U.S. patent application number 13/957601 was filed with the patent office on 2015-02-05 for methods of reducing salmonella in poultry.
The applicant listed for this patent is The United States of America, as represented by the Secretary of Agriculture, The United States of America, as represented by the Secretary of Agriculture, WISCONSIN ALUMNI RESEARCH FOUNDATION. Invention is credited to MARK ERIC COOK, MIKE H. KOGUT, JORDAN MARSHALL SAND, CHRISTINA L. SWAGGERTY.
Application Number | 20150037277 13/957601 |
Document ID | / |
Family ID | 51298992 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150037277 |
Kind Code |
A1 |
COOK; MARK ERIC ; et
al. |
February 5, 2015 |
METHODS OF REDUCING SALMONELLA IN POULTRY
Abstract
Described herein are methods of reducing Salmonella in the
intestines of poultry in need thereof by administering to a poultry
bird an effective amount of an interleukin-10 peptide or an
isolated antibody that specifically binds an interleukin-10
peptide. Administering may be performed within 1 to 4 weeks of
harvest of the poultry in order to reduce Salmonella transmission
to human consumers. Also included herein are finishing feeds that
include an interleukin-10 peptide or an isolated antibody that
specifically binds an interleukin-10 peptide.
Inventors: |
COOK; MARK ERIC; (MADISON,
WI) ; SAND; JORDAN MARSHALL; (MADISON, WI) ;
KOGUT; MIKE H.; (COLLEGE STATION, TX) ; SWAGGERTY;
CHRISTINA L.; (COLLEGE STATION, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary of
Agriculture
WISCONSIN ALUMNI RESEARCH FOUNDATION |
Washington
MADISON |
DC
WI |
US
US |
|
|
Family ID: |
51298992 |
Appl. No.: |
13/957601 |
Filed: |
August 2, 2013 |
Current U.S.
Class: |
424/85.2 ;
424/139.1 |
Current CPC
Class: |
A61K 2039/545 20130101;
A23K 20/195 20160501; A23K 20/147 20160501; A61K 2039/505 20130101;
C07K 2317/11 20130101; C07K 16/244 20130101; A61K 2039/542
20130101; A23K 50/75 20160501; A61K 39/3955 20130101; A61K 38/2066
20130101; C07K 2317/10 20130101 |
Class at
Publication: |
424/85.2 ;
424/139.1 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A23K 1/18 20060101 A23K001/18; A23K 1/16 20060101
A23K001/16; A61K 39/395 20060101 A61K039/395 |
Claims
1. A method of reducing Salmonella in the intestines of poultry in
need thereof, comprising administering to a poultry bird an
effective amount of an interleukin-10 peptide or an isolated
antibody that specifically binds an interleukin-10 peptide, wherein
administering is within 1 to 4 weeks of harvest of the poultry.
2. The method of claim 1, wherein the poultry bird is a Salmonella
incubatory or convalescent carrier.
3. The method of claim 1, wherein the interleukin-10 peptide is SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
or a combination thereof.
4. The method of claim 1, wherein the interleukin-10 peptide is SEQ
ID NO: 2 or SEQ ID NO: 3.
5. The method of claim 1, wherein the isolated antibody
specifically binds SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, or SEQ ID NO: 5.
6. The method of claim 1, wherein the isolated antibody
specifically binds SEQ ID NO: 2, or SEQ ID NO: 3.
7. The method of claim 1, wherein the poultry is a chicken, a
turkey, a duck, a quail, or a pheasant.
8. The method of claim 1, wherein the poultry is a chicken, and
wherein the interleukin-10 peptide or isolated antibody that
specifically binds the interleukin-10 peptide is administered at
weeks 3, 4, 5, 6, 7, and/or 8 after hatch.
9. The method of claim 1, wherein the poultry is a turkey, and
wherein the interleukin-10 peptide or isolated antibody that
specifically binds the interleukin-10 peptide is administered at
weeks 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and/or
22 after hatch.
10. The method of claim 1, wherein the peptide or isolated antibody
is administered by injection or oral administration.
11. The method of claim 1, comprising administering an isolated
antibody, wherein the isolated antibody is in the form of an avian
egg yolk antibody.
12. The method of claim 11, wherein the isolated antibody
specifically binds SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID
NO: 4, or SEQ ID NO: 5.
13. The method of claim 11, wherein the isolated antibody
specifically binds SEQ ID NO: 2, or SEQ ID NO: 3.
14. The method of claim 1, wherein the peptide or isolated antibody
is administered in the form of a poultry feed composition.
15. The method of claim 14, wherein the poultry feed composition is
a finishing feed comprising no added antibiotics.
16. The method of claim 14, wherein the peptide or isolated
antibody is present in the poultry feed composition in an amount of
0.15 mg/kg of animal feed to 100 mg/kg of poultry feed.
17. A poultry finishing feed composition comprising a basal poultry
finishing feed composition, an amount of an interleukin-10 peptide
or an isolated antibody that specifically binds an interleukin-10
peptide, and no added antibiotics, wherein the basal poultry
finishing feed composition is suitable for feeding during the final
1 to 4 weeks prior to slaughter.
18. The poultry feed finishing composition of claim 17, wherein the
isolated antibody is in the form of an egg yolk antibody.
19. The poultry feed finishing composition of claim 18, wherein the
isolated antibody specifically binds SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
20. The poultry feed finishing composition of claim 18, wherein the
isolated antibody specifically binds SEQ ID NO: 2, or SEQ ID NO:
3.
21. The poultry feed finishing composition of claim 17, wherein an
interleukin-10 peptide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, or a combination thereof.
22. The poultry feed finishing composition of claim 17, wherein an
interleukin-10 peptide is SEQ ID NO: 2, SEQ ID NO: 3, or a
combination thereof.
Description
FIELD OF THE DISCLOSURE
[0001] The present disclosure is related to methods of reducing
Salmonella in the intestines of animals, including poultry.
BACKGROUND
[0002] Livestock such as poultry that are infected with pathogens,
or serve as incubatory or convalescent carriers of pathogens,
present problems both for the animals as well as humans who consume
meat from the animals. Antibiotics are generally added to the
nursery and grower feeds of poultry to promote growth and/or reduce
disease occurrence during all phases of food production. The
addition of antibiotics reduces the passage of pathogens such as S.
Enteritidis to humans and has advantages for the health and weight
of the poultry. Antibiotics promote growth through the reduction of
biological stress, the decrease of bacteria, and by promoting the
health of the poultry. Poultry that are healthy and disease free
eat more food, and more effectively convert the food into muscle or
meat. Thus, the antibiotic supplementation of poultry diet has
numerous benefits.
[0003] Despite the advantages, the practice of supplementing
poultry diet with antibiotics is increasingly problematic.
Sub-therapeutic doses of antibiotics are linked to the increased
presence of antibiotic-resistant bacterial strains in humans,
animals and in the environment. It is also possible for residual
antibiotics to appear in food that is meant for human consumption.
To prevent residues, antibiotics are generally withdrawn from the
feed of the poultry at least two weeks prior to slaughter to allow
for drug clearance, and hence to prevent the antibiotics
sequestered in the poultry from ingestion by humans.
[0004] Of particular importance to human consumers is Salmonellosis
(Salmonella infection), associated with the pathogen Salmonella
enterica subspecies enterica serovar Enteritidis (S. Enteritidis).
Poultry products, including eggs and poultry birds, serve as a
reservoir for S. Enteritidis. Poultry birds can be asymptomatic for
Salmonella infection, thus making it difficult to isolate infected
birds. The S. Enteritidis pathogen can pass to humans via
contaminated poultry products through the food production chain.
Undercooked or raw eggs and poultry meat thus constitute a high
risk of infection for humans. In fact, poultry carcasses entering
the processing environment are generally contaminated with bacteria
such as Salmonella.
[0005] As one example of an attempt to prevent the transmission of
Salmonella to human consumers, ground turkey samples are routinely
tested for Salmonella. For example, 25 gram samples of ground
turkey are tested and 50% of the samples must be free of
Salmonella. Recent outbreaks of food poisoning associated with
ground turkey have prompted proposals to increase the standards for
ground turkey, which with current production methods may greatly
increase the number of samples testing positive for Salmonella.
What is needed are improved antibiotic-free methods of treating
poultry to reduce the incidence of Salmonella in poultry
products.
BRIEF SUMMARY
[0006] In one aspect, a method of reducing Salmonella in the
intestines of poultry in need thereof comprises administering to a
poultry bird an effective amount of an interleukin-10 peptide or an
isolated antibody that specifically binds an interleukin-10
peptide, wherein administering is within 1 to 4 weeks of harvest of
the poultry.
[0007] In another aspect, a poultry finishing feed composition
comprises a basal poultry finishing feed composition, an amount of
an interleukin-10 peptide or an isolated antibody that specifically
binds an interleukin-10 peptide, and no added antibiotics, wherein
the basal poultry finishing feed composition is suitable for
feeding during the final 1 to 4 weeks prior to slaughter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1 shows the Salmonella counts vs. days post challenge
at 0.341 g of antibody/kg of feed.
[0009] FIG. 2 shows the Salmonella counts vs. days post challenge
at 1.705 g of antibody/kg of feed.
[0010] FIG. 3 shows the Salmonella counts vs. days post challenge
at 3.41 g of antibody/kg of feed.
[0011] The above-described and other features will be appreciated
and understood by those skilled in the art from the following
detailed description, drawings, and appended claims.
DETAILED DESCRIPTION
[0012] Described herein are methods of reducing Salmonella in the
intestines of animals, particularly poultry. Certain interleukin-10
(IL-10) peptides and anti-IL-10 peptide antibodies have been
described as useful in the treatment of Coccidiosis, a protozoal
infection, in animals such as poultry. The principal downside to
Coccidiosis is the growth suppression and other disease effects in
the birds as Coccidia is generally not transferrable to humans
consuming poultry products. Because young birds are more
susceptible to Coccidiosis than more mature birds, Coccidiosis is
generally treated by providing anti-Coccidial agents in starter
feeds. In commercial chicken production, for example, starter feeds
are generally used only in the first 3 weeks of a chick's life due
to the early susceptibility to Coccidiosis, the expense of feed
containing additives, and concerns about carry-over of agents into
produced meat. In turkeys, starter feeds may be used for 6-8 weeks
after hatch. In contrast to Coccidia, infection with Salmonella
bacteria often does not produce symptoms in the infected poultry,
but is a serious problem when passed to humans through consumption
of poultry products. An additive that can reduce Salmonella in the
intestines of poultry when used in the final 1 to 4 weeks prior to
harvest would greatly reduce the passage of Salmonella from poultry
to humans through consumption of poultry meat. It was thus found by
the present inventors that IL-10 peptides and anti-IL-10 peptide
antibodies are also effective against Salmonella and represent a
new strategy to prevent transmission of Salmonella to humans.
[0013] In one aspect, described herein is a method of reducing
Salmonella in the intestines of poultry in need thereof comprising
administering to a poultry bird an effective amount of an
interleukin-10 peptide or an isolated antibody that specifically
binds an interleukin-10 peptide, wherein administering is within 1
to 4 weeks of harvest of the poultry. As used herein, poultry means
domesticated fowl raised for meat or eggs. In general, broiler
chickens are harvested at 5 to 8 weeks after hatch, thus the
interleukin-10 peptide or an isolated antibody that specifically
binds an interleukin-10 peptide would be administered at weeks 3,
4, 5, 6, 7 and/or 8 after hatch. Turkeys are harvested between
weeks 10-22 after hatch, thus the interleukin-10 peptide or an
isolated antibody that specifically binds an interleukin-10 peptide
would be administered at weeks 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21 and/or 22 after hatch.
[0014] The IL-10 peptides and isolated antibodies that specifically
bind to IL-10 peptides described herein reduce the Salmonella
burden in chickens. Specifically, isolated antibodies that
specifically bind to IL-10 peptides reduced the Salmonella burden
in the ceca of chickens challenged with Salmonella enteritidis. In
one aspect, the poultry to be treated is a Salmonella-tolerant
bird, meaning that the bird is asymptomatic for Salmonellosis.
[0015] As used herein, the term "peptide" includes the peptide as
well as pharmaceutically acceptable salts of the peptide. "Amino
acid residue" means the individual amino acid units incorporated
into the peptides of the disclosure. As used herein, the term
"amino acid" means a naturally occurring or synthetic amino acid,
as well as amino acid analogs, stereoisomers, and amino acid
mimetics that function similarly to the naturally occurring amino
acids.
[0016] As used herein, the term "antibody", or "immunoglobulin",
encompasses naturally occurring antibodies, such as polyclonal and
monoclonal antibodies, as well as artificial or synthetic
antibodies or genetically engineered forms of antibodies, including
single chain antibodies, chimeric, and bifunctional antibodies, as
well as fragments thereof.
[0017] The term "isolated antibody" as used herein, refers to an
antibody that is substantially free of other naturally associated
molecules, or substantially free of antibodies having different
antigenic specificities.
[0018] The IL-10 peptide of the present disclosure includes the
amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ
ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and combinations
thereof (see Table 1). In particularly suitable embodiments, the
IL-10 peptide has an amino acid sequence of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5.
TABLE-US-00001 TABLE 1 Sequence ID NO. and Corresponding Amino Acid
Sequence. SEQ ID NO: AMINO ACID SEQUENCE Organism SEQ ID NO: 1
DDELNIQL Chicken, quail, turkey SEQ ID NO: 2 VLPRAMQT chicken SEQ
ID NO: 3 VLPRAMKT Quail and turkey SEQ ID NO: 4 EKMDENGI Chicken,
quail, turkey SEQ ID NO: 5 EPTCLHFS Chicken, quail, turkey SEQ ID
NO: 6 DQMGDLL pig SEQ ID NO: 7 DQLHSLL cow SEQ ID NO: 8 VMPKAESD
pig SEQ ID NO: 9 VMPQAENH Cow/sheep SEQ ID NO: 10 SKLQERGV pig SEQ
ID NO: 11 SELQERGV cow SEQ ID NO: 12 ENSCIHFP pig SEQ ID NO: 13
DSSCIHLP cow SEQ ID NO: 14 DQLNSML sheep SEQ ID NO: 15 NMLQERGV
sheep SEQ ID NO: 16 DSSCTHFP sheep SEQ ID NO: 17 DDLEIGL fish SEQ
ID NO: 18 VLPTAIADMTEE fish SEQ ID NO: 19 TQMEGKGP fish SEQ ID NO:
20 NQCCRFV fish
[0019] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
and/or SEQ ID NO: 5 are amino acid sequences corresponding to
peptides of the IL-10 cytokine in poultry, such as chickens, quail,
and turkeys. The IL-10 peptides of the present disclosure should
reduce the Salmonella burden in the ceca of chickens challenged
with Salmonella enteritidis by interfering with IL-10 signaling and
Salmonella tolerance.
[0020] The present disclosure further includes antibodies that
specifically bind to the IL-10 peptides (also referred to herein as
"anti-IL-10 antibody"). These antibodies have surprisingly been
found to reduce the Salmonella burden in the ceca of chickens
challenged with Salmonella enteritidis when isolated and
administered thereto. The antibodies of the present disclosure
specifically bind to IL-10 peptides including the amino acid
sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:
4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,
SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO: 18, SEQ ID NO:19, SEQ ID NO: 20, and combinations thereof. In
some embodiments, the isolated antibodies specifically bind to
IL-10 peptides having an amino acid sequence of SEQ ID NO: 1, SEQ
ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 5. In other
embodiments, the isolated antibodies bind SEQ ID NO: 2, SEQ ID NO:
3, or both.
[0021] The present disclosure is further directed to generating
antibodies that specifically bind to the IL-10 peptides. In one
embodiment, an antibody is generated by administering the IL-10
peptides described above to an animal. Suitable animals to
administer the IL-10 peptides for generating the antibodies
include, for example, poultry. Exemplary poultry include chickens,
turkeys, ducks, quail, and pheasant. Specific poultry include
turkeys and chickens. Additional animals include livestock animals
such as cows, pigs, sheep, and fish.
[0022] Exemplary methods for administering the IL-10 peptides to
the animal include injection and oral administration. Injection and
oral administration optionally include use of an adjuvant such as,
for example, Freund's Complete adjuvant and Cholera toxin.
Administration optionally further includes conjugation of the IL-10
peptide to a carrier protein such as, for example, bovine gamma
globulin or keyhole limpet hemocyanin.
[0023] In one embodiment, antibodies to the IL-10 peptides are
generated by an animal (referred to herein as the "producer
animal"). When the animal is an avian animal, as know by those
skilled in the art, the antibodies generated are passed to the egg,
and may specifically be concentrated in the egg yolk of the avian
producer animal. Alternatively, antibodies of the present
disclosure may be isolated from the animal itself such as from
serum.
[0024] In one embodiment, the antibody is an avian egg yolk
antibody. Egg yolks derived from a laying hen are inexpensive,
convenient and can be safer to handle as compared to the
hyperimmunized mammalian sera. Also, egg yolk antibodies are able
to stand up to the scrutiny under modern animal protection
regulations. Immunoglobulin Y (IgY) is an avian immunoglobulin.
[0025] To produce avian egg yolk antibodies, the IL-10 peptides are
injected into laying fowl, such as hens, preferably at various
intervals, to induce an immune response. The hens may be injected
intramuscularly or sub-cutaneously. The specific mode of injection
is not essential. It is well known that the IgY antibodies produced
by the hens in response to such an immune challenge are transferred
and concentrated in the egg yolk.
[0026] Once the eggs are harvested, the eggs may be further
processed to isolate the egg yolk, which itself may be further
processed. The liquid egg yolk may be encapsulated or otherwise
used in oral dosage forms. The egg yolk may be dried by spray or
refractant drying methods, and the resulting dried powder may be
encapsulated or otherwise used in oral dosage forms.
[0027] Alternatively, a procedure of partial purification or
fractionation may be carried out to remove the majority of the
non-aqueous bio-molecules and granules and optionally the majority
of other proteins in the egg yolk. Exemplary purification
techniques include the use of PEG, dextran sulfate or a natural
gum, such as sodium alginate, carrageenan and xanthan gum, to
coprecipitate the undesired substances, and the use of an aqueous
buffer or water to obtain an aqueous phase rich with
antibodies.
[0028] In a specific embodiment, the yolk is separated from the egg
white, and then washed with distilled water to remove as much
albumen as possible. The vitelline membrane encasing the yolk is
punctured, and the separated yolk fraction is then diluted with an
effective amount of an aqueous buffer or water to form a suspension
of the egg yolk. The collected egg yolk may be diluted with an
aqueous buffer solution or distilled water in a ratio of about 1:2
to about 1:40 v/v, and more specifically, in a ratio of about 1:5
to about 1:30 v/v. For efficient recovery of yolk antibodies, pH is
about 5-7. Desirably, the temperature in this step is within about
0.degree. C. to about 60.degree. C. The suspension of the egg yolk
is gently agitated to form a homogenous mixture, and then allowed
to stand for a period of time sufficient to form the aqueous and
non-aqueous phases. The water insoluble materials, including
non-aqueous bio-molecules such as lipoproteins, phospholipids,
sterols and the like, are then removed from the aqueous yolk
suspension by centrifugation. The resulting antibody-containing
supernatant may then be separated from the viscous precipitant by
decanting, suctioning, or other like methods known in the art.
[0029] Optionally, the yolk supernatant is further treated with a
high concentration of a non-denaturing salt to induce precipitation
of the antibodies. Examples of the salts useful for precipitation
of the yolk antibodies include, but are not limited to, NaCl,
Na.sub.z SO.sub.4, (NH.sub.4).sub.2SO.sub.4, KCl, CaCl.sub.2, and
MgSO.sub.4. Specific salts include Na.sub.2SO.sub.4 and
(NH.sub.4).sub.2SO.sub.4. The salt concentration for precipitating
antibodies depends on the type of the salt. In one embodiment, the
salt is present in an amount of higher than 15% and lower than 35%
by weight, specifically between 20% and 30% by weight of the salt,
on the basis of the final volume of the yolk supernatant.
[0030] Alternatively, the antibodies may be purified or isolated
using any conventional technique such as by immunoaffinity
purification.
[0031] In one embodiment, egg yolk antibodies are prepared by the
following method. Laying hens are inoculated with IL-10 peptide.
Optionally, an adjuvant is administered in conjunction with the
IL-10 peptide to enhance the immunization. An adjuvant useful for
this purpose is a water-in-oil emulsion adjuvant such as complete
Freund's adjuvant. The IL-10 peptide causes the hens to produce
anti-IL-10 antibodies which are passively transferred into the egg
yolk of eggs laid by the hens.
[0032] Egg yolks or whole eggs containing the anti-IL-10 antibody
can be collected and homogenized to form an emulsion. The resulting
emulsion can be dried to form a powder containing the anti-IL-10
antibody. This powder can then be formulated in a manner
appropriate to the administration route and then administered to
the desired animals using methods known in the art. The preparation
is preferably administered orally, such as in an oral dosage form
or in a supplement to the animal's diet.
[0033] The antibodies that specifically bind to IL-10 peptides may
be isolated and purified from animal serum or egg using a suitable
method known in the art. Such methods include affinity
chromatography, as well as other suitable methods for antibody
isolation and purification known in the art and described in U.S.
Pat. No. 6,608,172 and De Meulenaer et al., "Isolation and
Purification of Chicken Egg Yolk Immunoglobulins: A Review," Food
and Agricultural Immunology, Vol. 13(4), 2001, hereby incorporated
by reference to the extent that they are consistent herewith. In
one particularly suitable embodiment, the production animal is an
avian animal such as a chicken, turkey, duck, or quail, and the
antibody is isolated from the egg yolk of the egg of the avian
animal.
[0034] In one embodiment, the egg yolk or serum including the
antibodies are further dried to form a powder including the
antibodies. The whole egg, egg yolk or parts of the egg may be
spray dried. Serum may be separated from whole blood according to
methods known by those skilled in the art. Spray drying of egg and
serum may be performed using known spray drying methods and
commercially available spray drying equipment. Dry egg and serum
powders may also be prepared by lyophilization. The dried egg, egg
yolk or serum powder may then be introduced into animal feeds as a
feed additive to transfer antibodies to an animal.
[0035] The present disclosure is further generally directed to
animal feed additives including the IL-10 peptides, or isolated
antibodies which specifically bind to IL-10 peptides.
[0036] As used herein, the term "feed" broadly refers to a
material, liquid or solid, that is used for nourishing an animal,
and for sustaining normal or accelerated growth of an animal
including newborns or young and developing animals. The term
includes a compound, preparation, mixture, or composition suitable
for intake by an animal. Specifically, the feed is suitable for
poultry such as quail, ducks, turkeys, and chickens. A feed
composition comprises a basal food composition and one or more feed
additives. The term "basal food composition" refers to a food
composition combinable with additives such as the peptides and
antibodies described herein. Basal animal food compositions may
include components such as proteins, grains, flavor compositions,
vitamins, minerals, preservatives, and the like. Basal food
compositions can be suitable for ingestion by a target animal. The
term "feed additive" as used herein refers to components included
in small quantities for the purpose of fortifying basic feed with
nutrients, stimulants, medicine, or to promote feed intake or alter
metabolism. Feed additives include pre-mixes of biological
compositions, or in the present disclosure, pre-mixes of IL-10
peptide or isolated antibody that specifically binds to IL-10
peptide.
[0037] In one embodiment, the present disclosure includes an animal
feed additive including IL-10 peptides including the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20, and combinations thereof.
Particularly, the feed additive may include IL-10 peptides having
an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,
SEQ ID NO: 4, and/or SEQ ID NO: 5.
[0038] In another embodiment, the present disclosure includes an
animal feed additive including isolated antibodies that
specifically bind to the IL-10 peptide including the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ
ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19, SEQ ID NO: 20, and combinations thereof. In
particularly suitable embodiments, the feed additive includes
isolated antibodies that specifically bind to IL-10 peptides having
the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3, SEQ ID NO: 4, and/or SEQ ID NO: 5.
[0039] The IL-10 peptides or isolated antibodies which specifically
bind to IL-10 peptides may be added to an animal feed as a feed
additive or mixed into an animal feed by a method known in the art
for mixing feed additives and animal feed. In one embodiment, the
IL-10 peptide or isolated antibody which specifically binds to the
IL-10 peptide is directly added to the animal feed or mixed with
the animal feed just prior to feeding the animal. In another
embodiment, since feeds may be pelleted or extruded, the IL-10
peptide or isolated antibody which specifically binds to the IL-10
peptide may be coated on the surface of feed (pellet) after the
feed has been pelleted or extruded (post pelleted application) in
order to maintain functional properties of the IL-10 peptide or
isolated antibody which specifically binds to the IL-10 peptide.
The addition of the IL-10 peptide or isolated antibody which
specifically binds to the IL-10 peptide post pelleting can be aided
by mixing the IL-10 peptide or isolated antibody which specifically
binds to the IL-10 peptide in water, oil, or another suitable
carrier and spraying it on the pellets as they exit the pellet
die.
[0040] The amount of the IL-10 peptide or isolated antibody that
specifically binds to IL-10 peptide added and/or mixed with the
animal feed depends on the feeding regimen and the type of feed for
the animal, and may be determined by those skilled in the art.
Typically, the amounts of IL-10 peptides and/or isolated antibodies
to IL-10 peptide to be used in an animal feed are summarized in
Table 2 below. Antibody prepared using other sources may be
calculated as equivalents using Table 2.
TABLE-US-00002 TABLE 2 Dose of Anti-IL-10 Antibody in Animal Feed
(mg/Kg diet) prepared using egg yolk antibody. Source Low Dose High
Dose Affinity purified anti-peptide 0.0015 0.5 Anti-peptide IgY
0.015 50 Dry Immune Yolk 0.8 4000 Dried Immune Whole Egg 1.5
7500
[0041] The doses shown are based on the amount of epitope specific
antibody in total IgY (1 to 10%), the amount of IgY in egg (5-10
mg/Kg of feed), antibody losses due to drying storage and
gastrointestinal degradation.
[0042] An animal feed may further include optional ingredients
including vitamins, minerals, antibiotics, lipids, carbohydrates,
proteins, antioxidants, and amino acids.
[0043] Exemplary vitamins include Vitamin A, Vitamin B, Vitamin D,
Vitamin E, and Vitamin K. Exemplary minerals include calcium,
phosphorus, sodium, potassium, magnesium, chlorine, cobalt, iodine,
iron, manganese, copper, molybdenum, zinc and selenium. Common
mineral supplements used in poultry feed, for example, include
limestone, bone meal, oyster shell, sodium chloride, dicalcium
phosphate, manganese sulphate, potassium iodide, and
superphosphate.
[0044] In some embodiments, one or more antibiotics may be included
in the animal feed along with the feed additive. Exemplary
antibiotics include penicillin, streptomycin, tetracyclines, zinc
bacitracin and aureomycin.
[0045] Exemplary lipids include oil seeds, oils and lipids derived
from plants or animals. Sources of oilseeds, oils and lipids
include corn, soybean, cotton, lupin, peanut, sunflower, canola,
sesame seed oil, olive oil, copra and coconut oil, palm kernels and
palm oil, casein, butterfat, lard, fish oils, linseed and oil, tuna
oil, tallow and yellow grease, and mixtures thereof.
[0046] Exemplary carbohydrates include starch, cellulose,
pentosans, other complex carbohydrates, corn, milo, barley, rye,
oats, wheat, wheat middlings, and various grain-by-products.
[0047] Exemplary sources of protein include protein obtained from
meat meal or fish meal, liquid or powdered egg, fish solubles,
whey, milk protein, rice, milo, millet, corn, oats, barley, wheat,
rye, wheat bran and/or middlings, soybeans, sesame seeds, peas and
beans, sunflower seeds, wheat germ, alfalfa seed, flaxseed, yeast,
earthworms, and fish.
[0048] Exemplary amino acids include arginine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, threonine,
tryptophan, valine, tyrosine ethyl HCl, alanine, aspartic acid,
sodium glutamate, glycine, proline, serine, cystein ethyl HCl, and
analogs, and salts thereof.
[0049] Exemplary antioxidants include beta-carotene, Vitamin E,
Vitamin C, and tocopherol, or synthetic antioxidants.
[0050] Specifically, the animal feed including the feed additive of
either IL-10 peptide or isolated antibody is a feed for avian
species such as quail, ducks, turkeys, and chickens.
[0051] In a specific embodiment, the animal feed is a finishing
feed, that is, a feed composition used for poultry for consumption
that is generally used in the last 1 to 4 weeks prior to slaughter
to bring the poultry to market weight. Finishing feeds do not
contain antibiotics and other additives that might be transferred
to the meat produced upon slaughter. Finishing feeds can be
distinguished from starter feeds which contain, for example,
antibiotics and anti-coccidial agents which are crucial to prevent
disease prior to the development of immune defenses in the young
animal. Finishing feeds can also be referred to as withdraw
feeds.
[0052] In one aspect, poultry finishing feed composition comprises
a basal poultry finishing feed composition, an amount of an
interleukin-10 peptide or an isolated antibody that specifically
binds an interleukin-10 peptide, and no added antibiotics, wherein
the basal poultry finishing feed composition is suitable for
feeding during the final 1 to 4 weeks prior to slaughter. The
interleukin-10 peptides includes SEQ IDs: 1-5. In specific
embodiments, the peptide or isolated antibody is present in the
poultry feed composition in an amount of 0.15 mg/kg of animal feed
to 100 mg/kg of poultry feed.
[0053] The methods of the present disclosure are generally directed
to methods for treating gastrointestinal Salmonella infection in an
animal, or in an animal with a status of convalescence carriers of
pathogen. In one embodiment, the methods involve injecting or
orally administering an IL-10 peptide to an animal, thereby
producing antibodies within the animal that specifically bind to
the IL-10 peptide. IL-10 cytokine production is associated with
down regulation of inflammation, and the IL-10 cytokine functions
as an essential immunoregulator of the intestinal tract. The
antibody to IL-10 peptide prevents the IL-10 cytokine from down
regulating the immune system, thereby allowing the immune system to
eliminate the pathogen.
[0054] In some embodiments, the methods involve injecting or orally
administered an antibody to the IL-10 peptide to an animal. The
term "animal", as used herein to describe animals administered an
IL-10 peptide or isolated antibody to the IL-10 peptide in
accordance with the present disclosure, includes poultry such as
quail, ducks, turkeys, pheasants, and chickens.
[0055] In one aspect, the present disclosure is directed to methods
for preventing the transfer of human pathogens from poultry that
are infected or incubatory or convalescent carriers of human
pathogens to the human consumer of poultry products, or at risk of
being infected or incubatory or convalescent carriers of pathogens
by administering isolated antibodies that specifically bind to
IL-10 peptides including the amino acid sequences selected of SEQ
ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,
SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ
ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:
19, SEQ ID NO: 20, and combinations thereof. For example, the
methods may include administering isolated antibodies that
specifically bind to IL-10 peptides having the amino acid sequence
of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and/or
SEQ ID NO: 5. As used herein "at risk of" refers to having little
resistance to a certain condition or disease (i.e., bacterial
infection), including being genetically predisposed, having a
family history of, and/or having symptoms of the condition or
disease, or in a state of being a incubatory or convalescent
carrier of pathogens and being exposed to other animals that have
been exposed to or have the condition, disease or carrier
status.
[0056] In an aspect, described herein is a method of reducing
Salmonella in the intestines of a mammal or fish in need thereof
comprising administering an effective amount of an interleukin-10
peptide or an isolated antibody that specifically binds an
interleukin-10 peptide, wherein administering is within 1 to 4
weeks of harvest of the mammal or fish. When the animal is a
mammal, exemplary IL-10 peptides are SEQ ID NOs: 6-16. When the
animal is a fish, exemplary IL-10 peptides are SEQ ID NOs:
17-20.
[0057] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Detection of Antibody Production
[0058] In this Example, the concentration of anti-IL-10 antibody
production contained within the egg yolk of IL-10
peptide-administered producer hens was determined by using
Enzyme-linked immunosorbent assay (ELISA) techniques.
[0059] Specifically, each of four IL-10 peptides selected from the
group consisting of IL-10 Peptide #1 (SEQ ID NO: 1), IL-10 Peptide
#2 (SEQ ID NO: 2), IL-10 Peptide #3 (SEQ ID NO: 4), and IL-10
Peptide #4 (SEQ ID NO: 5) was conjugated to hen ovalbumin (OVA,
Sigma, St. Louis, Mo.) for ELISA using glutaraldehyde procedure. A
96-well Nunc.TM. immunosorbent F-series microplate (Sigma, St.
Louis, Mo.) was coated with 100 .mu.g/plate of peptide-specific OVA
conjugate in sodium carbonate coating buffer having a pH of 9.6.
The plate was allowed to coat overnight (100 .mu.l/well) at
4.degree. C. Dry egg yolk samples containing antibody to IL-10
Peptide #1, #2, #3, or #4 were diluted 1:10 in acidic PBS having a
pH of 4 and allowed to incubate overnight at 4.degree. C. After
overnight incubation, the antibody was extracted using
centrifugation and used as a source of antibody to determine
specificity for the peptide conjugated to the IL-10 peptide. The
plate coated with OVA-peptide conjugate was washed 6 times with
PBS/0.5% Tween solution, blocked with non-protein blocking buffer
(200 .mu.l/well, Pierce Scientific, Rockford, Ill.), and allowed to
incubate at room temperature for at least 1 hour. The plate was
washed 6 times and then samples of either adjuvant only injected
control or egg antibody (isolated as described above) were added at
a concentration of 100 .mu.l/well in duplicate at 10.times. serial
dilutions starting at 1:1000. Primary antibodies were incubated for
1 hour, the plate was washed 6 times, and then secondary antibody
(HRP-conjugated goat anti-chicken antibody, Bethyl Labs,
Montgomery, Tex.) was diluted in blocking buffer 1:5000 and added
at a concentration of 100 .mu.l/well. Secondary antibody was
incubated for 30 minutes, the plate was washed 6 times, and then
substrate solution containing 19.74 ml 0.05M sodium acetate, 100
.mu.l 20 mg/mL 3,3',5,5' Tetramethyl Benzidine (TMB), 128 .mu.l
0.5M H.sub.2O.sub.2 was added at a concentration of 125 .mu.l/well
and allowed to incubate until sufficient color development during
the linear phase of development (blue color indicates primary
antibody presence). A stop solution (0.5M sulfuric acid) was added
to produce a yellow stable color and the plate was read at 450 nm
on a Biotek EL800 plate reader. Duplicate optical densities were
averaged and blocking buffer background was subtracted to produce a
final optical density. The optical density of antibody to IL-10
peptides #1-4 and FCA control were compared to determine
specificity and dose level used in the final chick experiment (see
Table 3).
TABLE-US-00003 TABLE 3 Optical Densities of Anti-IL-10 Peptides.
Dilution IL-10 #1 IL-10 #2 IL-10 #3 IL-10 #4 10 1.0585 0.935 0.968
0.8822 100 1.049 1.006 0.973 0.9 1000 0.9705 1.033 1.001 0.678
10000 0.4795 0.6775 0.593 0.224 100000 0.075 0.129 0.115 0.059
1000000 0.028 0.03 0.037 0.03 10000000 0.059 0.016 0.027 0.028
100000000 0.022 0.0055 0.0181 0.033 FCA 0.562 0.499 0.60
Example 2
Anti-IL-10 Antibody Feed Study in Chickens
[0060] This study was performed to determine whether an anti-IL-10
antibody can decrease Salmonella carrier status in poultry,
specifically chickens. Chicks were challenged with Salmonella
enteritidis (10.sup.6 per chick on day 4 or 5 post-hatch) or not
challenged. This experimental protocol allows chick to become
recovered carriers and thus incubatory or convalescent carriers of
pathogens for humans. The antibody used was an egg yolk antibody to
peptide SEQ ID NO: 2. Control antibodies were made by injecting
chickens with adjuvant only (FCA antibody or isotype adjuvant
control antibody). Fourteen treatments were performed:
[0061] 1. normal diet, no challenge (60 chicks/pen)
[0062] 2. normal diet, SE challenge (60 chicks/pen)
[0063] 3. Control ab diet (0.341 g/Kg), no challenge (50
chicks/pen)
[0064] 4. Control ab diet (1.705 g/Kg), no challenge (50
chicks/pen)
[0065] 5. Control ab diet (3.41 g/Kg), no challenge (50
chicks/pen)
[0066] 6. Control ab diet (0.341 g/Kg), SE challenge (50
chicks/pen)
[0067] 7. Control ab diet (1.705 g/Kg), SE challenge (50
chicks/pen)
[0068] 8. Control ab diet (0.341 g/Kg), SE challenge (50
chicks/pen)
[0069] 9. IL-10 ab diet (0.341 g/Kg), no challenge (50
chicks/pen)
[0070] 10. IL-10 ab diet (1.705 g/Kg), no challenge (50
chicks/pen)
[0071] 11. IL-10 ab diet (3.41 g/Kg), no challenge (50
chicks/pen)
[0072] 12. IL-10 ab diet (0.341 g/Kg), SE challenge (50
chicks/pen)
[0073] 13. IL-10 ab diet (1.705 g/Kg), SE challenge (50
chicks/pen)
[0074] 14. IL-10 ab diet (3.41 g/Kg), SE challenge (50
chicks/pen)
[0075] Chicks were placed on their respective diet immediately
post-hatch. At baseline (2 days post-hatch) and each time-point (7,
11, 14, 18, 21, 30, 35 and 42 days post-hatch), five birds were
sacrificed. The samples collected include: Blood, cecal content for
Salmonella cfu; cecal for enrichment; tissues from ceca, liver and
muscle; intestine; and cecal tonsils
[0076] FIG. 1 shows the cecal Salmonella counts vs. days post
challenge at 0.341 g of antibody/kg of feed. FIG. 2 shows the cecal
Salmonella counts vs. days post challenge at 1.705 g of antibody/kg
of feed. FIG. 3 shows the cecal Salmonella counts vs. days post
challenge at 3.41 g of antibody/kg of feed. In chicks not infected
with Salmonella, Salmonella was not isolated (not shown). Shown in
FIGS. 1-3 is the colony forming units (CFU) per gram of cecum
contents. The first ten days post infection, there was no
difference in the CFU between treatment groups. All chicks
regardless of treatment had between 5 and 7 Log 10 Salmonella per
gram of cecal matter. This finding showed that the anti-IL-10
antibody was not effective at preventing infection. Beginning on
day 17 post-infection, the number of Salmonella per gram of cecal
matter decreased, such that by day 37 the chicks not provided with
either control or anti-IL-10 antibody stabilized at approximately 2
Log 10 CFU/gram cecal content, a level of bacteria that would be
consistent with an incubatory or convalescent carrier status for
Salmonella. The finding of no Salmonella in the ceca of control
birds (n=5) at day 30 was a sampling error, since at day 37 it was
clear that control chicks still had cecal Salmonella. For detecting
Salmonella, a level of Salmonella of 1 Log 10 or greater is
considered incubatory or convalescent carrier of Salmonella. None
of the chicks fed anti-IL-10 had a carrier status by day 37. Also
at day 37, 20% (1/5) of the birds not fed either control nor
anti-IL-10 antibody and 20% (3/15) of chicks fed control antibody
had a greater than 1 Log 10 Salmonella, whereas 0/15 of the
anti-IL-10 chicks had Salmonella. Hence the use of anti-IL-10
antibodies at all doses studied eliminated an incubatory or
convalescent carrier status for the chicks, where as 20% of those
fed the control antibody or no antibody continued to be incubatory
or convalescent carrier of Salmonella. Another objective estimate
of the effects of the anti-IL-10 antibody is that the anti-IL-10
antibody reduced the amount of Salmonella in the ceca of chickens
by at least 100-fold at all concentrations tested. These data
suggest that the amount of antibody may be lowered from the lowest
level of 0.341 g of antibody/kg of feed (0.341 g antibody/Kg of
feed is equivalent to 0.7 pounds of antibody-containing yolk
powder/ton of feed).
Example 3
Anti-IL-10 Antibody Feed Study in Turkeys
[0077] The USDA Food Safety and Inspection Service has decreased
the tolerance for naturally abundant Salmonella species on turkey
products. A study will be conducted to determine if the number of
positive tests following USDA guidelines can be decreased by the
use of anti-IL-10 antibody in the withdraw diet of turkeys. Turkey
Toms 14-18 weeks of age are placed either on a withdraw diet with
no added anti-IL-10 antibody, or a withdraw diet supplemented with
3.1 Kg IL-10 antibody in a dried egg yolk carrier per ton of feed.
Turkeys are fed the two diets for two weeks, turkeys are
slaughtered, and whole turkeys and their products are tested using
the USDA guidelines for the present of Salmonella species. The
incidence of Salmonella on turkey carcasses can be 2% in those
turkeys fed a control diet, and should be lower in those turkeys
fed a diet containing anti-IL-10 antibody. Ground turkey meat will
also be analyzed. Control fed turkeys can have 11% or more
Salmonella positive samples, while those turkeys fed a diet
containing anti-IL-10 antibody should have fewer than 11%
Salmonella positive samples.
[0078] The use of the terms "a" and "an" and "the" and similar
referents (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
terms first, second etc. as used herein are not meant to denote any
particular ordering, but simply for convenience to denote a
plurality of, for example, layers. The terms "comprising",
"having", "including", and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to")
unless otherwise noted. Recitation of ranges of values are merely
intended to serve as a shorthand method of referring individually
to each separate value falling within the range, unless otherwise
indicated herein, and each separate value is incorporated into the
specification as if it were individually recited herein. The
endpoints of all ranges are included within the range and
independently combinable. All methods described herein can be
performed in a suitable order unless otherwise indicated herein or
otherwise clearly contradicted by context. The use of any and all
examples, or exemplary language (e.g., "such as"), is intended
merely to better illustrate the invention and does not pose a
limitation on the scope of the invention unless otherwise claimed.
No language in the specification should be construed as indicating
any non-claimed element as essential to the practice of the
invention as used herein.
[0079] While the invention has been described with reference to an
exemplary embodiment, it will be understood by those skilled in the
art that various changes may be made and equivalents may be
substituted for elements thereof without departing from the scope
of the invention. In addition, many modifications may be made to
adapt a particular situation or material to the teachings of the
invention without departing from the essential scope thereof.
Therefore, it is intended that the invention not be limited to the
particular embodiment disclosed as the best mode contemplated for
carrying out this invention, but that the invention will include
all embodiments falling within the scope of the appended claims.
Any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
2018PRTGallus gallus 1Asp Asp Glu Leu Asn Ile Gln Leu 1 5
28PRTGallus gallus 2Val Leu Pro Arg Ala Met Gln Thr 1 5
38PRTMeleagris gallopavo 3Val Leu Pro Arg Ala Met Lys Thr 1 5
48PRTGallus gallus 4Glu Lys Met Asp Glu Asn Gly Ile 1 5 58PRTGallus
gallus 5Glu Pro Thr Cys Leu His Phe Ser 1 5 67PRTSus scrofa 6Asp
Gln Met Gly Asp Leu Leu 1 5 77PRTBos taurus 7Asp Gln Leu His Ser
Leu Leu 1 5 88PRTSus scrofa 8Val Met Pro Lys Ala Glu Ser Asp 1 5
98PRTBos taurus 9Val Met Pro Gln Ala Glu Asn His 1 5 108PRTSus
scrofa 10Ser Lys Leu Gln Glu Arg Gly Val 1 5 118PRTBos taurus 11Ser
Glu Leu Gln Glu Arg Gly Val 1 5 128PRTSus scrofa 12Glu Asn Ser Cys
Ile His Phe Pro 1 5 138PRTBos taurus 13Asp Ser Ser Cys Ile His Leu
Pro 1 5 147PRTOvis aries 14Asp Gln Leu Asn Ser Met Leu 1 5
158PRTOvis aries 15Asn Met Leu Gln Glu Arg Gly Val 1 5 168PRTOvis
aries 16Asp Ser Ser Cys Thr His Phe Pro 1 5 177PRTGadus morhua
17Asp Asp Leu Glu Ile Gly Leu 1 5 1812PRTGadus morhua 18Val Leu Pro
Thr Ala Ile Ala Asp Met Thr Glu Glu 1 5 10 198PRTGadus morhua 19Thr
Gln Met Glu Gly Lys Gly Pro 1 5 207PRTGadus morhua 20Asn Gln Cys
Cys Arg Phe Val 1 5
* * * * *