Note: Descriptions are shown in the official language in which they were submitted.
CA 02476367 2004-07-30
A FARM ANIMAL PRODUCT WITH PROBIOTIC
ENTEROCOCCUS BACTERIA
FIELD OF THE INVENTION
(001] The present invention relates to a farm animal product comprising
probiotic
Enterococcus bacteria and the use of this product to reduce the number of
pathogenic
Esche~ichia coli 0157:H7 cells in farm animals such as cattle.
BACKGROUND OF THE INVENTION
[002] The animal feed industry, such as the beef cattle industry, is
experiencing challenges
like never before, and one of the most critical challenges to the industry is
food safety. For
example, the consumer and governmental agencies are requiring that beef sold
in restaurants,
grocery stores and meat markets be as safe and pathogen free as possible. Meat
packing
companies are looking to the feed yards and the cattle producers to implement
strategies to
help achieve this goal.
[003] The demand for food industry control of potentially contaminating
pathogens starts,
as noted, at the consumer level, wha are stating, through their buying
patterns at the meat
case, that they need a product in which they can have confidence.
Subsequently, retailers
look to wholesalers and the packing companies. The packers are looking to the
feed yards
and, e.g., the cattle producers to take the necessary steps to help reduce
this problem by
adopting safety standards and procedures at all points along the production
chain. This issue
is key and it will take adjustments of management procedures by all entities
involved in beef
production to address this issue.
[004] It has been well documented through scientific and medical research that
the
predominant organism at the root of food safety issues is Esclaerichia Coli
(E. coli) 0157:H7,
otherwise known as enterohaemorrharic E. coli microorganism. E. coli 0157:H7
is one of
hundreds of strains of the bacterium. Although most strains are harmless and
live in the
intestines of healthy humans and animals, this strain produces a powerful
toxin and can cause
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2
severe illness. It also possesses other significant attributes, which
contribute to its ability to
cause disease. One of the more notable of its characteristics is the size of
the infectious dose,
which is incredibly small in comparison with those for most other food-borne
pathogens.
Figures as low as two bacteria per 25g food have been quoted capable of
creating a disease
condition.
[005] The strain E. coli 0157:H7 was first recognized as a cause of illness in
1982 during
an outbreak of severe bloody diarrhea; the outbreak was traced to contaminated
hamburgers.
Since then, most infections have resulted from eating undercooked ground beef.
The
combination of letters and numbers in the name of the bacterium refers to the
specific
markers found on its surface and distinguishes it from other types of E. coli.
Another
pathogen of concern includes strains of Salmonella, with both E. coli and
Salmonella
commonly existing in the gastrointestinal tracts of cattle. These organisms
are endemic and
commonly found in virtually all phases of production. While they may not cause
a problem
in the host animal they can cause illness and even death in humans. Cattle
become "infected"
with this organism through exposure in their natural envirorunent. After the
organism is
ingested it travels to the intestine where it adheres to the tract lining.
Meat is "contaminated"
by the organism during the slaughtering and processing stages when intestinal
contents can
come in contact with other meat surfaces and subsequently become mixed with
ground beef.
[006] In humans, an E. coli infection can lead to bloody diarrhea and even
kidney failure.
In some persons, particularly children under 5 years of age and the elderly,
the infection can
also cause a complication called hemolytic uremic syndrome, in which the red
blood cells are
destroyed and the kidneys fail. About two to seven percent of infections lead
to this
complication. In the United States, hemolytic uremic syndrome is the principal
cause of
acute kidney failure in children, and most cases of hemolytic uremic syndrome
are caused by
the strain E. coli 0157:H7.
[007j Most illnesses have been associated with eating undercooked,
contaminated ground
beef. In addition, however, person-to-person contact in families and childcare
centers is also
an important mode of transmission. Infection can also occur after drinking raw
milk and after
swimming in or drinking sewage-contaminated water. As an example of this, the
USDA
Food Safety and Inspection Service (FSIS) has estimated that consumption of
meat
contaminated with pathogenic bacteria annually results in thousands of deaths
and millions of
illnesses in the U. S. alone. The gavennment estimates the annual losses in
production and
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medical costs may reach as high as $35 billion. The problem is well documented
and
identified.
[008] Having recognized this problem and a need to solve it or at least
diminish it, recent
proactive efforts have been shown by the industry. The proactive efforts
exerted by, e.g., the
US beef industry have resulted in recommendations of expanded research and
accelerated use
of intervening methodologies by industry leaders. Control and treatment
techniques such as
the irradiation of beef products post slaughter, use of new vaccines in cattle
and direct
feeding of certain additives are all under serious investigation and
consideration as
contributing solutions. Of these; the use of feed additives has gained
significant interest,
largely due to simplicity of administration.
[009] One particular group of feed additives showing significant promise in
this area is
probiotic or Direct-Fed Microbial ("DFM") products. The use of DFM's has grown
significantly over recent years largely as a means of enhancing the health and
performance of
the animal. The use of bacterial-based DFM's in ruminant diets for specific
applications has
become widely recognized. Products of this nature often contain lactobacilli
with
Lactobacillus acidophilus being one of the most common.
(010] Most bacterial-based DFM's are beneficial because they have effects in
the lower gut
and not in the rumen. For example, Lactobacillus acidophidus produces lactic
acid, which
may lower the pH in small intestines to levels that inhibit the growth of
pathogenic microbes,
one of the reasons for the current interest. Early research with DFM in
ruminants first
involved applications for young calves fed milk, calves being weaned, or
cattle being
shipped. These animals, in many cases, are highly stressed or had a microbial
gut ecosystem
that was not fully mature. Young cattle have immature digestive tracts that
are obviously
more prone to upset by pathogenic bacteria. Cattle that are slopped are often
on limited feed
and water for prolonged periods of time during transit. During these periods
microbial
populations may decrease in numbers, thus resulting in digestive tracts that
are in less than
optimal condition. Large doses of beneficial organisms were thought to re-
colonize a
stressed intestinal environment and return gut function to normal.
(011 ] The American Meat Institute ("AMI") Foundation published in 2002 a
result of a
research study that was done by Mindy Brashears and Michael Galyean of Texas
Tech
University. At the filing date of the present application the st~xdy was
published on the
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4
Internet at the address: http:l/www.amiforgJProbioticsRe~ort042302.pdf.
According to the
study, the feeding of two different Lactobacillus acidophilus bacteria strains
gave a
significant reduction (P<.05) in the incidence of E. Coli 0157:H7 in the
faeces of finishing
cattle. The experimental design of the study was:
- Control - Cattle fed with a standard diet,
- NP 747 - Cattle fed with a standard diet with 1 x 109CFU Lactobacillus
acidophilus strain
NPC 747 mixed in water and added to the diet at the time of feeding;
- NP 750 - Cattle fed with a standard diet with 1 x 109CFU Lactobacillus
acidophilus strain
NPC 750 mixed in water and added to the diet at the time of feeding.
The result of the study was explained as: "Just 14 d after initiating
treatment, significant (P <
.05) differences were observed among the three treatment groups. At this
sampling time
56.6% of the control animals were positive, whereas only 20% of the animals
fed with the
NPC 747 sample and 11 % of those fed with the NPC 750 probiotic were positive.
Comparing the data based on a positive pen basis, significant (P < .OS)
differences were also
observed. Forty-one percent of the pens receiving the NPC 750 treatment had at
least one
positive animal, which was significantly (P < .05) the percentage of pens in
cattle receiving
NPC 747 (66% with at least one positive sample)." Expressed in log units, the
best data
reduces the number of positives by around 0.5 logs (1 log is a ten times
reduction).
[012] Ongoing work has shown that levels of E. coli increase in cattle during
the finishing
period. Feeding of specific strains of benef cial bacteria has shown to reduce
the levels of
pathogenic proliferation. Studies of this nature are eliciting positive
responses from a number
of meat packers. Many packers are making strong recommendations to their
supplying feed
yards to feed probiotics to help with this issue. Their position is that if
the level of E. coli
entering the facility via the animal is reduced, their ability to further
reduce contamination is
vastly improved.
[013] The research into this area is ongoing by universities and a number of
companies. In
particular several bacterial strains developed by Lallemand Animal Nutrition
("LAN;"
3o Milwaukee, WI) have shown significant results in reducing the
concentrations of E. coli
0157:H7 and Salmonella via a process known as competitive exclusion.
Competitive
exclusion is a process by which beneficial bacteria are used to colonize the
lining of the
intestinal walls, reducing the area available for attachment by pathogenic
microbes. The
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results so far confirm earlier theories that part of the effect noted through
the feeding of
beneficial bacteria results from this reduction in the area of the intestinal
lining available to
the pathogen for attachment.
5 [014] Certain specific strains of Lactobacilli and Propionibacterium
developed by LAN
have proven effective at reducing the numbers of these pathogens under
different
environmental conditions. Probiotic research has shown the effectiveness of
gut colonization
of beneficial bacteria in reducing pathogenic populations through competitive
exclusion of
these harmful organisms. In recent in vitro collaborative work by LAN and
AgTech
(Waukesha, WL, a 15 year-old biotech research company), it was found that
several bacterial
strains were highly effective in inhibiting the growth and development of
strains of
Salmonella and E. coli including E. coli 0157:H7. The results indicate that,
in particular, the
BG2F04 strain of Lactobacillus acidophidus was very effective in inhibiting
all strains of
pathogenic E. coli tested. It is also important to note that the inhibition
was a result of not
only competitive exclusion but also of the action of extracellular
bacteriocins produced by the
Lactobacillus. The results also indicated inhibition of several strains of
Salmonella. A
concluding result was that Lactobacillus acidophilus BG2F04 exhibits a high
degree of
pathogen oriented anti-microbial activity and is an excellent choice fox use
in beef cattle for
this purpose.
[015] U.S. Patent No. 5,718,894 ("the '894 patent") describes a formulation
for use in the
promotion of growth or weight gain in a farm animal. The '894 patent states
that the
formulation comprises two groups oFbacteria: a so-called first bacterium
capable of
producing lactic acid in the gastrointestinal tract of the animal; and a
second bacterium
capable of producing a bactericide to which the bacteria are resistant,
wherein said second
bacterium is a Bacillus. The ' 894 patent states that bactericidle produced by
the Bacillus
strain is capable of combating microorganisms that are the positive agent of
enteric disorders,
e.g. Staphylococcus aureus, E. coli and Salmonella (see column 2, lines 23-
31). The '894
patent states that examples of bacterium capable of producing lactic acid are
bacteria of the
genus Lactobacillus or Enterococcus. According to the '894 patent, they are
distinguished by
their ability to produce lactic acid and thus reduce the local pI-I in the
gastrointestinal tract of
the animal (see column 2, lines 8-22). A specific formulation for use in pigs
is described in
the '894 patent. It is composed of the four strains Lactobacillus,
Enterococcus faecalis,
Enterococcus faecium and Bacillus licheniformis. The '894 patent states that
each strain is
used at 109 cfu/g.
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6
[016] In summary, current research has already revealed and continues to
reveal useful
methodologies for the control of pathogenic bacterial populations in farm
animals such as
beef cattle. In relation to use of probiotic bacteria, relevant detailed
studies have mainly
focused on use of suitable Lactobacillus acidophilus strains.
SUMMARY OF THE INYENf'ION
[017) The problem to be solved by the present invention includes the provision
of a
composition (also referred to herein as "farm animal product") that has an
improved ability
with respect to decreasing the number of Escherichia coli 0157:H7 in farm
animals
(preferably cattle) when the farm animals are challenged with Escherichia coli
0157:H7
pathogen.
[018] One solution to this problem is that the present inventors have found
that a
composition comprising Enterococcus strains works better than a corresponding
composition
comprising similar amounts (CFU/g) of Lactobacillus acidophilus strains.
[019] Working examples herein demonstrate, inter alia, that cattle fed with
about 109 CFU
Enterococcus bacteria per day had a significant reduction of the number of
Escherichia coli
0157:H? quantified in the faeces of the challenged animals. The reduction was
about 1.5 to
2 logs in 10 to 14 days. 1 log unit denotes a ten times reduction and 2 log
units denotes a 100
times reduction.
[020] In the Bra,shears & Galyean study discussed supra, a corresponding
example of an
animal feed composition comprising Lactobacillus acidophilus resulted in
around 0.5 log
reduction. Without being limited by theory, it is believed that direct-fed
microbial ("DFM")
products currently being commercially sold in cattle to reduce the number of
Escherichia coli
are based on Lactobacillus acidophilus, and they reduce the number of the
target pathogen by
only 0.5 logs.
[021) In relation to the reduction of the number ofEscherichia coli 0157:H7,
Enterococcus
bacteria work better than Lactobacillus acidophilus bacteria. However, in one
embodiment
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7
of the present invention, apart from Enterococcus, the farm animal product may
comprise
smaller amounts of Lactobacillus acidophilus bacteria than those of
Enterococcus bacteria.
[022] Accordingly, one aspect of the invention relates to a farm animal
product comprising
at least 106 CFU/g of probiotie Enterococcus bacteria, characterized in that,
if the product
contains Lactobacillus acidophilus, the product has at least 2.5 times more of
Enterococcus
bacteria than Lactobacillus acidophilus bacteria measured as CFU/g.
[023] Another aspect of the invention relates to a method of feeding a farm
animal
1o comprising feeding the farm animal with a farm animal product comprising at
least 106
CFU/g of probiotic Enterococcus bacteria, characterized in that, if the
product contains
Lactobacillus acidophilus, the product has at least 2.5 times more of
Enterococcus bacteria
than Lactobacillus acidophilus bacteria measured as CFU/g.
15 [024] A further aspect of the invention relates to a method of feeding a
farm animal
comprising feeding the farm animal with a farm animal product comprising at
least 106
CFU/g of probiotic Enterococcus bacteria, wherein said product reduces the
number of
Escherichia coli 0157:H7 cells quantified in faeces of challenged animals by
at least 1.5
logs.
2o
DETAILED DESCRIPTION OF THE INVENTION
[025] As used herein, the term "probiotic" is a well-defined term in the art
and relates to a
class of microorganisms defined as live microbial organisms that confer health
benefit to
25 farm animal hosts or is not pathogenic to same. The beneficial effects
include improvement
of the microbial balance of the intestinal micro flora and the improvement of
the properties of
the indigenous micro flora when, for example, the microorganism is orally
administered.
[026] As used herein, the term "Enterococcus" is a well-known and well-defined
term for
3o this Enterococcus genus of bacteria species. For further details see, e.g.,
the standard
reference book Bergeys Manual of Systematic Bacteriology. Based on the general
knowledge of one of ordinary skill in the art, the skilled person is capable
of determining
whether or not a specific Enterococcus bacterium of interest is a bacterium of
the
Enterococcus genus.
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[027] In all embodiments of the invention, when the term "comprising" or
"including" is
used, the respective description also includes the same composition, product
or item which
consists essentially of or consists of the ingredients of the composition,
product or item.
(028] As used herein, the term "CFU" denotes Colony Forming Units. The term
"CFU/g"
refers to CFU/g of farm animal product.
Farm animal product
[029] In one embodiment of the present invention, the farms animal product
comprises
suitable farm animal feedstuff ingredients in addition to an Enterococcus
bacteria. The
skilled person is aware of selecting the adequate ingredients in relation to
the specific farm
animal of interest. Herein, such suitable farm animal feedstuff ingredients
may be termed
farm animal feedstuff ingredients known per se or farm animal feedstuff
ingredients.
[030] In this aspect of the invention, these ingredients should be in
concentrations adjusted
to meet animal's dietary requirements and may include nutrient ingredients
such as animal
protein products, at about 0 - about 95 weight percent; plant protein
products, at about 0 -
about 95 weight percent; poultry egg products, at about 0 - about 25 weight
percent.
[031] In a further embodiment of the invention, the farm animal product may
also comprise
other suitable ingredients such as antibiotics such as Sarafin, Romet,
Terramycin at about
0.01 - about 50 weight percent; cyanocobalamin at about 40 -~ about 60 mg/kg;
D-biotin at
about 5 - about 20 mg/kg; D-pantothenic acid at about 250 - about 350 mg/kg;
folic acid at
about 10 - about 30 mg/kg; L-ascorbyl-2-polyphosphate (STAY-C, stable form of
vitamin C)
at about 1,000 - about 4,000 mg/kg; myo-inositol at about 3,000 - about 4,000
mg/kg; niacin
at about 600 - about 800 mglkg; p-amino-benzoic acid at about 350 - about 450
mg/kg;
pyridoxine hydrochloride at about 40 - about 60 mglkg; riboflavin at about 125
- about 175
mg/kg; thiamine hydrochloride at about SO - about 80 mg/kg; choline chloride
at about 6,500
- about 7,500 mg/kg.
[032] In another embodiment of the invention, the farm animal product may be
present in
any suitable form, such as a powder, liquid or in form of pellets or tablets.
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9
[033] One embodiment of a farm animal product is a composition comprising
Enterococcus
bacteria in a bolus, or, in a further embodiment, in a gelatin bolus. A
particular embodiment
of a farm animal product is a composition comprising Enterococcus bacteria,
Glucidex IT12
(about 30%) and Type 4A Act Molecular Sieve Powder (about 10%).
[034] In another embodiment of the invention, the farm animal product may be
in form of,
e.g., two different compositions. Such an embodiment includes one composition
comprising
the suitable farm animal feedstuff ingredients and the other composition
comprising the
Enterococcus bacteria as described herein. In a further aspect of the
invention, the farm
animal product is comprised of such two compositions and accompanied by
suitable
instructions to administer them to the farm animal either simultaneously or
sequentially. In
other words, in an embodiment of the present invention, while the farm animals
are fed with
the suitable farm animal feedstuff ingredients, they should also be fed with
the probiotic
Enterococcus bacteria-containing product as described herein.
[035] In an alternative embodiment of the invention, the farm animal product
may be in
form of a composition comprising the suitable farm animal feedstuff
ingredients and the
probiotic Enterococcus bacteria as described herein. In a particular
embodiment of the
invention, the composition is in the form of a suitable powder, a liquid or in
the form of
pellets or tablets.
[036] In another embodiment of the invention, in order to improve some
stability aspects of
the probiotic bacteria, it may be advantageous to provide the farm animal
product as a stable
emulsion of solids in-oil comprised of lipid soluble bioactive comp~unds such
as inhibitory
furanones dissolved in lipid forms of the continuous phase and with dry feed
ingredients and
the probiotic bacteria of interest forming the dispersed phase of the stable
emulsion. See,
e.g., WO 02/00035 for further details.
[037] In a further embodiment of the present invention, the farm animal
product may be in
form of a capsule, e.g., a microencapsulated product.
[038] As described supra, one preferred embodiment of the invention is to use
Enterococcus bacteria instead of Lactobacillus acidophilus bacteria.
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CA 02476367 2004-07-30
[039] In an embodiment of the invention, the farm animal product comprises at
least 5 times
more of Enterococcus bacteria than Lactobacillus acidophilus bacteria measured
as CFU/g,
in a further embodiment comprises at least 50 times more of Enterococcus
bacteria than
Lactobacillus acidophilus bacteria measured as CFU/g, in another embodiment
comprises at
5 least 500 times more of Enterococcus bacteria than Lactobacillus acidophilus
bacteria
measured as CFU/g and in another further embodiment comprises at least 5000
times more of
Enterococcus bacteria than Lactobacillus acidophilus bacteria measured as
CFU/g.
[040] In other words, one embodiment of a farm animal product does not
comprise
io measurable amounts of Lactobacillus acidophilus. Such an embodiment of a
farm animal
product may be denoted a farm animal product consisting essentially of
probiotic
Enterococcus bacteria, or consisting essentially of at least 106 CFUIg of
probiotic
Enterococcus bacteria. In another embodiment, such an embodiment of a farm
animal
product may be denoted a farm animal product consisting of probiotic
Enterococcus bacteria,
i5 or consisting of at least 106 CFU/g of probiotic Enterococcus bacteria.
Another embodiment
of a farm animal product does not comprise significant amounts of
Lactobacillus
aeidophilus. In all embodiments including Enterococcus bacteria, one or more
strains of
such bacteria may be present.
[041] In one embodiment of the present invention, the farm animal product
comprises the
probiotic Enterococcus bacteria in a concentration of at least 106 CFU/g, in a
further
embodiment comprises the probiotic Enterococcus bacteria in a concentration of
at least 108
CFU/g, in a further embodiment comprises the probiotic Enterococcus bacteria
in a
concentration of at least 10'° CFU/g and in a further embodiment
comprises the probiotic
Enterococcus bacteria in a concentration of at least 1011 CFU/g. In
embodiments of the
invention, the farm animal product comprises the probiotic Enterococcus
bacteria in a
concentration of less than 1014 CFU/g. In an embodiment of the invention, the
farm animal
product comprises the probiotic Enterococcus bacteria in a concentration from
108 CFU/g to
10'2 CFU/g. In another embodiment of the invention, the farm animal product
comprises the
probiotic Enterococcus bacteria in a concentration of approximately 2 x 1011
CFU/g. In a
further embodiment of the invention, the faxm animal product comprises the
probiotic
Enterococcus bacteria in a concentration of approximately 5 x 109 CFU/g. In a
further
embodiment, the farm animal product comprises probiotic Enterococcus bacteria
in a
concentration of approximately 5 x 101° CFU/g. In another embodiment of
the present
invention, the farm animal product comprises probiotic Enterococcus bacteria
in a
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11
concentration of from approximately 5 x 109 CFU/g to approximately 2 x 1011
CFUIg. In
another embodiment of the invention, the farm animal product comprises
probiotic
Enterococcus bacteria in a concentration of from 106 CFU/g to approximately 2
x 1011
CFU/g.
Probiotic Enterococcus bacteria
[042] The probiotic Enterococcus bacteria described herein may be any
probiotic
Enterococcus bacteria. Based on the information disclosed herein, the skilled
person is
1o capable of selecting a specific Enterococcus strain of interest.
[043] In embodiments of the invention, the Enterococcus strain is an
Enterococcus faecium.
[044] In an embodiment of the present invention, Enterococcus faecium strains
are SF-273
i5 (CHCC 4202) and SF-301 (CHCC 3828). In a further embodiment, the farm
animal product
comprises both SF-273 (CHCC 4202) and SF-301 (CHCC 3828), and, in a further
embodiment the two strains are present, in a ratio of 50:50 (based on
potency). In another
embodiment, the two strains are present in the farm animal product in a ratio
of 60:40 or
40:60. Enterococcus faecium strain number SF-273 was deposited on April 14,
1998, under
20 ATCC 27273, in the American Type Culture Collection, 10801 University
Blvd., Manassas,
Virginia 20110-2209. Also, SF-273 was deposited in the Chr Hansen Culture
Collection
under CHCC 4202. Enterococcus faecium strain number SF-301 was deposited on
November 27, 1997, under DSM 4789, in the German Culture Collection, Deutsche
Sammlung von Mikoorganismen and Zellkulturen GmbH, Mascheroder Weg lb, D-38124
25 Braunschweig, Germany. Also, SF-301 was deposited in the Chr Hansen Culture
Collection
under CHCC 3828.
[04S] In an additional embodiment of the present invention, the Enterococcus
bacteria are
selected to be tolerant of the following conditions: high acid (pH 4.0), high
concentrations of
30 volatile fatty acids (200 to 400 mM mixtures of acetic, propionic and
butyric acids) and
complete anaerobiosis.
[046] Working Example 2 herein sets forth an example of one preferred assay to
test if an
Enterococcus bacteria is tolerant to these conditions.
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12
[047] In another embodiment of the present invention, the Enterococcus
bacteria are also
oxygen scavengers. Such bacteria are much more stable to oxygen exposure,
moisture and
heat than L. acidophilus.
S [048] Based on the information provided herein, combined with the knowledge
of the
skilled artisan, it is routine work to select Enterococcus bacteria tolerant
to the conditions
provided supra.
[049] In embodiments of the invention in which the product bacteria are
tolerant to the
1o conditions and/or are oxygen scavengers, such traits combined are useful to
confer a high
degree of survival within the gastrointestinal tract of feedlot cattle. In an
embodiment of the
invention, the product bacteria are metabolically active upon ingestion by the
animal. This
activity can have an immediate impact either on the environment within the
gastrointestinal
tract, or on the E. coli cells present or on the receptor sites/niches with
which the E. coli cells
15 associate. Or, in another embodiment of the invention, it could be any
combination of these
influences.
[050] In accordance with the present invention, the product containing the
Enterococcus
bacteria can be in many different forms. In one embodiment of the present
invention, the
2o product containing the Enterococcus bacteria is a feed product. In another
embodiment of
the present invention, the product containing the Enterococca~s bacteria is in
the form of a
direct dose to the animal. In an embodiment of the invention the product
containing the
Enterococcus bacteria is in the form of a direct dose to the animal through a
stomach tube.
25 Farm animal
[051) In an aspect of the present invention, the farm animal may be a pig, a
cow, a cattle, a
sheep, a chicken, a duck or an ostrich. In another embodiment of the
invention, the farm
animal is a ruminant animal, in particular a cattle or a cow. hn a further
embodiment of the
30 invention, the farm animals are cattle.
A method~or administering a farm animal nxoduct as described herein to a farm
animal
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13
[052] The feeding may be done according to the art, and the skilled artisan is
aware of how
to properly feed farm animals.
(053] In an embodiment of the present invention, the farm animals are fed with
an amount
of farm animal product that provides at least 10' CFU Enterococcus bacteria
per animal per
day, in another embodiment the farm animals are fed with an amount of farm
animal product
that provides at least 108 CFU Enterococcus bacteria per animal per day, in
another
embodiment the farm animals are fed with an amount of farm animal product that
provides at
least 109 CFU Enterococcus bacteria per animal per day and in another
embodiment the farm
animals are fed with an amount of farm animal product that provides at least
101 ° CFU
Enterococcus bacteria per animal per day. In embodiments of the invention, the
farm animals
are fed with an amount of farm animal product that provides less than 1013 CFU
Enterococcus bacteria per animal per day. In an additional embodiment of the
invention, the
farm animals are fed with an amount of farm animal product that provides
approximately 2 x
1011 CFU Enterococcus bacteria per animal per day. In another embodiment, the
farm
animals are fed with an amount of farm animal product that provides
approximately 5 x 109
CFU Enterococcus bacteria per animal per day. In a further embodiment of the
invention,
the farm animals are fed with an amount of farm animal product that provides
approximately
5 x 101° CFU Enterococcus bacteria per animal per day. In a further
embodiment of the
invention, the farm animals are fed with an amount of farm animal product that
provides from
108 CFU to 1011 Enterococcus bacteria per animal per day, in a fixrther
embodiment the farm
animals are fed with an amount of farm animal product that provides from 109
CFU to 1010
Enterococcus bacteria per animal per day. In a further embodiment of the
invention, the farm
animals axe fed with an amount of farm animal product that provides from
approximately
5 x 109 CFU to approximately 2 x 1011 CFU Enterococcus bacteria per animal per
day.
[054] In an embodiment of the invention, the animals are fed once a day. In an
alternative
embodiment of the invention, they may be fed twice a day or, in another
embodiment of the
invention, once every second day. The skilled artisan is aware of what is best
in relation to a
specific farm animal of interest.
[055] 1n another embodiment of the present invention, the farm animals are fed
with the
farm animal product as described herein at least once a day for at least 10
days, and in a
further embodiment the farm animals are fed with the farm animal product as
described
herein at least once a day for at least 20 days.
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14
[056] In a further embodiment of the invention, the farm animals are fed with
the farm
animal product as described herein until slaughter. In a particular
embodiment, the farm
animals are fed with the farm animal product as described herein at least once
a day for at
least the last 10 days until slaughter, and in another embodiment the farm
animals are fed
with the farm animal product as described herein at least once a day for at
least the last 20
days until slaughter.
(057J In a further embodiment of the present invention, the farm animals are
fed with the
farm animal product as described herein for about 26 days. In an additional
embodiment of
the present invention, the farm animals are fed for about 26 days with an
amount of farm
animal product that provides from approximately 5 x 109 CFU to approximately 2
x 1011
CFU Ercterococcus bacteria per animal per day. In an additional embodiment of
the present
invention, the farm animals are fed for about 26 days with an amount of farm
animal product
that provides from approximately 5 x 109 CFU to approximately 5 x 101°
CFU Er~terococcus
bacteria per animal per day. In an additional embodiment, the farm animals are
fed for about
26 days prior to slaughter with an amount of farm animal product that provides
from
approximately 5 x 109 CFU to approximately 5 x 101° CFU Eiaterococcus
bacteria per animal
per day. Additionally, in this embodiment, the animals can be fed the farm
animal product
set forth in Example 3.
[058] In another embodiment of the invention, the farm animal product is used
for feeding
the animals in an amount and for a number of days where the farm animal
product reduces
the number of Escherichia coli 0157:H7 cells quantified in the faeces of the
challenged
animals by at least 1.5 logs, or, in another embodiment, by at least 2 logs.
In another
embodiment of the present invention, the farm animal product reduces the
number of
Escherichia coli 0157:H7 cells quantified in the faeces of the challenged
animals by at least
1.5 logs in the first few days of treatment, or, in another embodiment, by at
least 2 logs in the
first few days of treatment. In another embodiment of the present invention,
the farm animal
product reduces the number of Escherichia coli 0157:H7 cells quantified in the
faeces of the
challenged animals by at least 1.5 logs in 3 days of treatment, or, in another
embodiment, by
at least 2 logs in 3 days of treatment. In another embodiment of the present
invention, the
farm animal product reduces the number of Escherichia coli ()157:H7 cells
quantified in the
faeces of the challenged animals by at least 1.5 logs in 10 to 14 days of
treatment, or, in
another embodiment, by at least 2 logs in 10 to 14 days of treatment. In yet
another
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embodiment of the present invention, the farm animal product reduces the
number of
Escherichia coli 0157:H7 cells quantified in the faeces of the challenged
animals by about
1.5 logs to about 2 logs. In yet another embodiment of the present invention,
the farm animal
product reduces the number of Escherichia call Ol 57:H7 cells quantified in
the faeces of the
challenged animals by about 1.5 logs to about 2 logs within :3 days of
treatment.
Additionally, in this embodiment, the animals can be fed the farm animal
product set forth in
Example 3.
[059] Working examples herein describe a suitable assay to quantitatively
measure this.
[060] In accordance with the present invention, the product containing the
Enterococcus
bacteria can be administered to the animal in many ways. In one embodiment of
the present
invention, the product containing the Enterococcrcs bacteria is fed to the
animal as a feed
product. In another embodiment of the present invention, the product
containing the
Enterococcus bacteria is dosed directly to the animal. In an embodiment of the
invention, the
product containing the Enterococcus bacteria is dosed directly to the animal
through a
stomach tube.
[061] The entire disclosure of each document cited (including patents, patent
applications,
journal articles, abstracts, laboratory manuals, books, or other disclosures)
in the Background
of the Invention, Summary, Detailed Description, and Examples is herein
incorporated by
reference.
EXAMPLES
[062] Example 1: Dose Titration Study to determine the ef~"ect of two
different dosing
regimens of a characterized Direct Fed Microbial Culture on the post-challenge
fecal
shedding of Escherichia coli 0157: H7
Bacteria containing products:
[063] CHB DFM: Two Enterococcus faecium strains present together at 50:50
(based on
potency) in a gelatin bolus. The strains are SF-273 (CHCC 4202) and SF-301
(CHCC 3828).
The two Enterococcus faecium strains each are present in the product at 2.5 x
109 CFU/g.
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16
[064] CHB Probios TC: A corresponding product, which comprises probiotic
Enterococcus
faecium bacteria in a similar CFU/g as for CHB DFM: This product also
comprised active dry
yeast at around 2.5 x 109 CFU/g.
Objective:
(065] The objective of this project was to explore the effect of two different
doses of CHB
DFM on the magnitude of fecal shedding of NalrE. coli 0157: H7 in beef cattle
fed a highly
fermentable complete ration.
Materials and Methods:
[066] Twelve beef steer calves weighing approximately 400 pounds were ranked
by body
weight and randomly allotted to one of three treatment groups. The treatment
groups were
control (no DFM) and DFM dosed at 2, or 20 g/head/day. All calves were orally
dosed with
DFM using gelatin boluses in order to assure that they received their daily
target dose of
DFM.
[067] A similar strategy was used for Probios TC, and Probios TC was dosed at
2, or 20
g/head/day.
(068] Cattle were housed together in a single isolation room and allowed to
commingle
throughout the study. All cattle were allowed to consume a ration formulated
with rolled
corn and corn gluten feed containing sodium rnonensin (30 grams/ton). Fecal
samples were
collected and provided to detection of E. coli 0157: H7 by enrichment
technique to assure
that calves were free of (Nal~ E. coli 0157: H7 prior to challenge.
[069] Prior to the initiation of the study, all cattle were identified using
duplicate unique
Temple~ ear tags and were vaccinated with a modified live virus vaccine
containing IBR,
BVD, PI3, and BRSV (Bovishield 4~, Pfizer), 2 ml intranasal vaccination
containing IBR and
PI3 (TSV-2~, Pfizer), a single 2 ml injection of a Clostridial vaccine (Vision-
7~, Intervet),
and a single injection of anthelmintic to control internal and external
parasites (Dectomax~,
Pfizer).
Experimental Design:
[070] The following txeatxnent groups were evaluated:
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~, .,~,~>.
CA 02476367 2004-07-30
17
~ Treatment A: Non-medicated Control (n = 4)
~ Treatment B: CHB - Direct Fed Microbial (CHB-DFM) at a rate of 2
grams/head/day (n
= 4)
~ Treatment C: CHB - Direct Fed Microbial (CHB-DFM) at a rate of 20
grams/head/day (n
= 4)
~ Treatment D: Probios TC at a rate of 2 grams/headlday
~ Treatment E: Probios TC at a rate of 20 grams/head/day
[071] Animals were inoculated with E. coli 0157:H7, strains FRIK 1123 and FRIK
2000
1o which were adapted to nalidixic acid (Nal~ in the laboratory (20 ~g/ml).
The organisms were
grown in GN broth (Difco laboratories, Detroit, ML) for 7 h (approx. 0.8 abs
at 600 nm), the
two cultures were pooled and colony counts of the pooled cultures were done by
spread plate
technique. Each animal was inoculated (day 0) by using a stomach tube through
a Frick
speculum with 60 ml of the pooled cultures containing 8.6 x 108 CFU/ml of Nalr
E. coli
0157:H7 (5.2 x 101° CFU/animal). Following administration of the
challenge material, the
tube was flushed with 120 mL of sterile phosphate buffered saline.
[072] Animals were evaluated daily for evidence of adverse reactions. Fecal
(rectal)
specimens were collected on 1, 3, 5, 8, 10, 12, 14, 17, 19, 23, 24, 26, and 30
days following
oral challenge. Fecal samples wexe placed in whirl packs, packed in ice and
provided for
detection and quantification of Nalr E. coli 0157: H7.
Detection and enumeration of Nadr E. coli D157: H7:
[073] One gram of faeces was added to 9.0 ml of GN broth containing 50 ~ul
(O.OSmg/liter)
of cefixime (C), 200 ~l (lOmg/liter) of cefsulodin (C), and 100 ~l (8mg/liter)
of vancomycin
(V). Samples were vortexed for 30 sec, serially diluted, and 100 ~.l of 10'1,
10'z, 10'3
dilutions was spread plated, in triplicate, onto sorbitol MacConkey agar
(SMAC) plates
containing 20 ~g/ml of Nal. The remaining GN broth was incubated as an
enrichment step in
the isolation procedure. After 6 h incubation at 37°C, 1.0 ml was
transferred into 9.0 ml of
3o GNccv broth and incubated an additional 18 to 24 h at 37°C. The
inoculated SMAC plates
were incubated for 24 h at 37°C and typical sorbitol-negative (gray
colored) colonies were
counted. A maximum of three colonies per sample per animal were collected,
streaked onto
blood agar plates, and incubated for 24 h at 37°C. The indole test was
done on colonies from
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~.,~",~,a~~,,,~~,"~,."~,- _. ~._,._ _ ,..,.__~_~.-__.,~.__._ _._... _ .._._
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CA 02476367 2004-07-30
18
the blood agar plates; indole positive colonies were tested for agglutination
specific for 0157
(Oxoid Diagnostic Reagents, Basingstock, Hampshire, England).
[074) If E. coli 0157:H7 colonies were not detected by direct plating
(detection limit >
102/g), GNccv broth incubated for 18 to 24 h was plated, in duplicate, on SMAC
plates
containing Nal (20 p.g/ml) and incubated for 24 h at 37°C. Following
incubation, three
colonies per sample with typical colony morphology (from the enriched samples)
were
streaked on blood agar plates and incubated for 24 h at 37°C. The
indole test was done on
colonies from the blood agar plates and indole positive colonies were tested
for agglutination
specific for 0157.
Statistical Analysis:
[075] The study had two outcomes of interest. First, the level of fecal E.
coli 0157:H7
shedding was compared between treatment groups. The independent variables were
treatment, day, and the treatment by day interaction. The comparison was done
using
repeated measures analysis of variance (MIXED procedure, SAS Institute Inc.).
The
outcome was the level of fecal shedding (CFU/g of faeces) with group as the
treatment and
day as the repeated measure. Day and treatment interaction was included as a
fixed effect.
Colony counts were log transformed prior to analysis.
Results:
[076] Cattle in all groups shed at least 102 CFU/g (range 10'' to 104) of Nalr
E. coli 0157:H7
in the faeces during the first week (days 1, 3 and 5) after inoculation. After
that there was a
general decrease in magnitude of shedding and numbers of Nalr E. coli 0157:H7
recovered
ranged from 102 to undetectable (see Table 1). After day 12, the shedding
pattern was
somewhat erratic with concentrations fluctuating from 10z to undetectable.
Treatment groups
fed DFM at 2 or 20 g shed lower concentrations of Nalr E. coli 0157:H7 in the
faeces
compared to the control (P values = 0.01 and 0.06, respectively). The extent
of reduction was
greater in the group dosed with 20 g compared to 2 g dose. Table 3 reports
data from a
3o similar study made during another period than the study behind the data of
Table 1. This
second data confirms the overall results described above.
Conclusions:
[077) DFM at 20 g per animal per day caused a significant reduction in the
level of
shedding of Nalr E. coli 0157:H7.
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(078] Tuble 1. Effects of Direct-fed microbials (DFM) on fecal shedding of
Nalidixic acid-
resistant E. coli 0157:H7 in cattle.
Sampling DFM, g/animal/day
days 0 2 20
0 3.5 x10' 3.5 x10'' 3.5 x10''
1 2.5x10''a 1.5x10''a 3.5x10'a
3 1.5 x10'' 9.1 x10' 2.3 x10'
a a a
6.5x10'a 1.6x10'a 2.2x10'a
8 7.6x10'a 6.Ox101a 3.Ox10'a
3.9x 10'a,b 5.7x10'a 5.4b
12 3.3x10'a 3.Ox10'a Ob
14 1.8a 2.9a 9.4x10'a
17 ?.4 xl a 9.3 a 5.3 x10 a
19 1.9x10'a 2.6x10'a 2.9b
23 4.5 x10' a 1.2 x10' 1.1 x10'
a a
24 5.4a 2.0a 1.6x10'a,b
26 6.3x10 a 9.5a 1.5a
30 Oa Oa 1.8a
5 Treatment effect P= 0.02
Days effect P < 0.01
Treatment by Days Interaction P = 0.6
a, b Means not sharing the same letters differ at P < 0.1.
10 (079] Table 2. Treatment Means.
Treatment Mean (CFU/g of faeces) Significance
Control, 0 1.9 x 10' Control vs. 2 g DFM P = 0.06
g/day Control vs. 20 g DFM P = 0.01
DFM, 2 g/day 4.5 x 10 ' ~ 2 g DFM vs. 20 g DFM P =
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0.54
DFM, 20 g/day2.8 x 10~ 20 g DFM vs. 200 g DFM
P =
0.01
P-value fect( = 0.02.
(treatment
ef
(080] Table 3. Effects of Direct-fed microbials (DFM) on fecal shedding of
Nalidixic acid-
resistant E. coli 0157:H7 in cattle.
5
DFM
Day Control 2gX DFM 20g TC 2g TC 20g
DO 37000003700000370000037000003700000
D1 3400000120000 2400000750000 370000()
D3 900000 120000 580000 100000 580000
DS 83000 33000 250000 60000 3800000
D8 5000 470 14000 160 35000
D10 2800 120 72 1300 520
D12 1400 110 104 11000 570
D15 2000 130 7 120 620
D19 120 350 19 1700 17
D22 17 17 21 18 9
D24 230 230 0 18 14
D26 9 61 0 0 0
[081] Example 2: Assay to select Enterococcus bacteria that are tolerant to
preferred
conditions.
10 [082] A number of publicly available Enterococcus bacteria were tested in
the assay
described below. The strains SF-273 (CHCC 4202) and SF-3t~1(CHCC 3828) were
both
tolerant to the below described testing conditions.
Exposure to Rumen Fluid
15 (083] The medium of Bryant MP and Burkey LA (J. Dairy Science 1960)
containing 40%
rumen fluid (RF medium) was prepared either under 100% C02 (with sodium
carbonate
solution as buffer) or under 80%:20% N2:CO2 headspace gas (with sodium
bicarbonate
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21
solution as buffer). This medium was used to score growth of the test strains
as 0 (no
growth) or +, ++, +++, or ++++ (excellent growth) at 37C after 48 to 72 h.
Exposure to Volatile Fatty Acids
[084] Medium 10 of Bryant MP and Robinson IM (J. Dairy Science 1966) was
prepared to
test the tolerance of each test strain to volatile fatty acids (acetic,
propionic and butyric acids,
200 to 400 mM). This medium was used to score growth of the test strains as 0
(no growth)
or +, ++, +++, or ++++ (excellent growth) at 37C after 48 to 72 h. Only those
strains having
a +++ or ++++ score advanced in the testing procedure are in present context
an
Enterococcus bacterium considered tolerant high concentrations of volatile
fatty acids (200 to
400 rnM mixtures of acetic, propionic and butyric acids).
Acid Tolerance
[085] Medium 10 Bryant MP and Robinson IM (J. Dairy Science 1966) was prepared
and
poised at pH 4.0, 5.0, or 6.0 using a 5 N solution of HCl (hydrochloric acid).
The medium
was autoclaved, cooled and then inoculated with each test strain. These media
were used to
score growth of the test strains as 0 (no growth) or +, ++, +++, or ++++
(excellent growth) at
37C after 48 to 72 h. Only those strains having a +++ or ++++ score in the pH
4.0 medium
advanced in the testing procedure are in present context an Enterococcus
bacterium
considered tolerant of high acid (pH 4.0) concentrations.
Oxygen Tolerance
[086] Medium 10 of Bryant MP and Robinson IM (J. Dairy Science 1966) was
prepared to
test the tolerance of each test strain to oxygen tolerance. In this test, the
medium was
modified as follows: 2% w/v agar was added to solidify the medium, the amount
of resazurin
(redox indicator) was doubled, and the amount of reductant, cysteine
hydrochloride solution,
was reduced by 50%. This medium was dispensed in 10 mL amounts and solidified
after
autoclaving in an upright position. Each test strain was grown in broth
culture overnight and
then stab-inoculated from top to bottom in the center of the 10 mL tubes of
modified Medium
10. The inoculated tubes were exposed to the atmosphere for 5 min then closed
and
incubated overnight (ca. 18 h) at 37C. After incubation the tap one-centimeter
of the medium
had oxidized (turned the redox indicator pink) and the resultant growth
pattern of each test
strain was scored. The strain was scored as a strict anaerobe if visible
growth along the stab
line was only in the reduced portion of the tube. The strain was scored as an
oxygen tolerant
anaerobe if strain growth extended into the pink (oxidized) portion of the
medium. The strain
was scored as a facultative anaerobe if growth was visible along the stab line
within both the
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22
reduced and oxidized portions of the tube. The strain was scored as an aerobe
if growth was
visible only within the oxidized portion of the medium. In the present context
an
Enterococcus bacterium is considered tolerant to complete anaerobiosis
conditions if it grew
in the reduced portion of the medium.
Oxygen scavenging
[087] Medium 10 as modified for oxygen tolerance testing was used. After
oxygen
tolerance capacity was scored, the stab-inoculated tubes were re-incubated at
37 C for an
additional 24 h. Then the oxidized zone of each tube was examined and scored
as follows. If
the pink zone was re-reduced to colorless, then the strain was scored as being
an oxygen
scavenger (characteristic present or absent). In the present context an
Enterococcus
bacterium is considered an oxygen scavenger if it re-reduced the medium.
[088] Example 3: Formulation of farm animal product.
[089] Table 4 sets forth the composition of one embodiment of a farm animal
product
according to the present invention. In this embodiment depicted in Table 4,
the farm animal
product is not less than 200 billion CFU/gram. The farm animal product in
Table 4 is
packaged in 250 gm foil pouches, with 14 pouches per case, and it is available
from Chr.
2o Hansen, Inc. in Milwaukee, Wisconsin under the name Probios FS.
[090] Table 4. Farm animal product containing probiotic Enterococcus bacteria.
Ingredients Typical Inclusion
Dried Enterococcus faecium Fermentation Product (45.0-75.0%)
(50:50 mix ofE. faecium strains SF-273 and SF-301)
Sodium Thiosulfate 0.12%
Sodium Aluminosilicate 10.00%
Maltodextrin (Balance) (44.88 - 14.88%)
Total 100.00%
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Z3
The present invention is not to be limited in scope by the specific
embodiments
described herein. Indeed, various modifications of the present invention, in
addition to those
described herein, will be apparent to those of ordinary skill in the art from
the foregoing
description. Thus, such modifications are intended to fall within the scope of
the following
appended claims. Further, although the present invention has been described
herein in the
context of a particular implementation in a particular environment for a
particular purpose,
those of ordinary skill in the art will recognize that its usefulness is not
limited thereto and
that the present invention can be beneficially implemented in any number of
environments
for any number of purposes. Accordingly, the claims set forth below should be
construed in
view of the full breadth and spirit of the present invention as disclosed
herein.
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