Note: Descriptions are shown in the official language in which they were submitted.
CA 02560283 2006-09-18
ANTIBODIES AS GROWTH PROMOTING AGENTS
PRIOR APPLICATION INFORMATION
This application claims the benefit of Canadian Patent Application
2,521,156, filed September 16, 2005.
BACKGROUND OF THE INVENTION
It is believed that feed additives which improve gut health and provide a
more balanced gut microflora will promote growth of an animal.
In recent years, passive immunotherapy by oral administration of
specific antibodies against gastrointestinal pathogens has been studied
extensively in
animals. Furthermore, there has been increasing interest in developing avian
polyclonal antibodies for treatment of gastrointestinal diseases caused by
various
pathogens. As orally administered antibodies would provide the advantage of
reduced
cost and ease of administration for the treatment of enteric diseases, as an
alternative
to antibiotics. The aim of efficient pork production is to maximize lean meat
yield while
minimizing production cost. It has been established that two important factors
contributing to lean growth are improving health status and the prevention of
specific
diseases. Enterotoxigenic E. coil (ETEC) strains that express K-88 fimbriae
are a
major cause of diarrhea and death in neonatal and newly weaned pigs resulting
in
major economic loss to the pork industry. It has been estimated that K-88-
mediated
ETEC are responsible for 50% of the 10 million baby pig deaths each year.
Historically, antibiotics and animal plasma proteins have helped to reduce the
detrimental effects of this condition. However, there is mounting pressure to
discontinue the use of antibiotics and animal plasma proteins in swine
industry, due to
the concerns of human health. Many antibiotics and plasma proteins are banned
for
use in swine diets in Europe and will most likely be restricted in North
America.
Therefore, researchers have taken keen interest in searching for alternative
therapies
to antibiotics that could be used in swine nutrition, particularly during the
nursery
phases, Passive immunotherapy has been shown to have prophylactic and
therapeutic benefits for controlling disease and improving livestock growth
CA 02560283 2013-04-02
2
performance. Avian (egg) antibodies developed against ETEC can be transferred
to the
recipient by oral administration through supplementation of the normal diet of
post-weaning
piglets, thereby preventing the ETEC disease in piglets and acting as an
effective growth
promoter. Orally administered antibodies provide the advantage of reduced cost
and ease of
administration for the treatment of enteric disease as well as for improvement
of gut health,
the key requirement for the improved growth performance, offering effective
and sustainable
replacements for both plasma proteins and antibiotics
SUMMARY OF THE INVENTION
According to a first aspect of the invention, there is provided a method of
increasing
growth performance in a piglet comprising: immunizing a population of hens
with K88
bacterial antigen; collecting eggs from the population of hens, said eggs
containing
antibodies raised against said K88 bacterial antigen; recovering antibody-
containing material
from said eggs; drying said antibody- containing material to a powder, thereby
producing
dried egg powder; confirming that the titer of the K88 antibodies is at least
1/256,000
compared to a control titer from eggs from a non-immunized hen of 1/1600;
supplementing
feed of a piglet with 0.1-1.0% (w/w) of the dried egg powder; and feeding said
feed to said
piglet ad libitum, wherein said piglet has increased growth compared to a
control piglet of
similar age and condition, fed a similar feed ration ad libitum and regimen
but said similar
feed ration being without supplementation with said antibody-containing egg
powder.
BRIEF DESCRIPTION OF THE DRAWINGS AND TABLES
Figure 1: bar graph of weight over time in groups fed control feed, feed +
0.1% SDEP
and feed + 0.4% SDEP.
Figure 2: bar graph of weight over time in groups fed antibiotics and control
feed,
feed + 0.1% SDEP and feed + 0.4% SDEP.
Figure 3 is a bar graph corresponding to Figure 1 but showing average daily
gain.
Figure 4 is a bar graph corresponding to Figure 2 but showing average daily
CA 02560283 2006-09-18
3
gain.
Figure 5 is a bar graph corresponding to Figure 1 but showing average daily
food intake (ADFI).
Figure 6 is a bar graph corresponding to Figure 2 but showing average daily
food intake (ADFI).
Figure 7 shows fecal E. coli isolation versus hours post infection.
Figure 8 is a bar graph showing E. coli challenge on phase 1 ADG of pigs.
Figure 9 is a bar graph of body weight changes during the study period.
Figure 10 is a bar graph showing the effect of E. coli challenge on phase 1
feed
disappearance of pigs.
Figure 11 is a bar graph showing the effect of E. coli challenge on phase 2
and
overall performance of pigs.
Figure 12 is a bar graph showing the effect of E. coil challenge on feed
efficiency of pigs.
Figure 13: Effect of diet on body weight increase. The increase in body weight
was higher in the test groups than control group, on day 10, 14, and 28,
except for
group 4 (ABDEX 2%, 0.1% antibody egg powder + dextrose). On day 28, the
increase
of body weight in piglets of the three test groups ranged from 1.7 ¨ 2.5kg,
over the
control group, and the difference was statistically significant (p<0.01).
Overall, the
increase ranged from 13-20% over the control group. There was a slight
increase in
body weight in piglets fed diet 4, compared to the control diet, but the
difference was
not significant.
Figure 14 is a bar graph showing the dietary effect on week 1 ADG of pigs.
Figure 15 is a bar graph showing the dietary effect on week 2 ADO of ETEC
challenged pigs.
Figure 16 is a bar graph showing dietary effects on week 1 to 2 ADO of pigs.
Figure 17 is a bar graph showing the dietary effects on pig performance during
weeks 3 to 4.
Table 1: Measures of growth performance in piglets fed diets supplemented
with or without antibiotics and/or SDEP.
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Table 2: Effect of avian antibodies on improvement of growth and performance
in
field studies of piglets fed diet supplemented with 0.05-0.3% of antibody-
containing spray-
dried egg powder (SDEP), measuring average daily gain (ADO), average daily
feed intake
(ADF1) and morbidity and mortality.
Table 3: Composition of experimental diets (Phase 1).
Table 4: Composition of experimental diets (Phase 2).
Table 5: Effect of egg antibody on the performance of early (18-d old) weaned
pigs.
Table 6: Diet formulation.
Table 7: ETEC K-88 dosing protocol.
Table 8: Clinical response of 18-day old piglets following challenge with
different
doses of E. coil K-88.
Table 9: Enumeration of ETEC in rectal swabs.
Table 10: Growth performance of pigs.
Table 11: Growth performance: body weight changes during the study period.
Table 12: Percentage body weight changes before and after feeding with diet
supplemented with 0.4% avian antibodies.
Table 13: Treatment schedule.
Table 14: Clinical response after prophylactic treatment of piglets with
control or
antibody egg powder following challenge with 1012 CFU doses of ETEC K-88.
Table 15: Enumeration of ETEC in rectal swabs.
Table 16: Growth performance based on body weight increase.
Table 17: Growth performance based on ADO, ADFI and G/F.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless defined otherwise, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which the
invention belongs. Although any methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention, the
preferred methods and materials are now described.
CA 02560283 2013-04-02
As used herein, "passive immunity" refers to the transfer of antibodies from
an
immunized animal to a non-immune recipient.
As used herein, "effective amount" refers to a dosage sufficient to have the
desired effect.
5 As
discussed above, improvements to gut health and that provide a more
balanced gut microflora will improve animal growth. As such, supplementing
animal feed with
polyclonal antibodies directed against one or more pathogens is shown herein
to improve
growth of the animals, as shown in the attached figures and tables. As will be
appreciated by
one of skill in the art, the pathogens do not necessarily have to be causing
disease, but may
still consume nutrients or cause nutrients to be used less efficiently by the
host animal,
thereby reducing growth. Furthermore, improving gut health will have several
beneficial
effects, for example, reducing inflammation, which will in turn improve for
example nutrient
absorption and uptake. Thus, while not wishing to be bound by a specific
hypothesis, it is
believed that for example gut inflammation promotes feed uptake which in turn
promotes
growth. Similarly, mortality and morbidity are decreased because of the
treatment and/or
removal of disease but also by the promotion of growth.
As will be appreciated by one of skill in the art, generation of polyclonal
antibodies using a variety of host animals is well-known in the art. In one
embodiment, the
polyclonal antibodies are egg yolk antibodies.
In a preferred embodiment, a population of hens are immunized or challenged
with one or more microbial antigens. That is, the entire population may be
immunized with an
antigen from a single microorganism or portions of the population may be
immunized with
different antigens from the same microorganism or portions of the population
may be
immunized with different antigens from different microorganisms.
Following immunization, the antibody-containing material is recovered once a
suitable
antibody titer has been obtained. As will be appreciated by one of skill in
the art, titers may
be maintained by rechallenge or reinoculation of hens over time.
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In some embodiments, the titer obtained from the eggs may be at least
1/256,000
compared to a control titer of 1/1600.
The antibody-containing material, for example, the whole egg or the egg
yolk or the egg white or fraction(s) thereof, is then combined and is dried,
for example
spray dried or freeze dried, to a powder using means known in the art.
In some embodiments, the antibodies may be a powder, for example, a
dried egg yolk powder.
The polyclonal antibodies may be a mixture of antibodies from several
host animals. In some embodiments, the polyclonal antibodies may be a mixture
of
antibodies against different antigens from one pathogenic organism or multiple
pathogenic organisms, as discussed above.
The pathogen may be selected from the group consisting of: Bacillus
cereus, Bacillus anthracis, Bacillus subtilis, Bacillus thuringiensis,
Bacillus
stearothermophilus, Vibrio parahemolyticus, Vibrio cholerae 01, Vibrio
cholerae non-
01, Vibrio vulnificus, Aeromonas hydrophilia, Salmonella enterica, Salmonella
typhi,
Salmonella paratyphi, Salmonella entertidis, Salmonella cholerasuis,
Salmonella
typhimurium, Clostridium difficile, Clostridium botulinum, Clostridium
perfringens,
Staphylococcus aureus, Escherichia coli (ETEC, EPEC, EHEC, EaggEC, UPEC and
EIEC), Campylobacter jejuni, Campylobacter coli, Campylobacter lari,
Campylobacter
fetus, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis,
Listeria
monocytogenes, Plesiomonas shigelloides, Shigella, Streptococcus, Giardia
lamblia,
Entamoeba histolytica, Cryptosporidium parvum, Cylcospora cayetanenis,
Amisakis,
Diphyllobothrium, Nanophyetus, Eustrongylides, Acanthamoeba, Ascaris
lumbricoides, Trichuris trichuris, Hepatitis A virus, Hepatitis E virus,
Rotavirus,
Norwalk virus group, Acinetobacter, Actinobacillus, Actinomycetes,
Actinomyces,
Aeromonas, Peptostreptococcus, Veillonella, Mobiluncus, Propionibacterium
acnes,
Lactobacillus, Eubacterium, Bifidobacterium, Bacteroides,
Prevotella,
Porphyromonas, Fusobacterium, Bordetella, Borrelia, BruceIla, Burkholderia,
Citrobacter, Corynebacterium, Edwardsiella, Enterobacter, Enterobacteriaceae,
Klebsiella, Morganella, Proteus, Providencia, Serratia, Enterococcus,
Erysipelothrix
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rhusopathiae, Francisella tularensis, Haemophilus, Helicobacter, Legionella
pneumophilia, Leptospira interrogans, Micrococcaceae, Moraxella catarrhalis,
Mycobacterium, Nocardia, Neissaria, Pasteurella multocida, Pasteurellaceae,
Pseudomonas aeruginosa, Rhodococcus, Serratia marcescens, Stenotrophomonas
maltophilia, Streptococcus pneumoniae, Streptomyces, Treponema and
combinations
thereof.
As will be appreciated by one of skill in the art, the antibody-containing
whole egg powder or egg yolk powder may be from hens immunized against a
single
pathogen or a mixture comprised of egg powder from hens immunized against
different pathogens. Furthermore, the antibody-containing egg powder may be
added
to feed at a range of about 0.01% to about 1.0% or about 0.05% to about 1.0%.
It is of
note that the powder may be added to the feed or may be used to coat portions
of the
feed or may be otherwise presented to the animal.
Thus, in some embodiments, hens are immunized against an antigen
from an organism or the organism itself. Eggs are then collected and the
antibodies
may be purified or concentrated using means known in the art.
As discussed above, supplementing feed with the polyclonal antibody
containing egg powder has the surprising benefit of promoting growth, that is,
of
increasing daily food intake, increasing weight gain, and/or increasing
average daily
weight gain in an animal fed a feed ration supplemented with the polyclonal
antibody
containing egg powder compared to a control which was fed the feed ration
only.
Specifically, the control may comprise an animal of similar age and condition,
fed a
similar feed ration and regimen but without supplementation with the egg
powder as
discussed herein.
In a preferred embodiment of the invention, there is provided a method
of increased growth in an animal comprising:
immunizing a population of hens with at least one bacterial antigen;
collecting eggs from the population of hens, said eggs containing
antibodies raised against said at least one bacterial antigen;
recovering antibody-containing material from said eggs;
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a
drying said antibody- containing material to a powder, thereby producing
dried egg powder; and
supplementing feed of an animal with 0.1-1.0% (w/w) of the dried egg
powder, wherein said animal has increased growth compared to a control animal
fed
6 unsupplemented feed.
As will be appreciated by one of skill in the art, improved or increased
growth can also be considered to be reduced gut inflammation, improved
nutrient
absorption and uptake, improved gut health, improved daily weight gain,
improved
weight gain over an extended time period or other suitable metrics as
discussed
herein.
The invention will now be described by way of examples. It is to be
understood however that the examples are for illustrative purposes and are not
necessarily limiting.
Example 1
Thirty-six 18 day old piglets (8.3 0.2 kg) were allocated to six
treatment groups, blocked by weight and given ad libitum access to feed and
water
(n=6/group)
Each group was administered varying doses of SDEP containing
antibodies to ETEC K88 and/or antibiotics for 28 days (Table 1).
Animals were weighed at the end of phase 1 (14 d) and phase 2 (28 d),
and average daily gains were calculated.
Group Treatment
A No antibiotics, no SDEP
Antibiotics*, no SDEP
C No antibiotics, 1 kg/T SDEP
No antibiotics, 4 kg/T SDEP
Antibiotics*, 1 kgfT SDEP
Antibiotics*, 4 kg/T SDEP
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9
*Antibiotic administered was Mecadox at a rate of 1.0%
Four individual field studies were conducted. In each study the control
group of piglets were administered a normal diet, while the test groups' diets
were
supplemented with a specified amount of antibody-containing egg powder (SDEP)
ranging from 0.5 kg/T to 3 kg/T. The piglets were monitored for growth
performance.
Growth performance of animals fed diet with and without antibiotics and
supplemented with SDEP:
Overall growth performance was improved in all groups supplemented
with SDEP (C, D, E & F ) compared with controls, with (B) or without
antibiotics (B).
= 10 A significant level (P < 0.05) of growth improvement
was achieved when
piglets were fed diet with only SDEP (4 kg/T) without antibiotics(D), compared
with a
diet that did not contain antibiotics (A) (Table 1).
In both phase 1 (0-14d) and phase 2 (14-28d), 4 kg/T SDEP increased
growth performance by 25.3- 27.6% (0) over the control group (A) while 1 kg/T
SDEP
increased growth performance by 9.2% (C) over the control group (A) in phase
2.
The combination with antibiotics, SDEP improved growth performance in
both phase 1 and phase 2. SDEP at 1 kg/T (E) increased by 7.5-25% and SDEP at
4kg/T (F) increased by 16,6-27.9% ,over the control group (B) (Table 1).
Growth performance and reduction of mortality for animals fed diet
supplemented with SDEP compared with controls:
Increased body weight gain by 1-3 kg
Improvement on average daily gain by 10.5-31.5% (Table 2)
Improvement on average daily feed intake by 15-20% (Table 2)
Improvement on mortality by 10-15% (Table 2B)
Improvement on morbidity by 25-40% (Table 2B)
It can be concluded from the experimental study that nursery diet
supplemented with SDEP in absence of antibiotics increases body weight of
piglets by
2.8 kg over the control. While. SDEP in presence of antibiotics increases body
weight
of piglets by 2.0 kg over the control.
Similar conclusions can be drawn from the four separate field studies as
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SDEP increases the live weight of piglets by 1-3 kg.
Based on the results obtained from both experimental and field studies,
it can be concluded that Chicken egg antibodies in SDEP used in swine nursery
diet
are proved to be as a viable alternative to antibiotic growth promoters.
5
Example 2
Experimental Design:
Thirty six crossbred (GAP Genetics, Winnipeg, MB, Canada) 18 ¨d old
pigs with an initial weight of 6.3 0.2 kg were allotted into six treatment
groups: (1)
10 control; (2) 0.1% SDEP; (3) 0.1% SDEP + antibiotics; (4) control +
antibiotics; (5)
0.4% SDEP; (6) 0.4% SDEP + antibiotics. Pigs were blocked by weight and
assigned
to pens, and thus treatments, such that the average weight across treatments
was
similar.
Pigs were housed in stainless steel pens equipped with nipple waterers,
stainless steel feeders, and plastic-coated expanded metal floors. The pens
were
located in an environmentally regulated building maintained at 25 2'C with a
12-h
light/dark cycle. Body weight and feed consumption were recorded after every
phase
and ad libitum access to feed and water was provided during the entire period
of the
study.
There were three pens per treatment and two pigs per pen. The pigs
were fed a standard pig starter diet in phases 1 (Table 3) and 2 (Table 4).
The diets
were with or without spray-dried egg powder (SDEP) containing E. Coli K-88
antibodies and with or without antibiotics. The diets were fed in two phases.
Both
phases were fed for 14 days each. Pigs were allotted to treatments based on
weaned
weights and dam. Feed and water was available always. Pigs and feeders were
weighed at the end of each phase to determine weight gains and feed
disappearance.
Pigs were observed every day for any changes in behavior due to the dietary
treatments.
Results:
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11
There was no mortality or morbidity and abnormal health problems
observed in animals of group treated with feed supplemented with Escherichia
coli
antibody, chicken egg origin (SDEP), at 0.1-0.4% level, during the 4-week
study
period. In contrast, the overall growth performance was found to be improved
in
groups treated with feed supplemented with Escherichia coli antibody, chicken
egg
origin (SDEP), at 0.1-0.4% level.
The growth performance data is presented in Table 5. The inclusion of
0.4% SDEP tended to improve performance in Phase 1. The combination of
antibiotics and SDEP tended to increase growth performance in Phase 1 by 30%
for
0.1% SDEP and 15% for 0.4% SDEP inclusion rates, In Phase 2, 0.1% SDEP
increased growth performance by 9.2% over the control group. Inclusion of 0.4%
SDEP in the diet increased growth performance by 27.6% over the control group.
Feed efficiency tended to be improved by 0.4% SDEP in the diet.
The results of this study demonstrated that the addition of 0.4% SDEP to the
diets of early weaned pigs improved growth performance. This can be seen on
review of Figures 1-6.
Example 3 Determination of infective dose of E. coli K-88
Animal Use and Feeding
Thirty crossbred 18-d old pigs (body weight 5.6-6.5 kg) were randomly allotted
to 15 pens with 2 pigs per pen. Pens were randomly assigned to 5 treatments
with 3
pens per treatment. Same regular nursery diet was fed to pigs across
treatments for
week 1 (Table 6). In week 2, pigs were switched to basal phase 1 diet
supplemented
with 0.4% antibody egg powder. In week 3 & 4, pigs were switched to phase 2
nursery
diets. Pigs were allowed ad libitum access to water and feed.
E coli Inoculums Preparation
The particular E. coli K-88 strain, different from the one used for producing
antigen in order to produce avian antibodies, was isolated and propagated at
the
Department of Veterinary Diagnostic Laboratory, College of Veterinary
Medicine,
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12
University of Minnesota, St. Paul.
E. coli K88 obtained from case D05-29173 was initially grown on a sheep blood
agar plate for 18 hours at 37 C. One isolated colony was aseptically removed,
placed in 2 ml sterile distilled water and mixed thoroughly. A sterile swab
was dipped
6 into the E. coil suspension and excess inoculums removed by rotating the
swab
against the inside wall of the tube above the fluid level. A 150 mm sheep
blood
Mueller-Hinton agar plate (BD Diagnostics, Sparks, MD 21152) was inoculated by
streaking the swab over the entire surface in three different directions
ensuring an
even distribution of the inoculum for a confluent lawn of growth. This process
was
used to inoculate 10 sheep blood Mueller-Hinton agar plates. The plates were
incubated for 15-18 hours at 37 C.
After incubation, a sterile tissue cell scraper was used to remove the
bacteria
growing on the agar plate by gently scraping the agar surface. Bacteria on the
scraper were placed into 200 ml sterile saline. This process was repeated with
all the
Mueller-Hinton agar plates. The E. coli saline was mixed thoroughly to obtain
a
homogeneous suspension.
Once a homogenous suspension was obtained, serial dilutions were made in
sterile saline until an optical density equal to a 0.5 McFarland was obtained.
The
concentration of the challenge suspension was adjusted to 1010 and 1012
cells/ml.
This was placed in sterile 5cc syringes, which were labeled and kept cold
until the
pigs were orally dosed.
To determine the challenge suspension colony forming unit concentration per
ml, an aliquot was serially diluted and fixed volume plated onto blood agar
plates
which were incubated at 37 C for 15-18 hours. After incubation, plates with
30-300
26 colonies were counted and the concentration of viable bacteria per ml
determined.
Treatment Arrangement & Dosing Scheme
As described in Table 7, pigs were given PBS solutions (control), or 5 mls of
PBS solution with 1010 or 1012 cfu/m1 of E.coli K-88 at 0 hr of day 1 (E-10
and E-12),
or 5 mls of PBS solution with 1010or 1012(cfu/m1) at 0 and 5 hr of day 1 (G-10-
10 and
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13
E-12-12).
Data Collection and Sampling
Pigs were weighed on day 1, 3, 7, 14, and 28 to determine growth rate. Feed
disappearance was recorded on day 7, 14, and 28. Average daily feed intake
(ADFI),
average daily gain (ADG), and feed efficiency (G/F) were calculated
accordingly.
Daily clinical observation was assured to record incidence of diarrhea, fecal
consistency score, appetite and mortality. Fecal scores were recorded as
0=norrnal,
1=light, 2=medium, and 3=severe, to indicate diarrhea severity.
Rectal swabbing (BBL culturette and transport swab, BD Diagnostics, Sparks,
MD, 21152) were taken at Ohr before infection, 6, 24, 48, 72, and 120hr after
infection.
Rectal swabs in cold pack were delivered within 24 hr to Veterinary Diagnostic
Laboratory, College of Veterinary Medicine, University of Minnesota, St. Paul,
for
isolation and identification of ETEC.
Laboratory Analysis
The swab portion of a BBL culturette and transport swab (BD Diagnostics,
Sparks, MD, 21152) was placed into 2 ml of sterile water and mixed thoroughly.
Serial 10 fold dilutions were made and each dilution plated onto separate
MacConkey
agar plate. After 15-18 hours of incubation at 370 C, the plates containing 30-
300
lactose positive colonies typical of E. coli were counted, averaged and the
total
bacteria per swab calculated.
Incidence of Diarrhea, Fecal Consistency and Pig Loss
During the first week post-infection, fecal score, enumeration of ETEC and pig
loss were recorded to establish incidence and severity of diarrhea. Piglets
infected
vvith higher doses of ETEC had incidence of diarrhea at 6hours post-infection;
while
piglets with lower dose of ETEC had diarrhea at later stages. However, piglets
from
single infected group with 1012 CFU, double infected group with 1010 and 1012
CFU of
ETEC had higher diarrhea incidents. The average fecal score was higher for
piglets
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14
from 1012 CFU of ETEC infected group, and 3 piglets were lost within 72hour
post-
infection with double dose of 1012 CFU. Numerically, 1012 CFU of ETEC caused
increased diarrhea and fecal score. In control group, 2 piglets had mild
diarrhea with
fecal score of 1-2. Similarly, at the lowest dose only 2 piglets had diarrhea
with a low
fecal score. The results are illustrated in Table 8.
Rectal ETEC Isolation
Rectal swabs from each piglet were cultured to enumerate ETEC concentration
at each time points, 0, 6, 24, 48 and 120hr post-infection. In all groups,
ETEC
numbers were changed with the hours of post -infection. Rectal swabs from
groups
infected with single dose of ETEC had the highest number bacteria at 48 hour
post-
infection, while double-infected piglets with 101 and 1012 CFU of ETEC had
the
highest peaks of ETEC concentration at 72 hours. All ETEC concentration
decreased
to undetectable level after 72 hr. The average ETEC count from each group at
each
time point is shown in Table 9 and Figure 7.
It has been established from this study that there is a definite relationship
between the K-88 ETEC infection dose with the incidence and severity of
diarrhea in
18-day old piglets. Increasing ETEC concentration decreased growth rate, and
increase diarrhea incidents. Double infection dose using 1012 CFU had
significant
impact on piglet growth, severity of diarrhea, mortality and presence of ETEC
in rectal
swab, while single dose of 1012 CFU is sufficient for inducing diarrhea in
pigs fed
regular diet.
Based on the results from the Study 3, it was concluded that to determine the
prophylactic efficacy of e. coli K-88 avian antibodies, piglets will be
infected with a
single dose of ETEC 1012 CFU (Study -2), and to determine the therapeutic
efficacy
of avian antibodies, piglets will be infected with a double dose of ETEC 1012
CFU (
Study-5).
Growth Performance
Four pigs died within the first week post ETEC infection (Table 10). ADG and
CA 02560283 2006-09-18
ADFI were calculated after corrected with pig loss. The corrected growth
performance
of pigs is shown in Table 10.
During the first 3 days post infection, pigs from all treatments lost weight
Pigs
double-dosed with 1012 CFU of ETEC had the greater weight loss than pigs from
5 other treatment. During 4 to 7 days post infection, similar trend was
observed in
growth, pigs recovered with increasing growth, pigs from control group and
double
1010 CFU-infected group had greater ADG compared to pigs from double 1012 CFU-
infected group. During the first week post infection, feed intake (ADFI) was
also lower
for pigs infected with double 1012 CFU (Figure 8).
10 Overall for the first week post- infection, pigs from control group
(without ETEC
challenge) had numerically greater ADG compared to pigs from other group
(P>.08),
especially greater gain and feed efficiency than pigs from double 1012 CFU-
infected
group (P.08).
As the animals were sick, to avoid further loss of animals, and to determine
the
15 effect of avian antibodies on recovery of the sick animals as well
as on growth
performance, on day 7, pigs were switched to diets supplemented with 0.4%
antibody
egg powder.
After feeding diet supplemented with 0.4% ETEC antibody, the growth rate of
piglets was determined to be similar or higher compared to the uninfected
control
animals, particularly during day 8-14 post-infection. Also, it can be
indicated from the
results that piglets infected with lower doses of ETEC recovered at faster
rate than
piglets infected with higher doses. The results are shown in Tables 11 and 12
and
Figures 9-12.
No difference in ADG was observed among treatments, which may indicate the
beneficial effect of avian antibody on post infection pig recovery. Pigs from
control
group ate more feed than pigs from double 1012 CFU-infected group (13.08).
Overall, during Phase 1, pigs from 2 x 1012 CFU-infected group had lower ADG
than pigs from 2 x 101 CFU-infected group, but not different compared with
pigs from
other groups.
During week 3 and 4, pigs were switched to Phase 2 diet with 0.4% antibody
CA 02560283 2006-09-18
16
egg powder. Continuing recovery of pigs was observed across treatments. There
was
no difference in ADG and ADFI among pigs from different treatments.
Increasing ETEC concentration decreased growth rate and after feeding diet
supplemented with 0.4% K-88 avian antibodies, piglets recovered at similar or
better
rate to the controls. Therefore, it can be suggested that piglets infected at
various
doses of ETEC can be recovered completely following feeding with diet
supplemented
with 0.4% E. coli K-88 avian antibodies.
Example 4 Prophylactive efficacy of E. coil K-88 Avian-antibody Products
Animal Use and Treatment
Thirty crossbred 18-d old pigs (body weight 5.5-6.5 kg) were randomly allotted
to 15 pens with 2 pigs per pen. Pens were randomly assigned to 5 treatments
with 3
pens per treatment. Five experimental diets were fed to pigs across treatments
for
week 1 and 2 (Table 8). The diets were made by adding either control egg
powder
from unimmunized chickens (group-1 EG) or E. coli K88 antibody, chicken
origin,
(Serial 12-04, Lot-003, Inovatech Egg Products, Lot # 5078) at different
concentrations (Test groups, 2-5, ABEG 2%, ABEG 4%, ADDEX 2% or ABDEX 4%),
to basal diets used in the study 3. In week 3 and 4, pigs were switched to
phase 2
nursery diets. Pigs were allowed ad libitum access to water and feed.
E. coli lnoculums Preparation and Challenging
At day 7, following feeding with control and test diets, piglets were
challenged
by orally dosing 5mL of ETEC K-88 at 1012 cfu/ml suspended in PBS, following
similar
procedure as described above.
Clinical Observation and Data Collection
Daily clinical observation was assured to record incidence of diarrhea, fecal
consistency score, appetite and mortality. Fecal scores were recorded as
0..normal,
1=light, 2=medium, and 3=severe, to indicate diarrhea severity.
CA 02560283 2006-09-18
17
Rectal swabs were taken at Ohr before infection, 6, 24, 48, 96, and 120hr
after
infection. Rectal swabs in cold pack were delivered within 24 hr to Veterinary
Diagnostic Laboratory, College of Veterinary Medicine, University of
Minnesota, St.
Paul, for isolation and enumeration of ETEC, following the procedure described
above.
Pigs were weighed on day 0, 7, and 10, 14, and 28 to determine growth rate.
Feed disappearance was recorded at day 7, 14, and 28. Average daily feed
intake
(ADFI), average daily gain (ADG), and feed efficiency (G/F) were calculated
accordingly.
Statistics
Data were analyzed by using the GLM procedures of SAS (1999) for LSMEAN.
For ETEC counting, fecal score and ADG, the statistical model uses individual
pigs as
experiment units. ADFI calculation uses pen as unit The body weight at day 7
was
used as covariate to analyze growth response. Following orthogonal contrasts
(Steel
and Torrie, 1980) were used to characterize main effects of treatments: 1) egg
powder vs. antibody egg powder; 2) egg powder vs. antibody-dextrose; 3)
antibody
egg powder vs. antibody dextrose. Polynomial regressions (Steel and Torrie,
1980)
for unequally spaced treatments were used to characterize the shape of the
ETEC
response to the timely change.
Incidence of Diarrhea, Fecal Consistency and Pig Loss
During the first week post-infection, fecal score, enumeration of ETEC and pig
loss were recorded to establish incidence and severity of diarrhea. Piglets in
the
control group, fed with 0.4% egg powder from unimmunized chicken for 7 days,
following infection with 1012 CFU ETEC had higher incidence of diarrhea,
compared to
the test groups, which were fed with various concentrations (0.1-0.4%) of
antibody
egg powder. However, severity and incidence of diarrhea among piglets varied
in test
groups. Piglets fed with either 0.4% antibody egg powder (ABEG 4%) or 0.2%
antibody egg powder and dextrose (0.4% Eggstend, ABDEX 4%) had the lowest
CA 02560283 2006-09-18
18
incidence of diarrhea. However, no mortality was observed in the test group,
when
piglets were fed with0.4% antibody egg powder ABDEX 4%. The highest incidence
of
diarrhea and mortality were observed in piglets, which were fed with 0.1%
antibody
egg powder and dextrose (0.2% Eggstend, ABDEX 2%) and 0.2% antibody egg
powder (ABDEX 2%). The results are illustrated in Table 14.
Isolation of ETEC from rectal swabs
Rectal swabs from each piglet were cultured to enumerate ETEC concentration
at each time points, 0, 6, 24, 48 and 120hr post-infection. In all groups,
ETEC
numbers were changed with the hours of post -infection. Rectal swabs from ETEC
infected piglets had the highest number bacteria at 48hour post-infection, as
observed
in the Study 3. At 48hr post infection, rectal ETEC isolation were
significantly higher
for piglets fed control diet than pigs fed 2 or 4% ABEG or 4% ABDEX diet.
The highest number of ETEC average count per swab was determined to be
29.6 x106 CFU in the control group, while in the test groups, the average
count
ranged from 0.16 - 2.13 x106 CFU. ETEC concentration decreased to undetectable
level after 96hr. However, at 120hr post infection, piglets fed with control
diet or 2%
ABDEX had rectal ETEC isolation. The average ETEC count at each time point is
shown in Table 16.
Growth Performance
Six pigs died within 1st week post infection. Growth performance was
calculated with correction for pig loss.
Through week 1 and 2, pigs were fed different experimental diets. In 1st week,
pigs from control group grew similar to the pigs from other groups. At day 7,
all pigs
were infected with 1012 CFU of ETEC. During three days post-infection, most of
pigs'
growth rates were reduced numerically, especially for pigs fed control diet.
During 4-7
days post- infection, pigs recovered in terms of ADG, except pigs fed control
diet (egg
powder from unimmunized chicken) and 2% ABDex diet (0.1% antibody egg powder +
dextrose). Overall, during 1st week post-infection, pigs fed 4% ABEG diets
(0.4%
CA 02560283 2013-04-02
19
antibody-egg powder) and 4% ABDex (0.2% antibody egg powder + dextrose) had
greater
ADG compared to pigs fed control diet, and numerically greater than 2% ABDex
diet (0.2%
antibody egg powder).
For period of week 1 and 2, pigs fed 4% ABDex and 4% AB diet had greater ADG,
ADFI and feed efficiency compared to pigs fed other diets.
In week 3 and 4, pigs were switched to Phase 2 treatment diets. There was no
difference in final weight, ADG and G/F, though pigs fed 2% ABEG and 4% ABDEX
gained
25% and 10% more daily than control diet. ADFI was 43.4% higher for pigs fed
4% ABDEX
during this period.
The results are illustrated in Tables 16 and 17, and Figures 13-17.
Feeding antibody egg powder and antibody dextrose did not positively impact
growth
rate, fecal consistency or diarrhea incidence of pigs in 1st week post
weaning. However,
feeding 0.4% antibody egg powder or 0.2% antibody plus dextrose improved the
growth
performance and recovery of pigs post ETEC infection. Piglets fed with lower
dose of
antibody egg powder at 0.1% with dextrose for 14 days did not improve the
growth
performance on day 28, but reduced the bacterial load in the swab samples
following
challenge with high dose of ETEC. While, piglets fed with 0.2% antibody egg
powder for 14
days improved the growth performance on day 28, and reduced the bacterial load
in the
swab samples following challenge with high dose of ETEC.
In contrast, piglets fed with control egg powder for the same time period, had
very
high count of ETEC in their swab samples and the body weight was found to be
the lowest,
compared to the test groups.
CA 02560283 2013-04-02
Therefore, based on the results, it can be suggested that K-88 antibody
containing
egg powder at 0.2 -0.4% level with or without the presence of dextrose can be
used for
prophylactic treatment of post-weaning diarrhea as well as improvement of
growth.
CA 02560283 2006-09-18
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Table 1: Measures of growth performance in piglets fed diets supplemented with
or
without antibiotics and/or SDEP.
Treatment Group
Measures A BCDEF
'it of Piglets 6 6 e 6 6 6
Mean initial weight (kg) 6.0 6.2 6.1 6.2 6.2 6.2
Mean finalweight (kg) 9.0 9.6 -8.9 10.0 9.8 11.6
Avg. daily gain (g) 213 240 198 267 258 307
Avg, daily intake (g) 310 328 325 356 364 376
>.
Mean feed/gain 1.44 1.63 1.40 1.38 1.31 1.22
_O. 6 __
-Mean final weight (kg) 15.6 15.4 16.0 18.4 17.2 17.4
Avg. daily gain (g) 466 -421 509 595 527 491
Avg. daily intake (g) 527 532 577 659 604 627
fa co _______________________________________________________
>.
ca v. Mean feed/gain 1.13 1.35 1.13 1.1 1.12 1.12
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Table 2: Effect of avian antibodies on improvement of growth and performance
in
field studies of piglets fed diet supplemented with 0.05-0.3% of antibody-
containing
spray-dried egg powder (SDEP), measuring average daily gain (ADG), average
daily
feed intake (ADFI) and morbidity and mortality.
A Field Study #1 Field Study #2
Control Test Control 'Test
(0.05%) (0.3%)
# Piglets 1156 1163 4338 1716
Days on feed 59.6 58.4 59.0 57.8
Initial weight (kg) 5.3 5.2 7.27 6.93
Final weight (kg) 28.0 28.8 23.81 27.22
ADG (g) 381 404 318 351
ADFI (g) 544 627 620 750
Field Study #3 Field Study #4 ¨
Control Test Control Test
(0.2%) (0.3%)
-# Piglets 20 20 20 ¨ ¨20 =
Days on feed 14 14 14 14
Initial weight (kg) 8.2 8.2 8.0 8.0
Final weight (kg) 12.7 14.1 11.5 12.4
ADG (g) 321 422 250 314
Morbidity (%) 30 5 50 10
Mortality (%) 10 0 15 0
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Table-3 : Composition of experimental diets (Phase 1)
________________________________________________ ---
Groups 1 2 3 4 5 6
Treatments Control 0.1% 0.13 Control 0.4%
0,4%
5DEP 5DEP MEP SDEP
+ AB + AB +AB
Ingredients
Corn 49.1 48.1 49.1 48.1 49.1 48,1
SBM 28.5 28.5 28.5 28.5
28.3 28.3
Fishmeal
7 69 6.9 7 6.8 6.8
SDEP' 0.1 0.1 0.4 0,4
Whey powder 12 12 12 12 12 12
Blended fat 2 2 2 2 2 2
Limestone 0.55 0.55 0.55 -0.55 0.55 0.55
Di-calcium Phosphate 0.1 0.1 0.1 0.1 0.1 0.1
Salt 0.25 0.25 0.26 0.25 0.25 0,25
Vitamins-Trace mineral premix 0.3 0.3 0.3 0.3 0.3 0.3
Mecadox 1.0 1.0 1.0
Choline chloride 0.2 0.2 0,2 0.2 0.2 0.2 ,
TOTAL 100 100 100 100 100 100
Nutrient Analyses
ME, Kcal/kg 3307 3308 3308 3309 3321 3320
Crude protein (%) 23.2 23.1 23.1 23.2 - 23.1 23,1
Calcium(%) 0.88 0.87 0.87 0.88 0.87 0.87
Total phosphorus(%) 0.69 _ 0.69 0.69 0.69 0.69 0.69
Lysine(%) 1.45 1,45 1,45 1.45 1.45 1.45
Methionine(%) 0,35 0.35 , 0.35 0.35 0.35 0.35
Threonine(%) 0.95 0.95 0.95 0.95 0.95 0.95
Tryptophan(%) 0.29 0.29 0.29 0.29
0.29 029
1SDEP; Spray-dried egg powder
=
CA 02560283 2006-09-18
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Table-4: Composition of experimental diets (Phase 2)
Groups 1 2 3 4 5 6
Treatments Control 0.1% 0.1%
Control 0.4% 0.4%
SDEP SDEP SDEP SDEP
+ AB .AB +AB
-.
Ingredients, %
Corn 612 81.2 60.2 60.2 61.2 60.2 II
SBM 25 25.0 25 25 iiimi 24.8 1111
Fishmeal 5 4.9 4.9 5 4.8 4.8
SDEP' 0.1 0.1 0.4 0.4
Whey powder 5 5 5 5 5 5 I
Blended fat 2 2 2 2 2 2
I
Limestone 0.8 0.8 0.8 0.8 0.8 0.8
Dicalcium Phosphate 0.2 0.2 0.2 0.2 0.2 0.2
1
Salt 0.26 0.25 0.25 0.25 0.25 0.25 II
Vitamins-Trace mineral premix 0.3 0.3 0.3 0,3 0.3 0.3 I.
Mecadox 1.0 1.0 1.0 II
Choline chloride 0,2 0,2 0.2 0.2 0.2
al
TOTAL 100 100 1 100 100 100 100 II
__ Nutrient Analyses
ME, Kcal/kg 3332 3334 3331 3330 3336 3332 II
Crude protein (%) 20.4 20.4 20.4 204 20.4 20.4
Calcium(%) 0.78 0.78 0.78 0.78 0,78 0.78
Total phosphorus(%) 0.59 0.59 0.59 0.59 0.59 0.58
Lysine(%) 1.21 1.21 1.21 1.21 1.21 1.21
Methionine(%) 0.32 0.32 0.32 0.32
0_32 0.32
Threonine(%) 0.81 0.81 0.81 0.81 0.81 0.81
Tryptophan(%) 0.24 0.24 0.25 0.24
0.24 0.25
'SDEP; Spray-dried egg powder
=
CA 02560283 2006-09-18
Table4 : Effect of egg antibody on the performance of early (184 old) weaned
pigs
Groups 1 2 3 4 5 6 SEM
Treatments Control 0.1% 0.1% Control 0.4% 0,4%
SOEP1 SDEP SDEP SDEP
+ AB2 + AB +AB
Phase 1
No. of pigs 6 6 6 6 6 6
Start weight (kg) 6.1 6.1 6.2 6.2 6.2 6.2
End Weight(kg) 9.0 8.9 9.8 9.6 10,0 11.6
ADFI (g) 310 325 364 328 356 376 20.9 NS
ADG (g) 213 198 258 240 267 307 27.2 NS
G:F 0.69 0.61 0,71 0.72 0.76 0_82 0.07 NS
Phase 2
No. of pigs 8 6 6 6 6 6
Start weight (kg) 9.0 8.9 9.8 9.6 10.0 11.6
End Weight(kg) 15.6 16.0 17.2 15.4 18,4 17,4
ADFI (g) 527 577 604 532 659 627 58.6 NS
ADG (g) 466 509 527 421 595 491 69.0 NS
G:F 0.88 0.88 0.89 0.74 0.90 0.79 0.08 NS
SDEP; spray dried egg product
2A8; antibiotic, mecadox
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Table 6 Diet Formulation
Ingredients Phase 1 Phase 2
Corn 48.02 58.43
SBM 28.33 25.00
Dried whey 15.00 10.00
Fishmeal 6.00 4.00
Premix base 0.50 0.50
Lysine -HCI 0.10 0.05
DL-methionine 0.05 0.02
Blended Animal fat 2.00 2.00
100 100
Calculated Analysis
Crude protein (%) 25.5 22.10
ME, kcal/kg 3300 3300
Lysine (%) 1.68 1.37
Methionine (cY0) 0.42 0.36
Tryptophan (%) 0.33 0.28
Threonine (%) 1.12 0.93
Ca (%) 0.90 0.81
P(%) 0.78 0.64
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Table 7: ETEC K-138 Dosing Protocol
Tr tm # of Pigs E.coli K-88 conc. Dosage in
Frequency
eaent
(du/m1) mi. (hr)
6 0 5 0
control
10" 5 0
E. coli 10"
6 1.0" 5 0
E. coli 1012
E. coli 6 1010 5 ______ 0 and 5
2 x 101 _______
E. coli 6 1012¨ 5 0 and 5 __ ¨
2 x 1012
Table 8:
Clinical response of 18-day old piglets following challenge with different
doses of E. coil k-88
Groups Number No. pigs with diarrhea (fecal score) with
time
of (hours) post-infection)
animals
with 6 24 48 72 96 120
diarrhea
Control 2/6 0 0 2(1.5) 1(1.0) 1(1.0) 0
E. coli 10" - 2/6 0 2(13) 0 2(1.0) 2(1.5) 1(1.0)
E. cOli 2 x 6/6 0 3(1.8) 3(1.0) 5(1,4) 5(1,4)
5(1.0)
101
E. coli 1012 5/6 1(3.0) 4(2.0) 4(1.8) 3(2.3) 3(1.7)
3(1.0)
E. coil 2 x10" 4/6 2(3,0) 4(2.25) 3(2,3) 2(23) 1(2.0)
1(2.0)
Fecal scores were recorded as 0=norrnal, 1=light, 2=mecilum, and 3=severe, to
indicate diarrhea
severity. 2(10) indicates 2 pig had diarrhea with an average score of 3,0.
*indicates that one piglet was dead at each time point. Another piglet in
group infected with 1010 was
dead on day 7 post-infection,
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Table 9: Enumeration of ETEC In Rectal Swabs
Control E. coil E. coil E. coil E. coli
, 1010 1012 , 2 x 10 2 x 1012
1st Pre swab, x106 cfu/ml 0.12 0.13 1.40 0.97 0.20
6hr swab, x106 cfu/ml 1.40 0.15 0.41 0.36 0.21
24hr swab, 106 cfu/ml 0.07 0.14 1.39 0.67 1.57
48hr swab 106cfu/m1 5.22 5.23 14.26 0.83 4,34
- 7-2hr swab 106 cfu/ml 0.23 449 2.01 4.92 20.91
120hr swab 106 du/ml 0.01 0.46 0.07 - 0.08 0.07
_ .
Table 10: Growth Performance of Pigs
Performance Control E-10 E-12 _E- 2
x,10_ E- 2 x 12
Phase 1
ADG 0-3, g/d 28.4a- -79.5a ..945a -47.7 a -
229.5b
ADG 4-7, g/d 143.86 57.5 ab 78.2ab 139.2 b - 753
ADG 0-70/d 45.43 -20.8 at' -20.6 ab 32.4 ab -
85.6 b '
ADFI 0-7,g/d 115.2 b 81.7a 93.1" 97.8 ab 75.0a
G/F wk1, kg/kg 3901' . -580'16 -350 ab 320 b -1090a
ADG wk2,g/d 269.6 284.9 239.3 353.0 234.6
ADFI wk2, gicl 339.8" 316.4ab 277.4' 270.4'4 244,2b
G/F wkZg/d 790 a 910 a - 960a 1320" 820 a
ADG wk12,g/d 165.0 ab 142.2 ab 118.0a6 - 203.4" ,
110.0 a
Phase 2
ADG wk34, g/d 539.0 389.1 428.1 455.8 356.2 ,
ADFI wk34,g/d 524.4 494.7 497.7 546.9 509.0
G/F wk34, g/kg 1100 810 900 810 740 ,
ADG1-4,g/d 338.6 256.8 270.8 310.1 224.3
1 Values in each row with different letters indicate significant difference in
means, P<0.04.
CA 02560283 2006-09-18
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Table 11. Growth performance: Body Weight changes during the study period
Study period Body weight kg Se in different groups
(Days) Control E. coil 1010 1012 2x 1010 2x 1012
0 6.06 0.21 6.05 0.15 6.06 0.15 6.02 0.16 6.01
0.15
4 5.94 0.20 5.69 0.13 5.68 0.25 5,75 0,23 5.30
0.30
7 6.37 0.28 5.66 0.31 5.91 0.38 6.00 0.32 5.90
0.30
_
15 8.53 0.51 8.24*0.21 7.82*0.46 8.19 1.06 7.77
0.51
_
28 15.53 1.03 13.29 1.17 12.96 1.20 15.46 1750. _ 12.40 0.12
Table 12
Percentage body weight changes before and after feeding with diet supplemented
with 0.4% avian antibodies
_ _____________________________________________________________________
Study period Grove
(Days) Control; ¨
E. Coil 1016 1012 2x 101 2x 1011
0-4 - ¨ -1.98 - -5.95 -6.27 -4.49 -11.81
5-7 7.24 -0.53 4.05 4.35 11.32
-
8-14 33.91 45.58 32.32 36.5 31.69
15-28 82.06 61.29 55.73 88.77 59.59
Table 13: Treatment Schedule
Treatment # of Pigs Abr. Name Diets for Phase 1 & 2
Groups
1 , 6 EG_ Basal + 0.4% control g powder
.
2 6 ABEG2% Basal + 0.2% antibody egg powder
,
3 6 ABEG4% Basal + 0.4% antibody egg powder
, ... .
4 6 ABDeac2% Basal + 0.2%Eggstend
(0.2% antibody
___________________________________________ egg powder-dextrose at 1:1)
5 6 ABDex40/0 Basal + 0.4%
Eggstend (0.4% antibody
_ egg powder-dextrose at 1:1)
.
CA 02560283 2006-09-18
Table 14
Clinical response after prophylactic treatment of piglets with control or
antibody egg powder following challenge with 1012CFU doses of ETEC K-
5 88
Groups Number No. pigs with diarrhea (fecal score) with time
of (hours) post-infection)
animals ___________________________________________________________
with 6 24 48 72 96 120
diarrhea
Control Egg 5/6 2 (3.75)* 4(2.5) 4(2.5) 3(2.5)
2 (2.5) 2(2.0)
Powder
(EG) _
ABEG 2% 4/6 2(3.0) 3(1.5)* 3(2.0) 2(1.5) 2(1.5)*
1(1.0)
ABEG 4% 2/6 2(1.7) 2(2.0) 2(2.0) 2(1.5) 2(1.5)
2(1.0)
ABDEX 2% 4/6 3(1.7)* 4(2,0) 3(2.0) 3(2.0) 3(1.7)* -
3(1.0)
ABDEX 4% 2/6 1(2.0) 2(2.0) 2(2.0) 2(2.0) 2(2.0)
1(2.0)*
Fecal scores were recorded as 0=normal, 1=light, 2=medium, and 3=severe, to
indicate diarrhea
severity. 2(3.0) indicates 2 pig had diarrhea with an average score of 3Ø
* indicates that one piglet was dead at each time point.
Table 15: Enumeration of ETEC in Rectal Swabs
Control ABEG ABEG ABDEX ABDEX
-EG 2% 4% 2% 4%
1st Pre swab, x106 0.03 0.02 0.02 0.06 0.02
cfu/swab
6hr swab, x106 cfu/swab 0.02 0.02 0.05 0.03 0.03
24hr swab, 106-cfu/swab 6.43 2.35 1.02 0.61 0.12
48hr swab 106cfu/swab 29.66 1.29 0.68 2.13 0.16
96hr swab 1O cfu/swab 0.18 0.07 0.03 0.24 0.16
120hr swab 106- 1.6 0.4 0.3 1.8 0.2
digswab
CA 02560283 2006-09-18
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Table 16: Growth performance based on Body Weight Increase
Body weight kgtSE in different groups
Study EG 4%
ABEG 2% ABEG 4% *ABDEX 2% ABDEX 4%
period (control egg (0.2% Ab (0.4% Ab (Eggstend
(Eggstend
powder Egg powder) Egg powder) 0.2%) OA%)
0.4%)
5.96 0.12
5.89 0.18 5.96+0.16 5.95+0.15 5.97+0.14
7.11 0.19
7 7.01+0.13 6.84+0.20 7.22+0.18 6.97 0.15
7.69*(1.33
7.10+0.22 7.12+0.39 7.65+0.25 6.78+0.44
9.41 0.23
14 8.01+0.25 8.61+0.50 9.27+0.60 7.83+0.95
16.45 1.35
28 12.89 0.90 14.59 0.69 14.65 1.88 13.14 1.03
CA 02560283 2006-09-18
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Table 17. Growth Performance based on ADG, ADFI and G/F
Performance * EG
ABEG2% ABEG4% ABDex2% ABDex4% P value
= _______________________________________________________________________
Phase l.
Wt day 0, kg 5.88 5.96 5-95 5.97 5.96
0.9093
Wt day 7, kg 7.01 7.06 7.22 6.98 7.11
0.9238
ADG 0-7, g/d " 126 -12-1:9- 14I i 97.5 127.4
0.8274
Wt day 10,kg 7.1 7.24 7.65 6.98 7.69
0.3626
Wt day 14, kg 8.018 8.6181' 9.27 b 7.83 9.41
0.1410
ADG 7-10, g/d 68 149.5 216 157.8 b
286.6
0.7221
ADG 11-14, g/d 226.8 296 405 174.5 356.4
0.3474
ADG wk2õg/d 173.9 b 278.3 ab , 342 ab 186.9 6b 375.5
b 0.2499
ADG wk12,g/d 141.5 190 221.4 104.4 .225.5
0.2331
ADFI wk12, g/d 232.2k 194.68 273.5c 157.3a 252c
0.0002
G/F wk12,g/d 600 918 810 627 888
0.4017
Phase 2 ________________________________________________________________ _
Wt day 14, kg 8.018 8.61 1' 9.27 b 7.838 9.41 b
0.1410
Wt day 28, kg 12.88 _14.59 14.66 13.13 15.45
0.7203
ADG wk34, g/d 374.7 460.1 414.1 408.4 464.7
0.9494
ADFI wk34,g/d 568.18 701.9 ab 642.68 535.48 8148b
0.0242
G/F wk34, kg/kg 630 686 609 722 573
0.9450
ADG1-4, g/d 249.8 315.4 310.9 245.5 336.4
0.7481
* Values in each row with different letters indicate difference in means, P<
.05
