U.S. patent application number 14/659452 was filed with the patent office on 2015-09-10 for methods of treating pigs with bacillus strains.
This patent application is currently assigned to DUPONT NUTRITION BIOSCIENCES APS. The applicant listed for this patent is DuPont Nutrition Biosciences ApS. Invention is credited to Ashley Baker, Mari Ellen Davis, Thomas G. Rehberger.
Application Number | 20150250831 14/659452 |
Document ID | / |
Family ID | 41267038 |
Filed Date | 2015-09-10 |
United States Patent
Application |
20150250831 |
Kind Code |
A1 |
Rehberger; Thomas G. ; et
al. |
September 10, 2015 |
METHODS OF TREATING PIGS WITH BACILLUS STRAINS
Abstract
Disclosed are methods of administering at least two Bacillus
strains to a pig, such as female breeding stock, nursery pigs, or
other pigs. The Bacillus strains inhibit Clostridium in litters
borne to the pig. The Bacillus strains also are useful when
administered to herds lacking symptoms of Clostridium infection.
Administration of the Bacillus strains improves performance of
female breeding stock and in piglets borne by the female breeding
stock.
Inventors: |
Rehberger; Thomas G.;
(Wauwatosa, WI) ; Davis; Mari Ellen; (Waukesha,
WI) ; Baker; Ashley; (Waukesha, WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DuPont Nutrition Biosciences ApS |
Copenhagen |
|
DK |
|
|
Assignee: |
DUPONT NUTRITION BIOSCIENCES
APS
Copenhagen
DK
|
Family ID: |
41267038 |
Appl. No.: |
14/659452 |
Filed: |
March 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13937826 |
Jul 9, 2013 |
9011836 |
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14659452 |
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|
13212409 |
Aug 18, 2011 |
8506951 |
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13937826 |
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12404149 |
Mar 13, 2009 |
8021654 |
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13212409 |
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61036741 |
Mar 14, 2008 |
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Current U.S.
Class: |
424/93.462 |
Current CPC
Class: |
A61K 2035/11 20130101;
A23K 50/30 20160501; A61K 35/74 20130101; A23K 50/10 20160501; A23K
10/18 20160501; A61P 31/04 20180101 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A23K 1/00 20060101 A23K001/00; A23K 1/18 20060101
A23K001/18 |
Claims
1. A method comprising administering an effective amount of at
least two Bacillus strains selected from strains 22CP1 (ATCC
PTA-6508), 3AP4 (ATCC PTA-6506), 15AP4 (ATCC PTA-6507), 2084 (NRRL
B-50013), LSSAOI (NRRL B-50104), and 27 (NRRL B-50105) to a
pig.
2. The method of claim 1, wherein the Bacillus strains are strains
3AP4 (ATCC PTA-6506) and LSSAOI (NRRL B-50104).
3. The method of claim 1, wherein the Bacillus strains are strains
15AP4 (ATCC PTA-6507) and LSSA01 (NRRL B-50104).
4. The method of claim 1, wherein the Bacillus strain is
administered to female breeding stock.
5. The method of claim 1, wherein the Bacillus strain is
administered during gestation of the pig.
6. The method of claim 5, wherein a total microbial count of
3.75.times.10.sup.5 CFU/g of feed/day is administered.
7. The method of claim 1, wherein the Bacillus strain is
administered during lactation of the pig.
8. The method of claim 7, wherein a total microbial count of
8.5.times.10.sup.8 CFU/g of feed/day is administered.
9. The method of claim 1, wherein administration of the Bacillus
strain inhibits Clostridium in litters borne to the pig.
10. The method of claim 9, wherein the Clostridium is Clostridium
peifringes.
11. The method of claim 9, wherein the Clostridium is Clostridium
difficile.
12. The method of claim 1, further comprising modifying the
Bacillus strains administered based on a change in Clostridium
strains to which the pig is exposed.
13. The method of claim 1, wherein administration of the Bacillus
strains provides at least one of the following in litters borne to
the pig relative to that in litters borne to pigs that have not
been administered the Bacillus strains: (A) lower average scour
scores in the first week after birth, and (B) a decrease in the
percentage of scouring litters.
14. The method of claim 1, wherein the pig is in a herd lacking
symptoms of i Clostridium infection.
15. The method of claim 14, wherein administration of the Bacillus
strains decreases clostridial load in the gastrointestinal tract of
piglets borne by the pig relative to that in piglets borne to pigs
that have not been. administered the Bacillus strains.
16. The method of claim 1, wherein the pig is in a unit of pigs
that are subclinical for clostridial scours.
17. The method of claim 16, wherein performance of at least one of
the pig and piglets borne by the pig is improved during lactation
relative to pigs and piglets that have not been administered the
Bacillus strains.
18. The method of claim 1, wherein the Bacillus strain is
administered when the pig is at least one of a nursery pig and a
feedlot pig.
19. A method comprising administering an effective amount of
Bacillus strains 3AP4 (ATCC PTA-6506) and LSSAO1 (NRRL B-50104) to
a pig.
20. The method of claim 19, wherein the pig is a gestating pig.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
13/937,826 filed Jul. 09, 2013, which is a continuation of U.S.
Ser. No. 13/212,409 filed Aug. 18, 2011 and issued as U.S. Pat. No.
8,506,951 on Aug. 13, 2013, which is a continuation of U.S. Ser.
No. 12/404,149 filed Mar. 13, 2009, and issued as U.S. Pat. No.
8,021,654 on Sep. 20, 2011, which claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 61/036,741,
filed Mar. 14, 2008. The entireties of all of the above-referenced
applications are incorporated by reference herein.
FIELD DESCRIBED HEREIN
[0002] The invention relates to controlling disease in pigs,
enhancing pig performance, and improving the health of pigs with
Bacillus strains.
DESCRIPTION OF THE RELATED ART
[0003] Enteric clostridial infections in swine occur predominantly
in the neonatal period but are also associated with hemorrhagic
bowel syndrome affecting pigs in the finishing period. Although
immunization against C. perfringens type C has greatly reduced
pre-weaning mortality, no commercial vaccines are currently
available for C. perfringens type A or C. difficile. C. perfringens
type A and C. difficile infections are now recognized with
increasing frequency in neonatal pigs and approaches to diagnosis
and prophylaxis are both different and more complex than those for
type C infections.
[0004] There is a lack of efficacious commercial vaccines for C.
perfringens type A and. C. difficile. Conventional control
strategies for these clostridial enteric disease include fecal
feed-back programs, antibiotics, oregano oil, and probiotics.
Unfortunately, the efficacy of these therapies has been
limited.
[0005] Feeding antibiotics such as bacitracin to pigs has many
drawbacks to feeding antibiotics to livestock, including consumer
acceptance. There is also concern about selection of
antibiotic-resistant bacteria. Antibiotics are also expensive and
have variable effectiveness. In addition, some countries have
banned the feeding of antibiotics to animals. For example, on Jan.
1, 2006, the European Union banned the feeding of all antibiotics
and related drugs to livestock for growth promotion purposes. The
sweeping new policy follows up a 1998 ban on the feeding of
antibiotics that are valuable in human medicine to livestock for
growth promotion. Now, no antibiotics can be used in European
livestock for growth promotion purposes.
[0006] Oregano oil has also been used to combat clostridial disease
in pigs and improve health of swine. Etheric oils from oregano
exert antibacterial effects. However, the use of phytogenic feed
additives is controversial, and the efficacy of this therapy has
been limited.
[0007] Pigs and piglets suffer from scours, that is, diarrhea,
which can be caused by bacteria such as Escherichia colibacillosis
(E. coli) and Clostridium perfringens Types A and C. Scours can
cause death losses and severe production losses, including weight
loss, if left untreated.
[0008] In view of the foregoing, it would be desirable to provide
methods of using one or more Bacillus strains to treat or prevent
Clostridium-based disease in pigs and to enhance performance of
pigs.
SUMMARY OF THE INVENTION
[0009] The invention, which is defined by the claims set out at the
end of this disclosure, is intended to solve at least some of the
problems noted above. Methods are provided. In one embodiment of
the method, an effective amount of at least two Bacillus strains is
administered to a pig. The Bacillus strains are selected from
strains 22CP1 (ATCC PTA-6508), 3AP4 (ATCC PTA-6506), 15AP4 (ATCC
PTA-6507), 2084 (NRRL B-50013), LSSAOI (NRRL B-50104), and 27 (NRRL
B-50105).
[0010] In another embodiment of the method, an effective amount of
Bacillus strains 3AP4 (ATCC PTA-6506) and LSSAOI (NRRL B-50104) is
administered to a pig. The pig can be a gestating pig, a lactating
pig, or any other pig.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Preferred exemplary embodiments described herein are
illustrated in the accompanying drawings, in which like reference
numerals represent like parts throughout and in which:
[0012] FIG. 1 is the dendrogram representing the genetic
relatedness among 30 isolates of C. perfringens recovered from a
first set of swabs.
[0013] FIG. 2 is the dendrogram representing the genetic
relatedness among C. perfringens isolates harvested from a second
set of swabs.
[0014] FIG. 3 is the dendrogram representing the genetic
relatedness among 40 isolates of C. perfringens recovered from a
third set of swabs.
[0015] FIG. 4 is a graph showing clostridia counts (CFU/g of
tissue) from the ileum and distal colon of pigs from control and
DFM supplemented sows (section x treatment interaction: P=0.14,
SE=5.5.times.10.sup.5; a,b Means without a common letter differ,
P>0.05).
[0016] Before explaining embodiments described herein in detail, it
is to be understood that the invention is not limited in its
application to the details of construction and the arrangement of
the components set forth in the following description or
illustrated in the drawings. The invention is capable of other
embodiments or being practiced or carried out in various ways.
Also, it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.
DETAILED DESCRIPTION
[0017] As used herein, "performance" refers to the growth of an
animal, such as a pig, measured by one or more of the following
parameters: average daily gain (ADG), weight, scours, mortality,
feed conversion, which includes both feed:gain and gain:feed, and
feed intake. "An improvement in performance" or "improved
performance" as used herein, means an improvement in at least one
of the parameters listed under the performance definition.
[0018] The present application provides methods of administering an
effective amount of one or more Bacillus strains to a pig. In one
embodiment, the methods improve performance of pigs. Thus, it may
be economical for a swine producer to routinely administer one or
more Bacillus strain, either individually or in combination with
other Bacillus strains, not only to treat and prevent disease, but
also to improve performance.
[0019] In another embodiment administration of one or more Bacillus
strains inhibit pathogenic Clostridium, such as C. perfringens and
C. difficile in pigs. The methods may also be used to reduce or
even prevent clostridial disease in pigs not currently infected
with clostridial pathogens.
[0020] In at least some embodiments of the method, feeding one or
more Bacillus strain to pigs also include the following: a decrease
in percent of scouring litters of pigs, improved 15 d piglet body
weight, improved piglet average daily gain (ADG), and decrease in
sow weight loss.
[0021] Methods of administering one or more Bacillus strain to a
piglet are also provided. Such methods may include feeding the one
or more Bacillus strain to a mother of a piglet. The strain(s) may
be fed during gestation, lactation, or both. The one or more
Bacillus strain may also be fed to nursery pigs and to grow-finish
pigs.
[0022] Bacillus Strains:
[0023] Bacillus strains have many qualities that make them useful
for compositions that are ingested by animals. For example,
Bacillus strains produce extracellular enzymes, such as proteases,
amylases, and cellulase. In addition, Bacillus strains produce
antimicrobial factors, such as gramicidin, subtilin, bacitracin,
and polymyxin. Bacillus strains are also spore formers and thus,
are stable. Additionally, several species of Bacillus have GRAS
status, i.e., they are generally recognized as safe. All B.
subtilis strains are GRAS. The Bacillus strains described herein
are aerobic and facultative sporeformers. Bacillus species are the
only sporefonners that are considered GRAS. Feeding microorganisms
that have GRAS status to livestock is an acceptable practice
amongst producers, veterinarians, and others in the livestock
industry.
[0024] Bacillus strains that can be used in the methods described
herein include the following B. subtilis strains: 22CP1, 3AP4,
15AP4, 2084, LSSAO1, and 27. Other Bacillus strains are included
within the scope described herein. On January 12, 2005, strains
22CPL 3AP4, and 15AP4 were deposited at the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209
and given accession numbers PTA-6508, PTA-6506 and PTA-6507,
respectively. Strains 2084, LSSAOI, and 27 were deposited on Mar.
8, 2007, Jan. 22, 2008, and Jan. 24, 2008, respectively, at the
Agricultural Research Service Culture Collection (NRRL), 1815 North
University Street, Peoria, Illinois, 61604 and given accession
numbers NRRL B-50013, NRRL B-50104, and NRRL B-50105, respectively.
All deposits were made under the provisions of the Budapest Treaty
on the International Recognition of the Deposit of Microorganisms
for the Purposes of Patent Procedure.
[0025] Bacillus strains 22CP1, 3AP4 and 15AP4 were isolated from
different geographical regions of North America and from different
environmental sources. Specifically, strain 22C-P1 was isolated
from a swine lagoon from the eastern United States, strain 3AP4 was
isolated from chicken litter from Canada, and strain 15AP4 was
isolated from turkey litter from the Western United States.
[0026] Bacillus strains described herein can be combined, such as
in the non-limiting examples of combinations of Bacillus strains
shown in Table 1 below and then fed to pigs. A combination can be
determined based on the Clostridium strains present in a specific
production facility or other environment. The combination of
Bacillus strains can be modified if the Clostridium strains change.
In addition, because the strains are also useful in asymptomatic
animals, the combination of Bacillus strains that are fed can be
independent of the Clostridium strains present in a specific
production facility or other environment.
TABLE-US-00001 TABLE 1 Formula Strains Counts and Application Rates
25%, 25%, 3AP4, 2084, 7.5 .times. 10.sup.8 CFU/g of product 25%,
25% 27, LSSAO1 Gestation-1 lb of product per ton of feed
Lactation-5.0 lb of product per ton of feed 25%, 25%, 3AP4, 15AP4,
1.5 .times. 10.sup.8 CFU/g of product 25%, 25% 27, LSSAO1
Gestation-1 lb of product per ton of feed Lactation-5.0 lb of
product per ton of feed 50%, 50% LSSAO1, 3AP4 7.5 .times. 10.sup.8
CFU/g of product Gestation-1 lb of product per ton of feed
Lactation-5.0 lb of product per ton of feed
[0027] Although not intended to be a limitation to the present
disclosure, it is believed that inhibition of Clostridium pathogens
is accomplished by the Bacillus strain(s) via the secretion of an
active metabolite from the Bacillus.
[0028] Preparation of the Bacillus Strains:
[0029] The Bacillus strains are grown in a liquid nutrient broth,
preferably to a level at The counts may be increased or decreased
from this number and still have complete efficacy. CFU or colony
forming unit is the viable cell count of a sample resulting from
standard microbiological plating methods. The term is derived from
the fact that a single cell when plated on appropriate medium will
grow and become a viable colony in the agar medium. Since multiple
cells may give rise to one visible colony, the term colony forming
unit is a more useful unit measurement than cell number.
[0030] The Bacillus strains of the present invention are produced
by fermentation of the bacterial strains. Fermentation is started
by scaling-up a seed culture. In one embodiment, Bacillus cultures
are grown in growth medium, such as TSB or ISA, for 24 to 48 hrs at
32.degree. C. with agitation in a shaking incubator to a final pH
of 7.3.+-.0.2. This involves repeatedly and aseptically
transferring the culture to a larger and larger volume to serve as
the inoculum for the fermentation, which is carried out in large
stainless steel fermentors in medium containing proteins,
carbohydrates, and minerals necessary for optimal growth. A
non-limiting exemplary medium is TSB. After the inoculum is added
to the fermentation vessel, the temperature and agitation are
controlled to allow maximum growth. Once the culture reaches a
maximum population density, the culture is harvested by separating
the cells from the fermentation medium. This is commonly done by
centrifugation.
[0031] The count of the culture can then be determined and is
important when combined with a carrier. At the time of manufacture,
the Bacillus count preferably is at least about 1.0.times.10''
CFU/g.
Use of the Bacillus Strains:
[0032] The Bacillus strains described herein can be used in
direct-fed microbials, that is they can be fed directly to swine.
In one embodiment, one or more Bacillus strains is fed to female
pig breeding stock during gestation and/or during lactation When
one or more Bacillus strains are fed to female pig breeding stock,
the strain(s) is transferred to piglets at least through the
oral-fecal route. In another embodiment, piglets can be fed one or
more Bacillus strains from the day of birth to weaning at about
17-24 days old. This can be done with an oral drench or any other
suitable form of delivering the Bacillus strains. In other
embodiments, nursery pigs are fed one or more Bacillus strains. The
strains can also be fed to grow/finish pigs and to pigs of
different ages.
[0033] Administration of one or more Bacillus strains to animals is
accomplished by any convenient method, including adding the
Bacillus strains to the animals' drinking water, to their feed, or
to the bedding, or by direct oral insertion, such as by an aerosol.
Bacillus strains preferably are administered as spores.
[0034] Bacillus strains described herein may be presented in
various forms, for example as a top dress, liquid drench, gelatin
capsule, or gels. In one embodiment of the top dress form,
freeze-dried Bacillus fermentation product is added to a carrier,
such as whey, maltodextrin, sucrose, dextrose, limestone (calcium
carbonate), rice hulls, yeast culture, dried starch, sodium silico
aluminate. In one embodiment of the liquid drench, freeze-dried
Bacillus fermentation product is added to a carrier, such as whey,
maltodextrin, sucrose, dextrose, dried starch, sodium silico
aluminate, and a liquid is added to form the drench. In one
embodiment of the gelatin capsule form, freeze-dried Bacillus
fermentation product is added to a carrier, such as whey,
maltodextrin, sugar, limestone (calcium carbonate), rice hulls,
yeast culture dried starch, and/or sodium silico aluminate. The
Bacillus and carrier are enclosed in a degradable gelatin capsule.
In a one embodiment of the gels form, freeze-dried Bacillus
fermentation product is added to a carrier, such as vegetable oil,
sucrose, silicon dioxide, polysorbate 80, propylene glycol,
butylated hydroxyanisole, citric acid, and artificial coloring to
form the gel. In all of the examples, multiple carriers can be
used.
[0035] To prepare direct-fed microbials, the cultures and the
carrier can he added to a ribbon or paddle mixer and mixed
preferably for about 15 minutes, although the timing can be
increased or decreased. The components are blended such that a
uniform mixture of the carrier and cultures result. The final
product is preferably a dry, flowable powder.
[0036] The following dosages are for all of the Bacillus strains
that are fed. That is, if a single strain is fed, then that single
strain is used at the listed dosage. If multiple strains are used,
substantially equal amounts of each strain are used for a total
dosage that is listed. For example, where two strains are used,
half of each is used to arrive at the total dosage.
[0037] When fed to female pig breeding stock during gestation, the
one or more Bacillus strains with a total microbial count of
7.5.times.10.sup.8 CFU/g of DFM including one or more strain
blended to this count in a carrier is fed at a rate of 1 lb of the
DFM per ton of feed, to provide 3.75.times.10.sup.5 CFU/g of feed.
When fed during lactation, the one or more Bacillus strains with a
total microbial count of 7.5.times.10.sup.8 CFU/g of product is fed
at a rate of 5 lbs of the DFM per ton of feed, to provide
8.5.times.10.sup.8 CFU/g of feed. In one embodiment, the DFM is top
dressed onto feed. In another embodiment, the DFM is blended into
the complete feed. The DFM can be administered in other ways known
in the art, at other dosages, and at other stages in the pig's
life.
[0038] In at least one embodiment of the liquid drench and gel,
each has about 1.times.10.sup.4 to about 1.times.10.sup.10 CFU/day.
In another embodiment of the liquid drench and gel, each has about
1.times.10.sup.8 CFU/day. In at least one embodiment of the top
dress, basemix, and premix, each includes about. 7.5.times.10.sup.8
CPU/g of top dress, basemix, or premix. This can be added to feed
at 1 lb/ton of feed, resulting in 3.75.times.10.sup.5 CFU/g of
feed. However, other dosages can also be used. In one embodiment of
a dosage for inclusion into water, about 1.times.10.sup.3
CFU/animal/day to about 1.times.10.sup.1.degree. CFU/animal/day is
used. Some embodiments of a dosage for inclusion into water use
about 1.times.10.sup.8 CFU/animal/day. While these examples use
freeze-dried Bacillus, it is not necessary to freeze-dry the
Bacillus before feeding it to animals. For example, spray-dried,
fluidized bed dried, or solid state fermentation Bacillus or
Bacillus in other states may be used. The strains can also be
administered in a wet cell slurry paste, with or without
preservatives, in concentrated, un.con.centrated, or diluted
form.
EXAMPLES
[0039] The following Examples are provided for illustrative
purposes only. The Examples are included herein solely to aid in a
more complete understanding of the presently described invention.
The Examples do not limit the scope described herein described or
claimed herein in any fashion.
Example 1
[0040] An initial pool of Bacillus strains were obtained from
various environmental samples and a library of Bacillus strains.
Bacillus strains were selected that inhibit representative members
from the clusters of Clostridium. For this, tubes were seeded, each
with a representative pathogen from a representative cluster.
Supernatant from a Bacillus strain was added to the seeded tubes
and incubated. After incubation, the optical density (OD) of
control and Bacillus supernatant treated tubes was measured for
each pathogen. Colonies of Bacillus that produced a lowered OD were
then picked, and the Bacillus isolates were grown.
[0041] Clostridium samples were obtained and screened as follows.
Clostridium samples were obtained from swabs taken from farrowing
units. The swabs were taken from piglets, sows, and the
environment. The samples were plated for Clostridium, and colonies
were isolated. DNA was isolated from the colonies, and multiplex
PCR was performed of the a-toxin gene of Clostridium was used to
identify pathogens. To understand the diversity of the Clostridium
pathogens, a comparison of the isolates was performed using
RAPD-PCR. From the RAPD-PCR results, in a dendogram, clusters of
Clostridium were identified.
[0042] Useful Bacillus strains were selected by identifying
representative Clostridium pathogens present in swine production
facilities, such as farrowing units. In general, once Clostridium
pathogens were identified, Bacillus strains were screened to
determine which of them inhibit growth of the identified pathogens.
Bacillus strains that were useful against both C. perfringensand C.
difficile were identified. Specifically, the Bacillus strains were
selected and tested as is described below.
[0043] A total of 30 presumptive C. perfringens isolates were
obtained from the nine rectal swabs. The results of multiplex PCR
identified all 30 of the isolates as C. perfringens type A.
Twenty-two of the isolates also contained the recently identified
.beta.2-toxin which has been correlated with GI disease in domestic
animals. RAPD PCR was performed on the pathogenic isolates to
determine the relatedness among the strains. The results were then
analyzed to construct a dendrogram (shown in FIG. 1), which was
used to select isolates for bacteriocin screening. Isolates
connected to the same branch on the right of the vertical line,
drawn at 80% similarity, were considered to be members of the same
family. Seven families were observed and are marked with arrows on
the left. The arrows on the right signify which isolates were
screened against Bacillus strains described herein.
[0044] The Bacillus strains listed in Table 2 highly significantly
(>90%) inhibited the growth of all six C. perfringens isolates.
These six C. perfringens isolates represent 96.7% of the total
diversity observed. Table 2 below includes the results of screening
performed on six C. perfringens isolates representative of unique
families isolated from the swabs. One family containing only
isolate 1-1, which failed to grow for the assay, was not tested.
All six families tested were significantly inhibited (>50%) by
the Bacillus strains listed in Table 2.
TABLE-US-00002 TABLE 2 Isolates from swabs % Inhibition C.
perfringens Bacillus strains Type A strain 22CP1 15A-P4 3AP4 BS2084
27 LSSA01 1-2 98.2 100 99.1 100 100 97.3 1-3 99.2 99.2 97.7 96.2
100 99.2 2-1 100 96.8 98.9 98.9 100 100 2-3 91.7 98.1 98.1 98.1
99.1 98.1 6-2 97.7 97.7 94.6 98.5 98.5 97.7 9-3 95.9 95.1 96.7 95.9
95.9 96.7
Example 2
[0045] Clostridium samples were obtained and screened as is
described above in Example 1. The Bacillus strains were selected
and tested as is described in this example.
[0046] A total of 194 C. perfringens isolates were obtained from
six sets of swab samples. The results of multiplex PCR identified
189 of the isolates as C. perfringens type A, four isolates as C.
perfringens type E, and one as C. perfringens type C. RAPD PCR was
performed on the pathogenic isolates to determine the relatedness
among the strains. The results were then used to construct a
dendrogram (shown in FIG. 2), which was used to identify unique
families for screening of Bacillus strains. Isolates connected to
the same branch on the right of the vertical line, drawn at 80%
similarity, were considered to be members of the same family. Each
of these families is indicated by an arrow.
[0047] Twenty-one representative isolates were chosen for
bacteriocin screening against the strains of Bacillus shown in
Table 3. The growth of all but two C. perfringens isolates was
significantly (>50%) inhibited by a combination of the six
Bacillus strains. More bacteriocin screening will be performed to
cover all unique families observed to date.
[0048] Table 3 below includes the results of bacteriocin screening
performed on 21 representative isolates of 21 unique families of C.
perfringens. All but two of the isolates were significantly
inhibited (>50%) by the six Bacillus strains shown in Table
3.
TABLE-US-00003 TABLE 3 Isolates from swabs % Inhibition Clostridium
Identity of Bacillus strains strain C. perfringens 22CP1 15A-P4
3AP4 BS2084 27 LSSA01 21-1 Type A 31.1 37.8 60 5.6 0 22.2 28-2 Type
A 0 0 0 9.7 0 3.2 31-1 Type A 17.3 14.7 9.3 6.7 6.7 0 2-2 Type A
98.3 100 33.3 98.3 99.2 99.2 5-3 Type A 98.5 98.5 97.7 100 99.2
99.2 10-2 Type A 98.3 99.2 37.5 99.2 100 98.3 3-2 Type A 98.9 98.9
57.9 100 98.9 98.9 4-2 Type A 99.1 100 86.4 100 99.1 99.1 6-2 Type
A 99.2 99.2 62.5 99.2 99.2 99.2 2-3 Type A 96.9 97.7 96.2 98.5 97.7
97.7 5-2 Type A 97.3 89.1 96.4 98.2 99.1 98.2 8-3 Type A 99.1 98.2
98.2 99.1. 98.2 97.3 8-4 Type A 96.2 91.5 97.2 99.1 97.2 98.1 10-1
Type A 98.4 98.4 98.4 96.8 96.8 96 11-2 Type E 97.7 98.5 98.5 98.5
98.5 98.5 7-1-1 Type E 94.5 96.4 96.4 96.4 95.5 95.5 7-3-3 Type E
42.3 41.5 50.8 75 42.3 73.8 7-4-1 Type A 97.7 98.5 96.9 98.5 98.5
97.7 13-1-2 Type A 97.5 96.7 95.8 99.2 98.3 96.7 13-4-2 Type C 5.9
17.6 0 31.8 0 77.6 13-9-1 Type A 99.1 99.1 98.2 99.1 99.1 99.1
[0049] Isolates in the first three rows were not subjected to heat
treatment, and are likely contaminated which explains their
abnormal resistance to the Bacillus strains of Table 3. All other
swabs from which Clostridium strains shown in Table 3 were obtained
were heat treated to kill any non-spore forming contaminants.
Example 3
[0050] Clostridium samples were obtained and screened as is
described above in Examples 1 and 2. The Bacillus strains were
selected and tested as is described in this example.
[0051] A total of 40 presumptive C. perfringens isolates were
obtained, using Perfringens Agar selective media, from sixteen
rectal swabs. The results of multiplex PCR identified all 40 of the
isolates as C. perfringens type A. Thirty-five of the isolates also
contained the recently identified .beta.2-toxin which has been
correlated with GI disease in domestic animals. RAPD PCR was
performed on the pathogenic isolates to determine the relatedness
among the strains. The results were then analyzed to construct a
dendrogram (shown in FIG. 3), which was used to select isolates for
bacteriocin screening. Isolates connected to the same branch on the
right of the vertical line, drawn at 80% similarity, were
considered to be members of the same family. Nine families were
observed and are marked with arrows. The representative isolates
screened against the Bacillus are listed in Table 4.
[0052] Nine representative isolates were chosen for screening
against bacteriocins produced by the strains of Bacillus listed in
Table 4. The growth of all nine isolates was highly significantly
(>90%) inhibited, and represent 100% of the diversity observed
to date. These results suggest that a Bacillus direct fed microbial
including these strains will be effective for the control of C.
perfringens in this system.
[0053] Table 4 below includes the results of bacteriocin screening
performed on nine C. perfringens isolates representative of unique
families isolated from the swab samples. All nine families tested
were significantly inhibited (>50%) by the Bacillus shown in
Table 4. These nine families cover all of the diversity observed
within this sample set.
TABLE-US-00004 TABLE 4 Clostridium isolates from swabs % Inhibition
Clostridium Identity of Bacillus strains strain C. perfringens 3AP4
27 2-2 Type A 97.1 88.9 2-3 Type A 100 100 4-1 Type A 94.5 97.3 4-3
Type A 94.7 96.8 6-2 Type A 95 97.5 6-3 Type A 98.9 95.8 7-3 Type A
100 100 11-1 Type A 97.8 100 11-2 Type A 100 100
Example 4
[0054] This example describes piglet performance trials and
demonstrates improvements in scour scores and decreased percent
scouring litters with the administration of Bacillus strains 3AP4
and LSSA01. The strains were administered as a dried B. subtilis
fermentation product developed. These strains were specifically
formulated to be effective against clostridial disease in neonatal
pigs. This study was run to determine if these strains may also
provide a performance benefit by decreasing the load of pathogenic
clostridia in a subclinical herd and to determine the efficacy of
these strains for enhancing sow and piglet performance during
lactation in a commercial sow unit. This research evaluated the
performance enhancing benefits of feeding these strains to sows
whose piglets were known to be infected with Clostridium
perfringens type A.
Materials and Methods:
[0055] A total of 121 sows (Genetiporc) and their respective
litters were evaluated in a commercial swine production facility.
At day 72 of gestation (last 6 weeks of gestation), sows were
allotted to three treatments by parity group (1 vs. 2+) and 114 day
farrowing date. Blocks consisted of three sows of the same parity
group. Three treatments were administered to the sows during
gestation and lactation: 1) a negative control (n=44) diet devoid
of BMD and Bacillus strains 3AP4 and LSSA01, 2) a positive control
diet as 1) supplemented with BMD (n=39), and 3) as 1) with Bacillus
strains 3AP4 and LSSA01 supplementation (n=38). Feed-grade
antibiotics (i.e. CTC, Pulmotil, etc.) were not used in the
gestation and lactation feeds during the trial period. Experimental
treatments were top-dressed one time daily to approximate 250 g/ton
BMD and 454 g/ton Bacillus strains 3AP4 and LSSA01. The Bacillus
strains 3AP4 and LSSA.01 top-dress was formulated by combining 10
lb of Bacillus strains 3AP4 and LSSA01 (7.5.times.10.sup.8 cfu/g
product) and 5 lb of cornstarch to provide the equivalent of 1
lb/ton when 1 teaspoon/day was top-dressed, which provided
2.25.times.10.sup.9 cfu/day. BMD was top-dressed beginning two
weeks prior to lactation until weaning, whereas Bacillus strains
3AP4 and LSSA01. was top-dressed beginning six weeks prior to
lactation until weaning.
[0056] Sows were weighed prior to farrowing and at weaning to
determine sow body weight change over the course of the lactation
period. Litters were cross-fostered within treatment groups only
and to a minimum of 9-10 pigs/litter within the first 24 hours
post-farrowing. Litter weights were determined after
standardization and at weaning to calculate piglet average daily
gain (ADG). Scouring litters were recorded to determine the
percentage of litters scouring for each treatment, and scouring
severity was determined for each litter daily based on subjective
visual evaluation using a 0 to 3 scale (0=no scours and 3=heavy
scours).
[0057] Data were analyzed using the PROC MIXED procedure of SAS
with litter considered the experimental unit with the exception of
percentage of scouring litters and scour severity scores which was
analyzed by the Kruskal-Wallis nonparametric test.
Results and Discussion:
[0058] Treatments impacted (P=0.008) the percentage of scouring
litters, such that litters horn to sows supplemented with Bacillus
strains 3AP4 and LSSA01 or BMD had a lower percentage of scouring
litters than negative control sows (Table 5). Furthermore, Bacillus
strains 3AP4 and LSSA01 supplementation tended to result in a lower
(P=0.08) percentage of scouring litters than treatment with BMD.
Treatments affected (P=0.01) severity of scours as indicated by
litter scour scores, such that sows provided Bacillus strains 3AP4
and LSSA01 had the lowest scour severity score of the three
treatments. Furthermore, scour severity tended to be less (P=0.06)
in litters born to sows provided Bacillus strains 3AP4 and LSSA01
compared to sows supplemented with BMD.
[0059] Conclusions:
[0060] These data indicate that BMD and Bacillus strains 3AP4 and
LSSA01 are effective in controlling neonatal piglet scours. In
addition, Bacillus strains 3AP4 and LSSA01 may provide additional
benefit over BMD by decreasing the severity of scouring events.
TABLE-US-00005 TABLE 5 Sow and piglet effects of Bacillus strains
3AP4 and LSSA01 or BMD supplementation to gestating sows prior to
farrowing. Parity, Litter Piglet scours Treatment n n size, n.sup.1
Severity.sup.3 %.sup.4 Bacillus strains 3AP4 38 3.66 11.5 0.11 10.5
and LSSA01 (6-wk) BMD (2-wk) 39 4.03 11.1 0.41 25.6 Negative
control (NC) 44 3.86 10.9 0.57 40.9 Pooled standard error 0.36 0.2
-- -- of the mean (SEM) Significance.sup.2 Treatment 0.779 0.139
0.011 0.008 Bacillus strains 0.960 0.159 -- -- 3AP4 and LSSA01
& BMD vs NC Bacillus strains 3AP4 0.481 0.156 0.064.sup.5
0.088.sup.5 and LSSA01 vs BMD .sup.1Litter size reflects the
average number of pigs/litter following standardization.
.sup.2Levels of significance (P-values) accorded to the main effect
of treatment and a single degree-of-freedom comparison of Bacillus
strains 3AP4 and LSSA01 and BMD supplementation. .sup.3Severity (0
= no scours, 3 = heavy scours) of scouring of pigs in a litter was
analyzed by the Kruskal-Wallis nonparametric test. .sup.4Percentage
of litters exhibiting signs of scouring was analyzed by the
Kruskal-Wallis nonparametric test. .sup.5Bacillus strains 3AP4 and
LSSA01 and BMD treatment data (excluding negative control data)
analyzed by the Kruskal-Wallis nonparametric test.
Example 5
[0061] This example describes research conducted to document that
Bacillus subtilis organisms fed to sows during gestation and
lactation were transferred to suckling piglets. Specifically, this
research identified the fecal-oral transfer of Bacillus spores from
Bacillus strains 3AP4 and LSSA01 into the nursing piglets from
supplemented sows. However, other Bacillus sporeformers are
believed to also would be transferred via the fecal-oral route.
This research also evaluated the effectiveness of these spores
isolated from sow and piglet feces against Clostridium
perfringens.
[0062] This example documented the presence of Bacillus spores from
supplemented sows in their feces and any transfer to their piglets
via the fecal-oral route of transmission. This example also
measured the inhibition of Clostridia by Bacillus organisms
isolated from sow and piglet feces from control- and fed sows fed
strains 3AP4 and LSSA01.
Materials and Methods:
[0063] Two treatments were used: 1) control and 2) strains 3AP4 and
LSSA01 supplemented to the sow 2 weeks prior to and throughout
lactation. Gestation and lactation diets that comprised typical
farm management rations but without feed-grade antibiotics or
microbial products were used as the control basal diets in this
experiment. The strains 3AP4 and LSSA01 treatment was top-dressed
over the gestation ration administered to each sow and was mixed,
bagged and administered to treated sows during the lactation phase.
The strains were formulated to provide 3.75 CFU/g of feed with each
method of application. Nine sows were randomly assigned to the
control treatment and 27 sows represented the treatment with
strains 3AP4 and LSSA01. Fecal samples (.about.100 g) were
collected from 5 control sows and 15 treated sows before
supplementation with strains 3AP4 and LSSA01, on d -1 prior to
farrowing (d 0), and d 14 after farrowing. Samples were also
collected from piglets from each sows litter on d 3, 5, and 14
after farrowing. Fecal samples obtained from the pigs within the
same litter were pooled into a single sample bag (approximately
25-30 grams of fecal material from the litter). Fecal samples were
obtained fresh and if possible were taken directly from the sow or
piglet by rectal stimulation and placed in whirl-pak bags. (Note:
piglet feces were removed from the crate on a daily basis. However,
sow feces was not removed from the crate floor.) Fecal samples were
plated for the determination of the presence of strains 3AP4 and
LSSA01 through visual inspection of colony morphology. Furthermore,
Bacillus present in the fecal samples were isolated and screened
against C. perfringens isolates in vitro to determine if the
inhibitive activity of the Bacillus were maintained after passage
through the gastrointestinal tract. Strains 3AP4 and LSSA01 were
detected in the fecal samples of treated sows on d -1 and d 14 and
in litters from treated sows on d 5 and d 14, documenting the
fecal-oral transfer of strains 3AP4 and LSSA01 from sow to pig.
Laboratory Analyses:
Fecal Sample Assay--Bacillus Plating:
[0064] Eleven grams of the fecal sample was weighed and placed into
a whirlpak bag. Samples were masticated with 99 mL of peptone
(.about.1 dilution) and were spore treated for 10 minutes at
80.degree. C. A -2 dilution was made with 1.1 mL of the sample in a
9.9 mL tube of peptone as well as -3 and -4 dilutions. The -1 to -4
dilutions were pour-plated in duplicate with Tryptic Soy Agar and
incubated at 32.degree. C. for 24 hours. Plates from the treated
pigs were counted by hand for strains 3AP4 and LSSA01 strain
enumeration.
Fecal Sample Assay--Bacteriocin Screening Assay:
[0065] Eleven grams of fecal material was weighed, transferred to a
glass tube containing appropriate media for Bacillus, and incubated
for 24 hours. Following the 24-hour incubation, samples were
centrifuged and the supernatant was collected for use in a
bacteriocin screening assay. The bacteriocin screening was
conducted in a 48 well format, testing inhibition of the
supernatant produced by Bacillus isolated from fecal material of
control and treated sows or pigs against six different strains of
Clostridium perfringens. The plates were incubated at 37.degree. C.
for 24 hours and inhibition of clostridial growth was measured by
absorbance using a plate reader.
Results and Discussion:
Enumeration of Strains 3AP4 and LSSA01 Organisms:
[0066] Bacillus strains 3AP4 and LSSA01 were detected in the fecal
samples of treated sows on lactation day -1 and 14, documenting
that administration of strains 3AP4 and LSSA01 Bacillus in the feed
inoculates the sows' feces ranging from 10.sup.5 to10.sup.6 cfu/g
of feces (Table 6). Strains 3AP4 and LSSA01 organisms could not be
detected in the feces of pigs from treated sows at 3 d of age,
however they were present at 5 and 14 d of age at counts ranging
from 10.sup.3 to 10.sup.4 cfu of strains 3AP4 and LSSA01/g of
piglet feces. Detection of Bacillus strains 3AP4 and LSSA01 in the
feces of sows supplemented with strains 3AP4 and LSSA01 and their
piglets documents the transfer of Bacillus strains 3AP4 and LSSA01
from sow to piglet through the fecal-oral route of
transmission.
Inhibition of Clostridium perfringens by Strains 3AP4 and LSSA01
Organisms:
[0067] Total Bacillus isolated from the feces of sows supplemented
with strains 3AP4 and LSSA01 on d -1 of lactation tended to provide
greater (P=0.07) inhibition of C. perfringens growth than Bacillus
isolated from the feces of control sows (Table 7). Likewise,
Bacillus isolated from the feces of 5 d old pigs from Strains 3AP4
and LSSA01 supplemented sows exhibited greater (P=0.01) inhibition
of C. perfringens growth compared to bacteria isolated from the
feces of 5 d old pigs from control sows.
General Conclusions:
[0068] These data illustrate the presence of strains 3AP4 and
LSSA01 in the feces of supplemented sows and illustrates the
transfer of strains 3AP4 and LSSA01 to piglets from litters of
supplemented sows. Furthermore, total Bacillus growth from the
feces of sows supplemented with strains 3AP4 and LSSA01 and their
piglets exhibited greater inhibition of C. perfringens growth
compared to total Bacillus cultured from the feces of control sows
and their piglets, demonstrating that not only does transfer of
Bacillus strains 3AP4 and LSSA01 occur, but these strains'
effectiveness at inhibiting C. perfringens is maintained through
the transfer to the piglet.
TABLE-US-00006 TABLE 6 Counts of Strains 3AP4 and LSSA01 (CFU/g and
log 10 of feces) from sows and respective litters. Strains 3AP4 and
LSSA01 Lactation supplemented Item Day Control CFU/g log 10 Sow
Jan. 29, 2007 -1 N/D 1.1 .times. 10.sup.6 5.62 Feb. 19, 2007 14 N/D
3.7 .times. 10.sup.5 5.53 Piglets Jan. 31, 2007 3 N/D N/D N/D Feb.
2, 2007 5 N/D 6.0 .times. 10.sup.3 3.11 Feb. 19, 2007 14 N/D 1.5
.times. 10.sup.4 3.97 N/D = Not detectable
TABLE-US-00007 TABLE 7 Percent growth inhibition of six Clostridium
perfringens isolates by strains 3AP4 and LSSA01 isolated from fecal
samples of sows and their respective litters. Lactation Strains
3AP4 and Item Day Control LSSA01 supplemented P value.sup.a Sow
Jan. 29, 2007 -1 38.06 73.56 0.07 Piglets Feb. 2, 2007 5 24.37
93.13 0.01 .sup.aSingle degree of freedom contrasts between control
and sows supplemented with strains 3AP4 and LSSA01
Example 6
[0069] This example shows a decrease in percent scouring litters,
improved 15 d piglet body weight, improved piglet ADG, and decrease
in sow weight loss. Bacillus strains 15AP4 and LSSA01 were used in
this study to determine if these strains reduced scours and
improved piglet growth performance pre-weaning in a commercial sow
herd subclinical for clostridial disease.
[0070] Bacillus strains 15AP4 and LSSA01 were evaluated as a dried
Bacillus subtilis fermentation product that was developed to reduce
the incidence of disease associated with pathogenic Clostridium
perfringens and Clostridium difficile strains. Previous rectal
swabs taken at the research site where this study was conducted
indicate that C. perfringens and C. difficile were present, were
susceptible to the 15AP4 and LSSA01 Bacillus strain treatments, and
may be were contributing to litter mortality, morbidity, and
reduced growth rate pre-weaning although the herd was considered
subclinical for clostridial disease. This research evaluated if the
Bacillus strains 15AP4 and LSSA01 reduced scours and enhanced
pre-weaning piglet performance.
Experimental Procedures:
[0071] Animals: Data were gathered from 71 sows per treatment of
mixed parity and genotype. Sows were randomly allotted to one of
two treatments, either control or DFM topdressed with Bacillus
strains 15AP4 and LSSA01 the last five weeks of gestation and
throughout lactation.
[0072] Treatment Diets: Gestation and lactation diets were devoid
of antibiotics, and no supplemental nutrition (creep feed, milk
replacer, etc.) was provided to piglets during lactation. Topdress
treatments with Bacillus strains 15AP4 and LSSA01 were administered
once each day to provide each sow with 2.25.times.10.sup.9 cfu of
the strain combination each day. Sows were topdressed starting on
d-79 of gestation (5 weeks prior to farrowing) and treatment
continued throughout lactation.
[0073] Litters: Litters were cross-fostered within treatment group
within 24 hours after farrowing to a minimum of 10 and maximum of
12 pigs/litter. Litters were individually weighed at
standardization and 15 days post-standardization.
[0074] Sow Performance and Subsequent Litter Data: Identification
number, genotype, parity, replicate number, farrowing date,
treatment start date, wean date, date of standardization, and date
of litter weight were recorded for each sow on test. Native litter
information for each sow was also recorded, including number born
alive, stillbirths, and mummies. Sow body condition was determined
at standardization and 15 days post-standardization. Body condition
was recorded based on a subjective 3-point scale, with 1=thin,
2-desired condition, and 3=overweight.
[0075] Scour Scores: The percentage of scouring litters was
calculated as the total number of litters identified as
scouring/total number of litters per treatment x 100. Litters were
only considered scouring during the first two days after farrowing
if the scours persisted to d 3 post-farrowing to eliminate any
consideration of loose stools due to "milk scours" that clear up
within the first two days after birth. Each litter was assigned a
subjective scour score daily during lactation defined as follows:
0=no scours present in the litter; 1=less than 50% of the litter
exhibited signs of scours; 2=More than 50% of the litter exhibited
signs of scours.
[0076] Experimental Design: Data were analyzed as a randomized
complete block design with two treatments and sow (or litter) as
the experimental unit using the PROC MIXED procedure of SAS.
Results and Discussion:
[0077] Control sows tended (P=0.08) to have more live pigs born
than sows supplemented with Bacillus strains 15AP4 and LSSA01
(Table 8). Sows allotted to the treatment with Bacillus strains
15AP4 and LSSA01 entered the lactation phase with a slightly lower
body condition score than control sows but had a higher body
condition score 15 d after farrowing, indicating sows supplemented
with Bacillus strains 15AP4 and LSSA01 lost less weight during the
lactation period as evidenced by the significantly lower (P=0.006)
change in body condition score value compared to control sows.
TABLE-US-00008 TABLE 8 The effect of the DFM supplementation
pre-farrowing on litter size and body condition score of sows. Body
Condition Score.sup.1 Parity, Live Still- Mum., Ch. Treatment n n
born, n born, n n d 0 D 15 BCS DFM 71 4.97 10.82 1.65 0.35 1.99
1.96 0.028 Negative control 71 4.80 11.78 1.48 0.34 2.08 1.86 0.225
(NC) Pooled standard 0.17 0.57 0.68 0.11 0.05 0.06 0.058 error of
the mean (SEM) Significance (P 0.47 0.08 0.44 0.91 0.13 0.23 0.006
value) .sup.1Subjective body condition score based on a 3-point
scale: 1 = thin; 2 = good; 3 = overweight.
[0078] Supplementation with Bacillus strains 15AP4 and LSSA01 to
sows resulted in a tendency toward a greater (P=0.07) average
piglet body weight 15 d after farrowing compared to control sows
(Table 9). The improvement in d 15 body weights of pigs born to
sows supplemented with Bacillus strains 15AP4 and LSSA01 was
further supported by a tendency toward greater (P=0.11) ADG
compared to control pigs. The coefficient of variation associated
with individual piglet body weight within a litter tended to be
lower (P=0.06) in litters born to sows supplemented with Bacillus
strains 15AP4 and LSSA01 compared to control sows at birth (d 0),
with this trend remaining evident numerically (P=0.27) 15 d after
farrowing.
TABLE-US-00009 TABLE 9 The effect of DFM on piglet performance and
litter variation. d-0 Pig d-0 Pig d-15 d-15 CV, Pig ADG, Mortality
+ Treatment n wt., lbs CV, % wt., lbs % lbs/d Morbidity DFM 71 3.62
13.34 11.99 16.38 0.54 9.62 Negative control 71 3.55 14.45 11.50
17.27 0.51 9.75 (NC) Pooled standard 0.077 0.409 0.24 0.60 0.015
1.43 error of the mean (SEM) Significance (P 0.18 0.06 0.07 0.27
0.11 0.94 value)
[0079] Supplementation with Bacillus strains 15AP4 and LSSA01
decreased (P=0.02) the percentage of scouring litters from 14% to
2.8%, and litters born to sows provided the DFM had lower (P=0.02)
average scour scores in the first week after birth compared to
control litters (Table 10).
TABLE-US-00010 TABLE 10 The incidence of drug treatment and scour
in litters reared by sows supplemented DFM 5-weeks prior to
farrowing and during a 15-d lactation period. Ave. Litters d 0-7
Litters treated Trts. given scour scouring, for in scouring
Treatment n score.sup.1 %.sup.2 scours, %.sup.3 litters, n.sup.4
DFM 71 0.012 2.81 2.82 11.0 Negative 71 0.069 14.08 7.04 16.4
control (NC) Significance 0.02 0.02 0.25 0.54 (P value)
.sup.1Subjective scour score: 0 = no scours; 1 = less than 50% of
litter showing scours; 2 = More than 50% of the litter showing
signs of scour. .sup.2Calculated as the total number of litters
identified as scouring/total number of litters per treatment.
.sup.3Calculated as the number of litters treated for scours
(Tylan)/total number of litters per treatment .sup.4Average number
of Tylan injections given per litter.
Example 7
Introduction:
[0080] A Bacillus-based direct-fed microbial (DFM) was specifically
developed to aid in the prevention of clostridial-related scours in
neonatal pigs. In vitro analysis of the effects of the DEM strongly
supports its effectiveness against C. perfringens and C. difficile
isolated from scouring piglets. This study was devised to document
sow and litter performance responses and decreases in piglet
intestinal clostridia counts from the supplementation of the DFM to
sows in an asymptomatic herd.
Experimental Procedures:
[0081] A total of 208 mixed parity sows (and some gilts) were used
for the experiment and were fed one of two dietary treatments
during the gestation and lactation periods. One hundred and four
sows were fed standard control diets during gestation and
lactation, and the additional 104 sows were fed the standard diets
supplemented with a DFM including substantially equal CFU counts of
Bacillus strains LSSA01 and 3AP4 at a 1 lb/ton inclusion level of
product containing 7.5.times.10.sup.8 CFU/g of both of the strains
for six weeks prior to and throughout the lactation period.
Treatments were distributed in a randomized complete block design
with sows blocked by parity with the average parity being 1.78 for
DFM sows and 1.82 for control sows. Pulmotil (and other
antibiotics) were excluded from the experimental diets. Lactation
length was targeted at 21 days, but lengths from 18 to 25 days were
deemed acceptable.
[0082] Daily feed intakes are an estimate based on feed drop
information. In a previous experiment actual feed drop weight was
regressed against calibrated feed drop weight and gave the equation
y=0.6181.times..sup.1.2357 R.sup.2=0.9882, where y=actual output
and X=calibrated drop weight Data collected on their litters
included, 1) number born and number born alive, 2) number of
stillborns and mummies, 3) number weaned, 4) average piglet weight
at birth and weaning, 5) ADO during the lactation period, 6) scour
scores, which are presented as a percentage of litters scouring
during the first week of age. Scores range from 0 to 5 with 0 being
normal and 5 being severe. There were no scores above 2 reported
for this study, and 7) pre-weaning mortality.
[0083] From the individual piglets, data collected included 1)
individual piglet body weights on the last two farrowing
groups--litter variation, 2) enumeration of DFM Bacillus in the 01
tract of piglets at 3 d of age, and 3) enumeration of clostridia in
the GI tract of piglets at 3 and 10 d of age. A total of 50 litters
(523 piglets) for DFM sows and 48 litters (523 piglets) for control
sows were used for data analysis.
[0084] One piglet per litter was selected from the litters selected
per treatment for sampling on d 3 of age and d 10 of age from each
farrowing group. On each sampling day, piglets were euthanized by
electrical stunning and exsanguinated for collection of
gastrointestinal tissues for bacterial enumeration. Sampling
occurred in as many of the four farrowing groups as needed to
obtain adequate replication [-15 pigs/treatment for each age (d 3
and d 10)].
[0085] Sampling and Dissection: Following euthanization by
exsanguination, piglet intestinal samples were dissected to obtain
gastrointestinal tissue samples. Specific sections were obtained as
follows: The duodenal section for enumeration of clostridia and
Bacillus counts were ligated at the pyloric junction and 10 cm
distally, dissected and placed in a Whirl-Pak bag with .about.10 mL
of sterile saline. The jejunal sample for clostridia and Bacillus
enumeration was ligated 40 cm distally from the duodenum end and 20
cm distally from the first ligation, dissected and placed in a
Whirl-Pak bag with .about.10 mL of sterile saline. The ileal
section for clostridia and Bacillus enumeration was ligated at the
ileal-cecal junction and 15 cm proximally, dissected, and placed in
a Whirl-Pak bag with .about.10 mL of sterile saline. A section of
the distal colon was obtained by ligating the colon section at its
connection to the spiral colon and 8 cm distally for clostridia and
Bacillus enumeration. The ligated section was dissected and placed
in a Whirl-Pak bag with .about.10 mL of sterile saline. Samples
were transported to the Agtech lab on ice for processing.
[0086] Enumeration of intestinal Clostridia and Bacillus.
Gastrointestinal samples were plated to enumerate DFM Bacillus
strains in 3 d old piglets as well as clostridia counts in both 3
and 10 d old piglets in the small and large intestine. Samples were
dissected on-farm as described in the previous section. Two
dilutions of each intestinal section (duodenum, jejunum, ileum, and
large intestine) were plated in duplicate on Tryptic Soy Agar (TSA)
for Bacillus growth and C. perfringens Agar (CPA) for clostridia
growth. Each sample was rinsed with sterile peptone to remove
intestinal contents, dissected longitudinally, weighed, diluted in
99 niL of peptone and masticated for 60s. After mastication, a 10m1
aliquot was taken from the -1 dilution of each tissue sample and
placed in a sterile test tube. Samples were spore treated in a heat
block at 70.degree. C. for 30 minutes, after which a subsequent -3
dilution was made. The -1 and -3 dilutions were plated onto TSA for
the enumeration of Bacillus colonies and onto CPA to determine
clostridial counts. The cells from the -1 dilution were pelleted by
centrifugation, resuspended in 10m1 of TSB +10% glycerol, divided
into two aliquots in 15 mL conical tubes, and frozen for subsequent
analysis.
Results and Discussion:
[0087] Daily feed intake did not differ when comparing control and
DFM supplemented sows (Table 11). DFM supplemented sows farrowed
more (P>0.05) total pigs and pigs born alive than control sows,
although this response is likely a reflection of conception rate
that occurred before DFM treatment. Sows supplemented with DFM:
weaned more pigs (P=0.06) than control sows and, although not
significant (P=0.12), this response was reflected in the lower
percentage of piglet mortality with DFM supplementation.
TABLE-US-00011 TABLE 11 Performance of Sows Supplemented with DFM
in Late Gestation and Lactation.sup.1,2,3 Daily feed intake,
Wean-to-Estrus Treatment lbs.sup.4 Interval, d.sup.5 DFM 12.22 5.90
Control 11.99 5.60 SEM 0.37 0.60 Level of significance Dietary 0.54
0.55 treatment .sup.1The average parity was 1.78 for DFM sows and
1.82 for control sows. .sup.2Feed intake: n = 99 for DFM sows and n
= 101 for control sows; Wean-to-estrus interval: n = 91 for DFM
sows and n = 95 for control sows. .sup.3Outliers defined as any
data point plus or minus three standard deviations from the mean.
.sup.4Average feed intake over lactation period. Daily feed intake
is an estimate based on feed drop information (calculator
determined in JBS United experiment 07-S025). .sup.5There was not
effect f lactation length on wean-to-estrus interval.
[0088] Piglet performance determined by weighing pigs individually
from a subset of 50 litters from DFM supplemented sows and 48
litters from control sows is displayed in Table 12. No differences
in piglet performance (weaning weights, ADG, litter size, and
mortality) were observed between treatments when analyzing the data
from this subset of litters. However, when piglet performance was
evaluated based on litter and encompassing the entire dataset from
the trial (103 DFM litters and 102 control litters) litters nursing
DFM supplemented sows had greater (P=0.02) weaning weights compared
to control litters (Table 13). Although initial litter weight was
also greater (P=0.01) for litters nursing DFM supplemented sows,
the improvement in litter weaning weight was at least partially due
to the tendency toward improvement (P=0.09) in litter ADG of DFM
litters compared to controls.
TABLE-US-00012 TABLE 12 Native Litter Performance of Pigs Nursing
Sows Supplemented with DFM in Late Gestation and Lactation.
.sup.1,2,3 Treatment Total born Born alive Stillborn Mummies DFM
13.27 12.15 1.03 0.28 Control 12.39 11.09 1.21 0.14 SEM 0.43 0.37
0.17 0.09 Level of significance Dietary 0.04 <0.01 0.27 0.11
treatment .sup.1 The average parity was 1.78 for DFM sows and 1.82
for control sows. .sup.2Total born: n = 104 for DFM and control
sows; Born alive and mummies: n = 103 for DFM and control sows;
Stillborn: n = 101 for DFM and control sows; .sup.3Outliers defined
as any data point plus or minus three standard deviations from the
mean.
[0089] The high percentage of scouring litters was surprising as
the health status of the herd at the time of the trial was
considered to be relatively healthy (Table 13). More surprising was
the numerically higher (P=0.64) percentage of DFM litters scouring
compared to controls, as data from previous trials have
consistently reported decreases in the percentage of scouring
litters with administration of DFM. Scour severity was also scored
on a scale of 0 (no scours) to 5 (severe scours), and none of the
litters on test scored above a scour severity score of 2,
suggesting these scours were likely transient "milk" scours and not
indicative of a major health challenge.
TABLE-US-00013 TABLE 13 Standardized Litter Performance (Expressed
Per Piglet and Per Litter) of Sows Supplemented with DFM in Late
Gestation and Lactation .sup.1, 2, 3 Per Piglet Per Litter Initial
Weaning Initial Weaning weight, weight, Gain, ADG, Standardized
weight, weight, Gain, ADG, Wean lbs lbs lbs lbs Litter Size lbs lbs
lbs lbs % Scours.sup.4 litter size % mortality DFM 3.39 13.82 10.44
0.53 10.99 37.14 136.27 99.33 5.01 31.44 9.86 10.41 Control 3.09
13.43 10.41 0.52 10.95 33.97 128.76 94.91 4.74 28.66 9.56 12.76 SEM
0.12 0.25 0.25 0.01 0.06 1.28 3.15 3.21 0.16 5.86 0.16 1.54 Level
of 0.01 0.11 0.88 0.56 0.54 0.01 0.02 0.17 0.09 0.64 0.06 0.12
significance Dietary treatment .sup.1 Outliers defined as any data
point plus or minus three standard deviations from the mean. .sup.2
Lactation length ranged from 18 to 25 days. .sup.3 Growth
performance per piglet n = 101 for DFM litters and n = 100 for
control litters; Growth performance per litter: n = 103 for DFM
pigs and n = 102 for control pigs; Scour scores: n = 104 for DFM
and control pigs; Standardized litter size: n = 99 for DFM pigs and
n = 98 for control pigs; Mortality: n = 99 for DFM and control
pigs. .sup.4Percent of litters scouring at least once during wk 1
of age; n = 52 litters for DFM piglets, n = 48 litters for control
piglets. Scours were scored on a scale from 0 to 5 with 0 being no
scours and 5 being severe scours. There were no observations above
a 2 reported for this study.
[0090] Bacillus organisms from the DFM were not detected in the
intestinal tract of piglets sampled at 3 days of age. Intestinal
clostridia counts from a combination of ileal and distal colon
samples obtained from 3 day old piglets were reduced in piglets
nursing DFM supplemented sows compared to control sows
(2.9.times.10.sup.5 vs 1.0.times.10.sup.6.+-.4.6.times.10.sup.5,
P=0.07). Although DFM supplementation did not impact clostridia
counts in the ileum of piglets at 3 days of age, clostridia counts
were reduced (P>0.05) in the distal colon of pigs nursing DFM
supplemented sows compared to control pigs (section x treatment
interaction, P=0.14; FIG. 4). Although clostridia counts in the
intestinal tract of 10 d old pigs nursing DFM supplemented sows
were numerically lower compared to control pigs, there was no
statistical difference between treatments (1.5.times.10.sup.5 vs.
3.3.times.10.sup.5+1.6.times.10.sup.5, P=0.27).
[0091] In summary, this study demonstrates the benefits of DFM
supplementation to sows during gestation and lactation on number of
pigs weaned and piglet weight gain in a herd considered to have a
subclinical clostridia challenge. Additionally, the DFM decreased
the clostridial load in the gastrointestinal tract of 3 day old
piglets by specifically decreasing the clostridial load in the
large intestine. These data support previous findings in which DFM
improved sow and piglet performance in herds without clinical
clostridia challenges.
Example 8
Objective:
[0092] To determine the efficacy of a direct-fed microbial (DFM)
for enhancing sow and piglet performance during lactation in a
commercial sow unit considered subclinical for clostridia
scours.
Materials and Methods:
[0093] Sows (Genetiporc) and their respective litters were
evaluated in a 1200-sow commercial swine production facility. At
day 72 of gestation (last 6 weeks of gestation), sows were allotted
to three treatments by parity group (1 vs. 2+) and 114 day
farrowing date. Blocks consisted of three sows of the same parity
group. Three treatments were administered to the sows during
gestation and lactation: 1) a negative control diet devoid of BMD
and a DFM (described below), 2) a positive control diet consisting
of the negative control supplemented with BMD, and 3) the negative
control diet with DFM supplementation. Feed-grade antibiotics
(e.g., CTC, Pulmotil) were not used in the gestation and lactation
feeds during the trial period. Also, litters were not treated with
oral or injectable antibiotics for the duration of the trial unless
there were severe health challenges. If treatments occurred, all
individual pig treatments were recorded. Sows were vaccinated two
weeks prior to farrowing and treatments were top-dressed one time
daily to approximate 250 g/ton BMD and 454 g/ton DFM (Table 14).
The DFM included substantially equal CFU counts of Bacillus strains
LSSA01 and 3AP4. The DFM top-dress was formulated by combining 10
lb of DFM (7.5.times.10.sup.8 cfu/g product) and 5 lb of cornstarch
to provide the equivalent of 1 lb/ton when 1 teaspoon/day was
top-dressed, which provided 2.25.times.10.sup.9 cfu/day. BMD was
top-dressed beginning two weeks prior to lactation until weaning,
whereas the DFM was top-dressed beginning six weeks prior to
lactation until weaning. To avoid cross-contamination in gestation,
sows were grouped together by treatment. A "non-test" sow was
placed between treatment groups. Any residual feed from sows that
were "off-feed" was removed from the trough prior to watering to
prevent contamination.
TABLE-US-00014 TABLE 14 Summary of treatments administered,
vaccinations, and top-dress procedures performed during the
experiment. Treatments Vaccination.sup.1 BMD.sup.2 DFM.sup.3
Negative Yes No No Control Positive Yes 1 scoop/day BMD, No Control
last 2 weeks of (BMD) gestation through weaning DFM Yes No 1
teaspoon/day, last 6 Top-Dress weeks of gestation through weaning
.sup.1Vaccination included Beacon Scour Block (E-coli-4 +
Clostridium perfringens type C) distributed by Newport
Laboratories. Gilts were vaccinated 4 and 2 weeks prior to
farrowing. Sows were vaccinated 2 weeks prior to farrowing only.
.sup.2Each scoop was equivalent to 250 g/ton of BMD. .sup.3Each
teaspoon contained 3 g of DFM and provided 2.25 .times. 10.sup.9
cfu/day. This was equivalent to 454 g of the DFM/ton of feed.
[0094] Sows were weighed prior to farrowing and at weaning to
determine sow body weight change over the course of the lactation
period. Litters were cross-fostered within treatment groups only
and to a minimum of 9-10 pigs/litter within the first 24 hours
post-farrowing. Scouring litters were recorded to determine the
percentage of litters scouring for each treatment, and scouring
severity was determined for each litter daily based on visual
evaluation using a 0 to 3 scale (0=no scours and 3=heavy scours).
The following measurements were recorded for each litter:
[0095] Measurements: The following measurements were taken: 1. date
farrowed, 2. parity, 3. sow body weight (pre-farrow and weaning),
4. no. pigs at standardization and at weaning, 5. pig removals and
reason for removal, 7. individual pig treatments, and 8. scouring
litters.
Results and Discussion:
[0096] Average parity of sows during the second trial was between 4
and 5 for all three treatment groups (Table 15). Although there was
no significant treatment effect (P=0.60), DFM supplemented sows
averaged numerically fewer pigs born alive than control sows or
those supplemented with BMD. DFM and BMD supplemented sows had
lighter (P=0.02) prefarrowing body weights than negative control
sows, whereas there was no difference in body weight between the
treatments when sows were weighed 16 d into the lactation period.
As a result, DFM and BMD supplemented to sows reduced (P=0.08) body
weight loss during lactation compared to control sows. Sow weight
loss during lactation was also evaluated with prefarrow body weight
and live born pigs as covariates in the analysis, and while this
altered degrees of significance it did not change interpretation of
the data. Percentage of scouring litters and scour severity are not
reported, as only one litter was observed to be scouring during the
time this trial was conducted.
TABLE-US-00015 TABLE 15 Effects of DFM or BMD supplementation to
gestating sows prior to farrowing. d-16 Prefarrow lactation Sow Sow
Sow Live weight, sow weight, Weight weight weight Treatment N
Parity Born lbs lbs loss, lbs loss, lbs loss, lbs DFM (6-wk) 49
4.88 10.80 544.41 509.04 35.27 36.06 36.57 BMD (2-wk) 49 4.40 11.45
543.52 496.38 45.12 46.04 44.60 Negative control 52 4.79 11.20
569.23 521.36 50.35 48.53 50.25 (NC) Covariate -- Prefarrow Live
sow Born weight Pooled standard 0.41 0.56 13.30 12.14 6.96 6.70
6.62 error of the mean (SEM) Significance.sup.1 Treatment 0.65 0.60
0.08 0.19 0.08 0.16 0.12 DFM vs BMD 0.39 0.32 0.95 0.37 0.18 0.17
0.26 DFM & BMD vs 0.73 0.88 0.02 0.11 0.08 0.21 0.09 NC
.sup.1Levels of significance (P-values) accorded to the main effect
of treatment and a single degree-of-freedom comparison of DFM and
BMD supplementation.
[0097] The lack of scouring litters in this second trial documents
the varying health status of the herd at the time the two trials
were conducted. The herd had just gone through a PRRS break prior
to the start of the first trial, whereas the herd was PRRS stable
at the time the second trial was conducted.
Conclusion:
[0098] These data indicate that BMD and DFM decreased sow weight
loss during lactation when herd health status was good and when
clinical signs of clostridial scours were not present. This trial
and the results of the previous trial described in Example 4
conducted during health challenges (PRRS and scours) indicate a
beneficial effect of DFM supplementation regardless of herd health
status.
Example 9
Objective:
[0099] To determine the optimal level of supplementation with a
direct-fed microbial (DFM) in nursery pigs that results in enhanced
growth performance.
Materials and Methods:
[0100] There were five (5) dietary treatments with twelve (12)
replicates per treatment and eight pigs per pen. Pigs were allotted
by weight and sex. Sex ratio was equal within each replicate. A
basal diet was prepared for each nursery phase. The direct-fed
microbial (DFM) included strains 3AP4 and LSSA01 in substantially
equal amounts of CFUs. The DFM contained 3.75.times.10.sup.8 CPU/g
of each strain for a total of 7.5.times.10.sup.8 CFU/g of DFM
product. Additional ingredients in the DFM product that served as
carrier included calcium carbonate and rice hulls. Diets typical of
those used in the commercial swine industry were fed during each
nursery phase. Five premixes labeled A through. E were prepared for
each treatment and were used throughout the experiment to prepare
each treatment diet. Treatment diets defined below were formulated
by blending the appropriate premix into the basal diet at the
expense of corn.
[0101] The treatments were as follows: control, control diet +0.50
lb of product/ton of feed, control diet +1.00 lb of product/ton of
feed, control diet +2.00 lb of product/ton of feed, control diet
+4.00 lb of product/ton of feed. The duration of the treatments was
6 weeks, and there were four phases: Phase 1 (6 to 8 d), Phase 2 (1
wk), Phase 3 (1 wk), and Phase 4 (3 wk).
[0102] The following measurements were taken: feed samples at time
of manufacture, individual pig weights for allotment and weekly pen
weights thereafter, feed intakes weekly, mortality and morbidity,
medicinal treatments, scour scores, and feed samples collected out
of feeders. Scour scores were recorded using the following scale:
1=no scours, 2=any looseness evident in pen, and 3=considerable
looseness in pen.
[0103] EXCENEL.RTM. antibiotic (Pharmacia & Upjohn Co., North
Peacock, N.J.) was administered on the day of weaning. Mecadox.RTM.
antibiotic (Phibro Animal Health, Ridgefield Park, N.J.) was
supplemented in the diet during the nursery phases.
Results and Discussion:
[0104] Supplementation of DFM to nursery diets resulted in a
quadratic (P<0.03) average daily gain (ADG) response during
weeks 3 and 5 of the nursery period (Table 14). This improvement in
ADG translated to a quadratic increase (P<0.05) in pig body
weight at the end of weeks 3, 5, and 6, and pig body weight tended
(P<=0.08) to increase linearly during week 4 with DFM
supplementation. Average daily feed intake increased quadratically
(P<0.02) during week 3, with the greatest feed intake response
observed when DFM was supplemented at 0.5 lb/ton. Although the
greatest feed intake during week 5 was observed when pigs were fed
diets supplemented with 0.5 lb/ton of DFM, the only treatment that
differed from the control was a decrease (P<0.05) in feed intake
when pigs were supplemented with DFM at the 4 lb/ton inclusion
level.
[0105] Contrasts comparing the various levels of DFM
supplementation that were tested in this study indicated that DFM
supplementation at 0.5 and 1 lb/ton of feed resulted in greater
(P<0.05) ADG, average daily feed intake (AM) and pig body weight
in week 3 when compared to the average responses when DFM was
supplemented at higher levels (Table 14). During weeks 4 and 5,
supplementation with DFM at 0.5 lb/ton resulted in greater
(P.ltoreq.0.05) ADFI and pig body weight compared to the average of
pigs fed DFM supplemented at higher levels, whereas the same was
true for ADO and pig body weight when DFM was supplemented at 1
lb/ton in week 5 (P<0.05). Comparisons of DFM supplementation of
2 lb/ton verses 4 lb/ton tended (P.ltoreq.0.10) to indicate general
decreases in growth performance within weeks 2, 3, 5, and 6 when
DFM was supplemented at 4 lb/ton.
[0106] Cumulatively over the entire nursery trial, ADG and pig body
weight increased quadratically (P<0.05) with increasing DFM
supplementation, such that supplementation. with 0.5 lb/ton yielded
the greatest response (Table 15). Average daily feed intake
increased linearly (P=0.05) as DFM supplementation increased with
the greatest ADFI occurring in pigs supplemented with DFM at 0.5
lb/ton. Contrasts comparing levels of DFM supplementation in the
overall trial indicated ADG and ADFI were greater (P<0.05) when
pigs were fed DFM at 0.5 lb/ton compared to DFM supplementation at
higher levels. Scour scores were generally low in this study,
indicating minimal enteric challenge, and were not impacted by DFM
supplementation (Table 15).
[0107] In conclusion, DFM supplementation during the nursery period
improved growth performance in the middle and later nursery phases,
as well as over the cumulative trial. These improvements were
evidenced when DFM was supplemented at the 0.5 and 1.0 lb/ton
inclusion level. Specifically, DFM supplemented at 0.5 or 1.0
lb/ton improved ADG as a result of increased ADFI, and culminated
in an approximately 1 lb heavier pig at the end of the nursery
period.
TABLE-US-00016 TABLE 14 Efficacy of graded levels of DFM on weekly
growth performance in nursery pigs .sup.1 Pooled standard error of
0.5 1.0 the Contrast P-values lbs/ton lbs/ton Treatment 1 2 3 4 5
mean Diet P- vs. vs. 2.0 lbs/ton vs. DFM (lbs/ton) 0 0.5 1.0 2.0
4.0 (SEM) value Linear Quadratic higher higher 4.0 lbs/ton Week 1
Start wt. (lbs) 15.37 15.38 15.44 15.42 15.30 0.515 0.412 0.516
0.096 1.00 0.236 0.118 End wt. (lbs) 16.05 16.03 15.94 16.07 15.69
0.584 0.649 0.280 0.502 0.562 0.797 0.186 ADG (lb) 0.10 0.09 0.07
0.09 0.06 0.026 0.813 0.375 0.806 0.596 0.949 0.338 ADFI (lb) 0.19
0.18 0.17 0.19 0.16 0.016 0.553 0.431 0.902 0.988 0.604 0.187 F/G
(lb/lb) 1.90 2.00 2.43 2.11 2.67 0.870 0.325 0.359 0.665 0.295
0.209 0.363 Week 2 End wt. (lbs) 21.07 21.16 20.91 21.27 20.51
0.753 0.443 0.298 0.340 0.454 0.955 0.084 ADG (lb) 0.72 0.74 0.73
0.74 0.69 0.036 0.541 0.384 0.204 0.432 0.650 0.137 ADFI (lb) 0.74
0.75 0.74 0.76 0.70 0.035 0.362 0.240 0.210 0.443 0.687 0.064 F/G
(lb/lb) 1.03 1.03 1.02 1.03 1.03 0.027 0.995 0.967 0.712 1.00 0.717
0.981 Week 3 End wt. (lbs) 27.53.sup.ab 28.18.sup.a 27.88.sup.a
27.43.sup.ab 26.61.sup.b 0.909 0.037 0.025 0.023 0.039 0.051 0.109
ADG (lb) 0.902.sup.ab 0.968.sup.a 0.949.sup.a 0.858.sup.b
0.858.sup.b 0.054 0.004 0.014 0.028 0.007 0.004 1.000 ADFI (lb)
1.15.sup.ac 1.23.sup.b 1.18.sup.ab 1.12.sup.ac 1.08.sup.c 0.060
0.003 0.004 0.015 0.001 0.016 0.302 F/G (lb/lb) 1.29 1.27 1.24 1.32
1.27 0.031 0.418 0.899 0.751 0.804 0.150 0.196 Week 4 End wt. (lbs)
36.0.sup.ab 37.031.sup.b 36.27.sup.ab 35.64.sup.ab 35.00.sup.b
1.138 0.216 0.081 0.160 0.059 0.202 0.450 ADG (lb) 1.03 1.11 1.05
1.01 1.05 0.049 0.412 0.679 0.630 0.103 0.680 0.438 ADFI (lb) 1.50
1.60 1.53 1.46 1.51 0.070 0.192 0.336 0.524 0.044 0.331 0.347 F/G
(lb/lb) 1.47 1.45 1.46 1.45 1.44 0.026 0.932 0.506 0.993 0.885
0.623 0.777 Week 5 End wt. (lbs) 45.07.sup.ab 46.28.sup.a
45.80.sup.a 44.73.sup.ab 43.19.sup.b 1.334 0.039 0.022 0.026 0.049
0.038 0.128 ADG (lb) 1.30.sup.a 1.33.sup.a 1.33.sup.a 1.28.sup.a
1.18.sup.b 0.041 0.025 0.014 0.020 0.132 0.019 0.053 ADFI (lb)
1.91.sup.ab 1.97.sup.a 1.90.sup.ab 1.84.sup.bc 1.78.sup.c 0.054
0.034 0.006 0.166 0.012 0.091 0.324 F/G (lb/lb) 1.49 1.49 1.44 1.45
1.52 0.026 0.197 0.795 0.047 0.506 0.160 0.065 Week 6 End wt. (lbs)
55.56.sup.ab 56.69.sup.a 56.40.sup.a 55.66.sup.ab 53.57.sup.b 1.60
0.134 0.082 0.044 0.170 0.106 0.102 ADG (lb) 1.50 1.51 1.51 1.53
1.44 0.055 0.670 0.517 0.267 0.809 0.616 0.159 ADFI (lb) 2.24 2.31
2.29 2.24 2.18 0.082 0.651 0.342 0.242 0.350 0.285 0.536 F/G
(lb/lb) 1.49 1.53 1.51 1.47 1.52 0.020 0.281 0.812 0.964 0.213
0.424 0.109 .sup.1 There were 12 replicates pens per dietary
treatment. .sup.a-cMeans with different superscripts within a row
are significantly different (P < 0.05)
TABLE-US-00017 TABLE 15 Efficacy of graded levels of DFM on
cumulative growth performance and scour scores in nursery
pigs.sup.1, 2, 3 Pooled standard error of 0.5 1.0 the Contrast
P-values lbs/ton lbs/ton Treatment 1 2 3 4 5 mean Diet P- vs. vs.
2.0 lbs/ton vs. DFM (lbs/ton) 0 0.5 1.0 2.0 4.0 (SEM) value Linear
Quadratic higher higher 4.0 lbs/ton Cumulative Start wt. (lbs)
15.37 15.38 15.44 15.42 15.30 0.515 0.412 0.516 0.096 1.00 0.236
0.118 End wt. (lbs) 55.56.sup.ab 56.69.sup.a 56.40.sup.a
55.66.sup.ab 53.57.sup.b 1.60 0.134 0.082 0.044 0.170 0.106 0.102
ADG (lb) 0.91.sup.ab 0.95.sup.a 0.93.sup.a 0.91.sup.ab 0.88.sup.b
0.033 0.094 0.065 0.043 0.047 0.132 0.171 ADFI (lb) 1.27.sup.ab
1.33.sup.a 1.29.sup.ab 1.26.sup.ab 1.23.sup.b 0.046 0.097 0.050
0.113 0.019 0.186 0.442 F/G (lb/lb) 1.40 1.40 1.38 1.38 1.41 0.009
0.174 0.790 0.066 0.283 0.382 0.050 Scour Scores Week 1 score 1.36
1.39 1.51 1.62 1.40 0.104 0.352 0.328 0.193 0.290 1.000 0.137
Overall score 1.14 1.11 1.18 1.14 1.13 0.030 0.530 0.921 0.665
0.192 0.244 0.856 .sup.1There were 12 replicates pens per dietary
treatment. .sup.2Scour scores can were recorded as 1 = no scours; 2
= moderate looseness; 3 = considerable looseness. .sup.3Pigs were
fed dietary treatments from weaning to 42 d post-wean.
.sup.a-bMeans with different superscripts within a row are
significantly different (P < 0.05).
[0108] It is understood that the various preferred embodiments are
shown and described above to illustrate different possible features
described herein and the varying ways in which these features may
be combined. Apart from combining the different features of the
above embodiments in varying ways, other modifications are also
considered to be within the scope described herein. The invention
is not intended to be limited to the preferred embodiments
described above, but rather is intended to be limited only by the
claims set out below. Thus, the invention encompasses all alternate
embodiments that fall literally or equivalently within the scope of
these claims.
* * * * *