U.S. patent application number 16/369844 was filed with the patent office on 2019-07-25 for treatment of enteric stress from heat and infection in humans and animals by supplementation with zinc and butyric acid.
The applicant listed for this patent is KEMIN INDUSTRIES, INC.. Invention is credited to Venkatesh Mani, Mitchell Poss, Jon Rubach.
Application Number | 20190224235 16/369844 |
Document ID | / |
Family ID | 57609611 |
Filed Date | 2019-07-25 |
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United States Patent
Application |
20190224235 |
Kind Code |
A1 |
Mani; Venkatesh ; et
al. |
July 25, 2019 |
TREATMENT OF ENTERIC STRESS FROM HEAT AND INFECTION IN HUMANS AND
ANIMALS BY SUPPLEMENTATION WITH ZINC AND BUTYRIC ACID
Abstract
A method of improving intestinal integrity and reducing the
effects of heat stress, enteric disease challenges and other
intestinal stress conditions in humans and animals by feeding an
efficacious amount of a salt of zinc and butyric acid.
Inventors: |
Mani; Venkatesh; (Ames,
IA) ; Rubach; Jon; (West Des Moines, IA) ;
Poss; Mitchell; (Johnston, IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KEMIN INDUSTRIES, INC. |
Des Moines |
IA |
US |
|
|
Family ID: |
57609611 |
Appl. No.: |
16/369844 |
Filed: |
March 29, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15198833 |
Jun 30, 2016 |
10292999 |
|
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16369844 |
|
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62186831 |
Jun 30, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/12 20180101; A61K
33/30 20130101; A23K 20/105 20160501; A23K 50/20 20160501; A23K
20/158 20160501; A23K 20/30 20160501; A23K 50/75 20160501; A61K
31/19 20130101; A23K 50/30 20160501 |
International
Class: |
A61K 33/30 20060101
A61K033/30; A23K 20/158 20060101 A23K020/158; A23K 50/30 20060101
A23K050/30; A23K 50/75 20060101 A23K050/75; A61K 31/19 20060101
A61K031/19; A23K 20/20 20060101 A23K020/20; A23K 20/105 20060101
A23K020/105; A23K 50/20 20060101 A23K050/20 |
Claims
1. A method of improving intestinal integrity and reducing the
effects of heat stress, enteric disease challenges and other
intestinal stress conditions in humans and animals, comprising
feeding an efficacious amount of a salt of zinc and butyric acid
salt.
2. A method of improving feed intake, feed conversion and weight
gain during heat stress or enteric disease challenge in humans and
animals, comprising feeding an efficacious amount of zinc and
butyric acid salt.
3. The method of either claim 1 or claim 2, wherein said zinc and
butyric acid salt is fed between 50 ppm and 10,000 ppm, but most
optimally between 300 and 3000 ppm.
4. The method of claim 3, wherein said BPZ is encapsulated to
control the release of said zinc and butyric acid salt during
passage through the digestive system of the human or animal.
5. The method of claim 4, wherein the encapsulated from of said
zinc and butyric acid salt consists of at least 20% zinc and
butyric acid salt to 60% zinc and butyric acid salt, and optimally
at 35% to 45% zinc and butyric acid salt.
6. The method of claim 5, wherein the rate of release
characteristics of the salt of zinc and butyric acid salt is
controlled by additional excipients, selected from the group
consisting of salts, ionic surfactants, non-ionic surfactants, such
as glycerol, polyethylene glycol derivatives, simple or complex
sugar molecules such as glucose, dextran, and inulin added as
modifiers to hydrogenated vegetable fat used in the
encapsulation.
7. A method of shortening the recovery period after a heat stress
and/or disease challenge comprising feeding an efficacious amount
of a salt of zinc and butyric acid salt before or during the stress
condition.
8. A method of claim 1, wherein said animal is selected from swine,
poultry and horses.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S.
patent application Ser. No. 15/198,833, filed Jun. 30, 2016,
entitled TREATMENT OF ENTERIC STRESS FROM HEAT AND INFECTION IN
HUMANS AND ANIMALS BY SUPPLEMENTATION WITH ZINC AND BUTYRIC ACID,
which claims the benefit of priority of U.S. Provisional Patent
Application Ser. No. 62/186,831, filed Jun. 30, 2015, entitled
TREATMENT OF ENTERIC STRESS FROM HEAT AND INFECTION IN HUMANS AND
ANIMALS BY SUPPLEMENTATION WITH ZINC AND BUTYRIC ACID, the
disclosures of which are hereby incorporated herein by reference in
their entirety
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to reducing the
effect of stress in humans and animals and especially production
animals and, more specifically, to the feeding of a combination of
zinc and butyric acid for the treatment or amelioration of enteric
stress from heat stress or disease stress.
[0003] High summer temperatures result in enormous amounts of
physiological stress to production animals, leading to decreased
feed intake, decreased growth rate and decreased feed conversion.
During periods of rapid increasing temperature and humidity
(expressed as a high humidity index (HI)), animals adjust to the
increasing HI over a period of weeks. During this period, stress
from HI to the animal causes the body core temperature to rise,
resulting in an increase of the blood flow from the organs to the
skin of the animal which allows for rapid cooling of the inner body
core. Once the HI reaches a specific threshold, the animal is no
longer able to effectively dissipate heat from the body core. This
causes the animal's systemic blood flow to be diverted to the
peripheral tissues to mitigate the heat stress, which leads to
decreased blood flow to the intestine. These heat stressed animals
are not in a position to consume any feed. The resulting decreased
feed intake combined with lower blood flow to the intestine leads
to villi damage, a decreased absorptive area for nutrients and
sloughing of the intestinal epithelial cells. All of these combined
effects result in a loss of intestinal integrity.
[0004] Swine encounter multiple pathogens throughout their life
including viruses, bacteria, fungi and parasites. Any infection
during the early life stages, particularly in nursery and early
weaned piglets, can cause severe morbidity and mortality. It is
difficult to predict the occurrence of any such event, and any
disease outbreak results in severe economic losses. Therefore, it
is important to develop strategies for these health challenges to
decrease the mortality, maintain the growth of the animals and
mitigate the losses. Most infections of agricultural animals occur
through the gastrointestinal (GI) route. One example of such
infection with huge economic potential is porcine epidemic diarrhea
virus (PEDV). The PEDV coronavirus infects the enterocytes of the
small intestine, resulting in severe inflammation of the
gastrointestinal tract leading to diarrhea and decreased nutrient
and water absorption. In suckling pigs, a severe PEDV infection
commonly results in mortality. In newly weaned pigs, clinical signs
lasts for approximately 7-14 days. Clinical signs include:
decreased feed intake, decreased tissue accretion, and diarrhea;
however, mortality is rarely observed. Because the animal's growth
is affected significantly, they never regain their growth potential
and it leads to huge economic losses to the producers.
[0005] The pathogenesis of any enteric disease is complex and
involves different cellular pathways. These include inflammatory
and immune signaling, oxidative stress, energy sensing, osmotic and
microbial homeostasis pathways. Compounds which can counteract one
or more of these pathways have the potential to mitigate the
severity or prevent the negative effects of gastrointestinal
infections. Additionally, compounds that augment intestinal
restitution and recovery can reduce the impact that enteric
pathogens may have on intestinal function and integrity, pig
performance, wellbeing and may help the animals regain their growth
potential.
[0006] Heat stress, infection with enteric viruses or bacteria will
increase the permeability of the intestinal cells. This allows
bacterial compounds such as lipopolysaccharide (LPS) and/or other
noxious substances from the intestinal lumen to get through the
intestinal barrier and cause an unwarranted inflammatory and immune
response in animals. Compounds that improve or sustain intestinal
integrity can decrease the effect of heat stress or disease
challenges on intestinal integrity and can improve the growth of
the animals during heat stress or infection.
SUMMARY OF THE INVENTION
[0007] The present invention includes the use of a salt of zinc and
butyric acid, in a 1:2 ratio, (ZBA) which in a preferred embodiment
is an encapsulated form of zinc and butyric acid salt (EZBA), to
improve intestinal integrity and reduce the effects of heat stress,
enteric disease challenges and other intestinal stress conditions
in production animals. EZBA improves feed intake, feed conversion
and weight gain during heat stress or enteric disease challenge.
EZBA is fed between 50 ppm and 10,000 ppm, but most commonly
between 300 and 2000 ppm. It has been discovered that feeding EZBA
before or during heat stress and/or a disease challenge shortens
the recovery period after a stress condition.
[0008] EZBA preferably is encapsulated to control the release of
ZBA throughout the digestive system. An encapsulated form of EZBA
preferably contains at least 20% ZBA to 60% ZBA, optimally at 35%
to 45% ZBA. A method (co-pending U.S. patent application Ser. No.
15/198,775, filed Jun. 30, 2016, which claims priority to No.
62/186,787, filed Jun. 30, 2015, and which is incorporated herein
in its entirety by this reference) has been developed to control
the rate of release characteristics of the active ingredient, ZBA,
by the use of excipients, such as salts, aqueous salts, aqueous
organic salts, ionic surfactants, non-ionic surfactants, such as
glycerol, polyethylene glycol derivatives, simple or complex sugar
molecules such as glucose, dextran, and inulin, to modify
hydrogenated vegetable oil used in the encapsulation process.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 is a chart of the effect of 1 mM ZBA on the
transepithelial resistance (TER) of cultured pig ileal intestinal
cells (IPEC-J2 cells) under normal conditions; the Control
treatment has no added zinc.
[0010] FIG. 2 is a chart of the effect of 300 .mu.M concentration
of different test compounds on the transepithelial resistance (TER)
values of IPEC-J2 cells under heat stress conditions.
[0011] FIG. 3A is a chart of the average daily feed intake (ADFI)
for the control and EZBA fed pigs during the pre-heat stress period
of the trial; and FIG. 3B is a chart of the average daily feed
intake (ADFI) for the control and EZBA fed pigs during the heat
stress period of the trial.
[0012] FIG. 4A is a chart of the Gain:Feed for the control and EZBA
fed pigs during the pre-heat stress period of the trial; and FIG.
4B is a chart of the Gain:Feed for the control and EZBA fed pigs
during the heat stress period of the trial.
[0013] FIG. 5A is a chart of the Average Daily Gain (ADG) for the
control and EZBA fed pigs during the pre-heat stress period of the
trial; and FIG. 5B is a chart of the Average Daily Gain (ADG) for
the control and EZBA fed pigs during the heat stress period of the
trial.
[0014] FIG. 6 is a chart of the intestinal integrity (Papp)
measured by FITC-Dextran permeability of the ileum intestinal
samples for the control and EZBA fed pigs at the end of the heat
stress period.
[0015] FIG. 7 is a chart of viral shedding of Porcine Epidemic
Diarrhea Virus (PEDV) determined by qPCR on fecal swabs from pigs
inoculated with porcine epidemic diarrhea virus (PEDV) and
supplemented with EZBA in feed over 20 days post inoculation
(DPI).
[0016] FIG. 8A is a chart of the average daily gain and FIG. 8B is
a chart of the average daily feed intake of pigs inoculated with
porcine epidemic diarrhea virus (PEDV) and supplemented with EZBA
in feed over 20 days post inoculation (DPI).
[0017] FIG. 9A is a chart of body weight and FIG. 9B is a chart of
Gain: Feed of pigs inoculated with porcine epidemic diarrhea virus
(PEDV) and supplemented with EZBA in feed over 20 days post
inoculation (DPI).
[0018] FIG. 10A is a chart of PEDV immunohistochemistry (IHC) and
FIG. 10B is a chart of In situ hybridization (ISH) at 5 dpi of
jejunum and ileum of pigs inoculated with porcine epidemic diarrhea
virus (PEDV) and supplemented with EZBA in feed over 20 days post
inoculation (DPI).
[0019] FIG. 11A is a chart of the jejunum and FIG. 11B is a chart
of the ileum Ki67 immunohistochemistry of pigs inoculated with
porcine epidemic diarrhea virus (PEDV) and supplemented with EZBA
in feed over 20 days post inoculation (DPI).
[0020] FIG. 12A is a chart of lesion score of the jejunum and FIG.
12B is a chart of lesions score of the ileum from pigs inoculated
with porcine epidemic diarrhea virus (PEDV) and supplemented with
EZBA in feed over 20 days post inoculation (DPI).
[0021] FIG. 13A is a chart of the villus height of the jejunum and
FIG. 13B is a chart of the villus height of the ileum from pigs
inoculated with porcine epidemic diarrhea virus (PEDV) and
supplemented with EZBA in feed over 20 days post inoculation
(DPI).
[0022] FIG. 14A is a chart of the crypt depth of the jejunum and
FIG. 14B is a chart of the crypt depth of the ileum from pigs
inoculated with porcine epidemic diarrhea virus (PEDV) and
supplemented with EZBA in feed over 20 days post inoculation
(DPI).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] The compounds of this invention may be administered to
subjects (humans and animals, including companion animals, such as
dogs, cats and horses) in need of such treatment in dosages that
will provide optimal pharmaceutical efficacy. It will be
appreciated that the dose required for use in any particular
application will vary from subject to subject, not only with the
particular compound or composition selected, but also with the
route of administration, the nature of the condition being treated,
the age and condition of the subject, concurrent medication or
special diets then being followed by the subject, and other factors
which those skilled in the art will recognize.
[0024] A suitable dosage level of EZBA of the present invention is
about 50 to 10,000 ppm in animal feed. Preferably, the dosage range
will be about 500 ppm to 5000 ppm. Compositions of the present
invention may be provided in a formulation comprising about 25 ppm
to 5000 ppm of the active ingredient (ZBA).
[0025] The EZBA in a preferred embodiment is a combination of zinc
oxide and butyric acid, although other compounds of zinc,
particularly mineral and organic salts of zinc may be used.
Preferably, the EZBA is between about 10% and 90% of zinc and
between about 90% and about 10% of butyric acid.
[0026] "Efficacious amount" for the purposes of this application is
defined to be the amount of a compound or composition or
derivatives thereof of the present invention is an amount that,
when administered to a subject, will have the intended therapeutic
effect. The full therapeutic effect does not necessarily occur by
administration of one dose, and may occur only after administration
of a series of doses. Thus, a therapeutically effective amount may
be administered in one or more administrations. The precise
effective amount needed for a subject will depend upon, for
example, the subject's size, health and age, the nature and extent
of the impairment, and the therapeutics or combination of
therapeutics selected for administration, and the mode of
administration. The skilled worker can readily determine the
effective amount for a given situation by routine
experimentation.
[0027] In preferred embodiments of the present invention, the
effective amount of a salt of zinc and butyric acid ranges between
50 ppm and 10,000 ppm of the product being treated and all values
between such limits, including, for example, without limitation or
exception, 61 ppm, 237 ppm 1,011 ppm, 2,170 ppm, 6,573 ppm and 9999
ppm. Stated another way, in preferred embodiments of the invention,
the dosage can take any value "abcde" ppm wherein a is selected
from the numerals 0 and 1, and b, c, d and e are each individually
selected from the numerals 0, 1, 2, 3, 4, 5, 6, 7, 8 and 9, with
the exception that d cannot be less than 5 if a, b, c and d are all
0.
Example 1--Cell Culture Work
Materials and Methods
[0028] The data in FIG. 1 and corresponding table (Table 1) show
that growing IPEC-J2 epithelial cells in the presence of ZBA causes
them to have increased trans-epithelial resistance. This indicates
that the tight junctions between the cells are well formed when
compared to the alternate treatments and demonstrates improved
intestinal integrity.
TABLE-US-00001 TABLE 1 Effect of heat stress challenge on IPEC-J2
cells under various treatments Treatment 0 h 12 h 24 h 36 h 48 h
Control-TN 1.00 1.97 2.11 2.52 2.81 Control-HS 1.00 1.47 1.81 2.10
2.24 Zinc Sulfate-HS 1.00 1.67 1.89 2.19 2.31 ZBA-HS 1.00 2.04 2.24
2.60 2.76 Calcium Butyric Acid Salt- 1.00 1.58 1.74 1.96 2.10 HS
TN--Thermoneutral HS; Heat stress
[0029] When animals undergo heat stress, the increased temperature
leads to damage of the epithelial layer as well as withdrawal of
tight junction proteins to the cytoplasm of the epithelial cells,
thus compromising the tight junctions. The compromised tight
junctions allow for toxins and other inflammatory substances to
enter the systemic circulation. Compared to growing the cells under
thermoneutral conditions (FIG. 1), ZBA also improves the
trans-epithelial resistance when cells are grown under heat stress.
As reported in FIG. 2, cells were treated with the test compounds
and incubated at 41.5.degree. C. (Con-TN) Control-Thermoneutral
conditions--37.degree. C. (CON-HS)--Control-Heat Stress
conditions--41.5.degree. C. (Zn Sulf) 300 .mu.M Zinc Sulfate under
Heat Stress Conditions--41.5.degree. C. (ZBA) 300 .mu.M ZBA under
Heat Stress Conditions--41.5.degree. C. (CaBA) 300 .mu.M Calcium
and Butyric Acid Salt under Heat Stress Conditions--41.5.degree. C.
The presence of 300 .mu.M ZBA in the cell culture media improves
the trans-epithelial resistance to the level of cells grown that
are grown at thermoneutral conditions at 37.degree. C., which is
the normal temperature at which the cells are grown. The treatments
of zinc or butyric acid separately, as zinc sulfate or calcium and
butyric acid salt, do not improve the trans-epithelial resistance
as compared to the untreated heat stress cells (FIG. 2).
Example 2
Heat Stress Trial in Swine.
[0030] To determine the effect of feeding EZBA on intestinal
integrity during metabolic and physiological stress, an animal
trial was performed which uses pigs as the experimental model and
heat stress for the stress model. The effect of the EZBA was
determined by comparing the growth of the pigs treated with EZBA to
the growth of the untreated pigs.
[0031] During the acclimation phase, which comes before the heat
stress period, twenty four grower pigs weighing approximately 30 kg
were used for the study (n=12/trt) in a randomized complete block
design. The pigs were fed two treatments, a control treatment with
no ZBA added and an encapsulated ZBA treatment. The EZBA treatment
contained 3000 ppm of ZBA, fed in an encapsulated form of 40% ZBA
and 57.5% hydrogenated palm oil and 2.5% propylene glycol. The pigs
were blocked by weight and feed and water were provided ad libitum.
Pigs were on the respective treatments for 28 days at ambient
temperature to allow for enrichment of the test compounds. The pigs
were weighed and blood samples were collected every week.
[0032] After the collection, the animals were subjected to a
bi-phasic heat stress to simulate the natural heat stress model,
where during the day time the temperature was around 33.degree. C.
and during the night it was around 22.degree. C., for 7 days. Blood
was collected every twelve hours. Rectal temperature of each pig
was measured every two hours. At the end of this period six pigs
from each group were euthanized and Using chamber studies were
performed on fresh ileum and colon from each pig. Trans-epithelial
resistance and macromolecule transport using FITC labeled dextran
were determined to measure the effect of supplementation of the
different zinc compounds on intestinal integrity and health during
heat stress.
[0033] During both the acclimation phase and the heat stress
period, the EZBA fed pigs had a higher feed intake as compared to
the control pigs (FIG. 3B).
[0034] During the acclimation phase, the control and EZBA treated
pigs had similar Gain:Feed (FIG. 4A) and average daily gain (FIG.
5A), but during the heat stress period, the EZBA fed pigs had a
higher Gain:Feed (FIG. 4B) and average daily gain (FIG. 5B) as
compared to the control fed pigs.
[0035] Macromolecule transport through the intestinal tissue, as
measured by FITC-Dextran transport, was lower with the EZBA treated
group, as compared to the control group during heat stress (FIG.
6). This indicates that the intestines of the EZBA fed pigs have
more intact tight junctions than the intestines of the control fed
pigs.
[0036] The results of the swine trial show that feeding EZBA can
help alleviate the loss in feed intake that occurs during the
stress period. In addition, feeding EZBA can help increase both the
Gain:Feed ratio and weight gain when animals are being stressed,
such as being exposed to excessive heat or any pathogenic
challenges.
Example 3--Effectiveness of EZBA on Swine Infected with PEDV
Materials and Methods
Feed
[0037] Three nursery pig diets were manufactured at the Iowa State
University Swine Nutrition Farm. All pigs were on a common
corn-soybean meal basal diet that met or exceeded the NRC 2012
nutrient requirements for this size pig (Table 2). EZBA was
included in the feed at 5 grams per kg feed and hand mixed into the
feed, this supplied 550 ppm of supplemental zinc to the diet.
Animals, Housing, and Inoculation
[0038] A total of 32 mixed sex pigs were obtained from an Iowa
State University source and delivered after weaning at an average
initial body weight (BW) of 7.5.+-.1.2 kg. Pigs were allotted based
on sex and arrival BW across 2 treatments (Control and EZBA) with
16 pigs per treatment and 2 pigs per pen (8 pens per
treatment).
[0039] There were 2 rooms in which these pigs were housed and both
rooms contained equal numbers of pigs of each of treatment. Pigs
were provided the treatment diets for the duration of the study.
They were allowed a 7 or 8 day adaptation to the diet and housing
in which time, pre-challenge growth performance data were
collected. During this first week a starter pellet diet was
included at a 1:1 ratio with the treatment diet to ensure that the
pigs started well prior to challenge. All pigs were equally split
into two replications to allow for necropsies to be offset by 1
day. Therefore, the pre-challenge period was 7 days for replicate 1
and 8 days for replicate 2. Data were pooled for each replicate and
was included in the model as a covariate if it significantly
contributed to the model. Pigs were gastrically gavaged with 5 ml
of 10.sup.3 TCID.sub.50 of the PEDV plaque-cloned isolate from Iowa
(18984/2013) on 0 days post innoculation (DPI).
Growth Performance Calculations, Tissue Collection, and Analyses of
Intestinal Sections
[0040] Pigs and feeders were weighed at dpi -7/-8, 0, 5, 10, 15,
and 20 to calculate average daily gain (ADG), average daily feed
intake (ADFI), and gain to feed ratio (G:F). There were 2 pigs per
pen for data including and prior to 5 dpi and 1 pig per pen for
data including and after 10 days post infection (dpi). Upon
arrival, pigs were confirmed PEDV naive by fecal swab qPCR. To
characterize the pathogenesis of PEDV, at dpi 0, 5, 10, 15 and 20
fecal swabs from all pigs and blood (.about.10 mL) from 1 pig per
pen was obtained. The same pig was bled at each time point. All
fecal swabs were analyzed for PEDV by qPCR.
[0041] At dpi 5 and 20, 8 pigs per treatment (1 pig per pen) were
weighed, euthanized, and necropsied. Pigs were restrained and
euthanized by intravenous administration of a lethal dose of sodium
pentobarbital followed by exsanguination. Jejunum and ileum were
collected, flushed with phosphate buffered saline, and formalin
fixed. After 24 hours, fixed samples were removed from formalin and
placed in ethanol. Tissues were paraffin embedded and sectioned for
analysis.
TABLE-US-00002 TABLE 2 Ingredient composition of for the basal
diets Control EZBA Ingredient, % Corn 50.76 50.26 Soybean Meal
19.00 19.00 Soybean oil 1.73 1.73 Fishmeal, menhaden 4.50 4.50
Limestone 0.35 0.35 Whey, dried 20.00 20.00 Meat and Bone Meal 2.04
2.04 L-Lysine HCL 0.43 0.43 DL-Methionine 0.16 0.16 L-Threonine
0.13 0.13 L-Tryptophan 0.03 0.03 L-Valine 0.07 0.07 EZBA -- 0.5
Salt 0.40 0.40 SNF Grow-Fin Vit Premix 0.25 0.25 SNF Trace Mineral
Premix 0.15 0.15 Calculated Composition ME, kcal/kg 3,408 3,408 SID
Lysine, % 1.35 1.35 STTD Phos., % 0.40 0.40
[0042] Formalin-fixed, paraffin-embedded intestinal tissue sections
were mounted on positively charged glass slides and oven-dried for
20 min at 60.degree. C. Deparaffinization occurred with three
changes of xylene for 5 min each followed by rehydration with 100%
alcohol, 95% alcohol, 70% alcohol, tap water, and distilled water.
Slides were then placed in 3% hydrogen peroxide (Fisher Scientific,
Pittsburgh, Pa.), diluted in methanol, for 10 min followed by three
changes of distilled water. Antigen retrieval was accomplished with
0.05% protease XIV (Sigma-Aldrichl, St. Louis, Mo.) for 2 min
followed by three distilled water rinses. Approximately 200 ml of
murine monoclonal 6C8 primary antibody specific for the
nucleoprotein of PEDV (BioNote, Seoul, Korea), diluted 1:100, was
applied to each slide, then transferred to a 37.degree. C.
incubator for 1 h, removed, uncovered, and rinsed with Tris Buffer
Saline (TBS) (Fisher Scientific, Pittsburgh, Pa.) for 3 min.
LSAB2-HRP Link and Label (Dako, Carpinteria, Calif.) were then
applied to each slide for 10 mi and rinsed in TBS for 3 min,
respectively. DAB substrate chromogen (Dako, Carpinteria, Calif.)
was applied to the slides for 5 min followed by a distilled water
rinse for 3 min. The slides were then counterstained in
hematoxylin, dehydrated, cleared with Pro-Par clearant (Anatech
LTD, Battel Creek, Mich.), and cover-slipped. Immunohistochemistry
(IHC) slides were prepared for all five small intestine sections of
all challenged pigs. Antigen detection was semi-quantitatively
scored based on the following criteria: 0=no signal, 1=1-10% of
villous enterocytes within the section showing a positive signal,
2=11-50% of villous enterocytes showing a positive signal, and
3=greater than 50% of villous enterocytes showing a positive
signal. Semi-quantitative scores were recorded by a single blinded
veterinary pathologist (Madson, D. M., Magstadt, D. R., Arruda, P.
H., Hoang, H., Sun, D., Bower, L. P., Bhandari, M., Burrough, E.
R., Gauger, P. C., Pillatzki, A. E., Stevenson, G. W., Wilberts, B.
L., Brodie, J., Harmon, K. M., Wang, C., Main, R. G., Zhang, J.,
and Yoon, K. J., (2014). Pathogenesis of porcine epidemic diarrhea
virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs. Vet
Microbiol. 174: 60-8).
[0043] The ISH protocol uses an oligonucleotide probe targeting the
N gene of PEDV (5'-TGTTGCCATTACCACGACTCCTGC-3') obtained from a
commercial vendor (Invitrogen Custom Oligos, Life Technologies,
Carlsbad, Calif.) with a 5' fluorescein label. The probe is
reconstituted in a commercial hybridization buffer (Bond.TM.
Hybridization Solution, Leica Biosystems, Newcastle Upon Tyne, UK)
at 5 ng/.mu.l and the procedure is performed using a commercially
available system (Leica Bond-III, Leica Biosystems, Melbourne,
Australia). Tissue sections are dewaxed using a commercial dewaxing
solution and then treated with a commercially available enzymatic
pretreatment (Bond.TM. Enzyme Pretreatment Kit, Leica Biosystems,
Newcastle Upon Tyne, UK) for 5 min followed by 5 rinses with a
commercial wash solution (Bond.TM. Wash Solution, Leica Biosystems,
Newcastle Upon Tyne, UK). The diluted probe is applied and allowed
to hybridize at 45.degree. C. for 12 hours and then rinsed prior to
incubation with an anti-FITC antibody for 30 minutes followed by
application of a commercial chromagen system (Bond DAB Refine Kit,
Leica Biosystems, Newcastle Upon Tyne, UK).
[0044] Intestinal sections were also stained to detect Ki67, a
marker for stem cell proliferation. The monoclonal Ki67 antibody
was purchased from Dako (Glostrup, Denmark) and has been previously
published for pigs (Jung, Kwonil, Annamalai, Thavamathi, Lu,
Zhongyan, and Saif, Linda J., (2015). Comparative pathogenesis of
US porcine epidemic diarrhea virus (PEDV) strain PC21A in
conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs.
Veterinary Microbiology. 178: 31-40). Ki67 was quantified by
counting positive nuclei in 3 intact crypts that were orientated
properly and had approximate average depth for that specific
section.
[0045] Lesion scores were given for sections of ileum and jejunum
that were routinely stained with H&E. Scores were given from 0
to 3, where 0=normal villus:crypt ratio (>4:1), 1=mild villus
blunting (4:1-3:1) with rare fusion, 2=moderate villus blunting
(3:1-2:1) and moderate fusion with lymphoid infiltration, and
3=severe villus blunting (<2:1) with severe fusion and lymphoid
infiltration. The same person recorded lesion scores for all
sections to eliminate scorer biases. Sections, staining, and
scoring was done at the ISU Veterinary Diagnostic Lab using
internal standards and protocols.
Data Analysis
[0046] Growth performance data were analyzed to determine effect of
treatment, dpi, and treatment by dpi interaction and this model
included the fixed effects of treatment, dpi, and treatment by dpi
interaction. Start BW was used as a covariate for BW, ADG, and G:F
estimates and replicate was used as a covariate for ADG estimates.
Growth performance data were also analyzed over the entire 20 dpi
period. The model included treatment as the fixed effect and start
BW was used as a covariate for BW gain estimates. For all growth
performance data, the repeated statement was used with a pen as the
subject over time and the structure used was compound symmetry.
Results
Quantitative PCR for PEDV
[0047] All pigs were positive for PEDV at least once during the
study (FIG. 7). There was an effect of dpi where the lowest cycle
threshold (Ct) value was at 5 dpi and increased thereafter for both
PEDV treatments. PEDV values were not different between the two
treatments.
Growth Performance
[0048] The EZBA treatment had a higher ADG at day 20 and also
during the overall period whereas ADFI was higher on days 15 and 20
as well as during the overall period (FIGS. 8A and 8B).
[0049] At day 20 the EZBA group had a higher body weight than the
control group. Except for day 0 and day 5, Gain: Feed of EZBA group
was higher than the control group throughout the experimental
period (FIGS. 9A and 9B).
Immunohistochemistry for PEDV and Ki65, In Situ Hybridization for
PEDV, and Lesion Scores
[0050] Antigen and nucleic acid staining of PEDV was only observed
at 5 dpi for PEDV treatments, as expected. There was no difference
in jejunum or ileum PEDV IHC scores between the treatments. In both
tissues PEDV IHC was lower in EZBA group and the ileum PEDV ISH
scores for the EZBA treatment was lower than those of the control
treatment. There was no PEDV IHC or ISH detected at 20 dpi (FIGS.
10A and 10B).
[0051] The positive nuclei counts of the control group at dpi 20
were lower in both the jejunum and ileum compared to dpi 5. In both
the jejunum and ileum, EZBA pigs had greater Ki67 positive nuclei
at 20 dpi compared with 5 dpi. This demonstrates that the EZBA
treatment was able to increase the number of Ki67 positive nuclei
and sustain the increase at 20 dpi compared with the Control. In
the jejunum, the EZBA group had a significantly higher positive
nuclei count compared to the control at dpi 20 (FIGS. 11A and
11B).
[0052] There was no treatment by dpi interaction or treatment
effect of lesion score in both jejunum and ileum. There was a dpi
effect in jejunum and ileum for lesion scores where there was a
decrease in lesion scores at 20 dpi compared with 5 dpi regardless
of treatment. EZBA significantly reduced the lesion score in the
jejunum at dpi 5, and EZBA also reduced the lesion scores in the
ileum compared to the control at dpi 5 (FIGS. 12A and 12B).
Morphology of Jejunum and Ileum
[0053] There was a treatment by dpi interaction for villi height in
the jejunum and ileum. Regardless of treatment, jejunum and ileum
villi height were increased at 20 dpi compared with 5 dpi. Villi
height was numerically higher in the EZBA treatment at dpi 5 in
both jejunum and ileum (FIGS. 13A and 13 B).
[0054] Crypt depth was not different in the jejunum on both days
and in the ileum on dpi5. On dpi 20 crypt depth of the EZBA group
in ileum was significantly higher than the control group (FIGS. 14A
and 14 B).
DISCUSSION
[0055] PEDV has significant negative impact on the health and
growth of pigs. In this trial EZBA was studied to understand
whether it could give protection and alleviate the negative effects
during a PEDV infection in nursery pigs. Pigs were confirmed
positive for PEDV 5 dpi using qPCR. ADG, ADFI, body weight were
comparatively lower during the first ten days of infection in both
treatment groups but the EZBA pigs recovered quickly and by d20
they had a better ADG, ADFI and body weight compared to the control
pigs. G: F of EZBA pigs was also numerically higher from d10
onwards. Even though growth performance is not the focus of this
study, these results present clear evidence that EZBA could help
the ADFI in pigs which would improve the health as well as maintain
the growth during viral infection.
[0056] IHC and ISH were performed to confirm and quantify the
presence of PEDV protein and DNA in the jejunum and ileum. The EZBA
group of pigs had a lower IHC values in the jejunum indicating the
lesser presence of PEDV antigen and lower ISH values in both ileum
and jejunum indicating lesser presence of PEDV genetic material.
These results indicate that EZBA has the potential to decrease the
proliferation of the virus which would result in decreased
pathogenicity of the virus and help the animal recover quickly.
[0057] EZBA significantly reduced the lesion score in jejunum and
also reduced the lesion score in ileum at dpi 5. Decreased lesion
scores indicate that the intestinal tissues from EZBA pigs were
better protected against PEDV compared to the control group. Ki67
is a stem cell marker which specifically binds to proliferating
stem cells. Higher binding of Ki67 is an indication that the tissue
is regenerating faster. In the jejunum, there was no difference
between the Ki67 positive nuclei at dpi 5 but at dpi 20 EZBA pigs
had a significantly higher amount of positive nuclei. Also, the
positive nuclei value of the control group in both tissues
decreased compared to dpi 5 where as the EZBA group increased. This
indicates that EZBA group has the potential to stimulate the tissue
regeneration faster after the PEDV infection. Severity of any
infection can be contained by replacing the inflamed and infected
tissues faster. Both zinc and butyric acid, ingredients of EZBA,
have the potential to act on different cellular pathways to improve
the recovery and increase the cell number of the GI tract under
various inflammatory conditions which is evident from these
results.
[0058] Jejunum and ileum morphology results also supported the
beneficial effects of supplementing EZBA in nursery pig diets.
Villus height and crypt depth were increased during the peak
infection period, 5 dpi. This indicates that EZBA was able to blunt
the severity of PEDV infection.
[0059] All this evidence strongly suggests that EZBA can maintain
the gut health and barrier integrity and has the potential to
alleviate the negative effects during PEDV infection.
[0060] The foregoing description and drawings comprise illustrative
embodiments of the present inventions. The foregoing embodiments
and the methods described herein may vary based on the ability,
experience, and preference of those skilled in the art. Merely
listing the steps of the method in a certain order does not
constitute any limitation on the order of the steps of the method.
The foregoing description and drawings merely explain and
illustrate the invention, and the invention is not limited thereto,
except insofar as the claims are so limited. Those skilled in the
art that have the disclosure before them will be able to make
modifications and variations therein without departing from the
scope of the invention.
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