter in their ceca while
only 4 out of the 10 birds receiving S. boulardii were contaminated
by Campylobacter. The birds treated with S. cerevisiae also showed
reduced incidence of Campylobacter.
Examole 3
Test for Yeast ffS-3 \ Adaina S"lmon 77~
Chickens were obtained as in Example 2 above (30, day-of-
hatch) and placed in groups of 10 into three isolation units.
Positive control, negative control, and a treatment group were
designated. The treatment group received 0.2ml of S. boulardii by
daily oral gavage as described in Examples 1 and 2. Positive
control and treatment groups were challenged on day two with
approximately 1 x 106 Salmonella typnimurium (with induced resistance
for nalidixic acid) per bird. Daily yeast treatments as described
~ above in Example 2 were continued until day 8 when the chicks were
sacrificed and sampled as in Example 2. Salmonella colonization
was determined by plating serial dilutions on brilliant green sulfa
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~
(BGS) agar containing nalidixic acid. Results are presented below
in Table 2.
TARLE 9
_ --- 2 DIMINISHED CAMPYLOBACTER COLONIZATION OF CHr .KENS DUE TO
YEAST TREATMENT
Group Description Mean Log Difference, # Colonized
Campylobacter/g Positive Birds/10
Ctrl
1. Positive controlT 5.08 0 10
2. Negative control-,_ <10 0
3. S.b. gavage 3.36 1.72 7
A reduction in Salmonella colonization levels of 1.7 log was
observed in chicks_treated with S. boulardii as compared to the
positive controls_ .:The negative control birds showed no evidence
of Salmonella contamination as expected. Furthermore, an increase
in weight gain was observed in birds treated. with yeast as compared
to controls. The results are presented in Table 3 below.
TABLE 3
Group Description Mean Bird Weight (g)
1. Positive control 138.7
2. Negative control__ 141.4
3. S.b. gavage 147.5
If this weight gain persists through the life of the bird,
another benefit of th3s treatment will be increased production.
The foregoing __detailed description is fox the purpose of
illustration. Such detail is solely for that purpose and those
skilled in the art._ can make vaiiations therein without departing
from the spirit and scope of the invention.
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