U.S. patent application number 14/670636 was filed with the patent office on 2016-09-29 for compositions and methods of use of interleukin-10 peptides and anti-interleukin-10 antibodies.
The applicant listed for this patent is WISCONSIN ALUMNI RESEARCH FOUNDATION. Invention is credited to MARK ERIC COOK, SHEILA MARY McGUIRK, SARAH MARIE RAABIS, JANE ELLEN RIEMAN, JORDAN MARSHALL SAND.
Application Number | 20160280778 14/670636 |
Document ID | / |
Family ID | 55586417 |
Filed Date | 2016-09-29 |
United States Patent
Application |
20160280778 |
Kind Code |
A1 |
COOK; MARK ERIC ; et
al. |
September 29, 2016 |
COMPOSITIONS AND METHODS OF USE OF INTERLEUKIN-10 PEPTIDES AND
ANTI-INTERLEUKIN-10 ANTIBODIES
Abstract
Disclosed herein are methods of reducing a symptom of
respiratory disease in a pre-weaned milk-fed mammal by orally
administering an isolated antibody that specifically binds the
interleukin-10 peptide or an anti-interleukin-10 antibody. Also
included are methods of reducing mixing stress in human and
non-human mammals by administering an isolated antibody that
specifically binds the interleukin-10 peptide or an
anti-interleukin-10 antibody. Further included are milk and food
compositions including the interleukin-10 peptide or
anti-interleukin-10 antibody.
Inventors: |
COOK; MARK ERIC; (MADISON,
WI) ; SAND; JORDAN MARSHALL; (MADISON, WI) ;
McGUIRK; SHEILA MARY; (MADISON, WI) ; RIEMAN; JANE
ELLEN; (DAVIS, IL) ; RAABIS; SARAH MARIE;
(MADISON, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WISCONSIN ALUMNI RESEARCH FOUNDATION |
MADISON |
WI |
US |
|
|
Family ID: |
55586417 |
Appl. No.: |
14/670636 |
Filed: |
March 27, 2015 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 33/40 20160801;
A61K 2039/505 20130101; A23L 33/18 20160801; C07K 2317/23 20130101;
A23C 9/1526 20130101; A23V 2002/00 20130101; A61P 33/00 20180101;
C07K 2317/11 20130101; C07K 2317/10 20130101; A61K 2039/542
20130101; C07K 2317/34 20130101; C07K 16/244 20130101; A61K 38/08
20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24; A23L 1/305 20060101 A23L001/305; A23C 9/152 20060101
A23C009/152; A23L 1/29 20060101 A23L001/29 |
Claims
1. A method of reducing a symptom of respiratory disease in a
pre-weaned milk-fed mammal, comprising orally administering to the
pre-weaned milk-fed mammal an effective amount of an interleukin-10
peptide or an isolated antibody that specifically binds an
interleukin-10 peptide, wherein administration is initiated between
birth and weaning, and wherein administration is performed at least
once daily for a period of 7 days to 7 weeks.
2. The method of claim 1, wherein administration of the Il-10
peptide or anti-IL-10 antibody is stopped after the 7 day to 7 week
period and is not restarted for 1 to 14 days.
3. The method of claim 1, wherein the pre-weaned milk-fed mammal is
of a genus Bos, Ovis, Capra, Bubalus, or Sus.
4. The method of claim 3, wherein the pre-weaned milk-fed mammal is
a bovine 56 days of age or younger and the respiratory disease is
bovine respiratory disease complex.
5. The method of claim 4, wherein the symptom of respiratory
disease is elevated rectal temperature, cough, nasal discharge,
ocular discharge, ear droop, diarrhea, increased fecal pathogens
including Cryptosporidium parvum, reduced appetite, abnormal
attitude, dehydration, reduced weight gain, decreased fecal pH, or
a combination thereof.
6. The method of claim 1, wherein the isolated antibody that
specifically binds the interleukin-10 peptide is an egg yolk
antibody.
7. The method of claim 1, wherein the interleukin-10 peptide or the
isolated antibody that specifically binds the interleukin-10
peptide is administered in a milk composition.
8. 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,
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, or SEQ ID NO.
14.
9. A method of reducing mixing stress in a mammal, comprising
orally administering to the mammal an effective amount of an
interleukin-10 peptide or an isolated antibody that specifically
binds an interleukin-10 peptide, wherein administration is
initiated within four weeks of mixing the mammal with a new
population of mammals, and wherein administration is performed at
least once daily for a period of 7 days to 7 weeks.
10. The method of claim 9, wherein administration of the Il-10
peptide or an isolated anti-IL-10 antibody is stopped after the 7
day to 7 week period and is not restarted for 1 to 14 days.
11. The method of claim 9, wherein the isolated antibody that
specifically binds the interleukin-10 peptide is an egg yolk
antibody.
12. The method of claim 9, wherein the interleukin-10 peptide or
the isolated antibody that specifically binds the interleukin-10
peptide is administered in a food composition.
13. The method of claim 9, wherein the mammal is a non-human
mammal.
14. The method of claim 13, further comprising exposing the
non-human mammal to a shipping crate, new food, vaccination,
branding, tail docking, castration, dehorning, temperature change,
a means of transportation, a new handler, a new caregiver, or a
combination thereof.
15. The method of claim 13, wherein the interleukin-10 peptide is
any one of SEQ ID NOs. 1-34.
16. A method of reducing mixing stress in a human child, comprising
orally administering to the human child an effective amount of an
interleukin-10 peptide or an isolated antibody that specifically
binds an interleukin-10 peptide, wherein administration is
initiated within four weeks of mixing the human child with a new
population of human children, and wherein administration is
performed at least once daily for a period of 7 days to 7
weeks.
17. A milk or formula composition suitable for administration to a
pre-weaned mammal of a genus Bos, Ovis, Capra, Bubalus or Sus
comprising a basal milk composition and a therapeutically effective
amount of an interleukin-10 peptide or an isolated antibody that
specifically binds the interleukin-10 peptide.
18. The milk composition of claim 17, wherein the basal milk
composition comprises whole milk, waste milk, or a milk replacer
composition.
19. The milk composition of claim 17, wherein the therapeutically
effective amount of an interleukin-10 peptide or an isolated
antibody that specifically binds the interleukin-10 peptide is 3
g/kg to 0.0001 mg/kg of the milk composition as a dry matter
weight.
20. The milk composition of claim 17, wherein the peptide is of any
one 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, or SEQ
ID NO. 14. or the isolated anti-IL-10 antibody specifically binds a
peptide of and one 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, or SEQ ID NO. 14.
21. The milk composition of claim 17, wherein the isolated antibody
that specifically binds the interleukin-10 peptide is an egg yolk
antibody.
22. A food composition suitable for administration to a human,
comprising a basal food composition and a peptide of SEQ ID NO. 35,
SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38; or an isolated
anti-IL-10 antibody that specifically binds a peptide of SEQ ID NO.
35, SEQ ID NO. 36, SEQ ID NO. 37, or SEQ ID NO. 38.
23. The food composition of claim 22, wherein the basal food
composition is milk, juice, formula, or a solid food consumable by
humans.
24. The food composition of claim 22, wherein the isolated antibody
that specifically binds the interleukin-10 peptide is an egg yolk
antibody.
Description
FIELD OF THE DISCLOSURE
[0001] The present disclosure is related to compositions containing
interleukin-10 peptides and anti-interleukin-10 antibodies and to
their methods of use in the treatment of humans and other
mammals.
BACKGROUND
[0002] Interleukin-10 (IL-10) peptides and anti-IL-10 antibodies
have been described in U.S. Pat. No. 8,652,457, as well as their
use to treat gastrointestinal protozoan infections in animals. In
particular, isolated anti-IL-10 antibodies were shown to prevent
growth suppression due to Coccidiosis infection in chicks, and
injection of IL-10 peptides into hens was shown to passively
transfer anti-IL-10 antibodies to chicks to prevent growth
suppression caused by Coccidia infection. Animal feeds and animal
feed additives containing the IL-10 peptides and anti-IL-10
antibodies were described.
[0003] What is needed are additional methods for the treatment
and/or prevention of additional diseases and conditions in
animals.
BRIEF SUMMARY
[0004] In an aspect, a method of reducing a symptom of respiratory
disease in a pre-weaned milk-fed mammal comprises orally
administering to the pre-weaned milk-fed mammal an effective amount
of an interleukin-10 peptide or an isolated antibody that
specifically binds an interleukin-10 peptide, wherein
administration is initiated between birth and weaning, and wherein
administration is performed at least once daily for a period of 7
days to 7 weeks.
[0005] In another aspect, a method of reducing mixing stress in a
mammal comprises orally administering to the mammal an effective
amount of an interleukin-10 peptide or an isolated antibody that
specifically binds an interleukin-10 peptide, wherein
administration is initiated within four weeks of mixing the mammal
with a new population of mammals, and wherein administration is
performed at least once daily for a period of 7 days to 7
weeks.
[0006] In yet another aspect, a method of reducing mixing stress in
a human child comprises orally administering to the human child an
effective amount of an interleukin-10 peptide or an isolated
antibody that specifically binds an interleukin-10 peptide, wherein
administration is initiated within four weeks of mixing the human
child with a new population of human children, and wherein
administration is performed at least once daily for a period of 7
days to 7 weeks.
[0007] Also included herein is a milk composition suitable for
administration to a pre-weaned mammal of a genus Bos, Ovis, Capra,
Bubalus, or Sus that comprises a basal milk composition and a
therapeutically effective amount of an interleukin-10 peptide or an
isolated antibody that specifically binds the interleukin-10
peptide.
[0008] Further included herein is a food composition suitable for
administration to a human that comprises a basal food composition
and a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or
SEQ ID NO. 38; or an isolated anti-IL-10 antibody that specifically
binds a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, or
SEQ ID NO. 38.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows reduced respiratory disease and antibiotic
usage in pre-weaned calves fed an anti-IL-10 antibody in the field
trial of Example 2.
[0010] 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
[0011] Described herein are methods of reducing the incidence of
disease and stress in mammals by orally administering IL-10
peptides and/or anti-IL-10 antibodies. It has been unexpectedly
shown herein that oral administration of an anti-IL-10 antibody
increased hipometer measurement, reduced the incidence of bovine
respiratory disease, and decreased antibiotic use in pre-weaned
calves. Because the IL-10 peptides and anti-IL-10 antibodies can
improve respiratory immunity as well as gastrointestinal immunity,
the IL-10 peptides and anti-IL-10 antibodies can be used to reduce
the symptoms of a variety of stresses in mammals, such as the
stress associated with mixing populations of mammals.
[0012] Young pre-weaned mammals such as dairy calves are
susceptible to dairy calf pneumonia, referred to as Bovine
Respiratory Disease Complex. Bovine respiratory disease complex
(BRD) is a significant cause of morbidity, mortality and animal
welfare concern and costs the industry between $800-900 million
annually. Antibiotic treatment is costly, recurrence rates are
high, the development of refractory sequelae are common, and
antibiotic resistance is a concern. Thus, there is a need for
improved treatment of pre-weaned mammals that are susceptible to
respiratory infections, including mammals of a genus Bos
(calves/cows), Ovis (lambs/sheep), Capra (kids/dairy goats), Sus
(piglets/pigs) and Bubalus (calf/water buffalo), particularly
milk-fed mammals.
[0013] In one embodiment, a method of reducing a symptom of
respiratory disease in a pre-weaned milk-fed mammal comprises
orally administering to the pre-weaned milk-fed mammal an effective
amount of an interleukin-10 peptide or an isolated antibody that
specifically binds an interleukin-10 peptide, wherein
administration is initiated between birth and weaning, such as
within 1 to 3 days of birth, and wherein administration is
performed at least once daily for a period of 7 days to 7 weeks,
specifically 7 days to three, four, five or six weeks, more
specifically 7 days to 2 weeks. In one embodiment, the treated
mammal is a bovine that exhibits reduced evidence of respiratory
disease at 56 days of age compared to a control untreated
pre-weaned bovine (FIG. 1). In certain aspects, administration of
the IL-10 peptide or anti-IL-10 antibody is stopped after the
treatment period, e.g., a 7 day to 7 week treatment period. In
another aspect, administration of the IL-10 peptide or anti IL-10
antibody is not restarted for at least 1 to 14 days, specifically 7
to 14 days. In a specific aspect, the pre-weaned milk-fed mammal is
a Bovine (a calf), and the respiratory disease is bovine
respiratory disease. In other aspects, the pre-weaned milk-fed
mammal is a sheep, a dairy goat, or a water buffalo.
[0014] As used herein, the term weaning means the practice of
separating a mammal such as a calf from its source of milk. Calves
are generally separated from their mothers shortly after birth and
are fed whole milk or a milk replacer until weaning from their milk
source, generally at 28 to 56 days of age. Calves that are raised
apart from their mothers can be referred as housed calves and are
generally housed in individual pens or in small groups. Nursing
calves, however, can also be supplemented with milk or milk
replacer. Thus, a pre-weaned mammal is a baby mammal that receives
the majority of its nutrition from milk. As used herein, milk-fed
means that a mammal is fed either whole milk, waste milk or milk
replacer as their primary source of nutrition. Milk replacer, in
the case of calves, generally includes protein such as whey protein
or casein, but can also contain soy protein for example. In
addition, milk replacers include fat such as animal fat or
vegetable oil, essential amino acids, vitamins and minerals.
[0015] In one aspect, a pre-weaned calf is a calf that is 56 days
old or younger. In the case of goats and sheep, weaning is usually
based on weight, however, weaning generally occurs at 6-8 weeks of
age, or longer. Weaning of water buffalo is longer than cows,
generally at 90 days or more.
[0016] Symptoms of respiratory disease include elevated rectal
temperature, cough, nasal discharge, ocular discharge, and/or ear
droop. Diarrhea can be caused by fecal pathogens including
Cryptosporidium parvum and is frequently associated with a reduced
appetite, abnormal attitude, dehydration, reduced weight gain,
and/or decreased fecal pH. Respiratory disease and diarrhea are the
two most important diseases of preweaned calves.
[0017] Fecal pH, specifically a neutral fecal pH, may be an
indication of improved digestion and gastrointestinal health. In
certain aspects, fecal pH can be used as an indication of the
health of the gastrointestinal tract of a calf A pH of 5.5 to 7.4
is indicative of a healthy calf, while a pH of less than 5.5 or
greater than 7.4 may be indicative of digestive tract
dysfunction.
[0018] In one aspect, the IL-10 peptide or anti-IL-10 antibody is
administered in the form of a milk composition. As used herein, the
term milk includes whole milk, waste milk, or a milk replacer
composition.
[0019] It was unexpectedly found herein that oral administration of
anti-IL-10 antibodies, which had previously been shown to reduce
gastrointestinal parasites, can also be used to improve respiratory
immunity. Administration of IL-10 peptides and/or IL-10 antibodies
is expected to reduce the use of antibiotics in treated mammals,
resulting in a significant cost savings. Broad spectrum antibiotics
such as NUFLOR.RTM. (florfenicol), Excede.RTM. (ceftiofur),
Draxxin.RTM. (tulathromycin), Baytril.RTM. 100 (enrofloxacin) and
Zuprevo.RTM. (tildopirosin) are used to treat BRD associated with
Mannheimia (Pasteurella) haemolytica, Pasteurella multocida, and
Histophilus somni, for example. Because the immunity provided by
the IL-10 peptides and/or IL-10 antibodies is expected to last past
weaning, protection of post-weaned animals from BRD is
expected.
[0020] Effective amount of the interleukin-10 peptide or the
isolated antibody that specifically binds an interleukin-10 peptide
for the treatment of mammals are 3 g/kg (dry matter) milk to 0.001
mg/Kg (dry matter) milk, specifically 1 g/Kg (dry matter) milk to
0.341 g/Kg (dry matter) milk.
[0021] In another embodiment, young and adult mammals are subject
to stress related to introducing them to a new population of
mammals, a process referred to herein as "mixing." The stress
associated with mixing a mammal into a new population of mammals is
referred to herein as "mixing stress." The term mammals includes
both human and non-human mammals. In addition to the stress from
exposing the mammal to a new population of mammals and any illness
within that population, stress can be induced by moving a mammal
from familiar surroundings and exposing the mammal to any
combination of shipping crates, new foods, temperature changes,
means of transportation, and new handlers or caregivers. Stress can
result from transport, comingling, feeding changes, vaccination,
animal management procedures, crowding, auction, temperature
fluctuations, and the like. Stress can affect the immune system of
these animals resulting in weight loss (referred to as shrink in
cows), fatigue, lack of appetite, signs of depression, and even
death. All mammals, including for example, animals of genus Bos
(calves/cows), Ovis (lambs/sheep), Capra (kids/dairy goats), Sus
(piglets/pigs) and Bubalus (calf/water buffalo), are subject to
mixing stress. Additional animals that are subject to mixing stress
include horses, dogs, cats, rabbits, and guinea pigs. Mixing stress
can occur whether or not the new population is infected with a
known illness, such as a viral or bacterial infection.
[0022] Thus, a method of reducing mixing stress in a mammal
comprises orally administering to the mammal an effective amount of
an interleukin-10 peptide or an isolated antibody that specifically
binds an interleukin-10 peptide, wherein administration is
initiated within four weeks of mixing the mammal with a new
population of mammals, and wherein administration is performed at
least once daily for a period of 7 days to 7 weeks, specifically 7
days to three, four, five or six weeks, more specifically 7 days to
2 weeks. As used herein, within four weeks of mixing means that
administration of the IL-10 peptide or anti-IL-10 antibody is
initiated from 4 weeks prior to mixing with the new population to 4
weeks after mixing with the new population. In one embodiment,
administration of the IL-10 peptide or anti-IL-10 antibody is
stopped after the treatment period and is not restarted until 1 to
14 days, specifically 7 to 14 days. The IL-10 peptide or anti-IL-10
antibody can be administered in a food composition, such as a milk
composition or an animal feed composition.
[0023] In one embodiment, when the mammal is a non-human mammal,
the method further comprising exposing the non-human mammal to a
shipping crate, new food, temperature change, a means of
transportation, vaccination, castration, tail docking, branding,
dehorning, a new handler, a new caregiver, or a combination
thereof.
[0024] In the case of young mammals, calves for example, mixing
stress can result in respiratory disease, weight loss, and
behavioral changes. Mixing stress in pigs has been shown to have
long-term adverse effects on immune function. In companion animals,
moving of animals between shelters or adoption of an animal is
often associated with an increased incidence of infectious
diseases. Thus, methods of decreasing mixing stress in mammals is
applicable across all species of mammals that are subject to mixing
of populations.
[0025] Effective amount of the interleukin-10 peptide or the
isolated antibody that specifically binds an interleukin-10 peptide
for the treatment of mammals are 3 g/kg (dry matter) feed/food to
0.001 mg/Kg (dry matter) feed/food, specifically 1 g/Kg (dry
matter) feed/food to 0.341 g/Kg feed/food.
[0026] In a specific aspect, human children are exposed to stress
when they are mixed with a new population of children, such as in a
new school or home, a new child care facility, or a new classroom.
The move itself may weaken the immune system of the child and the
child may be exposed to illnesses in the new population that the
child was not previously exposed to. Introduction of a child into a
new population of children can result in a variety of illness,
including gastrointestinal and respiratory illnesses.
Cryptosporidiosis, for example, is a common cause of diarrhea in
child care settings. Viral and bacterial respiratory illnesses are
also prevalent in childcare situations and schools. Thus, methods
to reduce illness associated with mixing stress in human children
are also desirable.
[0027] In one embodiment, a method of reducing mixing stress in a
human child comprises orally administering to the human child an
effective amount of an interleukin-10 peptide or an isolated
antibody that specifically binds an interleukin-10 peptide, wherein
administration is initiated within four weeks of mixing the human
child with a new population of human children, and wherein
administration is performed at least once daily for a period of 7
days to 7 weeks, specifically 7 days to three, four, five or six
weeks, more specifically 7 days to 2 weeks.
[0028] In certain aspects, administration of the Il-10 peptide or
anti-IL-10 antibody is stopped after the treatment period. As used
herein, groups of children include school groups, such as a new
school classroom, or a population of children in a childcare
setting. The group of children can be as small as, for example,
four to six children, to much larger classroom situations.
[0029] Reduction of mixing stress may be accompanied by a reduction
in gastrointestinal and/or respiratory illness in the child,
particularly illnesses associated with mixing of populations of
children. Symptoms of illness include, for example, runny nose,
cough, fever, listlessness, vomiting, diarrhea, earaches, sore
throat, wheezing, and the like, and combinations thereof. Oral
administration of an IL-10 peptide or an anti-Il-10 antibody is
expected to improve the gastrointestinal and respiratory immunity
of the child, thus reducing the symptoms of illness when exposed to
new groups of children.
[0030] The IL-10 peptides and anti-IL-10 antibodies can be
administered in the form of a pharmaceutical composition, or in the
form of a food or beverage composition. Effective amount of the
interleukin-10 peptide or the isolated antibody that specifically
binds an interleukin-10 peptide for the treatment of mammals are
depend on the level of isolation. The range of effective doses are
shown in table 2.
[0031] 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.
[0032] 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 (domain) antibodies (e.g., camelid antibodies,
chimeric, and bifunctional antibodies, as well as fragments
thereof.
[0033] The term "isolated antibody" as used herein, refers to an
antibody that is at least partially purified from other naturally
associated molecules, or substantially free of antibodies having
different antigenic specificities. In some cases, particularly in
the case of egg yolk antibodies, the antibody may comprise 50-70%
or more of an isolated antibody preparation.
[0034] An 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, SEQ ID NO. 21, SEQ
ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO.
26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ
ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO.
35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, and combinations
thereof (see Table 1).
TABLE-US-00001 TABLE 1 Sequence ID NO. and Corresponding Amino Acid
Sequence. AMINO ACID SEQ ID NO. SEQUENCE Organism SEQ ID NO. 1
DQLHSLL cow SEQ ID NO. 2 VMPQAENH Cow/sheep/ goat/water buffalo SEQ
ID NO. 3 DSSCIHLP cow SEQ ID NO. 4 SELQERGV cow SEQ ID NO. 5
DQMGDLL pig SEQ ID NO. 6 VMPKAESD pig SEQ ID NO. 7 SKLQERGV pig SEQ
ID NO. 8 ENSCIHFP pig SEQ ID NO. 9 DQLNSML sheep/goat SEQ ID NO. 10
NMLQERGV sheep/goat SEQ ID NO. 11 DSSCTHFP sheep/goat SEQ ID NO. 12
DQLDSLL water buffalo SEQ ID NO. 13 SKLQDRGV water buffalo SEQ ID
NO. 14 DSSCTQFP water buffalo SEQ ID NO. 15 DQLDNMLL horse SEQ ID
NO. 16 VMPQAENH horse SEQ ID NO. 17 SKLQEKGV horse SEQ ID NO. 18
ENSCTHFP horse SEQ ID NO. 19 DKLDNILL dog, Canis lupis familiaris
SEQ ID NO. 20 VMPRAEN dog SEQ ID NO. 21 SKLQEKGV dog SEQ ID NO. 22
EDDCTHFP dog SEQ ID NO. 23 DELHSILL cat, Felis catus SEQ ID NO. 24
VMPQAENE cat SEQ ID NO. 25 SKLQEKGV cat SEQ ID NO. 26 EDNCTHFS cat
SEQ ID NO. 27 DQLNSMLL rabbit SEQ ID NO. 28 VMPQAENH rabbit SEQ ID
NO. 29 SKLQEEGV rabbit SEQ ID NO. 30 ENSCIHFP rabbit SEQ ID NO. 31
DQLDNVLL guinea pig SEQ ID NO. 32 VMPQAEKH guinea pig SEQ ID NO. 33
NKLQDQGV guinea pig SEQ ID NO. 34 EDSCAHFP guinea pig SEQ ID NO. 35
DQLDNLL human SEQ ID NO. 36 VMPQAENQ human SEQ ID NO. 37 NKLQEKGI
human SEQ ID NO. 38 ENSCTHFP human
[0035] SEQ ID NOs. 1-14 are amino acid sequences corresponding to
peptides of the IL-10 cytokine in pre-weaned animals according to
the present disclosure. SEQ ID NOs. 1-38 correspond to exemplary
mammals subject to mixing stress.
[0036] The present disclosure further includes antibodies that
specifically bind to the IL-10 peptides (also referred to herein as
"anti-IL-10 antibody"). 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, SEQ ID NO. 21, SEQ ID NO. 22,
SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID
NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31,
SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID
NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, and combinations thereof.
[0037] 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 cattle, pigs, sheep, and fish.
[0038] 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.
[0039] 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 known 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.2SO.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.
[0046] Alternatively, the antibodies may be purified or isolated
using any conventional technique such as by immunoaffinity
purification.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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 or vacuum drum
drying. 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.
[0051] In another aspect, isolated antibodies can include
antibodies in serum, or antibodies that have been purified to
varying degrees. Such antibodies may include polyclonal antibodies,
camelid antibodies, monoclonal antibodies, humanized or chimeric
antibodies, anti-idiotypic antibodies, single chain antibodies, Fab
fragments, fragments produced from a Fab expression library,
epitope-binding fragments of the above, and the like. Production of
antibodies is well-known in the art.
[0052] In yet another aspect, an antibody is isolated from the
colostrum of an animal such as from bovine colostrum.
[0053] The methods disclosed herein can be achieved using food/feed
additives including the IL-10 peptides, or isolated antibodies
which specifically bind to IL-10 peptides.
[0054] 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
herbivorous mammals such as cattle, horses, sheep and goats; for
fish; or for companion animals. 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.
[0055] In one embodiment, the methods disclosed herein are achieved
using animal feed additive including IL-10 peptides including the
amino acid sequence of any one of SEQ ID NOs. 1-34, and
combinations thereof.
[0056] In another embodiment, the methods of the present disclosure
utilize an animal feed additive including isolated antibodies that
specifically bind to the IL-10 peptide including the amino acid
sequence of SEQ ID NOs. 1-34, and combinations thereof.
[0057] 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.
[0058] 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
[0059] 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(dry matter)), antibody losses due to drying storage
and gastrointestinal degradation.
[0060] An animal feed may further include optional ingredients
including vitamins, minerals, antibiotics, lipids, carbohydrates,
proteins, antioxidants, and amino acids.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] Exemplary carbohydrates include starch, cellulose,
pentosans, other complex carbohydrates, corn, milo, barley, rye,
oats, wheat, wheat middlings, and various grain-by-products.
[0065] 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.
[0066] 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.
[0067] Exemplary antioxidants include beta-carotene, Vitamin E,
Vitamin C, and tocopherol, or synthetic antioxidants.
[0068] Specifically, the animal feed including the feed additive of
either IL-10 peptide or isolated anti-IL-10 antibody is a feed for
a non-human mammal such as a cow, horse, goat or sheep.
[0069] In one aspect, included herein is a milk composition
suitable for administration to a pre-weaned mammal of a genus Bos,
Ovis, Capra, Bubalus or Sus containing a basal milk composition and
a therapeutically effective amount of an IL-10 peptide or
anti-IL-10 antibody. Basal milk compositions include whole milk,
waste milk, or a milk replacer composition. As used herein, a
therapeutically effective amount of an IL-10 peptide or anti-IL-10
antibody is an amount effective to reduce the incidence of
respiratory or gastrointestinal disease in the animal when
administered daily for a period of 7 days to 7 weeks. Exemplary
therapeutically effective amounts of interleukin-10 peptide or the
isolated antibody that specifically binds an interleukin-10 peptide
include 3 g/kg (dry matter) milk to 0.001 mg/Kg (dry matter) milk,
specifically 1 g/Kg (dry matter) milk to 0.341 g/Kg (dry matter)
milk. Exemplary peptides for inclusion in the milk compositions
include the peptides of any one of SEQ ID Nos. 1-14, or
combinations thereof. Exemplary anti-IL-10 antibodies for inclusion
in milk compositions include antibodies that specifically bind a
peptide of any one of SEQ ID NOs. 1-14, or any combination of
antibodies.
[0070] In another aspect, included herein is a food composition
suitable for administration to humans such as human children
containing a basal food composition and an IL-10 peptide or
anti-IL-10 antibody. A food composition suitable for administration
to a human comprises a peptide of SEQ ID NO. 35, SEQ ID NO. 36, SEQ
ID NO. 37, or SEQ ID NO. 38; or an isolated anti-IL-10 antibody
that specifically binds a peptide of SEQ ID NO. 35, SEQ ID NO. 36,
SEQ ID NO. 37, or SEQ ID NO. 38. Exemplary basal food compositions
include milk, juice, formula, and solid foods such as snack food
consumable by humans including human children.
[0071] 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.
[0072] In some embodiments, the methods involve injecting or orally
administering 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.
[0073] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Anti-IL-10 Antibody Production
[0074] 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.
[0075] Specifically, IL-10 peptide SEQ ID NO: 2 was conjugated to
hen ovalbumin (OVA, Sigma, St. Louis, Mo.) for ELISA using a
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 SEQ ID NO. 2 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 1:20. 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. Triplicate optical densities were
averaged and blocking buffer background was subtracted to produce a
final optical density. The optical density of antibody to IL-10
peptide SEQ ID NO. 9 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 Antibody titer for cow anti-IL-10 antibody.
Antibody extracts from control eggs and eggs from hens injected
with an IL-10 peptide conjugate were serially diluted and added to
plate bound peptide and analyzed as described above. The antibody
to the IL-10 antibody could be diluted up to 1:320 (titer) before
the absorbance equaled background absorbance. Cow Anti- Control
Dilution IL-10 Antibody 1:20 2.114 1.298333333 1:40 1.791
1.141333333 1:80 1.308 0.932666667 1:160 0.905666667 0.737333333
1:320 0.720666667 0.553666667 1:640 0.375 0.409666667 1:1280
0.254333333 0.294666667
Example 2
Field Trial to Evaluate the Effect of an Anti-IL-10 Antibody on C.
parvum Oocyst Shedding and Other Health and Growth Parameters in
Pre-Weaned Dairy Calves
[0076] The study was a double-blinded, randomized, prospective
clinical trial. There were 134 calves randomly assigned to a
treatment group (72) and a control group (62) of primarily Holstein
calves. Calves were enrolled in the study on five separate days
within a 2-week period. On enrollment day, 23-28 calves (depending
on the number of calves that arrived on the farm) were randomly
assigned to a group designated by color (green=anti IL-10 IgY
treatment, purple=anti IL-10 IgY treatment, orange=egg powder
control group). Calf feeders, health screeners and investigators
were blinded to the color code until the study was completed.
Calves in this study originated from 12 surrounding dairy farms,
where colostrum and newborn calf care were administered. Calves
arrived between 1 and 3 days of age. On enrollment day, the data
collected included body weight (electronic scale), hipometer
(stall-side indirect measure of body weight), packed cell volume
(PCV) and serum total protein concentration (indicator of
dehydration and adequate passive transfer of immunoglobulins,
respectively), and fecal pH. Calves were identified by group with
color-coded zip ties on eartags and color-coded Duct Tape.RTM. on
their individual hutches. The calves were fed in milk either 0.96 g
of egg yolk powder containing anti IL-10 antibodies (treatment) or
0.96 g control egg yolk powder without anti-IL-10 antibodies
(control egg yolk powder from hens receiving a sham inoculation)
added to their milk daily (divided into 2 feedings) from Day 0 (day
after enrollment) through Day 10. Attitude, appetite and health
scores were recorded daily for 14 days using The Calf Health Scorer
App. Fecal pH and fecal pathogen testing (C. parvum RT-PCR,
Coronarvirus RT-PCR, Rotavirus RT-PCR and Salmonella culture) were
performed on Day 5 and Day 14 of the trial. Hydration status (PCV
and serum total protein) and hipometer measurements were recorded
on Day 7 and Day 14. Growth (average daily gain and hipometer)
measurements along with health score were performed on Day 56 (for
Calf Health Score) or when calves were moved out of the hutches. At
this time, antibiotic administration data (by farm personnel) was
also recorded.
[0077] The protocol deviations in the study were minor. The unequal
group sizes are based on randomization using 3 colors assigned on
the day that calves arrived. One calf died after enrollment and was
removed from the study. Two missing data points on calf health
scores and on enrollment fecal pH's, respectively were handled by
leaving those calves out of the analysis of those specific data
sets but not other parameters where the data collection was
complete.
[0078] Experimental procedure: In this trial, 0.96 g egg yolk
powder containing anti IL-10 IgY antibodies was added to the milk
of calves in the treatment group while 0.96 egg yolk powder without
anti IL-10 IgY antibodies was added to the milk of calves in the
control group for 11 days. The dose of egg yolk powder administered
in the milk was 1.2 gm/kg of milk dry matter, using an assumption
of 12.5% dry matter for whole milk. The egg product used is defined
by American Association of Feed Control Officials (AAFCO) 9.74.
[0079] Results: Average total health score and days of diarrhea
were not significantly different between the two groups. There was
a higher score for the diarrhea average score from Days 0 through
Day 4 in the treated group (p=0.03). Rotavirus was significantly
increased in the treatment group on Day 5 (p=0.02), but at Day 14
it was no longer significant. There was no significant difference
in C. parvum oocyst shedding between the two groups. However, on
Day 5 there was a trend (p=0.06) for increased C. parvum oocyst
shedding in the treatment group, whereas the trend was reversed on
Day 14 when there was decreased shedding of C. parvum oocysts in
the treatment group. The mean cycle threshold value for C. parvum
fecal PCR detection was higher in the treatment group than the
control group on Day 14, another indicator of reduced C. parvum
oocyst shedding in anti IL-10 antibody treated calves. Fecal pH was
significantly higher in the treatment group at Day 14 (p=0.004).
The differences in fecal pH from enrollment day to Day 5,
enrollment day to Day 14 and Day 5 to Day 14 were all significantly
higher in the treatment group. A more alkaline fecal pH is
associated with improved digestion and overall gastrointestinal
health.
TABLE-US-00004 TABLE 4 Fecal pH of control or anti-IL-10 antibody
treated calves and the change from start date (E) to day 5 and 14,
or from day 5 to 14. Control Treatment Day E 5.95.sup.a 5.78.sup.b
Day 5 5.32.sup.a 5.38.sup.a Day 14 6.35.sup.a 6.67.sup.b Day 5-E
-0.64.sup.a -0.40.sup.b Day 14-E 0.39.sup.a 0.89.sup.b Day 14-5
1.03.sup.a 1.30.sup.b
[0080] Average daily gain was significantly increased in the
control group (p=0.04) but the hipometer measurement from Day 0 to
Day 56 was significantly higher in the treated group (p=0.02). The
latter finding is indicative of a larger frame size (width between
the greater trochanter of the left and right femurs) in treated
calves, even though average daily gain was greater in the control
calves.
[0081] The prevalence of respiratory disease on Day 56, as
determined by the calculated total respiratory score, was
significantly higher in the control group (21%) versus the treated
group (8%) (p=0.04). In addition, use of antibiotics by farm
personnel for respiratory disease (investigators excluded
antibiotic treatment for joint or navel infections) was
significantly higher in the control group (20.7%) versus the
treated group (7.2%) (p=0.04).
[0082] General discussion and conclusions: Dairy calf health is
significantly affected by both enteric disease (most severely in
the preweaning period) and by pneumonia (most commonly in the
postweaning period). Antibiotic therapy is commonly used to treat
both enteric and respiratory pathogens, leading to issues regarding
antibiotic resistance, withdrawal times and residues.
Cryptosporidium parvum is an Apicomplexan protozoan that infects
intestinal cells and destroys villi, causing a malabsorptive
diarrhea. The shedding prevalence of C. parvum is reported in the
United States as 8 to 49% in calves and 6 to 13% in adult cows.
Within-farm prevalence can be high as 70% on some farms, and both
shedding and intensity of shedding have been significantly
associated with diarrhea. C. parvum and Rotavirus have been found
to be the most common enteric pathogens in diarrheic feces and most
calves with diarrhea have more than one pathogen isolated from
their feces. Oral IgY products have been successful in treating
gastrointestinal infections, including bovine rotavirus,
coronavirus, enterotoxigenic E. coli, and Salmonella spp. Although
orally administered IgY is proteolytically digested to Fab and Fc
fragments, both show neutralizing activity in the GI tract and can
be detected in feces. Previous studies have shown that IL-10
expression is increased in calves infected with C. parvum. In
addition, IL-10 knockout mice have been found to be significantly
resistant to C. parvum infection. The present study was conducted
to investigate the effects of feeding anti-IL-10 egg yolk
antibodies on fecal pathogen shedding, health, and weight gain in
dairy calves. The results showed a trend in reduced C. parvum
shedding when evaluating the cycle threshold values at day 14
(p=0.08) and a significant increase in fecal pH in the treatment
group. Fecal pH is an indicator of colonic acidity, as nutrients
that escape digestion in the small intestine ferment in the large
bowel. It has been shown that decreased fecal pH in dairy calves is
associated with elevated lactate production and reduced volatile
fatty acid concentrations in the colon (VFAs). Absorption of VFAs
from the large intestine is critical for promoting sodium and water
reabsorption in the gut. Without being held to theory, we associate
the significant increase in fecal pH and the trend towards reducing
fecal shedding of C. parvum in the treatment group as indicators of
improved gastrointestinal health. The increased shedding of
rotavirus could be related to increased pass-through of the virus
without attachment to the small intestinal villi.
[0083] An unanticipated benefit to the treatment group was a
decrease in respiratory disease and less antibiotic usage than the
control group during the trial (day 0-day 56) (FIG. 1). Without
being held to theory, it is proposed that the mechanism relating
gastrointestinal health with respiratory disease resistance is due
to cross-talk between the respiratory and gastrointestinal mucosal
surfaces. Mucosa-associated lymphoid tissue (MALT) directs antigen
processing, lymphoid cell trafficking, and local host defenses.
This resident lymphoid tissue within the respiratory and
gastrointestinal tracts has a common embryonic origin in the
primitive foregut. Thus, feeding anti IL-10 IgY antibodies to
neonatal calves may have far-reaching effects on overall calf
health, welfare, performance and profitability. Any ability to
reduce antibiotic use in neonatal dairy calves can promote a more
balanced gastrointestinal microbiome, improve the susceptibility of
pathogens when antibiotics are needed, and reduce antibiotic
resistance, a major goal of the food animal industry.
[0084] In summary, the results presented herein suggest that
improved gastrointestinal health is linked to improved respiratory
immunity utilizing the common mucosal immune system hypothesis.
From this study, there is evidence of improved gastrointestinal
tract health in enteric pH data; there is a trend for reduced C.
parvum oocyst shedding, and improved frame size in neonatal calves
given anti IL-10 antibody for 11 days. With improved digestive
function, there appear to be combined benefits of improved enteric
and respiratory mucosal immunity. The significant reduction in
respiratory disease in dairy calves at weaning has the potential to
decrease respiratory disease in post weaning group housing.
Respiratory disease in this age group has significant negative long
term economic and welfare consequences of recurring disease, poor
growth, delayed breeding, reduced milk production in the first
lactation and early culling.
Example 3
Second Field Trial
[0085] The proposed second field trial will investigate a dose
response to anti IL-10 antibody feeding (0, 0.5, 1 and 2 times the
dose used in trial 1 and a farm control group) and will prolong
inclusion in the diet for 14 days, when the risk of neonatal
diarrhea is greatly reduced. Parameters of health monitoring,
gastrointestinal function, fecal pathogen shedding, growth and
antibiotic usage will be similar to trial 1 but, to validate the
finding of reduced respiratory disease prevalence in trial 1,
health and respiratory disease parameters will be monitored twice
weekly for the entire 56-day clinical trial. Thoracic ultrasound
and respiratory pathogen detection by deep nasopharyngeal swab
sampling will confirm that respiratory disease is present and help
define the benefit of feeding anti IL-10 antibody.
[0086] 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.
[0087] 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
3817PRTBos taurus 1Asp Gln Leu His Ser Leu Leu 1 5 28PRTBos taurus
2Val Met Pro Gln Ala Glu Asn His 1 5 38PRTBos taurus 3Asp Ser Ser
Cys Ile His Leu Pro 1 5 48PRTBos taurus 4Ser Glu Leu Gln Glu Arg
Gly Val 1 5 57PRTSus scrofa 5Asp Gln Met Gly Asp Leu Leu 1 5
68PRTSus scrofa 6Val Met Pro Lys Ala Glu Ser Asp 1 5 78PRTSus
scrofa 7Ser Lys Leu Gln Glu Arg Gly Val 1 5 88PRTSus scrofa 8Glu
Asn Ser Cys Ile His Phe Pro 1 5 97PRTOvis aries 9Asp Gln Leu Asn
Ser Met Leu 1 5 108PRTOvis aries 10Asn Met Leu Gln Glu Arg Gly Val
1 5 118PRTOvis aries 11Asp Ser Ser Cys Thr His Phe Pro 1 5
127PRTBubalis bubalus 12Asp Gln Leu Asp Ser Leu Leu 1 5
138PRTBubalis bubalus 13Ser Lys Leu Gln Asp Arg Gly Val 1 5
148PRTBubalis bubalus 14Asp Ser Ser Cys Thr Gln Phe Pro 1 5
158PRTEquus caballus 15Asp Gln Leu Asp Asn Met Leu Leu 1 5
168PRTEquus caballus 16Val Met Pro Gln Ala Glu Asn His 1 5
178PRTEquus caballus 17Ser Lys Leu Gln Glu Lys Gly Val 1 5
188PRTEquus caballus 18Glu Asn Ser Cys Thr His Phe Pro 1 5
198PRTCanis familiaris 19Asp Lys Leu Asp Asn Ile Leu Leu 1 5
207PRTCanis familiaris 20Val Met Pro Arg Ala Glu Asn 1 5
218PRTCanis familiaris 21Ser Lys Leu Gln Glu Lys Gly Val 1 5
228PRTCanis familiaris 22Glu Asp Asp Cys Thr His Phe Pro 1 5
238PRTFelis catus 23Asp Glu Leu His Ser Ile Leu Leu 1 5 248PRTFelis
catus 24Val Met Pro Gln Ala Glu Asn Glu 1 5 258PRTFelis catus 25Ser
Lys Leu Gln Glu Lys Gly Val 1 5 268PRTFelis catus 26Glu Asp Asn Cys
Thr His Phe Ser 1 5 278PRTLepus curpaeums 27Asp Gln Leu Asn Ser Met
Leu Leu 1 5 288PRTLepus curpaeums 28Val Met Pro Gln Ala Glu Asn His
1 5 298PRTLepus curpaeums 29Ser Lys Leu Gln Glu Glu Gly Val 1 5
308PRTLepus curpaeums 30Glu Asn Ser Cys Ile His Phe Pro 1 5
318PRTCavia porcellus 31Asp Gln Leu Asp Asn Val Leu Leu 1 5
328PRTCavia porcellus 32Val Met Pro Gln Ala Glu Lys His 1 5
338PRTCavia porcellus 33Asn Lys Leu Gln Asp Gln Gly Val 1 5
348PRTCavia porcellus 34Glu Asp Ser Cys Ala His Phe Pro 1 5
357PRTHomo sapiens 35Asp Gln Leu Asp Asn Leu Leu 1 5 368PRTHomo
sapiens 36Val Met Pro Gln Ala Glu Asn Gln 1 5 378PRTHomo sapiens
37Asn Lys Leu Gln Glu Lys Gly Ile 1 5 388PRTHomo sapiens 38Glu Asn
Ser Cys Thr His Phe Pro 1 5
* * * * *