Note: Descriptions are shown in the official language in which they were submitted.
CA 02438698 2003-06-27
=
1
IMMUNOGEN ADHERENCE INHIBITOR AND METHOD OF MAKING AND USING SAME
FIELD OF THE INVENTION
This invention is directed to microbial adherence inhibitor, in the form of
fowl egg
antibodies, for substantially preventing the attachment or adherence of colony-
forming
immunogens or haptens in the rumen and intestinal tract of host food animals,
to the method of
producing such adherence inhibitors, and to the methods of using such
inhibitors to: (1) promote
the growth of food animals by improving feed conversion rates by decreasing
the waste of dietary
protein caused by the presence of certain colony-forming protein-wasting
organisms in food
animals, and (2) to substantially reduce or eliminate the incidence of
illnesses caused by the
/34
io presence of certain illness-causing colony-forming immunogens or haptens
in meat from food
animals, which are not themselves subjected to the targeted illness, and in
other food stuffs.
BACKGROUND OF THE INVENTION
Common bacterial immunogens which cause dramatic decreases in an animal's
ability to
utilize dietary protein include but are not limited to Peptostreptococcus
anaerobius, Clostridium
aminophilum, and Clostridium sticklandii. According to Russell (USDA-ARS, May
1993) these
organisms, and others disclosed therein, have been collectively responsible
for wasting up to 25
percent of the protein in cattle diets. This is a loss of as much as $25
billion annually to cattle
producers and is especially apparent in "grazing animals which are often
deficient in protein, even
though their protein intake appears to be adequate." As the host consumes
protein in the diet, these
deleterious organisms wastefully degrade the protein to ammonia which is
converted to urea by the
liver and kidneys and thus lost to the host when excreted as urine. These
deleterious organisms
also compete with beneficial organisms which the host needs for the efficient
utilization of
ammonia. In addition, they need other beneficial organisms in the rumen for
greater ammonia
utilization.
SYTEEr
CA 02438698 2003-06-26
WO 03/061693
PCT/US01/49588
2
The principal objective of the present invention is to substantially prevent
the colonization
of deleterious organisms such as P. anaerobius, C. sticklandii and C.
aminophilum as well as the
growth of such organisms in the rumen and the intestinal tracts of food
animals resulting in their
substantial elimination from the animal by the administration of the fowl egg
antibody to the
specific organisms.
Common bacterial immunogens which cause food borne illness in humans include
E. coli,
Listeria, Salmonella and Campylobacter, all of which produce flu-like symptoms
such as nausea,
vomiting, diarrhea and/or fever, and in some cases causes kidney damage or
death. In recent years
foodstuffs contaminated with these bacteria have caused gastro-intestinal
distress in tens or
hundreds of thousands of people and the recall and destruction of millions of
pounds of food. The
resulting economic loss has been staggering. Especially daunting as a public
health threat has been
E. coli 0157:H7, a pathogenic strain of the common gut bacterium, first
identified in 1982. The
bacteria are carried in the intestinal tracts of food animals and expelled in
their feces. From there,
the bacteria enter the food supply, not only in the meat of those animals, but
foods such as milk,
fruit juices, lettuce, alfalfa sprouts, radishes and others.
Haptens are partial or incomplete immunogens such as certain toxins, which
cannot by
themselves cause antibody formation but are capable of combining with specific
antibodies. Such
haptens may include bacterial toxin, yeast mold toxin, viruses, parasite
toxins, algae toxins, etc.
Other colony-forming organisms include Actinomycetes, Streptococcus,
Bacteriodes such
).0 as B. ruminicola, Crytococcus and yeast molds.
Another principal object of the present invention is to substantially prevent
the adherence of
immunogens, such as E. coli 0157:H7, or haptens, and the colonization and
growth of such
immunogens or haptens in the rumen or intestinal tracts of food animals, and
substantial
elimination of the immunogen or hapten from the feces of the animals, by the
administration to the
15 animals of fowl egg antibody to the specific immunogen or hapten.
CA 02438698 2003-06-26
WO 03/061693
PCT/US01/49588
3
PRIOR ART
The production of avian egg antibody for the diagnosis or treatment of
specific conditions
has been known. The production of avian egg antibody for the inhibition of
organisms, specifically
the colonization of non-illness-causing protein-wasting organisms, and the
adherence and
colonization of illness-causing immunogens is not suggested.
Representative prior art patents include the following:
Poison, U.S. Patent No. 4,550,019
Stolle et al, U.S. Patent No. 4,748,018
Tokoro, U.S. Patent No. 5,080,895
Carroll, U.S. Patent No. 5,196,193
Lee, U.S. Patent No. 5,367,054
Coleman, U.S. Patent No. 5,585,098
Stolle et al, U.S. Patent No. 5,753,268
Raun, U.S. Patent No. 3,794,732, discusses the uses of polyester antibiotics
in ruminant
rations to improve the utilization of feed in ruminant animals. This
specifically addresses the use of
antibiotics in ruminant animals as growth promotants.
Raun, U.S. Patent No. 3,947,836, discusses the use of specific antibiotic
compounds for
ruminant feed utilization improvement when give orally to the animal.
Specifically, the animal
develops rumen function where more propionates in relation to acetates are
produced thus
improving feed utilization.
Ivy et al, U.S. Patent No. 4,933,364, discusses an alternative process for
promoting growth
and feed efficiency of food producing mammals. They propose the use of zinc
antibiotic that can
be added in insoluble form to create a zinc antibiotic complex which enhances
feed efficiency of
food producing mammals. They reference two U.S. Patents, Nos. 3,501,568 and
3,794,732, that
cover monensin in great detail.
CA 02438698 2003-06-26
WO 03/061693
PCT/US01/49588
4
Other references on the use of additives such as monensin have mentioned the
need for wise
application of these materials because they can be toxic to some animals, such
as horses. These
antibiotics, which are not approved for use in dairy cows, must be
administered carefully. In
addition, feed intake is initially reduced as monensin cannot be added to
molasses based
supplements which are classic additives to cattle fees. (Pate, F., "Ionophores
Do Not Appear To
Work In Molasses Supplements", ONA Reports, November, 1966, 2 pages, Florida
Cattleman and
Livestock Journal; Lona, R. P. et al, J. Anim. Sci. 75(1): 2571-2579, 1979.)
Poison, U.S. Patent No. 4,550,019, is directed to the manufacture and use of
fowl egg yolk
antibodies for making immunological preparations for the passive immunizations
of animals,
0 including humans, as immuno reagents for immunosorbitive processes and in
particular for
quantitative analytical tests, especially micro assays for diagnostic,
pathological, forensic and
pharmacokinectic investigations.
Stolle et al, U.S. Patent No. 4,748,018, is directed to a method of passive
immunization of
mammals using avian egg yolk antibody against any of a variety of antigens
using various methods
5 of administration under various conditions and using various compositions
incorporating the
antibody, after first developing in the mammal a tolerance for the antibody.
Tokoro, U.S. Patent No. 5,080,895, is directed to a specific antibody
containing substance
from eggs and method of production and use thereof for the treatment of
infectious or other
diseases, and as additives in food for livestock and poultry, cosmetics, and
medicines, and in the
0 field of serodiagnosis. Although not explicitly stated, it is apparent
that the use of the egg antibody
in feeds is to provide an easy means of oral administration of the antibody
for the treatment of
intestinal infections in livestock or poultry.
Carroll, U.S. Patent No. 5,196,193, and divisional U.S. Patent No. 5,443,976,
are directed
to anti-venom compositions containing horse antibody or avian egg yolk
antibody for neutralizing
5 snake, spider, scorpion or jelly fish venom.
CA 02438698 2003-06-26
WO 03/061693
PCT/US01/49588
Lee, U.S. Patent No. 5,367,054, is directed to methods for large scale
purification of egg
immunoglobulin for the treatment of infections.
Coleman, U.S. Patent No. 5,585,098, is directed to a method of oral
administration of
chicken yolk immunoglobulins to lower somatic cell count in the milk of
lactating ruminants.
5 Stolle
et al, U.S. Patent No. 5,753,268, is directed to an anti-cholesterolemic egg
vaccine
and method for production and use as a dietary supplement for the treatment of
vascular disorders
in humans and other animals.
SUMMARY OF THE INVENTION
Broadly stated this invention is directed to a method for the production of a
microbial
adherence inhibitor for administration to host food animals to substantially
prevent the adherence of
colony-forming immunogens or haptens in the rumen and/or intestinal tracts of
the food animals by
first inoculating female birds, in or about to reach their egg laying age,
with the particular target
immunogen. Then, after a period of time sufficient to permit the production in
the bird of antibody
to the targeted immunogen, the eggs laid by the birds are harvested. The total
antibody-containing
contents of the eggs are separated from the shells and dried. The egg contents
may be dried on a
feed extender or carrier material. The dried separated egg antibody adherence
inhibiting material
may be stored or shipped for use when needed.
The target immunogen with which the bird is inoculated depends upon the
anticipated use
of the inhibitor, a non-disease-causing protein-wasting organism where
boosting of feed efficiency
is the objective, and a targeted disease-causing organism where the objective
is the substantial
reduction or elimination of illnesses.
The dried egg contents incorporating the antibody specific to the targeted
immunogen is
administered to the food animals by distributing the antibody material
substantially uniformly
throughout an animal feed and then supplying the resulting antibody-containing
animal feed to the
food animals. When improved feed utilization is the objective, the antibody-
containing animal feed
CA 02438698 2003-06-26
WO 03/061693
PCT/US01/49588
6
is supplied to food animals during the normal finishing schedule prior to
slaughter. The substantial
prevention of colonization of the targeted organism in the rumen or intestinal
tract of the animal
will ultimately permit elimination of the organism from the animal. This
repression of colonization
and elimination of the subject organisms will permit a significant decrease in
the wasteful
degradation of the dietary protein fed to food production animals. In
addition, the resulting
decrease in competition to the non-ammonia producing organisms will further
enhance the most
efficient utilization of feed by the host. (Russell, USDA-ARS, May 1993.) When
the objective is
the elimination of disease-causing organisms from the meat of food animals,
the antibody-
containing feed is supplied sufficiently before slaughter to substantially
prevent adherence of the
0 target immunogen or hapten in the intestinal tract of the animal, and
permit elimination of the
immunogen or hapten from the animal.
The invention is directed particularly to the production of an adherence
inhibitor specific to
E. coli 0157:H7 and to the substantial reduction or elimination of gastric
illnesses caused by this
bacterium. The invention is described with particular reference to elimination
of illnesses caused
5 by E. colt 0157:H7, but it is understood that the invention is not so
limited, but is equally
applicable to elimination of illnesses caused by the other colony-forming
immunogens and haptens.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is based on the concept of specifically inhibiting the
ability of colony-
forming protein-wasting organisms, such as P. anaerobius, C. sticklandii and
C. aminophilum, and
0 colony forming disease-causing organisms, such as E. colt 0157:H7,
Listeria, Salmonella and
Campylobacter, to adhere in the rumen or intestinal tracts of food animals and
thus reduce their
ability to multiply, grow and colonize. Dietary modifications may be designed
to make the rumen
and intestinal tract less receptive to the organisms over the lifetime of the
animal. While the
microbial inhibitor of the present invention may be administered at will by
the producer, it is
5 preferred for efficient animal feed utilization that a carefully
determined and managed course of
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
7
administration during the finishing period at the feedlot level be scheduled
and followed. Such a
predetermined period which takes advantage of the low dose, longer cumulative
effect of the
inhibitor and which is also easily integrated into current production
practices will provide the most
economically attractive rate of return through improved animal performance.
For the elimination of disease-causing organisms the inhibitor may be
administered either
immediately pre-slaughter or over some substantial period of the lifetime of
the animal. It is
preferred that a carefully determined and managed mid-term period course of
administration at the
feedlot level be followed. As described, a set pre-slaughter period takes
advantage of the low dose,
longer cumulative effect, is easily integratable into current production
practices and is the most
economical. It also allows the microorganism to naturally disappear from the
mud and manure on
the outside of the animal, a significant source of potential contamination at
slaughter. Under the
current feeding system, food animal feed efficiency is enhanced through the
use of ionophores such
as monesin, a feed additive marketed under the trade name Rumensin. These are
a class of
polyester antibiotics approved for feed given to beef cattle and dairy heifers
but not approved for
use with lactating diary cows. Most gram-positive organisms are non-
specifically vulnerable to the
ionophores, antibiotics which can also be quite toxic to the host animal if
used improperly. As
these antibiotics are not specific, many of the ruminal organisms required to
digest the cellulose of
ingested plant material may also be affected. The problem with carry over and
the development of
drug resistant strains of organisms are also major concerns to the industry.
The use of broad
spectrum antibiotics has further drawbacks including vulnerability to human
error, additional cost,
consumer resistance and the like. In addition, the monensin type additive
cannot be administered
with commonly used molasses based supplements.
Any organism that colonizes in the rumen or alimentary tract of its host must
possess the
capability of sticking or adhering to that surface in order to multiply and
grow. The specific
organisms addressed by this invention are no exception to the rule. As other
factors such as the
CA 02438698 2003-06-26
WO 03/061693
PCT/US01/49588
8
need of beneficial organisms for specific enzymes must also be considered,
specific reagents are
required to reduce the number of targeted organisms in the rumen or intestinal
tract while not
interfering with other normal flora. The organism inhibitor of this invention
strongly interferes
with adherence in a highly specific manner and, on a cumulative basis, thereby
prevents the
targeted organisms from multiplying, growing and colonizing. Through the
vehicle of a simple
daily feed supplement, the product essentially supplies the host with an
antibody preparation
designed not to cure any disease in the animal but to specifically dislodge
any resident bacteria in
the rumen or alimentary tract and to prevent attachment of any newly
introduced numbers of that
same bacteria. The microbial inhibitor has no direct effect whatsoever on the
ultimate food
0 products and leaves absolutely no undesirable residue in the animal or in
the ultimate food
products. In addition, since the deleterious organisms are prevented from
multiplying, they will
over time, for example the 120-day finishing period in the feedlot, disappear
through natural
degradation from the feedlot environment helping to eliminate that significant
potential source of
recontamination. The inhibitor product itself can be classified as a natural
material of animal origin
5 and as such can be used in almost any kind of feeding program. As the
active ingredients are
completely natural, they will work well with most feeds and feed additives
including molasses
based supplements.
All mammals and birds provide similar types of protection which allow for an
immediate
immune response in their very young offspring until they too acquire the
ability to make the
0 antibodies for themselves. More specifically called passive antibody
protection, this defense
mechanism is passed to the young of mammals through the placenta, the mother's
milk or through
both. The young of birds, however, receive their passive antibody protection
through the store of
antibodies placed in the eggs in which they develop from the embryonic stage.
Birds, in particular,
have the ability to "load up" their eggs as they are formed, with a very large
supply of antibodies
5 concentrated many fold over that which is present in the serum of the
mother. In addition, avian
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
9
antibodies are much more stable and resistant to inactivation through
digestion than mammalian
antibodies, especially under adverse conditions. Once immunized the hen layers
the unique IgY
types immunoglobulins in the yolk while depositing the common chicken 1gM and
IgA
immunoglobulins in the albumin. The albumin helps resistance to the whole egg
preparations and
helps protect the avian antibodies. Furthermore, the large quantities of
antibodies which are placed
in eggs are much more exclusively those specific for the antigens to which the
mother has most
recently been exposed to and challenged by. This all results in the eggs of
birds being a most ideal
source for large quantities of economically produced, highly specific and
stable antibodies. While
the invention is illustrated by the use of chickens to produce avian antibody,
other fowl including
turkeys, ducks, geese, etc. may be used.
Specifically, groups are obtained of young hen chickens typically Rhode Island
Reds, White
Leghorns, sex-linked hybrid crosses or other breeds suited to large egg size,
high volume egg
production and ease of handling which are about to reach laying age, about 19
weeks for chickens,
on a schedule predetermined by the amount and timing of final product desired
resulting in a steady
continuous production stream. After a suitable period of isolation and
acclimatization of about 2 to
4 weeks, each group will enter into an inoculation program using rehydrated
proprietary
preparations of specific antigens to which an antibody is desired. The
antigens may be obtained
from commercial sources such as the American Type Culture Collection (ATCC).
The antigen may
be injected intra-muscularly, but preferably injected sub-cutaneously. In
approximately four to five
weeks, the average egg collected will contain copious amounts of the desired
specific antibody in a
readily usable and stable form. The chickens may be reinoculated with the
targeted antigen
throughout the egg laying period to maintain the high antibody level.
Batches of eggs from predetermined groups of chickens are cracked, the
contents are
separated from the shells and mixed and preferably pasteurized (to eliminate
potential pathogenic
microorganism from the chicken and thus reduce potential contamination of
feed). The total egg
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
content is dried using standard commercial methods, such as spray drying using
ambient or hot air
up to 50 C and tested to determine overall titer or antibody level. The egg
contents may be dried
alone or on innocuous feed extenders such as dry soy or rice husks or the
like. Standard test
procedures are used, such as ELISA, or agglutination, or the like. The typical
batch is then blended
5 with batches from groups of chickens at other average production levels
resulting in a lot of
standardized active ingredient. The dried egg antibody microbial inhibitor
material may be stored
and shipped on carrier materials such as soy bean hulls, boluses and/or
tablets. Dependent on the
needs and specifications of the feed formulator and the final customer, the
final antibody product
may include some type of innocuous additive, such as dried whey or dried soy
protein powder,
10 dried soy or rice husks or the like for formulation with feed ration.
One egg produced and
processed by the above procedures will yield a product sufficiently active and
stable to provide at
least as many as 350 to 700 daily doses of managed protection against specific
microbial
colonization. This method provides for the first time, an economical, safe and
effective means for
controlling feed efficiency organisms in beef cattle and dairy herds, and an
economical, safe and
effective means for controlling E. coli 0157..H7 and other illness-causing
organisms in cattle herds.
The present invention specifically addresses feed efficiency as it relates to
beef cattle, and
by extension dairy cattle and dairy herds, and to the problem of eliminating
illness-causing
organisms from cattle. However, the concept of preventing microbial adherence
has great
economic potential for a number of diverse food safety and production
applications. One such field
of application is in feed and water targeting specific undesirable
microorganisms. An example of
this application would include products to actively inhibit pathogenic or even
spoilage
microorganisms in animal feed formulated for chickens and other poultry.
Another such field of
application is as rinse aid ingredients targeted to specific undesirable
microorganisms. Examples of
this application include products to actively dislodge pathogenic or even
spoilage microorganisms
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
11
for use in solutions for spot cleaning and rinsing beef carcasses or for
chilling poultry after they
have been dressed.
The most successful colonizing microorganisms, bacteria, viruses and parasite,
etc., have
evolved a number of different types of molecules, referred to as "adherins,"
on their surfaces which
can very tightly stick to one or more types of specific molecules that are
part of the host's various
surfaces. The adhesion inhibitor is an avian antibody of extraordinarily high
specific activity which
can very tightly bind to, coat, cover and obliterate these adherins which
attach themselves to their
hosts with a lock and key type of fit to very unique chemical structures. In
addition to this direct
attack, components of the complement system included in most biological
fluids, such as blood,
lymph, saliva, tears and to some extent intestinal secretions, recognize an
antibody attachment as
triggers for their many types of defensive activities. Specific antibody
attachment and coating
combined with the very likely mobilization of many other cellular defense
systems, therefore,
quickly culminates in the chemical inactivation and ultimately the destruction
of the targeted
microorganism.
The invention is further illustrated by the following examples:
Example 1: Selection of Egg Laying Avian Hens
The strain of egg laying hen may vary with needs and uses. Any egg laying fowl
hens may
be immunized including chickens, turkeys, ducks, emus or any other fowl. The
common strains of
egg laying chickens are the preferred and are usually selected for the number
of eggs laid per year,
size of egg and ease of housing. Rhode Island Red, White Leghorn and Red Sex
Linked hybrids
are the animals of choice based on egg size (large to ex-large, 50-65 gm) and
were used for the
immunization schedules. The ease of handling the animals and the size and
uniformity of the eggs
along with the number of eggs laid per hen per year were observed. Although
any avian egg laying
hen could be used, for cost and ease of use these chickens proved to work the
best. The Red Sex
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
12
Linked hybrid gave the most uniformity and greater number of eggs per animal.
These animals
produce a large to extra-large grade of egg (50-65 gm) and up to 300 eggs a
year per hen.
Example 2: Preparation of Stock Culture
The American Type Culture Collection E. coli 0157:H7 Stock #43895 was used as
the
model bacterium. The organism was isolated from raw hamburger and colonizes in
cattle. The
ATCC Method for rehydration of the stock was followed. The bacterium is
rehydrated in 1.0 ml of
TSB Broth (Tryptase Soy Broth, Becton Dickinson), transferred to 5 ml of TSB
sterile broth and
incubated overnight (approximately 18 hours) at 37 C. Nice turbid growth was
observed. This is
used as stock as needed. It was streaked on Sorbitol-MacConkey Agar (Difco)
for verification of
colony production.
Example 3: Preparation of H Antigens for Immunogens
The H antigens were selected for development into an immunogen for immunizing
the egg
laying hens. Certain conditions are used to maintain the optimum growth of the
H antigen during
culturing to give added concentrations for the prep. Veal Infusion Agar (VIS)
and Veal Infusion
Broth (VIB, Becton Dickinson) is preferred for H antigen production. Stock TSB
inoculated with
VIB is incubated at 22 to 24 C or room temperature for 18 hours. This
stimulates flagella
development on the bacteria. Flasks layered with VIA are inoculated with VIB
culture. Good
growth was seen after 22 hours. The product was harvested after 4 days. Flasks
are combined by
washing off the agar surface with Dulbecco's PSB solution (pH 7.3-7.4). The
product is collected
in tubes. Density is checked using spectrophotometer enumeration and McFarland
nephelometer
standards. Approximately 3 x 10/12/m1 in stock. Motility is checked with
motility agar slant
(Northeast Laboratory Services). Stock is diluted to concentration of
approximately 1 x 109 per ml
in PBS and stirred for 1 hour at room temperature. The flagella is removed
from the outside of the
bacteria. Supernatant is collected using centrifugation. Pellet of whole
bacteria is separated from
the supernatant. Dry weight approximately 14.7mg/m1 is determined and the
material is used as
CA 02438698 2010-01-29
13
stock immunogen for H antigen. It is diluted to 1 mg/m1 in PBS and heated for
30 minutes at 600 to
70 C. This helps keep contamination down to a minimum. Thiogylcollate broth
is inoculated to.
check for growth and animals are inoculated with immunogen. =
Fxample 4: Preparation of 0 Antigen for Immunogens
Brain Heart Infusion (BFI,=acumedia) is used to stimulate the 0 antigens on
the bacterium.
Stock TSB innoculate BI-II Broth is formed and incubated at 37 C for 18
hours. This stimulates
somatic antigen development on the bacteria. Flasks containing BHI Broth are
inoculated with BHT
Broth culture. While stirring slowly, flasks are incubated at 37 C. Good
growth is seen after 22
hours. Flasks are combined and the material is harvested using centrifugation
and sterile saline =
= 10 (0.9%) at approximately 3000 rpm for 30 minutes. The harvest is
collected in tubes. Density is
checked using spectrophotometer enumeration and McFarland nephelometer
standards. The
material is diluted to approximately 1 X 109 per ml. Four percent (4%) sodium
decixycholate
(Difco) solution is added as a 1:1 ratio with culture in 0.9% sterile saline
and _
stirred for approximately 18 hours at room temperature (22 to 24 C). The
material is centrifuged
15= = to remove whole cells. Supernatant is used as stock for 0 antigen.
Dry weight is determined at , =
approximately 14.9 mg/ml. The product is diluted in sterile PBS, pH 7,4 to 1
mg/ml for 0
Immunogen.
Example 5: Preparation of WC Antigen for Immunogens
Tryptic Soy Broth (TSB, Northeast Laboratory Services) plus Yeast Extract
(BBL) is used
20 for Whole Cell (WC) antigen production. TSB plus Yeast Extract 0.6%
Broth is inoculated with
TSB Stock and incubated at 37 C for 18 hours. This stimulates somatic and
other surface antigens
to development on the bacteria. Flasks are inoculated with TSB with Yeast
Extract Broth. While
== stirring slowly, it is incubated at 37 C. Good growth is seen after 22
hours. The flasks are
= combined and the product is harvested using centrifugation at
approximately 3000 rpm for 30
25 minutes and collected in tubes. The product is resuspended in sterile
PBS, pH 7.4. Density is
=
=
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
14
checked using spectrophotometer enumeration and McFarland nephelometer
standards. Dry weight
is approximately 19.7 mg/ml. The product is diluted to approximately 2 x 109
per ml or 2 mg/ml
dry weight, and 0.6% formaldehyde solution in PBS is added as a 1:1 ratio with
culture and stirred
for approximately 18 hours at room temperature (22 to 24 C) to fix cells.
Thiogylcollate broth is
inoculated to check for growth and pH of preparation (pH 7-7.4) is checked.
The supernatant is
used as stock for WC antigen. The stock is diluted in PHS, pH 7.4 to 1 mg/ml
for WC immunogen.
Example 6: Preparation of A Antigen for Immunogen
The Minca Medium is used for A antigen production. It is a standard medium for
stimulating the pilii and related adherin antigens. Stock TSB Minca Medium
Broth (Inf. Immun.,
Feb. 1977, 676-678) is inoculated and incubated at 37 C for 18 hours. This
stimulated adhesion
antigen development on the bacteria. Flasks are inoculated with Minca Medium
Broth and while
stirring slowing is incubated at 37 C. Good growth is seen after 18 hours.
The flasks are
combined and the product is harvested using centrifugation at approximately
2500 rpm for 30
minutes and collected in tubes. The pellet is resuspended in PBS and stirred
with a stir bar for one
hour at 22 to 24 C (room temperature). This removes the flagella. The
product is collected in
tubes and the pellet is resuspended in PBS and 0.01% Tween 2OTM, transferred
to Waring Blender
in cold (4 C) at low speed for 30 minutes. Density is checked using
spectrophotometer
enumeration and McFarland nephelometer standards. The product is centrifuged
to remove whole
cells. The supernatant is used as stock for A antigen. It may be heated at 60
C for 40 minutes to
inactivate if needed. Gentamycin is added at 50 i/m1 as preservative.
Thioglycollate broth is
inoculated to check for growth. Dry weight is determined at approximately 10.6
mg/ml. The
product is diluted with PBS, pH 7.4 to 1 mg/ml for A immunogen.
Example 7: Preparation of P Antigen for Immunogen
The Reinforced Clostridial Medium is used for P antigen production. It is a
standard
medium for stimulating adherence antigens for Peptostreptococcus anaerobius.
These cultures
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
must be grown under strict anaerobic conditions. The stock culture is grown
according to ATCC
for #49031. As with other organisms, subcultures are grown in small amounts.
Thioglycollate
Media (Difco) is inoculated with the stock and incubated for 48 hours. Flasks
are inoculated with
Reinforced Clostridia! Medium Broth. The medium is covered with a mixture of
anaerobic gas.
5 Flasks are combined and the product is harvested using centrifugation at
approximately 2500 rpm
for 30 minutes, collected in tubes and run at low speed for 30 minutes.
Density is checked. The
product is centrifuged to remove whole cells. The supernatant is used as stock
for P antigen. It is
heated at 60 C for 40 minutes to inactivate if needed. Dry weight is
determined. Approximately
20.5 mg/ml. The product is diluted with PBS, pH 7.4 to 1 mg/ml for P
immunogen.
10 Example 8: Preparation of CS Antigen for Immunogen
The Reinforced Clostridial Medium is used for CS antigen production. It is a
standard
medium for stimulating adherence antigens for Clostridium slicklandii. These
cultures must be
grown under strict anaerobic conditions. The stock culture is grown according
to ATCC for
#12662. As with other organisms, subcultures are grown in small amounts.
Thioglycollate Media
15 (Difco) is inoculated with the stock and incubated for 48 hours. Flasks
are inoculated with
Reinforced Clostridial Medium Broth. The medium is covered with a mixture of
anaerobic gas.
Flasks are combined and the product is harvested using centrifugation at
approximately 2500 rpm
for 30 minutes. The product is collected in tubes and spun at low speed for 30
minutes. Density is
checked using spectrophotometer enumeration and McFarland nephelometer
standards. The
product is centrifuged to remove whole cells. The supernatant is used as stock
for CS antigen. It is
heated at 60 C for 40 minutes to inactivate if needed. Dry weight is
determined at approximately
22 mg/ml. The product is diluted with PBS, pH 7.4 to 1 mg/ml for CS immunogen.
Example 9: Preparation for CA Antigen for Immunogen
The Reinforced Clostridial Medium is used for CA antigen production. It is a
standard
medium for stimulating adherence antigens for Clostridium aminophilius. These
cultures must be
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
16
grown under strict anaerobic conditions. The stock culture is grown according
to ATCC for
#49906. As with other organisms, subcultures are grown in small amounts.
Thioglycollate Media
(Difco) is inoculated with the stock and incubated for 48 hours. Flasks are
inoculated with
Reinforced Clostridial Medium Broth. The medium is covered with a mixture of
anaerobic gas.
Flasks are combined and the product is harvested using centrifugation at
approximately 2500 rpm
for 30 minutes. The product is collected in tubes and spun at low speed for 30
minutes. Density is
checked using spectrophotometer enumeration and McFarland nephelometer
standards. The
product is centrifuged to remove whole cells. The supernatant is used as stock
for CA antigen. It is
heated at 60 C for 40 minutes to inactivate if needed. Dry weight is
determined at approximately
to 20.5 mg/ml. The product is diluted with PBS, pH 7.4 to 1 mg/ml for CA
immunogen.
Example 10: Preparation of ELISA Plates Using H, 0, WC and A
Antigens for Monitoring Antibodies in Eggs, Chickens and Feed
H, 0, WC and A ELISA: Ninety-six well assay plate (flat bottom Costar ) were
coated
using 100 4iI/m1 with various concentration of antigens (H, A, 0, or WC or
combination: 10 pg -
200 4ug/m1) in carbonate buffer, ph 9.6. Plates were incubated between 22 to
37 C for up to 18
hours. The wells were aspirated to prevent cross-contamination. The plates
were blocked with 390
pl/well of 0.5% BSA and incubated at 37 C for 1 hour. Plates were coated
using alternative rows
of positive or negative for controls. Plates were rinsed one time with wash
buffer containing
TweenTm 20. One hundred microliters per well of diluted sample are added to
wells in duplicate
wells, and incubated at 37 C for one hour. Goat anti-Chicken IgG conjugate
with Horseradish
peroxidase (Kirkegard and Perry Laboratories; 1:1000 to 1:3000) was added.
After one hour
incubation, the substrate (TMB, KPL) was added according to manufacturer's
instructions and the
reaction is stopped after 10 minutes with 0.1 M phosphoric acid. Optical
densities of the wells
were determined in Dynatech ELISA Reader at 450 nm and the information was
recorded for
further data analysis.
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
17
Example 11: Analysis of Individual Eggs and Serum Over Time
Eggs were selected at various periods in the immunization period for
monitoring antibody
responses to the specific antigens. Selected chickens were monitored at day 0
and continued on a
monthly basis after the fourth month. The whole egg was collected from the
shell and then a 1 ml
sample was taken. This sample was then extracted with buffer to analyze the
antibody content.
The standard ELISAs for the H, 0, WC and A immunogens were used for analysis.
The negative
readings were subtracted form the OD readings. Serum samples were collected
from each animal
two weeks after the fourth immunogen injection.
The data given in the table below are examples of the results obtained over
the first four
months.
Egg Sample Date H Chicken 0 Chicken WC Chicken , A Chicken
.1 day:After first 0.030D = Neg 0.050D Neg
= injection
1 month 0.600D Neg 0.050D Neg _________
5 weeks 0.74
2 months 1.220D 1.110D 0.8801) 0.790D
3 months 1.000D .1.40D 0.990D 1.40D
4 months 1.160D 1.4013 0.940D 1.220D
Senuir 1 month 1.400. 0.910D _ 1.170D 0.970D
Example 12: Preparation of ELISA Plates Using P, CS and CA
Antigens for Monitoring Antibodies in Eggs, Chickens and Feed
P, CS and CA ELISA: Ninety-six well assay plate (flat bottom Costar ) were
coated using
100 p1/ml with various concentrations of antigens (P, CS, CA or combination:
10 pl - 200 pg/ml)
in carbonate buffer, pH 9.6. Plates were incubated between 22 to 37 C for up
to 18 hours. The
wells were aspirated to prevent cross-contamination. The plates were blocked
with 390 ,u1/well of
0.5% BSA and incubated at 37 C for one hour. Plates were coated using
alternative rows of
positive or negative for controls. Plates are rinsed one time with wash buffer
containing TweenTm
20. One hundred microliters per well of diluted sample are added to wells in
duplicate wells, and
incubated at 37 C for one hour. Goat anti-Chicken IgG conjugate with
Horseradish peroxidase
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
18
(Kirkegard and Perry Laboratories: 1:1000 to 1:3000) was added. After one hour
incubation, the
substrate (TMB, KPL) was added according to manufacturer's instructions and
the reaction is
stopped after 10 minutes with 0.1 M phosphoric acid. Optical densities of the
wells were
determined in Dynatech ELISA Reader at 450 nm and the information was recorded
for further
data analysis.
Example 13: Immunization of Chicken with H Immunogen
Six selected egg laying hens, three White Leghorns and three Rhode Island Reds
approximately 19 weeks old were injected with the stock H immunogen. Four
injections (500 dug,
100 pg, 200 pg and 250 pg) were given one week apart. A serum sample was
collected two weeks
after the last initial injection. If boosters were needed, 100 ,ug was given
in each booster (every six
months). Within four weeks, four out of six hens produced excellent antibodies
in the eggs.
ELISA H readings averaged 1.00 OD for 1:10,000 dilution and 0.265 OD for
1:50,000. Leghorn
hens did not do as well but all three Rhode Island Reds did well. After six
weeks the average
ELISA H reading was 1.40 OD for 1:20,000 dilution with all chickens
responding.
Example 14: Immunization of Chicken with 0 Immunogen
Six selected egg laying hens, six White Leghorns, approximately 19 weeks old
were
injected with the stock 0 immunogen. Four injections (500 pg, 100 pg, 200 pg
and 250 pg) were
given one week apart. A serum sample was collected two weeks after the last
initial injection. If
boosters were needed, 100 pg was given in each booster (every six months).
Within four weeks,
five out of the six hens produced excellent antibodies in the eggs. ELISA 0
readings averaged 1.42
OD for 1:10,000 dilution and 0.68 OD for 1:50,000. After six weeks the average
ELISA 0 reading
was 1.15 OD for 1:20,000 dilution with still five chickens responding.
Example 15: Immunization of Chicken with WC Immunogen
Six selected egg laying hens, six Rhode Island Reds, approximately 19 weeks
old were
injected with the stock WC immunogen. Four injections (500 pg, 100 pg, 200 pg
and 250 pg)
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
19
were given one week apart. A serum sample was collected two weeks after the
last initial injection.
If boosters were needed, 100 pg was given in each booster (every six months).
Within four weeks,
four out of the six hens produced excellent antibodies in the eggs. ELISA WC
readings averaged
0.95 OD for 1:10,000 dilution and 0.250 OD for 1:50,000. After six weeks the
average ELISA WC
reading was 0.95 OD for 1:20,000 dilution with still five chickens responding.
Example 16: Immunization of Chicken with A lmmunogen
Six selected egg laying hens, six White Leghorns, approximately 19 weeks old
were
injected with the stock A immunogen. Four injections (500 pg, 100 pg, 200 pg
and 250 dug) were
given one week apart. A serum sample was collected two weeks after the last
initial injection. If
boosters were needed, 100 pg were given in each booster (every six months).
Within four weeks,
five out of the six hens produced excellent antibodies in the eggs. ELISA A
readings averaged 1.40
OD for 1:10,000 dilution and 0.576 OD for 1:50,000. After six weeks the
average ELISA A
reading was 1.15 OD for 1:20,000 dilution with still all chickens responding.
Example 17: Immunization of Chicken with P lmmunogen
Six selected egg laying hens, White Leghorns, approximately 19 weeks old were
injected
with the stock P immunogen. Four injections (500 pg, 100 ,ug, 200 pg and 250
pg) were given one
week apart. A serum sample was collected two weeks after the last initial
injection. If boosters
were needed, 100 pg were given in each booster (every six months). Within four
weeks, five out of
the six hens produced excellent antibodies in the eggs.
Example 18: Immunization of Chicken with CS Immunogen
Six selected egg laying hens, White Leghorns, approximately 19 weeks old were
injected
with the stock CS immunogen. Four injections (500 pg, 100 ,ug, 200 pg and 250
pg) were given
one week apart. A serum sample was collected two weeks after the last initial
injection. If boosters
were needed, 100 pg was given in each booster (every six months). Within four
weeks, all five out
of six hens produced excellent antibodies in the eggs.
CA 02438698 2003-06-26
WO 03/061693
PCT/US01/49588
Example 19: Immunization of Chicken with CA lmmunogen
Six selected egg lay hens, Red Sex-Linked Hybrids, approximately 19 weeks old
were
injected with tht stock CA immunogen. Four injections (500 jig, 100 jig, 200
dug and 250 jig) were
given one week apart. A serum sample was collected two weeks after the last
initial injection. If
5 boosters were needed, 100 ,ug was given in each booster (every six
months). Within four weeks, all
six hens produced excellent antibodies in the eggs.
Example 20: Preparation of Stock Production Whole Egg Reagents
Selected hens were combined from all four immunogen groups for E. coli 0157:H7
or three
immunogen groups for anaerobes, to be used to produce production batches of
whole egg reagents.
10 Sterling (U.S. Patent No. 5,753,228) presents an excellent review of
uses for the selection of eggs
and storage of the same. The eggs were randomized and shell removed. The whole
egg is mixed
well and pasteurized using standard conditions (60 C (140 F) for 3.5
minutes) Charley, H. and C.
Weaver, 3rd Edition, Foods: a scientific approach, Merrill-Prentice Hall, p.
350, 1998). Once
pasteurized, samples were tested for activity and store at 4 C until dried or
sprayed onto carriers.
15 Samples of 250 pl were analyzed.
Examples of results for ELISAs are given:
Pasteurized Whole Egg: E. coli 0157:H7
Immunogen . Dilution O.D.
WC 500 = 0.532
WC = = = = 2500 . 0.113 = = = =
H . = . 0.466 "
20 H 2500 - = . 0.115
0 = = 500 *0,338 =
o . 2500 . 0.128
A 500 . . 0.588 . = .=
A = 2500 = . = 0.155 . =
Pasteurized Whole Egg: Anaerobes
Inmiunogen _ Dilution .latch in = . . Batch#2 Batch #3 =
CA = 100 0.339 = 0.275 = 0.627 =
CA 500 = 0.104 = = 0.296 0.201 =
=
100 0.724 = 0.882 0.576
500 . 0.248 0.594 . 0:651 =
100 Ø457 Ø268
= 0.650
[CC8S 500 = 0.304 0.143 = 0.476
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
21
Example 21: Coating of Feed Additive Carriers
Although whole egg can be dispensed in water supplies, or in a dried format as
whole
powdered egg, use of a carrier helps distribute the material in a uniform
method. This makes it
easier for mixing with standards feeds. A number of carriers can be used to
provide a vehicle as a
feed additive as needed. Soy hulls in crude, refined and pelted format, rice
hulls, corn, cottonseed
hulls, distilled dried grains, beet pulp or any other. The production
pasteurized whole egg prep is
coated onto the carrier and either fed directly to the animals or dried to 10-
15% moisture.
Approximately 1000 ml of whole, pasteurized egg is sprayed on 50 lbs of
pelleted soybean hulls.
The preferred carrier for cattle is pelleted soybean hulls while for young
swineAhe fines from
pelleted soybean hulls. The feed additive is mixed with the standard animal
feed. The preferred
level is 10-15 lbs of feed additive to 2000 lbs of animal feed.
Example 22: Analysis of Feed Additive Samples After Coating with Reagents
Samples were collected from batches of feed additive after they were coated on
to the
carriers. The samples were analyzed and the results are as follows:
.Product Name Moisture % Protein % Fat % Fiber, crude %
Crude Soybean 11.59 .26.76:. 9.10 18.63.
Hulls, uncoated
CAMAS EYE 12.35 25.67 8,26 19.46
0157
Crude soybean
Hulls . . _ =
CAMAS EYE 4 12.06 24.89 9.92 20.38
Control Crude
Soybean hulls
Soybean Pellets 11.65 9.89 " 243 33.47
uncoatod
CAMAS EYE. 12.37 10.19 2.57 33.12
Efficiency . = =
Pellets
*CAMAS EYE identifies inhibitors produced according to the present invention
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
22
Example 23: Analysis of Production Eggs Over Time-- E. coli 0157:H7
Samples of the whole egg preparations were analyzed using the ELISA systems
for H, 0,
WC and A immunogens to monitor activity over time after the initial
immunization schedule was
completed. Selected animals from each group were placed into the production
group. The average
ELISA OD readings (negative subtracted) for the fourth through the sixth
months are given in the
table below. The eggs were sampled using 250 pl of the whole eggs and diluted
1:500 and 1:2,500
in PBS buffer and then run in the appropriate ELISA to determine the average
OD reading at each
dilution. The negative control readings are subtracted from each reading. The
immunogens
showed different responses in animals along with good specificity. The A
immunogen gave the
to best responses in these tests. Data for these immunogens over time is
given below:
Irnmuogen Fourth Month Fifth Month Six Month
H: 1:500 0.388 0.848 '0.718
1:2500 0.085 0.237 0.195
0: 1:500 0.593 0.792 0.704 =
1:2500 0.147 0.294 0.184 =
:WC: 1:500 0.398 0.730 0.578
= 1:2500 0.062 . 0.273 0.130
A: 1:500 0:700 1.014 0.90 =
1:2500 0.102 0.305 = 0.224
Example 24: Analysis of Production Eggs Over Time -- Feed Efficiency
Samples of whole egg preparations were analyzed using the ELISA systems for P,
CS and
CA immunogens to monitor activity over time after the initial immunization
schedule was
completed. Selected animals from each group were placed into the production
group. The average
ELISA OD readings for the fourth through the sixth months are given in the
table below. The eggs
were sampled using 250 pi of the whole eggs and diluted 1:500 and 1:2,500 in
PBS buffer and then
run in appropriate ELISA to determine the average OD reading at each dilution.
The negative
control readings are subtracted from each reading. The immunogens showed
different responses in
the animals along with good specificity.
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
23
Immuogen Fourth Month = Fifth Month = = Six Month
P: 1:500 = 1.18.20D. . 1.1280D . = "
0.9420D
1:2500 . . = . 0.7850D = = = . ._ 0.4890D. = . - =
0.3430D =
CS: 1:500 0.8430D = = 0.9890D. = . 0..5820D '
. 1:2500 = 0.3180D 0.3560D 0.1870D =
CA: 1:500 = 1.1560D 1.0870D 0.9980D
1:2500 . 04090D = 0.2820D = .
Ø5070D
Example 25: Analysis of Feed Additives for Antibody Activity-- E. coli 0157:H7
Samples of the coated hulls were analyzed using the ELISA systems for H, 0, WC
and A
immunogens to monitor activity after pasteurizing, spraying, drying and
storage. Good antibody
response was recorded after the processing of the production whole egg batches
and drying on
crude soybean hulls. Data for two batches is given below:
= Batch; Coated WC H Immuogen 0 Immunogen A
immunogen
Hulls . Irnmunogen
Batch #1 1:10 0.6730D 1.1,03 0.D 1.10501) 1.299 OD
1:100 0.1060D 0.2360D 0.229.01) 0302 OD. . .
Batch #2 1:10 1.174 OD 1.291.0D. - 1.180 OD 1.224
OD .
.1:100 0.1770DØ3960D 0.327 OD _ 0.458 OD = .
Example 26: Analysis of Feed Additives for Antibody Activity -- Feed
Efficiency
Samples of the coated hulls were analyzed using the ELISA systems for P, CS
and CA
immunogens to monitor activity after pasteurizing, spraying, drying and
storage. Good antibody
response was recorded after the processing of the production whole egg batches
and drying on
crude soybean hulls. One gram samples of the 15 lbs of coated hulls were
extracted and analyzed.
Data for three batches is given in the table below:
Batch: Coated P Imtnunogen CS Ipimuogen CA Initnunogen
Mills = ...
Batch #1 1:100 0.06701) 0.289011 0:05 1 op.
1:500 0.0570D . 0.1310D 0.0370D=
Batch #2 1:100 0.0280D = 0.03901) 0.09501)
= . 1:500 0.0490D. 0.0150D =
0.0210D
Batch#3 1:100 0.0460D 0.11501) 0.13601)
1:500 0.01201) 0.0550D 0.01201) .
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
24
Example 27: Recovery of Active Antibody and Egg Protein After Feed Mix
Bags of coated soybean refined hulls were coated with the production whole egg
reagent
containing anti-E. coli 0157:H7 adherence inhibitors. One bag of feed additive
(15 lbs) was added
to 2000 lbs of standard cattle feed. Control feed additive was produced with
whole eggs from free
ranging chickens. Soybean hulls were coated with this preparation and mixed as
the test feed
additive containing the specific antibodies. Samples of the mixed feed were
collected and analyzed
for active antibody to the ELISA WC immunogen as well as commercial ELISA for
detecting egg
protein in food (Vertatox Quantitative Egg Allergen Test, Neogen). The data
is given in the chart
below for two batches of feed ration.
i 0 Mixed Feed First Batch = = Second Batch
Test Feed-Additive: 0.172 or) 0.1120D
= 1:6000 0.0000D 0.036
= 1:12000
Control Feed-No 0.049 = Net.
Additive 0.005 Neg.
=
1:6000 .
1:12000
.
Test Feed-Additive: 0.9580P :117Ppin 1.2680D >2.0ppra
Eg_g Protein
Control Feed-No 0.8000D 15 ppiii 1.0500D 20ppm
Additive: Egg
Protein = = .
Example 28: Feeding of Cattle
Two groups of cattle were fed either the E. coli 0157:H7 feed additive (coated
onto refined
soybean hulls) or control feed additive (coated with control eggs and no
specific adherence
inhibitors). The animals were fed at a rate of 15 lbs of feed additive per
2000 lbs of feed. They
averaged 10 lbs per animal per day. Animals weighed approximately 1000 lbs
when they started
and over 1400 lbs when sent to market. All animals looked very healthy with
the test animals
eating more feed during the 87 days. Five of the test animals were positive
during the start of the
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
experiment for E. coli 0157:H7 and only one of the control animals. Within 30
days on feed
additive all test animals were negative for E. coli 0157:H7 and stayed
negative for three
consecutive samples over a 30-day period. Standard protocols were followed for
sampling. All
animals were ear-tagged and placed in separate pens. Animals were sampled on a
weekly basis for
5 the first month and then bi-weekly after that until shipped to market.
Grab samples were taken
from the rectum and placed into sterile labeled bags. All samples were held on
ice until processed
in the lab. All samples were processed within four hours of collection each
day. The fecal samples
were diluted with TSB with 0.6% yeast extract. Dilutions of the mixture were
streaked into
Sorbitol-MacConkey's agar with or without cefixime-tellurite supplement
(Dynale). Colorless
10 colonies are picked for further testing. A latex agglutination test was
used to identify E. coli
serogroup 0157 (Oxoid dry SpotTM E. coli 0157). If positive, then individual
colonies were
selected for further isolation on SMC agar streak plates. Isolated colonies
were run on the
commercial EIA for EH E. coli 0157 (Binax, NOW EH E. coli 0157). Biochemical
confirmation
can be done with API-20E (Analytab Products). (Appl. Environ. Microbiol.,
62(7) 2567-2570,
15 1966; J. Clin. Micro. 36(10): 3112, 1998.)
One of the most startling and distressing characteristics of E. coli 0157:H7
is the small
number of microorganisms necessary to produce cases of human illness. By way
of example, at
least 10,000 of the more virulent Salmonella serotypes but as few as ten E.
coli 0157:H7 are
required to cause a person to become symptomatic. Therefore, one animal
hosting or externally
20 contaminated with the microorganism can, when slaughtered, affect as
much as 16 tons of ground
beef to the extent that a single helping of the product could result in
illness if improperly prepared.
Although the probability of any one animal hosting the microorganism at any
one time is low, the
probability of its presence in any one particular feedlot is high.
There are presently three different methods for protecting the consumer from
the E. coli
25 0157:117 threat which have been officially recognized. The three methods
are (1) thorough
CA 02438698 2003-06-26
WO 03/061693 PCT/US01/49588
26
cooking, (2) steam pasteurization and (3) irradiation, all of which have
specific drawbacks,
including human and mechanical error, cost, consumer resistance, and the like.
Any microorganism which colonizes the alimentary tract of its host must
possess the
capability of sticking or adhering to that surface in order to multiply. E.
coli 0157:H7 is no
exception to this rule. The adherence inhibitor of this invention strongly
interferes with adherence
and, on a cumulative basis, thereby prevents the specific targeted
microorganism from colonizing
and multiplying. Through the vehicle of a simple daily feed additive, the
product essentially
supplies the host with a specific antibody preparation designed not to cure
any disease in the animal
(cattle are essentially unaffected by E. coli 0157:H7 being only transitory
hosts) but merely to
dislodge any resident bacteria and to prevent the attachment of any newly
introduced bacteria in the
alimentary tract. The adherence inhibitor has no direct effect on the host
itself, leaves absolutely no
undesirable residue in the animals and thus has no effect whatsoever on the
ultimate food products.
In addition, since the microorganism is prevented from multiplying, it will
over time (for example
the 120 day finishing period in the feedlot) disappear through natural
degradation from the mud and
manure coating the animal, eliminating this significant potential source of
contamination at
slaughter. Properly managed, the risk of cross contaminating other food
sources through feedlot
runoff or by the application of manure as fertilizer is also essentially
eliminated.
It is apparent that many modifications and variations of this invention as
hereinbefore set
forth may be made without departing from the spirit and scope thereof. The
specific embodiments
described are given by way of example only and the invention is limited only
by the terms of the
appended claims.
