WO2016083481A1 - Souches et procédés permettant une segmentation énergétique chez les ruminants - Google Patents

Souches et procédés permettant une segmentation énergétique chez les ruminants Download PDF

Info

Publication number
WO2016083481A1
WO2016083481A1 PCT/EP2015/077704 EP2015077704W WO2016083481A1 WO 2016083481 A1 WO2016083481 A1 WO 2016083481A1 EP 2015077704 W EP2015077704 W EP 2015077704W WO 2016083481 A1 WO2016083481 A1 WO 2016083481A1
Authority
WO
WIPO (PCT)
Prior art keywords
animal
strain
nrrl
bacillus pumilus
cows
Prior art date
Application number
PCT/EP2015/077704
Other languages
English (en)
Inventor
Elizabeth GALBRAITH
Keith Mertz
Ajay AWATI
Original Assignee
Dupont Nutrition Biosciences Aps
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US14/554,726 external-priority patent/US10172892B2/en
Application filed by Dupont Nutrition Biosciences Aps filed Critical Dupont Nutrition Biosciences Aps
Priority to US15/528,660 priority Critical patent/US20170258111A1/en
Priority to AU2015352558A priority patent/AU2015352558A1/en
Priority to BR112017011067A priority patent/BR112017011067A2/pt
Priority to CN201580074305.XA priority patent/CN107205435A/zh
Priority to EP15808549.8A priority patent/EP3267802A1/fr
Priority to MX2017006886A priority patent/MX2017006886A/es
Priority to CA2968734A priority patent/CA2968734A1/fr
Publication of WO2016083481A1 publication Critical patent/WO2016083481A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants

Definitions

  • the invention relates to strains and methods for controlling acidosis. More particularly, the invention relates to bacterial strains useful for improving ruminant health and/or performance and methods of making and using the strains.
  • Ionophores inhibit intake and reduce the production of lactic acid in the rumen by reducing the ruminal populations of gram-positive, lactic acid-producing organisms such as Streptococcus bovis and Lactobacillus spp. (Muir et al. 1981).
  • direct-fed microbials as a method to modulate ruminal function and improve cattle performance has been gaining increased acceptance over the past 10 years.
  • the first approach i.e., using lactic acid producing DFM technology, attempts to increase the rate of ruminal lactic acid utilization by stimulating the native ruminal microbiota.
  • the addition of relatively slow growing lactic acid producing bacteria, such as species of Enterococcus produces a slightly elevated concentration of ruminal lactic acid.
  • the gradual increase forces the adaptation of the ruminal microflora to a higher portion of acid tolerant lactic acid utilizers.
  • these Enterococcus strains failed to adequately control and prevent acidosis.
  • the second approach i.e., adding specific bacterial species capable of utilizing ruminal lactic acid, is based on the finding that species of Propionibacterium significantly minimize the accumulation of ruminal lactic acid during an acidosis challenge with a large amount of Readily Fermentable Carbohydrate (RFC).
  • RRC Readily Fermentable Carbohydrate
  • Propionibacterium are natural inhabitants of the rumen in both dairy and beef cattle and function in the rumen by using lactic acid to produce important volatile fatty acids like acetate and propionate.
  • Isolated strains are provided, including Enterococcus faecium strain 8G-1 (NRRL B-50173), a strain having all of the identifying characteristics of Enterococcus faecium strain 8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172), a strain having all of the identifying characteristics of Enterococcus faecium strain 8G-73 (NRRL B-50172), Bacillus pumilus strain 8G-134 (NRRL B- 50174), a strain having all of the identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174), and combinations thereof.
  • the administration of the one or more strain to the animal provides at least one of the following benefits in or to the animal when compared to an animal not administered the strain: (a) reduces acidosis, (b) stabilizes ruminal metabolism as indicated by delayed lactic acid accumulation and prolonged production of volatile fatty acids, (c) recovers more quickly from acidosis challenge as measured by pH recovery and lactic acid decline, (d) reduces exhibition of clinical signs associated with acidosis (e) increased milk production in lactating dairy cows, (f) increased milk fat content in lactating dairy cows, (g) decreased somatic cell count (SCC) in lactating dairy cows, (h) improved immunological response and health as evidenced by decreased SCC and (i) increased efficiency of milk production in lactating dairy cows.
  • benefits in or to the animal when compared to an animal not administered the strain: (a) reduces acidosis, (b) stabilizes ruminal metabolism as indicated by delayed lactic acid accumulation and prolonged production of volatile fatty acids, (c) recovers more quickly
  • the strains, compositions, and methods can be used with any animal including but not limited to a postpartum animal, a peripartum animal, bovine, cattle, bull, steer, heifer, calf, cow, dairy cow, postpartum dairy cow, peripartum dairy cow, ovine, sheep, llama, alpaca, caprine, goat, ruminants, porcine, pig, swine, hog, avian species, turkey, fowl, chicken, and hen.
  • a postpartum animal a peripartum animal, bovine, cattle, bull, steer, heifer, calf, cow, dairy cow, postpartum dairy cow, peripartum dairy cow, ovine, sheep, llama, alpaca, caprine, goat, ruminants, porcine, pig, swine, hog, avian species, turkey, fowl, chicken, and hen.
  • a strain selected from the group consisting of Enterococcus faecium strain 8G-1 (NRRL B- 50173), Enterococcus faecium strain 8G-73 (NRRL B-50172), and Bacillus pumilus strain 8G- 134 (NRRL B-50174) is grown in a liquid nutrient broth.
  • the strain is separated from the liquid nutrient broth to make the direct-fed microbial.
  • the strain is freeze dried.
  • a method of making a direct-fed microbial is provided.
  • a strain selected from the group consisting of Enterococcus faecium strain 8G-1 (NRRL B-50173), Enterococcus faecium strain 8G-73 (NRRL B-50172), and Bacillus pumilus strain 8G-134 (NRRL B-50174) is grown in a liquid nutrient broth.
  • the strain is separated from the liquid nutrient broth to make the direct-fed microbial.
  • Monensin is added to the direct- fed microbial.
  • the disclosure relates to a method for reducing negative energy balance in an animal comprising: (a) identifying an animal at risk for negative energy balance; and (b) administering to the animal identified in step (a) an effective amount of Bacillus pumilus strain 8G-134 (NRRL B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174) to reduce negative energy balance.
  • a method for reducing negative energy balance in an animal comprising: (a) identifying an animal at risk for negative energy balance; and (b) administering to the animal identified in step (a) an effective amount of Bacillus pumilus strain 8G-134 (NRRL B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174) to reduce negative energy balance.
  • the disclosure relates to a method for reducing incidence of disease in an animal comprising: (a) administering to the animal an effective amount of Bacillus pumilus strain 8G-134 (NRRL B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174) to reduce incidence of disease, wherein the disease is selected from the group consisting of ketosis, displaced abosmasums, retained placenta, mastitis, metritis, and pyometra.
  • the disclosure relates to a method for reducing inflammation in an animal comprising: (a) identifying an animal at risk for, or showing signs of, inflammation; and (b) administering to the animal identified in step (a) an effective amount of Bacillus pumilus strain 8G-134 (NRRL B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus strain 8G-134 (NRRL B-50174) to reduce inflammation.
  • An advantage of the strains, compositions and methods disclosed herein is that administration of B. pumilus 8G-134 to animals improves energy balance.
  • An advantage of the strains, compositions and methods disclosed herein is that administration of B. pumilus 8G-134 to animals reduces negative energy balance.
  • An advantage of the strains, compositions and methods disclosed herein is that feeding B. pumilus 8G-134 reduces the levels of nonesterified fatty acids ( EFA) in animals while maintaining high milk production.
  • An advantage of the strains, compositions and methods disclosed herein is that feeding B. pumilus 8G-134 promotes changes in immune cell populations of an animal.
  • An advantage of the strains, compositions and methods disclosed herein is that feeding B. pumilus 8G-134 improves the immune response of an animal.
  • An advantage of the strains, compositions and methods disclosed herein is that feeding B. pumilus 8G-134 influences the energy partitioned between milk and body tissue promoting an appropriately balanced body condition, which may promote health and fertility in subsequent lactations.
  • Figure 1 is a graph showing pH differences between tester (non-acidotic; Cluster 2) and driver (acidotic; Cluster 1) populations.
  • Figure 2 is a graph showing lactic acid accumulation differences between tester (non- acidotic; Cluster 2) and driver (acidotic; Cluster 1) populations.
  • Figure 3 is a graph showing in vitro glucose by treatment over time.
  • Figure 4 is a graph showing in vitro lactic acid accumulation by treatment over time.
  • Figure 5 is a graph showing total VFA (acetate+propionate+butyrate) accumulation over time.
  • Figure 6 is a graph showing mean ruminal pH over time in control and candidate DFM cattle.
  • Figure 7 is a graph showing mean ruminal lactate over time in control and candidate DFM cattle.
  • Figure 10 is a bar graph depicting mean fluorescence intensity of T cell surface markers at approximately 60 days in milk in multiparous dairy cows fed B. pumilus 8G-134 at 5 x 10 9 CFU/cow/day or control (no DFM). Means within group with different superscript differ P ⁇ 0.05.
  • Figure 11 is a bar graph depicting mean fluorescence intensity of T cell surface markers at approximately 60 days in milk in multiparous dairy cows fed B. pumilus 8G-134 at 5 x 10 9 CFU/cow/day or control (no DFM). Means within group with different superscript differ P ⁇ 0.05.
  • Figure 12 is a bar graph depicting percentage milk fat in multiparous cows categorized by days in milk (DIM) over a 12 week lactation period.
  • Days in milk (DIM) class was a covariate ( ⁇ 100 DIM (62 Control cows, 75 TRT cows), 101-200 DIM (77 Control cows, 58 TRT cows), and > 201 DIM (6 Control cows, 16 TRT cows). Numerical decreases in fat % were observed in early and late lactation groups with DFM treatment.
  • Figures 13 A-D are line graphs depicting the association between dietary starch and Bacillus pumilus concentration on postpartum serum metabolites.
  • BIOLOGY 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill with a general dictionary of many of the terms used in this disclosure.
  • CD cluster differentiation
  • administer is meant the action of introducing at least one strain and/or supernatant from a culture of at least one strain described herein into the animal's gastrointestinal tract. More particularly, this administration is an administration by oral route. This administration can in particular be carried out by supplementing the feed intended for the animal with the at least one strain, the thus supplemented feed then being ingested by the animal. The administration can also be carried out using a stomach tube or any other way to make it possible to directly introduce the at least one strain into the animal's gastrointestinal tract.
  • the term "effective amount” refers to the amount of the strain, strain combination or composition that will elicit the response including but not limited to the biological or medical response of a tissue, system or animal that is being sought.
  • the term “effective amount” includes that amount of the strain, strain combination or composition that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated.
  • the effective amount may vary depending on the strain, strain combination or composition, the disorder or condition and its severity.
  • the phrase "improving reproductive performance” refers to enhancing traits beneficial to reproduction and/or reducing traits, conditions, and/or diseases that impede reproduction. Improving reproductive performance includes but is not limited to reducing metritis, placental retention, mastitis, and postpartum diseases. Improving reproductive performance also includes reducing the reproductive cycle time.
  • negative energy balance is a frequent condition occurring in dairy cows. It consists of an imbalance between diet energy supply and production requirement.
  • performance refers to the growth of an animal, such as a pig or poultry, measured by one or more of the following parameters: average daily gain (ADG), weight, scours, mortality, feed conversion, which includes both feed: gain and gain: feed, and feed intake.
  • ADG average daily gain
  • weight weight
  • scours weight
  • mortality feed conversion
  • feed conversion which includes both feed: gain and gain: feed
  • feed intake feed intake
  • prevent refers to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or condition and/or one or more of its attendant symptoms or barring an animal from acquiring or reacquiring a disorder or condition or reducing an animal's risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.
  • the term "reducing" in relation to a particular trait, characteristic, feature, biological process, or phenomena refers to a decrease in the particular trait, characteristic, feature, biological process, or phenomena.
  • the trait, characteristic, feature, biological process, or phenomena can be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater than 100%.
  • T cell population includes regulatory T cells and effector T cells.
  • T cell population includes both activated and un-activated T-cells.
  • Cells within the T cell population may express a marker including but not limited to CD1, CD la, CD lb, CDlc, CD Id, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8, CD8a, CD8b, CD9, CD11 , CD 11 a, CD l ib, CD1 lc, CD1 Id, CD 13, CD 16, CD 17, CD 18, CD20, CD21 , CD23, CD25, CD26, CD27, CD28, CD29, CD30, CD31 , CD32, CD32b, CD35, CD37, CD38, CD39, CD40L (also known as CD40 ligand), CD43, CD44, CD45, CD45R, CD45RA, CD45RB, CD45RC, CD45R0, CD45RHi, CD46, CD47, CD48, CD49
  • T reg cells refer to T cells (T lymphocytes) that regulate the activity of other T cell(s) and/or other immune cells, usually by suppressing their activity.
  • the T reg cells are CD4 + , CD25 + , FoxP3 + T-cells (but it will be appreciated by persons skilled in the art that T reg cells are not fully restricted to this phenotype).
  • effector T cells or "T e ff cells” refer to T cells (T lymphocytes) that carry out the function of an immune response, such as killing tumor cells and/or activating an anti- tumour immune-response that can result in clearance of the tumour cells from the body.
  • the T e ff cells are CD3 + with CD4 + or CD8 + .
  • T e ff cells may secrete, contain or express effector markers such as IFN-gamma, granzyme B and ICOS (but it will be appreciated by persons skilled in the art that T e ff cells are not fully restricted to these phenotypes)
  • the terms “treat,” “treating,” “treatment” and grammatical variations thereof includes partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, pallatively or remedially.
  • strains Provided herein are strains. Methods of making and using the strains are also provided.
  • a direct-fed microbial (DFM) made with one or more of the strains provided herein allows beef and dairy producers to continue managing feeding regimens to optimize growth and performance without sacrificing health due to digestive upset associated with ruminal acidosis.
  • DFMs were selected on the basis of managing ruminal lactate concentrations via lactate utilization or priming the rumen to maintain lactate utilizing microflora.
  • At least some embodiments of the DFMs were developed to manage ruminal energy concentrations. Unlike the current DFMs marketed to cattle producers to alleviate acidosis, at least some of the embodiments of the invention were not developed to manage a problem after it occurs, but rather to alleviate the problem before it happens.
  • the strains provided herein include Enterococcus faecium strain 8G-1 , Enterococcus faecium strain 8G-73, and Bacillus pumilus strain 8G-134, which are also referred to herein as 8G-1 , 8G-73, and 8G-134, respectively.
  • strains Enterococcus faecium strain 8G-1 , Enterococcus faecium strain 8G-73, and Bacillus pumilus strain 8G-134 were deposited on August 29, 2008 at the Agricultural Research Service Culture Collection ( RRL), 1815 North University Street, Peoria, Illinois, 61604 and given accession numbers B-50173, B-50172 , and B-50174, respectively. The deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
  • One or more strain provided herein can be used as a direct- fed microbial (DFM).
  • a "biologically pure strain” means a strain containing no other bacterial strains in quantities sufficient to interfere with replication of the strain or to be detectable by normal bacteriological techniques. "Isolated” when used in connection with the organisms and cultures described herein includes not only a biologically pure strain, but also any culture of organisms which is grown or maintained other than as it is found in nature. In some embodiments, the strains are mutants, variants, or derivatives of strains 8G-1 , 8G-73, or 8G-134 that also provide benefits comparable to that provided by 8G-1 , 8G-73, and 8G-134.
  • the strains are strains having all of the identifying characteristics of strains 8G-1 , 8G-73, or 8G-134. Further, each individual strain (8G-1 , 8G-73, or 8G-134) or any combination of these strains can also provide one or more of the benefits described herein. It will also be clear that addition of other microbial strains, carriers, additives, enzymes, yeast, or the like will also provide control of acidosis and will not constitute a substantially different DFM.
  • Bacillus strains have many qualities that make them useful as DFMs. For example, several Bacillus species also have GRAS status, i.e., they are generally recognized as safe by the US Food and Drug Administration and are also approved for use in animal feed by the Association of American Feed Control Officials (AAFCO). The Bacillus strains described herein are aerobic and facultative sporeformers and thus, are stable. Bacillus species are the only sporeformers that are considered GRAS.
  • a Bacillus strain found to prevent or treat acidosis is Bacillus pumilus strain 8G-134 .
  • Enterococcus strains also have many qualities that make them useful as DFMs.
  • Enterococcus strains are known to inhabit the gastrointestinal tract of monogastrics and ruminants and would be suited to survive in this environment. Enterococcus have been shown to be facultatively anaerobic organisms, making them stable and active under both aerobic and anoxic conditions. Enterococcus faecium strain 8G-1 and Enterococcus faecium strain 8G-73 were identified by the inventors as being useful for these purposes.
  • each one of the strains described herein is cultured individually using conventional liquid or solid fermentation techniques.
  • the Bacillus strain and Enterococcus strains are grown in a liquid nutrient broth, in the case of the Bacillus, to a level at which the highest number of spores are formed.
  • the Bacillus strain is produced by fermenting the bacterial strain, which can be started by scaling-up a seed culture. This involves repeatedly and aseptically transferring the culture to a larger and larger volume to serve as the inoculum for the fermentation, which can be carried out in large stainless steel fermentors in medium containing proteins, carbohydrates, and minerals necessary for optimal growth.
  • Non-limiting exemplary media are MRS or TSB. However, other media can also be used.
  • the temperature and agitation are controlled to allow maximum growth.
  • the strains are grown at 32° to 37° under agitation.
  • the culture is harvested by separating the cells from the fermentation medium. This is commonly done by centrifugation.
  • the Bacillus strain is fermented to a 5 x 10 8 CFU/ml to about 4 x 10 y CFU/ml level. In at least one embodiment, a level of 2 x 10 CFU/ml is used.
  • the bacteria are harvested by centrifugation, and the supernatant is removed. The pelleted bacteria can then be used to produce a DFM.
  • the pelleted bacteria are freeze-dried and then used to form a DFM. However, it is not necessary to freeze-dry the Bacillus before using them.
  • the strains can also be used with or without preservatives, and in concentrated, unconcentrated, or diluted form.
  • 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.
  • one or more strain is used to form a DFM.
  • One or more carriers including, but not limited to, sucrose, maltodextrin, limestone, and rice hulls, can be added to the strain.
  • strain(s) and carriers (where used), they can be added to a ribbon or paddle mixer and mixed preferably for about 15 minutes, although the timing can be increased or decreased.
  • the components are blended such that a uniform mixture of the cultures and carriers result.
  • the final product is preferably a dry, fiowable powder, and may be formulated based upon the desired final DFM concentration in the end product.
  • a strain described herein is grown in a medium, such as a liquid nutrient broth.
  • the strain is separated from the liquid nutrient broth to make the direct-fed microbial.
  • the strain can be freeze dried after it is separated from the broth.
  • Ente vcoccus faecium strain 8G- 1 Enterococcus faecium strain 8G-73
  • Bacillus pumilus strain 8G-134 can be fed to animals to reduce or even eliminate the occurrence of acidosis. For this, an effective amount of one or more of these strains is administered to the animals.
  • the strain(s) Upon administration to the animals, the strain(s) provides at least one of the following benefits in or to the animals: (a) reduces acidosis in the animals, (b) stabilizes ruminal metabolism as indicated by delayed lactic acid accumulation and prolonged production of volatile fatty acids, (c) recovers more quickly from acidosis challenge as measured by pH recovery and lactic acid decline, and (d) does not exhibit clinical signs associated with acidosis.
  • the animals can be cattle, including both beef cattle and dairy cattle, that is, one or more bull, steer, heifer, calf, or cow; goats; sheep; llamas; alpacas; other four-compartment stomached, and ruminant animals that may encounter ruminal imbalance when fed readily fermentable carbohydrate (RFC).
  • cattle including both beef cattle and dairy cattle, that is, one or more bull, steer, heifer, calf, or cow; goats; sheep; llamas; alpacas; other four-compartment stomached, and ruminant animals that may encounter ruminal imbalance when fed readily fermentable carbohydrate (RFC).
  • RRC readily fermentable carbohydrate
  • the strain when Enterococcus faecium strain 8G-1 or Enterococcus faecium strain 8G-73 is fed, the strain is administered to the animals at a level such that the animals are dosed daily with about 5 x 10 8 CFU/ani ⁇ mal/day to about 5 x 1010 CFU/animal/day. In at least one embodiment, when Bacillus pumilus strain 8G-134 is fed, the strain is
  • two or more strains of Enterococcus faecium strain 8G-1 , Enterococcus faecium strain 8G-73 and Bacillus pumilus strain 8G-134 are fed, and the strains are administered to the animals at a level such that the animals are dosed daily with about 5 x 10 8 CFU/animal/day to about 5 x 10 10
  • CFU/animal/day as the total dose of the combined strains.
  • Other levels of one or more strains can be fed to the animals.
  • the strain can be administered to the animals from about 30 days of age through the remainder of the adult ruminant productive life or for other time periods.
  • the strain is fed as a direct-fed microbial (DFM), and the DFM is used as a top dressing on a daily ration.
  • the strain can be fed in a total mixed ration, pelleted feedstuff, mixed in with liquid feed, mixed in a protein premix, delivered via a vitamin and mineral premix.
  • the strain is fed as a DFM, and the DFM is fed in
  • Type A Medicated Article monensin (Rumensin®), with a daily dose about 50mg to 660 mg per head.
  • Monensin is fed to increase feed efficiency.
  • Monensin, as an ionophore creates permeability in bacterial cell membrane creating an ion imbalance between the intracellular and extracellular spaces. This response affects ruminal microbiota populations and influence feedstuff fermentation to improve livestock feed efficiency.
  • the strain is fed as a DFM, and the DFM is fed in combination with Type A Medicated Article tylosin phosphate (Tylan®), with a daily dose of about 60 to 90 mg/head.
  • Tylosin phosphate is fed to beef cattle to reduce liver abscesses caused by Fusobacterium necrophorum and Actinomyces pyogenes.
  • the strains, methods, and compositions disclosed herein can be used to reduce incidence of disease in an animal.
  • the strain is Bacillus pumilus 8G-134.
  • the disclosure relates to a method for reducing incidence of disease in an animal comprising: administering to the animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to reduce incidence of disease.
  • the animal is an animal selected from the group consisting of: a peripartum animal, a postpartum animal, or an animal in labor.
  • the disclosure relates to a method for reducing incidence of disease in an animal comprising: administering to a peripartum animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to reduce incidence of disease, wherein Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for two weeks prior to labor and delivery. In another embodiment, Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for four weeks prior to labor and delivery.
  • NRRL-B-50174 Bacillus pumilus 8G-134
  • NRRL-B-50174 Bacillus pumilus 8G-134
  • Bacillus pumilus 8G-134 (NRRL-B- 50174) is administered for six to eight weeks prior to labor and delivery. In still another embodiment, Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for a time period including but not limited 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, and 16 weeks prior to labor and delivery.
  • the disclosure relates to a method for reducing incidence of disease in an animal comprising: administering to a peripartum animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to reduce incidence of disease, wherein Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for a period of time prior to labor and delivery and for a period of time after labor and delivery.
  • the strain is administered for a time period including but not limited 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, and 16 weeks prior to labor and delivery.
  • the strain is administered for a time period including but not limited 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 weeks after labor and delivery.
  • the disclosure relates to a method for reducing incidence of disease in an animal comprising: administering to a peripartum animal, a postpartum animal, or an animal in labor and delivery an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B- 50174) to reduce incidence of disease, wherein Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for two weeks postpartum. In another embodiment, Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for four weeks postpartum.
  • Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for six to eight weeks postpartum. In still another embodiment, Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for a time period including but not limited 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 weeks postpartum.
  • the animal is a ruminant animal. In still another embodiment, the animal is a dairy cow. In yet another embodiment, the animal is a peripartum dairy cow, a dairy cow in the process of calving, or a postpartum dairy cow.
  • the disease is selected from the group consisting of: ketosis
  • acetonaemia displaced abomasums, retained placenta, metritis, an inflammatory disease, inflammation, and mastitis.
  • Ketosis is a metabolic state where most of the body's energy supply comes from ketone bodies in the blood, in contrast to a state of glycolysis where blood glucose provides most of the energy. It is almost always generalized, with hyperketonemia, that is, an elevated level of ketone bodies in the blood throughout the body. Ketone bodies are formed by ketogenesis when liver glycogen stores are depleted. The main ketone bodies used for energy are acetoacetate and ⁇ - hydroxybutyrate, and the levels of ketone bodies are regulated mainly by insulin and glucagon- Most cells in the body can use both glucose and ketone bodies for fuel, and during ketosis free fatty acids and glucose synthesis (gluconeogenesis) fuel the remainder.
  • ketosis is sometimes referred to as the body's "fat burning" mode.
  • ketosis is a common ailment that usually occurs during the first weeks after giving birth to a calf. Ketosis is in these cases sometimes referred to as acetonemia.
  • a study from 2011 revealed that whether ketosis is developed or not depends on the lipids a cow uses to create butterfat. Animals prone to ketosis mobilize fatty acids from adipose tissue, while robust animals create fatty acids from blood phosphatidylcholine (lecithin). Healthy animals can be recognized by high levels of milk glycerophosphocholine and low levels of milk
  • the abomasum (or true stomach) normally lies on the floor of the abdomen, but can become filled with gas and rise to the top of the abdomen, when it is said to be “displaced.”
  • the abomasum is more likely to be displaced to the left (LDA) than the right (RDA).
  • LDA left
  • RDA right
  • the majority of cases occur soon after calving.
  • the uterus displaces the abomasum, so that after calving the abomasum has to move back to its normal position, increasing the risk of displacement.
  • LDA Fifty to eighty percent of LDA are diagnosed within two weeks postpartum. Eighty to ninety percent of LDA are diagnosed within one month postpartum. Cows with LDA were at increased risk 50X for ketosis.
  • Displaced abomasum can also be caused by atony of the abomasum. If the abomasum stops contracting and turning over its contents, accumulation of gas will occur and the abomasum will tend to move up the abdomen. This tends to be a cause of inadequate nutrition.
  • Symptoms of displaced abomasum include but are not limited to loss of appetite; drop in milk yield; reduced rumination; and mild diarrhea.
  • RP Retained Placenta
  • RP is also known as retained fetal membrane or retained cleansing. RP occurs when the calf s side of the placenta (the fetal membranes) fails to separate from the mother's side. Separation of the membranes normally occurs after the calf is born (early separation is one cause of stillbirth). RP is usually defined as the failure to expel fetal membranes within 24 hr after parturition.
  • Retained placenta is most commonly associated with dystocia, milk fever (metabolic diseases) and twin births.
  • the single sign associated with RP is degenerating, discolored, ultimately fetid membranes hanging from the vulva.
  • the retained membranes may remain within the uterus and not be readily apparent, in which case their presence may be signaled by a foul-smelling discharge.
  • Cows with retained fetal membranes are at increased risk of developing metritis, ketosis, mastitis, and even abortion in a subsequent pregnancy.
  • Mastitis occurs when white blood cells (leukocytes), are released into the mammary gland, usually in response to an invasion of bacteria of the teat canal. Milk-secreting tissue, and various ducts throughout the mammary gland can be damaged by toxins from bacteria. Mastitis can also occur as a result of chemical, mechanical, or thermal injury.
  • the inflammatory response results in an increase in the blood proteins and white blood cells in the mammary tissue, which can then pass into the milk product.
  • This response aims to destroy the irritant, repair the damaged mammary tissue and return the udder to its normal function.
  • mastitis a loss in milk output is often experience.
  • Negative energy balance occurs for a variety of reasons including but not limited to a decrease in dry matter intake (DMI); an increase in energy demands due to milk production; and a lagging increase in DMI after parturition.
  • Higher blood NEFA andbeta-hydroxybutyrate (BHBA) are typical in the early postpartum period when cows are partitioning energy toward milk production, and are known to be negatively correlated with energy balance.
  • cows with greater NEFA are mobilizing more adipose tissue to support milk production and are losing more body condition (BC) relative to cows with lower NEFA levels.
  • Adipose is mobilized as NEFA and transported to the liver to be oxidized or re-esterified into triglycerides. When re-esterification of triglycerides is increased, production of ketone bodies is increased.
  • Ketone bodies include BHBA, acetoacetate, and acetone.
  • NEFA and/or BHBA lower levels of NEFA and/or BHBA likely indicate normal adjustment to the new energy demands, however, higher levels of NEFA and/or BHBA can be predictive of greater incidence of disease such as ketosis and displaced abomasums.
  • the strains, methods, and compositions disclosed herein can be used to reduce negative energy balance of an animal.
  • the strain is Bacillus pumilus 8G-134.
  • the disclosure relates to a method for reducing negative energy of an animal comprising: administering to the animal an effective amount of a Bacillus pumilus 8G- 134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to reduce negative energy balance of an animal.
  • a Bacillus pumilus 8G- 134 NRRL-B-50174
  • a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 NRRL-B-50174
  • the animal is a ruminant animal. In yet another embodiment, the animal is a peripartum animal. In another embodiment, the animal is a postpartum animal. In still another embodiment, the animal is an animal in the process of labor and delivery. In still another embodiment, the animal is a dairy cow, a peripartum dairy cow, a postpartum dairy cow, or a dairy cow in the process of calving.
  • the disclosure relates to a method for reducing negative energy of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) results in better milk production and body condition score as compared to animals not administered the strain.
  • NRRL-B-50174 Bacillus pumilus 8G-134
  • NRRL-B-50174 a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134
  • the disclosure relates to a method for reducing negative energy of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) results in lower levels of NEFA as compared to animals not administered the strain.
  • administration of Bacillus pumilus 8G-134 (NRRL-B-50174) results in lower levels of BHBA as compared to animals not administered the strain.
  • NRRL-B-50174 results in lower levels of BHBA as compared to animals not administered the strain.
  • administering results in lower levels of NEFA and BHBA as compared to animals not administered the strain.
  • the disclosure relates to a method for reducing negative energy of an animal comprising: obtaining a sample from an animal; determining the level of NEFA and/or BHBA in the sample; administering to an animal, with a level of NEFA and/or BHBA indicative of negative energy balance, an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B- 50174).
  • the sample is blood.
  • the sample is collected from a peri-parturient animal (from 14 days prepartum to 14 days postpartum).
  • samples are collected at time points to avoid diurnal and postprandial variations.
  • the disclosure relates to a method for reducing negative energy of an animal comprising: obtaining a sample from numerous animals in a herd; determining the level of NEFA and/or BHBA in each sample obtained; administering an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to the herd when 10-25% of the sampled animals had NEFA and/or BHBA concentrations indicative of disease.
  • the strain is administered when 15% of the sampled animals had NEFA and/or BHBA concentrations indicative of disease.
  • a level of NEFA in a prepartum animal greater than 0.27 mEq/1 is indicative of negative energy balance.
  • a level of NEFA in a postpartum animal greater than 0.70 mEq/1 is indicative of negative energy balance.
  • a level of BHBA in a postpartum animal greater than 12 mg/dL is indicative of negative energy balance.
  • the disclosure relates to a method for reducing negative energy of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) to the animal results in lower levels of NEFA and better milk production as compared to animals not administered the strain.
  • administration of Bacillus pumilus 8G-134 (NRRL-B-50174) results in lower levels of BHBA and better milk production as compared to animals not administered the strain.
  • administration of Bacillus pumilus 8G-134 (NRRL-B-50174) results in lower levels of NEFA and BHBA and better milk production as compared to animals not administered the strain.
  • administration of Bacillus pumilus 8G-134 can increase milk production as compared to animals not administered the strain by a percentage selected from the group consisting of 0.1-0.5%, 0.5-1%, 1-3%, 3-5%, 5-7%, 7-9, 10%, 10-12%, 12-15%, 15-20%, 20-25%, 25-30%, 30-35%, and greater than 35%.
  • administration of Bacillus pumilus 8G-134 can increase milk production as compared to animals not administered the strain by greater than 0.1%, greater than 0.3%, greater than 0.5%, greater than 1%, greater than 3%, greater than 5%, greater than 7%, or greater than 10%.
  • administration of Bacillus pumilus 8G-134 (NRRL-B-50174) to an animal can decrease levels of NEFA in the animal as compared to an animal not administered the strain by a percentage selected from the group consisting of 0.1-1%, 1 -5%, 5-10%, 10-15%, 15- 20%, 20-25%, 25-30% 35-40%, 40-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, and greater than 95%.
  • administering can decrease levels of NEFA in the animal as compared to animals not administered the strain by a percentage selected from the group consisting of 1 -10%, 10-20%, 20-40%, 40-60%, 60-80%, 80-100% and greater than 100%.
  • administration of Bacillus pumilus 8G-134 (NRRL-B-50174) to an animal can decrease levels of BHBA in the animal as compared to an animal not administered the strain by a percentage selected from the group consisting of 0.1-1%, 1 -5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% 35-40%, 40-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, and greater than 95%.
  • administering can decrease levels of BHBA in the animal as compared to animals not administered the strain by a percentage selected from the group consisting of 1 -10%, 10-20%, 20-40%, 40-60%, 60-80%, 80-100% and greater than 100%.
  • the disclosure relates to a method for reducing negative energy of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) to the animal reduces incidence of disease associated with negative energy balance.
  • administration of Bacillus pumilus 8G-134 (NRRL-B-50174) results in lower incidence of a disease including but not limited to subclinical ketosis, retained placenta, displaced abomasums, and mastitis.
  • the strains, methods, and compositions disclosed herein can be used to improve an immune response of an animal.
  • improving an immune response of an animal comprises changing the immune cell population of an animal.
  • improving an immune response of an animal comprises increasing expression of T- cell markers.
  • the strains, methods, and compositions disclosed herein can be used for changing an immune cell population in an animal. In another embodiment, the strains, methods, and compositions disclosed herein can be used for inducing changes in the immune cell population of a ruminant animal. In one embodiment, the strain is Bacillus pumilus 8G-134.
  • changing the immune cell population of an animal comprises altering the expression pattern of the immune cells without an increase in total immune cell population.
  • the disclosure relates to a method for inducing a change in the in immune cell population of an animal comprising: (a) identifying an animal in need of a change in immune cell population; and (b) administering to the animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to induce a change in the immune cell population.
  • an animal in need of a change in immune cell population is an animal at risk for disease and/or infection or an animal showing signs of disease and/or infection.
  • the immune cell population is leukocytes.
  • Leukocytes (white blood cells) are classified into two major lineages: the myeloid leukocytes and the lymphocytes.
  • the white cells of the myeloid lineage include neutrophils, monocytes, eosinophils and basophils.
  • the white cells of the lymphocytes include T cells, B cells and natural killer cells.
  • the ruminant animal is a dairy cow. In yet another embodiment, the ruminant animal is a postpartum dairy cow.
  • a ruminant animal in need of a change in leukocyte population is an animal with a disease or infection.
  • the disease or infection can be acute or chronic.
  • the disease is selected from the group consisting of ketosis (acetonaemia), displaced abomasums, retained placenta, and mastitis.
  • strains, methods, and compositions are disclosed for changing T-cell population subsets. In another embodiment, strains, methods, and compositions are disclosed for inducing changes in T-cell population subsets. In one embodiment, a change in T-cell population is reflected by an increase in expression of T cell CD markers, including but not limited to CD4, CD8, CD25, CD45R, CD45R0, CD62L, and CD45RHi. In one embodiment, the strain is Bacillus pumilus 8G-134. In one embodiment,
  • T cells are one of the cell populations playing major roles in the immune system as a biodefence system against various pathogens. Such T cells are roughly classified into CD4 positive helper T cells and CD8 positive cytotoxic T cells, where the former relates to the promotion of immune response and the latter relates to the exclusion of virus-infected cells and tumor cells. Helper T cells are further classified into Type I helper T cells for promoting cellular immunity and Type II helper T cells for promoting humoral immunity. These T cells with such diversified properties have a function of excluding pathogens and gaining infection resistance under a well-balanced immune response.
  • the disclosure relates to a method for inducing a change in the T cell population of an animal comprising: (a) identifying an animal in need of a change in T cell population; and (b) administering to the animal an effective amount of a Bacillus pumilus 8G- 134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to induce a change in the T cell population.
  • a change in T cell population is evident by an increase in expression of T cell CD markers, including but not limited to CD4, CD8, CD25, CD45R, CD45R0, CD62L, and CD45RHi.
  • the disclosure relates to a method for inducing a change in the T cell population of an animal comprising: (a) obtaining a sample from an animal; (b) determining an initial T cell population in the sample from the animal; and (c) administering to the animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to induce a change in the T cell population.
  • the method further comprises (d) determining the T-cell population in a second sample from the animal after administration of Bacillus pumilus 8G-134 (NRRL-B-50174).
  • a change in T-cell population is shown by a change in cell surface markers.
  • the cell surface marker is selected from the group consisting of CD4, CD8, CD45R, CD45R0, CD62L, and CD45RHL
  • Immunoglobulin A in its secretory form, is the main effector of the mucosal immune system and provides an important first line of defense against most pathogens that invade the body at a mucosal surface.
  • SIgA Secretory IgA
  • body secretions such as saliva, tears, colostrum and gastrointestinal secretions.
  • the molecular stability and effector immune functions make SIgA particularly well suited to provide mucosal protection against pathogens.
  • IgA mediates a variety of protective functions. Luminal SIgA is believed to interfere with pathogen adherence to mucosal epithelial cells, a process called immune exclusion. In addition, IgA appears to have two other defense functions: intracellular neutralization, and virus excretion. IgA is also found as a monomer in the serum where it may function as a second line of defence by eliminating pathogens that have breached the mucosal surface. Serum IgA interacts with an Fc receptor called FcaRl triggering antibody-dependent-cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent-cell-mediated cytotoxicity
  • the strains, methods, and compositions disclosed herein can be used to improve immune response of an animal to an infection.
  • the strain is Bacillus pumilus 8G-134.
  • the disclosure relates to a method for improving immune response of an animal to an infection comprising: administering to the animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying
  • Bacillus pumilus 8G-134 (NRRL-B-50174) to improve the immune response of an animal to an infection.
  • the strain is administered for a period of time prior to labor and delivery including but not limited to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, and 16 weeks prior to labor and delivery.
  • the strain is administered for a period of time post labor and delivery including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 weeks post labor and delivery.
  • the strain is administered within the first two weeks post-calving. In yet another embodiment, the strain is administered within the first week post calving.
  • the strain is administered two weeks prior to calving. In yet another embodiment, the strain is administered one week prior to calving.
  • the disclosure relates to a method for improving immune response of an animal comprising: administering to the animal an effective amount of a Bacillus pumilus 8G- 134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174), wherein an increase in IgA as compared to animals not administered the strain is indicative of an improved immune response.
  • An increase of IgA in the milk during the first week post-calving can provide improved immune defense in the mammary gland to prevent bacterial adhesion and invasion of mammary epithelial cells. It would also be expected to provide a benefit to the calf, boosting calf health through passive transfer of immunity.
  • the disclosure relates to a method for improving immune response of an animal comprising: obtaining a sample from an animal; determining the level of IgA in the sample; administering to an animal, with a level of IgA indicative of a suppressed immune response, an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174).
  • the sample is blood.
  • the sample is collected from a peri- parturient animal (from 14 days prepartum to 14 days postpartum).
  • administering to an animal can increase levels of IgA in the animal as compared to an animal not administered the strain by a percentage selected from the group consisting of 1-5%, 5-10%, 10-15%, 15-20%, 20- 25%, 25-30% 35-40%, 40-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, and greater than 95%.
  • administering to an animal can increase levels of IgA in the animal as compared to an animal not administered the strain by a percentage selected from the group consisting of 1-10%, 10-20%, 20-40%, 40- 60%, 60-80%, 80-100% and greater than 100%.
  • the level of IgA in a treated animal as compared to an untreated animal is increased by 10-30%. In one embodiment, the level of IgA in a treated animal as compared to an untreated animal is increased by at least 15%.
  • the strains, methods, and compositions disclosed herein can be used to improve reproductive performance an animal.
  • the strain is Bacillus pumilus 8G-134.
  • the disclosure relates to a method for improving reproductive performance of an animal comprising: administering to the animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying
  • the animal is a ruminant animal. In still another embodiment, the animal is a dairy cow. In yet another embodiment, the animal is a postpartum dairy cow.
  • the disclosure relates to a method for improving reproductive performance of an animal comprising: administering to a postpartum animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to improve reproductive performance of an animal.
  • the postpartum animal had been diagnosed with a disease including but not limited to ketosis (acetonaemia), displaced abomasums, retained placenta, and mastitis.
  • Bacillus pumilus 8G-134 (NRRL-B-50174) is administered for a time period including but not limited 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16- 20, 20-24, 24-28, 28-32, 32-36, 36-40, 40-44, 44-48, 48-52, and greater than 52 weeks postpartum.
  • postpartum anestrous After calving, it typically takes about 60 to 90 days for a cow to resume cycles. This period is called postpartum anestrous. In first calf heifers, postpartum anestrous lasts longer than mature cows. It normally takes about 90 to 120 days for first calf heifers to resume cycles.
  • the disclosure relates to a method for improving reproductive performance of an animal comprising: administering to a postpartum animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to improve reproductive performance of an animal, wherein animals administered Bacillus pumilus 8G-134 (NRRL-B- 50174) resume cycles sooner than animals not administered the strain.
  • NRRL-B-50174 Bacillus pumilus 8G-134
  • NRRL-B-50174 a Bacillus pumilus 8G-134
  • animals administered the strain resume cycles 1 -5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35- 40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, and 75-80 days sooner than animals not administered the strain.
  • animals administered Bacillus pumilus 8G-134 achieve a subsequent fertilization quicker than animals not administered the strain.
  • the animal administered the strain achieves a subsequent fertilization 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60- 65%, 65-70%, 70-75%, 75-80%, 80-85%, and 85-90%, 90-95%, 95-100%, and greater than 100% sooner than animals not administered the strain.
  • the disclosure relates to a method for improving reproductive performance of an animal comprising: administering to a postpartum animal an effective amount of a Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174), wherein animals administered Bacillus pumilus 8G-134 (NRRL-B-50174) have an increased number of conceptions as compared to animals not administered the strain.
  • animals administered Bacillus pumilus 8G-134 have 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30- 35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, 95-100%, and greater than 100% more conceptions than animals not administered the strain.
  • Acute -phase proteins are a class of proteins whose plasma concentrations increase (positive acute -phase proteins) or decrease (negative acute-phase proteins) in response to inflammation. This response is called the acute-phase reaction (also called acute -phase response).
  • Two positive acute -phase proteins are haptoglobin and serum amyloid A.
  • Haptoglobin is an inflammatory marker, and when an animal has an infection or inflammation that causes tissue damage, the animal's liver produces haptoglobin in abundance. High haptoglobin levels have been reported in the blood of animals with mastitis, metritis, pyometra, traumatic reticulitis, absomasal displacement, traumatic pericarditis, bacterial nephritis, and hepatic lipidosis.
  • the strains, methods, and compositions disclosed herein can be used to reduce inflammation in an animal.
  • the strain is Bacillus pumilus 8G-134.
  • the disclosure relates to a method for reducing inflammation of an animal comprising: administering to the animal an effective amount of a Bacillus pumilus 8G- 134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G-134 (NRRL-B-50174) to reduce inflammation of an animal.
  • a Bacillus pumilus 8G- 134 NRRL-B-50174
  • NRRL-B-50174 a Bacillus pumilus 8G- 134
  • NRRL-B-50174 a strain having all of the identifying characteristics of Bacillus pumilus 8G-134
  • the animal is a ruminant animal. In still another embodiment, the animal is a dairy cow. In yet another embodiment, the animal is a peripartum dairy cow, a dairy cow in the process of calving, or a postpartum dairy cow.
  • the disclosure relates to a method for reducing inflammation of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) reduces inflammation as compared to animals not administered the strain.
  • the disclosure relates to a method for reducing inflammation of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) increases levels of negative acute phase proteins as compared to animals not administered the strain.
  • the disclosure relates to a method for reducing inflammation of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) reduces levels of positive acute phase proteins as compared to animals not administered the strain.
  • the positive acute phase proteins are haptoglobin and serum amyloid A.
  • the disclosure relates to a method for reducing inflammation of an animal comprising: administering to an animal an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134 (NRRL-B-50174), wherein administration of Bacillus pumilus 8G-134 (NRRL-B-50174) results in a decrease in haptoglobin and/or serum amyloid A levels as compared to animals not administered the strain.
  • the disclosure relates to a method for reducing inflammation of an animal comprising: obtaining a sample from an animal; determining the level of one or more acute phase proteins in the sample; administering to an animal, with a level of one or more acute phase proteins indicative of inflammation, an effective amount of Bacillus pumilus 8G-134 (NRRL-B-50174) or a strain having all of the identifying characteristics of Bacillus pumilus 8G- 134 (NRRL-B-50174).
  • the sample is blood.
  • the sample is collected from a peri -parturient animal (from 14 days prepartum to 14 days postpartum).
  • samples are collected at time points to avoid diurnal and postprandial variations.
  • administration of Bacillus pumilus 8G-134 (NRRL-B-50174) to an animal can decrease levels of one or more acute phase proteins in the animal as compared to an animal not administered the strain by a percentage selected from the group consisting of 0.1-1%, 1-5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% 35-40%, 40-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, and greater than 95%.
  • administering can decrease levels of one or more acute phase proteins in the animal as compared to animals not administered the strain by a percentage selected from the group consisting of 1-10%, 10-20%, 20-40%, 40-60%, 60-80%, 80-100% and greater than 100%.
  • administering to an animal can decrease levels of haptoglobin in the animal as compared to an animal not administered the strain by a percentage selected from the group consisting of 0.1-1%, 1-5%, 5- 10%, 10-15%, 15-20%, 20-25%, 25-30% 35-40%, 40-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, and greater than 95%.
  • administering can decrease levels of haptoglobin in the animal as compared to animals not administered the strain by a percentage selected from the group consisting of 1-10%, 10-20%, 20-40%, 40-60%, 60-80%, 80-100% and greater than 100%.
  • administering can decrease levels of serum amyloid A in the animal as compared to an animal not administered the strain by a percentage selected from the group consisting of 0.1-1%, 1-5%, 5- 10%, 10-15%, 15-20%, 20-25%, 25-30% 35-40%, 40-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, and greater than 95%.
  • administering can decrease levels of serum amyloid A in the animal as compared to animals not administered the strain by a percentage selected from the group consisting of 1-10%, 10-20%, 20-40%, 40-60%, 60-80%, 80-100% and greater than 100%.
  • Concentrate diet treatments consisted of highly fermentable carbohydrate sources of steam flaked corn on a 90% as fed basis. After fasting for 24 hours, the concentrate diet was fed ad libitum at 100 lbs/pen to all pens (Oh). Challenge diet consumption was visually monitored and additional feed added on an as needed basis.
  • Rumen fluid samples were obtained from individual animals via oral intubation using a collection tube attached to a vacuum flask. Different flasks and collection tubes were used for each pen to minimize cross contamination of microbiota between treatments. Ruminal fluid collected in the vacuum flasks was decanted into sterile 50 ml Falcon tubes labeled with sample time and animal identification number (ear tag number). Ruminal samples were collected from all pens at -36h, -24h, and -12h. Time -36h and -24h samples represented the physiological baseline for each animal. Time -12h samples represented rumen fluid in the fasted state for each animal. Time Oh was designated as the beginning of the feeding challenge. Ruminal samples were collected from all animals every 4 hours from +6 to +22 hours. All pens were sampled at +28, +36, and +48 hours. The pH from individual ruminal samples were analyzed immediately after acquisition. All samples were frozen and prepared for shipment to Agtech Products, Inc. (Waukesha, WI) for further analysis.
  • Volatile fatty acids and carbohydrate concentrations were measured in individual ruminal samples. Samples were prepared for HPLC analysis by aseptically removing duplicate 1.0 ml samples from the rumen fluid collected from each animal at each time period. Samples were placed in a 1.5 ml microcentrifuge tube and the debris was pelleted by centrifugation (10 minutes, at 12,500 rpm). The supernatant fluid (750 ⁇ ) was transferred to a clean tube and acidified with an equal volume of 5 mM H 2 SO 4 . The acidified fluid was thoroughly mixed and filtered through 0.2 ⁇ filter directly into a 2 ml HPLC autosampler vial and capped.
  • Samples were analyzed using a Waters 2690 HPLC system (Waters Inc., Milford, MA). The sample were injected into 5 mM H 2 SO 4 mobile phase heated to 65 °C and separated using a BioRad HPX-87H Column (Bio-Rad Laboratories, Inc., Hercules, CA). The HPLC was standardized using a set of concentrations for each compound of interest. Compounds used as standards were include dextrose (glucose), lactate, methylglyoxal, butyrate, propionate, and acetate.
  • the Genome Subtraction Kit (Clontech, Palo Alto, CA) was utilized to determine microbial population differences between two sets of pooled ruminal samples.
  • Hierarchal clustering analysis was performed to determine similarities and differences between animals based on pH and lactic acid profiles over time.
  • Cluster analysis positioned cattle 2069, 2071 , 2078, 2113, and 2127 in Cluster 1 and cattle 2107, 2115, 2088, 2133, and 2124 in Cluster 2.
  • Repeated measures analysis was performed to compare pH and lactic acid from Cluster 1 to Cluster 2. All variables were analyzed separately.
  • the rumen fluid from individual animal was pooled within cluster for suppressive subtractive hybridization (SSH) procedures.
  • SSH suppressive subtractive hybridization
  • SSH Suppressive subtraction hybridization
  • DNA sequences that are recovered after subtraction were cloned for further analysis.
  • DNA sequences were inserted into the pCR2.1 vector (Invitrogen) and transformed into E. coli chemically competent TOP 10 cells.
  • the transformation mixture was plated onto 22 x 22 cm LB agar plates containing 50 ⁇ g/ml kanamycin and overlaid with 40 mg/ml X-gal in DMF. Plates were incubated at 37°C for 24h. Recombinant colonies (white colonies) were picked into sterile microtitre plates containing LB medium and kanamycin at 50 ⁇ g/ml. All wells containing recombinant PCR products were separated into 1 ml aliquots.
  • Hybridizations were conducted on cloned inserts. At each time period, subtraction was performed, SSH 6, 10, 14, and 18. From SSH 6, 10, 14, and 18, there were 12, 29, 105, and 29 cloned inserts, respectively, that were tester specific.
  • the DNA sequence from each tester positive insert was determined (Lark Technologies; Houston, TX). Sequence from each insert was compared with sequences from the NCBI database using the blastX function. Nucleotide sequences were translated and gene function was deduced by comparing sequences to those found in the NCBI database using the blastX function. Gene function was placed in a gene category using the Clusters of Orthologous Groups (COG) web site. Specific COG genes identified were used to construct oligonucleotide probes for colony hybridization and slot-blot hybridization experiments. Four genes of the twenty-nine were selected from SSH 10 to be utilized for colony hybridization based upon functional attributes based on selection from non-acidotic cattle.
  • COG Clusters of Orthologous Groups
  • the genes were selected from clones 79, 84, 94, and 110 were identified via using the NCBI blastX function with assigned functions: beta-xylosidase, glucose/galactose transporter, 4-alpha-glucanotransferase, and 4-alpha- glucanotransferase, respectively. All genes selected for colony hybridization had assigned properties as identified by COG as Carbohydrate and Transport Metabolism function, which would have provided bacteria containing these genes an advantage at metabolizing excess energy such as that found in the rumen when challenged with RFC. Colony Hybridization:
  • NLA sodium lactate agar
  • LPSA Lactate Propionibacterium Selective Agar
  • RCS
  • Tubes showing growth were separated into two separate aliquots of 9 ml and 1 ml.
  • the 1 ml aliquot was utilized for DNA isolation procedures utilizing the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals; Mannheim, Germany).
  • the 9 ml aliquot was transferred to a sterile 15 ml Falcon Tube and centrifuged until a solid pellet was formed.
  • the pellet was then reconstituted in NLB or RCS broths containing 10% glycerol.
  • the reconstituted sample was placed in the -80°C for future use.
  • the extracted DNA was then used for RAPD- PCR analysis of individual isolates to determine phylogenic relationships.
  • PCR product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). Purified product was analyzed by gel electrophoresis. When sufficient product was available, the purified sample was sent overnight on ice for single pass sequencing (Lark Technologies, Houston, TX). The 16s sequences from each cluster were compared with sequences from the NCBI database using the blastn function. Organisms of interest brought forward from this comparison consisted of Enterococcus faecium strain 8G-1 , Enterococcus faecium strain 8G-73, andBacillus pumilus strain 8G-134.
  • Example 2 Example 2
  • Rumen fluid was collected for in vitro trials from two yearling Hereford heifers. Heifers were identified by identification tags and were referred to as 101 and 133. Heifers were fed 6 lbs/head/day of dried distillers grain (DDGS) and had access to free choice haylage.
  • DDGS dried distillers grain
  • rumen fluid was collected from each heifer and placed into marked, pre -warmed thermoses. Thermoses were transported to Agtech Products, Inc. for processing. Rumen fluid was added in duplicate to bottles containing McDougall's Buffer and 3.0% glucose (final concentration after McDougall's Buffer and rumen fluid have been mixed to a volume of 180 ml), which had been tempered to 39 °C.
  • Candidate DFM strains, Enterococcus faecium strain 8G-1 , Enterococcus faecium strain 8G-73, and Bacillus pumilus strain 8G-134 were added to designated bottles at 1.0 x 10 7 CFU/ml (final concentration). The unit of observation was the bottle, and treatments were performed in quadruplicate. Treatments consisted of Control (glucose added but no DFM), Enterococcus faecium strain 8G-1 , Enterococcus faecium strain 8G-73, and Bacillus pumilus strain 8G-134. Bottles were then purged with of C0 2 and capped.
  • Bottles were maintained in a shaking water bath at 39°C and 140 rpm. Approximately 10 minutes prior to sampling, bottles were briefly vented to release gases produced as a byproduct of fermentation. Rumen fluid was withdrawn from each bottle initially and every 6 hours until the 36 hour mark. Rumen pH and volatile fatty acids were measured and recorded. Statistical analysis was performed using repeated measures analysis to determine DFM effects over time or one-way ANOVA to determine treatment affects at a specific points in time.
  • the daily feed ration for all treatment groups prior to challenge consisted of 62.5% roughage and 30% cracked corn and 7.5% protein supplement (Table 3) below.
  • the protein supplement contained monensin (Rumensin®) fed at 375 mg/head/day.
  • the protein supplement also contained tylosin phosphate (Tylan®).
  • Cattle were fed 15 lbs/head/day of the ration once in the morning and had any remaining feed pushed closer to the feeding stanchion late in the afternoon.
  • Treatment groups were fed candidate DFMs at the dose designated in Table 2 above as a top dressing on the daily ration.
  • Bacillus pumilus strain 8G-134 was fed at a minimum of 5 x 10 y CFU/Head/Day.
  • the Enterococcus candidates 8G-1 and 8G-73 were fed at 5 x 10 1U CFU/Head/Day.
  • Candidate DFM strains previously selected for the challenge trial, were Enterococcus spp. 8G-1 and 8G-73; and Bacillus pumilus strain 8G-134. Strains were stored at -80°C. Each culture was inoculated into 10 ml broth tubes containing MRS (Man, Rogosa and Sharp) or TSB (tryptic soy broth). Broth tubes were incubated for 24 hours at 32° and 37°C for the Bacillus and Enterococcus candidates, respectively. Cultures were struck for isolation on respective agar medium and incubated. An isolated colony was picked into 10 ml of broth and allowed to grow to mid log phase (18 to 24 h) and transferred into fresh broth (10% vol/vol transfer).
  • MRS Man, Rogosa and Sharp
  • TSB tryptic soy broth
  • Enterococcus candidates were grown at 37°C in MRS broth. Bacillus was grown at in a shaking incubator at 130 rpm at 32°C in horizontal TSB tubes. For the growth of Enterococcus , 2 ml were transferred into a 250 ml bottle containing 198 ml of broth and incubated for 18hrs.
  • the 200 ml of culture was inoculated into a 2 L bottle containing 1.8 L of broth and allowed to incubate for 18hr.
  • 5 ml were transferred into a 250 flask containing 50 ml of TSB and then was incubated at 32°C in a shaking incubator at 130 rpm for 24hr.
  • the 50 ml was used to inoculate a 1L flask containing 600 ml and allowed to incubate for another 24 hours.
  • the optical density (OD) of the 18hr culture of Enterococcus candidates was taken before harvesting the cells. The OD was compared to previous growth curves to determine the cfu/ml of culture. Samples were plated for enumeration and genetic fingerprinting. Quality control was ensured between each fermentation batch via RAPD-PCR analysis. With a target minimum of 5.0el0 cfu/head/day for Enterococcus candidates, the calculated amount of culture was dispensed into 250 ml Nalgen centrifuge bottle and spun at 4°C for lOmin at 4500 rpm.
  • Target minimum for Bacillus candidate was 5.0e9 cfu/head/ day, and a total of 100 ml of the Bacillus culture was spun down similar to the Enterococcus. Supernatant was discarded. The pellet was resuspended in 30 ml of growth media containing 10% glycerol. This amount was transferred to a 50 ml conical tube. The centrifuge bottles were then rinsed with 10 ml of broth and transferred to the same conical tube. Samples were labeled with strain, date the candidate was harvested, and fermentation batch number. Plate counts were used to determine the total cfu in each tube. Tubes were combined to deliver counts of a minimum of 5.0el0 cfU/head/day for Enterococcus candidates and 5.0e9 cfu/head/day for Bacillus candidates. All conical tubes were frozen at - 20°C
  • Rumen fluid samples were obtained from individual animals via ruminal intubation using a collection tube fitted with a strainer and attached to a vacuum source through a vacuum flask. The pH was immediately measured after rumen fluid acquisition and samples were frozen to be transported to Agtech Products, Inc. for VFA analysis. Samples were collected from all cattle at sample times -12h, +6h, +10h, +14h, +18h, +22h, +30h, +36h, and +48h, with time Oh representing the initiation of the challenge. All feed was removed from the cattle at time -24h to initiate the fast and encourage cattle to engorge the challenge ration at time 0.
  • the concentrate diet consisted of 28 lb flake weight steam fiaked corn (Table 3 above).
  • the challenge ration was fed to deliver 20 lbs/head. Challenge ration consumption was visually monitored and additional feed added on an as needed basis through the remainder of the trial.
  • Ruminal samples were collected every 4 hours from all cattle from +6 to +22 hours. Each rumen sample pH was analyzed immediately after acquisition. Rumen fluid was then frozen and transported to Agtech Products, Inc. for VFA analysis via HPLC. Repeated measures analysis was performed on rumen pH, VFAs, and glucose levels using individual animal as the unit of observation. Pairwise comparisons were performed over time between each candidate DFM treatment pens and the control pen to determine the candidates' effectiveness to alter ruminal fermentation patterns.
  • Average feed consumption/steer was calculated as a percentage of the average steer weight for that pen
  • lactic acid profiles for all treatment groups are shown in Figure 7.
  • Candidate DFM strains 8G-1, 8G-73, and 8G-134 again exhibited visible mean numeric differences in lactate accumulation in comparison to that of the control pen.
  • Mean lactic acid accumulation was similar between the control cattle and the 8G-1 treated cattle through the first 14 hours of the challenge. Subsequent accumulation levels for the remainder of the trial were much less in the 8G-1 treated cattle although not significant (P 0.1892).
  • Treatment pens 8G-73 demonstrated decreased levels of lactic acid accumulation at times 30 and remained lower than the control pen for the remainder of the trial.
  • Candidate strain 8G-134 also showed decreased levels of lactic acid starting at +22h and remained consistently lower than the control pen through +48h.
  • VFA Volatile fatty acid
  • VFA concentration or the individual VFA (consisting of acetate, propionate, or butyrate) levels.
  • Acute acidosis is marked by the accumulation of lactic acid and the decline in VFA production.
  • Proper rumen function is a combination of managing the available energy and nitrogen components available in feedstuffs.
  • digestive upset typically follows and can manifest in the form acidosis.
  • strains 8G-1 , 8G-73, and 8G-134 enhanced the recovery of rumen function as indicated by ruminal fermentation parameters.
  • Cattle fed 8G-1 Enterococcus faecium, on average recovered more quickly from the acidosis challenge as measured by pH recovery and lactic acid decline.
  • cattle fed candidate DFM 8G-1 did not exhibit clinical signs associated with acidosis.
  • Mean lactic acid levels were the lowest for all candidate strains tested at +48h at 12.54 mM.
  • a corresponding increase in pH was also associated with the recovery with a final pH of 6.02, which was 1.08 pH units higher than that of the control.
  • Candidate strain 8G-134 Bacillus pumilus, also enhanced ruminal recovery during the acidotic challenge.
  • Mean lactic acid levels, in cattle fed 8G-134 peaked at 89mM at time +22h, while the control pen continued to increase and peaked at 105mM at time +30 hours.
  • TMR basal total mixed ration
  • Treatment 2 and Treatment 3 which received basal total mixed ration TMR and were fed Bacillus pumilus 8G-134 at 5 x 10 9 and 1 x 10 10 CFU/head/day, respectively, from 3 weeks prepartum to 22 week after parturition.
  • the primary objective was to determine the effects of B. pumilus 8G-134 on dairy cow milk production and performance above control cattle during this time period.
  • the secondary objective was to determine if there was a dose response associated with feeding B.
  • B. pumilus 8G-134 The B. pumilus 8G-134 regimens significantly increased milk production, milk fat, and decreased somatic cell count. These significant B. pumilus 8G-134 production effects did not come at the expense of cow body condition score, body weight, increases in dry matter intake or significantly change blood metabolite profiles, and would indicate B. pumilus 8G-134 also provided dairy cow efficiency benefits.
  • TMRs total mixed rations
  • NeL mcal/kg 1.60 1.74
  • Cows were milked twice a day, and milk volume was recorded electronically at each milking and am-pm amounts summed for daily total. Once a week milk samples from am and pm milkings were composited for analysis of content of fat, protein, somatic cells, solids not fat, and milk urea nitrogen (MUN) by Dairy One milk laboratory in State College, PA using a
  • Fossamatic 4000 (FOSS; Eden Prairie, MN). Animals were on study from approximately three weeks prepartum through 22 weeks postpartum. Animal weight was estimated by heart girth circumference on weeks 1 , 3, 7, 1 1 , 15 and 18 postpartum. Body condition was assessed by two independent observers at the same time as body weight was collected.
  • Blood samples were collected from the coccygeal vein, serum harvested, frozen and analyzed for glucose, beta-hydroxy butyrate (BHB), and non-esterified fatty acids ( EFA) at weeks 2 and 8 postpartum.
  • BHB beta-hydroxy butyrate
  • EFA non-esterified fatty acids
  • Glucose and BHB were analyzed using an Abbott Precision XtraTM meter (Abbott Diabetes Care Inc., Alameda, CA).
  • a Randox assay kit Cat. HN 1530, Randox Laboratories, Northern Ireland
  • NEFA non-esterified fatty acid
  • ELIS A enzyme linked immunosorbant assay
  • the Randox kit uses Acyl CoA synthetase and oxidase to convert NEFA to 2,3-trans-Enoyl-CoA plus peroxide; peroxide plus N-ethyl-N-(2 hydroxy-3-sulphopropyl) m-toluene leads to a purple product, which is the indicator of NEFA concentration in serum.
  • Milk production and content, body weight, and body condition score were analyzed using the mixed procedure in SAS statistical software. Cow was the repeated subject with the co variance matrix set to type 1 correlation structure. Daily milk observations were aggregated by week postpartum.
  • the statistical model was as follows:
  • TRT j jth treatment effect, 1 , 2, 3;
  • Lact k kth lactation, 1 , 2+;
  • Weeki lth week, 1..22;
  • Blood concentrations of glucose, BHB, and NEFA were analyzed using the general linear models in SAS statistical software. Class variables were cow, week and treatment.
  • Treatment was nested in cow and was the error term for testing treatment significance. Treatment by week interaction was tested for statistical significance using the residual error.
  • TMR composition for dry and lactating TMRs is presented in Table 6 over the course of the study. Composition was not different between the treatment groups.
  • Table 7 Least square mean milk production in Holstein cows from calving through 22 weeks postpartum fed a microbial additive.
  • Treatment 1 33.12 a 0.65 0.00 0.66
  • Milk fat and yield are presented in Table 8 below. Milk fat was significantly increased by Treatments 2 and 3 above the control. Yield responses followed milk yield with fat yield increased in the Bacillus pumilus 8G-134 fed treatment groups. Bacillus pumilus 8G-134 cattle for treatment 2 and 3 yielded significantly higher fat percentage above that of the control with 0.24% and 0.31% higher levels, respectively (Table 8). Coupled with the significant increase in milk production, daily fat yield for both treatment 2 and 3 provided significant increases in daily fat production above that of the control (Table 8). Table 8. Milk fat content by treatment groups.
  • Log of the linear Somatic cell count (LogSCC) scores were different by treatment and lactation.
  • the Bacillus pumilus 8G-134 treatments (Treatments 2 and 3) had significantly lower log linear score than the control cows (Table 9 below).
  • Treatments 2 and 3 cows had LogSCC of 4.97 and 4.96, respectively compared to those of the control cows at 5.92. Parity two cows had significantly higher log linear score than first lactation cows. Somatic cell counts are associated with infection as well as immunological status and health of the lactating dairy cow.
  • SCC is indicative of inflammatory responses to infection. Decreases in SCC demonstrated here may indicate that cows fed the Bacillus pumilus 8G-134 are better immunologically to handle infectious challenge during lactation and maintain udder and cow health.
  • Table 10 Least square means for group feed intake, serum glucose, beta-hydroxy butyrate, esterified fatty acids, by treatment group. Treatment group ⁇
  • Beta-OH butyrate mg/dl 1. .05 0, .15 0.88 0, .15 1.15 0, .15
  • BHB beta-hydroxy butyrate
  • LS pre andpostpartum + inert limestone carrier postpartum LSCO
  • LSCO LS pre and postpartum + Bacillus pumilus 8G-134 (BP) postpartum
  • HSCO HS pre and postpartum + inert limestone carrier postpartum
  • HSBP HS pre and postpartum +BP postpartum
  • Serum calcium concentrations on d -7 and d 1 relative to calving were measured using a calcium (Arsenazo) reagent set (Point Scientific, Inc., Canton, MI). Serum haptoglobin concentrations were determinedby a colorimetric procedure as described by Hulbert et al. (201 1). Absorbance for EFA, glucose, calcium, and haptoglobin assays was quantified using a microplate spectrophotometer (Eon TM, BioTek Instruments Inc., Winooski, VT). Serum BHBA concentrations were quantified using the Precision Xtra® ketone monitoring system direct electrochemical test (Abbot Laboratories Inc., Abbott Park, IL).
  • a droplet of serum was placed on a ketone test strip containing the enzyme ⁇ -hydroxybutyrate dehydrogenase, which oxidizes BHBA to acetoacetate.
  • the enzyme ⁇ -hydroxybutyrate dehydrogenase reduces nicotinamide adenine dinucleotide (NAD+) to NADH.
  • NAD+ nicotinamide adenine dinucleotide
  • the NADH is then reoxidized to NAD+ by an electron transfer mediator molecule.
  • the electrical current generated by this conversion is measured by the meter and is directly proportional to the BHBA concentration (Oetzel and McGuirk, 2007).
  • Postpartum data were analyzed as a randomized complete block design with a 2 X 2 factorial arrangement of treatments using MIXED models procedure of SAS.
  • Model included effects of breed, starch concentration, BP, time, and interactions between starch, BP, and time.
  • On pre- and postpartum periods repeated measures over time were modeled with autoregressive [AR (1)], and denominator degrees of freedom were estimated using Kenwards-Rogers method. Single measurements were modeled with autoregressive [AR (1)], and denominator degrees of freedom were estimated using Satterthwaite methods.
  • Least squares means for starch, BP, time and all interactions were separated by use of PDIFF statement when the overall F-test was P ⁇ 0.05. Trends were indicated when P ⁇ 0.10.
  • Feeding B. pumilus 8G-134 reduces the levels of nonesterified fatty acids (NEFA) in the blood of dairy cows in the early postpartum period
  • BHBA beta-hydroxybutyrate
  • NEFA nonesterified fatty acids
  • DM dry matter
  • CP crude protein
  • RUP rumen undegradable
  • ADF acid detergent fiber
  • NDF neutral detergent fiber
  • EB Energy balance
  • measures of milk production quantity and composition
  • dietary intake quantity and composition
  • body condition has been associated with postpartum health and fertility.
  • high milk production can come at the expense of body condition score, as fat and muscle are mobilized to support production, particularly in high yielding dairy cows.
  • Loss of body condition is associated with altered blood metabolite and hormone profiles which may influence fertility and it has been demonstrated that cows with a low body condition score at 7-10 weeks postpartum take longer to conceive.
  • negative energy balance is associated with metabolic diseases such as ketosis.
  • pumilus 8G-134 is influencing the energy balance of dairy cows through an immunomodulatory mechanism, having downstream beneficial impacts on metabolic markers (including NEFA and BHBA), metabolic diseases (SCK and RP), and eventually milk production. It is anticipated that greater reproductive efficiency will also be a result of B. pumilus 8G-134 administration.
  • Treatments were mixed with 0.45 kg ground corn and top-dressed on the close-up or lactation TMR once daily for each cow. Treatments were applied from 21 ⁇ 1 d before expected calving date to 154 d after calving.
  • Cows were randomly assigned to treatments and balanced for initial body weight (BW; 719 ⁇ 9.59 kg vs 715 ⁇ 9.78 kg for CON and DFMt, respectively), parity (2.53 ⁇ 0.42 vs 2.03 ⁇ 0.36 for CON and DFMt, respectively), and previous lactation (305 d) milk production (11,703 ⁇ 543 kg vs 1 1,389 ⁇ 473 kg for CON and DFMt, respectively).
  • Feed ingredients and TMR samples were obtained weekly and analyzed for dry matter (DM) content (AO AC, 1995) by drying for 24 h in a forced-air oven at 110°C. Dietary DM was adjusted weekly for changes in DM content. Total mixed ration samples were taken weekly, and stored at -20°C until submitted for analysis. Monthly composite samples were analyzed for contents of DM, crude protein (CP), acid-detergent fiber (ADF), neutral-detergent fiber (NDF), lignin, starch, fat, ash, Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, and S using wet chemistry methods (Dairy One, Ithaca, NY). Values for RFV, TDN, NE1, NEm, NEg, ME, and DE were provided by the lab and were based on NRC (2001). Intake from each cow was measured and DMI was recorded daily.
  • Cows were milked 3 times daily at 0600, 1400, and 2100 h. Milk weights were recorded daily and samples were obtained from 3 consecutive milkings weekly. Consecutive weekly samples were composited in proportion to milk yield at each sampling and preserved (800 Broad Spectrum Microtabs II; D&F Control Systems, Inc., San Ramon, CA). Composite milk samples were analyzed for fat, protein, lactose, urea N (MUN), total solid and somatic cell count (SCC) using mid-infrared procedures (AO AC, 1995) at a commercial laboratory (Dairy Lab Services, Dubuque, IA).
  • MUN lactose
  • SCC total solid and somatic cell count
  • RP retained placenta
  • DA displaced abomasum
  • CK clinical ketosis
  • MAST mastitis
  • MET metritis
  • General appearance was scored as (Krause et al., 2009): 1 : bright and alert; 2: depressed; 3: reluctant to rise.
  • Cows with fecal score ⁇ 2 were classified as experiencing transient digestive problems (FS ⁇ 2) whereas cows with FS > 2 were classified as healthy (HEALTHY). Cows with GA > 2 were classified as sick (ALTERED) whereas cows with GA ⁇ 2 were classified as healthy
  • Body weight was measured and body condition score (BCS) was assigned in quarter unit increments (Ferguson et al., 1994) for each cow weekly. More than one individual assigned BCS independently at each time of scoring throughout the experiment.
  • BCS body condition score
  • BHBA Beta-hydroxybutyrate
  • NEFA non-esterified fatty acids
  • the immunoglubulins, IgA, IgG and IgM were quantified in milk collected during the first week after calving and serum collected on d 5 and d 14 by an ELISA assay (Bethyl laboratories, Montgomery, TX) according to the manufacturer's protocol. Haptoglobin was quantified in serum samples by an ELISA assay (ALPCO, SALEM, NH) according to the manufacturer's protocol. Cows that had haptoglobin serum concentrations lower than 20 ⁇ g/ml were classified as negative (NEGATIVE) whereas cows that had serum haptoglobin concentrations higher than 20 ⁇ g/ml were classified as positive (POSITIVE), according to cut-off points previously established by Eckersall et al. (2010). D. Statistical Analyses
  • the data were analyzed using SAS (v 9.3; SAS Institute Inc., Cary, NC).
  • the MIXED models procedure was used for the outcomes of interest DMI, BW, BCS, milk parameters, and composed variables (e.g. FE), which were averaged weekly.
  • the model contained the fixed effects of treatment, week, and the interaction of treatment by week.
  • Initial measurements, before treatment administration, were used as covariates when analyzing the dependent variables BW and BCS.
  • Variables were subjected to 5 covariance structures: compound symmetry, auto regressive order 1, autoregressive heterogeneous order 1, unstructured, and Toeplitz.
  • the covariance structure that yielded the lowest corrected Akaike information criterion was used in the model (Littell et al., 1998).
  • Cow was the experimental unit and considered as a random effect. Week was included in the model as a repeated measurement with cow as subject. All performance variables were analyzed as weekly averages. Least squares means were calculated and are presented with standard errors of means (SEM). Degrees of freedom were estimated by using the Kenward-Roger method in the model statement (Littell et al., 1998). Residual distribution was evaluated for normality and homoscedasticity.
  • Dry matter intake change from 3 wk before and after calving, milk yield change from calving to 3 wk, blood BHBA, plasma NEFA, serum and milk IgA, Ig M, IgG and serum haptoglobin concentrations were analyzed as continuous variables using the MIXED procedure.
  • GLIMMIX procedure multivariable logistic mixed models
  • SCK, NEFA, RP, DA, CK, MET, MAST, FS, GA, and HAPTOGLOBIN dichotomized variables
  • the procedure used for each variable is also indicated in the results section for each outcome of interest.
  • a log transformation was used for the variables NEFA, SCC, and haptoglobin for better homogeneity of the distribution of residuals. Means shown in tables and graphs for these variables are back-transformed.
  • A. Feeding B. pumilus 8G-134 reduces disease associated with negative energy balance in early postpartum dairy cows.
  • Muciparous dairy cows fedS. pumilus 8G-134 at 5 x 10 9 CFU/cow/day had a tendency for lower incidence of subclinical ketosis (SCK) at d 5 postpartum (P 0.09) as measured by blood BHBA level than cows fed a control diet with no DFM (Table 14).
  • Cows fed B. pumilus 8G-134 at 5 x 10 9 CFU/cow/day also tended (P 0.13) to have lower incidence of retained placenta (RP) than cows fed a control diet with no DFM (Table 15).
  • Cows that received CON showed signs of excessive adipose tissue mobilization as indicated by higher levels of BHBA and NEFA after calving when compared to DFMt cows (Table 16).
  • DFMt improved immunity in the mammary gland and reduced the incidence of an elevated haptoglobin response. All the measured health occurrences are shown in Table 16.
  • Bacillus pumilus 8G-134 (DFMt) or placebo (CON) from week 4 before calving through week 22 after calving.
  • Immunoglobulin A in milk of Holstein cows top-dressed with Bacillus pumilus 8G-134 (DFMt) Immunoglobulin A have protective effects that would aid in the defense against pathogens in the mammary gland.
  • Immunoglobulin A normally found at mucosal sites, binds to bacterial cells and prevents their adhesion to epithelial cells.
  • the additional immune defense provided by DFMt could explain the difference in haptoglobin levels between DFMt and CON cows.
  • serum haptoglobin concentration is below 20 ⁇ g/ml.
  • haptoglobin serum concentrations can increase 100 fold or more.
  • IgA in the mammary gland may provide greater protection against infection, reducing the incidence of disease and elevated haptoglobin concentrations. Elevated milk immunoglobulins also have the added benefit of boosting neonatal calf health through passive transfer of immunity. IgA in particular provides mucosal protection in the calf gastrointestinal tract, aiding in the development of a healthy immune system and gut microbiota.
  • Table 18 Least squares means of IgA in milk from Holstein cows top-dressed with Bacillus pumilus 8G-134 (DFM) or placebo (CON) from wk 4 before calving through wk 22 after calving at 5 and 14 d relative to calving.
  • DFM Bacillus pumilus 8G-134
  • CON placebo
  • T cell surface markers CD4, CD8, CD62L, CD25, and CD45RO Figures 10 and 11
  • the enhanced expression of T cell markers may help dairy cows recover faster from immune suppression that naturally occurs during parturition, increasing resistance to mastitis and other common postpartum diseases.
  • cows must restore the body condition lost during early lactation to be able to partition energy toward fetal growth of a calf.
  • care must be taken to avoid overconditioning or fatness, as cows with high body condition score precalving showed higher losses in body condition postparturition that negatively affected pregnancy rates and may spend more time in a negative energy balance, resulting in metabolic disorders and reproductive problems leading to economic consequences.
  • Production and component least-squares means (+/- SE) by treatment with Days in Milk (DEVI) category included in the statistical model for cows on study for 12 weeks
  • B. pumilus 8G-134 is influencing the energy balance of dairy cows through an immunomodulatory mechanism, having downstream beneficial impacts on metabolic markers (including NEFA and BHBA), metabolic diseases (SCK and RP), and milk production. It is anticipated that greater reproductive efficiency will be a result of B. pumilus 8G- 134 administration.
  • HSCO High starch pre- and postpartum + Bacillus pumilus postpartum
  • Prepartum diets LS (12 % starch), and HS (19 % starch).
  • Postpartum diets LS (20 % starch), and HS (27
  • NEFA concentration was consistently greater for LSCO and HSCO compared with LSBP and HSBP, respectively, throughout the 28 d after calving ( Figure 13).
  • Cows assigned to LSBP tended to have 2.5 mg/dl lower BHBA concentration than LSCO, and HSBP had 6.5 mg/dl lower BHBA concentration than HSCO.
  • BHBA concentration was consistently greater for LSCO and HSCO compared with LSBP and HSBP, respectively, throughout the 28 d after calving (Figure 13).
  • Bacillus pumilus supplementation could be advantageous when fed to early lactation cows due to a reduction in NEFA and a reduction in BHBA, which demonstrates a decrease in body lipid mobilization.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Husbandry (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Birds (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Physiology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Fodder In General (AREA)
  • Feed For Specific Animals (AREA)

Abstract

La présente invention concerne des souches comprenant Enterococcus faeciumstrain 8G-1 (NRRL B-50173), Enterococcus faeciumstrain 8G-73 (NRRL B-50172), Bacillus pumilusstrain 8G-134 (NRRL B-50174) et des souches ayant toutes les caractéristiques d'identification de chacune de ces souches. Une ou plusieurs souches peuvent être utilisées pour réduire un bilan énergétique négatif chez un ruminant. Elles peuvent également être utilisées pour améliorer d'autres mesures de la santé et/ou des performances des ruminants. L'invention concerne également des procédés d'utilisation des souches, seules et en combinaison. L'invention porte également sur des procédés de fabrication des souches.
PCT/EP2015/077704 2008-12-02 2015-11-25 Souches et procédés permettant une segmentation énergétique chez les ruminants WO2016083481A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
US15/528,660 US20170258111A1 (en) 2014-11-26 2015-11-25 Strains and methods for energy partitioning in ruminants
AU2015352558A AU2015352558A1 (en) 2014-11-26 2015-11-25 Strains and methods for energy partitioning in ruminants
BR112017011067A BR112017011067A2 (pt) 2008-12-02 2015-11-25 cepas e métodos para a partição de energia em ruminantes
CN201580074305.XA CN107205435A (zh) 2008-12-02 2015-11-25 用于在反刍动物中进行能量分配的菌株和方法
EP15808549.8A EP3267802A1 (fr) 2014-11-26 2015-11-25 Souches et procédés permettant une segmentation énergétique chez les ruminants
MX2017006886A MX2017006886A (es) 2008-12-02 2015-11-25 Cepas y métodos para el reparto de energía en rumiantes.
CA2968734A CA2968734A1 (fr) 2014-11-26 2015-11-25 Souches et procedes permettant une segmentation energetique chez les ruminants

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US14/554,726 2014-11-26
US14/554,726 US10172892B2 (en) 2008-12-02 2014-11-26 Strains and methods for energy partitioning in ruminants

Publications (1)

Publication Number Publication Date
WO2016083481A1 true WO2016083481A1 (fr) 2016-06-02

Family

ID=54849912

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2015/077704 WO2016083481A1 (fr) 2008-12-02 2015-11-25 Souches et procédés permettant une segmentation énergétique chez les ruminants

Country Status (5)

Country Link
EP (1) EP3267802A1 (fr)
AU (1) AU2015352558A1 (fr)
CA (1) CA2968734A1 (fr)
MX (1) MX2020013361A (fr)
WO (1) WO2016083481A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107526945A (zh) * 2016-06-22 2017-12-29 黑龙江八农垦大学 一种血清nefa评价奶牛乳成分及饲料转化水平的方法
CN113712121A (zh) * 2021-08-31 2021-11-30 河南牧业经济学院 尼克酰胺在改善泌乳期奶山羊乳成分中的应用
CN114727585A (zh) * 2019-11-27 2022-07-08 利拉伐控股有限公司 用于动物健康状况指示的挤奶装置

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100172873A1 (en) * 2008-12-02 2010-07-08 Danisco A/S Strains and methods for improving ruminant health and/or performance
US20130064927A1 (en) * 2011-08-24 2013-03-14 Dupont Nutrition Biosciences Aps Enzyme producing bacillus strains
US20140323430A1 (en) * 2011-03-03 2014-10-30 The Governors Of The University Of Alberta Use of bacterial endotoxins and lipoteichoic acids to improve postpartal health and productivity of dairy cows and their newborns

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100172873A1 (en) * 2008-12-02 2010-07-08 Danisco A/S Strains and methods for improving ruminant health and/or performance
US20140323430A1 (en) * 2011-03-03 2014-10-30 The Governors Of The University Of Alberta Use of bacterial endotoxins and lipoteichoic acids to improve postpartal health and productivity of dairy cows and their newborns
US20130064927A1 (en) * 2011-08-24 2013-03-14 Dupont Nutrition Biosciences Aps Enzyme producing bacillus strains

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
H IWASAKI ET AL: "Comparative studies of lipoteichoic acids from several Bacillus strains", JOURNAL OF BACTERIOLOGY, 1 August 1986 (1986-08-01), UNITED STATES, pages 508 - 516, XP055256978, Retrieved from the Internet <URL:http://jb.asm.org/content/167/2/508.full.pdf> [retrieved on 20160310] *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107526945A (zh) * 2016-06-22 2017-12-29 黑龙江八农垦大学 一种血清nefa评价奶牛乳成分及饲料转化水平的方法
CN107526945B (zh) * 2016-06-22 2021-01-29 黑龙江八一农垦大学 一种血清nefa评价奶牛乳成分及饲料转化水平的方法
CN114727585A (zh) * 2019-11-27 2022-07-08 利拉伐控股有限公司 用于动物健康状况指示的挤奶装置
CN113712121A (zh) * 2021-08-31 2021-11-30 河南牧业经济学院 尼克酰胺在改善泌乳期奶山羊乳成分中的应用
CN113712121B (zh) * 2021-08-31 2023-08-15 河南牧业经济学院 尼克酰胺在改善泌乳期奶山羊乳成分中的应用

Also Published As

Publication number Publication date
CA2968734A1 (fr) 2016-06-02
AU2015352558A1 (en) 2017-06-15
MX2020013361A (es) 2021-03-09
EP3267802A1 (fr) 2018-01-17

Similar Documents

Publication Publication Date Title
US20190134112A1 (en) Strains and methods for energy partitioning in ruminants
Saleem et al. Growth performance, nutrients digestibility, and blood metabolites of lambs fed diets supplemented with probiotics during pre-and post-weaning period
He et al. Supplementing a yeast probiotic to pre-weaning Holstein calves: Feed intake, growth and fecal biomarkers of gut health
US11622569B2 (en) Bacillus microbial terroir for pathogen control in swine
Hussein Effect of biological additives on growth indices and physiological responses of weaned Najdi ram lambs.
Luan et al. Effects of direct-fed Bacillus pumilus 8G-134 on feed intake, milk yield, milk composition, feed conversion, and health condition of pre-and postpartum Holstein cows
Carpinelli et al. Effects of peripartal yeast culture supplementation on lactation performance, blood biomarkers, rumen fermentation, and rumen bacteria species in dairy cows
Lu et al. Effects of feed additives on the development on the ileal bacterial community of the broiler chicken
US20170258111A1 (en) Strains and methods for energy partitioning in ruminants
EP3267802A1 (fr) Souches et procédés permettant une segmentation énergétique chez les ruminants
Satık Effects of kefir as a probiotic source on the performance and health of young dairy calves
Wojnicki et al. Immunomodulatory effects of whole yeast cells and capsicum in weanling pigs challenged with pathogenic Escherichia coli
Ye et al. Oral administration of Megasphaera elsdenii to Jersey cows during early lactation
AU2019210497B2 (en) Strains and methods for improving ruminant health and/or performance
EP3984376A1 (fr) Lactase pour utilisation
Shetawy et al. EFFICACY OF USING PROBIOTICS CONTAINING Bacillus subtilis AND Bacillus licheniformis SPORES ON PERFORMANCE AND HEALTH OF HOLSTEIN SUCKLING CALVES
AU2012207046A1 (en) Strains and methods for improving ruminant health and/or performance
WO2017081193A1 (fr) Complément alimentaire comprenant du bêta-glucane et un hydrolysat de caséine pour améliorer la santé et les performances de croissance chez un mammifère
Amanullah et al. Effects of feeding probiotic on daily gain, fecal characteristics and blood metabolites in calf
WO2024112863A1 (fr) Compositions et procédés pour augmenter l&#39;efficacité alimentaire chez des animaux
JP2023530671A (ja) ブタの健康及び発育成績を向上させるブタ由来のプロバイオティクス
Conklin Evaluating the impact of Bovamine® on performance, nutrient digestibility, and digestive function in lactating dairy cows

Legal Events

Date Code Title Description
REEP Request for entry into the european phase

Ref document number: 2015808549

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15808549

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 15528660

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2968734

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: MX/A/2017/006886

Country of ref document: MX

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112017011067

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2015352558

Country of ref document: AU

Date of ref document: 20151125

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112017011067

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20170525