U.S. patent application number 12/629497 was filed with the patent office on 2010-07-08 for strains and methods for improving ruminant health and/or performance.
This patent application is currently assigned to DANISCO A/S. Invention is credited to Keith J. Mertz, Thomas G. Rehberger.
Application Number | 20100172873 12/629497 |
Document ID | / |
Family ID | 42233590 |
Filed Date | 2010-07-08 |
United States Patent
Application |
20100172873 |
Kind Code |
A1 |
Mertz; Keith J. ; et
al. |
July 8, 2010 |
STRAINS AND METHODS FOR IMPROVING RUMINANT HEALTH AND/OR
PERFORMANCE
Abstract
Described are strains including Enterococcus faecium strain 8G-1
(NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172),
Bacillus pumilus strain 8G-134 (NRRL B-50174) and strains having
all of the identifying characteristics of each of these strains.
One or more of the strains can be used to treat or prevent
acidosis. They can also be used to improve other measures of
ruminant health and/or performance. Methods of using the strains,
alone and in combination, are described. Methods of making the
strains are also provided.
Inventors: |
Mertz; Keith J.; (Neosho,
WI) ; Rehberger; Thomas G.; (Wauwatosa, WI) |
Correspondence
Address: |
WHYTE HIRSCHBOECK DUDEK S.C.;INTELLECTUAL PROPERTY DEPARTMENT
33 East Main Street, Suite 300
Madison
WI
53703-4655
US
|
Assignee: |
DANISCO A/S
Copenhagen
DK
LALLEMAND, INC.
Rexdale
CA
|
Family ID: |
42233590 |
Appl. No.: |
12/629497 |
Filed: |
December 2, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61119256 |
Dec 2, 2008 |
|
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|
Current U.S.
Class: |
424/93.3 ;
424/93.44; 424/93.46; 435/252.4; 435/252.5; 435/253.4 |
Current CPC
Class: |
C12R 1/46 20130101; A61K
35/744 20130101; A23K 10/16 20160501; A61P 39/02 20180101; A61K
35/742 20130101; C12R 1/07 20130101; A61P 1/00 20180101; A23K 10/18
20160501; A23K 50/10 20160501; A61K 35/742 20130101; A61K 2300/00
20130101; A61K 35/744 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/93.3 ;
435/252.5; 435/253.4; 435/252.4; 424/93.44; 424/93.46 |
International
Class: |
A61K 35/74 20060101
A61K035/74; C12N 1/20 20060101 C12N001/20 |
Claims
1. An isolated strain selected from the group consisting of
Enterococcus faecium strain 8G-1 (NRRL B-50173), a strain having
all of the identifying characteristics of Enterococcus faecium
strain 8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL
B-50172), a strain having all of the identifying characteristics of
Enterococcus faecium strain 8G-73 (NRRL B-50172), Bacillus pumilus
strain 8G-134 (NRRL B-50174), a strain having all of the
identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL
B-50174), and combinations thereof.
2. The strain of claim 1, wherein the strain is Enterococcus
faecium strain 8G-1 (NRRL B-50173).
3. The strain of claim 1, wherein the strain is a strain having all
of the identifying characteristics of Enterococcus faecium strain
8G-1 (NRRL B-50173).
4. The strain of claim 1, wherein the strain is Enterococcus
faecium strain 8G-73 (NRRL B-50172).
5. The strain of claim 1, wherein the strain is a strain having all
of the identifying characteristics of Enterococcus faecium strain
8G-73 (NRRL B-50172).
6. The strain of claim 1, wherein the strain is Bacillus pumilus
strain 8G-134 (NRRL B-50174).
7. The strain of claim 1, wherein the strain is a strain having all
of the identifying characteristics of Bacillus pumilus strain
8G-134 (NRRL B-50174).
8. A combination comprising: an isolated strain selected from the
group consisting of Enterococcus faecium strain 8G-1 (NRRL
B-50173), a strain having all of the identifying characteristics of
Enterococcus faecium strain 8G-1 (NRRL B-50173), Enterococcus
faecium strain 8G-73 (NRRL B-50172), a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-73
(NRRL B-50172), Bacillus pumilus strain 8G-134 (NRRL B-50174), a
strain having all of the identifying characteristics of Bacillus
pumilus strain 8G-134 (NRRL B-50174), and combinations thereof; and
monensin.
9. A method comprising administering to an animal an effective
amount of a strain selected from the group consisting of a strain
selected from the group consisting of Enterococcus faecium strain
8G-1 (NRRL B-50173), a strain having all of the identifying
characteristics of Enterococcus faecium strain 8G-1 (NRRL B-50173),
Enterococcus faecium strain 8G-73 (NRRL B-50172), a strain having
all of the identifying characteristics of Enterococcus faecium
strain 8G-73 (NRRL B-50172), Bacillus pumilus strain 8G-134 (NRRL
B-50174), a strain having all of the identifying characteristics of
Bacillus pumilus strain 8G-134 (NRRL B-50174), and combinations
thereof.
10. The method of claim 9, wherein upon administration to the
animal, the strain provides at least one of the following benefits
in or to the animal when compared to an animal not administered the
strain: (a) reduces acidosis, (b) stabilizes ruminal metabolism as
indicated by delayed lactic acid accumulation and prolonged
production of volatile fatty acids, (c) recovers more quickly from
acidosis challenge as measured by pH recovery and lactic acid
decline, and (d) reduces exhibition of clinical signs associated
with acidosis, (e) increases milk production, (f) increases milk
fat content, (g) decreases somatic cell count (SCC), (h) improves
immunological response and health as evidenced by decreased SCC,
and (i) increases efficiency of milk production.
11. The method of claim 9, wherein the animal is a ruminant.
12. The method of claim 9, wherein the animal is a bull, steer,
heifer, cow or calf.
13. The method of claim 9, wherein the strain is Enterococcus
faecium strain 8G-1 (NRRL B-50173), Enterococcus faecium strain
8G-73 (NRRL B-50172), or combinations thereof, and wherein the
strain is administered to the animal at a level such that the
animal is dosed daily with about 5.times.10.sup.8 CFU/animal/day to
about 5.times.10.sup.10 CFU/animal/day.
14. The method of claim 9, wherein the strain is Bacillus pumilus
strain 8G-134 (NRRL B-50174), and wherein the strain is
administered to the animal at a level such that the animal are
dosed daily with about 5.times.10.sup.8 CFU/animal/day to about
5.times.10.sup.10 CFU/animal/day.
15. The method of claim 11, wherein the strain is administered to
the animal starting from about 30 days of age.
16. The method of claim 9, wherein the animal is a beef animal.
17. The method of claim 9, wherein the animal is a dairy cow.
18. The method of claim 17, wherein upon administration to the
dairy cow, the strain provides at least one of the following
benefits in or to the dairy cow when compared to a dairy cow not
administered the strain: (a) the strain increases daily fat yield
and milk fat percent in the dairy cow administered the strain, (b)
the strain lowers the log Somatic cell count (LogSCC) scores in the
dairy cow administered the strain, (c) improves immunological
response and health as evidenced by decreased LogSCC, (d) increases
efficiency of milk production in dairy cow administered the strain,
and (a) when the dairy cows are second parity or greater cows, the
strain increases milk production of milk in the dairy cow
administered the strain.
19. The method of claim 18, wherein the increase in milk production
in the dairy cow administered the strain is achieved with
substantially no increase in dry matter intake in the dairy cow
administered the strain.
20. The method of claim 9, wherein the strain is Enterococcus
faecium strain 8G-1 (NRRL B-50173) or a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-1
(NRRL B-50173).
21. The method of claim 9, wherein the strain is Enterococcus
faecium strain 8G-73 (NRRL B-50172) or a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-73
(NRRL B-50172).
22. The method of claim 9, wherein the strain is Bacillus pumilus
strain 8G-134 (NRRL B-50174) or a strain having all of the
identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL
B-50174).
23. A method comprising administering to an animal a combination
comprising: an effective amount of a strain selected from the group
consisting of Enterococcus faecium strain 8G-1 (NRRL B-50173), a
strain having all of the identifying characteristics of
Enterococcus faecium strain 8G-1 (NRRL B-50173), Enterococcus
faecium strain 8G-73 (NRRL B-50172), a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-73
(NRRL B-50172), Bacillus pumilus strain 8G-134 (NRRL B-50174), a
strain having all of the identifying characteristics of Bacillus
pumilus strain 8G-134 (NRRL B-50174), and combinations thereof; and
monensin.
24. A method of making a direct-fed microbial, the method
comprising: (a) growing in a liquid nutrient broth a strain
selected from the group consisting of Enterococcus faecium strain
8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL
B-50172), Bacillus pumilus strain 8G-134 (NRRL B-50174), a strain
having all of the identifying characteristics of Enterococcus
faecium strain 8G-1 (NRRL B-50173), a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-73
(NRRL B-50172), and a strain having all of the identifying
characteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174);
and (b) separating the strain from the liquid nutrient broth to
make the direct-fed microbial.
25. The method of claim 24, further comprising freeze drying the
strain.
26. The method of claim 24, wherein the strain is Enterococcus
faecium strain 8G-1 (NRRL B-50173) or a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-1
(NRRL B-50173).
27. The method of claim 24, wherein the strain is Enterococcus
faecium strain 8G-73 (NRRL B-50172) or a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-73
(NRRL B-50172).
28. The method of claim 24, wherein the strain is Bacillus pumilus
strain 8G-134 (NRRL B-50174) or a strain having all of the
identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL
B-50174).
29. A method of making a direct-fed microbial, the method
comprising: (a) growing in a liquid nutrient broth a strain
selected from the group consisting of Enterococcus faecium strain
8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL
B-50172), Bacillus pumilus strain 8G-134 (NRRL B-50174), a strain
having all of the identifying characteristics of Enterococcus
faecium strain 8G-1 (NRRL B-50173), a strain having all of the
identifying characteristics of Enterococcus faecium strain 8G-73
(NRRL B-50172), and a strain having all of the identifying
characteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174);
(b) separating the strain from the liquid nutrient broth to make
the direct-fed microbial; and (c) adding monensin to the direct-fed
microbial.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Patent Application No. 61/119,256,
filed Dec. 2, 2008, the entirety of which is incorporated by
reference herein.
BIBLIOGRAPHY
[0002] Complete bibliographic citations of the references referred
to herein by the first author's last name and year of publication
can be found in the Bibliography section, immediately preceding the
claims.
FIELD OF THE INVENTION
[0003] The invention relates to strains and methods for controlling
acidosis. More particularly, the invention relates to bacterial
strains useful for improving ruminant health and/or performance and
methods of making and using the strains.
DESCRIPTION OF THE RELATED ART
[0004] The feeding of high concentrations of fermentable
carbohydrate to ruminants has become a common practice in the beef
and dairy cattle industry over the last 50 years. The need for
improving the production efficiency and quality of meat has led to
this trend. Improvements in production have not occurred without
certain difficulties. Increasing the ruminant consumption of
fermentable carbohydrate by feeding higher levels of cereal grains
has resulted in increased incidence of metabolic disorders such as
acidosis. The relationship between high concentrate consumption and
ruminal acidosis has been well documented in reviews (Dunlop, 1972;
Slyter, 1976). Many researchers have shown a decline in ruminal pH
following the feeding of high levels of readily fermentable
carbohydrate (RFC) to cattle and the subsequent disruption of
ruminal microbiota and physiological changes occurring in the
animal (Allison, et. al., 1975, Hungate et. al., 1952; Elam, 1976).
Most have attributed this decline to an over production of organic
acids by ruminal bacteria such as Streptococcus bovis. However, the
effect of excessive carbohydrate on the ruminal microbiota that
initiates this response has not been well documented.
[0005] In the past, intensive management of feeding has been the
only method to combat acidosis. More specifically, grains are
diluted with roughage and the increase in dietary concentrate
percentage is carefully controlled in a step-wise method to ensure
smooth transition to high levels of concentrate over a 14-21 day
period. Most commercial feedlots formulate and deliver several
"adaptation" diets that contain different ratios of grain to
forage.
[0006] Although intensive feeding management is usually quite
effective in controlling acidosis, it is very costly to the
producer due to the high cost of producing, transporting, chopping
forage, disposing of increased animal waste, and lower production
efficiencies. Producers and feedlot managers need to implement
strategies that will allow for efficient production of livestock
fed high concentrate rations.
[0007] Other strategies have been to combine the use of adaptation
diets with feeding antimicrobial components such as ionophores.
Ionophores inhibit intake and reduce the production of lactic acid
in the rumen by reducing the ruminal populations of gram-positive,
lactic acid-producing organisms such as Streptococcus bovis and
Lactobacillus spp. (Muir et al. 1981).
[0008] Although the usage of ionophores have reduced the incidence
of acute acidosis in feedlots, consumer concern about the use of
antibiotics in meat production and the need for feedlot managers to
continually find ways to reduce costs while improving animal
performance and carcass composition has lead to the examination of
alternative methods to reduce acidosis and improve feedlot cattle
performance.
[0009] The use of direct-fed microbials as a method to modulate
ruminal function and improve cattle performance has been gaining
increased acceptance over the past 10 years. There are two basic
direct-fed microbial technologies that are currently available to
the beef industry for the control of ruminal acidosis: (1) using
lactic acid producing DFM technology and (2) adding specific
bacterial species capable of utilizing ruminal lactic acid. While
the reported mode of action of each of these technologies is
different, they both attempt to address the accumulation of ruminal
lactic acid.
[0010] The first approach, i.e., using lactic acid producing DFM
technology, attempts to increase the rate of ruminal lactic acid
utilization by stimulating the native ruminal microbiota. As
reported, the addition of relatively slow growing lactic acid
producing bacteria, such as species of Enterococcus, produces a
slightly elevated concentration of ruminal lactic acid. The gradual
increase forces the adaptation of the ruminal microflora to a
higher portion of acid tolerant lactic acid utilizers. However,
these Enterococcus strains failed to adequately control and prevent
acidosis.
[0011] The second approach, i.e., adding specific bacterial species
capable of utilizing ruminal lactic acid, is based on the finding
that species of Propionibacterium significantly minimize the
accumulation of ruminal lactic acid during an acidosis challenge
with a large amount of Readily Fermentable Carbohydrate (RFC).
Propionibacterium are natural inhabitants of the rumen in both
dairy and beef cattle and function in the rumen by using lactic
acid to produce important volatile fatty acids like acetate and
propionate.
[0012] Current DFM technologies developed to date have been
developed based upon an antiquated microbiological understanding of
the incidence of acidosis in the rumen. Until recently, methods of
studying the microbial ecology of the rumen have relied on
cultivation techniques. These techniques have been limited due to
unknown growth requirements and unsuitable anaerobic conditions for
many of the rumen microorganisms. Thus, ecological studies relying
on these cultivation techniques have been based on a limited
understanding of the rumen microbiota.
[0013] Current DFMs when used alone or with yeast to minimize the
risk of ruminal acidosis and to improve utilization of a feedlot
cattle diet containing high concentrate provide mixed results.
However, a study of DFM strains Propionibacterium P15, and
Enterococcus faecium EF212, and E. faecium EF212, fed alone or fed
combined with a yeast, Saccharomyces cerevisiae, indicated that
addition of DFM combined with or without yeast had no effect on
preventing ruminal acidosis (Yang, W., 2004).
[0014] In view of the foregoing, it would be desirable to provide
one or more strains to prevent and/or treat acidosis. It would be
advantageous if the one or more strains also improved other
measures of ruminant health and/or performance. It would also be
desirable to provide methods of making and using the strains.
SUMMARY OF THE INVENTION
[0015] 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. Isolated strains are provided, including
Enterococcus faecium strain 8G-1 (NRRL B-50173), a strain having
all of the identifying characteristics of Enterococcus faecium
strain 8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL
B-50172), a strain having all of the identifying characteristics of
Enterococcus faecium strain 8G-73 (NRRL B-50172), Bacillus pumilus
strain 8G-134 (NRRL B-50174), a strain having all of the
identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL
B-50174), and combinations thereof.
[0016] Additionally provided is a combination including one or more
of the strains listed above and monensin.
[0017] Also provided is a method of administering an effective
amount of one or more of the strains listed above to an animal and
a method of administering a combination including an effective
amount of one or more of the strains listed above and monensin to
an animal.
[0018] In at least some embodiments, the administration of the one
or more strain to the animal provides at least one of the following
benefits in or to the animal when compared to an animal not
administered the strain: (a) reduces acidosis, (b) stabilizes
ruminal metabolism as indicated by delayed lactic acid accumulation
and prolonged production of volatile fatty acids, (c) recovers more
quickly from acidosis challenge as measured by pH recovery and
lactic acid decline, (d) reduces exhibition of clinical signs
associated with acidosis (e) increased milk production in lactating
dairy cows, (f) increased milk fat content in lactating dairy cows,
(g) decreased somatic cell count (SCC) in lactating dairy cows, (h)
improved immunological response and health as evidenced by
decreased SCC and (i) increased efficiency of milk production in
lactating dairy cows.
[0019] Also provided is a method of making a direct-fed microbial.
In the method, a strain selected from the group consisting of
Enterococcus faecium strain 8G-1 (NRRL B-50173), Enterococcus
faecium strain 8G-73 (NRRL B-50172), and Bacillus pumilus strain
8G-134 (NRRL B-50174) is grown in a liquid nutrient broth. The
strain is separated from the liquid nutrient broth to make the
direct-fed microbial. In at least some embodiments of the method,
the strain is freeze dried.
[0020] Additionally provided is a method of making a direct-fed
microbial. In the method, a strain selected from the group
consisting of Enterococcus faecium strain 8G-1 (NRRL B-50173),
Enterococcus faecium strain 8G-73 (NRRL B-50172), and Bacillus
pumilus strain 8G-134 (NRRL B-50174) is grown in a liquid nutrient
broth. The strain is separated from the liquid nutrient broth to
make the direct-fed microbial. Monensin is added to the direct-fed
microbial.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Preferred exemplary embodiments described herein are
illustrated in the accompanying drawings, in which like reference
numerals represent like parts throughout and in which:
[0022] FIG. 1 is a graph showing pH differences between tester
(non-acidotic; Cluster 2) and driver (acidotic; Cluster 1)
populations.
[0023] FIG. 2 is a graph showing lactic acid accumulation
differences between tester (non-acidotic; Cluster 2) and driver
(acidotic; Cluster 1) populations.
[0024] FIG. 3 is a graph showing in vitro glucose by treatment over
time.
[0025] FIG. 4 is a graph showing in vitro lactic acid accumulation
by treatment over time.
[0026] FIG. 5 is a graph showing total VFA
(acetate+propionate+butyrate) accumulation over time.
[0027] FIG. 6 is a graph showing mean ruminal pH over time in
control and candidate DFM cattle.
[0028] FIG. 7 is a graph showing mean ruminal lactate over time in
control and candidate DFM cattle.
[0029] FIG. 8 is a graph showing ruminal VFA concentrations over
time treatment. (Total VFA=acetate+propionate+butyrate).
[0030] 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
[0031] Provided herein are strains. Methods of making and using the
strains are also provided.
[0032] In at least some embodiments, a direct-fed microbial (DFM)
made with one or more of the strains provided herein allows beef
and dairy producers to continue managing feeding regimens to
optimize growth and performance without sacrificing health due to
digestive upset associated with ruminal acidosis. At least some
embodiments of the DFMs were selected on the basis of managing
ruminal lactate concentrations via lactate utilization or priming
the rumen to maintain lactate utilizing microflora. At least some
embodiments of the DFMs were developed to manage ruminal energy
concentrations. Unlike the current DFMs marketed to cattle
producers to alleviate acidosis, at least some of the embodiments
of the invention were not developed to manage a problem after it
occurs, but rather to alleviate the problem before it happens.
Strains:
[0033] The strains provided herein include Enterococcus faecium
strain 8G-1, Enterococcus faecium strain 8G-73, and Bacillus
pumilus strain 8G-134, which are also referred to herein as 8G-1,
8G-73, and 8G-134, respectively.
[0034] Strains Enterococcus faecium strain 8G-1, Enterococcus
faecium strain 8G-73, and Bacillus pumilus strain 8G-134 were
deposited on Aug. 29, 2008 at the Agricultural Research Service
Culture Collection (NRRL), 1815 North University Street, Peoria,
Ill., 61604 and given accession numbers B-50173, B-50172, and
B-50174, respectively. The 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. One
or more strain provided herein can be used as a direct-fed
microbial (DFM).
[0035] For purposes of this disclosure, a "biologically pure
strain" means a strain containing no other bacterial strains in
quantities sufficient to interfere with replication of the strain
or to be detectable by normal bacteriological techniques.
"Isolated" when used in connection with the organisms and cultures
described herein includes not only a biologically pure strain, but
also any culture of organisms which is grown or maintained other
than as it is found in nature. In some embodiments, the strains are
mutants, variants, or derivatives of strains 8G-1, 8G-73, or 8G-134
that also provide benefits comparable to that provided by 8G-1,
8G-73, and 8G-134. In some embodiments, the strains are strains
having all of the identifying characteristics of strains 8G-1,
8G-73, or 8G-134. Further, each individual strain (8G-1, 8G-73, or
8G-134) or any combination of these strains can also provide one or
more of the benefits described herein. It will also be clear that
addition of other microbial strains, carriers, additives, enzymes,
yeast, or the like will also provide control of acidosis and will
not constitute a substantially different DFM.
[0036] Bacillus strains have many qualities that make them useful
as DFMs. For example, several Bacillus species also have GRAS
status, i.e., they are generally recognized as safe by the US Food
and Drug Administration and are also approved for use in animal
feed by the Association of American Feed Control Officials (AAFCO).
The Bacillus strains described herein are aerobic and facultative
sporeformers and thus, are stable. Bacillus species are the only
sporeformers that are considered GRAS. A Bacillus strain found to
prevent or treat acidosis is Bacillus pumilus strain 8G-134.
[0037] Enterococcus strains also have many qualities that make them
useful as DFMs. Enterococcus strains are known to inhabit the
gastrointestinal tract of monogastrics and ruminants and would be
suited to survive in this environment. Enterococcus have been shown
to be facultatively anaerobic organisms, making them stable and
active under both aerobic and anoxic conditions. Enterococcus
faecium strain 8G-1 and Enterococcus faecium strain 8G-73 were
identified by the inventors as being useful for these purposes.
Preparation of the Strains:
[0038] In at least one embodiment, each one of the strains
described herein is cultured individually using conventional liquid
or solid fermentation techniques. In at least one embodiment, the
Bacillus strain and Enterococcus strains are grown in a liquid
nutrient broth, in the case of the Bacillus, to a level at which
the highest number of spores are formed. The Bacillus strain is
produced by fermenting the bacterial strain, which can be started
by scaling-up a seed culture. This involves repeatedly and
aseptically transferring the culture to a larger and larger volume
to serve as the inoculum for the fermentation, which can be carried
out in large stainless steel fermentors in medium containing
proteins, carbohydrates, and minerals necessary for optimal growth.
Non-limiting exemplary media are MRS or TSB. However, other media
can also be used. After the inoculum is added to the fermentation
vessel, the temperature and agitation are controlled to allow
maximum growth. In one embodiment, the strains are grown at
32.degree. to 37.degree. under agitation. 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.
[0039] In one embodiment, to prepare the Bacillus strain, the
Bacillus strain is fermented to a 5.times.10.sup.8 CFU/ml to about
4.times.10.sup.9 CFU/ml level. In at least one embodiment, a level
of 2.times.10.sup.9 CFU/ml is used. The bacteria are harvested by
centrifugation, and the supernatant is removed. The pelleted
bacteria can then be used to produce a DFM. In at least come
embodiments, the pelleted bacteria are freeze-dried and then used
to form a DFM. However, it is not necessary to freeze-dry the
Bacillus before using them. The strains can also be used with or
without preservatives, and in concentrated, unconcentrated, or
diluted form.
[0040] The count of the culture can then be determined. 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.
Using the Strains:
[0041] In at least some embodiments, one or more strain is used to
form a DFM. One or more carriers, including, but not limited to,
sucrose, maltodextrin, limestone, and rice hulls, can be added to
the strain.
[0042] To mix the strain(s) and carriers (where used), they can be
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 cultures
and carriers result. The final product is preferably a dry,
flowable powder, and may be formulated based upon the desired final
DFM concentration in the end product.
[0043] In at least one embodiment of a method of making a DFM, a
strain described herein is grown in a medium, such as a liquid
nutrient broth. The strain is separated from the liquid nutrient
broth to make the direct-fed microbial. The strain can be freeze
dried after it is separated from the broth.
[0044] One or more of Enterococcus faecium strain 8G-1,
Enterococcus faecium strain 8G-73, and Bacillus pumilus strain
8G-134 can be fed to animals to reduce or even eliminate the
occurrence of acidosis. For this, an effective amount of one or
more of these strains is administered to the animals. Upon
administration to the animals, the strain(s) provides at least one
of the following benefits in or to the animals: (a) reduces
acidosis in the animals, (b) stabilizes ruminal metabolism as
indicated by delayed lactic acid accumulation and prolonged
production of volatile fatty acids, (c) recovers more quickly from
acidosis challenge as measured by pH recovery and lactic acid
decline, and (d) does not exhibit clinical signs associated with
acidosis.
[0045] The animals can be cattle, including both beef cattle and
dairy cattle, that is, one or more bull, steer, heifer, calf, or
cow; goats; sheep; llamas; alpacas; other four-compartment
stomached, and ruminant animals that may encounter ruminal
imbalance when fed readily fermentable carbohydrate (RFC).
[0046] In at least one embodiment, when Enterococcus faecium strain
8G-1 or Enterococcus faecium strain 8G-73 is fed, the strain is
administered to the animals at a level such that the animals are
dosed daily with about 5.times.10.sup.8 CFU/animal/day to about
5.times.10.sup.10 CFU/animal/day. In at least one embodiment, when
Bacillus pumilus strain 8G-134 is fed, the strain is administered
to the animals at a level such that the animals are dosed daily
with about 5.times.10.sup.8 CFU/animal/day to about
5.times.10.sup.10 CFU/animal/day. In at least one embodiment, two
or more strains of Enterococcus faecium strain 8G-1, Enterococcus
faecium strain 8G-73 and Bacillus pumilus strain 8G-134 are fed,
and the strains are administered to the animals at a level such
that the animals are dosed daily with about 5.times.10.sup.8
CFU/animal/day to about 5.times.10.sup.10 CFU/animal/day as the
total dose of the combined strains. Other levels of one or more
strains can be fed to the animals.
[0047] The strain can be administered to the animals from about 30
days of age through the remainder of the adult ruminant productive
life or for other time periods.
[0048] In at least one embodiment, the strain is fed as a
direct-fed microbial (DFM), and the DFM is used as a top dressing
on a daily ration. In addition, the strain can be fed in a total
mixed ration, pelleted feedstuff, mixed in with liquid feed, mixed
in a protein premix, delivered via a vitamin and mineral
premix.
[0049] In at least one embodiment, the strain is fed as a DFM, and
the DFM is fed in combination with Type A Medicated Article
monensin (Rumensin.RTM.), with a daily dose about 50 mg to 660 mg
per head Monensin is fed to increase feed efficiency. Monensin, as
an ionophore, creates permeability in bacterial cell membrane
creating an ion imbalance between the intracellular and
extracellular spaces. This response affects ruminal microbiota
populations and influence feedstuff fermentation to improve
livestock feed efficiency.
[0050] In at least one embodiment, the strain is fed as a DFM, and
the DFM is fed in combination with Type A Medicated Article tylosin
phosphate (Tylan.RTM.), with a daily dose of about 60 to 90
mg/head. Tylosin phosphate is fed to beef cattle to reduce liver
abscesses caused by Fusobacterium necrophorum and Actinomyces
pyogenes.
Examples
[0051] 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
Acidosis Model Experimental Design:
[0052] Ten crossbred steers were blocked by weight and assigned to
two pens. The daily feed ration for all treatment groups prior to
challenge consisted of 45% roughage and 55% concentrate on a dry
matter basis. Cattle were fed 15 lbs/head/day of the ration once in
the morning and had remaining feed pushed closer to the feeding
stanchion late in the afternoon. Both pens were fasted for 24 hours
before challenge with the concentrate diet treatments. Concentrate
diet treatments consisted of highly fermentable carbohydrate
sources of steam flaked corn on a 90% as fed basis. After fasting
for 24 hours, the concentrate diet was fed ad libitum at 100
lbs/pen to all pens (0 h). Challenge diet consumption was visually
monitored and additional feed added on an as needed basis.
[0053] Rumen fluid samples were obtained from individual animals
via oral intubation using a collection tube attached to a vacuum
flask. Different flasks and collection tubes were used for each pen
to minimize cross contamination of microbiota between treatments.
Ruminal fluid collected in the vacuum flasks was decanted into
sterile 50 ml Falcon tubes labeled with sample time and animal
identification number (ear tag number). Ruminal samples were
collected from all pens at -36 h, -24 h, and -12 h. Time -36 h and
-24 h samples represented the physiological baseline for each
animal. Time -12 h samples represented rumen fluid in the fasted
state for each animal. Time 0 h was designated as the beginning of
the feeding challenge. Ruminal samples were collected from all
animals every 4 hours from +6 to +22 hours. All pens were sampled
at +28, +36, and +48 hours. The pH from individual ruminal samples
were analyzed immediately after acquisition. All samples were
frozen and prepared for shipment to Agtech Products, Inc.
(Waukesha, Wis.) for further analysis.
[0054] Volatile fatty acids and carbohydrate concentrations were
measured in individual ruminal samples. Samples were prepared for
HPLC analysis by aseptically removing duplicate 1.0 ml samples from
the rumen fluid collected from each animal at each time period.
Samples were placed in a 1.5 ml microcentrifuge tube and the debris
was pelleted by centrifugation (10 minutes, at 12,500 rpm). The
supernatant fluid (750 .mu.l) was transferred to a clean tube and
acidified with an equal volume of 5 mM H.sub.2SO.sub.4. The
acidified fluid was thoroughly mixed and filtered through 0.2 .mu.m
filter directly into a 2 ml HPLC autosampler vial and capped.
Samples were analyzed using a Waters 2690 HPLC system (Waters Inc.,
Milford, Mass.). The sample were injected into 5 mM H.sub.2SO.sub.4
mobile phase heated to 65.degree. C. and separated using a BioRad
HPX-87H Column (Bio-Rad Laboratories, Inc., Hercules, Calif.). The
HPLC was standardized using a set of concentrations for each
compound of interest. Compounds used as standards were include
dextrose (glucose), lactate, methylglyoxal, butyrate, propionate,
and acetate.
Discovery of Bacterial Genes in Non-Acidotic Cattle Ruminal
Microflora:
[0055] Suppressive Subtractive Hybridization:
[0056] The Genome Subtraction Kit (Clontech, Palo Alto, Calif.) was
utilized to determine microbial population differences between two
sets of pooled ruminal samples. Hierarchal clustering analysis was
performed to determine similarities and differences between animals
based on pH and lactic acid profiles over time. Cluster analysis
positioned cattle 2069, 2071, 2078, 2113, and 2127 in Cluster 1 and
cattle 2107, 2115, 2088, 2133, and 2124 in Cluster 2. Repeated
measures analysis was performed to compare pH and lactic acid from
Cluster 1 to Cluster 2. All variables were analyzed separately.
Cluster 1 had a significantly higher mean lactic acid profile than
Cluster 2 (P=0.0004) accompanied with lower mean pH (P=0.0075)
throughout the course of the challenge diet period (FIGS. 1 and 2).
The rumen fluid from individual animal was pooled within cluster
for suppressive subtractive hybridization (SSH) procedures.
[0057] Suppressive subtraction hybridization (SSH) strategies were
developed to compare pooled ruminal DNA samples from cattle in
Cluster 1 to those in Cluster 2 at sample times +6 h, +10 h, +14 h,
and +18 h. Suppressive subtraction hybridization was performed
utilizing Cluster 2 as the tester (non-acidotic cattle) and Cluster
1 as the driver (acidotic cattle). The SSH was hypothesized to
result in unique DNA fragments from organisms that resulted in
lower levels of lactic acid and a higher pH (ruminal energy
modulating organism). By performing subtractions using samples from
time +10 h, the DNA fragments (genes) found, were from organisms
that were able to modulate the utilization of excess energy in the
ruminal environment in the form of RFC and alleviate potential
effects of acidosis.
[0058] Cloning and Screening of Unique Tester Sequence Library:
[0059] Strain specific DNA sequences that are recovered after
subtraction were cloned for further analysis. DNA sequences were
inserted into the pCR2.1 vector (Invitrogen) and transformed into
E. coli chemically competent TOP10 cells. The transformation
mixture was plated onto 22.times.22 cm LB agar plates containing 50
.mu.g/ml kanamycin and overlaid with 40 mg/ml X-gal in DMF. Plates
were incubated at 37.degree. C. for 24 h. Recombinant colonies
(white colonies) were picked into sterile microtitre plates
containing LB medium and kanamycin at 50 .mu.g/ml. All wells
containing recombinant PCR products were separated into 1 ml
aliquots. One aliquot was purified using the Qiaquick PCR
Purification Kit (Qiagen), with the second aliquot pelleted via
centrifugation, resuspended in LB+Kan+10% glycerol and stored at
-80.degree. C.
Southern Hybridization:
[0060] Slot-blot hybridizations were conducted using standard
protocols. To confirm the specificity of the cloned DNA inserts,
positively charged Zeta-Probe.RTM. Blotting Membranes (Bio-Rad
Laboratories; Hercules, Calif.) were hybridized with probes made
from the original tester and driver DNA digested with Alu I and
labeled with the DIG High Prime DNA labeling kit (Roche Diagnostics
Corporation, Indianapolis, Ind.). Recombinant inserts showing
sequence homology to the tester DNA but not the driver DNA was
selected for sequence analysis. Hybridizations were conducted on
cloned inserts. At each time period, subtraction was performed, SSH
6, 10, 14, and 18. From SSH 6, 10, 14, and 18, there were 12, 29,
105, and 29 cloned inserts, respectively, that were tester
specific.
[0061] The DNA sequence from each tester positive insert was
determined (Lark Technologies; Houston, Tex.). Sequence from each
insert was compared with sequences from the NCBI database using the
blastX function. Nucleotide sequences were translated and gene
function was deduced by comparing sequences to those found in the
NCBI database using the blastX function. Gene function was placed
in a gene category using the Clusters of Orthologous Groups (COG)
web site. Specific COG genes identified were used to construct
oligonucleotide probes for colony hybridization and slot-blot
hybridization experiments. Four genes of the twenty-nine were
selected from SSH 10 to be utilized for colony hybridization based
upon functional attributes based on selection from non-acidotic
cattle. The genes were selected from clones 79, 84, 94, and 110
were identified via using the NCBI blastX function with assigned
functions: beta-xylosidase, glucose/galactose transporter,
4-alpha-glucanotransferase, and 4-alpha-glucanotransferase,
respectively. All genes selected for colony hybridization had
assigned properties as identified by COG as Carbohydrate and
Transport Metabolism function, which would have provided bacteria
containing these genes an advantage at metabolizing excess energy
such as that found in the rumen when challenged with RFC.
Colony Hybridization:
[0062] Rumen fluid collected during the acidosis trial from cattle
at times +10 h, +14 h, and +18 h was utilized. Cattle 2107, 2124,
2115, 2088, and 2133 were selected from each of these time periods.
These cattle are representative of animals that were previously
selected for the "tester population" or non-acidotic group.
Individual rumen samples were taken from -20.degree. C. and allowed
to thaw at room temperature. Thawed rumen samples were individually
plated on three separate mediums in duplicate. Media utilized
consisted of sodium lactate agar (NLA), Lactate Propionibacterium
Selective Agar (LPSA), and modified reinforced Clostridial media
(RCS). The RCS was prepared similar to commercially available
reinforced Clostridial media sans glucose. Thus, the major
carbohydrate source in RCS is starch. Table 1 below indicates the
incubation conditions and dilutions of rumen fluid plated on each
media.
TABLE-US-00001 TABLE 1 Incubation conditions and dilutions plated
on each media. Incubation Conditions Incubation Dilutions Media O2
Conditions Incubation Time Temperature Plated LPSA Anaerobic 7 Days
32.degree. C. 10-1, 10-2 NLA Anaerobic 5 Days 37.degree. C. 10-2,
10-3 RCS Anaerobic 48 Hours 37.degree. C. 10-1, 10-2
[0063] After incubation, individual colonies were picked off of
each plate and inoculated into 10 ml broth tubes consisting of the
respective media, except LPSA, which was inoculated into NLB.
Colonies were selected from each time period and each animal (five
cattle.times.three time periods). For the RCS media, five colonies
were picked for each animal-time period. The LPSA exhibited less
colonies and diversity on the plates and number of colonies
selected per animal-time period was variable. Two colonies per
animal-time period were selected from the NLA media, except animal
2107 at time period 18. Six colonies were picked from this
animal-time period due to increased visible diversity. Not all
inoculated tubes exhibited growth after incubation.
[0064] Tubes showing growth were separated into two separate
aliquots of 9 ml and 1 ml. The 1 ml aliquot was utilized for DNA
isolation procedures utilizing the High Pure PCR Template
Preparation Kit (Roche Molecular Biochemicals; Mannheim, Germany).
The 9 ml aliquot was transferred to a sterile 15 ml Falcon Tube and
centrifuged until a solid pellet was formed. The pellet was then
reconstituted in NLB or RCS broths containing 10% glycerol. The
reconstituted sample was placed in the -80.degree. C. for future
use. The extracted DNA was then used for RAPD-PCR analysis of
individual isolates to determine phylogenic relationships. Analysis
was performed using Bio-Numerics (Applied Maths Inc., Austin, Tex.)
on the RAPD DNA banding patterns to determine the relatedness of
the isolates. The similarity coefficient of isolates was determined
using the Dice coefficient and an un-weighted pair group method
(UPGMA). A similarity of 80% or greater was used to group the 109
isolates into 65 separate clusters. Of the 65 clusters, 23 grew
only on RCS, 11 grew only on LPSA, 14 grew only on NLA, 4 clusters
grew both on RCS and LPSA, 6 grew on both RCS and NLA, 3 grew on
both LPSA and NLA, and 4 clusters were found to be present on all
three media.
[0065] Slot blot hybridizations were prepared utilizing the Bio-Dot
SF Microfiltration Apparatus (BIO-RAD; Hercules, Calif.). The
genomic DNA of a single isolate within a cluster was selected to
represent the cluster and blotted onto membranes. Probes were
prepared for hybridization using the PCR DIG Probe Synthesis Kit
(Roche Molecular Biochemicals; Mannheim, Germany). Probes selected
were derived from the cloned insert analysis described above and
consisted of four clone inserts (Clones 79, 84, 94, and 110) from
SSH10. Labeled probes were pooled prior to hybridizations.
Hybridizations were conducted at 45.degree. C. for 5 hours.
Colorimetric reactions were allowed to develop overnight on the
membranes. Thirty of the 37 isolates (clusters) on the RCS membrane
exhibited hybridization as identified by colorimetric reaction and
25 of the 28 isolates on the LPSA/NLA membrane
[0066] Isolates exhibiting hybridization were then prepared for 16
s rRNA sequencing. Briefly, the 16 s rRNA of each of the 55
isolates was amplified via PCR using the primers 8F
(AGAGTTTGATYMTGGCTCAG) and 1406R (ACGGGCGGTGTGTRC). The PCR product
was purified using the QIAquick PCR purification kit (Qiagen,
Valencia, Calif.). Purified product was analyzed by gel
electrophoresis. When sufficient product was available, the
purified sample was sent overnight on ice for single pass
sequencing (Lark Technologies, Houston, Tex.). The 16 s sequences
from each cluster were compared with sequences from the NCBI
database using the blastn function. Organisms of interest brought
forward from this comparison consisted of Enterococcus faecium
strain 8G-1, Enterococcus faecium strain 8G-73, and Bacillus
pumilus strain 8G-134.
Example 2
In Vitro Strain Testing:
[0067] Rumen fluid was collected for in vitro trials from two
yearling Hereford heifers. Heifers were identified by
identification tags and were referred to as 101 and 133. Heifers
were fed 6 lbs/head/day of dried distillers grain (DDGS) and had
access to free choice haylage.
[0068] The in vitro protocol was followed as closely as possible to
decrease experimental error between each trial. Briefly, rumen
fluid was collected from each heifer and placed into marked,
pre-warmed thermoses. Thermoses were transported to Agtech
Products, Inc. for processing. Rumen fluid was added in duplicate
to bottles containing McDougall's Buffer and 3.0% glucose (final
concentration after McDougall's Buffer and rumen fluid have been
mixed to a volume of 180 ml), which had been tempered to 39.degree.
C. Candidate DFM strains, Enterococcus faecium strain 8G-1,
Enterococcus faecium strain 8G-73, and Bacillus pumilus strain
8G-134, were added to designated bottles at 1.0.times.10.sup.7
CFU/ml (final concentration). The unit of observation was the
bottle, and treatments were performed in quadruplicate. Treatments
consisted of Control (glucose added but no DFM), Enterococcus
faecium strain 8G-1, Enterococcus faecium strain 8G-73, and
Bacillus pumilus strain 8G-134. Bottles were then purged with of
CO.sub.2 and capped. Bottles were maintained in a shaking water
bath at 39.degree. C. and 140 rpm. Approximately 10 minutes prior
to sampling, bottles were briefly vented to release gases produced
as a byproduct of fermentation. Rumen fluid was withdrawn from each
bottle initially and every 6 hours until the 36 hour mark. Rumen pH
and volatile fatty acids were measured and recorded. Statistical
analysis was performed using repeated measures analysis to
determine DFM effects over time or one-way ANOVA to determine
treatment affects at a specific points in time.
[0069] The focus of the ruminal in vitro experiments was to
determine if candidate DFM strains, Enterococcus faecium strain
8G-1, Enterococcus faecium strain 8G-73, and B. pumilus strain
8G-134, could positively influence ruminal fermentation in an
energy excess environment. Excess glucose was added to each ruminal
in vitro, to replicate cattle engorgement with a readily
fermentable carbohydrate. As shown in FIG. 3, the addition of each
of the candidate strains significantly increased the utilization of
glucose over time (P=0.0001). In comparison to the control (FIG.
4), the influence on lactic acid production over time was also
significantly impacted by the addition of the candidate DFM to the
challenged in vitro model (P=0.0025). By time point 36 hours, there
was 17% less lactic acid production in the B. pumilus and 32% less
lactic acid accumulation in both the Enterococcus candidates.
[0070] Volatile fatty acid analysis was performed via HPLC. Total
VFA (acetate+propionate+butyrate) were significantly affected by
addition of the Enterococcus candidates (P=0.0279) (FIG. 5). The
Enterococcus candidates, 8G-1 and 8G-73, appeared to increase the
amount of total VFA produced over time. There was no significant
affect on total VFA production when comparing the B. pumilus
candidate to that of the control.
[0071] The in vitro results indicated that the candidate DFMs 8G-1,
8G-73, and 8G-134 positively affected ruminal fermentation by
increasing glucose utilization without a corresponding increase in
lactic acid production in comparison to that of the control
treatment. Excess glucose in the rumen is typically fermented
rapidly with the production lactic acid. It is the accumulation of
lactic acid which drives an acute acidotic response. By utilizing
glucose without the concomitant production of lactic acid, the
candidate DFMs have demonstrated the potential to ameliorate the
affects of acidosis. The ruminal in vitro model suggested that
these strains may be able to successfully modulate excess ruminal
energy in cattle fed high amounts of readily fermentable
carbohydrates.
Example 3
Candidate DFM Testing in Cattle Fed a Readily Fermentable
Carbohydrate--an Acute Acidosis Challenge:
[0072] Materials and Methods:
[0073] Cattle and Pens Assignments:
[0074] Twenty cross-bred beef steers were purchased at local sale
barns. Cattle were housed at the research facility for a period of
two weeks prior to trial initiation for observation of morbidity or
mortality. Cattle were randomly blocked across treatment by weight.
Five head of cattle were assigned to a pen and pens designated to
one of four treatments. Treatments consisted of 3 pens each
receiving a different DFM as is detailed below with the fourth pen
receiving no DFM (control). Treatment assignments can be seen in
Table 2 below.
TABLE-US-00002 TABLE 2 Treatment assignments by pen. Candidate
Minimum Dose Pen ID DFM (TX) (CFU/Head/Day) 16s rRNA Identification
1 None 0 None (Control) 2 8G-1 5 .times. 10.sup.10 Enterococcus
spp. 3 8G-73 5 .times. 10.sup.10 Enterococcus spp. 4 8G-134 5
.times. 10.sup.9 Bacillus pumilus
The daily feed ration for all treatment groups prior to challenge,
consisted of 62.5% roughage and 30% cracked corn and 7.5% protein
supplement (Table 3) below. The protein supplement contained
monensin (Rumensin.RTM.) fed at 375 mg/head/day. The protein
supplement also contained tylosin phosphate (Tylan.RTM.).
TABLE-US-00003 TABLE 3 Challenge Ration Composition Ingredient % of
Diet (DM) Pre-challenge diet Ground Hay 62.5 Cracked Corn 30
Steakmaker .RTM. K+ 45-25 7.5 R500 T180* Challenge Diet Steam
Flaked Corn 87.4 Alfalfa Pellets 5.1 Steakmaker .RTM. K+ 45-25 7.5
R500 T180* *Both rations contain Rumensin and Tylan.
Cattle were fed 15 lbs/head/day of the ration once in the morning
and had any remaining feed pushed closer to the feeding stanchion
late in the afternoon. Fourteen days prior to fasting, treatment
groups were fed candidate DFMs at the dose designated in Table 2
above as a top dressing on the daily ration.
[0075] Bacillus pumilus strain 8G-134 was fed at a minimum of
5.times.10.sup.9 CFU/Head/Day. The Enterococcus candidates 8G-1 and
8G-73 were fed at 5.times.10.sup.10 CFU/Head/Day.
Candidate DFM Preparation:
[0076] Candidate DFM strains, previously selected for the challenge
trial, were Enterococcus spp. 8G-1 and 8G-73; and Bacillus pumilus
strain 8G-134. Strains were stored at -80.degree. C. Each culture
was inoculated into 10 ml broth tubes containing MRS (Man, Rogosa
and Sharp) or TSB (tryptic soy broth). Broth tubes were incubated
for 24 hours at 32.degree. and 37.degree. C. for the Bacillus and
Enterococcus candidates, respectively. Cultures were struck for
isolation on respective agar medium and incubated. An isolated
colony was picked into 10 ml of broth and allowed to grow to mid
log phase (18 to 24 h) and transferred into fresh broth (10%
vol/vol transfer). Enterococcus candidates were grown at 37.degree.
C. in MRS broth. Bacillus was grown at in a shaking incubator at
130 rpm at 32.degree. C. in horizontal TSB tubes. For the growth of
Enterococcus, 2 ml were transferred into a 250 ml bottle containing
198 ml of broth and incubated for 18 hrs.
[0077] The 200 ml of culture was inoculated into a 2 L bottle
containing 1.8 L of broth and allowed to incubate for 18 hr. For
the Bacillus candidates, 5 ml were transferred into a 250 flask
containing 50 ml of TSB and then was incubated at 32.degree. C. in
a shaking incubator at 130 rpm for 24 hr. The 50 ml was used to
inoculate a 1L flask containing 600 ml and allowed to incubate for
another 24 hours.
[0078] The optical density (OD) of the 18 hr culture of
Enterococcus candidates was taken before harvesting the cells. The
OD was compared to previous growth curves to determine the cfu/ml
of culture. Samples were plated for enumeration and genetic
fingerprinting. Quality control was ensured between each
fermentation batch via RAPD-PCR analysis. With a target minimum of
5.0e10 cfu/head/day for Enterococcus candidates, the calculated
amount of culture was dispensed into 250 ml Nalgen centrifuge
bottle and spun at 4.degree. C. for 10 min at 4500 rpm. Target
minimum for Bacillus candidate was 5.0e9 cfu/head/day, and a total
of 100 ml of the Bacillus culture was spun down similar to the
Enterococcus. Supernatant was discarded. The pellet was resuspended
in 30 ml of growth media containing 10% glycerol. This amount was
transferred to a 50 ml conical tube. The centrifuge bottles were
then rinsed with 10 ml of broth and transferred to the same conical
tube. Samples were labeled with strain, date the candidate was
harvested, and fermentation batch number. Plate counts were used to
determine the total cfu in each tube. Tubes were combined to
deliver counts of a minimum of 5.0e10 cfu/head/day for Enterococcus
candidates and 5.0e9 cfu/head/day for Bacillus candidates. All
conical tubes were frozen at -20.degree. C.
Challenge Diet and Rumen Fluid Collection Phase:
[0079] Rumen fluid samples were obtained from individual animals
via ruminal intubation using a collection tube fitted with a
strainer and attached to a vacuum source through a vacuum flask.
The pH was immediately measured after rumen fluid acquisition and
samples were frozen to be transported to Agtech Products, Inc. for
VFA analysis. Samples were collected from all cattle at sample
times -12 h, +6 h, +10 h, +14 h, +18 h, +22 h, +30 h, +36 h, and
+48 h, with time 0 h representing the initiation of the challenge.
All feed was removed from the cattle at time -24 h to initiate the
fast and encourage cattle to engorge the challenge ration at time
0.
[0080] All pens were fasted for 24 hours before challenge with the
concentrate diet (Time 0). The concentrate diet consisted of 28 lb
flake weight steam flaked corn (Table 3 above). The challenge
ration was fed to deliver 20 lbs/head. Challenge ration consumption
was visually monitored and additional feed added on an as needed
basis through the remainder of the trial.
[0081] Ruminal samples were collected every 4 hours from all cattle
from +6 to +22 hours. Each rumen sample pH was analyzed immediately
after acquisition. Rumen fluid was then frozen and transported to
Agtech Products, Inc. for VFA analysis via HPLC. Repeated measures
analysis was performed on rumen pH, VFAs, and glucose levels using
individual animal as the unit of observation. Pairwise comparisons
were performed over time between each candidate DFM treatment pens
and the control pen to determine the candidates' effectiveness to
alter ruminal fermentation patterns.
[0082] Results and Discussion:
[0083] Twenty head of crossbred beef cattle weighing on average
731.95 lbs were randomly blocked by weight across treatments such
that there were no significant differences by weight between
treatment groups (Table 4). There were 3 treatment pens and one
control pen with five head/pen. Treatment assignment per pen can be
seen in Table 2 above.
TABLE-US-00004 TABLE 4 Treatment assignments by pen. Average Feed
Ave. Pen Weight in lbs Consumption/Steer.sup.1 Pen Treatment N (SD)
(% of Body Weight) 1 Control 5 731.6 (79.94) 4.8 2 8G-1 5 741.2
(94.03) 3.9 3 8G-73 5 731.6 (70.12) 3.7 4 8G-134 5 727.6 (72.71)
3.6 .sup.1Average feed consumption/steer was calculated as a
percentage of the average steer weight for that pen
[0084] Cattle were fed challenge ration at time 0 and rumen fluid
was collected at designated time points to measure ruminal
fermentation values. Feed consumption per pen was monitored and
recorded after 24 hours. Average feed consumption/steer was
calculated as a percentage of the average steer weight for that pen
(Table 4 above). The control pen (pen 1) appeared to have the
highest consumption of feed in comparison to the other treatment
groups with the average steer consuming 4.8% of its body weight.
The lowest challenge ration consumption/pen was in pen 4 with the
average steer eating approximately 3.6% of its body weight. Cattle
in all pens on average would have been consuming approximately
5.625 lbs of concentrate/day as part of the pre-challenge ration,
which on average would have constituted 0.8% of the average steers'
body weight. Despite the pen variation of challenge ration
consumption, the difference was not greater than the increase in
concentrate consumption from the pre-challenge ration and would not
be causative in fermentation differences between pens.
[0085] After 24 hours, feed was removed from the cattle and ground
prairie hay was fed ad libitum. Cattle were given free choice hay
as a precaution against the continually decreasing rumen pHs. The
addition of hay would stimulate additional cud chewing and help to
buffer the rumen. Despite the addition of hay, the ruminal pH still
continued to decline.
[0086] Immediately after rumen fluid collection, sample pH was
analyzed. All treatment groups exhibited a decline in ruminal pH as
can be observed in FIG. 6. The pH for the control group achieved
nadir at time 30 hours and began to gradually climb thereafter. By
the last rumen sample collection the mean pH for the control pen
was still acutely acidotic with a pH of 4.94. Acute acidosis is
associated with pH that remains below 5.2 and chronic or subacute
acidosis characterized by a pH below 5.6 (Owens, et. al., 1998).
Mean numerically higher trends appeared for strains 8G-1, 8G-73,
and 8G-134 in pens 2, 3, and 4 when compared to that of the control
from time +22 to +48. This suggests that cattle treated with the
candidate DFMs in these pens recovered more quickly from the
acidotic challenge. Mean pH for the for cattle treated with 8G-1,
8G-73, and 8G-134 at time +48 was 5.96, 6.02, and 6.14,
respectively, which is greater than 1.0 pH unit above the control
pen. Repeated measures analysis of these three strain over time (+6
to +48) did not exhibit significant differences when compared to
the control pen. However when pH comparisons of pens treated with
8G-1, 8G-73, and 8G-134 to the control pen from time +22 h to +48 h
were performed, differences were or approached significance
(P=0.1562, 0.0965, and 0.0466 for 8G-1, 8G-73, and 8G-134,
respectively).
[0087] Average lactic acid profiles for all treatment groups are
shown in FIG. 7. Mean ruminal lactic acid accumulation peaks at 105
mM for the control pen 30 hours after receiving challenge ration.
Candidate DFM strains 8G-1, 8G-73, and 8G-134 again exhibited
visible mean numeric differences in lactate accumulation in
comparison to that of the control pen. Mean lactic acid
accumulation was similar between the control cattle and the 8G-1
treated cattle through the first 14 hours of the challenge.
Subsequent accumulation levels for the remainder of the trial were
much less in the 8G-1 treated cattle although not significant
(P=0.1892). Treatment pens 8G-73 demonstrated decreased levels of
lactic acid accumulation at times 30 and remained lower than the
control pen for the remainder of the trial. Candidate strain 8G-134
also showed decreased levels of lactic acid starting at +22 h and
remained consistently lower than the control pen through +48 h.
[0088] Individual VFAs were measured and analyzed. Volatile fatty
acid (VFA) concentrations increased in the control pen and
treatments 8G-73 and 8G-134 and peaked at six hours (FIG. 8). After
six hours each of these treatment pens showed declining levels of
total VFA (acetate, propionate and butyrate). There were no
significant differences between these treatments and the control.
Treatment 8G-1, however, exhibited a delay in VFA decline which did
not occur until +14 h. Over the course of the trial there were no
significant differences in total VFA concentration or the
individual VFA (consisting of acetate, propionate, or butyrate)
levels.
[0089] In addition to monitoring and measuring ruminal fermentation
characteristics over the course of the acidotic trial, cattle were
observed throughout the trial for visible clinical effects
associated with acidosis. Early in the acidotic challenge (+0 h to
+14 h), the effects of the challenge diet were minimal. Cattle did
not show signs of depression and continued to feed on the challenge
ration. By +22 hours post receiving the challenge ration all cattle
except those in receiving Treatment 8G-1) were showing signs of
soreness, depression, and had loose, liquid fecal excretion. Cattle
in pen 2 were no longer consuming feed, but did not exhibit
clinical symptoms, despite similar having similarly declining pH
levels.
[0090] Acute ruminal acidosis by definition is the decline in
ruminal pH to levels deleterious not only to rumen function but
also livestock health. Acute acidosis is marked by the accumulation
of lactic acid and the decline in VFA production. Proper rumen
function is a combination of managing the available energy and
nitrogen components available in feedstuffs. When imbalances in
ruminal metabolism occur, digestive upset typically follows and can
manifest in the form acidosis. In this trial, strains 8G-1, 8G-73,
and 8G-134 enhanced the recovery of rumen function as indicated by
ruminal fermentation parameters.
[0091] Cattle fed 8G-1, Enterococcus faecium, on average recovered
more quickly from the acidosis challenge as measured by pH recovery
and lactic acid decline. In addition to the measured ruminal
fermentation patterns, cattle fed candidate DFM 8G-1 did not
exhibit clinical signs associated with acidosis.
[0092] Candidate strain 80-73, Enterococcus faecium, improved
ruminal fermentation through the course of the trial. Mean lactic
acid levels were the lowest for all candidate strains tested at +48
h at 12.54 mM. A corresponding increase in pH was also associated
with the recovery with a final pH of 6.02, which was 1.08 pH units
higher than that of the control.
[0093] Candidate strain 8G-134, Bacillus pumilus, also enhanced
ruminal recovery during the acidotic challenge. Mean lactic acid
levels, in cattle fed 8G-134, peaked at 89 mM at time +22 h, while
the control pen continued to increase and peaked at 105 mM at time
+30 hours. Mean lactic acid levels had dropped to 57 mM by +30
hours. As with lactic acid accumulation, ruminal pH in cattle fed
8G-134 recovered more quickly than that of the control and was
found to be significantly different from +22 to +48 h
(P=0.0466).
Example 4
[0094] Summary:
[0095] Thirty prima and multiparous Holstein cows were blocked by
previous lactation and predicted producing ability (PPA) and
assigned to one of three treatments. Ten cows were assigned per
treatment and treatments consisted of a control group (Treatment 1)
which received a basal total mixed ration (TMR), Treatment 2 and
Treatment 3 which received basal total mixed ration TMR and were
fed Bacillus pumilus 8G-134 at 5.times.10.sup.9 and
1.times.10.sup.10 CFU/head/day, respectively, from 3 weeks
prepartum to 22 week after parturition. The primary objective was
to determine the effects of B. pumilus 8G-134 on dairy cow milk
production and performance above control cattle during this time
period. The secondary objective was to determine if there was a
dose response associated with feeding B. pumilus 8G-134. The B.
pumilus 8G-134 regimens significantly increased milk production,
milk fat, and decreased somatic cell count. These significant B.
pumilus 8G-134 production effects did not come at the expense of
cow body condition score, body weight, increases in dry matter
intake or significantly change blood metabolite profiles, and would
indicate B. pumilus 8G-134 also provided dairy cow efficiency
benefits.
[0096] Materials and Methods:
Livestock:
[0097] Thirty Holstein cows were randomly assigned to one of three
dietary treatments in a continuous lactation trial from 3 weeks
prior to parturition through 22 weeks postpartum. There were no
significant differences for previous milk yield for second and
older cows or for predicted producing ability (PPA) for first
lactation cows for the different treatment groups. The numbers of
first and second lactation animals deviated between the groups, but
did not influence overall mean production, as lactation was
adjusted in the statistical model. Animals were on study from
approximately three weeks prepartum through 22 weeks
postpartum.
Nutrition:
[0098] Dietary ingredients and formulated composition of total
mixed rations (TMRs) are presented in Table 5 below for dry and
lactating cows, respectively. The base TMR was the same for each
group and differed by top dress treatment. Each group received a
top dress of 8 ounces of finely ground corn to which was added 1
ounce of maltodextrin (Treatment 1, control), Bacillus spp at
5.times.10.sup.9 CFU/head/day (Treatment 2), and Bacillus spp at
1.times.10.sup.10 CFU/head/day (Treatment 3).
TABLE-US-00005 TABLE 5 Formulated composition of the TMR offered to
dry and lactating cows. Dry Period Lactating Period Ingredients, %
DM basis Corn Silage 52.57 39.14 Ryelage 18.54 Alfalfa haylage --
16.52 Grass hay 14.45 1.77 SBM48 5.74 10.77 Blood Meal 4.41
AminoPlus -- 6.01 Corn 2.41 21.07 Fat 0.65 1.42 Limestone -- 1.26
Sodium bicarbonate -- 0.84 MagOx 0.65 0.42 Salt 0.32 0.53 TMin Vit
0.27 0.26 Composition, % DM CP 16.50 16.64 SP, % CP 32.29 28.58 NDF
41.06 30.65 Starch 19.56 29.82 Sugar 3.19 2.68 NFC 33.47 41.87 Fat
3.82 4.15 Ca 0.31 0.87 P 0.29 0.34 Mg 0.54 0.42 K 1.72 1.33 NeL,
mcal/kg 1.60 1.74
Sample and Data Collection:
[0099] Daily TMR samples and refusals were collected and composited
weekly, weekly composites combined monthly, and monthly samples
were analyzed for dry matter (DM), crude protein (CP), acid
detergent fiber bound protein (ADF-CP), neutral detergent fiber
bound protein (NDF-CP), soluble protein (SP), acid detergent fiber
(ADF), neutral detergent fiber (NDF), lignin, fat, starch, sugar,
ash, calcium (Ca), phosporus (P), magnesium (Mg), potassium (K),
sulfur (S), sodium (Na), chlorine (Cl), iron (Fe), manganese (Mn),
zinc (Zn), and copper (Cu) by Cumberland Valley Analytical
Services, Maugansville, Md.
[0100] Cows were milked twice a day, and milk volume was recorded
electronically at each milking and am-pm amounts summed for daily
total. Once a week milk samples from am and pm milkings were
composited for analysis of content of fat, protein, somatic cells,
solids not fat, and milk urea nitrogen (MUN) by Dairy One milk
laboratory in State College, Pa. using a Fossamatic 4000 (FOSS;
Eden Prairie, Minn.).
[0101] Animals were on study from approximately three weeks
prepartum through 22 weeks postpartum. Animal weight was estimated
by heart girth circumference on weeks 1, 3, 7, 11, 15 and 18
postpartum. Body condition was assessed by two independent
observers at the same time as body weight was collected.
[0102] Blood samples were collected from the coccygeal vein, serum
harvested, frozen and analyzed for glucose, beta-hydroxy butyrate
(BHB), and non-esterified fatty acids (NEFA) at weeks 2 and 8
postpartum. Glucose and BHB were analyzed using an Abbott Precision
Xtra.TM. meter (Abbott Diabetes Care Inc., Alameda, Calif.). A
Randox assay kit (Cat. HN 1530, Randox Laboratories, Northern
Ireland) was used to measure non-esterified fatty acid (NEFA)
concentration in serum adopted to an enzyme linked immunosorbant
assay (ELISA) plate reader at a wave length of 550 nm for multiple
samples. The Randox kit uses Acyl CoA synthetase and oxidase to
convert NEFA to 2,3-trans-Enoyl-CoA plus peroxide; peroxide plus
N-ethyl-N-(2 hydroxy-3-sulphopropyl) m-toluene leads to a purple
product, which is the indicator of NEFA concentration in serum.
Statistical Models.
[0103] Milk production and content, body weight, and body condition
score were analyzed using the mixed procedure in SAS statistical
software. Cow was the repeated subject with the covariance matrix
set to type 1 correlation structure. Daily milk observations were
aggregated by week postpartum. The statistical model was as
follows:
Y.sub.i=u.sub.i+TRT.sub.j+Lact.sub.k+Week.sub.1+TRT.sub.j*Lact.sub.k+TRT-
.sub.j*Week.sub.1+TRT.sub.j*Lact.sub.k*Week.sub.1+e.sub.jklm
[0104] Where [0105] Y.sub.i=least square mean of the production
dependent variables; [0106] u.sub.i=overall mean of the various
production variables; [0107] TRT.sub.j=jth treatment effect, 1, 2,
3; [0108] Lact.sub.k=kth lactation, 1, 2+; [0109] Week.sub.1=1th
week, 1 . . . 22; [0110] interaction terms
(TRTj*Lactk+TRTj*Week1+TRTj*Lactk*Week1) [0111]
e.sub.jklm=error
[0112] Monthly samples of TMR and feed refusals for each treatment
were tested for difference using means procedure in SAS.
[0113] Blood concentrations of glucose, BHB, and NEFA, were
analyzed using the general linear models in SAS statistical
software. Class variables were cow, week and treatment. Treatment
was nested in cow and was the error term for testing treatment
significance. Treatment by week interaction was tested for
statistical significance using the residual error.
[0114] Results:
[0115] Mean TMR composition for dry and lactating TMRs is presented
in Table 6 over the course of the study. Composition was not
different between the treatment groups.
TABLE-US-00006 TABLE 6 Analyzed composition of TMR for dry cows and
lactating cows Treatment, % DM basis Item 1 2 3 SEM Dry TMR N 3 3 3
CP 13.64 13.17 13.11 0.14 ADF 29.55 28.37 27.88 0.53 NDF 48.54
48.04 47.14 0.93 Lignin 3.64 3.52 3.58 0.09 Starch 18.74 18.19
17.97 0.87 Sugar 6.71 6.15 6.57 0.23 Ash 8.44 7.56 7.79 0.10 NFC
39.49 36.93 38.20 0.86 Ca 0.49 0.46 0.47 0.01 P 0.37 0.35 0.35
0.004 Lactating TMR N 9 9 9 CP, % 14.54 14.81 14.37 0.10 ADF, %
21.54 21.38 21.64 0.25 NDF, % 33.51 33.21 33.30 0.32 Lignin, % 3.27
3.27 3.26 0.06 Starch, % 25.36 26.52 25.48 0.38 Sugar, % 6.25 6.15
6.25 0.15 Fat, % 3.69 3.65 3.62 0.04 Ash, % 8.73 8.67 8.60 0.09
NFC, % 41.22 42.02 41.44 0.34 Ca, % 0.95 0.97 0.96 0.005 P, % 0.36
0.36 0.36 0.003 standard error means (SEM), NFC, nonfiber
carbohydrate NDF, neutral detergent fiber ADF, acid detergent fiber
Treatment 1 control; Treatment 2, Bacillus pumilus at 8G-134 5
.times. 10.sup.9; Treatment 3, Bacillus pumilus 8G-134 at 1 .times.
10.sup.10.
[0116] One control (Treatment 1) cow exhibited abnormal milk
production and her data was removed from the data analysis. The
analysis of milk production was repeated on 29 cows. Milk
production was significantly influenced by treatment (Table 7
below). Cows fed the Bacillus pumilus 8G-134 at 5.times.10.sup.9
CFU/head/day (Treatment 2), and Bacillus pumilus 8G-134 at
1.times.10.sup.10 CFU/head/day (Treatment 3) produced significantly
more milk than the cows fed the placebo control. Cows on Treatment
2 and 3 produced approximately 2 kg more milk than treatments 1
(Table 7). There was a significant interaction with parity.
Production increases were significant in second parity cows by 5.2
kg, but no significant differences in milk production in first
parity cows.
TABLE-US-00007 TABLE 7 Least square mean milk production in
Holstein cows from calving through 22 weeks postpartum fed a
microbial additive. Change Milk, relative to kg/d control, Effect
Treatment Lactation kg/d sem kg/d sem Treatment 1 33.12.sup.a 0.65
0.00 0.66 Treatment 2 35.38.sup.b 0.60 2.30* 0.60 Treatment 3
35.08.sup.b 0.61 1.99* 0.61 Lactation 1 31.59.sup.a 0.47 -0.83 0.47
Lactation 2 36.84.sup.b 0.39 3.09* 0.39 Interaction 1 1 32.44.sup.a
1.07 0.00 1.07 2 1 31.70.sup.a 0.93 -0.72 0.93 3 1 31.36.sup.a 0.93
-1.07 0.93 1 2 33.80.sup.b 0.74 0.00 0.74 2 2 39.06.sup.c 0.76
5.24* 0.76 3 2 38.79.sup.c 0.79 5.17* 0.79 Means within group with
different superscript differ, P < 0.05 Mean change with * differ
significantly from 0 Treatment 1 control; Treatment 2, Bacillus
pumilus at 8G-134 5 .times. 10.sup.9; Treatment 3, Bacillus pumilus
8G-134 at 1 .times. 10.sup.10.
[0117] Milk fat and yield are presented in Table 8 below. Milk fat
was significantly increased by Treatments 2 and 3 above the
control. Yield responses followed milk yield with fat yield
increased in the Bacillus pumilus 8G-134 fed treatment groups.
Bacillus pumilus 8G-134 cattle for treatment 2 and 3 yielded
significantly higher fat percentage above that of the control with
0.24% and 0.31% higher levels, respectively (Table 8). Coupled with
the significant increase in milk production, daily fat yield for
both treatment 2 and 3 provided significant increases in daily fat
production above that of the control (Table 8).
TABLE-US-00008 TABLE 8 Milk fat content by treatment groups. Fat,
Effect Treatment Lactation % sem Fat Yield, kg/d sem Treatment 1
3.57.sup.a 0.09 1.206.sup.a 0.042 2 3.81.sup.b 0.08 1.351.sup.b
0.039 3 3.88.sup.b 0.08 1.351.sup.b 0.039 Lactation 1 3.76 0.06
1.159.sup.a 0.030 2 3.78 0.05 1.395.sup.b 0.025 Means within column
with different superscript differ by P < 0.05 Treatment 1
control; Treatment 2, Bacillus pumilus at 8G-134 5 .times.
10.sup.9; Treatment 3, Bacillus pumilus 8G-134 at 1 .times.
10.sup.10.
[0118] Log of the linear Somatic cell count (LogSCC) scores were
different by treatment and lactation. The Bacillus pumilus 8G-134
treatments (Treatments 2 and 3) had significantly lower log linear
score than the control cows (Table 9 below). Treatments 2 and 3
cows had LogSCC of 4.97 and 4.96, respectively compared to those of
the control cows at 5.92. Parity two cows had significantly higher
log linear score than first lactation cows. Somatic cell counts are
associated with infection as well as immunological status and
health of the lactating dairy cow. Additionally increased SCC is
indicative of inflammatory responses to infection. Decreases in SCC
demonstrated here may indicate that cows fed the Bacillus pumilus
8G-134 are better immunologically to handle infectious challenge
during lactation and maintain udder and cow health.
TABLE-US-00009 TABLE 9 Treatment effects on Somatic Cell Count
(SCC). Effect Treatment Lactation LogSCC sem Treatment 1 5.92.sup.a
0.32 2 4.97.sup.b 0.30 3 4.96.sup.b 0.30 Lactation 1 5.27.sup.a
0.23 2 5.86.sup.b 0.19 LogScc = log of somatic cell count Treatment
1 control; Treatment 2, Bacillus pumilus at 8G-134 5 .times.
10.sup.9; Treatment 3, Bacillus pumilus 8G-134 at 1 .times.
10.sup.10.
[0119] Data for mean DMI for groups for dry and lactating periods
in Table 10 below. Dry matter intake for dry cows ranged from 10.79
to 11.64 kg/d across the groups. Lactating groups consumed 22.02,
21.31 and 21.48 kg/d for treatment groups 1, 2, and 3, respectively
(Table 10). Predicted DMI based on the NRC equation for cows by
week postpartum is presented in Table 10. Intake for treatment 1
was 0.83 kg higher than predicted; intake for treatment 2 was -1.37
kg lower than predicted; intake for treatment 3 was -1.04 kg lower
than predicted. The increase in milk production on the Treatments 2
and 3 was accomplished with no increase in DMI. In fact the
predicted or expected DMI based on NRC predictions compared to the
group mean intake suggests these cows consumed 1.0 to 1.5 kg/d less
DMI. Thus, the efficiency of DM utilization was increased on
Treatments 2 and 3.
TABLE-US-00010 TABLE 10 Least square means for group feed intake,
serum glucose, beta-hydroxy butyrate, non-esterified fatty acids,
by treatment group. Treatment group Item 1 sem 2 sem 3 sem Dry
Matter Intake, kg/d Dry cows 10.79 0.27 11.64 0.25 11.12 0.25
Lactating cows 22.02 0.11 21.31 0.11 21.48 0.11 Predicted DMI, kg/d
21.19 0.58 22.68 0.57 22.52 0.58 Serum values Glucose, mg/dl 53.95
1.98 51.5 1.98 53.5 1.98 Beta-OH butyrate, mg/dl 1.05 0.15 0.88
0.15 1.15 0.15 NEFA, ueq/ml 0.23 0.04 0.16 0.04 0.17 0.04 Serum
values by week, 2, 8; Glucose, mg/dl, 52.90 2.80 47.00 2.80 48.90
2.80 55.00 2.80 56.00 2.80 58.10 2.80 BHB, mg/dl 0.92 0.21 0.86
0.21 1.39 0.21 1.17 0.21 0.89 0.21 0.91 0.21 NEFA, ueq/ml 0.41 0.05
0.23 0.05 0.28 0.05 0.07 0.05 0.10 0.05 0.07 0.05 BHB =
beta-hydroxy butyrate Treatment 1 control; Treatment 2, Bacillus
pumilus at 8G-134 5 .times. 10.sup.9; Treatment 3, Bacillus pumilus
8G-134 at 1 .times. 10.sup.10. Predicted DMI = (.372 * FCM + 0.0968
* BWT(kg){circumflex over ( )}.75) * (1 - exp(-0.192 * (Week +
3.67)))
[0120] The DMI differences and production values could suggest that
cows on Treatments 2 and 3 could have mobilized more body tissue
than the control cows to produce more milk and eat less than
expected. However, serum NEFA, glucose, and BHB suggest these cows
were in similar energy status as control cows (Table 10 above).
Additionally, body weight and BCS were similar for the Bacillus
groups relative to the control group (Table 11 below). This
suggests they did not mobilize more body tissue to produce the
additional milk volume, indicating feed conversion efficiency in
cows feed Treatments 2 and 3 regardless of dose.
TABLE-US-00011 TABLE 11 Least square mean body weight (lb) and body
condition score by treatment groups. Body condition score is the
average score of two observers. Wt, Effect Treatment Lactation (lb)
sem BCS sem Treatment 1 1343.10 28.49 2.92 0.03 2 1324.60 27.54
3.13 0.03 3 1388.02 27.73 3.06 0.03 Lactation 1 1223.31 21.87 3.04
0.02 Lactation 2 1476.62 17.94 3.01 0.02 Interaction Treatment
.times. lactation 1 1 1170.78 27.72 3.06 0.05 2 1 1242.64 25.26
3.17 0.05 3 1 1208.50 24.00 2.94 0.05 1 2 1486.02 19.33 2.78 0.04 2
2 1406.42 19.96 3.10 0.04 3 2 1547.85 20.79 3.19 0.04 Wt = weight,
lbs BCS = body condition score, scale 1 to 5 by 0.25 points; 1 =
emaciated, 2 = thin, 3 = average, 4 = fat, 5 = obese Treatment 1
control; Treatment 2, Bacillus pumilus at 8G-134 5 .times.
10.sup.9; Treatment 3, Bacillus pumilus 8G-134 at 1 .times.
10.sup.10.
[0121] 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.
BIBLIOGRAPHY
[0122] Allison, M. J., M. Robinson, R. W. Dougherty, and J. A.
Bucklin. 1975. Grain overload in cattle and sheep: Changes in
microbial populations in the cecum and rumen. Amer. J. Vet Res.
36:181.
[0123] Dunlop, R. H. 1972. Pathogenesis of ruminant lactic
acidosis. Adv. Vet Sci. Comp Med. 16:259.
[0124] Elam, C. J. 1976. Acidosis in feedlot cattle: Practical
observations. J. Anim. Sci. 43:898.
[0125] Hungate, R. E., R. W. Dougherty, M. P. Bryant, and R. M.
Cello. 1952. Microbiological and physiological changes associated
with acute indigestion in sheep. Cornell Vet. 42:423.
[0126] Muir, L. A., E. L. Rickes, P. F. Duquette, and G. E. Smith.
1981. Prevention of induced lactic acidosis in cattle by
thiopeptin. J. Anim. Sci. 52:635.
[0127] Owens, F. N., Secrist, D. S., Hill, W. J., Gill, D. R. 1998.
Acidosis in cattle: a review. J. Anim. Sci. 76:275-286.
[0128] Slyter, L. L. 1976. Influence of acidosis on rumen function.
J. Anim. Sci. 43:910.
[0129] Yang, W., 2004. Effects of direct-fed microbial
supplementation on ruminal acidosis, digestibility, and bacterial
protein synthesis in continuous culture. Animal Feed Science and
Technology, 114(4): 179-193.
Sequence CWU 1
1
2120DNAArtificial SequenceSynthetic 1agagtttgat ymtggctcag
20215DNAArtificial SequenceSynthetic 2acgggcggtg tgtrc 15
* * * * *