U.S. patent application number 14/179926 was filed with the patent office on 2014-06-12 for sporulation-deficient b. texasporus cells and methods for efficient and cost-effective inactivation and use thereof.
The applicant listed for this patent is MYGALAXY LIMITED COMPANY. Invention is credited to Yiwei Jiang.
Application Number | 20140162343 14/179926 |
Document ID | / |
Family ID | 46753436 |
Filed Date | 2014-06-12 |
United States Patent
Application |
20140162343 |
Kind Code |
A1 |
Jiang; Yiwei |
June 12, 2014 |
SPORULATION-DEFICIENT B. TEXASPORUS CELLS AND METHODS FOR EFFICIENT
AND COST-EFFECTIVE INACTIVATION AND USE THEREOF
Abstract
Novel strains and methods for their use are provided.
Particularly, foods and other oral products or treatments
containing sporulation-deficient Brevibacillus strain when
administered to a subject can inhibit or reduce the number of
pathogens in the subject and improve the health of the subject.
Inventors: |
Jiang; Yiwei; (Fort Worth,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MYGALAXY LIMITED COMPANY |
FORT WORTH |
TX |
US |
|
|
Family ID: |
46753436 |
Appl. No.: |
14/179926 |
Filed: |
February 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13406202 |
Feb 27, 2012 |
8673290 |
|
|
14179926 |
|
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|
61447703 |
Mar 1, 2011 |
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Current U.S.
Class: |
435/252.1 |
Current CPC
Class: |
A61P 3/00 20180101; A61P
31/04 20180101; A61P 3/04 20180101; A61P 31/12 20180101; Y02A
50/481 20180101; A61P 3/10 20180101; A61P 33/02 20180101; A61P
11/00 20180101; A61P 1/00 20180101; A61P 31/16 20180101; C12R 1/07
20130101; C12N 1/36 20130101; Y02A 50/30 20180101; A61P 31/14
20180101; A61K 35/742 20130101; A61P 13/02 20180101; C12N 1/20
20130101 |
Class at
Publication: |
435/252.1 |
International
Class: |
C12N 1/36 20060101
C12N001/36 |
Claims
1. A sporulation-deficient B. texasporus strain having a survival
rate less than about 3.5.times.10.sup.-3, wherein
sporulation-deficiency may be measured by subjecting a culture to a
temperature of about 50.degree. C. for about 5 minutes and
measuring the survival rate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/447,703, filed Mar. 1, 2011, the entirety of
which is incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates in general to the field of
biotechnology, more specifically, to a stress-sensitive or
sporulation-deficient strain of Brevibacillus texasporus, a method
for efficient and cost-effective cell inactivation of the B.
texasporus organism, and a feed or water additive derived from such
strain.
BACKGROUND
[0003] Citation of any document herein is not an admission that the
document is prior art, or considered material to patentability of
any claim herein, and any statement regarding the content or date
of any document is based on the information available to the
application at the time of filing and does not constitute an
affirmation or admission that the statement is correct.
[0004] Healthy animals or animals that are not infected by
pathogenic organisms, such as pathogenic bacteria, grow faster and
gain more weight per kilogram of feed. As a result, antimicrobial
compounds have been used as growth promoters in farm animals since
the 1940's. Typically, the antimicrobial compounds are administered
in feed at a subtherapeutic or low dose.
[0005] While subtherapeutic doses of antimicrobial compounds have
long been used to help farm animals maintain health and grow
faster, recent reports demonstrate a link between the use of
antibiotics and the presence of drug-resistant bacteria on the meat
produced from these animals. As a result the European Commission,
U.S. Department of Agriculture (USDA) as well as U.S. Food and Drug
Administration (FDA) have all instituted bans and guidelines on the
use of certain antibiotics as growth promoters. Currently, the
regulations typically focus on the use of antibiotics that are the
same as or similar to antibiotics used to treat humans. However,
there is growing opposition in general to the use of all antibiotic
drugs to enhance the growth of farm animals. Furthermore, the
market for organically raised meat is increasing and to be
certified organic, U.S. meat must come from animals raised without
antibiotics. As a result there is a need for a new growth enhancer
for farm animal feed.
[0006] In addition, companion animal health is a fast-growing
market. Maintaining the health of companion animals (e.g., aging
companion animals) via stimulation of innate immunity is an
attractive approach to health maintenance in companion animals, and
an orally delivered immune stimulant would be highly valued.
[0007] Brevibacillus texasporus (BT) (e.g., ATCC PTA-5854) is a
recently identified soil bacterium that produces a group of
cationic NRPS peptides (see, WO 2005/074626, and WU et al. 2005).
The cationic peptides from BT display a broad-spectrum of
antibacterial activity in vitro, killing gram positive and negative
bacteria, fungi and protozoa (WO 2005/074626).
[0008] Despite the in vitro antibacterial activity, the BT peptides
seem to lack antibacterial activity in vivo. Vancomycin-resistant
enterococci are highly sensitive to the BT peptides in vitro.
However, the BT peptides at concentrations well above the minimal
inhibition concentrations fail to decolonize commensal VRE from the
mouse GI track.
[0009] However, an isolated peptide was shown to be effective in
preventing colibacillosis and salmonellosis in chickens when used
as a feed additive. In addition, this isolated peptide was also
shown to be effective in promoting growth and increasing feed
conversion in chickens.
[0010] Perhaps more importantly, the in vivo effect of the BT
peptides appears to be independent of its in vitro antibiotic
activities, as it is effective in preventing infections in chickens
by E. coli and Salmonella at concentrations below its in vitro
minimal inhibition concentrations (JIANG et al. 2005; KOGUT et al.
2007; KOGUT et al. 2010). It is also noted that blood heterophils
and monocytes are primed for activation in chickens fed the BT
peptides, pointing to innate immunostimulation as a likely mode of
action. These features make the BT peptides an ideal feed additive
and alternative to antibiotic compounds in farm animal
production.
[0011] In addition, since innate immunity is now also known to play
key roles in controlling viral and fungal infections as well as in
preventing non-infectious diseases such as obesity/Metabolic
Syndrome and type 2 diabetes mellitus (VIJAY-KUMAR et al. 2010),
the BT innate immunity modulator should also have important
applications in these therapeutic areas.
[0012] However, the economical value of the BT peptides as a feed
additive is severely limited by the need to isolate the peptides,
which increases the cost of production to a point where it is no
longer economically viable. As an alternative to purifying the
peptides, it is possible that the entire organism (PTA-5854), as
discussed in international patent publication WO 2005/074626, could
be used directly for the production of a feed additive.
[0013] Typically, a direct-fed microbial (DFM) or probiotic strain
needs to reach the intestine in a viable form and in sufficient
numbers, which requires the survival of the strain during feed
processing and digestion (see, U.S. Pat. No. 5,480,641 and U.S.
Patent Publication 20040247568). Since feed pellet production
typically involves enough heat to significantly reduce viability,
most probiotic strains are selected for their resistance to heat
and the pH conditions found in the stomach. Currently the most
stable probiotic strains are Bacillus spores, since bacterial
spores are heat resistant and stay viable during long-term
storage.
[0014] However, since the BT organism is not believed to be a
symbiotic bacterium normally found in the intestinal track of farm
animals, it is desirable and/or necessary to inactivate the
organism before use. However, in spore producing strains this is
problematic. In addition, at least one governmental regulatory
authority considers the following criteria when assessing novel
feeds involving microbial sources: safety of the production of the
microorganism; safety of the microbial product to humans, animals
and the environment; potential impact of horizontal gene transfer;
interactions with gastrointestinal microflora; persistence in the
gut; potential impact on humans and the environment due to shedding
of viable microorganisms, particularly if there are perceived
health impacts due to contamination of the meat (Directive on
Guidelines for the Assessment of Novel Feeds: Microbial Sources,
Draft--June 2007, The Canadian Food Inspection Agency). Therefore,
when using microorganisms as a food additive there are two
conflicting desired outcomes. The first is to maintain the organism
in a viable condition throughout processing and digestion and the
second diametrically opposed desire is to completely or nearly
completely inactivate the organism without inactivation of the
active peptides. An additional benefit of inactivation is that it
removes or reduces the chances of horizontal gene transfer between
B. texasporus and microorganisms in the gut and in the environment;
eliminates or limits potential interactions with the
gastrointestinal (GI) microflora; eliminates or reduces the
potential risk on humans and the environment through shedding of
viable cells; and/or eliminates or reduces contamination of the
meat derived from the animal consuming the feed. Therefore,
continuous spore formation by PTA-5854 severely limits the ability
to use this strain as a DFM, since the spores are extremely
resistant to most methods used to inactivate vegetative cells,
mandating harsh and expensive methods for their removal.
[0015] As a result, there is a need in the art for a strain of
Brevibacillus texasporus that can be used effectively as an
inactivated DFM.
SUMMARY OF THE INVENTION
[0016] In one embodiment, the current invention is a feed or water
additive that includes inactivated cells or an inactivated culture
of a sporulation-deficient B. texasporus bacterial strain. The
invention also relates to the use of inactivated dried cells or
cultures (e.g., lyophilized or spray-dried cells or cultures) of
the invention that may be added to a feed or drinking water for one
or more animal, including, but not limited to, poultry, livestock,
cattle, swine, chicken, horse, turkey, sheep, goat, duck, quail,
Cornish game hen, pigeon, farm-raised fish, crabs, shrimp,
fresh-water turtles, dog and cat. For example, cells of a culture
of a sporulation-deficient B. texasporus strain may be inactivated
via pasteurization, starvation, temperature (heat, cold or
freezing), dehydration (e.g., heat-dry, freeze-dry, spray-dry,
sun-dry, air-dry or vacuum-dry), acidification, akalination,
alcohols, detergents, lysozyme, mechanical forces, quaternary
ammonium cations, oxidizing agents (e.g. chlorine oxide, hydrogen
peroxide, hypochlorite or ozone) and/or irradiation (e.g., UV,
X-rays or gamma rays), the inactivated cells or culture then may be
included in drinking water or an animal feed, such as a
cereal-based feed, e.g., a feed containing at least one cereal
selected from the group consisting of barley, soy, wheat,
triticale, rye, maize and combinations thereof. In fact, the
present invention may be added to a large variety of feeds. The
inactivated cells of the invention or isolated peptides may be
mixed with drinking water or a feed for livestock selected from the
group consisting of a milk replacer, a grower feed, a finisher
feed, a pre-starter feed, a starter feed, water and combinations
thereof.
[0017] The present invention also includes a method for increasing
body weight gain and feed conversion in farm and companion animals,
by inactivating (e.g., heat treatment or dehydration)
sporulation-deficient B. texasporus cells or a cell culture and
providing the inactivated sporulation-deficient B. texasporus cells
or culture media plus cells to an animal in an amount sufficient to
increase growth. Optionally, an effective amount of
sporulation-deficient B. texasporus cells or a culture media
containing the cells may be dried (dehydrated) and/or pelleted (a
form of heat treatment) into an animal feed or added to drinking
water.
[0018] Examples of feed ingredients also include cereal, soybean
meal, isolated soybean protein, isolated soybean oil, isolated
soybean fat, skimmed milk, fish meal, meat meal, bone meal, blood
meal, blood plasma protein, whey, rice bran, wheat bran, and may
further include a sweetener, a mineral, a vitamin, salt, and
grass.
[0019] In an exemplary embodiment, sporulation-deficient B.
texasporus cells are grown in a medium containing a suspension of
feed ingredients (such as corn flour and soy flour). The culture
may then be subject to cell inactivation and/or drying/dehydration
to make a feed additive (wherein the particulates in the culture
function as carriers for the BT peptides) or an enhanced feed.
[0020] The present invention also includes a method for preventing
and/or treating microbial infections in animals, by providing
inactivated sporulation-deficient B. texasporus cells or culture in
an effective amount sufficient to treat and/or prevent microbial
infections in the animal.
[0021] The present invention also includes a method for promoting
weight gain and feed conversion in growing animals, by providing
inactivated sporulation-deficient B. texasporus cells or culture in
an effective amount sufficient to treat and/or prevent microbial
infections in the animal.
[0022] The present invention also includes the use of
sporulation-deficient cells as a human food or water additive.
[0023] In an exemplary embodiment, the invention provides
sporulation-deficient strains of B. texasporus having survival
rates less than about 3.5.times.10.sup.-3, less than about
1.times.10.sup.-4, less than about 1.times.10.sup.-5, less than
about 1.times.10.sup.-6, less than about 1.times.10.sup.-7, less
than about 1.times.10.sup.-8, or less than about 1.times.10.sup.-9,
which may be measured by subjecting a culture to a temperature of
about 50.degree. C. for about 5 minutes and determining the
survival rate or colony forming ability.
[0024] In an exemplary embodiment, a sporulation-deficient B.
texasporus strain also displays a decreased survival, such as a
survival rate less than about 1.times.10.sup.-9, when a culture is
subjected to an inactivation treatment, such as starvation,
freezing (in the absence of a effective amount of a
cryoprotectant), dehydration (with a speed vacuum at 23.degree.
C.), pH extremes (e.g., pH 1.0 or pH 13.0), saturating butanol,
addition of detergent (e.g., 1% SDS) or a lysing agent (e.g.,
lysozyme), sonication, mechanical force (e.g., a French press of
animal feed pelleting machine) or hydrogen peroxide (e.g., greater
than or equal to 1% H.sub.2O.sub.2).
[0025] The invention also relates to a method of increasing the BT
peptide yield in a B. texasporus preparation by using cells derived
from a sporulation-deficient B. texasporus strain. The present
invention also relates to a method of increasing the stability and
economic utility of the BT peptides in a B. texasporus preparation
by using cells derived from a sporulation-deficient B. texasporus
strain without purification or isolation of the BT peptides.
[0026] The invention also relates to a powdered form of B.
texasporus cells, wherein the powder is substantially free of
spores. In an exemplary embodiment, a culture of
sporulation-deficient B. texasporus is grown in a liquid medium to
a sufficient cell density and the culture is dehydrated or dried to
produce a powder that is substantially free of spores. The powder
may then be mixed with water or feed ingredients to produce an
enhanced animal drink or food that does not contain a significant
number of viable spores from the B. texasporus bacteria.
[0027] The invention also relates to a method of removing or
decontaminating B. texasporus cells from the apparatus and/or
facility used for B. texasporus production by treating the
apparatus and/or facility to inactivate or kill the
sporulation-deficient B. texasporus strain, thereafter the cells
may simply be washed away. For example, steam or high temperature
water may be used to clean the apparatus and/or facility, where the
water would both remove the cells and kill them (due to the
temperature), thereby effectively removing the B. texasporus cells
from the apparatus and/or facility.
[0028] The invention also relates to a method of stimulating the
immune system in an animal or human, by administering an effective
amount of a sporulation-deficient B. texasporus strain. For
example, a sporulation-deficient B. texasporus strain may be grown,
the cells may be harvested, with or without the culture media, and
the cells may be admixed with water or additional food ingredients
to produce an enhanced drink or food that stimulates the immune
system of the animal that it is administered to. For example, a
sporulation-deficient B. texasporus strain may be inactivated and
directly fed to an animal or human to stimulate the immune system
of the animal or human. In another exemplary embodiment, the
invention relates to an enhanced food that stimulates the immune
system of an animal or human by administering an effective amount
of a sporulation-deficient B. texasporus strain and a food carrier
to the animal or human.
[0029] A sporulation-deficient B. texasporus strain may be grown
and admixed with water or food ingredients to produce an enhanced
drink or food that stimulates the immune system, for example, it
may boost the immune response to a vaccine that is administered to
the animal or human either in combination with the enhanced drink
or food or subsequent to delivering the enhanced drink or food. In
addition, a sporulation-deficient B. texasporus strain may be
inactivated and fed to an animal or human, for example the
inactivated cells may be dried, shipped to a desired processing
and/or administration site and admixed with water or food that is
to be consumed by an animal or human, wherein the food or water
does not contain viable spores from the B. texasporus strain.
[0030] The present invention also relates to a composition for
stimulating the immune system of an animal, including a human, by
administering an effective amount of a sporulation-deficient B.
texasporus strain in combination with one or more vaccines. For
example, a sporulation-deficient strain may be grown, the cells
harvested (with or without the culture media, e.g., the cells may
be washed after centrifugation), and administered to an animal
either prior to or in combination with a vaccine, wherein the cells
prime or boost the immune system's response to the vaccine.
[0031] The present invention also relates to a composition and/or
method of preventing and/or treating a disease in an animal,
comprising administering a sporulation-deficient B. texasporus
strain to an animal. For example, the treatment may involve
reducing a pathogenic microbial population in an animal. Exemplary
pathogens that may be reduced include, but are not limited to,
Acinetobacter, Bacilli, Borrelia, Campylobacter, Clostridia, E.
coli, Enterococcus, Foot-and-mouth disease virus, Gonorrhea,
Haemophilus, Influenza virus, Mannheimia, Mycobacteria, Mycoplasma,
Pasteurella, Pseudomonas, Salmonella, Staphylococcus and
Streptococcus. Exemplary diseases include, but are not limited to,
colibacillosis, salmonellosis, necrotic enteritis, coccidiosis,
influenza, foot-and-mouth disease, porcine reproductive &
respiratory syndrome, bovine shipping fever pneumonia, urinary
track infection, obesity/Metabolic Syndrome and type 2 diabetes
mellitus.
[0032] Sporulation-deficient B. texasporus strains, designated as
PTA-12307 (Strain MYG110), PTA-12308 (Strain MYG113), PTA-12309
(Strain MYG107) and PTA-12310 (Strain MYG11) were deposited on Dec.
7, 2011. All of these strains have been deposited under the
provisions of the Budapest Treaty, with the American Type Culture
Collection (ATCC), 10801 University Blvd., Manassas, Va.
20110-2209, USA) and incorporated by reference.
[0033] The present invention also relates to at least one bacterial
strain belonging to the genus Brevibacillus selected from the group
consisting of at least one of PTA-12307, PTA-12308, PTA-12309 and
PTA-12310.
[0034] One approach that can be used to inactivate the cells of the
invention involves growing the cells in an appropriate medium, then
adding feed ingredients to the culture or the culture to the feed
ingredients to dehydrate the cells.
[0035] Another approach (beside heat shock) that can be used to
inactivate the cells of the invention involves subjecting the cells
to a pH extreme, such as a pH of about 1, about 2, about 3, about
4, about 5, about 9, about 10, about 11, about 12, about 13, or
about 14.
[0036] Changes in pH sufficient to inactivate the cells of the
invention can be accomplished by addition of a base, acid and/or
other composition to effectively shift the pH beyond a
physiologically tolerated range. For example, carbon dioxide can
dissolve in water to produce carbonic acid and lower the pH to
cause inactivation of the cells.
[0037] Another approach that can be used to inactivate cells of the
invention is high pressure processing (also known as "high pressure
treatment" or "ultra-high pressure treatment" or "ultra-high
pressure sterilization"), which is a process that may involve the
application of pressures in the range of 100-1,000 MPa
(14,500-145,000 psi), or 150-600 MPa (25,000 to 90,000 psi) to
eliminate vegetative cells of bacteria, mould and the like from
products where these cells exist.
[0038] An example of a high pressure treatment is the French press
approach (French pressing), which disrupts cell by applying a
pressure to a cell suspension (up to 40,000 psi) and then suddenly
releasing the pressure. Thus, the sporulation-deficient strain of
the invention may be inactivated by the application and release of
a pressure of about 40,000 psi, 20,000, 10,000, 5,000, 2,000, 1,000
psi, and any combination thereof.
[0039] Yet another approach that can be used to inactivate the
cells of the invention involves subjecting the cells to an alcohol
at a final concentration of about 70%, about 60%, about 50%, about
40%, about 30%, about 20%, about 10%, about 5%, about 2%, or about
1% (v/v).
[0040] Yet another approach that can be used to inactivate the
cells of the invention involves subjecting the cells to a detergent
at a final concentration of about 10%, about 5%, about 2%, about
1%, about 0.5%, about 0.2%, or about 0.1% (w/v).
[0041] Yet another approach that can be used to inactivate the
cells of the invention involves subjecting the cells to dehydration
by reducing the water content in the environment surrounding B.
texasporus cells to a level below about 80%, about 70%, about 60%,
about 50%, about 45%, about 40%, about 35%, about 30%, about 25%,
about 20%, about 15%, about 10%, about 5%, about 2% or about
1%.
[0042] In an exemplary embodiment, a culture of
sporulation-deficient cells according to the invention are
prepared, treated by one or more inactivation methods, and applied
to an animal feed. For example, a culture of sporulation-deficient
cells may be grown using a high yield system and then spray dried
to produce a powder. In this scenario, spray drying is used to
inactivate the cells and produce a product that may then be added
to dried feed stocks. Alternatively, the cell culture may be
inactivated by addition of heat and/or pressure, such as during the
process of preparing pelletized feed.
[0043] In another exemplary embodiment, a culture of
sporulation-deficient cells according to the invention, such as
MYG107, MYG110, or MYG113, are grown in a liquid medium and then
added to a substantially dry mixture of animal feed ingredients or
to animal feed grain to produce a mixture having a final moisture
content between about 10% and about 25% (relative to the solids),
the moistened mixture is then run through a pellet mill to produce
pellets of the appropriate size, which are then dried to a final
moisture content, for example, about 5%. In this example, addition
of the cells to the substantially dry mixture of animal feed
ingredients provides a form of dehydration that is then accentuated
by addition of heat and the drying of the pellets. Under these
conditions it is believed that all or virtually all of the cells
will be inactivated.
[0044] In another exemplary embodiment, feed ingredients, including
#2 yellow corn, soy meal, rice hulls, rendered protein products,
salt, powdered limestone phosphate, liquid animal fat, and liquid
choline are used to produce a feed containing inactivated cells of
the invention. A batch of feed is generally prepared by mixing the
dry powdered or ground ingredients into a homogenous state and then
adding the liquid ingredients, which may include a
sporulation-deficient culture of B. texasporus cells (a form of
dehydration). The moistened mixture is conveyed to a conditioning
chamber, where steam is added (providing a form of heat shock) and
the feed becomes a mash. This mash is mixed thoroughly before
pelleting and entry into the pellet mill. In the pellet mill, the
soft hot mash is forced through a die and formed into cylinder
shaped pellets. The pellets are sent to a cooler/dryer, where a fan
pulls ambient air through the bed of pellets to remove moisture
(further dehydrate) and heat from the pellets. A conveyor and
bucket elevator may then be used to move the feed to a liquid
coating process, where animal fat is sprayed on the feed at about
2% by weight. The finished feed is then ready for use.
[0045] In another exemplary embodiment, about 1.0.times.10.sup.10,
1.0.times.10.sup.11, 1.0.times.10.sup.12, 1.0.times.10.sup.13,
1.0.times.10.sup.14, or 1.0.times.10.sup.15 inactivated cells of a
sporulation-deficient B. texasporus strain are added to about 1000
kg of animal feed.
[0046] In yet another exemplary embodiment, an effective dose of
sporulation-deficient B. texasporus cells are administered to an
animal. In yet another exemplary embodiment, an effective in-water
or in-feed dose of sporulation-deficient B. texasporus cells
comprises BT peptides between about 5 ppm and about 1000 ppm,
between about 15 ppm and about 1000 ppm, between about 20 ppm and
about 1000 ppm, between about 5 ppm and about 100 ppm, between
about 10 ppm and about 100 ppm, between about 20 ppm and about 100
ppm, between about 5 ppm and about 75 ppm, between about 10 ppm and
about 75 ppm, between about 20 ppm and about 75 ppm, between about
5 ppm and about 50 ppm, between about 10 ppm and about 50 ppm or
between about 20 ppm and about 50 ppm.
[0047] In another exemplary embodiment, dehydration of a
sporulation-deficient B. texasporus strain of the invention is
performed by; freeze drying, vacuum drying, spray drying, osmotic
dehydration, fluidized bed dehydration, solvent evaporation
dehydration, sonication assisted dehydration, microwave-assisted
dehydration, RF-assisted dehydration, and/or combinations
thereof.
[0048] In another exemplary embodiment, dehydration of the
sporulation-deficient B. texasporus cells or culture is done by
addition of a water-absorbing substance (e.g., a feed ingredient),
heat (such as generated in a animal feed pelleting process) and/or
evaporation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIG. 1 illustrates consumption of the BT peptides by spores
of PTA-5854. PTA-5854 was inoculated into LB and grown in a
37.degree. C. air shaker. Samples were taken every day and the BT
peptides were extracted and assayed for activities. One artificial
unit is 0.8 ug/ml of BT peptides. On Day 3, the culture was split
into three aliquots (#1, #2 and #3). Aliquot #1 was untreated.
Aliquot #2 was UV-irradiated in a UV cross-linker for 10 minutes.
Aliquot #3 was boiled for 10 minutes. The aliquots were then
incubated in the 37.degree. C. air shaker.
[0050] FIG. 2 shows the growth of birds during a first (1) and
second (2) trial.
[0051] FIG. 3 shows the average weight of birds combined from both
trials at day 20 (mean.+-.SE). N=39-42 of challenged birds per
treatment and N=16-19 of non-challenged birds per treatment.
*p.ltoreq.0.05
[0052] FIG. 4 shows the average feed conversion ratios from both
trials as measured for the entire feeding treatment (mean.+-.SE,
N=3 pens/treatment/trial challenged birds, N=1 pens/treatment/trial
non challenged birds) (1=trial 1, 2=trial 2). *p.ltoreq.0.05,
**p.ltoreq.0.01
[0053] FIG. 5 shows the average of the length of the small
intestine (mean.+-.SE) in Eimeria-challenged birds (N=20-24) and
for non-challenged birds (N=8) from both trials (1=trial 1, 2=trial
2).
[0054] FIG. 6 shows the average ratio of the bursa weight to the
bird weight (mean.+-.SE) in the Eimeria-challenged (N=10-12) and
non-challenged birds (N=4).
[0055] FIG. 7 shows the results of Eimeria lesion scoring
(mean.+-.SE) for the control (N=8) and in BT peptide (N=12) group.
There was statistically significant difference between treatments
(p=0.098).
[0056] FIG. 8 shows the average percentage of dry matter of the
ileum digesta (mean.+-.SE) in challenged (N=5-6 pools per
treatment) and from non-challenged birds (N=2 pools per
treatment).
[0057] FIG. 9 shows the average percentage of dry mater of the
caecal digesta (mean.+-.SE) in challenged (N=4-6 pools per
treatment) and in non-challenged birds (N=2 pools per
treatment).
[0058] FIG. 10 shows the total microbial counts (mean.+-.SE) in the
ileum from challenged birds (N=5-6 pools per treatments) and from
non-challenged birds (N=2 pools per treatment).
[0059] FIG. 11 shows the total microbial counts in the caecum
(mean.+-.SE) from challenged birds (N=5-6 pools per treatments)
and, from non-challenged birds (N=2 pools per treatment).
[0060] FIG. 12 shows the quantity of different microbes in ileum of
Eimeria-challenged birds from both trials N=5-6 pools per
treatment). P-value Control vs. BT, Trial 1=0.037.
[0061] FIG. 13 shows the quantity of different microbes in ileum of
unchallenged birds from both trials (N=2 pools per treatment). No
significant differences between the groups.
[0062] FIG. 14 shows the quantity of different microbes in caecum
of Eimeria-challenged birds from both trials N=5-6 pools per
treatment). No significant differences between the groups.
[0063] FIG. 15 shows the quantity of different microbes in caecum
of unchallenged birds from both trials N=2 pools per treatment). No
significant differences between the groups.
DETAILED DESCRIPTION OF THE INVENTION
[0064] As used herein and in the appended claims, the singular
forms, for example, "a", "an", and "the," include the plural,
unless the context clearly dictates otherwise. For example,
reference to "a B. texasporus bacteria" includes a plurality of
such bacteria, and reference to a "cell" is also a reference to a
plurality of similar cells, and equivalents thereof.
[0065] As used herein, "about" means reasonably close to,
approximately, or a little more or less than, the stated number or
amount.
[0066] As used herein, "animal" means any invertebrate or
vertebrate, including horses, goats, sheep, cattle, swine,
chickens, turkeys, game hens, geese, ducks, dogs, cats, parrots,
fish, crabs, shrimp, fresh-water turtles, humans and the like.
[0067] As used herein, "feed" or "food" means any substance or
mixture of substances containing amino acids, anti-oxidants,
carbohydrates, condiments, enzymes, fats, minerals, non-protein
nitrogen products, proteins, vitamins, and/or binders and may
contain pelletizing, coloring, foaming and/or flavoring agents,
manufactured, sold or presented for consumption by animals, such as
livestock and domestic animals, or a human, to provide at least a
part of the nutritional requirements of the animal or human, and/or
for the purpose of preventing or treating nutritional disorders in
the animal. As a vital nutrient, water is considered a feed
ingredient and an aqueous drink is a feed or food.
[0068] As used herein, "inactivate" or "inactivation" means any
treatment or stress, such as starvation, temperature shock,
dehydration/lyophilization/drying (e.g., heat-drying,
freeze-drying, spray-drying, sun-drying, air-drying and/or
vacuum-drying), pH changes (e.g., acidification or alkalination),
alcohol treatment, treatment with a detergent, filtration,
treatment with metals such as silver and zinc, pressure treatment
such as high pressure processing, enzymatic disruption (e.g.,
treatment with lysozyme), mechanical force (e.g., sonication),
treatment with quaternary ammonium cations, oxidizing agents (e.g.,
chlorine oxide, hydrogen peroxide, hypochlorite and/or ozone),
salinization, electrical field treatment, microwave treatment,
electron beam treatment, irradiation and/or combinations thereof,
that is generally unable to kill spores and that reduces the
relative colony forming efficiency (survival rate) of a culture to
less than about 1.times.10.sup.-3, less than about
1.times.10.sup.-4, less than about 1.times.10.sup.--5, less than
about 1.times.10.sup.-6, less than about 1.times.10.sup.-7, less
than about 1.times.10.sup.-8, or less than about
1.times.10.sup.-9.
[0069] As used herein, "inactivated cells or culture" of a
sporulation-deficient B. texasporus strain means a mixture of
non-viable B. texasporus cells, at least partially released
cellular content and/or culture medium ingredients. It should be
noted that inactivation of a sporulation-deficient strain of the
invention may result in the lysis of many, most or all of the
cells, depending on the inactivation conditions used. Therefore, an
"inactivated cell," "inactivated culture" and equivalent phrases,
includes within their meaning a cell lysate produced, at least in
part, by the inactivation process.
[0070] As used herein, "mutation" means any change in the sequence
of a nucleic acid, including insertions, deletions, transitions and
transvertions of one or more nucleotides. The size of the deletion
or insertion can vary from a single nucleotide to many genes.
[0071] As used herein, "phenotype" means the observed biochemical,
physiological, and/or morphological characteristics of a cell or
culture of cells.
[0072] As used herein, "sporulation-deficient" refers to a
bacterial strain that exhibits any detectable defect in the spore
formation process or product as compared to the fully
sporulation-competent, wild-type (wt) counterpart strain. The term
"sporulation-deficient" thus refers to any strain having a
sensitivity to a stress such as starvation, temperature shock,
dehydration, acidification, alkalination, alcohols, detergents,
lysozyme, mechanical forces, quaternary ammonium cations, oxidizing
agents and/or irradiation, that reduces the relative colony forming
efficiency (survival rate) of a culture to less than about
8.1.times.10.sup.-7, less than about 3.5.times.10.sup.-3, less than
about 1.times.10.sup.-4, less than about 1.times.10.sup.-5, less
than about 1.times.10.sup.-6, less than about 1.times.10.sup.-7,
less than about 1.times.10.sup.-8, or less than about
1.times.10.sup.-9, which includes sporulation-incompetent and
substantially sporulation-impaired cells, or phrased another way,
cells wherein spores are not formed because the strain is not
capable, or the cells have a diminished capability of forming
spores or cells wherein spores are formed, but the spores may not
be viable or the spores are sensitive to an inactivation stress and
rendered nonviable upon exposure thereto. Sporulation-deficiencies
may be measured by any of the methods described herein, for
example, an actively growing culture may be subject to a heat shock
of about 50.degree. C. or about 70.degree. C. for about five
minutes and the relative colony forming efficiency measured against
an untreated culture or aliquot of the culture that has not been
subject to heat shock.
[0073] "Starvation" is a nutrient deprivation that causes a
reduction in the number of active or vegetative cells (and
stimulates spore formation and/or cell death). Wild-type B.
texasporus cells undergo sporulation at least in part to survive
starvation, but sporulation-deficient B. texasporus cells cannot
avoid cell death caused by starvation. Thus, an inactivated culture
of a sporulation-deficient B. texasporus strain may be simply
obtained by inoculating the strain into a growth medium and
incubating the strain for an extended period of time such that the
cells die of starvation after the initial growth phase.
[0074] As used herein "temperature shock" means a heat of about
50.degree. C. to about 100.degree. C., cold of about 0.degree. C.
to about 16.degree. C. or freezing.
[0075] "Dehydration" is defined as any process or treatment leading
to a reduction of the water content in the environment surrounding
B. texasporus cells, osmotic shock, for example, reducing the water
content in the surrounding environment to a level below about 80%,
about 70%, about 60%, about 50%, about 45%, about 40%, about 35%,
about 30%, about 25%, about 20%, about 15%, about 10%, about 5%,
about 2% or about 1%.
[0076] An effective amount of the cells of the invention is
preferably determined by a physician or veterinarian based on the
factors such as the animal's size, age, weight and/or medical
condition.
[0077] Spore formation in PTA-5854 limits the yield level of the BT
peptides, since PTA-5854 spores appear to degrade the BT peptides.
Without wishing to be bound by theory, it appears that spore
formation and/or spore activation reduces the production of the BT
peptides by PTA-5854. As shown in FIG. 1, continued culture of
PTA-5854 in LB culture beyond Day 3 at 37.degree. C., at which time
nutrient depletion has at least begun, resulted in a depletion of
the BT peptides (#1). This BT peptide depletion could be prevented
by UV-irradiation at Day 3, which killed both active (vegetative)
cells and spores (#2). However, the BT depletion could not be
prevented by boiling at Day 3, which only killed the active cells
but did not inactivate the spores (#3).
[0078] In addition to the reduction in BT peptide levels, spore
formation in PTA-5854 prevents the most economical method of using
the strain as a feed additive. In particular, the use of the BT
peptides as a feed additive requires a low production cost, which
practically rules out any peptide purification steps. As a result,
the most economical BT-based feed additive would be a dehydrated B.
texasporus culture. Dehydration can only inactivate vegetative
cells but not the spores of B. texasporus, therefore, dehydration
cannot inactivate a sporulation proficient strain. Furthermore, the
spores from such a strain can germinate and deplete surrounding BT
peptides, where degradation of the BT peptides by the germinating
spores would also pose a serious problem for the stability of any
feed product.
[0079] Finally, a feed additive containing B. texasporus spores may
not meet regulatory standards and causes concerns regarding
spreading the producer strain. As a result it is highly desirable
to be able to effectively inactivate or eliminate spores.
[0080] However, B. texasporus continuously forms spores that are
difficult to eliminate or inactivate. Common techniques that could
be used to inactivate B. texasporus and the resulting spores
include, gamma irradiation, autoclaving and other extreme
treatments. Although gamma irradiation is effective in eliminating
spores without damaging the BT peptides in particular physical
settings, it is generally considered neither practical nor
cost-effective by industry. Autoclaving is another effective method
to eliminate spores, but the process damages the BT peptides, thus
rendering this method undesirable.
[0081] The most practical and cost-effective method of cell
inactivation in an industrial setting is a brief treatment of
moderate heat (such as Pasteurization, e.g., 70.degree. C. for 30
minutes) and/or dehydration, which the BT peptides can withstand.
Since B. texasporus spores are quite resistant to heat treatment
(the spores can withstand significant stress levels, such as
boiling at 100.degree. C. for 60 minutes, Table 1, and dehydration,
Table 2), a sporulation-deficient strain sensitive to moderate
temperature and/or dehydration, is needed for the practical and/or
cost-effective use of B. texasporus, such as for a DFM. The present
invention provides a sporulation-deficient B. texasporus strain
that can be inactivated using BT peptide-safe techniques, such as
pasteurization, pelletization, lyophilization and/or drying,
wherein the inactivation conditions are capable of inactivating
vegetative cells, but generally not wild-type spores.
[0082] Examples of techniques useful for inactivation of vegetative
cells include, but are not limited to, starvation, temperature
shock (heat such as pasteurization or cold such as freezing),
dehydration/lyophilization/drying (e.g., heat-drying,
freeze-drying, spray-drying, sun-drying, air-drying and/or
vacuum-drying), pH changes (e.g., acidification or alkalination),
alcohol treatment, treatment with a detergent, filtration,
treatment with metals such as silver and zinc, pressure treatment
such as high pressure processing, enzymatic disruption (e.g.,
treatment with lysozyme), mechanical force (e.g., sonication),
treatment with quaternary ammonium cations, oxidizing agents (e.g.,
chlorine oxide, hydrogen peroxide, hypochlorite and/or ozone),
salinization, (pulse) electrical field treatment using radio
frequency (RF) energy, microwave treatment, electron beam
treatment, irradiation (e.g. UV, X-rays or gamma rays) and
combinations thereof. In an exemplary embodiment one or more of
these techniques may be used to inactivate sporulation-deficient
cells of the invention.
[0083] According to the inventor's best knowledge, there is no
publication documenting a sporulation deficiency meeting the
perceived regulatory standard for an inactivated DFM at a stress
survival rate of <10.sup.-6, <10.sup.-7, <10.sup.-8, or
<10.sup.-9. For example, in academic publications, a bacillus
stress survival rate at 10.sup.-2 is called sporulation-deficient
(LEE et. al., 2001); whereas methods published in issued U.S. Pat.
Nos. 4,302,544, 4,450,235, 4,450,236, 4,465,773, 6,284,490 and
7,655,452 could only produce sporulation-deficient ("asporugenous"
or "asporugenic") bacillus strains with a stress survival rate on
the order of 10.sup.-7. Therefore, the art to produce bacillus
strains with a sporulation deficiency at a stress survival rate of
<10.sup.-9 seemed to be lacking. The need to create such art was
great considering the fact that the more potent gene deletion
method is not a suitable choice (and the only usable method of
mutagenesis is a chemical one which mostly causes silent or "leaky"
point mutations) for two reasons. First, B. texasporus is a
non-genetically tractable organism and a gene deletion cannot be
performed. Second, a resultant gene knockout strain would be a
genetically modified organism. (GMO) which is prohibited in human
food in regions such as European Union. In another exemplary
embodiment, the inactivation method is sufficient to reduce the
survival rate of a sporulation-deficient strain according to the
invention to less than about 1.times.10.sup.-4, less than about
1.times.10.sup.-5, less than about 1.times.10.sup.-6, less than
about 1.times.10.sup.-7, less than about 1.times.10.sup.-8, or less
than about 1.times.10.sup.-9, but wherein the inactivation method
is insufficient to reduce the survival rate of
sporulation-competent PTA-5854 below about 3.3.times.10.sup.-1 or
below about 4.3.times.10.sup.-1.
Example 1
[0084] An attempt to isolate a sporulation-deficient mutant of
PTA-5854 was made as follows. PTA-5854 cells were mutagenized with
EMS and then plated onto LB-agar. About 50,000 colonies were
screened for sensitivity to treatment at 75.degree. C. for 1 hour,
and 42 temperature sensitive candidate strains were isolated. These
initial candidate strains were colony-purified and retested for
heat sensitivity. The PTA-5854 strain and the temperature sensitive
candidate strains were grown in liquid LB medium at 37.degree. C.
for three days. The cells were then dispensed into sterile
microfuge tubes as 100 .mu.l aliquots and incubated at various
temperatures (75, 70, 65, 60, 55, 50 or 37.degree. C.) for
different lengths of time (5, 15, 30 or 60 minutes). The treated
cells were plated onto LB-agar and incubated at 37.degree. C.
overnight to determine plating efficiency. The survival rate after
a heat shock treatment was calculated as the plating efficiency
after heat shock divided by the plating efficiency at 37.degree. C.
without heat shock.
[0085] Of the initial 42 isolates, several temperature sensitive
mutants were confirmed. The strain B7 showed the best temperature
sensitive phenotype (Table 1). A brief 5-minute treatment at
50.degree. C. resulted in a survival rate of 8.1.times.10.sup.-7
(about 1.times.10.sup.-7), and higher temperatures and longer
incubations did not decrease the survival rate significantly. This
level of cell inactivation is considered inadequate for purpose of
using B. texasporus as a DFM. Repeated mutagenesis and mutant
isolations of PTA-5854 did not produce a strain having a better
temperature sensitive phenotype than B7. It was reasoned that more
than one mutation might be necessary for a tighter temperature
sensitive phenotype to allow more efficient cell inactivation.
However, mutagenesis of B7 did not yield mutants with an improved
temperature sensitive phenotype, possibly due to the poor health of
B7 (relative to the parental PTA-5854).
TABLE-US-00001 TABLE 1 Temperature sensitivities of B. texasporus
cells (as two-day old L-Broth cultures) Temperature sensitivity
Geno- Temperature Duration Survival Strain type (.degree. C.) (min)
Rate PTA-5854 Spo+++ 100 60 3.3 .times. 10.sup.-1 PTA-5854 Spo+++
75 60 4.3 .times. 10.sup.-1 B7 Spo- 50 5 8.1 .times. 10.sup.-7 B7
Spo- 75 5 1.2 .times. 10.sup.-6 MYG11 (PTA-12310) Spo+/- 50 5 3.5
.times. 10.sup.-3 MYG11 (PTA-12310) Spo+/- 75 5 3.7 .times.
10.sup.-3 MYG107 (PTA-12309) Spo--- 50 5 <10.sup.-9 MYG110
(PTA-12308) Spo--- 50 5 <10.sup.-9 MYG113 (PTA-12307) Spo--- 50
5 <10.sup.-9
[0086] A new bacterial strain (MYG11, accession number PTA-12310)
was isolated from a soil sample. S16 rDNA sequencing of this strain
shows that it is of the same species as PTA-5854, but possesses a
significantly different phenotype with regard to heat treatment.
MYG11 grows rapidly in L-Broth media at 37.degree. C. and it shows
an initial sensitivity to high temperature, or a
sporulation-deficiency. A treatment at 50.degree. C. for 5 minutes
resulted in a survival rate of 3.5.times.10.sup.-3 (Table 1).
Therefore, MYG11 was mutagenized and about 50,000 colonies were
screened for mutants displaying increased temperature sensitivity.
At least three mutants, MYG107 (accession number PTA-12309), MYG110
(accession number PTA-12308) and MYG113 (accession number
PTA-12307), were isolated by this method and found to show severe
sporulation-deficiencies. In sharp contrast to PTA-5854 and B7,
treatment at a temperature of 50.degree. C. for 5 minutes resulted
in survival rates for MYG107, MYG110 and MYG113 lower than
10.sup.-9. Thus, the invention provides for B. texasporus strains
that have a survival rate lower than about 10.sup.-8 or about
10.sup.-9 when exposed to a temperature of at least 50.degree. C.
for at least 5 minutes.
[0087] MYG107 has a doubling time of about 45 minutes in a
log-phase L-Broth culture and was further characterized. Given that
the survival rate of MYG107, MYG110 and MYG113 are at or below the
detectable limit of the cultures used in this experiment, it is
believed that MYG107, MYG110 and MYG113 have a fundamental defect
in sporulation, are highly sporulation-deficient strains, which is
further supported by the fact that the BT yield from MYG107 is
believed to be more than tripled in comparison to PTA-5854.
[0088] Moreover, typical lateral endospores are detectable in a
culture of PTA-5854 at nearly all stages. Such lateral endospores
have not been observed in cultures of MYG107 at any stage,
supporting the notion that MYG107 has a fundamental defect in
sporulation. Furthermore, MYG107 displays sensitivities to other
types of stress that inactivate vegetative cells but not spores
(Table 2).
TABLE-US-00002 TABLE 2 Stress sensitivities of B. texasporus cells
(as two-day old L-Broth cultures) Survival Stress Strain Rate
Freezing (-20.degree. C. for 1 h) PTA-5854 4.2 .times. 10.sup.-1
MYG107 <10.sup.-9 pH 1.0 (transient, at room temperature)
PTA-5854 4.5 .times. 10.sup.-1 MYG107 <10.sup.-9 pH 13.0
(transient, at room temperature) PTA-5854 5.1 .times. 10.sup.-1
MYG107 <10.sup.-9 1% Butanol (transient, at room temperature)
PTA-5854 5.6 .times. 10.sup.-1 MYG107 <10.sup.-9 1% SDS
(transient, at room temperature) PTA-5854 3.3 .times. 10.sup.-1
MYG107 <10.sup.-9 1% hydrogen peroxide (transient, at room
PTA-5854 3.3 .times. 10.sup.-1 temperature) MYG107 <10.sup.-9
Lysozyme (1 mg/ml at 37.degree. C. for 1 h) PTA-5854 7.2 .times.
10.sup.-1 MYG107 <10.sup.-9 French pressuring (three times, at
room PTA-5854 1.5 .times. 10.sup.-1 temperature) MYG107
<10.sup.-9 Starvation (for 7 days at 37.degree. C.) PTA-5854 3.3
.times. 10.sup.-3 MYG107 <10.sup.-9 Dehydration (in speed vacuum
at 23.degree. C. for 1 h) PTA-5854 3.9 .times. 10.sup.-1 MYG107
<10.sup.-9
[0089] In a freezing sensitivity test, the wild-type PTA-5854
strain and MYG107 were grown in liquid LB medium at 37.degree. C.
for two days, and the cells were then dispensed into sterile
microfuge tubes as 100 .mu.l aliquots and incubated at -20.degree.
C. for 60 minutes (or upon formation of ice). The freeze-treated
and untreated cells were plated onto LB-agar and incubated at
37.degree. C. overnight to determine plating efficiency. The
survival rate for freezing was calculated (the plating efficiency
for treated cells/the plating efficiency of untreated cells).
Freezing resulted in a survival rate about less than 10.sup.-9 for
MYG107 in comparison to the survival rate of PTA-5854 at about
4.2.times.10.sup.-1.
[0090] In an acidification sensitivity test, the wild-type PTA-5854
strain and MYG107 were grown in liquid LB medium at 37.degree. C.
for two days, and the cells were then dispensed into sterile
microfuge tubes as 100 .mu.l aliquots. The pH was adjusted to about
1.0 (at room temperature) by adding 10 .mu.l of 1 M HCl to each
aliquot and then immediately neutralized by adding 10 .mu.l of 1 M
NaOH. The acid-treated and untreated cells were plated onto LB-agar
and incubated at 37.degree. C. overnight to determine plating
efficiency. The acidification resulted in a survival rate about
less than 10.sup.-9 for MYG107 in comparison to a survival rate of
PTA-5854 at about 4.5.times.10.sup.-1.
[0091] An alkalination sensitivity test was performed in a similar
fashion except that NaOH was first added to adjust pH to 13.0 then
HCl was added for neutralization. The alkalination resulted in a
survival rate about less than 10.sup.-9 for MYG107 in comparison to
the survival rate of PTA-5854 at about 5.1.times.10.sup.-1.
[0092] An alcohol sensitivity test was performed in a similar
fashion except that butanol was added to 1% (v/v). The butanol
treatment resulted in a survival rate about less than 10.sup.-9 for
MYG107 in comparison to the survival rate of PTA-5854 at about
5.6.times.10.sup.-1.
[0093] A detergent sensitivity test was performed in a similar
fashion except that SDS was added to 1% (w/v). The SDS treatment
resulted in a survival rate about less than 10.sup.-9 for MYG107 in
comparison to the survival rate of PTA-5854 at about
3.3.times.10.sup.-1.
[0094] An oxidation sensitivity test was performed in a similar
fashion except that hydrogen peroxide was added to 1% (w/v). The
hydrogen peroxide treatment resulted in a survival rate about less
than 10.sup.-9 for MYG107 in comparison to the survival rate of
PTA-5854 at about 3.3.times.10.sup.-1.
[0095] In a lysozyme sensitivity test, the wild-type PTA-5854
strain and MYG107 were grown in liquid LB medium at 37.degree. C.
for two days, and the cells were then dispensed into sterile
microfuge tubes as 100 .mu.l aliquots. Lysozyme was added to a
concentration of 1 mg/ml and the cells were incubated at 37.degree.
C. for 60 minutes. The lysozyme-treated and untreated cells were
plated onto LB-agar and incubated at 37.degree. C. overnight to
determine plating efficiency. The lysozyme treatment resulted in a
survival rate about less than 10.sup.-9 for MYG107 in comparison to
a survival rate of PTA-5854 at about 7.2.times.10.sup.-1.
[0096] In a pressure sensitivity test, the wild-type PTA-5854
strain and MYG107 were grown in liquid LB medium at 37.degree. C.
for two days. A portion of the culture was pressure-treated (at
40,000 psi) in a French Press three times at room temperature. The
survival rate for French pressuring was calculated (the plating
efficiency of treated cells/the plating efficiency of untreated
cells). The French pressuring treatment resulted in a survival rate
about less than 10.sup.-9 for MYG107 in comparison to a survival
rate of PTA-5854 at about 1.5.times.10.sup.-1.
[0097] In a starvation sensitivity test, the wild-type PTA-5854
strain and MYG107 were grown in liquid LB medium at 37.degree. C.
for seven days. The survival rate for starvation was calculated
(the plating efficiency at Day 7/the plating efficiency at Day 2).
The starvation treatment resulted in a survival rate about less
than 10.sup.-9 for MYG107 in comparison to a survival rate of
PTA-5854 at about 3.3.times.10.sup.-1.
[0098] In a dehydration sensitivity test, the wild-type PTA-5854
strain and MYG107 were grown in liquid LB medium at 37.degree. C.
for two days, and the cells were then dispensed into sterile
microfuge tubes as 100 .mu.l aliquots and subject to a speed-vacuum
for 60 minutes at room temperature. The vacuum-dried cells
(re-hydrated with 100 .mu.l sterile distilled water) and untreated
cells were plated onto LB-agar and incubated at 37.degree. C.
overnight to determine plating efficiency. The survival rate for
vacuum-drying was calculated (the plating efficiency for treated
cells/the plating efficiency of untreated cells). The vacuum-drying
resulted in a survival rate about less than 10.sup.-9 for MYG107 in
comparison to the survival rate of PTA-5854 at about
3.9.times.10.sup.-1.
[0099] In another dehydration sensitivity test, the wild-type
PTA-5854 strain and MYG107 cells were inoculated into growth media
containing 10% and 20% soybean meal respectively. Cell growth was
achieved for both strains in medium containing 10% soybean meal but
not in the medium containing 20% soybean meal. Dilution of the 20%
soybean meal cultures with sterile distilled water (1:1) restored
growth for PTA-5854 but not MYG107, indicating that cells of a
sporulation-deficient B. texasporus strain require a water content
level of at least about 80% in the environment to sustain
viability.
[0100] The characteristics of MYG107, MYG110 and MYG113 and the
method of creating such strains, allow sporulation-deficient
strains of B. texasporus to be used as an economically feasible
DFM. In particular, the strains of the invention provide for the
use of multiple inactivation methods that are incompatible with
inactivation of wild-type B. texasporus cells (e.g., pasteurization
and/or dehydration), which provides strains that may be used as an
economically feasible DFM.
[0101] Inactivated B. texasporus cells were compared to purified
peptides to demonstrate that the inactivated cells functioned
equivalently to the purified peptides. BT peptides are produced by
actively dividing B. texasporus cells, indicating that BT peptides
are not "secondary metabolites" produced in response to starvation.
In an LB culture, 10.sup.6 BT cells contain about 10 microgram of
BT peptides. In other words, 10.sup.11 BT cells will contain about
1 gram of BT peptides.
[0102] The BT peptide concentration in inactivated B. texasporus
cells was determined and an amount of inactivated cells equivalent
to a desired concentration of purified peptide was used in the
study. Newly hatched chicks were fed with diets with different
concentrations of purified BT peptides or inactivated B. texasporus
cells for two days. On Day 3, the chicks were inoculated with an
invasive Salmonella enterica serovar Enteritidis (SE) strain. On
Day 4, the chicks were sacrificed. The liver and spleen were
harvested and homogenized. The presence or absence of the SE strain
in the homogenate was determined. Birds receiving no BT peptide or
cells were found to have an SE infection rate of 60%, Birds
receiving 12 ppm of purified peptide or an amount of the
inactivated cells equivalent to 12 ppm had SE infection rates of
28% and 52% for the purified peptide and 35% for the inactivated
cells. Birds receiving 24 ppm or the equivalent amount of the
inactivated cells had infection rates of 36% and 16% for purified
peptide and 25% with the inactivated cells. Birds receiving 48 ppm
or the equivalent amount of the inactivated cells had infection
rates of 27% and 24% for the purified peptide and 10% for the
inactivated cells.
[0103] Non-purified BT peptides in the inactivated cells displayed
the same in vivo efficacy in preventing Salmonella organ invasion
as purified proteins. Therefore, inactivated B. texasporus cells
are equivalent to purified peptides.
Example 2
[0104] In order to assess the effect of veterinary-grade BT
peptides (comprising inactivated B. texasporus cells which contain
immunomodulatory BT cationic peptides) in preventing necrotic
enteritis caused by an oral challenge of Clostridium peifringens
(type A), necrotic enteritis lesions and/or mortality were obtained
in challenge birds.
[0105] In Experiment 1, veterinary-grade BT at about 24 ppm and
about 48 ppm delivered in feed reduced necrotic enteritis lesion
scores from 2.3 to 0.6 and 0.5 respectively, mortality from 17% to
6% and 7%, respectively, and intestinal recovery of C. perfringens
from 3.60 to 2.36 and 2.48 (log.sub.10 cfu/g) respectively
(p.ltoreq.0.05).
[0106] In Experiment 2, veterinary-grade BT at about 24 ppm and
about 48 ppm delivered in feed reduced necrotic enteritis lesion
scores from 2.8 to 0.8 and 0.6 respectively, mortality from 21% to
5% and 1%, respectively, and intestinal recovery of C. perfringens
from 3.12 to 1.88 and 1.31 (log.sub.10 cfu/g) respectively
(p.ltoreq.0.05).
[0107] These results demonstrate that orally delivered inactivated
B. texasporus cells are effective in preventing necrotic enteritis
in broilers.
[0108] Results:
TABLE-US-00003 TABLE 3 Prevention necrotic enteritis in broiler
chickens with oral delivery of inactivated B. texasporus cells
Lesion Treatment.sup.1 Dosage Score.sup.2 Mortality.sup.3
Log.sub.10 cfu/g.sup.4 Experiment 1: Control diet NA 2.3 17/100
(17%) 3.60.sup.A Veterinary-grade BT 24 ppm 0.6 10/150* (6%)
2.36.sup.B Veterinary-grade BT 48 ppm 0.5 11/150* (7%) 2.48.sup.B
Experiment 2: Control diet NA 2.8 21/100 (21%) 3.12.sup.A
Veterinary-grade BT 24 ppm 0.8 5/100* (5%) 1.88.sup.B
Veterinary-grade BT 48 ppm 0.6 1/100* (1%) 1.31.sup.B
.sup.1Treatment groups represented by the 24 and 48 ppm are BT
concentrations administered from day 1. .sup.2Lesion scores are
represented by the mean of the treatment subset *n = 25) with mean
square error. .sup.3Mortality is represented by incidence data
compared to the positive control (control diet) (p < 0.05).
.sup.4Log10 cfu/g is represented by the mean of the treatment
subset (n = 10). .sup.A-BMeans within the same column with no
common superscripts differ significantly (p < 0.05).
[0109] Materials and Methods:
[0110] On day of hatch, chicks were obtained from Sanderson Farms
(Bryan) and randomly divided into experimental groups and placed
into individual floor rearing pens on clean pine shavings. All
animals received a commercial whole-wheat based broiler starter
diet that met or exceeded NRC guidelines and water ad libitum.
Birds fed either control diet, or diets containing either 24 ppm or
48 ppm of veterinary-grade BT peptides from day 1 through day
24.
[0111] Multiple isolates of C. perfringens (type A) obtained from
active field cases in Virginia, North Carolina and Georgia was used
in this investigation. The isolate was grown in thioglycollate
medium. Sterile thioglycollate medium was used to inoculate the
chickens; birds were challenged once a day for three days (days
17-19 post-hatch) by oral gavage (1.5 ml/challenge).
[0112] Each batch of C. perfringens produced for challenge was
serially diluted and plated on SFP agar plates and then placed in
the incubator at 37.degree. C. for 24 hrs. The plates were then
counted and recorded in order to determine the amount of C.
perfringens given to the birds. To quantitative measure the
recovery of C. perfringens, a section of the small intestine was
removed; this piece of intestine was cranial to Meckel's
diverticulum. Once the intestine was removed it was placed in a
whirl pak bag with 10 ml of thioglycollate and stomached for 30
seconds. Then 0.5 ml was removed and place in an anaerobic
thioglycollate and serially diluted and placed on SFP agar. The SFP
agar was prepared as per label instructions. This media is an
overlay media the initial layer was poured several days prior to
the experiment and incubated to test for contamination. The second
layer was applied the day of the necropsy in aerobic conditions,
after the sample had been plated. The plates were then transported
into the coy box, and the plates were incubated for 24 hrs and read
the following day.
[0113] To evaluate gross lesions associated with NE, the jejunum
and ileum of the small intestine were examined. Lesion scores were
recorded using the following criteria:
[0114] 0=No gross lesions, normal intestinal appearance.
[0115] 1=Thin-walled or friable, gray appearance
[0116] 2=Thin-walled, focal necrosis, gray appearance, small
amounts of gas production.
[0117] 3=Thin walled, sizable patches of necrosis, gas filled
intestine, small flecks of blood.
[0118] 4=Severe extensive necrosis, marked hemorrhage, large
amounts of gas in intestine.
[0119] This study demonstrates the effectiveness of inactivated B.
texasporus cells in priming the immune system of an animal prior to
a bacterial challenge and that a primed immune system responds more
rapidly or more effectively to such a challenge.
Example 3
[0120] Veterinary grade BT (inactivated B. texasporus cells) are
actually non-antibiotic, although the cells carry the natural BT
peptides that appear to have an antibiotic activity when measured
with an in vitro assay. However, the in vitro assay is believed to
produce an artificial result. BT does not act as an antibiotic in
vivo, instead it is believed to enhance/prime the immune system of
the animal, explaining why beneficial and commensal bacterial
levels remain relatively unaffected but the levels of detrimental
bacteria are reduced in animals treated with BT peptides or cells.
This also explains why orally delivered veterinary grade BT (in
inactivated cells) at 96 ppm had no impact in vivo on what
appeared, using the in vitro assay, to be BT-sensitive beneficial
and commensal gut bacteria (see Example 4).
Example 4
[0121] The aim of this experiment was to evaluate the safety of
feeding veterinary grade BT (inactivated Brevibacillus texasporus
cells which contain BT peptides, referred to as "BT peptide") at a
high, level of 96 ppm in broiler chickens with or without the
stress of an Eimeria maxima challenge. The feeding trial was
performed as two sequential separate trials.
[0122] Material and Methods:
[0123] Ross 508 type broiler chicks were hatched on two separate
days approximately one month apart in a commercial hatchery. In
both trials 66 cock chicks were transported to the chicken house.
Chicks were weighted and caged in groups of four or five. At day 13
after arrival in the chicken hours the birds were moved to four
rooms each with two pens, with each pen housing eight birds. One
control group and one BT peptide group was present in each room.
After the birds had been moved to the pens the E. maxima oocysts
(tap water for control birds) were tube fed into the birds'
crops.
[0124] Temperature and relative moisture were measured
automatically 24 hours a day. During the first and second
experimental day the temperature was over 30.degree. C. and then
was dropped down about 0.5.degree. C. daily until it reached
20.degree. C. Light programme was 24 h daily lights throughout the
experiment.
TABLE-US-00004 TABLE 4 The amount of birds per treatment Control BT
peptide Trial 1 32 + 2 extra bird 32 Trial 2 32 + 2 extra bird
32
[0125] The diets consisted of wheat, soybean meal, barley, rapeseed
meal, rapeseed oil, vitamins, minerals and amino acids. The
unmodified feed was obtained from Agrifood Research Finland MTT,
Jokioinen. The feed was in mesh form and no coccidiostat or enzymes
were added. Veterinary grade BT was added to the feed to the
equivalent of a BT peptide concentration of 96 ppm. The treatment
groups got the same diet throughout the experiment. The feed and
water was offered ad libitum.
TABLE-US-00005 TABLE 5 Diet composition Composition of experimental
feed g/kg diet Wheat 549.8 Barley 40.00 Soybean meal 288.00 Rapseed
meal 39.00 Monocalcium 39.00 phosphate Ca 19.00 Salt 14.00
Limestone 4.20 Vit/Min 2.00 Methionine 2.00 Lysine 1.60 Calculated
chemical composition g/kg diet DM 890.00 Crude protein 220.80 Crude
fat 60.45 Crude fiber 31.31 Ash 59.44 Ca 10.11 P 8.32 Digestible P
4.79 Na 1.72 Lys 11.91 Met 4.82 Cys 3.87 Thr 8.24 Calculated energy
content MJ/kg DM ME 12.20 Feed 1 = control feed Feed 2 = control
feed + veterinary grade BT at the equivalent of 0.096 g peptides
per kg
[0126] All the birds in three of the four rooms received E. maxima
oocysts at day 13, while the fourth room was a control room for
non-challenged birds. The dosage was approximately 25,000
sporulated oocysts per bird. The oocysts were tube-fed into the
crop in a 2 ml of tap water. The Eimeria oocysts were purchased
from the Veterinary Laboratories Agency, Surrey, United Kingdom.
One room was without the challenge.
[0127] The birds were weighed at cage basis at arrival, 6, 13, 17,
(18, and 19 in trial 2) and 20 days of age. The feed intake was
measured at 6, 13 and 20 days of age. Feed intake was measured on a
cage or pen basis. The condition of the birds was checked at least
twice a day. The mortality rate and the weight of birds that died
were recorded. Birds that were very ill or damaged birds were
humanly euthanized. At the age of 20 days, chicks were killed by
cervical dislocation and sampled for ileal and caecal digesta,
yielding 32 digesta samples per treatment and trial.
[0128] Microbial Analysis of the Digesta Samples and Cell Lysis and
Isolation of Chromosomal DNA
[0129] The recovered bacterial DNA was analysed by quantitative
PCR, allowing the quantity of different bacterial groups to be
calculated. The following bacteria were analysed Bifidobacterium
spp, E. coli, Lactobacillus spp. and the C. perfringes group
cluster I. For the determination of DNA, triplicate samples were
used, and the mean quantity of bacteria per gram fresh digesta
weight was calculated.
[0130] The total bacterial cell counts in the digesta samples were
determined by flow cytometry.
[0131] Eimeria lesion scoring was performed only for the first
trial and unbiased expert identified the lesions. The
identification of Eimeria lesions was made to the last part of the
jejunum and whole ileum. The lesions are scored in a scale from 0-4
where 0 indicates a normal intestine and 4 indicates a seriously
injured intestine.
[0132] The weight of bursas was measured from two birds from every
pool. The bursas were weighted only for the second trial.
[0133] The total length of the small intestine (jejunum+ileum) was
measured from every bird.
[0134] T-tests were calculated and used to measure the statistical
differences between the treatments.
[0135] Results:
[0136] The Eimeria challenge significantly decreased the growth of
the control birds. However, the BT peptide-supplemented diet
indicated somewhat increased growth of the birds, both in the
non-challenged and Eimeria-challenged groups compared to the
control group (FIG. 2). At day 20 the birds in BT peptide group
appeared slightly heavier but no statistically significant
difference could be identified (FIG. 3). If the weight of the
non-challenged birds at day 20 was examined separately from the
first and second trial, the weight of birds in the second trial was
significantly (p=0.026) increased by the BT peptide supplemented
diet when compared to the control group.
[0137] During the Eimeria challenge (between day 13 and 20 d) the
feed conversion ratio (FCR) was improved in the BT peptide group
when compared to the control group (p=0.02 for the first trail and
p=0.006 for the second trial) (FIG. 4).
[0138] The Eimeria challenge increased the length of small
intestine as expected. The increased length of the small intestine
may indicate compensatory growth due to the decreased nutrient
absorption. However, there was no statistically significant
difference in the average lengths of the small intestine between
the treatments (FIG. 5).
[0139] The bursa of Fabricius is an epithelial and lymphoid organ
found only in birds. The bursa is the site of hematopoiesis, and is
a specialized organ essential for the development of B cells. Bursa
develops as a dorsal diverticulum of the proctadael region of the
cloaca. For the second trial, the bursae were weighed and compared
to the birds live weight. The proportion of the weight of the
bursae when compared to the live weight of the birds appeared
slightly bigger in the non-challenged birds than the
Eimeria-challenged birds. No differences were noted between the BT
peptide supplemented and the control group. However, the bursae of
the non-challenged birds appeared somewhat bigger in the control
group than in the BT peptide group (FIG. 6).
[0140] The E. maxima mainly infect the middle region of the small
intestine, the distal jejunum and proximal ileum. No high infection
scores were detected probably because the peak of the infection was
already surpassed, when the lesion scoring was preformed, at day
seven day after the E. maxima inoculation. The average life cycle
of E. maxima is between five to six days. The average score was
lower in the BT peptide supplemented birds (p=0.098) as an
indication of faster recovery or reduced inflammation (FIG. 7).
[0141] There was no mortality in unchallenged birds with either
diet in both trials. The mortality of the Eimeria-challenged birds
was relatively high. During first trial the mortality was the
highest in control group and the lowest in the BT peptide group
(p=0.011), while the situation was reversed during the second
trial. When the mortality of both trials was combined, the over-all
mortality appeared somewhat lower in the BT peptide group than in
the control group, but the difference was not significant (Table
6). The reason for why the mortality rate was different between the
treatments in trial 1 than trial 2 could be that in trial 2, very
severely sick birds were promptly euthanized. In trial 1 none of
the birds from the BT peptide group and three birds from the
control feed group were sacrificed before the end of the trial. In
trial 2 three birds from the BT peptide group and one bird form the
control feed group were sacrificed before the end of the trial.
TABLE-US-00006 TABLE 6 Mortality (%) Mortality from Treatment Trial
1 Trial 2 both trials Control 23.5 11.8 17.6 BT 3.13 15.6 9.4
[0142] The percentage of digesta dry matter is an important
background parameter, as it indicates the health of the gut and
balance of the microbiota. The Eimeria challenge inflicted
diarrhoea to the birds and decreased the dry mater content of the
ileal and caecum digesta. These results suggest that the challenged
birds had some problem with intestinal health and that the Eimeria
challenge affects the balance of the intestinal microbiota. The
effect of the BT peptide on the ileum dry matter of Eimeria
challenged and non-challenged birds was not significant (FIG.
8).
[0143] The digesta dry mater contents of the caeca were lower in
the challenged than in the non-challenged birds. The dry mater of
the caeca of Eimeria-challenged birds may have been slightly
increased towards normal values by the BT peptide supplementation
(FIG. 9).
[0144] The total number of microbes was higher in the caecum than
ileum. The Eimeria challenge increased the total number of microbes
in the ileum in both the control and the BT peptide supplemented
groups. The numbers of microbes in the caecum decreased in both
trials and both treatment groups due to the Eimeria challenge. No
statistically significant difference between the two feed
treatments in either trial could be detected (FIGS. 10 and 11).
[0145] During these two trials, the Eimeria challenge tended to
increase the numbers of Lactobacillus spp. and Bifidobacteria spp.
in both the ileal and caecal digesta in both the control feed and
the BT peptide supplemented groups. In trial 2, there was a
statistically non-significant trend towards decreased bacterial
levels in ileum in the BT peptide group, but in trial 1, there was
an increase in the ileal levels of the C. coccoides-E. rectale
group in the BT peptide group (FIG. 12). No differences in the
unchallenged birds were observed (FIGS. 13 and 15). Bacterial
content of the caecum was unaffected by the BT (FIGS. 14 and 15).
No Salmonella spp. was detected in either trial. Taken together,
the microbiota analyses suggest that at the current dose, BT
peptide has little or no effect on native (non-pathogenic)
bacterial populations, which indicates that the BT peptides are
well tolerated by the treated animals and do not exhibit antibiotic
effects.
[0146] This experiment demonstrates that no adverse effects on
native or desired gut bacteria were detected in chickens
supplemented with the BT peptide at 96 ppm. FCR was significantly
improved in the BT peptide-supplemented group in Eimeria-challenged
broiler chicken when compared to the control group (p=0.02 and
p=0.006 for the first and second trial, respectively). The weight
of birds in the second trial was significantly (p=0.026) improved
by the BT peptide supplemented diet when compared to the control
group. The mortality of the Eimeria-challenged broilers in the
first trial was significantly (p=0.011) decreased by the BT peptide
treatment when compared to the control group. Furthermore,
supplementation of the feed by the BT peptide reduced the average
lesion scores in the infected tissue (p=0.098).
Example 5
[0147] This study is to test the use of veterinary grade BT
(inactivated B. texasporus cells) as a feed additive in swine for
the effect on weight gain, Salmonella colonization and leukocyte
function.
[0148] Materials and Methods
[0149] Experimental Design.
[0150] At weaning (17-21 days of age), piglets were randomly placed
in pens with heat lamps for additional warmth in groups of 5
individuals. Piglets were provided with water and an un-medicated
Phase I diet ad libitum that meets the National Research Council
(1994) guidelines. Body weights were recorded on Days 0, 3, 5, and
7 post-weaning. Pigs were placed into 4 experimental groups. Groups
1 and 2 each contained 5 pigs fed a control balanced unmedicated
corn and soybean meal-based diet that contained 0 ppm or 12, ppm
(repetition 1) or 24 ppm (repetitions 2, 3) BT peptides,
respectively. Groups 3 and 4 were fed diets that contained 0 or 12
(or 24) ppm BT peptides, but pigs in these groups were challenged
with Salmonella typhimurium (ST). Pigs were fed these diets
throughout the experiments. The BT peptides were delivered in the
form of inactivated B. texasporus cells as a premix.
[0151] Salmonella enterica Serovar Typhimurium (ST).
[0152] An isolate of S. enterica serovar typhimurium (ST) (National
Veterinary Sciences Laboratory [NVSL]), Ames, Iowa (PT 24) was used
and it is a strain that has been selected for resistance to
carbenicillin and novobiocin (CN) and is maintained in TSB or
tryptic soy agar at 4.degree. C. Brilliant Green Agar (BGA), a
selective culture media for Salmonella, was used to culture the
resistant isolate in experimental studies and contained 100
.mu.g/ml carbenicillin and 25 .mu.g/ml novobiocin (i.e., BGA+CN) to
inhibit growth of other bacteria. Inoculum for challenge was
prepared from 18 to 24 h (TSB+CN cultures maintained at 41.degree.
C. and diluted in sterile PBS (pH 7.2). A stock solution
(1.times.10.sup.9 cfu/ml) was prepared for challenge
experiments.
[0153] At day 3 post-weaning, piglets in challenge groups were
administered, by oral gavage, 10.sup.7 cfu ST. Five days
post-challenge (7 days post-wean), pigs were euthanized and 1.0 g
samples of cecal contents from each pig were collected aseptically.
The cecal samples were serially diluted and spread-plated on
brilliant green agar (BGA) plates. The plates were then incubated
for 24 h at 37 C and the number of cfu of ST per gram of cecal
contents determined. In addition, tissues (liver, spleen,
ileo-cecal lymph nodes, cecum and rectum will be cultured for the
presence of ST using established methods. Suspect Salmonella
colonies were confirmed by biochemical tests on triple sugar agar
and lysine iron agar and further confirmed as ST by using
Salmonella O antiserum. Salmonella colony plate counts are
expressed as log.sub.10 Salmonella per gram of cecal contents.
[0154] Innate Immune Response in Weaned Pigs.
[0155] Leukocytes were isolated using density gradient separation
as previously described (KOGUT et al. 2010). Functional assays were
performed to assess the capabilities of neutrophils isolated from
pigs receiving the different treatments. Two methods of microbial
killing used by neutrophils were investigated. Specifically,
neutrophil degranulation, the release of bactericidal products from
granules inside the neutrophil, and the oxidative burst, the
production of bactericidal reactive oxygen species (ROS) were
assayed. Briefly, the assays are described below.
[0156] Functional Assay.
[0157] Leukocyte oxidative burst was measured by oxidation of
DCFH-DA to fluorescent DCF as described by (HE et al. 2003).
Leukocytes are stimulated with phorbol A-myristate 13 acetate (PMA
[2.0 .mu.g/ml], Sigma) or RPMI 1640 media for 60 minutes prior to
measurement. Cells were placed in 96-well plates and fluorescence
measured using a GENios Plus Fluorescence Microplate Reader
[(485/530 nm) Tecan US Inc., Research Triangle Park, N.C.
[0158] Statistical Analysis:
[0159] Statistical analysis was performed on data using
SigmaStat.RTM. statistical software (Jandel Scientific, San Rafael,
Calif., USA). Differences between the experimental groups will be
determined using the Student T test. P.ltoreq.0.05 was considered
to be statistically significant.
[0160] Results.
[0161] From the data presented below, it appears pigs fed the BT
cells had greater average daily gains in weight (ADG) than did pigs
fed the control diet, even in the presence of a Salmonella
infection (at 24 ppm) (Table 7).
TABLE-US-00007 TABLE 7 Average daily gain. Rep. 3, 24 ppm BT.
Values in pounds (lbs). Data from weights collected on days 0, 3,
5, and 7 post weaning from individual pigs was pooled for each time
point. Data represents the average gain +/- standard deviation.
Average daily gain (lbs) Treatment Repetition 2 Repetition 3 Cont
1.7 .+-. 0.3 0.04 .+-. 0.12 Cont Pept 24 ppm 4.2 .+-. 0.7 0.15 .+-.
0.06 ST Cont 3.2 .+-. 0.4 0.14 .+-. 0.09 ST Pept 24 ppm 4.6 .+-.
0.5 0.18 .+-. 0.06
[0162] Some reductions in fecal shedding of ST were observed as
shown in the rectal swab data (Table. 8). In Rep. 1, no difference
was seen in fecal shedding at 12 ppm BT, except on Day 5. In Rep.
2, control and BT at 24 ppm were similar in ST shedding, except on
Day 4, where the BT 24 ppm group had more pigs positive than the
control group. In Rep. 3, the BT group at 24 ppm had less pigs
shedding ST than did the control group on all days measured. In
Rep. 3, pigs were followed for 7 days post-infection instead of 5
days, as in Rep. 1 and 2.
TABLE-US-00008 TABLE 8 Daily Rectal swab data Number of pigs
positive for ST Rep 1 Rep 2 Rep 3 Days Post- BT 12 BT 24 BT 24
infection Control ppm Control ppm Control ppm 1 2 3 4 3 5 2 2 5 5 4
4 3 1 3 5 5 3 4 4 2 4 4 5 2 5 4 1 5 3 1 1 1 5 2 6 -- -- -- -- 5 1 7
-- -- -- -- 4 3
[0163] Pigs fed the peptide had less Salmonella in the
organs--lymph nodes, liver, spleen--as compared to the control pigs
overall, but did not show reductions in the gut tissues--ileum
(except rep 3), cecum, rectum. In Rep. 1, the BT group at 12 ppm
had fewer pigs positive for ST in both the liver and the spleen,
but recovery of ST was the same in the lymph nodes and the cecum
and rectum (Table 9). In Rep. 2, the BT group had fewer pigs
positive in all tissues, except the cecum. In Rep. 3, fewer pigs in
the BT group were positive for ST in the lymph nodes, spleen,
ileum, cecum, and rectum. Colonization data (CFUs) was
indeterminate due to low recovery of ST (data not shown). It
appears the peptide had some positive effects on Salmonella
invasion and colonization in weaned pigs.
TABLE-US-00009 TABLE 9 Salmonella isolated from tissues Number of
pigs positive for ST Rep 1 Rep 2 Rep 3 BT 12 BT 24 BT 24 Tissue
Control ppm Control ppm Control ppm Ileocecal 3 3 5 1 4 2 lymph
node Liver 3 1 5 1 2 2 Spleen 2 0 3 1 1 2 Ileum -- -- 5 4 4 0 Cecum
4 4 5 5 5 4 Rectum 5 5 5 4 4 3
[0164] Leukocytes from pigs fed the BT sporulation-deficient strain
had a significantly higher oxidative burst response (Table 10) than
did leukocytes from control pigs on days 3, 5, and 7 of being fed
the BT peptide (P<0.05).
TABLE-US-00010 TABLE 10 Swine leukocyte oxidative burst activity.
Pigs were fed respective diets throughout study period (24 ppm BT).
Pigs were bled on days 3, 5, and 7 post-weaning. Data represents
two repetitions, pooled from 10 pigs/group. Data expressed as the
mean +/- standard deviation. RFU = reflective fluorescent units (in
1,000). Treatment Day 3 Day 5 Day 7 Cont 0.8 .+-. 0.1 1.2 .+-. 0.1
1.5 .+-. 0.3 Pept 1.5 .+-. 0.2 1.7 .+-. 0.3 2.2 .+-. 0.2 Cont PMA
23.0 .+-. 1.3 14.2 .+-. 1.0 30.4 .+-. 3.3 Pept PMA 39.2 .+-. 4.8
38.0 .+-. 3.5 40.5 .+-. 5.3
[0165] The data suggest that inactivated B. texasporus cells are an
effective immunomodulator in swine with potential positive effects
on weight gain and the carriage of Salmonella in the gut and
tissues.
REFERENCES
[0166] All references, including publications, patents, patent
applications and deposited strains, cited herein are hereby
incorporated by reference to the same extent as if each reference
were individually and specifically indicated to be incorporated by
reference and were set forth in its entirety herein, including, but
not limited to: [0167] JIANG, Y. W., M. D. SIMS and D. P. CONWAY,
2005 The efficacy of TAMUS 2032 in preventing a natural outbreak of
colibacillosis in broiler chickens in floor pens. Poult Sci 84:
1857-1859; [0168] KOGUT, M. H., K. J. GENOVESE, H. HE, M. A. LI and
Y. W. JIANG, 2007 The effects of the BT/TAMUS 2032 cationic
peptides on innate immunity and susceptibility of young chickens to
extraintestinal Salmonella enterica serovar Enteritidis infection.
Int Immunopharmacol 7: 912-919; [0169] WU, X., J. BALLARD and Y. W.
JIANG, 2005 Structure and biosynthesis of the BT peptide antibiotic
from Brevibacillus texasporus. Appl Environ Microbiol 71:
8519-8530; [0170] KOGUT, M. H., H. HE, K. J. GENOVESE, and Y. W.
JIANG, 2010 Feeding the BT Cationic Peptides to Chickens at Hatch
Reduces Cecal Colonization by Salmonella enterica Serovar
Enteritidis and Primes Innate Immune Cell Functional Activity.
Foodborne Pathog Dis. 7(1): 23-30; [0171] VIJAY-KUMAR, M., AITKEN,
J., CARVALHO, F., CULLENDER, T., MWANGI, S., SRINIVASAN, S.,
SITARAMAN, S., KNIGHT, R., LEY, R., & GEWIRTZ, A. (2010).
Metabolic Syndrome and Altered Gut Microbiota in Mice Lacking
Toll-Like Receptor 5 Science, 328 (5975), 228-231; [0172] HE, H.,
M. B. FARNELL, M. H. KOGUT, 2003 Inflammatory agonist stimulation
and signal pathway of oxidative burst in neonatal chicken
heterophils. Comparative. Biochemistry and Physiology A, 135,
177-184.
* * * * *