U.S. patent application number 13/887792 was filed with the patent office on 2013-11-07 for microorganisms and methods for treating poultry.
The applicant listed for this patent is Tammy Baltzley, Shelly Gebert, Firmin Lago, Tony Neumann, Thomas Rehberger. Invention is credited to Tammy Baltzley, Shelly Gebert, Firmin Lago, Tony Neumann, Thomas Rehberger.
Application Number | 20130295067 13/887792 |
Document ID | / |
Family ID | 38444250 |
Filed Date | 2013-11-07 |
United States Patent
Application |
20130295067 |
Kind Code |
A1 |
Baltzley; Tammy ; et
al. |
November 7, 2013 |
MICROORGANISMS AND METHODS FOR TREATING POULTRY
Abstract
An isolated Bacillus strain LSSAO1 is provided. When fed to a
bird, this and other Bacillus strains described herein provide
benefits to the birds. For example, administration of the one or
more Bacillus strain can increase low G+C, gram positive bacteria
in the gastrointestinal flora of the bird. These type of bacteria
are increased by antibiotics and include beneficial Clostridium.
Administration of the one or more Bacillus strain can also inhibit
pathogen in the bird, such as E. coli, Salmonella, and Clostridium.
These benefits can enhance feed conversion in poultry. Useful
combinations of Bacillus strains and methods of using one or more
Bacillus strain are also provided.
Inventors: |
Baltzley; Tammy; (Milwaukee,
WI) ; Lago; Firmin; (Milwaukee, WI) ; Neumann;
Tony; (Wauwatosa, WI) ; Rehberger; Thomas;
(Wauwatosa, WI) ; Gebert; Shelly; (Hartford,
WI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baltzley; Tammy
Lago; Firmin
Neumann; Tony
Rehberger; Thomas
Gebert; Shelly |
Milwaukee
Milwaukee
Wauwatosa
Wauwatosa
Hartford |
WI
WI
WI
WI
WI |
US
US
US
US
US |
|
|
Family ID: |
38444250 |
Appl. No.: |
13/887792 |
Filed: |
May 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12829013 |
Jul 1, 2010 |
8455238 |
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13887792 |
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|
11565474 |
Nov 30, 2006 |
7754469 |
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12829013 |
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60740709 |
Nov 30, 2005 |
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Current U.S.
Class: |
424/93.46 |
Current CPC
Class: |
A61K 35/742 20130101;
C12N 1/20 20130101; A61P 1/02 20180101; C12R 1/07 20130101 |
Class at
Publication: |
424/93.46 |
International
Class: |
C12N 1/20 20060101
C12N001/20; A61K 35/74 20060101 A61K035/74 |
Claims
1. A method comprising administering an effective amount of at
least one isolated Bacillus strain chosen from at least one of
strains 3A-P4 ATCC Accession No. PTA-6506, 15A-P4 ATCC Accession
No. PTA-6507, 22C-P1 ATCC Accession No. PTA-6508, BS27 Accession
No. NRRL B-50105, 2084 Accession No. NRRL B-50013, and LSSAO1
Accession No. NRRL B-50104 to one or more bird, the administration
of the strain providing at least one benefit chosen from increasing
low G+C, gram positive bacteria in the gastrointestinal flora of
the bird and inhibiting a pathogen chosen from at least one of E.
coli, Salmonella, and Clostridium in the bird.
2. The method of claim 1, wherein the Bacillus strain is 2084
Accession No. NRRL B-50013.
3. The method of claim 1, wherein Bacillus strains 22C-P1 ATCC
Accession No. PTA-6508, 2084 Accession No. NRRL B-50013, and BS27
Accession No. NRRL B-50105 are administered.
4. The method of claim 1, wherein Bacillus strains LSSAO1 Accession
No. NRRL B-50104 and 3A-P4 ATCC Accession No. PTA-6506 are
administered.
5. The method of claim 1, wherein Bacillus strains LSSAO1 Accession
No. NRRL B-50104 and 2084 Accession No. NRRL B-50013 are
administered.
6. The method of claim 1, wherein from 7.5.times.10.sup.3 CFU of
the Bacillus strain per gram of feed to 7.5.times.10.sup.5 CFU of
the Bacillus strain per gram of feed is administered.
7. The method of claim 6, wherein about 7.5.times.10.sup.4 CFU of
the Bacillus strain per gram of feed is administered.
8. The method of claim 1, wherein the bird is a turkey.
9. The method of claim 8, wherein the turkey is a finishing
turkey.
10. The method of claim 9, wherein Bacillus strains 22C-P1 ATCC
Accession No. PTA-6508, 2084 Accession No. NRRL B-50013, and BS27
Accession No. NRRL B-50105 are administered.
11. The method of claim 10, wherein the administering step alters
the gastrointestinal flora of the finishing turkey similar to
alteration provided by the growth promoting antibiotic
virginiamycin.
12. The method of claim 9, wherein Bacillus strains 15A-P4 ATCC
Accession No. PTA-6507, 2084 Accession No. NRRL B-50013, and LSSAO1
Accession No. NRRL B-50104 are administered.
13. The method of claim 12, wherein the administering step reduces
levels of C. perfringens type A in the turkey.
14. The method of claim 1, wherein the bird is a broiler.
15. The method of claim 14, wherein Bacillus strains 15A-P4 ATCC
Accession No. PTA-6507, 2084 Accession No. NRRL B-50013, and LSSAO1
Accession No. NRRL B-50104 are administered.
16. The method of claim 15, wherein the administering step reduces
the level of Salmonella present in the litter of the broiler.
17. The method of claim 1, wherein the bird is a layer.
18. The method of claim 17, wherein Bacillus strains 2084 Accession
No. NRRL B-50013, LSSAO1 Accession No. NRRL B-50104, and BS27
Accession No. NRRL B-50105 are administered.
19. The method of claim 18, wherein the administering step reduces
the level of avian pathogenic E. coli in the layer.
20. The method of claim 19, wherein the reduced level of avian
pathogenic E. coli in the layer reduces mortality and morbidity in
the layer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
Utility patent application Ser. No. 12/829,013 filed Jul. 1, 2010,
which is a continuation application of U.S. Utility patent
application Ser. No. 11/565,474, filed Nov. 30, 2006, and issued as
U.S. Pat. No. 7,754,469 on Jul. 13, 2010, which claims priority
under 35 U.S.C. .sctn.119(e) to U.S. Provisional Patent Application
No. 60/740,709, filed Nov. 30, 2005, the entirety of each of the
above-referenced applications are incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] This invention relates to compositions of novel
microorganisms for treating and preventing poultry diseases and
enhancing feed conversion in poultry.
SUBMISSION OF SEQUENCE LISTING
[0003] The contents of the electronic submission text file sequence
listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0004] Several infections and diseases in poultry are caused by
pathogenic bacteria, including E. coli, Clostridium, and
Salmonella. Infections and diseases caused by pathogens result in
increased mortality, decreased performance, and increased cost of
turkey production. In addition, many of these pathogens can be
transmitted to humans.
[0005] Avian colibacillosis is a systemic infection caused by E.
coli and occurs most commonly in young broilers and poults. Avian
pathogenic E. coli (APEC) comprise a specific subset of pathogenic
E. coli that cause extraintestinal diseases of poultry.
(Snoeyenbos, G. H., et al. 1977 Avain Diseases. 22:273-287.) APEC
consists mainly of enteropathogenic E. coli (EPEC) and
enterotoxigenic E. coli (ETEC) serovars, i.e., subdivisions of a
species or subspecies distinguishable from other strains therein on
the basis of antigenicity. (Wiard, T., et al. 2003. Poult. Sci.
Asso. Supplement 1:58-59.) APEC is found in the intestinal
microflora of healthy birds and infections are enhanced or
initiated by secondary environmental and host predisposing factors.
Colibacillosis is a common systemic infection caused by APEC, and
occurs most commonly as acute septicemia or subacute aerosacculitis
and polyserositis in chickens, turkeys, and other avian species
(Zhu, X. Y., et al. 2002. Appl. Environ. Microbial.
68:124-137.).
[0006] Clostridium affecting poultry include C. perfringens, C.
septicum, and C. botulinum which are anaerobic, gram-positive,
spore-forming rods that produce potent toxins. Gangrenous
dermatitis and cellulitis have reemerged recently as a significant
concern for poultry producers in the U.S. Characterized by necrosis
of the skin, subcutaneous tissue, and often the underlying
musculature, the disease occurs suddenly and progresses rapidly
with death occurring often before the first symptoms are observed.
Crepitus tissue caused by gas accumulation under the affected skin
is also commonly observed in cases of GD and cellulitis. The
anaerobic, spore-forming, gram-positive rod Clostridium has been
implicated in numerous cases of necrotizing soft-tissue infections
in poultry (Carr, D., et al. 1996. Avian Dis 40:736-41; Hofacre, C.
L., et al. 1986. Avian Dis 30:620-2; Willoughby, D. H., et al.
1996. J Vet Diagn Invest 8:259-61). The two most commonly isolated
species have been C. perfringens and C. septicum. C. perfringens is
ubiquitous in nature, commonly found in the soil and
gastrointestinal tracts of warm blooded animals. It produces
.alpha.-toxin and .theta.-toxin that work synergistically to
produce the pathology observed in C. perfringens associated
clostridial myonecrosis (Awad, M. M., et al. 2001. Infect Immun
69:7904-10.). C. septicum is a very virulent, but poorly understood
pathogen that is recognized as the causative agent of atraumatic
myonecrosis. It also produces .alpha.-toxin, distinct from C.
perfringens, which acts as a pore-forming cytolysin and is
essential for virulence (Kennedy, C. L., et al. 2005. Mol Microbiol
57:1357-66.). In order to better understand the etiology of this
disease and the diversity of the Clostridium implicated,
microbiological analysis of affected birds' organs and tissues was
performed. The results suggest a clostridial bacteremia present
prior to death with both species and multiple strains involved.
[0007] The spores of C. botulinum are heat-resistant and can
survive in foods that are incorrectly or minimally processed. Food
borne botulism (as distinct from wound botulism and infant
botulism) is a severe type of food poisoning caused by the
ingestion of foods containing the potent neurotoxin formed during
growth of the organism. The toxin is heat labile and can be
destroyed if heated at 80.degree. C. for 10 minutes or longer. The
incidence of the disease is low, but the disease is of considerable
concern because of its high mortality rate if not treated
immediately and properly. Most of the 10 to 30 outbreaks that are
reported annually in the United States are associated with
inadequately processed, home-canned foods, but occasionally
commercially produced foods have been involved in outbreaks.
[0008] Strains of Salmonella cause Salmonellosis, which occurs in
animals, including humans. It is an enteric disease of varying
severity, usually involving diarrhea. With poultry, however, most
Salmonella infections are symptomless. Many strains of Salmonella
are zoonotic agents, spreading to humans from contaminated food
products. In humans, Salmonellosis is one of the most common causes
of food poisoning.
[0009] Thus, pathogenic bacteria are a major problem for poultry
producers. Further complicating this situation is the fact that
pathogen populations in poultry production facilities typically
fluctuate in terms of both levels and types of pathogens, making
control of the pathogens difficult. An adequate disease prevention
program is essential to a profitable commercial poultry operation.
Chronic diseases can reduce efficiency and increase costs.
[0010] To prevent disease in and stimulate growth of turkeys,
antibiotics have been used. Fed at a sub-therapeutic level,
antibiotics have been found to have beneficial effects. For
example, antibiotics reduce levels of lactic acid bacteria, which
in adult animals are not needed and can be problematic. In
addition, antibiotics increase low G+C, gram positive bacteria,
including beneficial Clostridium. See, e.g, WO 2004/104175 to Lee,
M. D. et al.
[0011] However, there are many drawbacks associated with antibiotic
use, such as consumer acceptance and selection of resistant
bacteria. For instance, mounting scientific evidence shows that
using antibiotics in livestock feed can lead to
antibiotic-resistant bacteria that can be transferred to people,
making it harder to treat certain infections.
[0012] Concerns over antibiotics have led the European Union on
Jan. 1, 2006 to ban 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.
[0013] In the United States, a bill banning low-level feeding of
seven antimicrobials (bacitracin, erythromycin, lincomycin,
penicillin, tetracycline, tylosin, and virginiamycin) was
introduced into the House of Representatives in November 1999 (H.R.
3266).
[0014] Accordingly, there is a recognized need for alternatives,
such as microorganisms and methods of using microorganism for
treating or preventing disease in poultry. Furthermore, there is an
important need for improving performance in and health of
poultry.
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. An isolated Bacillus strain LSSAO1 is
provided. When fed to a bird, this and other Bacillus strains
described herein provide benefits to the birds. For example,
administration of the one or more Bacillus strain can increase low
G+C, gram positive bacteria in the gastrointestinal flora of the
bird. These type of bacteria are increased by antibiotics and
include beneficial Clostridium. Administration of the one or more
Bacillus strain can also inhibit pathogen in the bird, such as E.
coli, Salmonella, and Clostridium. These benefits can enhance feed
conversion in poultry.
[0016] Also provided is a combination comprising two or more
isolated Bacillus strains chosen from at least one of strains
LSSAO1, 3A-P4, 15A-P4, 22C-P1, BS27, and 2084.
[0017] In addition, a method of using one or more of the Bacillus
strains provided herein is provided. In the method, an effective
amount of at least one isolated Bacillus strain is administered to
one or more bird. The administration of the strain providing at
least one of the following benefits: an increasing low G+C, gram
positive bacteria in the gastrointestinal flora of the bird and an
inhibition of a pathogen, such as E. coli, Salmonella, and
Clostridium in the bird.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Preferred exemplary embodiments of the invention are
illustrated in the accompanying drawings, in which like reference
numerals represent like parts throughout and in which:
[0019] FIG. 1 shows a BioNumerics.TM. output file showing the
presence or absence of bands in jejunal sections on denaturing
gradient gel electrophoresis (DGGE) gels.
[0020] FIG. 2 shows a BioNumerics.TM. output file showing the
presence or absence of bands in illeal sections on denaturing
gradient gel electrophoresis (DGGE) gels.
[0021] FIG. 3 is a sequence alignment of DNA sequences for the 16S
rDNA of the B1328-7, BB28-16, and BB28-6 ribotypes, which represent
samples obtained from DGGE gels from turkeys treated with a
Bacillus CSI product of the invention and two Clostridia spp. (NCBI
Accession # X73447 and M59107) known to be promoted by the growth
promoting antibiotic virginiamycin. These sequences were aligned
using ClustalW.
[0022] FIG. 4 is a graph showing the average Avian Pathogenic E.
coli (APEC) Counts in control birds versus treated birds and
demonstrates the reduction of bacterial APEC in gastrointestinal
tract samples over a 10-week Bacillus CSI product feeding period in
trial #1 of Example 5.
[0023] FIG. 5 is a graph showing cumulative percent mortality of
treated versus paired control flocks of Example 5 and represents
the cumulative percent mortality of a composite of all the control
flocks compared to all treated flocks. Each tick mark on the graph
would represent a week.
[0024] FIGS. 6 and 7 are graphs showing the percent of confirmed
Salmonella at various sites described in Example 9 from phase 1
(FIG. 6) and phase 2 (FIG. 7) of a feeding trail. A total of 120
drag swabs were taken for each phase of the feeding trial.
DETAILED DESCRIPTION
Definitions
[0025] The following definitions are intended to assist in
providing a clear and consistent understanding of the scope and
detail of the terms:
[0026] As used herein, "active metabolite" means a substance
produced by bacteria and that has antibacterial activity towards
other bacteria.
[0027] As used herein "basemix" or "concentrated basemix" refers to
Bacillus strains added to a carrier to make a basemix form. The
concentrated form is composed of the Bacillus strains added the
carrier in a more concentrated form. The basemix or concentrated
basemix forms are then be added to the feed at a desired inclusion
rate and fed to the animal.
[0028] As used herein, "performance" refers to the growth of an
animal, such as a bird, measured by the following parameters:
average daily weight gain, total weight gain, feed conversion,
which includes both feed:gain and gain:feed, feed efficiency,
mortality, and feed intake.
[0029] "An improvement in performance" as used herein, means an
improvement in at least one of the parameters listed above under
the performance definition.
[0030] In accordance with the present invention there may be
employed conventional molecular biology and microbiology within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular
Cloning: A Laboratory Manual, Third Edition (2001) Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0031] Microorganisms for treating and preventing poultry diseases
and improving performance in poultry are provided. In one
embodiment of the invention, microorganisms are Bacillus. One or
more Bacillus strains can be used. The Bacillus strain(s) can be
fed to poultry as a direct-fed microbial. Feeding one or more
Bacillus strains described herein to poultry reduces or prevents
poultry diseases and improves performance and health in
poultry.
[0032] Bacillus strains were selected based on their ability to
inhibit pathogenic bacteria and/or to mimic the effects of
antibiotics, such as virginiamycin.
[0033] Bacillus Strains
[0034] Characterization and Screening of Bacillus Strains:
[0035] Bacillus strains described herein were obtained from various
environmental samples and a library of Bacillus strains. The
Bacillus strains described herein were selected by identifying
representative pathogens present in a poultry flock. Once pathogens
were identified, Bacillus strains were screened to determine which
of them inhibit growth of the identified pathogens. Aerobic and
facultative spore-formers of the genus Bacillus were isolated.
[0036] Through necropsy sessions, pathogenic E. coli, Salmonella,
and Clostridium isolates were identified from multiple birds.
Colonies were then isolated. DNA was isolated from the colonies,
and multiplex PCR was performed. Multiplex PCR was used to
determine if the isolates are pathogenic. Through the detection of
virulence factors, the isolates were compared using RAPD-PCR. From
the RAPD-PCR results, clusters of E. coli, Salmonella, and
Clostridium were identified.
[0037] Bacillus strains were selected that inhibited representative
members from the clusters of pathogenic bacteria. For this, plates
were seeded, each with a specific pathogen from a flock. The seeded
plates were overlaid with a Bacillus strain and incubated to
determine if the Bacillus strain inhibited the pathogen that had
been seeded in the plate. After incubation, the plates were
observed for zones of inhibition for each pathogen. Colonies of
Bacillus that produced a zone of inhibition were then picked. The
isolates were grown.
[0038] 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. Furthermore, Bacillus strains are spore-formers and
thus are stable. Additionally, several species of Bacillus have
GRAS status, i.e., they are generally recognized as safe. Bacillus
species are the only spore-formers that are considered GRAS.
[0039] The Bacillus strains described herein inhibit one or more
strains of pathogenic bacteria, including E. coli, Salmonella, and
Clostridium. Multiple Bacillus strains can be combined for control
of various pathogens such as E. coli, Salmonella, and
Clostridium.
[0040] Although not intended to be a limitation to the present
disclosure, it is believed that inhibition of pathogens is
accomplished via the secretion of an active metabolite from the
Bacillus. While applicants do not wish to be restricted to a
particular theory of how an active metabolite would inhibit
microbial growth and do not intend to limit the present disclosure,
it is believed that a proteinaceous and bacteriocidal or
bacteriostatic active metabolite is secreted.
[0041] In addition to the direct inhibition of the pathogenic
bacteria in the gastrointestinal tract and production environment,
feeding Bacillus strains has also been shown to modulate the
gastrointestinal microbial communities. The changes in the
gastrointestinal communities associated with feeding Bacillus
strains has been characterized using molecular DNA techniques
including denaturing gradient gel electrophoresis. Feeding Bacillus
to poultry has been shown to result in an increase in the
gram-positive low G/C microorganisms. These low G/C gram-positive
organisms are generally related to a number of species of
non-pathogenic clostridial organisms. Identical results have been
found when feeding the antibiotic virginiamycin and may explain the
growth promoting effect of this antibiotic.
[0042] Bacillus strains identified as being useful against poultry
pathogens and for mimicking effect of antibiotics, such as
virginiamycin, include strains 3A-P4, 15A-P4, 22C-P1, BS27, 2084,
and LSSA01. These strains can be fed individually or in combination
with each other. Other Bacillus strains are also included within
the scope of the invention.
[0043] On Jan. 12, 2005, strains 3A-P4, 15A-P4, and 22C-P1 were
deposited at the American Type Culture Collection (ATCC), 10801
University Blvd., Manassas, Va. 20110-2209 and given accession
numbers PTA-6506 (3A-P4), PTA-6507 (15A-P4), and PTA-6508 (22C-P1),
respectively. Strains 2084, LSSAO1, and BS 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, Ill., 61604 and given accession
numbers NRRL B-50013, NRRL B-50104, and NRRL B-50105, 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.
[0044] Bacillus strains 3A-P4, 15A-P4, and 22C-P1 were isolated
from different geographical regions of North America and from
different environmental sources. Specifically, strain 3A-P4 was
isolated from chicken litter from Canada, strain 15A-P4 was
isolated from turkey litter from the Western United States, and
strain 22C-P1 was isolated from a swine lagoon from the Eastern
United States.
[0045] For all the Bacillus strains, growth times were determined
for production of an optimal level of the active metabolite using
the broth activity method. For this, active metabolite was added to
a culture of Clostridia or E. coli and optical densities (ODs) were
read at various time points.
[0046] Bacillus Strains as Direct-Fed Microbials:
[0047] Bacillus strains of the invention can enhance performance in
poultry. Therefore, it is economical for a producer to routinely
include one or more Bacillus strain, either individually or in
combination with other Bacillus strains, in feed not only to treat
and prevent disease but also to improve performance.
[0048] Bacillus strains can be directly fed to poultry also to
inhibit pathogenic poultry disease. Feeding microorganisms that
have GRAS status, such as Bacillus strains of the invention, to
livestock is an acceptable practice amongst producers,
veterinarians, and others in the livestock industry. By inhibiting
pathogens in poultry, the Bacillus strain(s) reduces and even
prevents disease in poultry.
[0049] Bacillus strains can be administered as a preventative to
poultry not currently infected with pathogens. In one embodiment,
one-day-old poults or chicks are fed one or more Bacillus isolates
throughout brood phase to inhibit or even prevent outbreaks of
disease and to improve performance. One or more Bacillus isolate
can also be fed at other phases also. Routine administration of the
microorganisms can dramatically reduce and even eliminate outbreaks
of disease at animal production facilities and enhance
performance.
[0050] Preparation and Feeding Bacillus Direct-Fed Microbials:
[0051] Bacillus strains of the invention can be administered as a
direct-fed microbial. Administration of one or more microorganisms
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 litter, or by direct oral
insertion, such as by an aerosol. Bacillus strains preferably are
administered as spores.
[0052] Bacillus strains 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 of the strain, 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 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.
[0053] The Bacillus strains are grown in a liquid nutrient broth,
preferably to a level at which the highest number of spores are
formed. In a preferred embodiment, the strains are grown to an OD
where the spore yield is at least 1.times.10.sup.9 colony forming
units (CFU) per ml of culture. The Bacillus strains of the present
invention are produced by fermentation of the bacterial strains.
Fermentation is 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
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 Trypticase Soy
Broth. 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. The count of the culture
can then be determined.
[0054] The count of the bacteria is important when combined with a
carrier. At the time of manufacture of the composition, the
Bacillus count preferably is at least about 1.0.times.10.sup.11
CFU/g. The counts may be increased or decreased from these base
numbers 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.
[0055] To prepare compositions, the cultures and the carrier can be
added to a ribbon or paddle mixer and mixed preferably for about 15
minutes. The components are blended such that a uniform mixture of
the carrier and cultures result. The final product is preferably a
dry flowable powder.
[0056] The preferred dosage range of the liquid drench and gel is
about 1.times.10.sup.4CFU/g or ml/day to about 1.times.10.sup.10
CFU/g or ml/day, and more preferably about 1.times.10.sup.6 CFU/g
or ml/day. The preferred dosage range of the top dress, basemix,
and premix is about 1.times.10.sup.3 CFU/g of feed to about
1.times.10.sup.8 CFU/g of feed, and more preferably about
1.times.10.sup.5CFU/g of feed. The preferred dosage range for
inclusion into water is about 1.times.10.sup.3 CFU/animal/day to
about 1.times.10.sup.10 CFU/animal/day, and more preferably about
1.times.10.sup.7CFU/animal/day. While these examples use
freeze-dried Bacillus as an ingredient in the top dress, liquid
drench, gels, water, and feed forms, 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
microorganisms can also be administered in a wet cell slurry paste,
with or without preservatives, in concentrated, unconcentrated, or
diluted form.
[0057] In one embodiment, one or more of the following Bacillus
strain are used with the strain having a count of 1.times.10.sup.5
CFU/g: strain 3A-P4, 15A-P4, 22C-P1, strain BS 27, strain 2084, and
strain LSSA01. The Bacillus strains can be combined in different
ratios to determine the best ratio to inhibit poultry pathogens and
improve performance. When used in combination, the following
exemplary, non-limiting ratios of Bacillus strains can be used: 1/3
each of three different strains; 1/4 each of four different
strains; 40% of a first strain, 40% of a second strain, and 20% of
a third strain. Other combinations of strains can also be used. In
addition, a combination having 50% more CFU per gram can be used to
boost the amount of microorganism fed to the animal. A final
bacteria count of about 1.times.10.sup.5 CFU/g to about
1.times.10.sup.6 CFU/g can be used. In one embodiment, about
7.5.times.10.sup.4 CFU/g is used.
[0058] In one embodiment, Bacillus subtilis strains BS 27, 2084,
and 22CP1 are used. These B. subtilis strains can be supplied in a
dry granular form, a liquid concentrate form, or other forms. An
exemplary count of the strains in the dry granular form is
1.18.times.10.sup.9 CFU/gram (5.34.times.10.sup.9 CFU/lb). The dry
granular form can be supplied as freeze-dried spores in a free
flowing carrier. An exemplary count of the strains in the liquid
concentrate is 1.0.times.10.sup.9 CFU/ml (6.6.times.10.sup.10
CFU/ton). The liquid concentrate can be supplied as stabilized
spores in water. The B. subtilis strains BS 27, 2084, and 22CP1 are
mixed at 33% of each strain and configured to apply
7.5.times.10.sup.4 CFU/g of the strains in the final feed. In one
embodiment, over 95% of the B. subtilis strains are in spore form.
All B. subtilis strains are GRAS. All strains are heat resistant to
160.degree. F. and can be pelleted in feed.
EXAMPLES
[0059] 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 of the invention described or
claimed herein in any fashion.
Example 1
[0060] Design.
[0061] Gastrointestinal samples from finishing phase turkeys within
a production system were sampled to examine pathogen populations
and microbial diversity.
[0062] Within the turkeys sampled were two treatments: an
experimental group that was treated with a CSI-Bacillus DFM and a
control, no treatment group. The Bacillus CSI consisted of strains
22CP1, 2084 and BS 27, with each strain consisting of one-third the
total count of 7.5.times.10.sup.4 CFU/g of feed. Three birds from
each of four houses (2 treated, 2 control) were sampled. Bird ages
for treated houses were 13 weeks and 6 weeks, and the controls
birds were 18 weeks and 14 weeks.
[0063] Bird/Intestinal Collection.
[0064] Turkey poults were sacrificed from each of the 25 sampling
houses. The birds ranged in age from 23 to 32 days. Intestinal
tracts were removed, tied off at the esophageal and cecal ends, and
immediately placed in sterile whirl-pak bags with sterile saline
covering the entire tract. Tract samples were shipped overnight to
Agtech Products for further testing. Upon arrival at Agtech, each
section of the gastrointestinal tract (D, J, and I) was treated as
an individual sample.
[0065] DNA Isolation, PCR, and DGGE.
[0066] Total genomic DNA was isolated from two 500 .mu.l aliquots
of each sample using the Roche Applied Science High Pure PCR
Template Preparation Kit per the manufacture's instructions
(Mannheim, Germany). Genomic DNA was visualized on a 0.7% agarose
gel stained with ethidium bromide (500 ng per liter ddH20). DNA
template concentration was measured using a fluorometer with
PicoGreen quantitation reagents from Molecular Probes, Inc.
(Eugene, Oreg.). The V6-V8 region of the 16S rDNA corresponding to
positions 968 to 1401 in Escherichia coli (Brosius, J., et al.
1981. Proc. Natl. Acad. Sci. USA 75:4801-4805) was amplified by
polymerase chain reaction (PCR) using the Platinum Taq DNA
polymerase from Invitrogen Life Technologies (Carlsbad, Calif.).
PCR mixtures (50 .mu.l) contained 75 ng genomic DNA, 2.5 units of
Taq polymerase, 1.times.PCR Buffer, 2.0 mM MgCl2, 200 .mu.M each
deoxynucleoside triphosphate, 50 pmol of each primer (968f+G/C
clamp 5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGAACGCGAAGAAC
CTTAC-3' (SEQ ID NO: 1) and 1401r 5'-CGGTGTGTACAAGACCC-3' (SEQ ID
NO: 2)) (Zhu et al., 2002), and water. PCR was carried out on a MJ
Research DNA Engine Opticon 2 thermocycler (Reno, Nev.). The PCR
amplification program started with an initial denaturation at
95.degree. C. for 5 min, followed by 30 cycles of incubations at
95.degree. C. for 30 s, 59.5.degree. C. for 45 s, and 72.degree. C.
for 1 min, and completed with a final extension at 72.degree. C.
for 7 min. PCR products were confirmed by electrophoresis on a 1%
agarose gel stained with ethidium bromide as previously
described.
[0067] With a BioRad Dcode Universal Mutation Detection System
(Hercules, Calif.) the PCR products were separated by DGGE
according to Muyzer et al. with the following modifications.
Electrophoresis was performed on a 5% polyacrylamide gel (37.5:1
acrylamide-bisacrylamide; dimensions 16 cm by 16 cm by 1 cm) using
a 15 to 45% linear denaturing gradient (100% denaturing solution
contained 40% (vol/vol) formamide and 7 M urea). Gels were
electrophoresed for 4 h at 130 V in 0.5.times.TAE buffer at a
constant temperature of 60.degree. C. Each gel was then stained for
10 min in ethidium bromide (250 ng per 500 ml 0.5.times.TAE) and
destained for 10 min in 0.5.times.TAE. Digital photographs were
taken using the Syngene BioImaging system (Cambridge, UK).
[0068] Analysis.
[0069] DGGE images were analyzed using Applied Maths BioNumerics
software package (Austin, Tex.). Ribotype (band) counts were
compared across treatments.
[0070] MANOVA discriminative analysis was used to compare the four
treatment groups and three ages to identify possible targets.
[0071] Target Identification.
[0072] Ribotypes identified by discriminative analysis as important
in relation to treatment were excised from the gel. The band was
purified using QIAGEN's QIAEXII kit (Valencia, Calif.) according to
manufacture's protocol. DNA quantity was increased by nested-PCR
(N-PCR) with similar PCR conditions as stated previously,
substituting the forward primer with 968f 5'-AACGCGAAGAACCTTAC-3'
(SEQ ID NO: 3). Each N-PCR sample was purified before visualization
using QIAGEN's PCR Purification Kit (Valencia, Calif.). Samples
were then sent to Lark Technologies (Houston, Tex.) for sequencing.
Resulting sequences were compared to the NCBI database with a Blast
search for preliminary identification.
[0073] Target Strains Identification.
[0074] The targets were identified using the BioLog microbial
identification system (Hayward, Calif.) according to manufactures
protocol. Each of the targets was also identified using 16S rDNA
sequencing. Briefly, primers corresponding to the region from 8 to
1406 of the 16S rDNA of Escherichia coli were amplified using the
Platinum Taq DNA polymerase from Invitrogen Life Technologies
(Carlsbad, Calif.). PCR mixtures contained 2 .mu.l of genomic DNA,
2.5 units of Taq polymerase, 1.times.PCR Buffer, 1.5 mM MgCl2, 200
.mu.M each deoxynucleoside triphosphate, 10 pmol of each primer (8f
5'-AGAGTTTGATYMTGGCTCAG-3' (SEQ ID NO: 4) and 1406r
5'-ACGGGCGGTGTGTRC-3' (SEQ ID NO: 5)), and water to a final volume
of 50 .mu.l. The PCR amplification program started with an initial
denaturation at 95.degree. C. for 5 min, followed by 32 cycles of
incubations at 94.degree. C. for 30 s, 57.5.degree. C. for 45 s,
and 72.degree. C. for 2 min, and completed with a final extension
at 72.degree. C. for 7 min on an ABI 2720 thermocycler. PCR
products were confirmed by electrophoresis on a 1% agarose gel
stained with ethidium bromide as previously described. PCR products
were then handled as previously mentioned.
[0075] The resulting DGGE images were analyzed using the
BioNumerics software using discriminative characters. The analysis
shows presence or absence of ribotypes (bands) across all selected
samples. Interestingly, the results revealed a group of ribotypes
that were more often present in the CSI-Bacillus DFM treated birds
then the controls. (Highlighted with the boxes on FIGS. 1 and 2).
This group of ribotypes was discovered in both the jejunal (FIG. 1)
and illeal (FIG. 2) portions of the tract.
[0076] DNA sequences for the 16S rDNA of the BB28-7, BB28-16, and
BB28-6 ribotypes, which represent samples obtained from DGGE gels
from turkeys treated with the CSI-Bacillus DFM, were obtained as is
described above. Referring now to FIG. 3, these sequences were
aligned using ClustalW, and were also aligned with two Clostridia
spp. (NCBI Accession # X73447 and M59107) known to be promoted by
the growth promoting antibiotic virginiamycin. See WO 2004/104175
to Lee, M. D. et al. These Clostridia spp. are beneficial bacteria.
Asterisks in the alignment denote identical sequences. The bacteria
that are associated with the CSI-Bacillus DFM treatment have a high
amount of homology to these Clostridia spp. Therefore, evidence is
provided that feeding a CSI-Bacillus DFM to finishing turkeys
naturally alters their gastrointestinal microflora similar to the
growth promoting antibiotic Virginiamycin. In summary, feeding the
CSI-Bacillus DFM to finishing turkeys naturally alters their
gastrointestinal flora similar to the growth promoting antibiotic
virginiamycin.
Example 2
Utilization of Strain 2084 in the Diets of Male Broiler
Chickens
[0077] Introduction.
[0078] A battery trial was conducted to determine the effects of
strain 2084, a Bacillus based direct-fed microbial feed additive,
on the performance of male broilers grown to 28 days of age.
[0079] Materials and Methods.
[0080] A starter and grower basal diet utilizing corn and soybean
meal as the primary ingredients were formulated to contain 105% of
the 1994 National Research Councils recommended nutrient
requirement for male broilers. Diet compositions and analyzed
nutrient content are shown in Tables 1 and 2. The test ingredient
was then added to each of these diets and blended with BMD-50 at
either 0 or 50 grams/ton drug activity. The levels of Bacillus
delivered per gram of feed for the 0.25% and 0.5% inclusion rates
were 3.75.times.10.sup.4 and 7.5.times.10.sup.4 colony forming
units. Dietary treatments were arranged in a 3.times.2 factorial to
give a total of 6 treatments. All diets were mixed for
approximately five minutes and then pelleted at 175.degree. F. The
starter diet was also crumbled. Each diet was fed to ten
replications of six male broilers of a commercial high yield strain
(Cobb 500.times.Cobb 500). The starter diets were fed from day one
to day fourteen and the grower diets were fed from day fourteen to
day twenty-eight. At the end of the starter period, the remaining
starter feed was removed and weighed before the grower feed was
added to each pen.
TABLE-US-00001 TABLE 1 Diet Composition Starter Diets Grower Diets
Ingredient g/kg g/kg Yellow Corn 583.39 627.45 Soybean Meal 312.58
285.49 Poultry Oil 39.85 45.79 Corn Gluten Meal 13.27 0.0 Dicalcium
Phosphate 17.04 15.05 Limestone 13.06 12.15 Salt 4.36 4.45 Vitamon
Premix 5.00 5.00 Trace Mineral Premix 1.00 1.00 DL Methionine 2.44
2.20 Lysine HCI 1.53 0.0 BMD-50.sup.1 0.50 0.50 Total 1000.0 1000.0
.sup.1In diets that do not contain BMD-50, corn was used to replace
the amount of BMD-50 removed from the diet.
TABLE-US-00002 TABLE 2 Analyzed Nutrient Content of the Test Diets
Diet Crude Protein Dry Matter Ash Crude Fat (BMD, strain 2084) (%)
(%) (%) (%) Starter 0, 0 23.2 88.5 4.9 5.6 Starter 0, .25 24.0 88.5
5.2 5.8 Starter 0, .50 23.6 88.1 5.0 5.6 Starter 50, 0 . . . .
Starter 50, .25 24.3 87.9 5.0 6.0 Starter 50, .5 23.6 88.3 5.0 5.5
Grower 0, 0 21.3 87.9 5.2 6.8 Grower 0, .25 21.0 87.9 5.0 6.7
Grower 0, .50 21.7 88.3 5.1 7.1 Grower 50, 0 20.0 88.0 5.1 7.3
Grower 50, .25 21.8 87.3 4.9 6.8 Grower 50, .5 21.8 87.4 4.8
6.5
[0081] Birds were group weighed by pen on days one, fourteen and
twenty-eight. Starter and grower feed consumption was also measured
by pen. All birds which died during the trial were weighed and this
weight was used to determine an adjusted feed conversion rate.
[0082] Birds were reared in start to finish battery units. Each
experimental unit was equipped with a stainless steel feed trough
and a plastic water cup. Bird comfort was maintained throughout the
trial utilizing a thermostatically controlled forced air heater and
exhaust fan. Initial room temperature was 88.degree. F.
[0083] Results were analyzed using the General Linear Means
procedure of SAS. Pen means served as the experimental unit in all
analysis. Treatment means were generated using the LSMeans
procedure and all significant means (Pr<0.05) were separated
using the repeated t-test. Mortality data was transformed using the
square root transformation to normalize data distribution.
[0084] Results:
[0085] Average weights, feed conversion, fed consumption and
mortality for day 14 and 28 are shown in Table 3. No differences
were found in initial group weights and therefore this information
was omitted. No significant interactions were observed for the
BMD-50 x strain 2084 factorial. Therefore results are summarized by
diet and by the main effects of BMD-50 and strain 2084. The
fourteen day feed-to-gain ratio was significantly influenced by
strain 2084 with the best results occurring for the 0.25% inclusion
rate. Fourteen day average body weight, feed consumption and
mortality were not significantly influenced by BMD-50 or strain
2084. Both twenty-eight day feed-to-gain and feed consumption were
significantly impacted by the inclusion of strain 2084 with the
0.50% inclusion rate supporting a significantly better feed-to-gain
rate as compared to diets which contained 0% or 0.25% strain 2084.
Results support the conclusion that the strain 2084 does improve
the feed conversion of male broiler chicks regardless of the
inclusion of dietary BMD-50. The diets used in this trial contained
a generous level of nutrients (105% of NRC recommendation) and it
may be beneficial to test the effects of dietary strain 2084 on
broilers receiving diets with a smaller safety margin of excess
nutrients.
TABLE-US-00003 TABLE 3 Table 3. Results of feeding Bacillus strain
2084 to Male Broiler Chickens Grown to 28 days of age Day 14 Day 28
Average Feed- Feed Average Feed- Feed BMD 2084 Weight to-Gain
Consumed Mortality Weight to-Gain Consumed Mortality (g/ton) (%)
(g) (g:g) (g) (%) (g) (g:g) (g) (%) 0 0 379 1.266 484 0 1235 (17)
1.510.sub.ab 1883.sub.a (29) 3.33 0 0.25 391 1.235 481 3.7 1206
(17) 1.470.sub.a 1817.sub.abc (30) 3.7 0 0.5 378 1.296 492 0 1231
(16) 1.434.sub.d 1737.sub.c (38) 7.57 50 0 398 1.253 495 1.67 1216
(16) 1.520.sub.a 1848.sub.ab (29) 1.67 50 0.25 391 1.233 482 0 1228
(19) 1.489.sub.bc 1825.sub.abc (35) 2.08 50 0.5 385 1.283 494 0
1229 (17) 1.460.sub.cd 1771.sub.bc (35) 1.67 SEM 8 0.02 8 1 0.01 2
Pr > F 0.4159 0.2017 0.6911 0.1754 0.8398 0.0001 0.048 0.3384 0
382 1.266 486 1.18 1225 1.472 1815 4.94 50 391 1.256 491 0.67 1224
1.489 1812 1.75 SEM 4.6 0.011 4 0.7 10 0.006 18 1.2 Pr > F
0.1477 0.5559 0.4706 0.6066 0.9375 0.0674 0.9147 0.0766 0 388
1.260.sub.ab 489 0.833 1226 1.515.sub.a 1865.sub.a (20) 2.5 0.25
391 1.234.sub.b 482 1.94 1217 1.480.sub.b 1820.sub.ab (22) 2.85 0.5
381 1.289.sub.a 493 0 1231 1.448.sub.c 1755.sub.b (25) 4.68 SEM 5
0.014 5 0.9 12 0.008 1 Pr > F 0.4591 0.0302 0.3532 0.3006 0.7114
0.0001 0.0065 0.541
Example 3
[0086] Bacillus subtilis Strain 2084 Broiler Pen Research
Trial.
[0087] Introduction:
[0088] In a trial conducted previously with male broiler chicks
reared in battery cages to 28 d, supplementation with strain 2084
resulted in improved feed conversion compared to birds not provided
strain 2084, and compared to those provided antibiotic (BMD)
supplementation. The current study was conducted to further
evaluate the effects of strain 2084 on the performance of broilers
to market age.
[0089] Materials and Methods:
[0090] A total of 500 male broiler chicks (Ross.times.Cobb 500)
were divided into 10 pens and fed one of two dietary treatments (5
pens/treatment) in a completely randomized design in a 48 d study.
Dietary treatments included: a control--typical commercial diet and
a treatment--control diet supplemented with strain 2084 at an
inclusion rate of 7.5.times.10.sup.4 CFU/g.
[0091] Diets were fed in four phases: a starter diet from d 0 to
14, a finisher diet from d 15 to 32, a withdrawal diet from d 33 to
43, and a final withdrawal diet from d 44 to 48. All diets were
pelleted at 85.degree. C. (185.degree. F.), and the starter diet
was crumbled. Dietary treatments were administered throughout each
phase of the trial.
[0092] Pen weights were obtained on d 0, 14, 32, 42, and 48 of the
trial, and the total number of birds within each pen was used to
determine an average body weight for individual birds. Feed
consumption for each pen was determined from d 0 to 14, d 15 to 32,
d 33 to 42, d 43 to 48. Birds were checked for mortality twice
daily, and dead weights were used to correct for feed utilization
for each pen.
[0093] Birds were maintained in an environmentally controlled room
containing pens with concrete flooring and kiln pine shavings for
litter. Room temperature was maintained at 31.degree. C.
(88.degree. F.) using thermostatically controlled heaters.
[0094] Results:
[0095] Supplementation with strain 2084 did not affect body weight,
feed intake, or feed conversion of broilers at the 14 and 32 d time
periods compared to control birds (Tables 4 and 5). However,
broilers provided Bacillus subtilis Strain 2084 had greater
(P=0.056) body weight than birds fed the control diet at d 43 of
the trial (Table 6), and improved feed/gain compared to control
birds at d 48 (Table 7).
TABLE-US-00004 TABLE 4 Evaluation of broiler chickens fed a program
with Bacillus subtilis 2084 on d 14 of the experiment. Treatment
Weight (g) Feed/Gain.sup.1 Feed Intake (g) Mortality % Control 385
1.127 437 2.40 Bacillus 2084 378 1.183 451 1.25 P Value 0.37 0.33
0.46 0.64
TABLE-US-00005 TABLE 5 Evaluation of broiler chickens fed a program
with Bacillus subtilis 2084 on d 32 of the experiment. Treatment
Weight (g) Feed/Gain.sup.1 Feed Intake (g) Mortality % Control 1604
1.434 2326 5.77 Bacillus 2084 1607 1.417 2303 3.99 P Value 0.87
0.49 0.43 0.41
TABLE-US-00006 TABLE 6 Evaluation of broiler chickens fed a program
with Bacillus subtilis 2084 on d 43 of the experiment. Treatment
Weight (g) Feed/Gain.sup.1 Feed Intake (g) Mortality % Control 2371
1.798 4430 8.87 Bacillus 2084 2413 1.753 4355 6.99 P Value 0.059
0.14 0.23 0.57
TABLE-US-00007 TABLE 7 Evaluation of broiler chickens fed a program
with Bacillus subtilis 2084 on d 48 of the experiment. Treatment
Weight (g) Feed/Gain.sup.1 Feed Intake (g) Mortality % Control 2675
1.956 6045 16.00 Bacillus 2084 2732 1.921 5594 10.52 P Value 0.77
0.024 0.34 .0.31 .sup.1Adjusted feed/gain; meaning the weight of
all mortality was included in the adjusted feed/gain
calculation.
[0096] High environmental temperatures and humidity during the last
10 days of the experiment resulted in higher mortality due to heat
stress at the d 43 and d 48 time points (Table 6 and 7). Although
not statistically significant, mortality was numerically decreased
with strain 2084 supplementation at each time point during the
trial, with final mortality decreasing by 34% in strain
2084-supplemented broilers compared to control birds (Tables
4-7).
[0097] Conclusion:
[0098] Strain 2084 supplementation improved feed/gain in broilers
evaluated to market age, and confirmed the improvement in feed
conversion observed with strain 2084 supplementation in a previous
study. Additionally, strain 2084 supplementation increased body
weight of broilers in the current experiment, and may have provide
a health benefit as indicated by numerically lower mortality rates
compared to control birds.
Example 4
[0099] The Effect of a Bacillus subtilis Feed Additive on
Improvement of Turkey Performance.
Introduction
[0100] Maintaining optimum gut health is a critical step in the
production of commercial turkeys. Poor gut health and enteritis
caused by pathogens such as E. coli and Clostridium perfringens
often the result of reduced nutrient absorption. Additionally,
managing feed costs is essential to the overall profitability of
turkeys. This evaluation demonstrated the ability of a Bacillus
based direct-fed microbial (DFM) to improve livability and body
weight of commercial turkeys. The Bacillus DFM consisted of three
strains of Bacillus subtilis selected for their ability to control
pathogenic E. coli and clostridia.
Scope of Investigation
[0101] Production data was collected. Trial 1, below, consisted of
84 barns of non-treated control turkey hens and 70 houses of hen
turkeys fed the Bacillus DFM.
TABLE-US-00008 Trial 1 Effect of Bacillus DFM feed additive on
performance of Turkeys. Treatments Performance Variable Control
Bacillus DFM Number hens finished 905,725 741,145 Livability (%)
94.16 94.66 Body Weight (lb.) 16.61 16.85 Wt. adjusted Feed 2.00
2.01 Conversion Days to Market 95.7 95.8
[0102] Trial 2, below, consisted of 140 barns of non-treated
control turkey hens and 105 houses of hen turkeys fed the Bacillus
DFM. During both trials the hen turkeys fed the Bacillus DFM did
not get the regular growth promotion antibiotic. The Bacillus feed
additive contained strains 2084, 15AP4 and LSSAO1 (added at equal
amounts) fed at a rate of 7.5.times.10.sup.4 CFU/g of feed. The
Bacillus DFM was added at the rate of one pound per ton of feed
from start to finish.
Results are presented.
TABLE-US-00009 Trial 2 Effect of Bacillus DFM feed additive on
performance of Turkeys. Treatments, Performance Variable Control
Bacillus DFM Number hens finished 1,489.285 1,130,258 Livability
(%) 94.35 94.80 Body Weight (lb.) 15.90 16.00 Wt. adjusted Feed
2.06 2.06 Conversion Days to Market 92.1 92.3
Conclusion:
[0103] This study demonstrated the ability of Bacillus DFM to
improve growth and performance. In trial 1, the improved
livability, body weight and medication cost resulted in 1:3.8
return on investment (ROI). In trial 2, the improved livability,
body weight and condemnation resulted in 1:2.7 ROI.
Example 5
Effect of Bacillus CSI on Layer Production
Bacillus CSI Formulation Development:
[0104] Gastrointestinal tracts (GIT) were collected from numerous
barns across three production sites. The GIT samples were plated
and E. coli isolates were obtained. E. coli isolates were then
tested using a multiplex PCR, those that contained two or more of
the targeted genes were determined to be pathogenic and selected
for further analysis. Eight hundred and thirty-five E. coli
isolates were deemed to be avian pathogenic and were ran analyzed
with RAPD PCR. The resulting fingerprint from each strain was then
compiled into a dendrogram and clusters based on similarity were
formed. Isolated that represent each cluster was tested to
determine inhibition by Bacillus strains. Three Bacillus strains
were found that were able to inhibit 96% of the total population of
APEC isolates represented in the dendrogram. The average inhibition
of these strains was 64%. The three strains 2084, LSSAO1, and BS27
were therefore chosen for the Bacillus CSI formula at an inclusion
rate of 7.5.times.10.sup.4 CFU/g. The strains were added at equal
amounts in this and all examples using a combination of strains.
This combination was then tested in a feeding trial situation.
Bacillus CSI Trials:
[0105] Two individual CSI trials were completed. Objectives of
these trials were to establish performance reference points and
determine the impact of Bacillus CSI in order to quantify benefits
of the Bacillus CSI program while also monitoring the bacterial
APEC challenges.
Trial 1:
[0106] Trial 1 was a trial that consisted of 9 control flocks that
did not receive any Bacillus CSI product and 10 Bacillus CSI
product treated flocks. Bovan and W36 breeds were used for this
feeding trial. Flocks included in this trial ranged in age from 16
wks to 72 wks at the start. Paired control and treated flocks that
were similar in age and breed were paired. Table 8, below, provides
information regarding the flocks used within this trial. Shown are
the complex, age, and breed of each flock along with its paired
control.
TABLE-US-00010 TABLE 8 Flock information for Trial 1 Treated Flocks
Age Paired Control Flocks Complex House Breed (weeks) Complex House
Breed BL 10 W36 72 BL 15 W36 BL 11 Bovan 54 BL 14 Bovan BL 32 Bovan
25 BL 29 Bovan BL 31 W36 17 BL 30 W36 BL 18 W36 16 BL 19 W36 BR 17
Bovan 60 BR 19 Bovan BR 2 Bovan 35 BR 32 Bovan BR 22 Bovan 31 BR 21
Bovan HP 54 Bovan 16 No pair HP 61 W36 16 HP 57 W36 HP 58 Bovan 16
HP 60 Bovan
Treated birds were fed the Bacillus CSI product for 10 consecutive
weeks. During the 10 weeks of feeding production data was gathered,
and gastrointestinal tract samples (GIT) were collected at week 2,
4, 6, 8, and 10 of the feeding period from five of the treated
flocks and 4 of their paired control flocks in order to assess
microbiological differences. Production data continued to be
gathered after the withdrawal of the Bacillus CSI product for the
remaining life of the flock in order to assess any carry over value
of use. Historical production data was also collected for Michael
Foods in order to make appropriate comparisons.
Trial 2:
[0107] Trial 2 consisted of 8 control flocks that did not receive
any Bacillus CSI product and 4 Bacillus CSI product treated flocks.
Bovan and W36 breeds were used for this feeding trial. Flocks
included in this trial ranged in age from 13 wks to 15 wks of age
at the start. Control and treated flocks were again paired based on
age and breed. Table 9, below, provides information regarding the
flocks used within this trial. Shown are the complex, age, and
breed of each flock along with its paired control.
TABLE-US-00011 TABLE 9 Flock information for Trial 2 Age Complex
House Breed (weeks) Complex House Breed HP 2 W36 15 HP 15 W36 BL 4
W36 13 BL 12 W36 HP 17 Bovan 14 HP 6 Bovan BL 15 W36 13 BL 10
W36
Treated birds were fed the Bacillus CSI product for 10 consecutive
weeks. During the 10 weeks of feeding production data was gathered,
and gastrointestinal tract samples (GIT) were collected at week 2,
4, 6, 8, and 10 of the feeding period from the four treated flocks
and the four control flocks in order to assess microbiological
differences. Historical production data continued to be collected
for Michael Foods in order to make appropriate comparisons.
[0108] Production results were combined for both feeding trials.
Production components of evaluation were: the monitoring of E. coli
populations from the gastrointestinal tracts (GIT), benchmarking
MFI historical performance, and the economic modeling of Bacillus
CSI product impact. Performance reference points included were
paired flocks of similar age and breed within the same complex. The
period of evaluation was 10 weeks of dosing plus any carry over
effect. Benchmarking was done against relative life of the flock
performance for MFI average, paired flocks and control flocks. The
life of flock impact was based on MFI average performance, which
was required to accurately assess the value of reduced early
mortality.
Results:
Microbiological:
[0109] FIG. 4 demonstrates the reduction of bacterial Avian
Pathogenic E. coli in the GIT samples over the 10 week Bacillus CSI
product feeding period in trial #1.
[0110] FIG. 5 represents the cumulative percent mortality of a
composite of all the control flocks compared to all treated flocks.
Each tick mark on the graph would represent a week.
[0111] The gross revenue generated by the use of the Bacillus CSI
product due to the reduction in early mortality due to bacterial
avian pathogenic E. coli is shown in Table 10.
TABLE-US-00012 TABLE 10 Revenue Generated: Treated versus Control
Flocks MFI/Control Variable Flocks MFI: CSI Treated Flocks Hens
Housed 140,000 140,000 Feed Cost (wk.17-110) 852,144 858,703
(increases cost due cost of CSI product) Pullet Cost 371,000
371,000 Egg Revenue through 110 wks. 2,513,974 2,543,154 (Based on
$/Dozen) Egg Revenue through 110 wks. 2,66,,560 2,685,791 (Based on
$/lb.) Gross Margin (Dozen) 1,290,831 1,313,451 Gross Margin (lbs.)
1,438,417 1,456,088 Benefit of using CSI/dozen $22,620 generated of
eggs above the control flocks Benefit of using CSI/lb of $17,671
generated eggs above the control flocks
Summary:
[0112] The Bacillus based product was successful at reducing
bacterial APEC levels in the gastrointestinal tract when directly
compared to similar control flocks. This reduction in pathogenic
organisms led to reduced mortality and morbidity in the treated
flocks. This reduction directly led to a $22,620 profit per treated
flock based on dozen per eggs produced and a $17,671 profit per
treated flock based on lbs. per egg produced
Example 6
The Effect of a Bacillus CSI Feed Additive on Reducing Necrotic
Enteritis and Improving Performance of Broilers.
Introduction
[0113] Maintaining optimum gut health is critical. Poor gut health
and enteritis, caused by pathogens such as Clostridium perfringens,
are often the result of reduced nutrient absorption. Additionally,
managing feed costs is essential to the overall profitability of
poultry. This evaluation demonstrated the ability of Bacillus CSI
product, a biological feed additive, to lower the incidence of
necrotic enteritis and improve production performance of
broilers.
Scope of Investigation
[0114] Production data was collected from a farm with four houses.
This farm had a history of necrotic enteritis and poor performance
over the last 5 years. Two houses of 30,000 broilers were fed
Bacillus CSI feed additive at one pound per ton, from start to
market (62 days). The Bacillus feed additive contained strains
2084, 15A-P4 and LSSAO1 fed at a rate of 7.5.times.10.sup.4 CFU/g
of feed. Equal amounts of the strains were used. The other two
houses of 30,000 broilers were fed a regular diet. Results are
presented in Tables 11 and 12.
TABLE-US-00013 TABLE 11 Effect of the Bacillus CSI on performance
of broilers. Treatment Performance Variable Control Bacillus CSI
Livability (%) 85.55 89.70 Body Weight (lb.) 8.25 8.14 Feed
Conversion 2.28 2.24 (after condemnation (%)) Total Condemnation
2.40 1.88 (DOA, WB and parts (%)) Days to Market 62 62
TABLE-US-00014 TABLE 12 The Economic Advantage. Bacillus CSI
Financial Performance Variable Advantage Advantage $$ Livability
(%) 4.2 $3,830 Feed Conversion 4 points $772 Condemnation 0.6 $555
Total Advantage $5,157
Conclusion
[0115] This study demonstrated the ability of the Bacillus CSI
product to control necrotic enteritis, improve livability, feed
efficiency and condemnation of broilers. Birds fed the Bacillus CSI
product did not break with clinical disease of Necrotic Enteritis.
The observed benefits of Bacillus CSI resulted in a per bird
improvement of $0.1770 over the non treated birds.
Example 7
Introduction
[0116] The incidence of cellulitis in turkeys has been increasing
in the last three years causing significant financial loss to the
turkey industry. It is a disease that affects mature turkeys close
to market age with quick onset and sudden mortality. It is caused
by Clostridium perfringens and Clostridium septicum. This
evaluation demonstrated the ability of a Bacillus CSI feed additive
to reduce the incidence of cellulitis and improve livability and
performance.
Scope of Investigation
[0117] Production data was collected from 10 barns of treated
turkey toms and 40 houses of non-treated turkey toms. During the
trial the CSI fed turkeys did not get the regular growth promotion
antibiotic. The Bacillus subtilis based CSI feed additive contained
strains 2084, LSSAO1 and 15A-P4 in equal amounts and was added at
the rate of one pound per ton of feed from start to finish to
supply the Bacillus at a rate of 4.75.times.10.sup.4 CFU/g of final
feed. The results are presented below in Tables 13 and 14.
TABLE-US-00015 TABLE 13 Effect of the Bacillus CSI feed additive on
performance of Turkeys. Treatments Performance Variable Control
Bacillus CSI Number toms finished 240,000 60,000 Livability (%)
86.23 87.04 Condemnation (%.) 2.20 1.81 Wt. adjusted Feed 2.66 2.64
Conversion
TABLE-US-00016 TABLE 14 Effect of the Bacillus CR feed additive on
performance of Turkeys. Treatments, Bacillus CSI treated
Performance Variable Previous 3 flock average flocks Number hens
finished 180.000 60,000 Livability (%) 84.86 87.04 Condemnation (%)
2.51 1.81 Wt. adjusted Feed 2.66 2.64 Conversion
Conclusion:
[0118] This study demonstrated the ability of CSI feed additive to
reduce the incidence of cellulitis and replace conventional growth
promotion antibiotics in the feed with equal or improved
performance.
Example 8
Development of a Bacillus CSI Direct-Fed Microbial and Application
for Litter Salmonella Reduction in Broilers
Introduction
[0119] The use of a direct-fed microbial product can stimulate a
host's microflora and, indirectly, its environment. A reduction in
gram-negative bacterial challenges in a host's environment can help
improve performance. In the poultry industry, Salmonella is a
serious environmental and food safety challenge. The objective of
this study is to determine if a Bacillus CSI product can impact the
Salmonella levels in the litter of broilers.
Bacillus CSI Product Development
[0120] The Bacillus CSI product \was based on the E. coli and
Clostridium perfringens that were previously collected at five
farms. A total of 1115 E. coli were collected of which, 441
contained two or more virulence genes and, therefore, were
considered to be avian pathogenic E. coli (APEC). A total of 232
Clostridium was also collected and 154 typed out as Clostridium
perfringens type A. The pathogenic E. coli and Clostridium were
used in a bacteriocin assay to develop a Bacillus feed product that
would help control these pathogens. Bacillus subtilis strains
LSSAO1, 2084 and 15A-P4 showed the best potential as a product
against both the APEC and type A Clostridium, in vitro.
Feeding Trial
[0121] A total of five farms were tested (three from the West
production system and two from the East production system) with
four houses on each farm (two treated and two control). Classically
good and poor performing farms were chosen for this study. The
Bacillus CSI was added to a standard poultry diet at an inclusion
rate of one pound per ton of finished feed. The product contained
1.5.times.10.sup.8 CFU/g, giving a final count of
4.75.times.10.sup.4 CFU/g of treated feed. The feeding trial was
repeated immediately after the first phase was completed using the
same farms on the West production system, but two different farms
on the East production system.
Salmonella Drag Swab Analysis
[0122] The incidence of Salmonella in litter was determined by
using a drag swab technique. All 20 houses were divided into three
sections and sampled in duplicate for a total of six drag swabs per
house. The swabs were added to mason jars with 90 ml sterile
peptone water and shipped overnight to Agtech at ambient
temperature. A 10 ml sample from each jar was enriched in 90 ml
Tetrothianiate broth for 24 hours at 42.degree. C. After
incubation, the enrichment was struck onto Xylose-lysine-Tergitol 4
agar (XLT4) and Brilliant Green agar with novobiocin (BGAN) and
incubated for 24 hours at 37.degree. C. Typical Salmonella colonies
appear black with a yellow halo on the XLT4 media and as a grayish
colony on the BGAN agar. Suspect Salmonella colonies were incubated
in selenite broth for 24 hours at 37.degree. C. and tested for
agglutination using the Wellcolex Colour Salmonella test to confirm
and identify the colony as Salmonella.
Salmonella Drag Swab Results
[0123] A total of 120 drag swabs were taken for each phase of the
feeding trial. FIGS. 6 and 7 show the percent of confirmed
Salmonella at each site.
Summary
[0124] The Bacillus CSI product was developed using the E. coli and
Clostridium that were previously collected at five farms. This
product was implemented into a two phase feeding trial and
Salmonella drag swabs were collected to determine if the product
could reduce Salmonella load in the litter.
[0125] In phase 1, sites 1, 2 and 5 showed a dramatic reduction in
percent Salmonella in the treated houses compared to the control
houses. In phase 2, sites 1 and 2 continued to show a reduction in
percent of Salmonella in the treated houses compared to the control
houses. The average Salmonella in phase 1 was 34.8% lower in the
treated houses compared to the control houses. The average
Salmonella in phase 2 was 13.3% lower in the treated houses
compared to the control houses. While the average Salmonella
challenge was not as high in phase 2, the treated houses continued
to show a reduction in Salmonella.
[0126] With the continued use of this Bacillus CSI product,
pathogen levels will continue to be reduced, allowing the birds an
opportunity to improve its overall gut microbial population. This
in turn can improve the bird's performance and environment and is
an important food safety measure.
Example 9
Development of a Bacillus CSI Direct-Fed Microbial and Application
for Improving Turkey Performance
CSI Development
[0127] The Bacillus CSI formulation was developed based on the E.
coli and Clostridium collected from the gastrointestinal tract of
turkeys at several of their farms. A total of 510 E. coli were
collected during three separate necropsy sessions of which, 194
contained two or more virulence genes and, therefore, are
considered to be avian pathogenic E. coli (APEC). A total of 92
Clostridium was also collected and 23 typed out as Clostridium
perfringens type A. The pathogenic E. coli and Clostridium were
used in a bacteriocin assay to develop a CSI product that would
help control these pathogens. Bacillus subtilis strains LSSAO1,
2084 and 15A-P4 showed the best potential as a product against both
the APEC and type A Clostridium, in vitro. The strains were used in
equal amounts at an inclusion rate of 7.5.times.10.sup.4 CFU/g.
Feeding Trial
[0128] The Bacillus CSI product was added to the feed. Feed mill A
manufactured diets 1 and 2 for all turkeys; therefore, all turkeys
initially received the Bacillus CSI. Feed mill B made diets 3
through market for a portion of the birds while feed mill A
produced Bacillus CSI treated feed for the remainder of the
turkeys. The turkeys fed from feed mill A throughout the trial were
considered true Bacillus CSI treated turkeys. The turkeys fed from
feed mill B that initially received Bacillus CSI for diets 1 and 2
only were the control turkeys. Bacillus CSI was added to a standard
turkey diet at an inclusion rate of one pound per ton of finished
feed. The Bacillus CSI product contained 1.5.times.10.sup.8 CFU/g,
giving a final count of 4.75.times.10.sup.4 CFU/g of treated
feed.
Performance Results
[0129] A total of ten flocks received the full Bacillus CSI
treatment and seven flocks, which are considered control, received
Bacillus CSI in diets 1 and 2 only, and then received Stafac or BMD
in the remaining diets. Table 15 shows the performance data for the
average of the treated and control flocks.
TABLE-US-00017 TABLE 15 Performance summary of average treated and
control flocks. Treated Control 14 day mortality (%) 4.06 3.99 Age
(days) 130.4 131.3 Gross weight (LB) 36.48 35.90 Average weight
(LB)* 36.82 35.90 Average Daily Gain (LB/day) 0.2797 0.2733
Adjusted Feed Conversion (LB:LB) 2.5581 2.5725 Gross Livability (%)
82.85 82.93 Net Livability (%) 82.22 82.53 Total Condemnation (%)
1.92 1.78 *Adjusted weight to 131.3 days
Microbiological Results
[0130] The gastrointestinal tract of both treated and control birds
were analyzed at 9 and 18 weeks of age to enumerate for E. coli and
Clostridium. FIG. 6 shows the total E. coli count and the confirmed
APEC count for the treated and control birds at 9 and 18 weeks of
age. At 9 weeks of age the control and treated birds had similar E.
coli counts and confirmed APEC counts. The percent of APEC in the
control birds was 21.7% and 42.0% in the treated birds. At 18 weeks
of age the E. coli counts and confirmed APEC counts were three logs
lower in the treated birds compared to control birds. The percent
of APEC in the control birds was 55.3% and 38.7% in the treated
birds.
[0131] FIG. 7 shows the total Clostridium and confirmed Clostridium
perfringens type A count for the treated and control birds at 9 and
18 weeks of age. At 9 weeks of age the control and treated birds
had similar Clostridium counts and confirmed C. perfringens type A
counts. The percent of C. perfringens type A in the control birds
was 66.0% and 27.5% in the treated birds. At 18 weeks of age the
Clostridium counts and confirmed C. perfringens type A counts were
three logs lower in the treated birds compared to control birds.
The percent of C. perfringens type A in the control birds was 90.9%
and 83.6% in the treated birds.
Summary
[0132] The comparison between treated and control flocks shows that
the Bacillus CSI product improved feed conversion by 2 pts and 0.92
lbs. This improvement may be due to a three log reduction in APEC
and Clostridium perfringens type A at 18 weeks of age. Bacillus CSI
fed the entire production phase has provided a biological tool that
can improve performance by controlling and maintaining microbial
gut populations.
[0133] The invention is not intended to be limited to the preferred
embodiments described above. Thus, the invention encompasses all
alternate embodiments that fall literally or equivalently within
the scope of these claims.
Sequence CWU 1
1
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57217DNAArtificial SequenceSynthetic DNA - primer 2cggtgtgtac
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3aacgcgaaga accttac 17420DNAArtificial SequenceSynthetic DNA -
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DNA - primer 5acgggcggtg tgtrc 156374DNAUnknownBB28-7 6tcgggaacat
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cgcaacgagc gcaaccctta tcttcagtag ccagcaattc ggttgggcac
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cagttcggat tgtagtctgc aactcgacta 300catgaagctg gaatcgctag
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg 360tcttgtacac accg
3747361DNAUnknownBB28-16 7agacaggtgg tgcatggttg tcgtcagctc
gtgtcgtgag atgttgggtt aagtcccgca 60acgagcgcaa cccttatctt cagtagccag
cgnttcggcc gggcactctg gagagactgc 120cagggataac ctggaggaag
gtggggatga cgtcaaatca tcatgcccct tatgggcagg 180gctacacacg
tgctacaatg gcgtaaacaa agggaggcan agccgcgagg ccgagcaaat
240ctcaaaaata acgtctcagt tcggattgta gtctgcaact cgactacatg
aagctggaat 300cgctagtaat cgcgaatcag aatgtcgcgg tgaatacgtt
cccgggtctt gtacacaccg 360a 3618357DNAUnknownBB28-6 8attgtttgca
tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 60gcgcaacccn
tatctttagt agccagcatt taaggtgggc actctagaga gactgccagg
120gataacttgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg
accagggcta 180cacacgtgct acaatggcgt aaacaaaggg aagcganacc
gcgaggtgga gcaaatccca 240aaaataacgt ctcagttcgg attgtagtct
gcaactcgac tacatgaagc tggaatcgct 300agtaatcgcg aatcagaatg
tcgcggtgaa tacgttcccg ggtcttgtac acaccga 3579416DNAClosterium sp.
9acatccttct gactctcctt aatcggagat ttccttcggg gacagaagtg acaggtggtg
60catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc
120cttgccttta gttgccagca ttaagttggg cactctagag ggactgccag
ggtaacctgg 180aggaaggtgg ggatgacgtc aaatcatcat gccccttatg
cttagggcta cacacgtgct 240acaatgggtg gtacagaggg cagccaagtc
gtgaggcgga gctaatccct taaagccatt 300ctcagttcgg attgtaggct
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ctgcggtgaa tgcgttcccg ggtcttgtac acaccgcccg tcacac
41610419DNAClosterium sp.misc_feature(173)..(173)n is a, c, g, or t
10acatcctttt gacctctccc taatcggaga tttcccttcg gggacagaag tgacaggtgg
60tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa
120cccttgcctt tagttgccag cattaagttg ggcactctag agggactgcc
agngataacc 180tggaggaagg tggggatgac gtcaaatcat catgcccctt
atgcttaggg ctacacacgt 240gctacaatgg gtggtacaga gggcagccaa
gtcgtgaggc ggagctaatc ccttaaagcc 300attctcagtt cggattgtag
gctgaaactc gcctacatga agctggagtt actagtaatc 360gcagatcaga
atgctgcggt gaatgcgttc ccgggtcttg tacacaccgc ccgtcacac 419
* * * * *