Patent Application
20230139325
The present disclosure relates to isolated and biologically pure microorganisms that have applications, inter alia, in dairy production. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into compositions. Furthermore, the disclosure provides microbial ensembles, containing at least two members of the disclosed microorganisms, as well as methods of utilizing said microbial ensembles. Furthermore, the disclosure provides for methods of modulating the rumen microbiome.
The sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is ASBI_021_02WO_ST25.txt. The text file is ˜901 kb, was created on Mar. 31, 2021, and is being submitted electronically via EFS-Web.
The global population is predicted to increase to over 9 billion people by the year 2050 with a concurrent reduction in the quantity of land, water, and other natural resources available per capita. Projections indicate that the average domestic income will also increase, with the projected rise in the GDP of China and India. The desire for a diet richer in animal-source proteins rises in tandem with increasing income, thus the global livestock sector will be charged with the challenge of producing more milk using fewer resources. The Food and Agriculture Organization of the United Nations predict that 70% more food will have to be produced, yet the area of arable land available will decrease. It is clear that the food output per unit of resource input will have to increase considerably in order to support the rise in population.
Milk and milk components from lactating ruminants are predominantly utilized in the preparation of foodstuffs in many different forms. Nevertheless, milk and milk components find numerous alternative applications in non-food areas such as the manufacture of glues, textile fibers, plastic materials, or in the production of ethanol or methane. There have been many strategies to improve milk production and content in ruminants through nutritional modulations, hormone treatments, changes in animal management, and selective breeding; however, the need for more efficient production of milk and milk components per animal is required.
Identifying compositions and methods for sustainably increasing milk production and modulating milk components of interest while balancing animal health and wellbeing have become imperative to satisfy the needs of every day humans in an expanding population. Increasing the worldwide production of milk and further modulating desirable milk components by scaling up the total number of livestock on dairy farms would not only be economically infeasible for many parts of the world, but would further result in negative environmental consequences.
Thus, meeting global milk and milk component yield expectations, by simply scaling up current high-input agricultural systems—utilized in most of the developed world—is simply not feasible.
There is therefore an urgent need in the art for improved methods of increasing milk production and further increasing yield of desirable milk components.
In some embodiments, the present disclosure provides an orally deliverable composition for increasing milk production or improving milk compositional characteristics in a ruminant, comprising: (a) Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108; and (b) a carrier suitable for oral ruminant administration. In some embodiments, the Ruminococcus bovis is deposited as TSD-225 or NCTC 14479. In some embodiments, the composition comprises: (a) one or more bacteria comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 1-30 and 2045-2103; and/or (b) one or more fungi comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 31-60 and 2104-2107. In some embodiments, the composition comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 32; and/or (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2067. In some embodiments, the composition comprises: (a) a Clostridium sp. with a deposit accession number of NRRL B-67248; (b) a Pichia sp. with a deposit accession number of NRRL Y-67249; and/or (c) a Butyrivibrio sp. with a deposit accession number of NRRL B-67347.
In some embodiments, the Ruminococcus bovis comprises one or more mutations in the whole genome. In some embodiments, the one or more mutations are selected from the group consisting of: (a) a G→T substitution at position 297 of SEQ ID NO: 2109; (b) a CC→TA substitution at positions 301-302 of SEQ ID NO: 2111; (c) a T→G substitution at position 307 of SEQ ID NO: 2111; (d) a −A deletion at position 300 of SEQ ID NO: 2113; (e) a CCA→TTC substitution at positions 116-118 of SEQ ID NO: 2115; (f) a +T insertion between positions 105-106 of SEQ ID NO: 2117; (g) a C→T substitution at position 298 of SEQ ID NO: 2119; (h) a C→A substitution at position 298 of SEQ ID NO: 2121; and (i) a +AC insertion between positions 43-44 of SEQ ID NO: 2123.
In some embodiments, the present disclosure provides an orally deliverable composition for increasing milk production or improving milk compositional characteristics in a ruminant, comprising: (a) Ruminococcus bovis comprising one or more mutations selected from the group consisting of: (i) a G→T substitution at position 297 of SEQ ID NO: 2109; (ii) a CC→TA substitution at positions 301-302 of SEQ ID NO: 2111; (iii) a T→G substitution at position 307 of SEQ ID NO: 2111; (iv) a −A deletion at position 300 of SEQ ID NO: 2113; (v) a CCA→TTC substitution at positions 116-118 of SEQ ID NO: 2115; (vi) a +T insertion between positions 105-106 of SEQ ID NO: 2117; (vii) a C→T substitution at position 298 of SEQ ID NO: 2119; (viii) a C→A substitution at position 298 of SEQ ID NO: 2121; and (ix) a +AC insertion between positions 43-44 of SEQ ID NO: 2123; and (b) a carrier suitable for oral ruminant administration. In some embodiments, the composition comprises: (a) one or more bacteria comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 1-30 and 2045-2103; and/or (b) one or more fungi comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 31-60 and 2104-2107. In some embodiments, the composition comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 32; and/or (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2067. In some embodiments, the composition comprises: (a) a Clostridium sp. with a deposit accession number of NRRL B-67248; (b) a Pichia sp. with a deposit accession number of NRRL Y-67249; and/or (c) a Butyrivibrio sp. with a deposit accession number of NRRL B-67347.
In some embodiments, the present disclosure provides an orally deliverable composition for increasing milk production or improving milk compositional characteristics in a ruminant, comprising: (a) Ruminococcus bovis comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 2110, 2112, 2114, 2116, 2118, 2120, 2122, or 2124; and (b) a carrier suitable for oral ruminant administration. In some embodiments, the composition comprises: (a) one or more bacteria comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 1-30 and 2045-2103; and/or (b) one or more fungi comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 31-60 and 2104-2107. In some embodiments, the composition comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 32; and/or (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2067. In some embodiments, the composition comprises: (a) a Clostridium sp. with a deposit accession number of NRRL B-67248; (b) a Pichia sp. with a deposit accession number of NRRL Y-67249; and/or (c) a Butyrivibrio sp. with a deposit accession number of NRRL B-67347.
In some embodiments, the present disclosure provides an orally deliverable composition for increasing milk production or improving milk compositional characteristics in a ruminant, comprising: (a) a Ruminococcus bovis with the deposit accession number PTA-125917, NRRL B-67764, TSD-225, or NCTC 14479; and (b) a carrier suitable for oral ruminant administration. In some embodiments, the composition comprises: (a) one or more bacteria comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 1-30 and 2045-2103; and/or (b) one or more fungi comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 31-60 and 2104-2107. In some embodiments, the composition comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO; 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 32; and/or (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2067. In some embodiments, the composition comprises: (a) a Clostridium sp. with a deposit accession number of NRRL B-67248; (b) a Pichia sp. with a deposit accession number of NRRL Y-67249; and/or (c) a Butyrivibrio sp. with a deposit accession number of NRRL B-67347.
In some embodiments, the ruminant is a cow.
In some embodiments, a ruminant administered the composition exhibits an increase in milk production that leads to an increase in milk yield or an increase in energy-corrected milk.
In some embodiments, a ruminant administered the composition exhibits an improved milk compositional characteristic selected from the group consisting of: an increase in milk fat(s), an increase in milk protein(s), an increase of carbohydrates in milk, an increase of vitamins in milk, an increase of minerals in milk, or combinations thereof.
In some embodiments, a ruminant administered the composition exhibits at least one improved phenotypic trait, selected from the group consisting of: an improved efficiency in feed utilization, improved digestibility, an increase in polysaccharide and lignin degradation, an increase in fatty acid concentration in the rumen, pH balance in the rumen, a reduction in methane emissions, a reduction in manure production, improved dry matter intake, an improved efficiency of nitrogen utilization, or combinations thereof.
In some embodiments, the composition is formulated to protect Ruminococcus bovis from external stressors prior to entering the gastrointestinal tract of the ruminant. In some embodiments, the composition is formulated to protect the Ruminococcus bovis from oxidative stress. In some embodiments, the composition is formulated to protect the Ruminococcus bovis from moisture.
In some embodiments, the composition is combined with food. In some embodiments, the composition is combined with cereal, starch, oilseed cake, or vegetable waste. In some embodiments, the composition is combined with hay, haylage, silage, livestock feed, forage, fodder, beans, grains, micro-ingredients, fermentation compositions, mixed ration, total mixed ration, or a mixture thereof.
In some embodiments, the composition is formulated as a solid, liquid, or mixture thereof. In some embodiments, the composition is dry. In some embodiments, the composition is formulated as a pellet, capsule, granulate, or powder. In some embodiments, the composition is encapsulated. In some embodiments, the composition is encapsulated in a polymer or carbohydrate.
In some embodiments, the composition is combined with water, medicine, vaccine, or a mixture thereof.
In some embodiments, the Ruminococcus bovis is present in the composition in an amount of at least 102 cells.
In some embodiments, the present disclosure provides a method for increasing milk production or improving milk compositional characteristics in a ruminant, the method comprising orally administering to a ruminant an effective amount of any of the compositions disclosed herein.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement exhibits an increase in milk production that leads to a measured increase in milk yield.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement exhibits an increase in milk production and improved milk compositional characteristics that leads to a measured increase in energy-corrected milk.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement exhibits an improved milk compositional characteristic selected from the group consisting of: an increase in milk fat(s), an increase in milk protein(s), an increase of carbohydrates in milk, an increase of vitamins in milk, an increase of minerals in milk, or combinations thereof.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement exhibits at least a 1% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement exhibits at least a 10% increase in the average production of: milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement exhibits at least a 20% increase in the average production of; milk fat(s), milk protein(s), energy-corrected milk, or combinations thereof.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement, further exhibits at least one improved phenotypic trait, selected from the group consisting of: an improved efficiency in feed utilization, improved digestibility, an increase in polysaccharide and lignin degradation, an increase in fatty acid concentration in the rumen, pH balance in the rumen, a reduction in methane emissions, a reduction in manure production, improved dry matter intake, an improved efficiency of nitrogen utilization, or combinations thereof.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement, further exhibits a shift in the microbiome of the rumen.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement, further exhibits a shift in the microbiome of the rumen, wherein a population of microbes present in the rumen before administration of the ruminant supplement increase in abundance after administration of the ruminant supplement.
In some embodiments, the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen, wherein a population of microbes present in the rumen before administration of the ruminant supplement decrease in abundance after administration of the ruminant supplement.
In some embodiments, wherein the ruminant administered the effective amount of the ruminant supplement, further exhibits: a shift in the microbiome of the rumen, wherein a first population of microbes present in the rumen before administration of the ruminant supplement increase in abundance after administration of the ruminant supplement, and wherein a second population of microbes present in the rumen before administration of the ruminant supplement decrease in abundance after administration of the ruminant supplement.
In some embodiments, the present disclosure provides a composition comprising: (a) a Ruminococcus bovis with a deposit accession number of TSD-225 or NCTC 14479; (b) a Clostridium sp. with a deposit accession number of NRRL B-67248; (c) a Pichia sp. with a deposit accession number of NRRL Y-67249; and/or (d) a Butyrivibrio sp. with a deposit accession number of NRRL B-67347.
In some embodiments, the present disclosure provides a composition that performs the same or better than recombinant bovine growth hormone for increasing milk production or improving milk compositional characteristics in a ruminant, wherein the composition comprises: (a) Ruminococcus bois comprising a 16S nucleic acid sequence of SEQ ID NO: 2108; and (b) a carrier suitable for oral ruminant administration. In some embodiments, the composition comprises: (a) one or more bacteria comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 1-30 and 2045-2103; and/or (b) one or more fungi comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to any one of SEQ ID NOs: 31-60 and 2104-2107. In some embodiments, the composition comprises: (a) a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 28; (b) a Pichia sp. comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 32; and/or (c) a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2067. In some embodiments, the composition comprises: (a) a Clostridium sp. with a deposit accession number of NRRL B-67248; (b) a Pichia sp. with a deposit accession number of NRRL Y-67249; and/or (c) a Butyrivibrio sp. with a deposit accession number of NRRL B-67347.
Some microorganisms described in this application were deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) Culture Collection (NRRL®), located at 1815 N. University St., Peoria, Ill. 61604, USA. Some microorganisms described in this application were deposited with the Bigelow National Center for Marine Algae and Microbiota, located at 60 Bigelow Drive, East Boothbay, Me. 04544, USA. Some microorganisms described in this application were also deposited with the American Type Culture Collection (ATCC®), located at 10801 University Blvd., Manassas, Va. 20110, USA. Some microorganisms described in this application were deposited with the National Collection of Type Cultures operated by the Public Health England, located at Porton Down, Salisbury, SP4 0JG, United Kingdom.
The deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and/or type strain rules and procedures. The deposit accession numbers for the Budapest Treaty deposits and type strains are provided in Table 2. The depository, accession numbers, and corresponding dates of deposit for the microorganisms described in this application are separately provided in Table 4.
In Table 1, the closest predicted hits for taxonomy of the microbes are listed in columns 2, and 5. Column 2 is the top taxonomic hit predicted by BLAST, and column 5 is the top taxonomic hit for genus+species predicted by BLAST. Table 3 shows the top taxonomic hit predicted by BLAST. The compositions of the present disclosure may contain any one, or any combination, of the microbes listed below in Table 1 and Table 3. The microbes of Table 1 and Table 3 may be administered as a single microbial product and/or as a microbial consortia comprising a combination of microbes from Table 1 and Table 3.
The strains designated in the tables below have been deposited in the labs of Native Microbials, Inc. since at least before the date of filing of the present application and before the date of deposit with the noted depository institutions.
Ruminococcus bovis
Ruminococcus
bromii
Ruminococcus bovis
Ruminococcus
bromii
Ruminococcus
Ruminococcus
bromii
Clostridium IV
Intestinimonas
butyriciproducens
Roseburia (Genus)
Pseudobutyrivibrio
ruminis
Hydrogenoanaero-
Roseburia
bacterium (Genus)
inulinivorans
Clostridium XIVa
Eubacterium
Eubacterium
ventriosum
ventriosum
Saccharofermentans
Faecalibacterium
prausnitzii
Saccharofermentans
Saccharofermentans
Saccharofermentans
acetigenes
Butyricicoccus
Clostridium sp.
Ruminococcus
flavefaciens
Papillibacter
Clostridium
saccharolyticum
Ruminococcus
Clostridium
lentocellum
Hydrogenoanaero-
Ruminococcus
bacterium (Genus)
flavefaciens
Pelotomaculum
Faecalibacterium
Faecalibacterium
prausnitzii
Saccharofermentans
Saccharofermentans
acetigenes
Blautia luti
Butyricicoccus
Clostridium
sensu stricto (Genus)
lentocellum
Coprococcus catus
Anaeroplasma
Anaeroplasma
Anaeroplasma
varium
varium
Clostridium sensu
Clostridium
stricto (Genus)
stercorarium
Butyricicoccus
Aminiphilus
circumscriptus
Butyricicoccus
Aminiphilus
circumscriptus
Rikenella
Bacteroides sp.
Alistipes shahii
Tannerella
Alistipes shahii
Alistipes shahii
Howardella
Oscillibacter
valericigenes
Prevotella
Odoribacter
splanchnicus
Butyricimonas
Tannerella forsythia
Clostridium sensu
Hydrogenoanaero-
bacterium
stricto (Genus)
saccharovorans
Clostridium sensu
Clostridium
Clostridium
stricto (Genus)
butyricum
butyricum
Saccharofermentans
Faecalibacterium
prausnitzii
Roseburia
Roseburia
intestinalis
intestinalis
Succinivibrio
Succinivibrio
Succinivibrio
dextrinosolvens
dextrinosolvens
Prevotella
Prevotella
ruminicola
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Ruminobacter
Ruminobacter sp.
Ruminobacter
amylophilus
Syntrophococcus
Blautia producta
Blautia producta
Succinivibrio
Succinivibrio
Succinivibrio
dextrinosolvens
dextrinosolvens
Pseudobutyrivibrio
Butyrivibrio
Butyrivibrio
fibrisolvens
fibrisolvens
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Prevotella
Prevotella albensis
Roseburia
inulinivorans
Syntrophococcus
Ruminococcus
Ruminococcus
gnavus
gnavus
Ruminobacter
Ruminobacter sp.
Ruminobacter
amylophilus
Butyrivibrio
Butyrivibrio sp.
Butyrivibrio
hungatei
Clostridium_XlVa
Eubacterium
Eubacterium
oxidoreducens
oxidoreducens
Prevotella
Prevotella brevis
Prevotella
Prevotella sp.
Prevotella copri
Eubacterium
Eubacterium
ruminantium
ruminantium
Roseburia
xylanovorans
Lachnospira
Lachnospira
pectinoschiza
pectinoschiza
Butyrivibrio
Butyrivibrio
Butyrivibrio
fibrisolvens
fibrisolvens
Pseudobutyrivibrio
Pseudobutyrivibrio
Pseudobutyrivibrio
ruminis
Turicibacter
Sinimarinibacterium
Sinimarinibacterium
flocculans
flocculans
Butyrivibrio
fibrisolvens
Pseudobutyrivibrio
Pseudobutyrivibrio
Pseudobutyrivibrio
ruminis
ruminis
Anaerolinea
Anaerolinea
thermophila
Roseburia
Eubacterium rectale
Propionibacterium
Propionibacterium
Propionibacterium
acnes
acnes
Clostridium_XIVa
Pseudobutyrivibrio
ruminis
Olsenella
Olsenella profusa
Streptococcus
Streptococcus
Streptococcus
dentirousetti
dentirousetti
Clostridium_XlVa
Butyrivibrio sp.
Butyrivibrio
proteoclasticus
Clostridium_XlVa
Butyrivibrio
hungatei
Roseburia
Eubacterium
oxidoreducens
Clostridium_IV
Ruminococcus
bromii
Clostridium_XICa
Clostridium
algidixylanolyticum
Eubacterium
ruminantium
Catenisphaera
Catenisphaera
adipataccumulans
adipataccumulans
Solobacterium
Solobacterium
moorei
Eubacterium
Eubacterium
ruminantium
ruminantium
Clostridium_XlVa
Butyrivibrio
Butyrivibrio
proteoclasticus
proteoclasticus
Ralstonia
Ralstonia sp. 94
Ralsonia insidiosa
Clostridium_XlVa
Butyrivibrio sp.
Butyrivibrio
proteoclasticus
Eubacterium
Casaltella
massiliensis
Lachnobacterium
Eubacterium
xylanophilum
Acholeplasma
Acholeplasma
Acholeplasma
brassicae
brassicae
Selenomonas
Mitsuokella
Mitsuokella
jalaludinii
jalaludinii
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Clostridium_XlVa
Butyrivibrio sp.
Butyrivibrio
fibrisolvens
Succinivibrio
Succinivibrio
Succinivibrio
dextrinosolvens
dextrinosolvens
Ruminobacter
Ruminobacter sp.
Ruminobacter
amylophilus
Sharpea
Sharpea azabuensis
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Prevotella
Prevotella sp.
Prevotella
ruminicola
Prevotella
Prevotella
ruminicola
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Prevotella
Prevotella
Prevotella
ruminicola
ruminicola
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Candida xylopsoc
Pichia kudriavzevii
Pichia kudriavzevii
Orpinomyces
Orpinomyces sp.
Neocallimastix
frontalis
Orpinomycs
Neocallimastix
Neocallimastix
frontalis
frontalis
Orpinomyces
Orpinomyces sp.
Neocallimastix
frontalis
Candida apicol
Candida apicola
Candida apicola
Candida rugosa
Candida
Candida
akabanensis
akabanensis
Neocallimastix
Neocallimastix sp.
Neocallimastix
frontalis
Orpinomyces
Orpinomyces sp.
Orpinomyces
joyonii
Orpinomyces
Neocallimastix
Neocallimastix
frontalis
frontalis
Neocallimastix
Neocallimastix sp.
Neocallimastix
frontalis
Neocallimastix
Neocallimastix
Neocallimastix
frontalis
frontalis
Ascomycota
Basidiomycota sp.
Sugiyamaella
lignohabitans
Piromyces
Caecomyces sp.
Cyllamyces
aberensis
Orpinomyces
Orpinomyces sp.
Orpinomyces
joyonii
Cyllamyces
Caecomyces sp.
Caecomyces
communis
Piromyces
Caecomyces sp.
Caecomyces
communis
Cyllamyces
Cyllamyces sp.
Cyllamyces
aberensis
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Piromyces
Caecomyces sp.
Cyllamyces
aberensis
Neocallimastix
Neocallimastix sp.
Neocallimastix
frontalis
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Neocallimastix
Neocallimastix sp.
Neocallimastix
frontalis
Candida ethanolica
Candida ethanolica
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Orpinomyces
Neocallimastix sp.
Neocallimastix
frontalis
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Phyllosticta
Tremellales sp.
Tremella giraffa
capitalensis
Orpinomyces
Neocallimastix
Neocallimastix
frontalis
frontalis
Orpinomyces
Neocallimastix
Neocallimastix
frontalis
frontalis
Orpinomyces
Orpinomyces
joyonii
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Piromyces
Piromyces sp.
Neocallimastix
frontalis
Ruminococcus
Streptococcus
bovis sp. nov.
Ruminococcus
Clostridium_XlVa
bovis sp. nov.
Ruminococcus
Clostridium_XlVa
Clostridium IV
Roseburia
Roseburia
Clostridium_IV
Hydrogenoanaero-
Clostridium_XICa
bacterium (Genus)
Clostridium XIVa
Saccharofermentans
Saccharofermentans
Solobacterium
Butyricicoccus
Papillibacter
Clostridium_XlVa
Ruminococcus
Ralstonia
Hydrogenoanaero-
Clostridium_XlVa
bacterium (Genus)
Pelotomaculum
Eubacterium
Saccharofermentans
Lachnobacterium
Acholeplasma
Butyricicoccus
Selenomonas
sensu stricto
Prevotella
Anaeroplasma
Clostridium_XlVa
Clostridium sensu
Succinivibrio
stricto (Genus)
Butyricicoccus
Ruminobacter
Butyricicoccus
Sharpea
Rikenella
Prevotella
Tannerella
Prevotella
Howardella
Prevotella
Prevotella
Prevotella
Butyricimonas
Prevotella
Clostridium sensu
Piromyces
stricto
Clostridium sensu
Candida xylopsoc
stricto
Saccharofermentans
Orpinomyces
Orpinomycs
Succinivibrio
Orpinomyces
Prevotella
Candida apicol
Prevotella
Candida rugosa
Prevotella
Neocallimastix
Ruminobacter
Orpinomyces
Syntrophococcus
Orpinomyces
Succinivibrio
Neocallimastix
Pseudobutyrivibrio
Neocallimastix
Prevotella
Ascomycota
Prevotella
Piromyces
Prevotella
Orpinomyces
Prevotella
Cyllamyces
Piromyces
Syntrophococcus
Cyllamyces
Ruminobacter
Piromyces
Butyrivibrio
Piromyces
Clostridium_XlVa
Neocallimastix
Prevotella
Piromyces
Prevotella
Neocallimastix
Roseburia
Piromyces
Orpinomyces
Butyrivibrio
Piromyces
Pseudobutyrivibrio
Phyllosticta
capitalensis
Turicibacter
Orpinomyces
Orpinomyces
Pseudobutyrivibrio
Orpinomyces
Anaerolinea
Piromyces
Roseburia
Piromyces
Propionibacterium
Piromyces
Clostridium_XIVa
Olsenella
1Deposit number applicable to Budapest treaty and/or type stain rules and procedures
2Deposit number applicable to Budapest treaty and/or type stain rules and procedures
3Deposit number applicable to Budapest treaty and/or type stain rules and procedures
4Deposit number applicable to Budapest treaty and/or type stain rules and procedures
Corynebacterium
Prevotella
Comamonas
Clostridium_XlVa
Hippea
Anaerovorax
Clostridium_XlVa
Rummeliibacillus
Clostridium_XlVa
Prevotella
Anaerovorax
Pseudoflavonifractor
Prevotella
Clostridium_XlVa
Clostridium_XlVa
Coprococcus
Pyramidobacter
Syntrophococcus
Prevotella
Clostridium_XlVa
Prevotella
Roseburia
Clostridium_XlVa
Clostridium_XlVa
Acidothermus
Adlercreutzia
Prevotella
Proteiniclasticum
Anaerovorax
Prevotella
Bacteroides
Clostridium_III
Prevotella
Acinetobacter
Erysipelothrix
Bacteroides
Clostridium_XlVa
Butyrivibrio
Eubacterium
Prevotella
Eubacterium
Prevotella
Coprococcus
Prevotella
Clostridium_XlVa
Prevotella
Catonella
Methanobrevibacter
Ruminococcus
Coprococcus
Clostridium_XlVa
Prevotella
Anaerovorax
Asteroleplasma
Clostridium_XlVa
Caulobacter
Roseburia
Clostridium_XlVa
Acinetobacter
Bacteroides
Erysipelothrix
Coprococcus
Clostridium_XlVa
Bacteroides
Coprococcus
Anaerovorax
Pseudoflavonifractor
Pseudoflavonifractor
Prevotella
Roseburia
Prevotella
Coprococcus
Prevotella
Clostridium_XlVa
Prevotella
Catonella
Methanobrevibacter
Ruminococcus
Coprococcus
Clostridium_XlVa
Prevotella
Anaerovorax
Asteroleplasma
Clostridium_XlVa
Caulobacter
Roseburia
Clostridium_XlVa
Acinetobacter
Bacteroides
Erysipelothrix
Coprococcus
Clostridium_XlVa
Bacteroides
Coprococcus
Anaerovorax
Pseudoflavonifractor
Pseudoflavonifractor
Prevotella
Roseburia
Prevotella
Ruminococcus
Atopobium
Eubacterium
Robinsoniella
Neisseria
Ruminococcus
Prevotella
Slackia
Prevotella
Clostridium_XlVa
Bacteroides
Anaerorhabdus
Bacteroides
Prevotella
Corynebacterium
Atopobium
Streptophyta
Prevotella
Roseburia
Prevotella
Prevotella
Eubacterium
Rhodocista
Prevotella
Clostridium_XlVa
Prevotella
Prevotella
Streptophyta
Ochrobactrum
Mogibacterium
Adlercreutzia
Prevotella
Riemerella
Prevotella
Roseburia
Slackia
Clostridium_IV
Syntrophococcus
Prevotella
Treponema
Prevotella
Anaerovorax
Prevotella
Methanobrevibacter
Corynebacterium
Clostridium_XlVa
Alkaliphilus
Ruminococcus
Clostridium_XlVa
Eubacterium
Bacteroides
Roseburia
Lentisphaera
Eubacterium
Roseburia
Clostridium_IV
Hahella
Butyricicoccus
Clostridium_IV
Prevotella
Clostridium_IV
Desulfovibrio
Sphingobacterium
Roseburia
Bacteroides
Ruminococcus
Prevotella
Asteroleplasma
Syntrophococcus
Victivallis
Lachnobacterium
Clostridium_IV
Anaerorhabdus
Altererythrobacter
Clostridium_XlVa
Clostridium_XlVa
Proteiniclasticum
Bifidobacterium
Clostridium_XlVa
Clostridium_XlVa
Desulfovibrio
Clostridium_XlVa
Nitrobacter
Enterorhabdus
Clostridium
—
sensu
—
stricto
Oscillibacter
Nautilia
Corynebacterium
Ruminococcus
Coprococcus
Eubacterium
Rikenella
Clostridium_XlVa
Paenibacillus
Ruminococcus
Prevotella
Haematobacter
Prevotella
Clostridium_XlVa
Enterorhabdus
Blautia
Sporobacter
Oscillibacter
Clostridium_XlVa
Atopobium
Sporobacter
Clostridium_XlVa
Oscillibacter
Clostridium_XlVa
Clostridium_IV
Mogibacterium
Roseburia
Pelotomaculum
Pelotomaculum
Clostridium_XlVa
Robinsoniella
Coprococcus
Wautersiella
Planctomyces
Treponema
Coprococcus
Paracoccus
Ruminococcus
Atopobium
Prevotella
Clostridium_IV
Clostridium_XlVa
Clostridium_XlVa
Clostridium_XlVa
Prevotella
Dethiosulfovibrio
Clostridium_XI
Clostridium_IV
Saccharofermentans
Clostridium
—
sensu
—
stricto
Roseburia
Hydrogenoanaerobacterium
Victivallis
Clostridium_IV
Pelotomaculum
Clostridium_XlVa
Saccharofermentans
Coprococcus
Clostridium_XlVa
Clostridium_XlVb
Papillibacter
Bartonella
Clostridium_IV
Eubacterium
Asaccharobacter
Clostridium_IV
Blautia
Prevotella
Ruminococcus
Selenomonas
Treponema
Adlercreutzia
Butyricicoccus
Pseudoflavonifractor
Corynebacterium
Adlercreutzia
Selenomonas
Coraliomargarita
Paraprevotella
Oscillibacter
Anaerovorax
Clostridium_XlVa
Saccharofermentans
Erysipelothrix
Agaricicola
Denitrobacterium
Armatimonadetes
Asaccharobacter
Anaeroplasma
Prevotella
Clostridium_IV
Streptococcus
Cellulosilyticum
Asaccharobacter
Enterorhabdus
Treponema
Roseburia
Victivallis
Prevotella
Roseburia
Ruminococcus
Mogibacterium
Prevotella
Clostridium
—
sensu
—
stricto
Victivallis
Cyanobacteria
Treponema
Stenotrophomonas
Clostridium_XlVa
Sphingobium
Oscillibacter
Methylobacterium
Zhangella
Oscillibacter
Clostridium_III
Coraliomargarita
Eubacterium
Enterorhabdus
Clostridium_XlVa
Saccharofermentans
Clostridium_IV
Clostridium
—
sensu
—
stricto
Victivallis
Coprococcus
Pseudoflavonifractor
Anaeroplasma
Anaeroplasma
Bacteroides
Acinetobacter
Victivallis
Victivallis
Mogibacterium
Oscillibacter
Butyricimonas
Dethiosulfovibrio
Pseudoflavonifractor
Clostridium_IV
Anaeroplasma
Oscillibacter
Herbiconiux
Eubacterium
Armatimonadetes
Selenomonas
Clostridium_IV
Mogibacterium
Clostridium_IV
Roseburia
Anaerovibrio
Clostridium_III
Saccharofermentans
Saccharofermentans
Prevotella
Clostridium_XlVa
Robinsoniella
Brevundimonas
Anaerotruncus
Victivallis
Bacteroides
Clostridium_XlVb
Prevotella
Ruminococcus
Pelobacter
Clostridium_XlVa
Clostridium_XlVa
Clostridium_XlVb
Coprococcus
Clostridium_IV
Clostridium_IV
Coprococcus
Victivallis
Clostridium_III
Anaerovibrio
Anaerovorax
Proteiniclasticum
Anaerovorax
Selenomonas
Hydrogenoanaerobacterium
Acetanaerobacterium
Clostridium_XlVa
Asaccharobacter
Clostridium_XlVa
Saccharofermentans
Prevotella
Anaeroplasma
Spirochaeta
Alkaliphilus
Paraprevotella
Hippea
Prevotella
Prevotella
Hydrogenoanaerobacterium
Clostridium
—
sensu
—
stricto
Paraeggerthella
Clostridium_XlVa
Clostridium_XlVa
Clostridium_IV
Clostridium_XlVa
Adhaeribacter
Syntrophococcus
Clostridium
—
sensu
—
stricto
Saccharofermentans
Clostridium_IV
Clostridium_IV
Clostridium
—
sensu
—
stricto
Coraliomargarita
Sharpea
Clostridium_IV
Anaerovorax
Blautia
Clostridium_XlVa
Clostridium_IV
Anaerovorax
Coraliomargarita
Aquiflexum
Pedobacter
Robinsoniella
Pelomonas
Saccharofermentans
Paracoccus
Enterorhabdus
Beijerinckia
Sporobacter
Clostridium_IV
Bacillus
Saccharofermentans
Spirochaeta
Prevotella
Eubacterium
Herbiconiux
Brevundimonas
Mogibacterium
Anaerorhabdus
Victivallis
Prevotella
Anaerovorax
Aquiflexum
Oscillibacter
Altererythrobacter
Hydrogenoanaerobacterium
Clostridium_III
Clostridium_XlVb
Saccharofermentans
Roseburia
Anaeroplasma
Planctomyces
Ruminococcus
Selenomonas
Anaeroplasma
Anaerovorax
Rummeliibacillus
Clostridium_XlVa
Anaeroplasma
Butyrivibrio
Anaerotruncus
Syntrophococcus
Paraeggerthella
Papillibacter
Prevotella
Papillibacter
Streptococcus
Methanobrevibacter
Prevotella
Prevotella
Prevotella
Coraliomargarita
Prevotella
Thermotalea
Clostridium_XlVa
Atopobium
Prevotella
Mogibacterium
Clostridium_XlVa
Clostridium_XlVa
Eggerthella
Blautia
Vampirovibrio
Papillibacter
Beijerinckia
Bacteroides
Desulfotomaculum
Acidobacteria
Clostridium_XlVa
Clostridium_XlVa
Clostridium_XlVa
Cryptanaerobacter
Prevotella
Syntrophomonas
Erysipelothrix
Selenomonas
Clostridium_III
Flavobacterium
Thermotalea
Mucilaginibacter
Bacteroides
Ruminococcus
Clostridium_XlVa
Asaccharobacter
Blautia
Mucilaginibacter
Coprococcus
Butyricimonas
Treponema
Clostridium
—
sensu
—
stricto
Clostridium_XlVa
Anaerovorax
Saccharofermentans
Clostridium_XlVa
Clostridium_III
Clostridium_IV
Ruminococcus
Clostridium_XlVa
Clostridium_XI
Clostridium_XlVa
Eubacterium
Clostridium_IV
Ruminococcus
Clostridium_IV
Faecalibacterium
Anaerovibrio
Asaccharobacter
Pelotomaculum
Spirochaeta
Prevotella
Anaerovorax
Clostridium_IV
Victivallis
Syntrophococcus
Syntrophococcus
Desulfovibrio
Clostridium_IV
Prevotella
Victivallis
Clostridium_XlVa
Selenomonas
Bacteroides
Clostridium_XlVa
Eggerthella
Selenomonas
Mogibacterium
Armatimonadetes
Clostridium_XlVa
Victivallis
Paraprevotella
Brevundimonas
Prevotella
Prevotella
Robinsoniella
Clostridium_III
Butyricimonas
Spirochaeta
Hydrogenoanaerobacterium
Proteiniclasticum
Roseburia
Clostridium_XlVa
Anaerofustis
Succiniclasticum
Anaeroplasma
Oscillibacter
Escherichia/Shigella
Bacteroides
Clostridium_XlVa
Clostridium_XlVa
Clostridium_IV
Clostridium_III
Prevotella
Coprococcus
Oscillibacter
Parabacteroides
Bacteroides
Mogibacterium
Solobacterium
Bacteroides
Clostridium_III
Victivallis
Saccharofermentans
Saccharofermentans
Olivibacter
Thermotalea
Proteiniclasticum
Clostridium_III
Anaeroplasma
Treponema
Clostridium_XlVa
Clostridium_III
Desulfotomaculum
Bacillus
Anaerovorax
Ruminococcus
Agarivorans
Anaerotruncus
Papillibacter
Clostridium_XlVa
Clostridium_III
Bacteroides
Clostridium_XlVa
Ruminococcus
Clostridium_XlVa
Oscillibacter
Nitrobacter
Clostridium_XlVa
Limibacter
Desulfovibrio
Coprococcus
Anaerovorax
Spirochaeta
Cyanobacteria
Saccharofermentans
Anaeroplasma
Clostridium_III
Victivallis
Enterorhabdus
Clostridium_IV
Erysipelothrix
Clostridium_III
Clostridium
—
sensu
—
stricto
Gelidibacter
Roseburia
Neisseria
Prevotella
Cyanobacteria
Oscillibacter
Prevotella
Saccharofermentans
Spirochaeta
Clostridium_XlVa
Clostridium_XlVb
Clostridium_XlVa
Adlercreutzia
Clostridium_XlVa
Clostridium_IV
Adlercreutzia
Prevotella
Syntrophococcus
Treponema
Prevotella
Clostridium_III
Pseudoflavonifractor
Clostridium_IV
Sharpea
Dongia
Eubacterium
Prevotella
Clostridium_IV
Parabacteroides
Brevundimonas
Clostridium_XlVa
Ruminococcus
Thermotalea
Victivallis
Anaeroplasma
Oscillibacter
Ruminococcus
Clostridium_XlVa
Clostridium_XlVa
Clostridium_IV
Roseburia
Eggerthella
Clostridium_III
Clostridium_XlVa
Lactobacillus
Bacteroides
Cellulosilyticum
Brevundimonas
Clostridium_IV
Prevotella
Helicobacter
Clostridium_IV
Proteiniclasticum
Brevundimonas
Clostridium_XlVa
Prevotella
Desulfovibrio
Coraliomargarita
Eubacterium
Sphingomonas
Prevotella
Clostridium_IV
Paraprevotella
Ruminococcus
Saccharofermentans
Clostridium_III
Clostridium_III
Turicibacter
Prevotella
Clostridium_XlVa
Fusibacter
Clostridium_XlVa
Clostridium_IV
Rummeliibacillus
Mogibacterium
Bacteroides
Pelospora
Eggerthella
Eubacterium
Blautia
Clostridium_XlVb
Ehrlichia
Eubacterium
Prevotella
Clostridium_XlVa
Treponema
Hydrogenoanaerobacterium
Selenomonas
Saccharofermentans
Clostridium_IV
Clostridium
—
sensu
—
stricto
Anaerovorax
Spirochaeta
Brevundimonas
Eubacterium
Clostridium_XlVa
Anaerovorax
Ruminococcus
Papillibacter
Clostridium_IV
Hydrogenoanaerobacterium
Asaccharobacter
Clostridium_XlVa
Rhodocista
Clostridium_XlVa
Beijerinckia
Lactobacillus
Cryptanaerobacter
Prevotella
Anaerovibrio
Anaerovorax
Enterorhabdus
Clostridium_XlVb
Selenomonas
Eubacterium
Thermotalea
Enterorhabdus
Clostridium_III
Acetanaerobacterium
Treponema
Clostridium_XlVa
Enterorhabdus
Prevotella
Desulfovibrio
Aminobacter
Clostridium_IV
Rikenella
Gordonibacter
Papillibacter
Syntrophococcus
Clostridium
—
sensu
—
stricto
Hahella
Vampirovibrio
Coprococcus
Coraliomargarita
Clostridium_III
Clostridium_XlVa
Desulfotomaculum
Helicobacter
Syntrophococcus
Clostridium_IV
Paludibacter
Adhaeribacter
Clostridium_IV
Cryptanaerobacter
Idiomarina
Clostridium_IV
Selenomonas
Acetanaerobacterium
Bifidobacterium
Clostridium_XlVb
Asaccharobacter
Eubacterium
Anaeroplasma
Saccharofermentans
Ruminococcus
Clostridium_III
Acholeplasma
Pedobacter
Sphingomonas
Verrucomicrobia
Anaerovorax
Spirochaeta
Paraeggerthella
Bacteroides
Paenibacillus
Prevotella
Bacteroides
Clostridium_XlVa
Clostridium_XlVa
Roseburia
Clostridium_XlVa
Clostridium_III
Pedobacter
Robinsoniella
Anaeroplasma
Clostridium_XlVa
Hydrogenoanaerobacterium
Turicibacter
Papillibacter
Clostridium_XlVa
Saccharofermentans
Clostridium_XlVb
Sporobacter
Asaccharobacter
Bacteroides
Anaeroplasma
Sporobacter
Streptomyces
Arcobacter
Clostridium_XlVa
Barnesiella
Lactobacillus
Flavobacterium
Victivallis
Clostridium_XlVa
Ureaplasma
Acetanaerobacterium
Slackia
Oscillibacter
Prevotella
Proteiniphilum
Spirochaeta
Ruminococcus
Prevotella
Butyricicoccus
Devosia
Anaeroplasma
Oscillibacter
Barnesiella
Atopobium
Clostridium_XlVa
Methanobrevibacter
Butyricimonas
Butyricimonas
Asaccharobacter
Enhydrobacter
Treponema
Clostridium_XlVa
Adlercreutzia
Prevotella
Pseudoflavonifractor
Syntrophococcus
Clostridium_IV
Demequina
Saccharofermentans
Sphaerisporangium
Anaeroplasma
Geobacillus
Prevotella
Clostridium_XlVa
Victivallis
Bacteroides
Demequina
Paraeggerthella
Paraprevotella
Pseudoflavonifractor
Roseburia
Gelidibacter
Clostridium_IV
Rhizobium
Acholeplasma
Clostridium_XlVa
Bacteroides
Bacteroides
Papillibacter
Fusibacter
Coraliomargarita
Papillibacter
Clostridium_XlVa
Acholeplasma
Catenibacterium
Clostridium_IV
Clostridium_IV
Clostridium_IV
Nitrobacter
Victivallis
Selenomonas
Enterorhabdus
Eubacterium
Roseburia
Prevotella
Asaccharobacter
Bacteroides
Clostridium_XlVa
Gelidibacter
Brevundimonas
Clostridium_XlVa
Prevotella
Oscillibacter
Asteroleplasma
Anaeroplasma
Oscillibacter
Bilophila
Oscillibacter
Clostridium_IV
Prevotella
Geosporobacter
Butyricimonas
Pseudoflavonifractor
Barnesiella
Selenomonas
Prevotella
Enterorhabdus
Oscillibacter
Pelotomaculum
Cellulosilyticum
Clostridium_IV
Parabacteroides
Papillibacter
Bacteroides
Prevotella
Hydrogenoanaerobacterium
Clostridium_XlVa
Prevotella
Clostridium_IV
Howardella
Slackia
Methylobacter
Treponema
Clostridium_XlVa
Devosia
Ruminococcus
Clostridium_III
Methanobrevibacter
Paraprevotella
Desulfobulbus
Butyricicoccus
Clostridium_XlVa
Dialister
Selenomonas
Spirochaeta
Clostridium_IV
Cellulosilyticum
Prevotella
Pseudoflavonifractor
Clostridium_III
Oscillibacter
Faecalibacterium
Clostridium_XlVb
Eubacterium
Clostridium_III
Prevotella
Paenibacillus
Pedobacter
Butyricicoccus
Clostridium_XlVa
Roseburia
Hydrogenoanaerobacterium
Adhaeribacter
Eubacterium
Bacteroides
Victivallis
Roseburia
Treponema
Prevotella
Prevotella
Hydrogenoanaerobacterium
Clostridium_XlVa
Bacteroides
Bacteroides
Lactobacillus
Adlercreutzia
Dethiosulfovibrio
Lutispora
Turicibacter
Cyanobacteria
Clostridium
—
sensu
—
stricto
Cyanobacteria
Bulleidia
Aquiflexum
Clostridium_III
Roseburia
Glaciecola
Clostridium_XlVa
Hydrogenoanaerobacterium
Clostridium_IV
Sphaerobacter
Cyanobacteria
Prevotella
Turicibacter
Ruminococcus
Clostridium_IV
Clostridium_XlVa
Saccharofermentans
Clostridium_XlVb
Ruminococcus
Fibrobacter
Proteiniclasticum
Anaeroplasma
Cyanobacteria
Algoriphagus
Clostridium_XlVa
Howardella
Clostridium_XlVa
Barnesiella
Clostridium_IV
Prevotella
Clostridium_XlVa
Butyricimonas
Blautia
Prevotella
Clostridium_XlVa
Blautia
Clostridium_IV
Flavobacterium
Prevotella
Clostridium_XlVa
Clostridium_XlVa
Eubacterium
Butyricicoccus
Fluviicola
Anaerovibrio
Blautia
Verrucomicrobia
Clostridium
—
sensu
—
stricto
Spirochaeta
Clostridium_XI
Anaerovorax
Roseburia
Mucilaginibacter
Clostridium_XI
Prevotella
Clostridium_III
Coprococcus
Acholeplasma
Clostridium_III
Lactobacillus
Clostridium_IV
Prevotella
Bifidobacterium
Adhaeribacter
Hydrogenoanaerobacterium
Acetivibrio
Cyanobacteria
Flammeovirga
Dethiosulfovibrio
Hippea
Faecalibacterium
Spirochaeta
Brevundimonas
Mucilaginibacter
Hydrogenoanaerobacterium
Asaccharobacter
Clostridium_IV
Mogibacterium
Clostridium_IV
Oscillibacter
Clostridium_XlVa
Faecalibacterium
Altererythrobacter
Gelidibacter
Prevotella
Anaerovorax
Riemerella
Sphingobacterium
Syntrophococcus
Bacteroides
Papillibacter
Butyricicoccus
Clostridium_IV
Hydrogenoanaerobacterium
Marvinbryantia
Brevibacillus
Clostridium_IV
Prevotella
Clostridium_IV
Aminobacter
Sporotomaculum
Clostridium_IV
Pedobacter
Victivallis
Gelidibacter
Prevotella
Wautersiella
Slackia
Pyramidobacter
Clostridium_XlVa
Prevotella
Lentisphaera
Desulfoluna
Clostridium_III
Clostridium
—
sensu
—
stricto
Prevotella
Clostridium_III
Clostridium_IV
Prevotella
Cyanobacteria
Helicobacter
Clostridium_XlVa
Coprococcus
Bradyrhizobium
Clostridium_IV
Sphingobacterium
Gelidibacter
Vasilyevaea
Eubacterium
Clostridium_XlVa
Eubacterium
Syntrophococcus
Prevotella
Treponema
Anaerovorax
Sulfurovum
Clostridium_IV
Papillibacter
Paracoccus
Hydrogenoanaerobacterium
Adhaeribacter
Bacteroides
Hydrogenoanaerobacterium
Telmatospirillum
Clostridium_XlVa
Hydrogenoanaerobacterium
Clostridium_IV
Vasilyevaea
Anaeroplasma
Sporotomaculum
Clostridium_IV
Enterorhabdus
Bacteroides
Anaerotruncus
Rhodopirellula
Clostridium_XlVa
Gelidibacter
Anaerofustis
Butyricicoccus
Butyricicoccus
Clostridium_XlVa
Cryptanaerobacter
Clostridium_XlVa
Mogibacterium
Syntrophococcus
Bacteroides
Treponema
Coraliomargarita
Ruminococcus
Prevotella
Pseudaminobacter
Prevotella
Treponema
Syntrophococcus
Clostridium_IV
Tenacibaculum
Parabacteroides
Luteimonas
Eubacterium
Roseburia
Oscillibacter
Cyanobacteria
Prevotella
Clostridium_IV
Treponema
Clostridium_IV
Victivallis
Clostridium_XlVa
Oscillibacter
Papillibacter
Cellulosilyticum
Treponema
Ruminococcus
Coraliomargarita
Butyricicoccus
Blautia
Prevotella
Clostridium_IV
Clostridium_IV
Clostridium_III
Neptunomonas
Clostridium_IV
Howardella
Clostridium_IV
Roseburia
Oscillibacter
Clostridium_XlVa
Clostridium_IV
Sporobacter
Clostridium_XlVa
Butyricicoccus
Clostridium_XlVa
Filomicrobium
Bacteroides
Clostridium_XlVa
Brevundimonas
Clostridium_IV
Paracoccus
Schlegelella
Clostridium_XI
Diaphorobacter
Clostridium
—
sensu
—
stricto
Saccharopolyspora
Prevotella
Eggerthella
Gelidibacter
Prevotella
Pseudomonas
Prevotella
Prevotella
Prevotella
Brevundimonas
Bacteroides
Clostridium_XlVa
Photobacterium
Clostridium_XlVa
Clostridium_XlVb
Prevotella
Clostridium_IV
Anaeroplasma
Caldilinea
Clostridium_XlVa
Victivallis
Brevundimonas
Cyanobacteria
Prevotella
Slackia
Pedobacter
Prevotella
Trueperella
Oscillibacter
Cyanobacteria
Victivallis
Bacteroides
Micrococcus
Olivibacter
Anaerophaga
Selenomonas
Megasphaera
Clostridium_XlVa
Clostridium_XlVa
Eubacterium
Cyanobacteria
Clostridium_XlVa
Treponema
Cryptanaerobacter
Xanthomonas
Asteroleplasma
Cyanobacteria
Sporotomaculum
Bacteroides
Asaccharobacter
Clostridium_IV
Cyanobacteria
Clostridium_XlVa
Treponema
Prevotella
Turicibacter
Clostridium_IV
Clostridium_IV
Clostridium_IV
Oscillibacter
Deinococcus
Pedobacter
Anaerovorax
Clostridium_IV
Bacteroides
Clostridium_IV
Rhodococcus
Treponema
Mucilaginibacter
Clostridium_XlVa
Olivibacter
Clostridium_XlVa
Barnesiella
Clostridium_XlVb
Gelidibacter
Methanobrevibacter
Anaerotruncus
Mesorhizobium
Clostridium_XI
Planctomyces
Aerococcus
Victivallis
Cyanobacteria
Bacteroides
Clostridium_XI
Clostridium_XlVa
Ruminococcus
Saccharofermentans
Oscillibacter
Fibrobacter
Kiloniella
Olivibacter
Clostridium_IV
Spirochaeta
Prevotella
Olivibacter
Prevotella
Parabacteroides
Prevotella
Leifsonia
Clostridium_IV
Victivallis
Treponema
Cyanobacteria
Sporotomaculum
Spirochaeta
Clostridium_III
Clostridium_XlVa
Anaerovorax
Oscillibacter
Victivallis
Spirochaeta
Clostridium_XlVb
Oscillibacter
Prevotella
Anaeroplasma
Adlercreutzia
Clostridium_XlVa
Beijerinckia
Prevotella
Coprococcus
Lentisphaera
Clostridium_XlVa
Saccharofermentans
Porphyrobacter
Rhodobacter
Oscillibacter
Roseburia
Prevotella
Aquiflexum
Rhodopirellula
Bacteroides
Bacteroides
Clostridium_XlVa
Clostridium_IV
Prevotella
Mogibacterium
Prevotella
Clostridium_XlVa
Prevotella
Capnocytophaga
Acholeplasma
Clostridium_IV
Succinivibrio
Pseudonocardia
Clostridium_XlVa
Butyricimonas
Anaerovorax
Prevotella
Butyricimonas
Parabacteroides
Clostridium_XlVa
Clostridium_XlVb
Bacteroides
Cyanobacteria
Riemerella
Anaeroplasma
Ruminococcus
Verrucomicrobia
Syntrophococcus
Clostridium_IV
Barnesiella
Olivibacter
Clostridium_XlVa
Cryptanaerobacter
Saccharofermentans
Clostridium_IV
Coprococcus
Barnesiella
Clostridium
—
sensu
—
stricto
Hydrogenoanaerobacterium
Clostridium_XlVb
Selenomonas
Prevotella
Hydrogenoanaerobacterium
Spirochaeta
Enterorhabdus
Thermoanaerobacter
Armatimonadetes
Syntrophococcus
Sphingobium
Clostridium_XlVa
Geosporobacter
Enterorhabdus
Verrucomicrobia
Clostridium_XlVa
Parabacteroides
Cryptanaerobacter
Anaeroplasma
Spirochaeta
Prevotella
Roseburia
Pedobacter
Pedobacter
Eggerthella
Prevotella
Rikenella
Anaerophaga
Spirochaeta
Clostridium_IV
Weissella
Butyricicoccus
Hahella
Acholeplasma
Clostridium_XlVa
Cellulosilyticum
Verrucomicrobia
Clostridium_XlVa
Pseudoflavonifractor
Calditerricola
Clostridium_IV
Clostridium_IV
Adlercreutzia
Bulleidia
Mucilaginibacter
Victivallis
Anaerovorax
Clostridium_XlVb
Clostridium_XlVa
Prevotella
Bacteroides
Schwartzia
Pyramidobacter
Eubacterium
Clostridium_XlVa
Roseburia
Clostridium_XlVb
Enterorhabdus
Pedobacter
Clostridium
—
sensu
—
stricto
Clostridium_XlVa
Clostridium_III
Desulfotomaculum
Clostridium_IV
Proteiniclasticum
Prevotella
Faecalibacterium
Microbacterium
Leucobacter
Prevotella
Sphingobacterium
Fusibacter
Howardella
Pedobacter
Caldilinea
Turicibacter
Clostridium_IV
Alistipes
Clostridium_XlVa
Clostridium_XlVa
Prevotella
Clostridium_XlVa
Butyricimonas
Anaerovibrio
Prevotella
Pseudoflavonifractor
Corynebacterium
Leucobacter
Kerstersia
Slackia
Lactococcus
Prevotella
Clostridium_IV
Prevotella
Bacteroides
Lactobacillus
Prevotella
Clostridium_XlVa
Clostridium
—
sensu
—
stricto
Syntrophococcus
Clostridium_XlVa
Victivallis
Bacteroides
Acidobacteria
Clostridium_XlVa
Prevotella
Verrucomicrobia
Clostridium_XlVa
Treponema
Pyramidobacter
Robinsoniella
Clostridium_XI
Bifidobacterium
Bacteroides
Gordonibacter
Enterorhabdus
Lactobacillus
Bacteroides
Prevotella
Tannerella
Bacteroides
Prevotella
Clostridium_XlVb
Gelidibacter
Cyanobacteria
Rhodoplanes
Selenomonas
Escherichia/Shigella
Rikenella
Coprococcus
Clostridium
—
sensu
—
stricto
Hyphomicrobium
Verrucomicrobia
Staphylococcus
Verrucomicrobia
Victivallis
Selenomonas
Desulfobulbus
Clostridium_III
Spirochaeta
Kordia
Bosea
Enterococcus
Clostridium_III
Xanthobacter
Lactobacillus
Prevotella
Acidaminococcus
Eubacterium
Bacteroides
Clostridium_XlVa
Lactobacillus
Devosia
Pedobacter
Clostridium_IV
Clostridium_XlVa
Corynebacterium
Spirochaeta
Anaeroplasma
Clostridium_XlVa
Saccharofermentans
Slackia
Limibacter
Sphingobium
Clostridium_XlVa
Riemerella
Saccharofermentans
Bacteroides
Prevotella
Selenomonas
Victivallis
Howardella
Pelospora
Clostridium
—
sensu
—
stricto
Selenomonas
Fibrobacter
Clostridium_III
Sphingomonas
Selenomonas
Eggerthella
Treponema
Mogibacterium
Adlercreutzia
Selenomonas
Methylomicrobium
Leuconostoc
Pyramidobacter
Butyrivibrio
Bacteroides
Butyricimonas
Ruminococcus
Clostridium
—
sensu
—
stricto
Butyrivibrio
Corynebacterium
Proteiniborus
Spirochaeta
Acetitomaculum
Selenomonas
Altererythrobacter
Atopobium
Clostridium_IV
Clostridium_XlVa
Clostridium_XlVa
Clostridium_IV
Clostridium_III
Desulfotomaculum
Pedobacter
Bacteroides
Asaccharobacter
Microbacterium
Treponema
Dethiosulfovibrio
Oscillibacter
Selenomonas
Eubacterium
Ruminococcus
Treponema
Spirochaeta
Roseburia
Ruminococcus
Butyricimonas
Pedobacter
Spirochaeta
Parabacteroides
Methylococcus
Enterorhabdus
Clostridium
—
sensu
—
stricto
Gelidibacter
Sporobacter
Pedobacter
Cyanobacteria
Syntrophococcus
Slackia
Mogibacterium
Prevotella
Pseudoflavonifractor
Veillonella
Clostridium_XlVa
Bacillus
Pedobacter
Clostridium_IV
Fibrobacter
Paenibacillus
Brevundimonas
Desulfovibrio
Clostridium_XI
Helicobacter
Prevotella
Clostridium_XlVa
Prevotella
Herbiconiux
Clostridium_IV
Rikenella
Clostridium_XlVa
Hippea
Lactobacillus
Eubacterium
Clostridium_IV
Clostridium_III
Lactobacillus
Lactobacillus
Desulfotomaculum
Prevotella
Staphylococcus
Tenacibaculum
Parabacteroides
Clostridium_XlVa
Clostridium_IV
Clostridium_IV
Pedobacter
Helicobacter
Proteiniclasticum
Anaplasma
Bacteroides
Clostridium_IV
Mucilaginibacter
Verrucomicrobia
Selenomonas
Parabacteroides
Eubacterium
Coprococcus
Weissella
Pedobacter
Clostridium_XI
Sphingomonas
Treponema
Geobacter
Clostridium_XlVa
Filomicrobium
Prevotella
Pedobacter
Pedobacter
Clostridium_XlVa
Bifidobacterium
Saccharofermentans
Ruminococcus
Flavobacterium
Rhodopirellula
Roseburia
Prevotella
Limibacter
Saccharofermentans
Clostridium
—
sensu
—
stricto
Clostridium_III
Prevotella
Pseudoxanthomonas
Anaerorhabdus
Clostridium_III
Streptomyces
Pedobacter
Cellulomonas
Clostridium_XlVa
Olivibacter
Treponema
Gelidibacter
Ruminococcus
Clostridium_IV
Gemmatimonas
Prevotella
Ethanoligenens
Leucobacter
Clostridium_XlVa
Clostridium_XlVa
Eggerthella
Prevotella
Prevotella
Solobacterium
Xanthobacter
Verrucomicrobia
Desulfovibrio
Microbacterium
Oscillibacter
Blautia
Papillibacter
Prevotella
Lentisphaera
Ruminococcus
Bacteroides
Catonella
Clostridium_XlVa
Clostridium_IV
Verrucomicrobia
Clostridium_XI
Prevotella
Mogibacterium
Clostridium_XlVa
Ruminococcus
Eubacterium
Clostridium_IV
Rhodomicrobium
Butyricicoccus
Saccharofermentans
Prevotella
Mannheimia
Lactobacillus
Clostridium_IV
Clostridium_IV
Adlercreutzia
Selenomonas
Paenibacillus
Clostridium_IV
Paenibacillus
Butyricimonas
Wandonia
Puniceicoccus
Lactonifactor
Selenomonas
Brevundimonas
Prevotella
Gelidibacter
Mogibacterium
Clostridium_XlVa
Coprococcus
Verrucomicrobia
Barnesiella
Verrucomicrobia
Clostridium_XlVa
Anaerovorax
Bacteroides
Parasporobacterium
Prevotella
Parapedobacter
Streptomyces
Thermotalea
Alkaliflexus
Oscillibacter
Anaerotruncus
Spirochaeta
Clostridium_XI
Sporotomaculum
Sporacetigenium
Bulleidia
Clostridium_IV
Syntrophomonas
Desulfatiferula
Hydrogenoanaerobacterium
Clostridium_XlVa
Mogibacterium
Spirochaeta
Prevotella
Treponema
Spiroplasma
Clostridium_XlVa
Bacteroides
Treponema
Selenomonas
Butyricicoccus
Gelidibacter
Acetitomaculum
Proteiniclasticum
Papillibacter
Prevotella
Elusimicrobium
Devosia
Roseburia
Mucilaginibacter
Mogibacterium
Saccharofermentans
Paenibacillus
Anaerotruncus
Leucobacter
Clostridium_XlVa
Eubacterium
Beijerinckia
Prevotella
Clostridium_III
Cyanobacteria
Pseudoflavonifractor
Butyrivibrio
Acholeplasma
Filomicrobium
Clostridium_III
Pseudoflavonifractor
Anaerophaga
Asaccharobacter
Kordia
Ruminococcus
Clostridium_III
Ethanoligenens
Clostridium_XlVa
Barnesiella
Eubacterium
Prevotella
Anaerophaga
Acetitomaculum
Prevotella
Clostridium_III
Marinoscillum
Pedobacter
Prevotella
Prevotella
Anaerovorax
Clostridium_XlVa
Clostridium_IV
Clostridium
—
sensu
—
stricto
Lishizhenia
Pedobacter
Howardella
Roseburia
Clostridium_XlVa
Anaerovorax
Lentisphaera
Prevotella
Saccharofermentans
Cyanobacteria
Proteiniphilum
Schwartzia
Anaerorhabdus
Robinsoniella
Clostridium_IV
Flavobacterium
Pedobacter
Clostridium_III
Selenomonas
Rhizobium
Victivallis
Butyricimonas
Parabacteroides
Adhaeribacter
Eubacterium
Acidobacteria
Treponema
Clostridium_XlVa
Clostridium_XlVa
Schwartzia
Prevotella
Selenomonas
Beijerinckia
Eubacterium
Adhaeribacter
Verrucomicrobia
Desulfobulbus
Bacteroides
Rummeliibacillus
Agarivorans
Clostridium_XlVa
Selenomonas
Verrucomicrobia
Prevotella
Spirochaeta
Selenomonas
Spiroplasma
Pedobacter
Clostridium_XlVa
Cyanobacteria
Lactobacillus
Clostridium_XlVa
Prevotella
Prevotella
Marinobacter
Butyricimonas
Prevotella
Dongia
Anaerovorax
Butyricimonas
Cryptanaerobacter
Papillibacter
Clostridium
—
sensu
—
stricto
Escherichia/Shigella
Butyricicoccus
Prevotella
Thermotalea
Cohaesibacter
Clostridium_XVIII
Spirochaeta
Clostridium_XlVa
Hydrogenoanaerobacterium
Clostridium_IV
Papillibacter
Sporosarcina
Selenomonas
Papillibacter
Clostridium_XlVa
Saccharofermentans
Clostridium_IV
Clostridium_IV
Desulfotomaculum
Pedobacter
Anaeroplasma
Clostridium_IV
Treponema
Mogibacterium
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6Deposit number applicable to Budapest treaty and/or type stain rules and procedures
7Deposit number applicable to Budapest treaty and/or type stain rules and procedures
8Deposit number applicable to Budapest treaty and/or type stain rules and procedures
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
The term “a” or “an” may refer to one or more of that entity, i.e. can refer to plural referents. As such, the terms “a” or “an”, “one or more” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.
Reference throughout this specification to “one embodiment”, “an embodiment”, “one aspect”, or “an aspect” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments.
As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%.
As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, Bacteria and Archaea, eukaryotic fungi and protists, as well as viruses. In some embodiments, the disclosure refers to the “microbes” of Table 1 or Table 3, or the “microbes” incorporated by reference. This characterization can refer to not only the predicted taxonomic microbial identifiers of the table, but also the identified strains of the microbes listed in the table.
The term “microbial consortia” or “microbial consortium” refers to a subset of a microbial community of individual microbial species, or strains of a species, which can be described as carrying out a common function, or can be described as participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest (e.g. increased milk production in a ruminant). The community may comprise two or more species, or strains of a species, of microbes. In some instances, the microbes coexist within the community symbiotically.
The term “microbial community” means a group of microbes comprising two or more species or strains. Unlike microbial consortia, a microbial community does not have to be carrying out a common function, or does not have to be participating in, or leading to, or correlating with, a recognizable parameter, such as a phenotypic trait of interest (e.g. increased milk production in a ruminant).
As used herein, “isolate,” “isolated,” “isolated microbe,” and like terms, are intended to mean that the one or more microorganisms has been separated from at least one of the materials with which it is associated in a particular environment (for example soil, water, animal tissue).
Microbes of the present disclosure may include spores and/or vegetative cells. In some embodiments, microbes of the present disclosure include microbes in a viable but non-culturable (VBNC) state. See Liao and Zhao (US Publication US2015267163A1). In some embodiments, microbes of the present disclosure include microbes in a biofilm. See Merritt et al. (U.S. Pat. No. 7,427,408).
Thus, an “isolated microbe” does not exist in its naturally occurring environment; rather, it is through the various techniques described herein that the microbe has been removed from its natural setting and placed into a non-naturally occurring state of existence. Thus, the isolated strain or isolated microbe may exist as, for example, a biologically pure culture, or as spores (or other forms of the strain) in association with an acceptable carrier.
As used herein, “spore” or “spores” refer to structures produced by bacteria and fungi that are adapted for survival and dispersal. Spores are generally characterized as dormant structures, however spores are capable of differentiation through the process of germination. Germination is the differentiation of spores into vegetative cells that are capable of metabolic activity, growth, and reproduction. The germination of a single spore results in a single fungal or bacterial vegetative cell. Fungal spores are units of asexual reproduction, and in some cases are necessary structures in fungal life cycles. Bacterial spores are structures for surviving conditions that may ordinarily be nonconductive to the survival or growth of vegetative cells.
As used herein, “microbial composition” refers to a composition comprising one or more microbes of the present disclosure, wherein a microbial composition, in some embodiments, is administered to animals of the present disclosure.
As used herein, “carrier”, “acceptable carrier”, or “pharmaceutical carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin; such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, in some embodiments as injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. The choice of carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice. See Hardee and Baggo (1998. Development and Formulation of Veterinary Dosage Forms. 2nd Ed. CRC Press. 504 pg.); E.W. Martin (1970. Remington's Pharmaceutical Sciences. 17th Ed. Mack Pub. Co.); and Blaser et al. (US Publication US20110280840A1).
In certain aspects of the disclosure, the isolated microbes exist as isolated and biologically pure cultures. It will be appreciated by one of skill in the art, that an isolated and biologically pure culture of a particular microbe, denotes that said culture is substantially free (within scientific reason) of other living organisms and contains only the individual microbe in question. The culture can contain varying concentrations of said microbe. The present disclosure notes that isolated and biologically pure microbes often “necessarily differ from less pure or impure materials.” See, e.g. In re Bergstrom, 427 F.2d 1394, (CCPA 1970) (discussing purified prostaglandins), see also, In re Bergy, 596 F.2d 952 (CCPA 1979) (discussing purified microbes), see also, Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 (S.D.N.Y. 1911) (Learned Hand discussing purified adrenaline), aff'd in part, rev'd in part, 196 F. 496 (2d Cir. 1912), each of which are incorporated herein by reference. Furthermore, in some aspects, the disclosure provides for certain quantitative measures of the concentration, or purity limitations, that must be found within an isolated and biologically pure microbial culture. The presence of these purity values, in certain embodiments, is a further attribute that distinguishes the presently disclosed microbes from those microbes existing in a natural state. See, e.g., Merck & Co. v. Olin Aathieson Chemical Corp., 253 F.2d 156 (4th Cir. 1958) (discussing purity limitations for vitamin B12 produced by microbes), incorporated herein by reference.
As used herein, “individual isolates” should be taken to mean a composition, or culture, comprising a predominance of a single genera, species, or strain, of microorganism, following separation from one or more other microorganisms. The phrase should not be taken to indicate the extent to which the microorganism has been isolated or purified. However, “individual isolates” can comprise substantially only one genus, species, or strain, of microorganism.
As used herein, “microbiome” refers to the collection of microorganisms that inhabit the digestive tract or gastrointestinal tract of an animal (including the rumen if said animal is a ruminant) and the microorgansims' physical environment (i.e. the microbiome has a biotic and physical component). The microbiome is fluid and may be modulated by numerous naturally occurring and artificial conditions (e.g., change in diet, disease, antimicrobial agents, influx of additional microorganisms, etc.). The modulation of the microbiome of a rumen that can be achieved via administration of the compositions of the disclosure, can take the form of: (a) increasing or decreasing a particular Family, Genus, Species, or functional grouping of microbe (i.e. alteration of the biotic component of the rumen microbiome) and/or (b) increasing or decreasing volatile fatty acids in the rumen, increasing or decreasing rumen pH, increasing or decreasing any other physical parameter important for rumen health (i.e. alteration of the abiotic component of the rumen microbiome).
As used herein, “probiotic” refers to a substantially pure microbe (i.e., a single isolate) or a mixture of desired microbes, and may also include any additional components that can be administered to a mammal for restoring microbiota. Probiotics or microbial inoculant compositions of the invention may be administered with an agent to allow the microbes to survive the environment of the gastrointestinal tract, i.e., to resist low pH and to grow in the gastrointestinal environment. In some embodiments, the present compositions (e.g., microbial compositions) are probiotics in some aspects.
As used herein, “prebiotic” refers to an agent that increases the number and/or activity of one or more desired microbes. Non-limiting examples of prebiotics that may be useful in the methods of the present disclosure include fructooligosaccharides (e.g., oligofructose, inulin, inulin-type fructans), galactooligosaccharides, amino acids, alcohols, and mixtures thereof. See Ramirez-Farias et al. (2008. Br. J. Nutr. 4:1-10) and Pool-Zobel and Sauer (2007. J. Nutr. 137:2580-2584 and supplemental).
The term “growth medium” as used herein, is any medium which is suitable to support growth of a microbe. By way of example, the media may be natural or artificial including gastrin supplemental agar, LB media, blood serum, and tissue culture gels. It should be appreciated that the media may be used alone or in combination with one or more other media. It may also be used with or without the addition of exogenous nutrients.
The medium may be amended or enriched with additional compounds or components, for example, a component which may assist in the interaction and/or selection of specific groups of microorganisms. For example, antibiotics (such as penicillin) or sterilants (for example, quaternary ammonium salts and oxidizing agents) could be present and/or the physical conditions (such as salinity, nutrients (for example organic and inorganic minerals (such as phosphorus, nitrogenous salts, ammonia, potassium and micronutrients such as cobalt and magnesium), pH, and/or temperature) could be amended.
As used herein, the term “ruminant” includes mammals that are capable of acquiring nutrients from plant-based food by fermenting it in a specialized stomach (rumen) prior to digestion, principally through microbial actions. Ruminants included cattle, goats, sheep, giraffes, yaks, deer, antelope, and others.
As used herein, the term “bovid” includes any member of family Bovidae, which include hoofed mammals such as antelope, sheep, goats, and cattle, among others.
As used herein, “energy-corrected milk” or “ECM” represents the amount of energy in milk based upon milk volume, milk fat, and milk protein. ECM adjusts the milk components to 3.5% fat and 3.2% protein, thus equalizing animal performance and allowing for comparison of production at the individual animal and herd levels over time. An equation used to calculate ECM, as related to the present disclosure, is:
ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.2×protein pounds)
As used herein, “improved” should be taken broadly to encompass improvement of a characteristic of interest, as compared to a control group, or as compared to a known average quantity associated with the characteristic in question. For example, “improved” milk production associated with application of a beneficial microbe, or consortia, of the disclosure can be demonstrated by comparing the milk produced by an ungulate treated by the microbes taught herein to the milk of an ungulate not treated. In the present disclosure, “improved” does not necessarily demand that the data be statistically significant (i.e. p<0.05); rather, any quantifiable difference demonstrating that one value (e.g. the average treatment value) is different from another (e.g. the average control value) can rise to the level of “improved.”
As used herein, “inhibiting and suppressing” and like terms should not be construed to require complete inhibition or suppression, although this may be desired in some embodiments.
The term “marker” or “unique marker” as used herein is an indicator of unique microorganism type, microorganism strain or activity of a microorganism strain. A marker can be measured in biological samples and includes without limitation, a nucleic acid-based marker such as a ribosomal RNA gene, a peptide- or protein-based marker, and/or a metabolite or other small molecule marker.
The term “metabolite” as used herein is an intermediate or product of metabolism. A metabolite in one embodiment is a small molecule. Metabolites have various functions, including in fuel, structural, signaling, stimulatory and inhibitory effects on enzymes, as a cofactor to an enzyme, in defense, and in interactions with other organisms (such as pigments, odorants and pheromones). A primary metabolite is directly involved in normal growth, development and reproduction. A secondary metabolite is not directly involved in these processes but usually has an important ecological function. Examples of metabolites include but are not limited to antibiotics and pigments such as resins and terpenes, etc. Some antibiotics use primary metabolites as precursors, such as actinomycin which is created from the primary metabolite, tryptophan. Metabolites, as used herein, include small, hydrophilic carbohydrates; large, hydrophobic lipids and complex natural compounds.
As used herein, the term “genotype” refers to the genetic makeup of an individual cell, cell culture, tissue, organism, or group of organisms.
As used herein, the term “allele(s)” means any of one or more alternative forms of a gene, all of which alleles relate to at least one trait or characteristic. In a diploid cell, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. Since the present disclosure, in embodiments, relates to QTLs, i.e. genomic regions that may comprise one or more genes or regulatory sequences, it is in some instances more accurate to refer to “haplotype” (i.e. an allele of a chromosomal segment) instead of “allele”, however, in those instances, the term “allele” should be understood to comprise the term “haplotype”. Alleles are considered identical when they express a similar phenotype. Differences in sequence are possible but not important as long as they do not influence phenotype.
As used herein, the term “locus” (loci plural) means a specific place or places or a site on a chromosome where for example a gene or genetic marker is found.
As used herein, the term “genetically linked” refers to two or more traits that are co-inherited at a high rate during breeding such that they are difficult to separate through crossing.
A “recombination” or “recombination event” as used herein refers to a chromosomal crossing over or independent assortment. The term “recombinant” refers to an organism having a new genetic makeup arising as a result of a recombination event.
As used herein, the term “molecular marker” or “genetic marker” refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, microsatellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location. Markers further include polynucleotide sequences encoding 16S or 18S rRNA, and internal transcribed spacer (ITS) sequences, which are sequences found between small-subunit and large-subunit rRNA genes that have proven to be especially useful in elucidating relationships or distinctions among when compared against one another. Mapping of molecular markers in the vicinity of an allele is a procedure which can be performed by the average person skilled in molecular-biological techniques.
The primary structure of major rRNA subunit 16S comprise a particular combination of conserved, variable, and hypervariable regions that evolve at different rates and enable the resolution of both very ancient lineages such as domains, and more modern lineages such as genera. The secondary structure of the 16S subunit include approximately 50 helices which result in base pairing of about 67% of the residues. These highly conserved secondary structural features are of great functional importance and can be used to ensure positional homology in multiple sequence alignments and phylogenetic analysis. Over the previous few decades, the 16S rRNA gene has become the most sequenced taxonomic marker and is the cornerstone for the current systematic classification of bacteria and archaea (Yarza et al. 2014. Nature Rev. Micro. 12:635-45).
A sequence identity of 94.5% or lower for two 16S rRNA genes is strong evidence for distinct genera, 86.5% or lower is strong evidence for distinct families, 82% or lower is strong evidence for distinct orders, 78.5% is strong evidence for distinct classes, and 75% or lower is strong evidence for distinct phyla. The comparative analysis of 16S rRNA gene sequences enables the establishment of taxonomic thresholds that are useful not only for the classification of cultured microorganisms but also for the classification of the many environmental sequences. Yarza et al. 2014. Nature Rev. Micro. 12:635-45).
As used herein, the term “trait” refers to a characteristic or phenotype. For example, in the context of some embodiments of the present disclosure, quantity of milk fat produced relates to the amount of triglycerides, triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, cholesterol, glycolipids, and fatty acids present in milk. Desirable traits may also include other milk characteristics, including but not limited to: predominance of short chain fatty acids, medium chain fatty acids, and long chain fatty acids; quantity of carbohydrates such as lactose, glucose, galactose, and other oligosaccharides; quantity of proteins such as caseins and whey; quantity of vitamins, minerals, milk yield/volume; reductions in methane emissions or manure; improved efficiency of nitrogen utilization; improved dry matter intake; improved feed efficiency and digestibility; increased degradation of cellulose, lignin, and hemicellulose; increased rumen concentrations of fatty acids such as acetic acid, propionic acid, and butyric acid; etc.
A trait may be inherited in a dominant or recessive manner, or in a partial or incomplete-dominant manner. A trait may be monogenic (i.e. determined by a single locus) or polygenic (i.e. determined by more than one locus) or may also result from the interaction of one or more genes with the environment.
In the context of this disclosure, traits may also result from the interaction of one or more mammalian genes and one or more microorganism genes.
As used herein, the term “homozygous” means a genetic condition existing when two identical alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism. Conversely, as used herein, the term “heterozygous” means a genetic condition existing when two different alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.
As used herein, the term “phenotype” refers to the observable characteristics of an individual cell, cell culture, organism (e.g., a ruminant), or group of organisms which results from the interaction between that individual's genetic makeup (i.e., genotype) and the environment.
As used herein, the term “chimeric” or “recombinant” when describing a nucleic acid sequence or a protein sequence refers to a nucleic acid, or a protein sequence, that links at least two heterologous polynucleotides, or two heterologous polypeptides, into a single macromolecule, or that re-arranges one or more elements of at least one natural nucleic acid or protein sequence. For example, the term “recombinant” can refer to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
As used herein, a “synthetic nucleotide sequence” or “synthetic polynucleotide sequence” is a nucleotide sequence that is not known to occur in nature or that is not naturally occurring. Generally, such a synthetic nucleotide sequence will comprise at least one nucleotide difference when compared to any other naturally occurring nucleotide sequence.
As used herein, the term “nucleic acid” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified nucleic acids such as methylated and/or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like. The terms “nucleic acid” and “nucleotide sequence” are used interchangeably.
As used herein, the term “gene” refers to any segment of DNA associated with a biological function. Thus, genes include, but are not limited to, coding sequences and/or the regulatory sequences required for their expression. Genes can also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.
As used herein, the term “homologous” or “homologue” or “ortholog” is known in the art and refers to related sequences that share a common ancestor or family member and are determined based on the degree of sequence identity. The terms “homology,” “homologous,” “substantially similar” and “corresponding substantially” are used interchangeably herein. They refer to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate gene expression or produce a certain phenotype. These terms also refer to modifications of the nucleic acid fragments of the instant disclosure such as deletion or insertion of one or more nucleotides that do not substantially alter the functional properties of the resulting nucleic acid fragment relative to the initial, unmodified fragment. It is therefore understood, as those skilled in the art will appreciate, that the disclosure encompasses more than the specific exemplary sequences. These terms describe the relationship between a gene found in one species, subspecies, variety, cultivar or strain and the corresponding or equivalent gene in another species, subspecies, variety, cultivar or strain. For purposes of this disclosure homologous sequences are compared. “Homologous sequences” or “homologues” or “orthologs” are thought, believed, or known to be functionally related. A functional relationship may be indicated in any one of a number of ways, including, but not limited to: (a) degree of sequence identity and/or (b) the same or similar biological function. Preferably, both (a) and (b) are indicated. Homology can be determined using software programs readily available in the art, such as those discussed in Current Protocols in Molecular Biology (F. M. Ausubel el al., eds., 1987) Supplement 30, section 7.718, Table 7.71. Some alignment programs are MacVector (Oxford Molecular Ltd, Oxford, U.K.), ALIGN Plus (Scientific and Educational Software, Pennsylvania) and AlignX (Vector NTi, Invitrogen, Carlsbad, Calif.). Another alignment program is Sequencher (Gene Codes, Ann Arbor, Mich.), using default parameters.
As used herein, the term “nucleotide change” refers to, e.g., nucleotide substitution, deletion, and/or insertion, as is well understood in the art. For example, mutations contain alterations that produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded protein or how the proteins are made.
As used herein, the term “protein modification” refers to, e.g., amino acid substitution, amino acid modification, deletion, and/or insertion, as is well understood in the art.
As used herein, the term “at least a portion” or “fragment” of a nucleic acid or polypeptide means a portion having the minimal size characteristics of such sequences, or any larger fragment of the full length molecule, up to and including the full length molecule. A fragment of a polynucleotide of the disclosure may encode a biologically active portion of a genetic regulatory element. A biologically active portion of a genetic regulatory element can be prepared by isolating a portion of one of the polynucleotides of the disclosure that comprises the genetic regulatory element and assessing activity as described herein. Similarly, a portion of a polypeptide may be 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, and so on, going up to the full length polypeptide. The length of the portion to be used will depend on the particular application. A portion of a nucleic acid useful as a hybridization probe may be as short as 12 nucleotides; in some embodiments, it is 20 nucleotides. A portion of a polypeptide useful as an epitope may be as short as 4 amino acids. A portion of a polypeptide that performs the function of the full-length polypeptide would generally be longer than 4 amino acids.
Variant polynucleotides also encompass sequences derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) PNAS 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zbang et al. (1997) PNAS 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458. For PCR amplifications of the polynucleotides disclosed herein, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.
The term “primer” as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and composition (A/T vs. G/C content) of primer. A pair of bi-directional primers consists of one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.
The terms “stringency” or “stringent hybridization conditions” refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimized to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridize to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or “conditions of reduced stringency” include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2×SSC at 40° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. Hybridization procedures are well known in the art and are described by e.g. Ausubel et al., 1998 and Sambrook et al., 2001. In some embodiments, stringent conditions are hybridization in 0.25 M Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, 0.5-20% sodium dodecyl sulfate at 45° C., such as 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by a wash in 5-SSC, containing 0.1% (w/v) sodium dodecyl sulfate, at 55° C. to 65° C.
As used herein, “promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence that can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of some variation may have identical promoter activity.
As used herein, a “constitutive promoter” is a promoter which is active under most conditions and/or during most development stages. There are several advantages to using constitutive promoters in expression vectors used in biotechnology, such as: high level of production of proteins used to select transgenic cells or organisms; high level of expression of reporter proteins or scorable markers, allowing easy detection and quantification; high level of production of a transcription factor that is part of a regulatory transcription system; production of compounds that requires ubiquitous activity in the organism; and production of compounds that are required during all stages of development. Non-limiting exemplary constitutive promoters include, CaMV 35S promoter, opine promoters, ubiquitin promoter, alcohol dehydrogenase promoter, etc.
As used herein, a “non-constitutive promoter” is a promoter which is active under certain conditions, in certain types of cells, and/or during certain development stages. For example, tissue specific, tissue preferred, cell type specific, cell type preferred, inducible promoters, and promoters under development control are non-constitutive promoters. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues.
As used herein, “inducible” or “repressible” promoter is a promoter which is under chemical or environmental factors control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions, certain chemicals, the presence of light, acidic or basic conditions, etc.
As used herein, a “tissue specific” promoter is a promoter that initiates transcription only in certain tissues. Unlike constitutive expression of genes, tissue-specific expression is the result of several interacting levels of gene regulation. As such, in the art sometimes it is preferable to use promoters from homologous or closely related species to achieve efficient and reliable expression of transgenes in particular tissues. This is one of the main reasons for the large amount of tissue-specific promoters isolated from particular tissues found in both scientific and patent literature.
As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked with a coding sequence when it is capable of regulating the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in a sense or antisense orientation. In another example, the complementary RNA regions of the disclosure can be operably linked, either directly or indirectly, 5′ to the target mRNA, or 3′ to the target mRNA, or within the target mRNA, or a first complementary region is 5′ and its complement is 3′ to the target mRNA.
As used herein, the phrases “recombinant construct”, “expression construct”, “chimeric construct”, “construct”, and “recombinant DNA construct” are used interchangeably herein. A recombinant construct comprises an artificial combination of nucleic acid fragments, e.g., regulatory and coding sequences that are not found together in nature. For example, a chimeric construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such construct may be used by itself or may be used in conjunction with a vector. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the disclosure. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others. Vectors can be plasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons, artificial chromosomes, and the like, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that is not autonomously replicating. As used herein, the term “expression” refers to the production of a functional end-product e.g., an mRNA or a protein (precursor or mature).
In some embodiments, the cell or organism has at least one heterologous trait. As used herein, the term “heterologous trait” refers to a phenotype imparted to a transformed host cell or transgenic organism by an exogenous DNA segment, heterologous polynucleotide or heterologous nucleic acid. Various changes in phenotype are of interest to the present disclosure, including but not limited to modifying the fatty acid composition in milk, altering the carbohydrate content of milk, increasing an ungulate's yield of an economically important trait (e.g., milk, milk fat, milk proteins, etc.) and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in organisms using the methods and compositions of the present disclosure.
As used herein, the term “MIC” means maximal information coefficient. MIC is a type of nonparamentric network analysis that identifies a score (MIC score) between active microbial strains of the present disclosure and at least one measured metadata (e.g., milk fat). Further, U.S. application Ser. No. 15/217,575, filed on Jul. 22, 2016 (issued as U.S. Pat. No. 9,540,676 on Jan. 10, 2017) is hereby incorporated by reference in its entirety.
The maximal information coefficient (MIC) is then calculated between strains and metadata 3021a, and between strains 3021b; as seen in
Based on the output of the network analysis, active strains are selected 3025 for preparing products (e.g., ensembles, aggregates, and/or other synthetic groupings) containing the selected strains. The output of the network analysis can also be used to inform the selection of strains for further product composition testing.
The use of thresholds is discussed above for analyses and determinations. Thresholds can be, depending on the implementation and application: (1) empirically determined (e.g., based on distribution levels, setting a cutoff at a number that removes a specified or significant portion of low level reads); (2) any non-zero value; (3) percentage/percentile based; (4) only strains whose normalized second marker (i.e., activity) reads is greater than normalized first marker (cell count) reads; (5) log 2 fold change between activity and quantity or cell count; (6) normalized second marker (activity) reads is greater than mean second marker (activity) reads for entire sample (and/or sample set); and/or any magnitude threshold described above in addition to a statistical threshold (i.e., significance testing). The following example provides thresholding detail for distributions of RNA-based second marker measurements with respect to DNA-based first marker measurements, according to one embodiment.
As used herein “shelf-stable” refers to a functional attribute and new utility acquired by the microbes formulated according to the disclosure, which enable said microbes to exist in a useful/active state outside of their natural environment in the rumen (i.e. a markedly different characteristic). Thus, shelf-stable is a functional attribute created by the formulations/compositions of the disclosure and denoting that the microbe formulated into a shelf-stable composition can exist outside the rumen and under ambient conditions for a period of time that can be determined depending upon the particular formulation utilized, but in general means that the microbes can be formulated to exist in a composition that is stable under ambient conditions for at least a few days and generally at least one week. Accordingly, a “shelf-stable ruminant supplement” is a composition comprising one or more microbes of the disclosure, said microbes formulated in a composition, such that the composition is stable under ambient conditions for at least one week, meaning that the microbes comprised in the composition (e.g. whole cell, spore, or lysed cell) are able to impart one or more beneficial phenotypic properties to a ruminant when administered (e.g. increased milk yield, improved milk compositional characteristics, improved rumen health, and/or modulation of the rumen microbiome).
In some aspects, the present disclosure provides isolated microbes, including novel strains of microbes, presented in Table 1 and Table 3.
In other aspects, the present disclosure provides isolated whole microbial cultures of the microbes identified in Table 1 and Table 3. These cultures may comprise microbes at various concentrations.
In some aspects, the disclosure provides for utilizing one or more microbes selected from Table 1 and Table 3 to increase a phenotypic trait of interest in a ruminant.
In some embodiments, the disclosure provides isolated microbial species belonging to taxonomic families of Clostridiaceae, Ruminococcaceae, Lachnospiraceae, Acidaminococcaceae, Peptococcaceae, Porphyromonadaceae, Prevotellaceae, Neocallimastigaceae, Saccharomycetaceae, Phaeosphaeriaceae, Erysipelotrichia, Anaerolinaeceae, Atopobiaceae, Botryosphaeriaceae, Eubacteriaceae, Acholeplasmataceae, Succinivibrionaceae, Lactobacillaceae, Selenomonadaceae, Burkholderiaceae, and Streptococcaceae.
In further embodiments, isolated microbial species may be selected from genera of family Clostridiaceae, including Acetanaerobacterium, Acetivibrio, Acidaminobacter, Alkaliphilus, Anaerobacter, Anaerostipes, Anaerotruncus, Anoxynatronum, Bryantella, Butyricicoccus, Caldanaerocella, Caloramator, Caloranaerobacter, Caminicella, Candidatus Arthromitus, Clostridium, Coprobacillus, Dorea, Ethanologenbacterium, Faecalibacterium, Garciella, Guggenheimella, Hespellia, Linmingia, Natronincola, Oxobacter, Parasporobacterium, Sarcina, Soehngenia, Sporobacter, Subdoligranulum, Tepidibacter, Tepidimicrobium, Thermobrachium, Thermohalobacter, and Tindallia.
In further embodiments, isolated microbial species may be selected from genera of family Ruminococcaceae, including Ruminococcus, Acetivibrio, Sporobacter, Anaerofilium, Papillibacter, Oscillospira, Gemmiger, Faecalibacterium, Fastidiosipila, Anaerotruncus, Ethanolingenens, Acetanaerobacterium, Subdoligranulum, Hydrogenoanaerobacterium, and Candidadus Soleaferrea.
In further embodiments, isolated microbial species may be selected from genera of family Lachnospiraceae, including Butyrivibrio, Roseburia, Lachnospira, Acetitomaculum, Coprococcus, Johnsonella, Catonella, Pseudobutyrivibrio, Syntrophococcus, Sporobacterium, Parasporobacterium, Lachnobacterium, Shuttleworthia, Dorea, Anaerostipes, Hespellia, Marvinbryantia, Oribacterium, Moryella, Blautia, Robinsoniella, Cellulosilyticum, Lachnoanaerobaculum, Stomatobaculum, Fusicatenibacter, Acetatifactor, and Eisenbergiella.
In further embodiments, isolated microbial species may be selected from genera of family Acidaminococcaceae, including Acidaminococcus, Phascolarctobacterium, Succiniclasticum, and Succinispira.
In further embodiments, isolated microbial species may be selected from genera of family Peptococcaceae, including Desulfotomaculum, Peptococcus, Desulfitobacterium, Syntrophobotulus, Dehalobacter, Sporotomaculum, Desulfosporosinus, Desulfonispora, Pelotomaculum, Thermincola, Cryptanaerobacter, Desulfitibacter, Candidatus Desulforudis, Desulfurispora, and Desulfitospora.
In further embodiments, isolated microbial species may be selected from genera of family Porphyromonadaceae, including Porphyromonas, Dysgonomonas, Tannerella, Odoribacter, Proteiniphilum, Petrimonas, Paludibacter, Parabacteroides, Barnesiella, Candidatus Vestibaculum, Butyricimonas, Macellibacteroides, and Coprobacter.
In further embodiments, isolated microbial species may be selected from genera of family Anaerolinaeceae including Anaerolinea, Bellilinea, Leptolinea, Levilinea, Longilinea, Ornatilinea, and Pelolinea.
In further embodiments, isolated microbial species may be selected from genera of family Atopobiaceae including Atopbium and Olsenella.
In further embodiments, isolated microbial species may be selected from genera of family Eubacteriaceae including Acetobacterium, Alkalibacter, Alkalibaculum, Aminicella, Anaerofustis. Eubacterium, Garciella, and Pseudoramibacter.
In further embodiments, isolated microbial species may be selected from genera of family Acholeplasmataceae including Acholeplasma.
In further embodiments, isolated microbial species may be selected from genera of family Succinivibrionaceae including Anaerobiospirillun, Ruminobacter, Succinatimonas, Succininonas, and Succinivibrio.
In further embodiments, isolated microbial species may be selected from genera of family Lactobacillaceae including Lactobacillus, Paralactobacillus, Pediococcus, and Sharpea.
In further embodiments, isolated microbial species may be selected from genera of family Selenomonadaceae including Anaerovibrio, Centipeda, Megamonas, Mitsuokella, Pectinatus, Propionispira, Schwartzia, Selenomonas, and Zymophilus.
In further embodiments, isolated microbial species may be selected from genera of family Burkholderiaceae including Burkholderia, Chitinimonas, Cupriavidus, Lautropia, Limnobacter, Pandoraea, Paraburkholderia, Paucimonas, Polynucleobacter, Ralstonia, Thermothrix, and Wautersia.
In further embodiments, isolated microbial species may be selected from genera of family Streptococcaceae including Lactococcus, Lactovum, and Streptococcus.
In further embodiments, isolated microbial species may be selected from genera of family Anaerolinaeceae including Aestuariimicrobium, Arachnia, Auraticoccus, Brooklawnia, Friedmanniella, Granulicoccus, Luteococcus, Mariniluteicoccus, Microlunatus, Micropruina, Naumannella, Propionibacterium, Propionicicella, Propioniciclava, Propioniferax, Propionimicrobium, and Tessaracoccus.
In further embodiments, isolated microbial species may be selected from genera of family Prevotellaceae, including Paraprevotella, Prevotella, hallella, Xylanibacter, and Alloprevotella.
In further embodiments, isolated microbial species may be selected from genera of family Neocallimastigaceae, including Anaeromyces, Caecomyces, Cyllamyces, Neocallimastix, Orpinomyces, and Piromyces.
In further embodiments, isolated microbial species may be selected from genera of family Saccharomycetaceae, including Brettanomyces, Candida, Citeromyces, CyWiclomyces, Debaryonyces, Issatchenkia, Kazachstania (syn. Arxiozyma), Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltoryma, Torulaspora, Williopsis, Zygosaccharomyces, and Zygotorulaspora.
In further embodiments, isolated microbial species may be selected from genera of family Erysipelotrichaceae, including Erysipelothrix, Solobacterium, Turicibacter, Faecalibaculum, Faecalicoccus, Faecalitalea, Holdemanella, Holdemania, Dielma, Eggerthia, Erysipelatoclostridium, Allobacterium, Breznakia, Bulleidia, Catenibacterium, Catenisphaera, and Coprobacillus.
In further embodiments, isolated microbial species may be selected from genera of family Phaeosphaeriaceae, including Barria, Bricookea, Carinispora, Chaetoplea, Eudarluca, Hadrospora, lsthmosporella, Katumotoa, Lautitia, Metameris, Mixtura, Neophaeosphaeria, Nodulosphaeria, Ophiosphaerella, Phaeosphaeris, Phaeosphaeriopsis, Setomelanomma, Stagonospora, Teratosphaeria, and Wilmia.
In further embodiments, isolated microbial species may be selected from genera of family Botryosphaeriaceae, including Amarenomyces, Aplosporella, Auerswaldiella, Botryosphaeria, Dichomera, Diplodia, Discochora, Dothidothia, Dothiorella, Fusicoccum, Granulodiplodia, Guignardia, Lasiodiplodia, Leptodothiorella, Leptodothiorella, Leptoguignardia, Macrophoma, Macrophomina, Nattrassia, Neodeightonia, Neofusicocum, Neoscytalidium, Otthia, Phaeobotryosphaeria, Phomatosphaeropsis, Phyllosticta, Pseudofusicoccum, Saccharata, Sivanesania, and Thyrostroma.
In some embodiments, the disclosure provides isolated microbial species belonging to genera of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. In further embodiments, the disclosure provides isolated microbial species belonging to the family of Lachnospiraceae, and the order of Saccharomycetales. In further embodiments, the disclosure provides isolated microbial species of Candida xylopsoci, Vrystaatia aloeicola, and Phyllosticta capitalensis.
In some embodiments, a microbe from the taxa disclosed herein are utilized to impart one or more beneficial properties or improved traits to milk in ruminants.
In some embodiments, the disclosure provides isolated microbial species, selected from the group consisting of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta.
In some embodiments, the disclosure provides novel isolated microbial strains of species, selected from the group consisting of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Mannerella, Prevotella, Butvricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallinastix, Ruminococcus, and Phyllosticta. Particular novel strains of these aforementioned taxonomic groups can be found in Table 1 and/or Table 3.
In some embodiments, the disclosure provides isolated microbial strains of Ruminococcus bovis. In some embodiments, the isolated microbial strain of Ruminococcus bovis comprises the 16S nucleic acid sequence of SEQ ID NO: 2108. In some embodiments, the isolated microbial strain of Ruminococcus bovis comprises the deposit accession number PTA-125917, NRRL B-67764, TSD-225, or NCTC 14479. In some embodiments, the isolated strain of Ruminococcus bovis comprises one or more mutations selected from the group consisting of: (a) a G→T substitution at position 297 of SEQ ID NO: 2109; (b) a CC→TA substitution at positions 301-302 of SEQ ID NO: 2111; (c) a T→G substitution at position 307 of SEQ ID NO: 2111; (d) a −A deletion at position 300 of SEQ ID NO: 2113; (e) a CCA→TTC substitution at positions 116-118 of SEQ ID NO: 2115; (f) a +T insertion between positions 105-106 of SEQ ID NO: 2117; (g) a C→T substitution at position 298 of SEQ ID NO: 2119; (h) a C→A substitution at position 298 of SEQ ID NO: 2121; and (i) a +AC insertion between positions 43-44 of SEQ ID NO: 2123. In some embodiments, the isolated strain of Ruminococcus bovis comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 2110, 2112, 2114, 2116, 2118, 2120, 2122, or 2124.
In some embodiments, the present disclosure provides an orally deliverable composition for increasing milk production or improving milk compositional characteristics in a ruminant, comprising: (a) Ruminococcus bovis comprising one or more mutations selected from the group consisting of: (i) a G→T substitution at position 297 of SEQ ID NO: 2109; (ii) a CC→TA substitution at positions 301-302 of SEQ ID NO: 2111; (iii) a T→G substitution at position 307 of SEQ ID NO: 2111; (iv) a −A deletion at position 300 of SEQ ID NO: 2113; (v) a CCA→TTC substitution at positions 116-118 of SEQ ID NO: 2115; (vi) a +T insertion between positions 105-106 of SEQ ID NO: 2117; (vii) a C→T substitution at position 298 of SEQ ID NO: 2119; (viii) a C→A substitution at position 298 of SEQ ID NO: 2121; and (ix) a +AC insertion between positions 43-44 of SEQ ID NO: 2123; and (b) a carrier suitable for oral ruminant administration.
In some embodiments, the present disclosure provides an orally deliverable composition for increasing milk production or improving milk compositional characteristics in a ruminant, comprising: (a) Ruminococcus bovis comprising a nucleic acid sequence selected from any one of SEQ ID NOs: 2110, 2112, 2114, 2116, 2118, 2120, 2122, or 2124; and (b) a carrier suitable for oral ruminant administration.
Furthermore, the disclosure relates to microbes having characteristics substantially similar to that of a microbe identified in Table 1 or Table 3.
The isolated microbial species, and novel strains of said species, identified in the present disclosure, are able to impart beneficial properties or traits to ruminant milk production. For instance, the isolated microbes described in Table 1 and Table 3, or consortia of said microbes, are able to increase total milk fat in ruminant milk. The increase can be quantitatively measured, for example, by measuring the effect that said microbial application has upon the modulation of total milk fat.
In some embodiments, the isolated microbial strains are microbes of the present disclosure that have been genetically modified. In some embodiments, the genetically modified or recombinant microbes comprise polynucleotide sequences which do not naturally occur in said microbes. In some embodiments, the microbes may comprise heterologous polynucleotides. In further embodiments, the heterologous polynucleotides may be operably linked to one or more polynucleotides native to the microbes.
In some embodiments, the heterologous polynucleotides may be reporter genes or selectable markers. In some embodiments, reporter genes may be selected from any of the family of fluorescence proteins (e.g., GFP, RFP, YFP, and the like), β-galactosidase, luciferase. In some embodiments, selectable markers may be selected from neomycin phosphotransferase, hygromycin phosphotransferase, aminoglycoside adenyltransferase, dihydrofolate reductase, acetolactase synthase, bromoxynil nitrilase, β-glucuronidase, dihydrogolate reductase, and chloramphenicol acetyltransferase. In some embodiments, the heterologous polynucleotide may be operably linked to one or more promoter.
Intestinimonas
Anaerolinea
Pseudobutyrivibrio
Olsenella
Eubacterium
Catenisphaera
Faecalibacterium
Solobacterium
Blautia
Ralsonia
Coprococcus
Casaltella
Anaeroplasma
Acholeplasma
Aminiphilus
Mitsuokella
Alistipes
Sharpea
Oscillibacter
Neocallimastix
Odoribacter
Pichia
Tannerella
Candida
Hydrogenoanaerobacterium
Orpinomyces
Succinivibrio
Sugiyamaella
Ruminobacter
Cyllamyces
Lachnospira
Caecomyces
Sinimarinibacterium
Tremella
Hydrogenoanaerobacterium
Turicibacter
Clostridium XlVa
Anaerolinea
Saccharofermentans
Piromyces
Butyricicoccus
Olsenella
Papillibacter
Clostridium XICa
Pelotomaculum
Lachnospiracea
Solobacterium
Anaeroplasma
Ralstonia
Clostridium
Eubacterium
Rikenella
Lachnobacterium
Tannerella
Acholeplasma
Howardella
Selenomonas
Butyricimonas
Sharpea
Succinivibrio
Phyllosticta
Ruminobacter
Candida xylopsoc
Syntrophococcus
Candida apicol
Pseudobutyrivibrio
Saccharomycetales
Ascomycota
Candida rugos
In some aspects, the disclosure provides microbial consortia comprising a combination of at least any two microbes selected from amongst the microbes identified in Table 1 and/or Table 3.
In certain embodiments, the consortia of the present disclosure comprise two microbes, or three microbes, or four microbes, or five microbes, or six microbes, or seven microbes, or eight microbes, or nine microbes, or ten or more microbes. Said microbes of the consortia are different microbial species, or different strains of a microbial species.
In some embodiments, the disclosure provides consortia, comprising: at least two isolated microbial species belonging to genera of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomacuium, Butnricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. Particular novel strains of species of these aforementioned genera can be found in Table 1 and/or Table 3.
In some embodiments, the disclosure provides consortia, comprising: at least two isolated microbial species, selected from the group consisting of species of the family of Lachnospiraceae, and the order of Saccharomycetales.
In particular aspects, the disclosure provides microbial consortia, comprising species as grouped in Tables 6-12. With respect to Tables 6-12, the letters A through I represent a non-limiting selection of microbes of the present disclosure, defined as:
A=Strain designation Ascusb_5 (SEQ ID NO: 1) identified in Table 1;
B=Strain designation Ascusb_3138 (SEQ ID NO: 28) identified in Table 1:
C=Strain designation Ascusb_5 (SEQ ID NO: 2108) identified in Table 1;
D=Strain designation Ascusb_826 (SEQ ID NO: 2067) identified in Table;
E=Strain designation Ascusf_22 (SEQ ID NO: 33) identified in Table 1;
F=Strain designation Ascusf_23 (SEQ ID NO: 34) identified in Table 1;
G=Strain designation Ascusf_24 (SEQ ID NO. 35) identified in Table 1;
H=Strain designation Ascusf_45 (SEQ ID NO: 38) identified in Table 1; and
I=Strain designation Ascusf_15 (SEQ ID NO: 32) identified in Table 1.
In some embodiments, the microbial consortia may be selected from any member group from Tables 6-12.
In some embodiments, the microbial consortia comprises: a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 28; a Pichia sp. comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 32; a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2108; and/or a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2067.
In some embodiments, the microbial consortia comprises: a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2108; and/or a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.
The microbes of the present disclosure were obtained, among other places, at various locales in the United States from the gastrointestinal tract of cows.
The microbes of Table 1 and Table 3 were matched to their nearest taxonomic groups by utilizing classification tools of the Ribosomal Database Project (RDP) for 16s rRNA sequences and the User-friendly Nordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences. Examples of matching microbes to their nearest taxa may be found in Lan et al. (2012. PLOS one. 7(3):e32491), Schloss and Westcott (2011. Appl. Environ. Microbiol. 77(10):3219-3226), and Koljalg et al. (2005. New Phytologist. 166(3):1063-1068).
The isolation, identification, and culturing of the microbes of the present disclosure can be effected using standard microbiological techniques. Examples of such techniques may be found in Gerhardt, P. (ed.) Methods for General and Molecular Microbiology. American Society for Microbiology, Washington, D.C. (1994) and Lennette, E. H. (ed.) Manual of Clinical Microbiology, Third Edition. American Society for Microbiology, Washington, D.C. (1980), each of which is incorporated by reference.
Isolation can be effected by streaking the specimen on a solid medium (e.g., nutrient agar plates) to obtain a single colony, which is characterized by the phenotypic traits described hereinabove (e.g., Gram positive/negative, capable of forming spores aerobically/anaerobically, cellular morphology, carbon source metabolism, acid/base production, enzyme secretion, metabolic secretions, etc.) and to reduce the likelihood of working with a culture which has become contaminated.
For example, for microbes of the disclosure, biologically pure isolates can be obtained through repeated subculture of biological samples, each subculture followed by streaking onto solid media to obtain individual colonies or colony forming units. Methods of preparing, thawing, and growing lyophilized bacteria are commonly known, for example, Gherna, R L. and C. A. Reddy. 2007. Culture Preservation, p 1019-1033. In C. A. Reddy, T. J. Beveridge, J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R. Snyder, eds. American Society for Microbiology, Washington, D.C., 1033 pages; herein incorporated by reference. Thus freeze dried liquid formulations and cultures stored long term at −70° C. in solutions containing glycerol are contemplated for use in providing formulations of the present disclosure.
The microbes of the disclosure can be propagated in a liquid medium under aerobic conditions, or alternatively anaerobic conditions. Medium for growing the bacterial strains of the present disclosure includes a carbon source, a nitrogen source, and inorganic salts, as well as specially required substances such as vitamins, amino acids, nucleic acids and the like. Examples of suitable carbon sources which can be used for growing the microbes include, but are not limited to, starch, peptone, yeast extract, amino acids, sugars such as glucose, arabinose, mannose, glucosamine, maltose, and the like; salts of organic acids such as acetic acid, fumaric acid, adipic acid, propionic acid, citric acid, gluconic acid, malic acid, pyruvic acid, malonic acid and the like; alcohols such as ethanol and glycerol and the like; oil or fat such as soybean oil, rice bran oil, olive oil, corn oil, sesame oil. The amount of the carbon source added varies according to the kind of carbon source and is typically between 1 to 100 gram(s) per liter of medium. Preferably, glucose, starch, and/or peptone is contained in the medium as a major carbon source, at a concentration of 0.1-5% (W/V). Examples of suitable nitrogen sources which can be used for growing the bacterial strains of the present disclosure include, but are not limited to, amino acids, yeast extract, tryptone, beef extract, peptone, potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia or combinations thereof. The amount of nitrogen source varies according to the type of nitrogen source, typically between 0.1 to 30 gram per liter of medium. The inorganic salts, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, ferric sulfate, ferrous sulfate, ferric chloride, ferrous chloride, manganous sulfate, manganous chloride, zinc sulfate, zinc chloride, cupric sulfate, calcium chloride, sodium chloride, calcium carbonate, sodium carbonate can be used alone or in combination. The amount of inorganic acid varies according to the kind of the inorganic salt, typically between 0.001 to 10 gram per liter of medium. Examples of specially required substances include, but are not limited to, vitamins, nucleic acids, yeast extract, peptone, meat extract, malt extract, dried yeast and combinations thereof. Cultivation can be effected at a temperature, which allows the growth of the microbial strains, essentially, between 20° C. and 46° C. In some aspects, a temperature range is 30° C.-39° C. For optimal growth, in some embodiments, the medium can be adjusted to pH 6.0-7.4. It will be appreciated that commercially available media may also be used to culture the microbial strains, such as Nutrient Broth or Nutrient Agar available from Difco, Detroit, Mich. It will be appreciated that cultivation time may differ depending on the type of culture medium used and the concentration of sugar as a major carbon source.
In some aspects, cultivation lasts between 24-96 hours. Microbial cells thus obtained are isolated using methods, which are well known in the art. Examples include, but are not limited to, membrane filtration and centrifugal separation. The pH may be adjusted using sodium hydroxide and the like and the culture may be dried using a freeze dryer, until the water content becomes equal to 4% or less. Microbial co-cultures may be obtained by propagating each strain as described hereinabove. In some aspects, microbial multi-strain cultures may be obtained by propagating two or more of the strains described hereinabove. It will be appreciated that the microbial strains may be cultured together when compatible culture conditions can be employed.
In some embodiments, the microorganisms of the present disclosure are subjected to a serial preservation challenge to improve microbial viability. In some embodiments, the microorganisms are subjected to a serial preservation challenge to improve stability. In some embodiments, the microorganisms of the present disclosure are subjected to at least one preservation challenge. In some embodiments, the microorganisms of the present disclosure are subjected to at least two, three, four, five, or more preservation challenges.
In some embodiments, the serial preservation method comprises: (a) subjecting a population of target microbial cells to a first preservation challenge to provide a first population of challenged microbial cells; (b) harvesting viable challenged microbial cells from the first population of challenged microbial cells to provide a first population of viable challenged microbial cells; (c) subjecting the first population of viable challenged microbial cells to a second preservation challenge to provide a second population of challenged microbial cells; (d) harvesting viable challenged microbial cells from the second population of challenged microbial cells to provide a second population of viable challenged microbial cells; (e) subjecting the second population of viable challenged microbial cells to a third preservation challenge to provide a third population of challenged microbial cells; (f) harvesting viable challenged microbial cells from the third population of challenged microbial cells to provide a third population of viable challenged microbial cells; (g) preserving the third population of viable challenged microbial cells to provide a population of preserved viability-enhanced microbial cells; and (h) preparing a product using the population of preserved viability-enhanced microbial cells.
In some embodiments, each of the preservation challenges are the same type of preservation challenge. For example, in some embodiments, the microorganisms of the present disclosure are subjected to two, three, four, five, or more preservation challenges before final preservation for storage and/or incorporation into a product, wherein each of the preservation challenges are of the same type. Types of preservation challenges include, but are not limited to, freeze drying/lyophilization, vitrification/glass formation, evaporation, foam formation, vaporization, cryopreservation, spray drying, adsorptive drying, extrusion, and fluid bed drying. Methods of microbial preservation are further described in PCT Application No. PCT/US2020/020311, herein incorporated by reference in its entirety.
In some embodiments, a population of target microbial cells is subjected to preservation by fluid bed drying. Fluid bed drying refers to a method in which particles are fluidized in a bed and dried. A fluidized bed is formed when a quantity of solid particulates are placed under conditions that cause a solid material to behave like a fluid. In a fluid bed drying system, inlet air provides significant air flow to support the weight of the particles.
In some embodiments, the preservation challenges are different types of preservation challenges. For example, in some embodiments, the microorganisms of the present disclosure are subjected to a first and a second preservation challenge, wherein the first and the second preservation challenges are different challenges types. For example, in some embodiments, the first preservation challenge is a cryopreservation challenge and the second preservation challenge is a freeze-drying preservation challenge.
In some embodiments, any one of the microorganisms listed in Table 1 or Table 3 may be subjected to serial preservation challenge. In some embodiments, a microorganism comprising a 16S nucleic acid sequence with at least 95% sequence identity to SEQ ID NOs: 1-30, 2045-2103, or 2108 is subjected to serial preservation challenge. In some embodiments, a microorganism comprising an ITS nucleic acid sequence with at least 95% sequence identity to SEQ ID NOs: 31-60 and 2104-2107 is subjected to serial preservation challenge. In some embodiments, the microorganism is a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, the microorganism is a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 32. In some embodiments, the microorganism is a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 2067. In some embodiments, the microorganism is a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 1 or 2108. In some embodiments, the microorganism is Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108.
In some embodiments, serial preservation results in one or more mutations in the genome of a microorganism. In some embodiments, serial preservation results in one or more mutations in the genome of any one of the microorganisms listed in Table 1 or Table 3. In some embodiments, serial preservation results in one or more mutations in a microorganism comprising a 16S nucleic acid sequence with at least 95% sequence identity to SEQ ID NOs: 1-30, 2045-2103, or 2108. In some embodiments, serial preservation results in one or more mutations in a microorganism comprising an ITS nucleic acid sequence with at least 95% sequence identity to SEQ ID NOs: 31-60 and 2104-2107. In some embodiments, the microorganism is a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 28. In some embodiments, the microorganism is a Pichia sp. comprising an ITS nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 32. In some embodiments, the microorganism is a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 2067. In some embodiments, the microorganism is a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least 95% sequence identity to SEQ ID NO: 1 or 2108. In some embodiments, the one or more mutations is a nucleotide substitution, deletion, and/or insertion in the whole genome of the microorganism.
In some embodiments, serial preservation results in one or more mutations in Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108. In some embodiments, the one or more mutations are located in the whole genome of Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108. In some embodiments, the one or mutations are not located in the 16S nucleic acid sequence of SEQ ID NO: 2108 of Ruminococcus bovis. Illustrative mutations in the whole genome of Ruminococcus bovis (SEQ ID NO: 2108) following serial preservation challenge are shown in Table 13 below and further described in Example 3 and Table 18. The mutations in Table 13 are in bold and underlined.
Ruminococcus bovis
Ruminococcus bovis
T
(SEQ ID NO: 2120)
A
(SEQ ID NO: 2122)
In some embodiments, serial preservation results in one or more mutations in Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108. In some embodiments, the one or more mutations is a G→T substitution at position 297 of SEQ ID NO: 2109. In some embodiments, the one or more mutations is a CC→TA substitution at positions 301-302 of SEQ ID NO: 2111. In some embodiments, the one or more mutations is a T→G substitution at position 307 of SEQ ID NO: 2111. In some embodiments, the one or more mutations is a −A deletion at position 300 of SEQ ID NO: 2113. In some embodiments, the one or more mutations is a CCA→TTC substitution at positions 116-118 of SEQ ID NO: 2115. In some embodiments, the one or more mutations is a +T insertion between positions 105-106 of SEQ ID NO: 2117. In some embodiments, the one or more mutations is a C→T substitution at position 298 of SEQ ID NO: 2119. In some embodiments, the one or more mutations is a C→A substitution at position 298 of SEQ ID NO: 2121. In some embodiments, the one or more mutations is a +AC insertion between positions 43-44 of SEQ ID NO; 2123.
In some embodiments, serial preservation results in one or more mutations in the genome of Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108. In some embodiments, the one or more mutations in Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108 results in a change in phenotype. In some embodiments, the one or more mutations in Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108 results in an increase in viability. In some embodiments, the one or more mutations in Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108 results in an increase in viability by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, or more. In some embodiments, the one or more mutations in Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108 results in an increase in stability. In some embodiments, the one or more mutations in Ruminococcus bovis comprising a 16S nucleic acid sequence of SEQ ID NO: 2108 results in an increase in stability by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 50%, or more.
In some embodiments, serial preservation of the microorganisms of the present disclosure results in an increase in microbial viability of at least 5%. In other words, the viability of the population of microbes present at the conclusion of the serial preservation challenges is increased by at least 5% compared to the viability of the population of microbes that were present prior to any preservation challenges. In some embodiments, serial preservation of a microbial culture results in an increase in microbial viability between about 5% and about 300%, about 5% and about 25%, about 5% and about 20%, about 5% and about 15%, about 5% and about 10%, about 10% and about 300%, about 15% and about 30%, about 20% and about 30%, or about 25% and about 30%. In some embodiments, serial preservation of a microbial culture results in an increase in microbial viability between about 10% and about 30%, about 15% and about 30%, about 20% and about 30%, about 25% and about 30%, about 10% and about 25%, about 10% and about 20%, or about 10% and about 15%. In some embodiments, serial preservation of a microbial culture results in an increase in microbial viability of at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more.
In some embodiments, serial preservation of the microorganisms of the present disclosure results in an increase in microbial stability of at least 5%. In other words, the stability of the population of microbes present at the conclusion of the serial preservation challenges is increased by at least 5% compared to the stability of the population of microbes that were present prior to any preservation challenges. In some embodiments, serial preservation of a microbial culture results in an increase in stability between about 5% and about 30%, about 5% and about 25%, about 5% and about 20%, about 5% and about 15%, about 5% and about 10%, about 10% and about 30%, about 15% and about 30%, about 20% and about 30%, or about 25% and about 30%. In some embodiments, serial preservation of a microbial culture results in an increase in stability between about 10% and about 30%, about 15% and about 30%, about 20% and about 30%, about 25% and about 30%, about 10% and about 25%, about 10% and about 20%, or about 10% and about 15%. In some embodiments, serial preservation of a microbial culture results in an increase in stability of at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% or more.
Microbes can be distinguished into a genus based on polyphasic taxonomy, which incorporates all available phenotypic and genotypic data into a consensus classification (Vandamme et al. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol Rev 1996, 60:407-438). One accepted genotypic method for defining species is based on overall genomic relatedness, such that strains which share approximately 70% or more relatedness using DNA-DNA hybridization, with 5° C. or less ΔTm (the difference in the melting temperature between homologous and heterologous hybrids), under standard conditions, are considered to be members of the same species. Thus, populations that share greater than the aforementioned 70% threshold can be considered to be variants of the same species. Another accepted genotypic method for defining species is to isolate marker genes of the present disclosure, sequence these genes, and align these sequenced genes from multiple isolates or variants. The microbes are interpreted as belonging to the same species if one or more of the sequenced genes share at least 97% sequence identity.
The 16S or 18S rRNA sequences or ITS sequences are often used for making distinctions between species and strains, in that if one of the aforementioned sequences share less than a specified percent sequence identity from a reference sequence, then the two organisms from which the sequences were obtained are said to be of different species or strains.
Thus, one could consider microbes to be of the same species, if they share at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.
Further, one could define microbial strains of a species, as those that share at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity across the 16S or 18S rRNA sequence, or the ITS1 or ITS2 sequence.
In one embodiment, microbial strains of the present disclosure include those that comprise polynucleotide sequences that share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, and 2108. In a further embodiment, microbial strains of the present disclosure include those that comprise polynucleotide sequences that share at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with any one of SEQ ID NOs:1-2108.
In one embodiment, microbial strains of the present disclosure include those that comprise polynucleotide sequences that share at least 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.60%, 99.7%, 99.8%, 99.9%, or 100% sequence identity with any one of SEQ ID NOs:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, and 2108.
Comparisons may also be made with 23S rRNA sequences against reference sequences.
Unculturable microbes often cannot be assigned to a definite species in the absence of a phenotype determination, the microbes can be given a candidatus designation within a genus provided their 16S or 18S rRNA sequences or ITS sequences subscribes to the principles of identity with known species.
One approach is to observe the distribution of a large number of strains of closely related species in sequence space and to identify clusters of strains that are well resolved from other clusters. This approach has been developed by using the concatenated sequences of multiple core (house-keeping) genes to assess clustering patterns, and has been called multilocus sequence analysis (MLSA) or multilocus sequence phylogenetic analysis. MLSA has been used successfully to explore clustering patterns among large numbers of strains assigned to very closely related species by current taxonomic methods, to look at the relationships between small numbers of strains within a genus, or within a broader taxonomic grouping, and to address specific taxonomic questions. More generally, the method can be used to ask whether bacterial species exist—that is, to observe whether large populations of similar strains invariably fall into well-resolved clusters, or whether in some cases there is a genetic continuum in which clear separation into clusters is not observed.
In order to more accurately make a determination of genera, a determination of phenotypic traits, such as morphological, biochemical, and physiological characteristics are made for comparison with a reference genus archetype. The colony morphology can include color, shape, pigmentation, production of slime, etc. Features of the cell are described as to shape, size, Gram reaction, extracellular material, presence of endospores, flagella presence and location, motility, and inclusion bodies. Biochemical and physiological features describe growth of the organism at different ranges of temperature, pH, salinity and atmospheric conditions, growth in presence of different sole carbon and nitrogen sources. One of ordinary skill in the art would be reasonably apprised as to the phenotypic traits that define the genera of the present disclosure.
In one embodiment, the microbes taught herein were identified utilizing 16S rRNA gene sequences and ITS sequences. It is known in the art that 16S rRNA contains hypervariable regions that can provide species/strain-specific signature sequences useful for bacterial identification, and that ITS sequences can also provide species/strain-specific signature sequences useful for fungal identification.
Phylogenetic analysis using the rRNA genes and/or ITS sequences are used to define “substantially similar” species belonging to common genera and also to define “substantially similar” strains of a given taxonomic species. Furthermore, physiological and/or biochemical properties of the isolates can be utilized to highlight both minor and significant differences between strains that could lead to advantageous behavior in ruminants.
Compositions of the present disclosure may include combinations of fungal spores and bacterial spores, fungal spores and bacterial vegetative cells, fungal vegetative cells and bacterial spores, fungal vegetative cells and bacterial vegetative cells. In some embodiments, compositions of the present disclosure comprise bacteria only in the form of spores. In some embodiments, compositions of the present disclosure comprise bacteria only in the form of vegetative cells. In some embodiments, compositions of the present disclosure comprise bacteria in the absence of fungi. In some embodiments, compositions of the present disclosure comprise fungi in the absence of bacteria.
Bacterial spores may include endospores and akinetes. Fungal spores may include statismospores, ballistospores, autospores, aplanospores, zoospores, mitospores, megaspores, microspores, meiospores, chlamydospores, urediniospores, teliospores, oospores, carpospores, tetraspores, sporangiospores, zygospores, ascospores, basidiospores, ascospores, and asciospores.
In some embodiments, spores of the composition germinate upon administration to animals of the present disclosure. In some embodiments, spores of the composition germinate only upon administration to animals of the present disclosure.
In some embodiments, the microbes of the disclosure are combined into microbial compositions.
In some embodiments, the microbial compositions include ruminant feed, such as cereals (barley, maize, oats, and the like); starches (tapioca and the like); oilseed cakes; and vegetable wastes. In some embodiments, the microbial compositions include vitamins, minerals, trace elements, emulsifiers, aromatizing products, binders, colorants, odorants, thickening agents, and the like.
In some embodiments, the microbial compositions of the present disclosure are solid. Where solid compositions are used, it may be desired to include one or more carrier materials including, but not limited to: mineral earths such as silicas, talc, kaolin, limestone, chalk, clay, dolomite, diatomaceous earth; calcium sulfate; magnesium sulfate; magnesium oxide; products of vegetable origin such as cereal meals, tree bark meal, wood meal, and nutshell meal.
In some embodiments, the microbial compositions of the present disclosure are liquid. In further embodiments, the liquid comprises a solvent that may include water or an alcohol, and other animal-safe solvents. In some embodiments, the microbial compositions of the present disclosure include binders such as animal-safe polymers, carboxymethylcellulose, starch, polyvinyl alcohol, and the like.
In some embodiments, the microbial compositions of the present disclosure comprise thickening agents such as silica, clay, natural extracts of seeds or seaweed, synthetic derivatives of cellulose, guar gum, locust bean gum, alginates, and methylcelluloses. In some embodiments, the microbial compositions comprise anti-settling agents such as modified starches, polyvinyl alcohol, xanthan gum, and the like.
In some embodiments, the microbial compositions of the present disclosure comprise colorants including organic chromophores classified as nitroso; nitro; azo, including monoazo, bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane, indamine, indophenol, methine, oxazine, phthalocyanine, thiazine, thiazole, triarylmethane, xanthene. In some embodiments, the microbial compositions of the present disclosure comprise trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.
In some embodiments, the microbial compositions of the present disclosure comprise an animal-safe virucide or nematicide.
In some embodiments, microbial compositions of the present disclosure comprise saccharides (e.g., monosaccharides, disaccharides, trisaccharides, polysaccharides, oligosaccharides, and the like), polymeric saccharides, lipids, polymeric lipids, lipopolysaccharides, proteins, polymeric proteins, lipoproteins, nucleic acids, nucleic acid polymers, silica, inorganic salts and combinations thereof. In a further embodiment, microbial compositions comprise polymers of agar, agarose, gelrite, gellan gumand the like. In some embodiments, microbial compositions comprise plastic capsules, emulsions (e.g., water and oil), membranes, and artificial membranes. In some embodiments, emulsions or linked polymer solutions may comprise microbial compositions of the present disclosure. See Harel and Bennett (U.S. Pat. No. 8,460,726B2).
In some embodiments, microbial compositions of the present disclosure occur in a solid form (e.g., dispersed lyophilized spores) or a liquid form (microbes interspersed in a storage medium).
In some embodiments, microbial compositions of the present disclosure comprise one or more preservatives. The preservatives may be in liquid or gas formulations. The preservatives may be selected from one or more of monosaccharide, disaccharide, trisaccharide, polysaccharide, acetic acid, ascorbic acid, calcium ascorbate, erythorbic acid, iso-ascorbic acid, erythrobic acid, potassium nitrate, sodium ascorbate, sodium erythorbate, sodium iso-ascorbate, sodium nitrate, sodium nitrite, nitrogen, benzoic acid, calcium sorbate, ethyl lauroyl arginate, methyl-p-hydroxy benzoate, methyl paraben, potassium acetate, potassium benzoate, potassium bisulphite, potassium diacetate, potassium lactate, potassium metabisulphite, potassium sorbate, propyl-p-hydroxy benzoate, propyl paraben, sodium acetate, sodium benzoate, sodium bisulphite, sodium nitrite, sodium diacetate, sodium lactate, sodium metabisulphite, sodium salt of methyl-p-hydroxy benzoic acid, sodium salt of propyl-p-hydroxy benzoic acid, sodium sulphate, sodium sulfite, sodium dithionite, sulphurous acid, calcium propionate, dimethyl dicarbonate, natamycin, potassium sorbate, potassium bisulfite, potassium metabisulfite, propionic acid, sodium diacetate, sodium propionate, sodium sorbate, sorbic acid, ascorbic acid, ascorbyl palmitate, ascorbyl stearate, butylated hydro-xyanisole, butylated hydroxytoluene (BHT), butylated hydroxyl anisole (BHA), citric acid, citric acid esters of mono- and/or diglycerides, L-cysteine, L-cysteine hydrochloride, gum guaiacum, gum guaiac, lecithin, lecithin citrate, monoglyceride citrate, monoisopropyl citrate, propyl gallate, sodium metabisulphite, tartaric acid, tertiary butyl hydroquinone, stannous chloride, thiodipropionic acid, dilauryl thiodipropionate, distearyl thiodipropionate, ethoxyquin, sulfur dioxide, formic acid, or tocopherol(s).
In some embodiments, microbial compositions of the present disclosure include bacterial and/or fungal cells in spore form, vegetative cell form, and/or lysed cell form. In one embodiment, the lysed cell form acts as a mycotoxin binder, e.g. mycotoxins binding to dead cells.
In some embodiments, the microbial compositions are shelf stable in a refrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable in a refrigerator (35-40° F.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.
In some embodiments, the microbial compositions are shelf stable at room temperature (68-72° F.) or between 50-77° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable at room temperature (68-72° F.) or between 50-77° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.
In some embodiments, the microbial compositions are shelf stable at −23-35° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable at −23-35° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.
In some embodiments, the microbial compositions are shelf stable at 77-100° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable at 77-100° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.
In some embodiments, the microbial compositions are shelf stable at 101-213° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the microbial compositions are shelf stable at 101-213° F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 weeks.
In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40° F.), at room temperature (68-72° F.), between 50-77° F., between −23-35° F., between 70-100° F., or between 101-213° F. for a period of about 1 to 100, about 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1 to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to 55, about 1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, about 1 to 30, about 1 to 25, about 1 to 20, about 1 to 15, about 1 to 10, about 1 to 5, about 5 to 100, about 5 to 95, about 5 to 90, about 5 to 85, about 5 to 80, about 5 to 75, about 5 to 70, about 5 to 65, about 5 to 60, about 5 to 55, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35, about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, about 5 to 10, about 10 to 100, about 10 to 95, about 10 to 90, about 10 to 85, about 10 to 80, about 10 to 75, about 10 to 70, about 10 to 65, about 10 to 60, about 10 to 55, about 10 to 50, about 10 to 45, about 10 to 40, about 10 to 35, about 10 to 30, about 10 to 25, about 10 to 20, about 10 to 15, about 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85, about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 65, about 15 to 60, about 15 to 55, about 15 to 50, about 15 to 45, about 15 to 40, about 15 to 35, about 15 to 30, about 15 to 25, about 15 to 20, about 20 to 100, about 20 to 95, about 20 to 90, about 20 to 85, about 20 to 80, about 20 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20 to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 20 to 35, about 20 to 30, about 20 to 25, about 25 to 100, about 25 to 95, about 25 to 90, about 25 to 85, about 25 to 80, about 25 to 75, about 25 to 70, about 25 to 65, about 25 to 60, about 25 to 55, about 25 to 50, about 25 to 45, about 25 to 40, about 25 to 35, about 25 to 30, about 30 to 100, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 80, about 30 to 75, about 30 to 70, about 30 to 65, about 30 to 60, about 30 to 55, about 30 to 50, about 30 to 45, about 30 to 40, about 30 to 35, about 35 to 100, about 35 to 95, about 35 to 90, about 35 to 85, about 35 to 80, about 35 to 75, about 35 to 70, about 35 to 65, about 35 to 60, about 35 to 55, about 35 to 50, about 35 to 45, about 35 to 40, about 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, about 40 to 80, about 40 to 75, about 40 to 70, about 40 to 65, about 40 to 60, about 40 to 55, about 40 to 50, about 40 to 45, about 45 to 100, about 45 to 95, about 45 to 90, about 45 to 85, about 45 to 80, about 45 to 75, about 45 to 70, about 45 to 65, about 45 to 60, about 45 to 55, about 45 to 50, about 50 to 100, about 50 to 95, about 50 to 90, about 50 to 85, about 50 to 80, about 50 to 75, about 50 to 70, about 50 to 65, about 50 to 60, about 50 to 55, about 55 to 100, about 55 to 95, about 55 to 90, about 55 to 85, about 55 to 80, about 55 to 75, about 55 to 70, about 55 to 65, about 55 to 60, about 60 to 100, about 60 to 95, about 60 to 90, about 60 to 85, about 60 to 80, about 60 to 75, about 60 to 70, about 60 to 65, about 65 to 100, about 65 to 95, about 65 to 90, about 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about 70 to 100, about 70 to 95, about 70 to 90, about 70 to 85, about 70 to 80, about 70 to 75, about 75 to 100, about 75 to 95, about 75 to 90, about 75 to 85, about 75 to 80, about 80 to 100, about 80 to 95, about 80 to 90, about 80 to 85, about 85 to 100, about 85 to 95, about 85 to 90, about 90 to 100, about 90 to 95, or 95 to 100 weeks
In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40° F.), at room temperature (68-72° F.), between 50-77° F., between −23-35° F., between 70-100° F., or between 101-213° F. for a period of 1 to 100, 1 to 95, 1 to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 to 85, 5 to 80, 5 to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, 5 to 10, 10 to 100, 10 to 95, 10 to 90, 10 to 85, 10 to 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 100, 25 to 95, 25 to 90, 25 to 85, 25 to 80, 25 to 75, 25 to 70, 25 to 65, 25 to 60, 25 to 55, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 100, 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 40 to 45, 45 to 100, 45 to 95, 45 to 90, 45 to 85, 45 to 80, 45 to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50, 50 to 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to 75, 50 to 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to 90, 55 to 85, 55 to 80, 55 to 75, 55 to 70, 55 to 65, 55 to 60, 60 to 100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 60 to 70, 60 to 65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, 65 to 80, 65 to 75, 65 to 70, 70 to 100, 70 to 95, 70 to 90, 70 to 85, 70 to 80, 70 to 75, 75 to 100, 75 to 95, 75 to 90, 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to 90, 80 to 85, 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95 to 100 weeks.
In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40° F.), at room temperature (68-72° F.), between 50-77° F., between −23-35° F., between 70-100° F., or between 101-213° F. for a period of about 1 to 36, about 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1 to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to 18, about 1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, about 1 to 8, about 1 to 6, about 1 one 4, about 1 to 2, about 4 to 36, about 4 to 34, about 4 to 32, about 4 to 30, about 4 to 28, about 4 to 26, about 4 to 24, about 4 to 22, about 4 to 20, about 4 to 18, about 4 to 16, about 4 to 14, about 4 to 12, about 4 to 10, about 4 to 8, about 4 to 6, about 6 to 36, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about 6 to 26, about 6 to 24, about 6 to 22, about 6 to 20, about 6 to 18, about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 6 to 8, about 8 to 36, about 8 to 34, about 8 to 32, about 8 to 30, about 8 to 28, about 8 to 26, about 8 to 24, about 8 to 22, about 8 to 20, about 8 to 18, about 8 to 16, about 8 to 14, about 8 to 12, about 8 to 10, about 10 to 36, about 10 to 34, about 10 to 32, about 10 to 30, about 10 to 28, about 10 to 26, about 10 to 24, about 10 to 22, about 10 to 20, about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 12 to 36, about 12 to 34, about 12 to 32, about 12 to 30, about 12 to 28, about 12 to 26, about 12 to 24, about 12 to 22, about 12 to 20, about 12 to 18, about 12 to 16, about 12 to 14, about 14 to 36, about 14 to 34, about 14 to 32, about 14 to 30, about 14 to 28, about 14 to 26, about 14 to 24, about 14 to 22, about 14 to 20, about 14 to 18, about 14 to 16, about 16 to 36, about 16 to 34, about 16 to 32, about 16 to 30, about 16 to 28, about 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20, about 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about 18 to 30, about 18 to 28, about 18 to 26, about 18 to 24, about 18 to 22, about 18 to 20, about 20 to 36, about 20 to 34, about 20 to 32, about 20 to 30, about 20 to 28, about 20 to 26, about 20 to 24, about 20 to 22, about 22 to 36, about 22 to 34, about 22 to 32, about 22 to 30, about 22 to 28, about 22 to 26, about 22 to 24, about 24 to 36, about 24 to 34, about 24 to 32, about 24 to 30, about 24 to 28, about 24 to 26, about 26 to 36, about 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28, about 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 30 to 36, about 30 to 34, about 30 to 32, about 32 to 36, about 32 to 34, or about 34 to 36 months.
In some embodiments, the microbial compositions of the present disclosure are shelf stable at refrigeration temperatures (35-40° F.), at room temperature (68-72° F.), between 50-77° F., between −23-35° F., between 70-100° F., or between 101-213° F. for a period of 1 to 36 1 to 34 1 to 32 1 to 30 1 to 28 1 to 26 1 to 24 1 to 22 1 to 20 1 to 18 1 to 16 1 to 14 1 to 12 1 to 10 1 to 8 1 to 6 1 one 4 1 to 2 4 to 36 4 to 34 4 to 32 4 to 30 4 to 28 4 to 26 4 to 24 4 to 22 4 to 20 4 to 184 to 164 to 144 to 124 to 10 4 to 8 4 to 6 6 to 36 6 to 34 6 to 32 6 to 30 6 to 28 6 to 26 6 to 24 6 to 22 6 to 20 6 to 186 to 166 to 146 to 126 to 10 6 to 8 8 to 36 8 to 34 8 to 32 8 to 30 8 to 28 8 to 26 8 to 24 8 to 22 8 to 20 8 to 18 8 to 16 8 to 14 8 to 12 8 to 10 10 to 36 10 to 34 10 to 32 10 to 30 10 to 28 10 to 26 10 to 24 10 to 22 10 to 20 10 to 18 10 to 16 10 to 14 10 to 12 12 to 36 12 to 34 12 to 32 12 to 30 12 to 28 12 to 26 12 to 24 12 to 22 12 to 20 12 to 18 12 to 16 12 to 14 14 to 36 14 to 34 14 to 32 14 to 30 14 to 28 14 to 26 14 to 24 14 to 22 14 to 20 14 to 18 14 to 16 16 to 36 16 to 34 16 to 32 16 to 30 16 to 28 16 to 26 16 to 24 16 to 22 16 to 20 16 to 18 18 to 36 18 to 34 18 to 32 18 to 30 18 to 28 18 to 26 18 to 24 18 to 22 18 to 20 20 to 36 20 to 34 20 to 32 20 to 30 20 to 28 20 to 26 20 to 24 20 to 22 22 to 36 22 to 34 22 to 32 22 to 30 22 to 28 22 to 26 22 to 24 24 to 36 24 to 34 24 to 32 24 to 30 24 to 28 24 to 26 26 to 36 26 to 34 26 to 32 26 to 30 26 to 28 28 to 36 28 to 34 28 to 32 28 to 30 30 to 36 30 to 34 30 to 32 32 to 36 32 to 34, or about 34 to 36.
In some embodiments, the microbial compositions of the present disclosure are shelf stable at any of the disclosed temperatures and/or temperature ranges and spans of time at a relative humidity of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98%.
In some embodiments, the microbes or microbial compositions of the disclosure are encapsulated in an encapsulating composition. An encapsulating composition protects the microbes from external stressors prior to entering the gastrointestinal tract of ungulates. Encapsulating compositions further create an environment that may be beneficial to the microbes, such as minimizing the oxidative stresses of an aerobic environment on anaerobic microbes. See Kalsta et al. (U.S. Pat. No. 5,104,662A), Ford (U.S. Pat. No. 5,733,568A), and Mosbach and Nilsson (U.S. Pat. No. 4,647,536A) for encapsulation compositions of microbes, and methods of encapsulating microbes.
In one embodiment, the encapsulating composition comprises microcapsules having a multiplicity of liquid cores encapsulated in a solid shell material. For purposes of the disclosure, a “multiplicity” of cores is defined as two or more.
A first category of useful fusible shell materials is that of normally solid fats, including fats which are already of suitable hardness and animal or vegetable fats and oils which are hydrogenated until their melting points are sufficiently high to serve the purposes of the present disclosure. Depending on the desired process and storage temperatures and the specific material selected, a particular fat can be either a normally solid or normally liquid material. The terms “normally solid” and “normally liquid” as used herein refer to the state of a material at desired temperatures for storing the resulting microcapsules. Since fats and hydrogenated oils do not, strictly speaking, have melting points, the term “melting point” is used herein to describe the minimum temperature at which the fusible material becomes sufficiently softened or liquid to be successfully emulsified and spray cooled, thus roughly corresponding to the maximum temperature at which the shell material has sufficient integrity to prevent release of the choline cores. “Melting point” is similarly defined herein for other materials which do not have a sharp melting point.
Specific examples of fats and oils useful herein (some of which require hardening) are as follows: animal oils and fats, such as beef tallow, mutton tallow, lamb tallow, lard or pork fat, fish oil, and sperm oil; vegetable oils, such as canola oil, cottonseed oil, peanut oil, corn oil, olive oil, soybean oil, sunflower oil, safflower oil, coconut oil, palm oil, linseed oil, tung oil, and castor oil; fatty acid monoglycerides and diglycerides; free fatty acids, such as stearic acid, palmitic acid, and oleic acid; and mixtures thereof. The above listing of oils and fats is not meant to be exhaustive, but only exemplary.
Specific examples of fatty acids include linoleic acid, γ-linoleic acid, dihomo-γ-linolenic acid, arachidonic acid, docosatetraenoic acid, vaccenic acid, nervonic acid, mead acid, erucic acid, gondoic acid, elaidic acid, oleic acid, palitoleic acid, stearidonic acid, eicosapentaenoic acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, margaric acid, stearic acid, nonadecyclic acid, arachidic acid, heneicosylic acid, behenic acid, tricosylic acid, lignoceric acid, pentacosylic acid, cerotic acid, heptacosylic acid, montanic acid, nonacosylic acid, melissic acid, henatriacontylic acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic acid, hexatriacontylic acid, heptatriacontanoic acid, and octatriacontanoic acid.
Another category of fusible materials useful as encapsulating shell materials is that of waxes. Representative waxes contemplated for use herein are as follows: animal waxes, such as beeswax, lanolin, shell wax, and Chinese insect wax; vegetable waxes, such as carnauba, candelilla, bayberry, and sugar cane; mineral waxes, such as paraffin, microcrystalline petroleum, ozocerite, ceresin, and montan; synthetic waxes, such as low molecular weight polyolefin (e.g., CARBOWAX), and polyol ether-esters (e.g., sorbitol); Fischer-Tropsch process synthetic waxes; and mixtures thereof. Water-soluble waxes, such as CARBOWAX and sorbitol, are not contemplated herein if the core is aqueous.
Still other fusible compounds useful herein are fusible natural resins, such as rosin, balsam, shellac, and mixtures thereof.
Various adjunct materials are contemplated for incorporation in fusible materials according to the present disclosure. For example, antioxidants, light stabilizers, dyes and lakes, flavors, essential oils, anti-caking agents, fillers, pH stabilizers, sugars (monosaccharides, disaccharides, trisaccharides, and polysaccharides) and the like can be incorporated in the fusible material in amounts which do not diminish its utility for the present disclosure.
The core material contemplated herein constitutes from about 0.10% to about 50%, about 1% to about 35%. or about 5% to about 30% by weight of the microcapsules. In some embodiments, the core material contemplated herein constitutes no more than about 30% by weight of the microcapsules. In some embodiments, the core material contemplated herein constitutes about 5% by weight of the microcapsules. The core material is contemplated as either a liquid or solid at contemplated storage temperatures of the microcapsules.
The cores may include other additives well-known in the pharmaceutical art, including edible sugars, such as sucrose, glucose, maltose, fructose, lactose, cellobiose, monosaccharides, disaccharides, trisaccharides, polysaccharides, and mixtures thereof; artificial sweeteners, such as aspartame, saccharin, cyclamate salts, and mixtures thereof; edible acids, such as acetic acid (vinegar), citric acid, ascorbic acid, tartaric acid, and mixtures thereof; edible starches, such as corn starch; hydrolyzed vegetable protein; water-soluble vitamins, such as Vitamin C; water-soluble medicaments; water-soluble nutritional materials, such as ferrous sulfate; flavors; salts; monosodium glutamate; antimicrobial agents, such as sorbic acid; antimycotic agents, such as potassium sorbate, sorbic acid, sodium benzoate, and benzoic acid; food grade pigments and dyes; and mixtures thereof. Other potentially useful supplemental core materials will be apparent to those of ordinary skill in the art.
Emulsifying agents may be employed to assist in the formation of stable emulsions. Representative emulsifying agents include glyceryl monostearate, polysorbate esters, ethoxylated mono- and diglycerides, and mixtures thereof.
For ease of processing, and particularly to enable the successful formation of a reasonably stable emulsion, the viscosities of the core material and the shell material should be similar at the temperature at which the emulsion is formed. In particular, the ratio of the viscosity of the shell to the viscosity of the core, expressed in centipoise or comparable units, and both measured at the temperature of the emulsion, should be from about 22:1 to about 1:1, desirably from about 8:1 to about 1:1, and preferably from about 3:1 to about 1:1. A ratio of 1:1 would be ideal, but a viscosity ratio within the recited ranges is useful.
Encapsulating compositions are not limited to microcapsule compositions as disclosed above. In some embodiments encapsulating compositions encapsulate the microbial compositions in an adhesive polymer that can be natural or synthetic without toxic effect. In some embodiments, the encapsulating composition may be a matrix selected from sugar matrix, gelatin matrix, polymer matrix, silica matrix, starch matrix, foam matrix, etc. In some embodiments, the encapsulating composition may be selected from polyvinyl acetates; polyvinyl acetate copolymers; ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinyl alcohol copolymers; celluloses, including ethylcelluloses, methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses and carboxymethylcellulose; polyvinylpyrolidones; polysaccharides, including starch, modified starch, dextrins, maltodextrins, alginate and chitosans; monosaccharides; fats; fatty acids, including oils; proteins, including gelatin and zeins; gum arabics; shellacs; vinylidene chloride and vinylidene chloride copolymers; calcium lignosulfonates; acrylic copolymers; polyvinylacrylates; polyethylene oxide; acrylamide polymers and copolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; and polychloroprene.
In some embodiments, the encapsulating shell of the present disclosure can be up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550 μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430 μm, 2440 μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510 μm, 2520 μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590 μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990 μm, or 3000 μm thick.
In some embodiments, compositions of the present disclosure are mixed with animal feed. In some embodiments, animal feed may be present in various forms such as pellets, capsules, granulated, powdered, liquid, or semi-liquid.
In some embodiments, compositions of the present disclosure are mixed into the premix at at the feed mill (e.g., Carghill or Western Millin), alone as a standalone premix, and/or alongside other feed additives such as MONENSIN, vitamins, etc. In one embodiment, the compositions of the present disclosure are mixed into the feed at the feed mill. In another embodiment, compositions of the present disclosure are mixed into the feed itself.
In some embodiments, feed of the present disclosure may be supplemented with water, premix or premixes, forage, fodder, beans (e.g., whole, cracked, or ground), grains (e.g., whole, cracked, or ground), bean- or grain-based oils, bean- or grain-based meals, bean- or grain-based haylage or silage, bean- or grain-based syrups, fatty acids, sugar alcohols (e.g., polyhydric alcohols), commercially available formula feeds, and mixtures thereof.
In some embodiments, forage encompasses hay, haylage, and silage. In some embodiments, hays include grass hays (e.g., sudangrass, orchardgrass, or the like), alfalfa hay, and clover hay. In some embodiments, haylages include grass haylages, sorghum haylage, and alfalfa haylage. In some embodiments, silages include maize, oat, wheat, alfalfa, clover, and the like.
In some embodiments, premix or premixes may be utilized in the feed. Premixes may comprise micro-ingredients such as vitamins, minerals, amino acids; chemical preservatives; pharmaceutical compositions such as antibiotics and other medicaments; fermentation products, and other ingredients. In some embodiments, premixes are blended into the feed.
In some embodiments, the feed may include feed concentrates such as soybean hulls, sugar beet pulp, molasses, high protein soybean meal, ground corn, shelled corn, wheat midds, distiller grain, cottonseed hulls, rumen-bypass protein, rumen-bypass fat, and grease. See Luhman (U.S. Publication US20150216817A1), Anderson el al. (U.S. Pat. No. 3,484,243) and Porter and Luhman (U.S. Pat. No. 9,179,694B2) for animal feed and animal feed supplements capable of use in the present compositions and methods.
In some embodiments, feed occurs as a compound, which includes, in a mixed composition capable of meeting the basic dietary needs, the feed itself, vitamins, minerals, amino acids, and other necessary components. Compound feed may further comprise premixes.
In some embodiments, microbial compositions of the present disclosure may be mixed with animal feed, premix, and/or compound feed. Individual components of the animal feed may be mixed with the microbial compositions prior to feeding to ruminants. The microbial compositions of the present disclosure may be applied into or on a premix, into or on a feed, and/or into or on a compound feed.
In some embodiments, the microbial compositions of the present disclosure are administered to ruminants via the oral route. In some embodiments the microbial compositions are administered via a direct injection route into the gastrointestinal tract. In further embodiments, the direct injection administration delivers the microbial compositions directly to the rumen. In some embodiments, the microbial compositions of the present disclosure are administered to animals anally. In further embodiments, anal administration is in the form of an inserted suppository.
In some embodiments, the microbial composition is administered in a dose comprise a total of, or at least, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL, or 1,000 mL.
In some embodiments, the microbial composition is administered in a dose comprising a total of, or at least, 1018, 1017, 1016, 1015, 1014, 1013, 1012, 1011, 1010, 109, 108, 107, 106, 105, 104, 103, or 102 microbial cells.
In some embodiments, the microbial compositions are mixed with feed, and the administration occurs through the ingestion of the microbial compositions along with the feed. In some embodiments, the dose of the microbial composition is administered such that there exists 102 to 1012, 103 to 1012, 104 to 1012, 105 to 1012, 106 to 1012, 107 to 1012, 108 to 1012, 109 to 1012, 1010 to 1012, 1011 to 1012, 102 to 1011, 103 to 1011, 104 to 1011, 105 to 1011, 106 to 1011, 107 to 1011, 108 to 1011, 109 to 1011, 1010 to 1011, 102 to 1010, 103 to 1010, 104 to 1010, 105 to 1010, 106 to 1010, 107 to 1010, 108 to 1010, 109 to 1010, 102 to 109, 103 to 109, 104 to 109, 105 to 109, 106 to 109, 107 to 109, 108 to 109, 102 to 108, 103 to 108, 104 to 108, 105 to 108, 106 to 108, 107 to 108, 102 to 107, 103 to 107, 104 to 107, 105 to 107, 106 to 107, 102 to 106, 103 to 106, 104 to 106, 105 to 106, 102 to 105, 103 to 105, 104 to 105, 102 to 104, 103 to 104, 102 to 103, 1012, 1011, 1010, 109, 108, 107, 106, 105, 104, 103, or 102 total microbial cells per gram or milliliter of the composition.
In some embodiments, the microbial compositions are mixed with feed, and the administration occurs through the ingestion of the microbial compositions along with the feed. In some embodiments, the dose of the microbial composition is administered such that there exists 102 to 1012, 103 to 1012, 104 to 1012, 105 to 1012, 106 to 1012, 107 to 1012, 108 to 1012, 109 to 1012, 1010 to 1012, 1011 to 1012, 102 to 1011, 103 to 1011, 104 to 1011, 105 to 1011, 106 to 1011, 107 to 1011, 108 to 1011, 109 to 1011, 1010 to 1011, 102 to 1010, 103 to 1010, 104 to 1010, 105 to 1010, 106 to 1010, 107 to 1010, 108 to 1010, 109 to 1010, 102 to 109, 103 to 109, 104 to 109, 105 to 109, 106 to 109, 107 to 109, 108 to 109, 102 to 108, 103 to 108, 104 to 108, 105 to 108, 106 to 108, 107 to 108, 102 to 107, 103 to 107, 104 to 107, 105 to 107, 106 to 107, 102 to 106, 103 to 106, 104 to 106, 105 to 106, 102 to 105, 103 to 105, 104 to 105, 102 to 104, 103 to 104, 102 to 103, 1012, 1011, 1010, 109, 108, 107, 106, 105, 104, 103, or 102 colony forming units per gram or milliliter of the composition.
In some embodiments, the administered dose of the microbial composition comprises 102 to 1018, 103 to 1018, 104 to 1018, 105 to 1018, 106 to 1018, 107 to 1018, 108 to 1018, 109 to 1018, 1010 to 1018, 1011 to 1018, 1012 to 1018, 1013 to 1018, 1014 to 1018, 1015 to 1018, 1016 to 1018, 1017 to 1018, 102 to 1012, 103 to 1012, 104 to 1012, 105 to 1012, 106 to 1012, 107 to 1012, 108 to 1012, 109 to 1012, 1010 to 1012, 1011 to 1012, 102 to 1011, 103 to 1011, 104 to 1011, 105 to 1011, 106 to 1011, 107 to 1011, 108 to 1011, 109 to 1011, 1010 to 1011, 102 to 1010, 103 to 1010, 104 to 1010, 105 to 1010, 106 to 1010, 107 to 1010, 108 to 1010, 109 to 1010, 102 to 109, 103 to 109, 104 to 109, 105 to 109, 106 to 109, 107 to 109, 108 to 109, 102 to 101, 103 to 108, 104 to 108, 105 to 108, 106 to 101, 107 to 108, 102 to 107, 103 to 107, 104 to 107, 105 to 107, 106 to 107, 102 to 106, 103 to 106, 104 to 106, 105 to 106, 102 to 100, 103 to 100, 104 to 105, 102 to 104, 103 to 104, 102 to 103, 1018, 1017, 1016, 1015, 1014, 1013, 1012, 1011, 1010, 109, 108, 107, 106, 105, 104, 103, or 102 total microbial cells.
In some embodiments, the administered dose of each microbe in the microbial composition is at least about, at least about 103 colony forming units (CFU), at least about 104 CFU, at least about 105 CFU, at least about 106 CFU, at least about 107 CFU, at least about 108 CFU, at least about 109 CFU, at least about 1010 CFU, at least about 1011 CFU, at least about 1012 CFU, at least about 1013 CFU, at least about 1014 CFU, at least about 1015 CFU, at least about 1016 CFU, at least about 1017 CFU, at least about 1018 CFU, at least about 1019 CFU, or at least about 1020 CFU.
In some embodiments, the composition is administered 1 or more times per day. In some aspects, the composition is administered with food each time the animal is fed. In some embodiments, the composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day.
In some embodiments, the microbial composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week.
In some embodiments, the microbial composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.
In some embodiments, the microbial composition is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per year.
In some embodiments, the feed can be uniformly coated with one or more layers of the microbes and/or microbial compositions disclosed herein, using conventional methods of mixing, spraying, or a combination thereof through the use of treatment application equipment that is specifically designed and manufactured to accurately, safely, and efficiently apply coatings. Such equipment uses various types of coating technology such as rotary coaters, drum coaters, fluidized bed techniques, spouted beds, rotary mists, or a combination thereof. Liquid treatments such as those of the present disclosure can be applied via either a spinning “atomizer” disk or a spray nozzle, which evenly distributes the microbial composition onto the feed as it moves though the spray pattern. In some aspects, the feed is then mixed or tumbled for an additional period of time to achieve additional treatment distribution and drying.
In some embodiments, the feed coats of the present disclosure can be up to 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm, 210 μm, 220 μm, 230 μm, 240 μm, 250 μm, 260 μm, 270 μm, 280 μm, 290 μm, 300 μm, 310 μm, 320 μm, 330 μm, 340 μm, 350 μm, 360 μm, 370 μm, 380 μm, 390 μm, 400 μm, 410 μm, 420 μm, 430 μm, 440 μm, 450 μm, 460 μm, 470 μm, 480 μm, 490 μm, 500 μm, 510 μm, 520 μm, 530 μm, 540 μm, 550 μm, 560 μm, 570 μm, 580 μm, 590 μm, 600 μm, 610 μm, 620 μm, 630 μm, 640 μm, 650 μm, 660 μm, 670 μm, 680 μm, 690 μm, 700 μm, 710 μm, 720 μm, 730 μm, 740 μm, 750 μm, 760 μm, 770 μm, 780 μm, 790 μm, 800 μm, 810 μm, 820 μm, 830 μm, 840 μm, 850 μm, 860 μm, 870 μm, 880 μm, 890 μm, 900 μm, 910 μm, 920 μm, 930 μm, 940 μm, 950 μm, 960 μm, 970 μm, 980 μm, 990 μm, 1000 μm, 1010 μm, 1020 μm, 1030 μm, 1040 μm, 1050 μm, 1060 μm, 1070 μm, 1080 μm, 1090 μm, 1100 μm, 1110 μm, 1120 μm, 1130 μm, 1140 μm, 1150 μm, 1160 μm, 1170 μm, 1180 μm, 1190 μm, 1200 μm, 1210 μm, 1220 μm, 1230 μm, 1240 μm, 1250 μm, 1260 μm, 1270 μm, 1280 μm, 1290 μm, 1300 μm, 1310 μm, 1320 μm, 1330 μm, 1340 μm, 1350 μm, 1360 μm, 1370 μm, 1380 μm, 1390 μm, 1400 μm, 1410 μm, 1420 μm, 1430 μm, 1440 μm, 1450 μm, 1460 μm, 1470 μm, 1480 μm, 1490 μm, 1500 μm, 1510 μm, 1520 μm, 1530 μm, 1540 μm, 1550 μm, 1560 μm, 1570 μm, 1580 μm, 1590 μm, 1600 μm, 1610 μm, 1620 μm, 1630 μm, 1640 μm, 1650 μm, 1660 μm, 1670 μm, 1680 μm, 1690 μm, 1700 μm, 1710 μm, 1720 μm, 1730 μm, 1740 μm, 1750 μm, 1760 μm, 1770 μm, 1780 μm, 1790 μm, 1800 μm, 1810 μm, 1820 μm, 1830 μm, 1840 μm, 1850 μm, 1860 μm, 1870 μm, 1880 μm, 1890 μm, 1900 μm, 1910 μm, 1920 μm, 1930 μm, 1940 μm, 1950 μm, 1960 μm, 1970 μm, 1980 μm, 1990 μm, 2000 μm, 2010 μm, 2020 μm, 2030 μm, 2040 μm, 2050 μm, 2060 μm, 2070 μm, 2080 μm, 2090 μm, 2100 μm, 2110 μm, 2120 μm, 2130 μm, 2140 μm, 2150 μm, 2160 μm, 2170 μm, 2180 μm, 2190 μm, 2200 μm, 2210 μm, 2220 μm, 2230 μm, 2240 μm, 2250 μm, 2260 μm, 2270 μm, 2280 μm, 2290 μm, 2300 μm, 2310 μm, 2320 μm, 2330 μm, 2340 μm, 2350 μm, 2360 μm, 2370 μm, 2380 μm, 2390 μm, 2400 μm, 2410 μm, 2420 μm, 2430 μm, 2440 μm, 2450 μm, 2460 μm, 2470 μm, 2480 μm, 2490 μm, 2500 μm, 2510 μm, 2520 μm, 2530 μm, 2540 μm, 2550 μm, 2560 μm, 2570 μm, 2580 μm, 2590 μm, 2600 μm, 2610 μm, 2620 μm, 2630 μm, 2640 μm, 2650 μm, 2660 μm, 2670 μm, 2680 μm, 2690 μm, 2700 μm, 2710 μm, 2720 μm, 2730 μm, 2740 μm, 2750 μm, 2760 μm, 2770 μm, 2780 μm, 2790 μm, 2800 μm, 2810 μm, 2820 μm, 2830 μm, 2840 μm, 2850 μm, 2860 μm, 2870 μm, 2880 μm, 2890 μm, 2900 μm, 2910 μm, 2920 μm, 2930 μm, 2940 μm, 2950 μm, 2960 μm, 2970 μm, 2980 μm, 2990 μm, or 3000 μm thick.
In some embodiments, the microbial cells can be coated freely onto any number of compositions or they can be formulated in a liquid or solid composition before being coated onto a composition. For example, a solid composition comprising the microorganisms can be prepared by mixing a solid carrier with a suspension of the spores until the solid carriers are impregnated with the spore or cell suspension. This mixture can then be dried to obtain the desired particles.
In some other embodiments, it is contemplated that the solid or liquid microbial compositions of the present disclosure further contain functional agents e.g., activated carbon, minerals, vitamins, and other agents capable of improving the quality of the products or a combination thereof.
Methods of coating and compositions in use of said methods that are known in the art can be particularly useful when they are modified by the addition of one of the embodiments of the present disclosure. Such coating methods and apparatus for their application are disclosed in, for example: U.S. Pat. Nos. 8,097,245, and 7,998,502; and PCT Pat. App. Publication Nos. WO 2008/076975, WO 2010/138522, WO 2011/094469, WO 2010/111347, and WO 2010/111565 each of which is incorporated by reference herein.
In some embodiments, the microbes or microbial consortia of the present disclosure exhibit a synergistic effect, on one or more of the traits described herein, in the presence of one or more of the microbes or consortia coming into contact with one another. The synergistic effect obtained by the taught methods can be quantified, for example, according to Colby's formula (i.e., (E)=X+Y−(X*Y/100)). See Colby, R. S., “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations,” 1967. Weeds. Vol. 15, pp. 20-22, incorporated herein by reference in its entirety. Thus, “synergistic” is intended to reflect an outcome/parameter/effect that has been increased by more than an additive amount.
In some embodiments, the microbes or microbial consortia of the present disclosure may be administered via bolus. In one embodiment, a bolus (e.g., capsule containing the composition) is inserted into a bolus gun, and the bolus gun is inserted into the buccal cavity and/or esophagus of the animal, followed by the release/injection of the bolus into the animal's digestive tract. In one embodiment, the bolus gun/applicator is a BOVIKALC bolus gun/applicator. In another embodiment, the bolus gun/applicator is a QUADRICAL gun/applicator.
In some embodiments, the microbes or microbial consortia of the present disclosure may be administered via drench. In one embodiment, the drench is an oral drench. A drench administration comprises utilizing a drench kit/applicator/syringe that injects/releases a liquid comprising the microbes or microbial consortia into the buccal cavity and/or esophagus of the animal.
In some embodiments, the microbes or microbial consortia of the present disclosure may be administered in a time-released fashion. The composition may be coated in a chemical composition, or may be contained in a mechanical device or capsule that releases the microbes or microbial consortia over a period of time instead all at once. In one embodiment, the microbes or microbial consortia are administered to an animal in a time-release capsule. In one embodiment, the composition may be coated in a chemical composition, or may be contained in a mechanical device or capsule that releases the microbes or microbial consortia all at once a period of time hours post ingestion.
In some embodiments, the microbes or microbial consortia are administered in a time-released fashion between 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, or 1 to 100 hours.
In some embodiments, the microbes or microbial consortia are administered in a time-released fashion between 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, or 1 to 30 days.
As used herein the term “microorganism” should be taken broadly. It includes, but is not limited to, the two prokaryotic domains, Bacteria and Archaea, as well as eukaryotic fungi, protists, and viruses.
By way of example, the microorganisms may include species of the genera of: Clostridium, Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, Prevotella, Butyricimonas, Piromyces, Pichia, Candida, Vrystaatia, Orpinomyces, Neocallimastix, and Phyllosticta. The microorganisms may further include species belonging to the family of Lachnospiraceae, and the order of Saccharomycetales. In some embodiments, the microorganisms may include species of any genera disclosed herein.
In certain embodiments, the microorganism is unculturable. This should be taken to mean that the microorganism is not known to be culturable or is difficult to culture using methods known to one skilled in the art.
In one embodiment, the microbes are obtained from animals (e.g., mammals, reptiles, birds, and the like), soil (e.g., rhizosphere), air, water (e.g., marine, freshwater, wastewater sludge), sediment, oil, plants (e.g., roots, leaves, stems), agricultural products, and extreme environments (e.g., acid mine drainage or hydrothermal systems). In a further embodiment, microbes obtained from marine or freshwater environments such as an ocean, river, or lake. In a further embodiment, the microbes can be from the surface of the body of water, or any depth of the body of water (e.g., a deep sea sample).
The microorganisms of the disclosure may be isolated in substantially pure or mixed cultures. They may be concentrated, diluted, or provided in the natural concentrations in which they are found in the source material. For example, microorganisms from saline sediments may be isolated for use in this disclosure by suspending the sediment in fresh water and allowing the sediment to fall to the bottom. The water containing the bulk of the microorganisms may be removed by decantation after a suitable period of settling and either administered to the GI tract of an ungulate, or concentrated by filtering or centrifugation, diluted to an appropriate concentration and administered to the GI tract of an ungulate with the bulk of the salt removed. By way of further example, microorganisms from mineralized or toxic sources may be similarly treated to recover the microbes for application to the ungulate to minimize the potential for damage to the animal.
In another embodiment, the microorganisms are used in a crude form, in which they are not isolated from the source material in which they naturally reside. For example, the microorganisms are provided in combination with the source material in which they reside; for example, fecal matter, cud, or other composition found in the gastrointestinal tract. In this embodiment, the source material may include one or more species of microorganisms.
In some embodiments, a mixed population of microorganisms is used in the methods of the disclosure.
In embodiments of the disclosure where the microorganisms are isolated from a source material (for example, the material in which they naturally reside), any one or a combination of a number of standard techniques which will be readily known to skilled persons may be used. However, by way of example, these in general employ processes by which a solid or liquid culture of a single microorganism can be obtained in a substantially pure form, usually by physical separation on the surface of a solid microbial growth medium or by volumetric dilutive isolation into a liquid microbial growth medium. These processes may include isolation from dry material, liquid suspension, slurries or homogenates in which the material is spread in a thin layer over an appropriate solid gel growth medium, or serial dilutions of the material made into a sterile medium and inoculated into liquid or solid culture media.
Whilst not essential, in one embodiment, the material containing the microorganisms may be pre-treated prior to the isolation process in order to either multiply all microorganisms in the material. Microorganisms can then be isolated from the enriched materials as disclosed above.
In certain embodiments, as mentioned herein before, the microorganism(s) may be used in crude form and need not be isolated from an animal or a media. For example, cud, feces, or growth media which includes the microorganisms identified to be of benefit to increased milk production in ungulates may be obtained and used as a crude source of microorganisms for the next round of the method or as a crude source of microorganisms at the conclusion of the method. For example, fresh feces could be obtained and optionally processed.
In some embodiments, the microbiome of a ruminant, including the rumen microbiome, comprises a diverse arrive of microbes with a wide variety of metabolic capabilities. The microbiome is influenced by a range of factors including diet, variations in animal metabolism, and breed, among others. Most bovine diets are plant-based and rich in complex polysaccharides that enrich the gastrointestinal microbial community for microbes capable of breaking down specific polymeric components in the diet. The end products of primary degradation sustains a chain of microbes that ultimately produce a range of organic acids together with hydrogen and carbon dioxide. Because of the complex and interlinked nature of the microbiome, changing the diet and thus substrates for primary degradation may have a cascading effect on rumen microbial metabolism, with changes in both the organic acid profiles and the methane levels produced, thus impacting the quality and quantity of animal production and or the products produced by the animal. See Menezes et al. (2011. FEMS Microbiol. Ecol. 78(2):256-265.)
In some aspects, the present disclosure is drawn to administering microbial compositions described herein to modulate or shift the microbiome of a ruminant.
In some embodiments, the microbiome is shifted through the administration of one or more microbes to the gastrointestinal tract. In further embodiments, the one or more microbes are those selected from Table 1 or Table 3. In some embodiments, the microbiome shift or modulation includes a decrease or loss of specific microbes that were present prior to the administration of one or more microbes of the present disclosure. In some embodiments, the microbiome shift or modulation includes an increase in microbes that were present prior to the administration of one or more microbes of the present disclosure. In some embodiments, the microbiome shift or modulation includes a gain of one or more microbes that were not present prior to the administration of one or more microbes of the present disclosure. In a further embodiment, the gain of one or more microbes is a microbe that was not specifically included in the administered microbial consortium.
In some embodiments, the administration of microbes of the present disclosure results in a sustained modulation of the microbiome such that the administered microbes are present in the microbiome for a period of at least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
In some embodiments, the administration of microbes of the present disclosure results in a sustained modulation of the microbiome such that the administered microbes are present in the microbiome for a period of at least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks.
In some embodiments, the administration of microbes of the present disclosure results in a sustained modulation of the microbiome such that the administered microbes are present in the microbiome for a period of at least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
In some embodiments, the presence of the administered microbes are detected by sampling the gastrointestinal tract and using primers to amplify the 16S or 18S rDNA sequences, or the ITS rDNA sequences of the administered microbes. In some embodiments, the administered microbes are one or more of those selected from Table 1 or Table 3, and the corresponding rDNA sequences are those selected from SEQ ID NOs:1-60, SEQ ID NOs: 2045-2108 and the SEQ ID NOs identified in Table 3.
In some embodiments, the microbiome of a ruminant is measured by amplifying polynucleotides collected from gastrointestinal samples, wherein the polynucleotides may be 16S or 18S rDNA fragments, or ITS rDNA fragments of microbial rDNA. In one embodiment, the microbiome is fingerprinted by a method of denaturing gradient gel electrophoresis (DGGE) wherein the amplified rDNA fragments are sorted by where they denature, and form a unique banding pattern in a gel that may be used for comparing the microbiome of the same ruminant over time or the microbiomes of multiple ruminants. In another embodiment, the microbiome is fingerprinted by a method of terminal restriction fragment length polymorphism (T-RFLP), wherein labelled PCR fragments are digested using a restriction enzyme and then sorted by size. In a further embodiment, the data collected from the T-RFLP method is evaluated by nonmetric multidimensional scaling (nMDS) ordination and PERMANOVA statistics identify differences in microbiomes, thus allowing for the identification and measurement of shifts in the microbiome. See also Shanks et al. (2011. Appl. Environ. Microbiol. 77(9):2992-3001), Petri et al. (2013. PLOS one. 8(12):e83424), and Menezes et al. (2011. FEMS Microbiol. Ecol. 78(2):256-265.)
In some embodiments, the administration of microbes of the present disclosure results in a modulation or shift of the microbiome which further results in a desired phenotype or improved trait.
According to the methods provided herein, a sample is processed to detect the presence of one or more microorganism types in the sample (
In one embodiment, the sample, or a portion thereof is subjected to flow cytometry (FC) analysis to detect the presence and/or number of one or more microorganism types (
In one embodiment, a sample is stained with one or more fluorescent dyes wherein a fluorescent dye is specific to a particular microorganism type, to enable detection via a flow cytometer or some other detection and quantification method that harnesses fluorescence, such as fluorescence microscopy. The method can provide quantification of the number of cells and/or cell volume of a given organism type in a sample. In a further embodiment, as described herein, flow cytometry is harnessed to determine the presence and quantity of a unique first marker and/or unique second marker of the organism type, such as enzyme expression, cell surface protein expression, etc. Two- or three-variable histograms or contour plots of, for example, light scattering versus fluorescence from a cell membrane stain (versus fluorescence from a protein stain or DNA stain) may also be generated, and thus an impression may be gained of the distribution of a variety of properties of interest among the cells in the population as a whole. A number of displays of such multiparameter flow cytometric data are in common use and are amenable for use with the methods described herein.
In one embodiment of processing the sample to detect the presence and number of one or more microorganism types, a microscopy assay is employed (
In another embodiment of in order to detect the presence and number of one or more microorganism types, the sample, or a portion thereof is subjected to fluorescence microscopy. Different fluorescent dyes can be used to directly stain cells in samples and to quantify total cell counts using an epifluorescence microscope as well as flow cytometry, described above. Useful dyes to quantify microorganisms include but are not limited to acridine orange (AO), 4,6-di-amino-2 phenylindole (DAPI) and 5-cyano-2,3 Dytolyl Tetrazolium Chloride (CTC). Viable cells can be estimated by a viability staining method such as the LIVE/DEAD® Bacterial Viability Kit (Bac-Light™) which contains two nucleic acid stains: the green-fluorescent SYTO 9™ dye penetrates all membranes and the red-fluorescent propidium iodide (PI) dye penetrates cells with damaged membranes. Therefore, cells with compromised membranes will stain red, whereas cells with undamaged membranes will stain green. Fluorescent in situ hybridization (FISH) extends epifluorescence microscopy, allowing for the fast detection and enumeration of specific organisms. FISH uses fluorescent labelled oligonucleotides probes (usually 15-25 basepairs) which bind specifically to organism DNA in the sample, allowing the visualization of the cells using an epifluorescence or confocal laser scanning microscope (CLSM). Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) improves upon the FISH method by using oligonucleotide probes labelled with a horse radish peroxidase (HRP) to amplify the intensity of the signal obtained from the microorganisms being studied. FISH can be combined with other techniques to characterize microorganism communities. One combined technique is high affinity peptide nucleic acid (PNA)-FISH, where the probe has an enhanced capability to penetrate through the Extracellular Polymeric Substance (EPS) matrix. Another example is LIVE/DEAD-FISH which combines the cell viability kit with FISH and has been used to assess the efficiency of disinfection in drinking water distribution systems.
In another embodiment, the sample, or a portion thereof is subjected to Raman micro-spectroscopy in order to determine the presence of a microorganism type and the absolute number of at least one microorganism type (
In yet another embodiment, the sample, or a portion thereof is subjected to centrifugation in order to determine the presence of a microorganism type and the number of at least one microorganism type (
In another embodiment, the sample, or a portion thereof is subjected to staining in order to determine the presence of a microorganism type and the number of at least one microorganism type (
In another embodiment, the sample, or a portion thereof is subjected to mass spectrometry (MS) in order to determine the presence of a microorganism type and the number of at least one microorganism type (
In another embodiment, the sample, or a portion thereof is subjected to lipid analysis in order to determine the presence of a microorganism type and the number of at least one microorganism type (
In the aspects of the methods provided herein, the number of unique first makers in the sample, or portion thereof (e.g., sample aliquot) is measured, as well as the abundance of each of the unique first markers (
Any marker that is unique to an organism strain can be employed herein. For example, markers can include, but are not limited to, small subunit ribosomal RNA genes (16S/18S rDNA), large subunit ribosomal RNA genes (23S/25S/28S rDNA), intercalary 5.8S gene, cytochrome c oxidase, beta-tubulin, elongation factor, RNA polymerase and internal transcribed spacer (ITS).
Ribosomal RNA genes (rDNA), especially the small subunit ribosomal RNA genes, i.e., 18S rRNA genes (18S rDNA) in the case of eukaryotes and 16S rRNA (16S rDNA) in the case of prokaryotes, have been the predominant target for the assessment of organism types and strains in a microbial community. However, the large subunit ribosomal RNA genes, 28S rDNAs, have been also targeted. rDNAs are suitable for taxonomic identification because: (i) they are ubiquitous in all known organisms; (ii) they possess both conserved and variable regions; (iii) there is an exponentially expanding database of their sequences available for comparison. In community analysis of samples, the conserved regions serve as annealing sites for the corresponding universal PCR and/or sequencing primers, whereas the variable regions can be used for phylogenetic differentiation. In addition, the high copy number of rDNA in the cells facilitates detection from environmental samples.
The internal transcribed spacer (ITS), located between the 18S rDNA and 28S rDNA, has also been targeted. The ITS is transcribed but spliced away before assembly of the ribosomes The ITS region is composed of two highly variable spacers, ITS1 and ITS2, and the intercalary 5.8S gene. This rDNA operon occurs in multiple copies in genomes. Because the ITS region does not code for ribosome components, it is highly variable.
In one embodiment, the unique RNA marker can be an mRNA marker, an siRNA marker or a ribosomal RNA marker.
Protein-coding functional genes can also be used herein as a unique first marker. Such markers include but are not limited to: the recombinase A gene family (bacterial RecA, archaea RadA and RadB, eukaryotic Rad51 and Rad57, phage UvsX); RNA polymerase p subunit (RpoB) gene, which is responsible for transcription initiation and elongation, chaperonins. Candidate marker genes have also been identified for bacteria plus archaea: ribosomal protein S2 (rpsB), ribosomal protein S10 (rpsJ), ribosomal protein L1 rplA), translation elongation factor EF-2, translation initiation factor IF-2, metalloendopeptidase, ribosomal protein L22, ffh signal recognition particle protein, ribosomal protein L4/Lle (rplD), ribosomal protein L2 (rplB), ribosomal protein S9 (rpsI), ribosomal protein L3 (rplC), phenylalanyl-tRNA synthetase beta subunit, ribosomal protein L14b/L23e (rplN), ribosomal protein S5, ribosomal protein S19 (rpsS), ribosomal protein S7, ribosomal protein L16/L10E (rplP), ribosomal protein S13 (rpsM), phenylalanyl-tRNA synthetase a subunit, ribosomal protein L15, ribosomal protein L25/L23, ribosomal protein L6 (rplF), ribosomal protein L11 (rplK), ribosomal protein L5 (rplE), ribosomal protein S12/S23, ribosomal protein L29, ribosomal protein S3 (rpsC), ribosomal protein S11 (rpsK), ribosomal protein L10, ribosomal protein S8, tRNA pseudouridine synthase B, ribosomal protein L18P/L5E, ribosomal protein S15P/S13e, Porphobilinogen deaminase, ribosomal protein S17, ribosomal protein L13 (rplM), phosphoribosylformylglycinamidine cyclo-ligase (rpsE), ribonuclease HII and ribosomal protein L24. Other candidate marker genes for bacteria include: transcription elongation protein NusA (nusA), rpoB DNA-directed RNA polymerase subunit beta (rpoB), GTP-binding protein EngA, rpoC DNA-directed RNA polymerase subunit beta′, priA primosome assembly protein, transcription-repair coupling factor, CTP synthase (pyrG), secY preprotein translocase subunit SecY, GTP-binding protein Obg/CgtA, DNA polymerase 1, rpsF 30S ribosomal protein S6, poA DNA-directed RNA polymerase subunit alpha, peptide chain release factor 1, rplI 50S ribosomal protein L9, polyribonucleotide nucleotidyltransferase, tsf elongation factor Ts (tsf), rplQ 50S ribosomal protein L17, tRNA (guanine-N(1)-)-methyltransferase (rplS), rplY probable 50S ribosomal protein L25, DNA repair protein RadA, glucose-inhibited division protein A, ribosome-binding factor A, DNA mismatch repair protein MutL, smpB SsrA-binding protein (smpB), N-acetylglucosaminyl transferase, S-adenosyl-methyltransferase MraW, UDP-N-acetylmuramoylalanine-D-glutamate ligase, rplS 50S ribosomal protein L19, rplT 50S ribosomal protein L20 (rplT), ruvA Holliday junction DNA helicase, ruvB Holliday junction DNA helicase B, serS seryl-tRNA synthetase, rplU 50S ribosomal protein L21, rpsR 30S ribosomal protein S18, DNA mismatch repair protein MutS, rpsT 30S ribosomal protein S20, DNA repair protein RecN, frr ribosome recycling factor (frr), recombination protein RecR, protein of unknown function UPF0054, miaA tRNA isopentenyltransferase, GTP-binding protein YchF, chromosomal replication initiator protein DnaA, dephospho-CoA kinase, 16S rRNA processing protein RimM, ATP-cone domain protein, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase, fatty acid/phospholipid synthesis protein PlsX, tRNA(Ile)-lysidine synthetase, dnaG DNA primase (dnaG), ruvC Holliday junction resolvase, rpsP 30S ribosomal protein S16, Recombinase A recA, riboflavin biosynthesis protein RibF, glycyl-tRNA synthetase beta subunit, trmU tRNA (5-methylaminomethyl-2-thiouridylate)-methyltransferase, rpmI 50S ribosomal protein L35, hemE uroporphyrinogen decarboxylase, Rod shape-determining protein, rpmA 50S ribosomal protein L27 (rpmA), peptidyl-tRNA hydrolase, translation initiation factor IF-3 (infC), UDP-N-acetylmuramyl-tripeptide synthetase, rpmF 50S ribosomal protein L32, rplL 50S ribosomal protein L7/L12 (rpIL), leuS leucyl-tRNA synthetase, ligA NAD-dependent DNA ligase, cell division protein FtsA, GTP-binding protein TypA, ATP-dependent Clp protease, ATP-binding subunit CIpX, DNA replication and repair protein RecF and UDP-N-acetylenolpyruvoylglucosamine reductase.
Phospholipid fatty acids (PLFAs) may also be used as unique first markers according to the methods described herein. Because PLFAs are rapidly synthesized during microbial growth, are not found in storage molecules and degrade rapidly during cell death, it provides an accurate census of the current living community. All cells contain fatty acids (FAs) that can be extracted and esterified to form fatty acid methyl esters (FAMEs). When the FAMEs are analyzed using gas chromatography-mass spectrometry, the resulting profile constitutes a ‘fingerprint’ of the microorganisms in the sample. The chemical compositions of membranes for organisms in the domains Bacteria and Eukarya are comprised of fatty acids linked to the glycerol by an ester-type bond (phospholipid fatty acids (PLFAs)). In contrast, the membrane lipids of Archaea are composed of long and branched hydrocarbons that are joined to glycerol by an ether-type bond (phospholipid ether lipids (PLELs)). This is one of the most widely used non-genetic criteria to distinguish the three domains. In this context, the phospholipids derived from microbial cell membranes, characterized by different acyl chains, are excellent signature molecules, because such lipid structural diversity can be linked to specific microbial taxa.
As provided herein, in order to determine whether an organism strain is active, the level of expression of one or more unique second markers, which can be the same or different as the first marker, is measured (
In one embodiment, if the level of expression of the second marker is above a threshold level (e.g., a control level) or at a threshold level, the microorganism is considered to be active (
Second unique markers are measured, in one embodiment, at the protein, RNA or metabolite level. A unique second marker is the same or different as the first unique marker.
As provided above, a number of unique first markers and unique second markers can be detected according to the methods described herein. Moreover, the detection and quantification of a unique first marker is carried out according to methods known to those of ordinary skill in the art (
Nucleic acid sequencing (e.g., gDNA, cDNA, rRNA, mRNA) in one embodiment is used to determine absolute abundance of a unique first marker and/or unique second marker. Sequencing platforms include, but are not limited to, Sanger sequencing and high-throughput sequencing methods available from Roche/454 Life Sciences, Illumina/Solexa, Pacific Biosciences, Ion Torrent and Nanopore. The sequencing can be amplicon sequencing of particular DNA or RNA sequences or whole metagenome/transcriptome shotgun sequencing.
Traditional Sanger sequencing (Sanger et al. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl. Acad. Sci. USA, 74, pp. 5463-5467, incorporated by reference herein in its entirety) relies on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication and is amenable for use with the methods described herein.
In another embodiment, the sample, or a portion thereof is subjected to extraction of nucleic acids, amplification of DNA of interest (such as the rRNA gene) with suitable primers and the construction of clone libraries using sequencing vectors. Selected clones are then sequenced by Sanger sequencing and the nucleotide sequence of the DNA of interest is retrieved, allowing calculation of the number of unique microorganism strains in a sample.
454 pyrosequencing from Roche/454 Life Sciences yields long reads and can be harnessed in the methods described herein (Margulies et al. (2005) Nature, 437, pp. 376-380; U.S. Pat. Nos. 6,274,320; 6,258,568; 6,210,891, each of which is herein incorporated in its entirety for all purposes). Nucleic acid to be sequenced (e.g., amplicons or nebulized genomic/metagenomic DNA) have specific adapters affixed on either end by PCR or by ligation. The DNA with adapters is fixed to tiny beads (ideally, one bead will have one DNA fragment) that are suspended in a water-in-oil emulsion. An emulsion PCR step is then performed to make multiple copies of each DNA fragment, resulting in a set of beads in which each bead contains many cloned copies of the same DNA fragment. Each bead is then placed into a well of a fiber-optic chip that also contains enzymes necessary for the sequencing-by-synthesis reactions. The addition of bases (such as A, C, G, or T) trigger pyrophosphate release, which produces flashes of light that are recorded to infer the sequence of the DNA fragments in each well. About 1 million reads per run with reads up to 1,000 bases in length can be achieved. Paired-end sequencing can be done, which produces pairs of reads, each of which begins at one end of a given DNA fragment. A molecular barcode can be created and placed between the adapter sequence and the sequence of interest in multiplex reactions, allowing each sequence to be assigned to a sample bioinformatically.
Illumina/Solexa sequencing produces average read lengths of about 25 basepairs (bp) to about 300 bp (Bennett et al. (2005) Pharmacogenomics, 6:373-382; Lange et al. (2014). BMC Genomics 15, p. 63; Fadrosh et al. (2014) Microbiome 2, p. 6; Caporaso et al. (2012) ISME J, 6, p. 1621-1624; Bentley et al. (2008) Accurate whole human genome sequencing using reversible terminator chemistry. Nature, 456:53-59). This sequencing technology is also sequencing-by-synthesis but employs reversible dye terminators and a flow cell with a field of oligos attached. DNA fragments to be sequenced have specific adapters on either end and are washed over a flow cell filled with specific oligonucleotides that hybridize to the ends of the fragments. Each fragment is then replicated to make a cluster of identical fragments. Reversible dye-terminator nucleotides are then washed over the flow cell and given time to attach. The excess nucleotides are washed away, the flow cell is imaged, and the reversible terminators can be removed so that the process can repeat and nucleotides can continue to be added in subsequent cycles. Paired-end reads that are 300 bases in length each can be achieved. An Illumina platform can produce 4 billion fragments in a paired-end fashion with 125 bases for each read in a single run. Barcodes can also be used for sample multiplexing, but indexing primers are used.
The SOLiD (Sequencing by Oligonucleotide Ligation and Detection, Life Technologies) process is a “sequencing-by-ligation” approach, and can be used with the methods described herein for detecting the presence and abundance of a first marker and/or a second marker (
The Ion Torrent system, like 454 sequencing, is amenable for use with the methods described herein for detecting the presence and abundance of a first marker and/or a second marker (
Pacific Biosciences (PacBio) SMRT sequencing uses a single-molecule, real-time sequencing approach and in one embodiment, is used with the methods described herein for detecting the presence and abundance of a first marker and/or a second marker (
In one embodiment, where the first unique marker is the ITS genomic region, automated ribosomal intergenic spacer analysis (ARISA) is used in one embodiment to determine the number and identity of microorganism strains in a sample (
In another embodiment, fragment length polymorphism (RFLP) of PCR-amplified rDNA fragments, otherwise known as amplified ribosomal DNA restriction analysis (ARDRA), is used to characterize unique first markers and the abundance of the same in samples (
One fingerprinting technique used in detecting the presence and abundance of a unique first marker is single-stranded-conformation polymorphism (SSCP) (Lee et al. (1996). Appl Environ Microbiol 62, pp. 3112-3120; Scheinert et al. (1996). J. Microbiol. Methods 26, pp. 103-117; Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64, pp. 4870-4876, each of which is incorporated by reference herein in its entirety). In this technique, DNA fragments such as PCR products obtained with primers specific for the 16S rRNA gene, are denatured and directly electrophoresed on a non-denaturing gel. Separation is based on differences in size and in the folded conformation of single-stranded DNA, which influences the electrophoretic mobility. Reannealing of DNA strands during electrophoresis can be prevented by a number of strategies, including the use of one phosphorylated primer in the PCR followed by specific digestion of the phosphorylated strands with lambda exonuclease and the use of one biotinylated primer to perform magnetic separation of one single strand after denaturation. To assess the identity of the predominant populations in a given consortium, in one embodiment, bands are excised and sequenced, or SSCP-patterns can be hybridized with specific probes. Electrophoretic conditions, such as gel matrix, temperature, and addition of glycerol to the gel, can influence the separation.
In addition to sequencing based methods, other methods for quantifying expression (e.g., gene, protein expression) of a second marker are amenable for use with the methods provided herein for determining the level of expression of one or more second markers (
In another embodiment, the sample, or a portion thereof is subjected to a quantitative polymerase chain reaction (PCR) for detecting the presence and abundance of a first marker and/or a second marker (
In another embodiment, the sample, or a portion thereof is subjected to PCR-based fingerprinting techniques to detect the presence and abundance of a first marker and/or a second marker (
In another embodiment, the sample, or a portion thereof is subjected to a chip-based platform such as microarray or microfluidics to determine the abundance of a unique first marker and/or presence/abundance of a unique second marker (
A protein expression assay, in one embodiment, is used with the methods described herein for determining the level of expression of one or more second markers (
In one embodiment, the sample, or a portion thereof is subjected to Bromodeoxyuridine (BrdU) incorporation to determine the level of a second unique marker (
In one embodiment, the sample, or a portion thereof is subjected to microautoradiography (MAR) combined with FISH to determine the level of a second unique marker (
In one embodiment, the sample, or a portion thereof is subjected to stable isotope Raman spectroscopy combined with FISH (Raman-FISH) to determine the level of a second unique marker (
In one embodiment, the sample, or a portion thereof is subjected to DNA/RNA stable isotope probing (SIP) to determine the level of a second unique marker (
In one embodiment, the sample, or a portion thereof is subjected to isotope array to determine the level of a second unique marker (
In one embodiment, the sample, or a portion thereof is subjected to a metabolomics assay to determine the level of a second unique marker (
According to the embodiments described herein, the presence and respective number of one or more active microorganism strains in a sample are determined (
The one or more microorganism strains are considered active, as described above, if the level of second unique marker expression at a threshold level, higher than a threshold value, e.g., higher than at least about 5%, at least about 10%, at least about 20% or at least about 30% over a control level.
In another aspect of the invention, a method for determining the absolute abundance of one or more microorganism strains is determined in a plurality of samples (
The absolute abundance values over samples are used in one embodiment to relate the one or more active microorganism strains, with an environmental parameter (
In one embodiment, determining the co-occurrence of one or more active microorganism strains with an environmental parameter comprises a network and/or cluster analysis method to measure connectivity of strains or a strain with an environmental parameter within a network, wherein the network is a collection of two or more samples that share a common or similar environmental parameter. In another embodiment, the network and/or cluster analysis method may be applied to determining the co-occurrence of two or more active microorganism strains in a sample (
In one embodiment, the cluster analysis method is a heuristic method based on modularity optimization. In a further embodiment, the cluster analysis method is the Louvain method. &e, e.g., the method described by Blondel et al. (2008). Fast unfolding of communities in large networks. Journal of Statistical Mechanics: Theory and Experiment, Volume 2008, October 2008, incorporated by reference herein in its entirety for all purposes.
In another embodiment, the network analysis comprises predictive modeling of network through link mining and prediction, collective classification, link-based clustering, relational similarity, or a combination thereof. In another embodiment, the network analysis comprises differential equation based modeling of populations. In another embodiment, the network analysis comprises Lotka-Volterra modeling.
In one embodiment, relating the one or more active microorganism strains to an environmental parameter (e.g., determining the co-occurrence) in the sample comprises creating matrices populated with linkages denoting environmental parameter and microorganism strain associations.
In one embodiment, the multiple sample data obtained at step 2007 (e.g., over two or more samples which can be collected at two or more time points where each time point corresponds to an individual sample), is compiled. In a further embodiment, the number of cells of each of the one or more microorganism strains in each sample is stored in an association matrix (which can be in some embodiments, an abundance matrix). In one embodiment, the association matrix is used to identify associations between active microorganism strains in a specific time point sample using rule mining approaches weighted with association (e.g., abundance) data. Filters are applied in one embodiment to remove insignificant rules.
In one embodiment, the absolute abundance of one or more, or two or more active microorganism strains is related to one or more environmental parameters (
In some embodiments described herein, an environmental parameter is referred to as a metadata parameter.
Other examples of metadata parameters include but are not limited to genetic information from the host from which the sample was obtained (e.g., DNA mutation information), sample pH, sample temperature, expression of a particular protein or mRNA, nutrient conditions (e.g., level and/or identity of one or more nutrients) of the surrounding environment/ecosystem), susceptibility or resistance to disease, onset or progression of disease, susceptibility or resistance of the sample to toxins, efficacy of xenobiotic compounds (pharmaceutical drugs), biosynthesis of natural products, or a combination thereof.
For example, according to one embodiment, microorganism strain number changes are calculated over multiple samples according to the method of
In a further embodiment, microorganism strains are ranked according to importance by integrating cell number changes over time and strains present in target clusters, with the highest changes in cell number ranking the highest.
Network and/or cluster analysis method in one embodiment, is used to measure connectivity of the one or more strains within a network, wherein the network is a collection of two or more samples that share a common or similar environmental parameter. In one embodiment, network analysis comprises linkage analysis, modularity analysis, robustness measures, betweenness measures, connectivity measures, transitivity measures, centrality measures or a combination thereof. In another embodiment, network analysis comprises predictive modeling of network through link mining and prediction, social network theory, collective classification, link-based clustering, relational similarity, or a combination thereof. In another embodiment, network analysis comprises differential equation based modeling of populations. In yet another embodiment, network analysis comprises Lotka-Volterra modeling.
Cluster analysis method comprises building a connectivity model, subspace model, distribution model, density model, or a centroid model.
Network and cluster based analysis, for example, to carry out method step 2008 of
In some aspects, the present disclosure is drawn to administering one or more microbial compositions described herein to cows to clear the gastrointestinal tract of pathogenic microbes. In some embodiments, the present disclosure is further drawn to administering microbial compositions described herein to prevent colonization of pathogenic microbes in the gastrointestinal tract. In some embodiments, the administration of microbial compositions described herein further clears pathogens from the integument and the respiratory tract of cows, and/or prevent colonization of pathogens on the integument and in the respiratory tract. In some embodiments, the administration of microbial compositions described herein reduce leaky gut/intestinal permeability, inflammation, and/or incidence of liver disease.
In some embodiments, the microbial compositions of the present disclosure comprise one or more microbes that are present in the gastrointestinal tract of cows at a relative abundance of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01%.
In some embodiments, after administration of microbial compositions of the present disclosure the one or more microbes are present in the gastrointestinal tract of the cow at a relative abundance of at least 0.5%, 1%, 5%, 10)%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
Pathogenic microbes of cows may include the following: Clostridium perfringens, Clostridium botulinum, Salmonella typi, Salmonella typhimurium, Salmonella enterica, Salmonella pullorum, Erysipelothrix insidiosa, Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Listeria monocytogenes, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium bovis, Mycoplasma sp., Citrobacter sp., Enterobacter sp., Pseudomonas aeruginosa, Pasteurella sp., Bacillus cereus, Bacillus licheniformis, Streptococcus uberis, Staphylococcus aureus, and pathogenic strains of Escherichia coli and Staphylococcus aureus. In some embodiments, the pathogenic microbes include viral pathogens. In some embodiments, the pathogenic microbes are pathogenic to both cows and humans. In some embodiments, the pathogenic microbes are pathogenic to either cows or humans.
In some embodiments, the administration of compositions of the present disclosure to cows modulate the makeup of the gastrointestinal microbiome such that the administered microbes outcompete microbial pathogens present in the gastrointestinal tract. In some embodiments, the administration of compositions of the present disclosure to cows harboring microbial pathogens outcompetes the pathogens and clears cows of the pathogens. In some embodiments, the administration of compositions of the present disclosure results in the stimulation of host immunity, and aid in clearance of the microbial pathogens. In some embodiments, the administration of compositions of the present disclosure introduce microbes that produce bacteriostatic and/or bactericidal components that decrease or clear the cows of the microbial pathogens. (U.S. Pat. No. 8,345,010).
In some embodiments, challenging cows with a microbial colonizer or microbial pathogen after administering one or more compositions of the present disclosure prevents the microbial colonizer or microbial pathogen from growing to a relative abundance of greater than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.01%. In further embodiments, challenging cows with a microbial colonizer or microbial pathogen after administering one or more compositions of the present disclosure prevents the microbial colonizer or microbial pathogen from colonizing cows.
In some embodiments, clearance of the microbial colonizer or microbial pathogen occurs in less than 25 days, less than 24 days, less than 23 days, less than 22 days, less than 21 days, less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, or less than 2 days post administration of the one or more compositions of the present disclosure.
In some embodiments, clearance of the microbial colonizer or microbial pathogen occurs within 1-30 days, 1-25 days, 1-20 day, 1-15 days, 1-10 days, 1-5 days, 5-30 days, 5-25 days, 5-20 days, 5-15 days, 5-10 days, 10-30 days, 10-25 days, 10-20 days, 10-15 days, 15-30 days, 15-25 days, 15-20 days, 20-30 days, 20-25 days, or 25-30 days post administration of the one or more compositions of the present disclosure.
In some aspects, the present disclosure is drawn to administering microbial compositions described herein to ruminants to improve one or more traits through the modulation of aspects of milk production, milk quantity, milk quality, ruminant digestive chemistry, and efficiency of feed utilization and digestibility.
In some embodiments, improving the quantity of milk fat produced by a ruminant is desirable, wherein milk fat includes triglycerides, triacylglycerides, diacylglycerides, monoacylglycerides, phospholipids, cholesterol, glycolipids, and free fatty acids. In further embodiments, free fatty acids include short chain fatty acids (i.e., C4:0, C6:0, and C8:0), medium chain fatty acids (i.e., C10:0, C10:1, C12:0, C14:0, C14:1, and C15:0), and long chain fatty acids (i.e., C16:0, C16:1, C17:0, C17:1, C18:0, C18:1, C18:2, C18:3, and C20:0). In further embodiments, it is desirable to achieve an increase in milk fat efficiency, which is measured by the total weight of milk fat produced, divided by the weight of feed ingested. The weight of milk fat produced is calculated from the measured fat percentage multiplied by the weight of milk produced.
In some embodiments, improving the quantity of carbohydrates in milk produced by a ruminant is desirable, wherein carbohydrates include lactose, glucose, galactose, and oligosaccharides. Tao et al. (2009. J. Dairy Sci. 92:2991-3001) disclose numerous oligosaccharides that may be found in bovine milk.
In some embodiments, improving the quantity of proteins in milk produced by a ruminant, wherein proteins include caseins and whey. In some embodiments, proteins of interest are only those proteins produced in milk. In other embodiments, proteins of interest are not required to be produced only in milk. Whey proteins include immunoglobulins, serum albumin, beta-lactoglobulin, and alpha-lactoglobulin.
In some embodiments, improving the quantity of vitamins in milk produced by a ruminant is desirable. Vitamins found in milk include the fat-soluble vitamins of A, D, E, and K; as well as the B vitamins found in the aqueous phase of the milk.
In some embodiments, improving the quantity of minerals in milk produced by a ruminant is desirable. Minerals found in milk include iron, zinc, copper, cobalt, magnesium, manganese, molybdenum, calcium, phosphorous, potassium, sodium, chlorine, and citric acid. Trace amounts of the following may be found in milk: aluminum, arsenic, boron, bromine, cadmium, chromium, fluorine, iodine, lead, nickel, selenium, silicon, silver, strontium, and vanadium.
In some embodiments, improving the milk yield and milk volume produced by a ruminant is desirable. In some embodiments, it is further desirable if the increase in milk yield and volume is not accompanied by simply an increase in solute volume.
In some embodiments improving energy-corrected milk (ECM) is desirable. In further embodiments, improving ECM amounts to increasing the calculated ECM output. In some embodiments, the ECM is calculated as follows: ECM=(0.327×milk pounds)+(12.95×fat pounds)+(7.2×protein pounds).
In some embodiments, improving the efficiency and digestibility of animal feed is desirable. In some embodiments, increasing the degradation of lignocellulosic components from animal feed is desirable. Lignocellulosic components include lignin, cellulose, and hemicellulose.
In some embodiments, increasing the concentration of fatty acids in the rumen of ruminants is desirable. Fatty acids include acetic acid, propionic acid, and butyric acid. In some embodiments, maintaining the pH balance in the rumen to prevent lysis of beneficial microbial consortia is desirable. In some embodiments, maintaining the pH balance in the rumen to prevent a reduction of beneficial microbial consortia is desirable.
In some embodiments, decreasing the amount of methane and manure produced by ruminants is desirable.
In some embodiments, improving the dry matter intake is desirable. In some embodiments, improving the efficiency of nitrogen utilization of the feed and dry matter ingested by ruminants is desirable.
In some embodiments, the improved traits of the present disclosure are the result of the administration of the presently described microbial compositions. It is thought that the microbial compositions modulate the microbiome of the ruminants such that the biochemistry of the rumen is changed in such a way that the ruminal liquid and solid substratum are more efficiently and more completely degraded into subcomponents and metabolites than the rumens of ruminants not having been administered microbial compositions of the present disclosure.
In some embodiments, the increase in efficiency and the increase of degradation of the ruminal substratum result in an increase in improved traits of the present disclosure.
The rumen is a specialized stomach dedicated to the digestion of feed components in ruminants. A diverse microbial population inhabits the rumen, where their primary function revolves around converting the fibrous and non-fibrous carbohydrate components into useable sources of energy and protein (
Individual fatty acids have been tested in ruminants in order to identify their impacts on varying aspects of production.
Acetate: Structural carbohydrates produce large amounts of acetate when degraded. An infusion of acetate directly into the rumen was shown to improve the yield of milk, as well as the amount of milk fat produced. Acetate represents at least 90% of acids in the peripheral blood—it is possible that acetate can be directly utilized by mammary tissue as a source of energy. See Rook and Balch. 1961. Brit. J. Nutr. 15:361-369.
Propionate: Propionate has been shown to increase milk protein production, but decrease milk yield. See Rook and Balch. 1961. Brit. J. Nutr. 15:361-369.
Butyrate: An infusion of butyrate directly into the rumen of dairy cows increases milk fat production without changing milk yield. See Huhtanen et al. 1993. J. Dairy Sci. 76:1114-1124.
A network and/or cluster analysis method, in one embodiment, is used to measure connectivity of the one or more strains within a network, wherein the network is a collection of two or more samples that share a common or similar environmental parameter. In one embodiment, network analysis comprises linkage analysis, modularity analysis, robustness measures, betweenness measures, connectivity measures, transitivity measures, centrality measures or a combination thereof. In another embodiment, network analysis comprises predictive modeling of network through link mining and prediction, social network theory, collective classification, link-based clustering, relational similarity, or a combination thereof. In another embodiment, network analysis comprises mutual information, maximal information coefficient (MIC) calculations, or other nonparametric methods between variables to establish connectivity. In another embodiment, network analysis comprises differential equation based modeling of populations. In yet another embodiment, network analysis comprises Lotka-Volterra modeling.
The environmental parameter can be a parameter of the sample itself, e.g., pH, temperature, amount of protein in the sample. Alternatively, the environmental parameter is a parameter that affects a change in the identity of a microbial community (i.e., where the “identity” of a microbial community is characterized by the type of microorganism strains and/or number of particular microorganism strains in a community), or is affected by a change in the identity of a microbial community. For example, an environmental parameter in one embodiment, is the food intake of an animal or the amount of milk (or the protein or fat content of the milk) produced by a lactating ruminant In one embodiment, the environmental parameter is the presence, activity and/or abundance of a second microorganism strain in the microbial community, present in the same sample. In some embodiments, an environmental parameter is referred to as a metadata parameter.
Other examples of metadata parameters include but are not limited to genetic information from the host from which the sample was obtained (e.g., DNA mutation information), sample pH, sample temperature, expression of a particular protein or mRNA, nutrient conditions (e.g., level and/or identity of one or more nutrients) of the surrounding environment/ecosystem), susceptibility or resistance to disease, onset or progression of disease, susceptibility or resistance of the sample to toxins, efficacy of xenobiotic compounds (pharmaceutical drugs), biosynthesis of natural products, or a combination thereof.
Bovine somatotropin (bST), also known as bovine growth hormone, is an animal drug approved by FDA to increase milk production in dairy cows. This drug is based on the somatotropin naturally produced in cattle. Somatotropin is a protein hormone produced in the pituitary gland of animals, including humans, and is essential for normal growth, development, and health maintenance.
FDA approved a bST product in 1993 with the brand name “Posilac™” (sometribove zinc suspension). Posilac™ is approved for over-the-counter use in dairy cows starting at around 2 months after the cow has a calf until the end of the lactation period. During this time, cows are injected with Posilac™ subcutaneously (under the skin) every 14 days. A cow's typical lactation period is approximately 10 months long, starting right after she has a calf Thus, treated dairy cows are typically given Posilac™ for about 8 months of the year.
Dairy cows treated with Posilac™ exhibit a milk yield of approximately 9.9 pounds and energy corrected milk of approximately 8.72 pounds post-peak (greater than 90 days in milk) compared to control. As shown below in Example 4, dairy cows supplemented with Treatment 2 resulted in improved milk yield and milk compositional characteristics compared to control animals in weeks 6 to 24 of the trial. The trial is still ongoing and it is expected that cows supplemented with Treatment 2 will reach milk yields similar to Posilac™ by the end of the trial. Therefore, microbial supplementation in dairy cows is the same or better than the industry standard Posilac™.
In some embodiments, the present disclosure provides microbial compositions that perform the same or better than bovine growth hormone (e.g., Posilac™) in dairy cows. In some embodiments, the composition comprises one or more microorganisms from Table 1 and/or Table 3. In some embodiments, the composition comprises: a Clostridium sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO; 28; a Pichia sp. comprising an ITS nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 32; a Ruminococcus sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2108; and/or a Butyrivibrio sp. comprising a 16S nucleic acid sequence sharing at least about 97% sequence identity to SEQ ID NO: 2067. In some embodiments, the composition comprises: a Clostridium sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 28; a Pichia sp. comprising an ITS nucleic acid sequence of SEQ ID NO: 32; a Ruminococcus sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2108; and/or a Butyrivibrio sp. comprising a 16S nucleic acid sequence of SEQ ID NO: 2067.
Applicant has identified a novel Ruminococcus sp. referred to herein as Ruminococcus bovis. This microorganism was recovered from the rumen content of a healthy, Holstein dairy cow and was taxonomically predicted to be Ruminococcus bromii based on sequencing of the 16S rRNA gene. However, upon further characterization, Applicant discovered that this was a novel species and named the species Ruminococcus bovis.
Applicant originally experienced difficulty obtaining a pure culture of the novel Ruminococcus sp. and therefore, it was deposited at the Bigelow depository as an enriched culture. The Bigelow deposit accession numbers and 16S rRNA sequence of this novel Ruminococcus sp. was first described in PCT Application No. PCT/US2017/012573 (incorporated by reference herein) and was identified in the application as SEQ ID NO: 1.
Applicant was eventually able to isolate Ruminococcus bovis into pure culture and characterized the isolate as described in Example 2 below. Isolated Ruminococcus bovis comprises a 16S rRNA sequence that differs by two nucleotides to the original Ruminococcus bovis of SEQ ID NO: 1. The isolate was deposited at the USDA, ATCC, and NCTC depositories. Isolated Ruminococcus bovis is described for the first time in the present application and is identified as SEQ ID NO: 2108.
Isolated Ruminococcus bovis (SEQ ID NO: 2108) underwent a series of preservation challenges and recoveries in order to improve yield, which is further described in Example 3 below. The Ruminococcus bovis strain recovered from this serial preservation challenge had a number of mutations in whole genome compared to the Ruminococcus bovis strain before the serial preservation challenge (see, Table 18 in Example 3). This novel Ruminococcus bovis strain exhibited a dramatic increase in survival compared to the Ruminococcus bovis strain before the serial preservation challenge (see, Table 17 in Example 3).
Therefore, the present application describes for the first time novel Ruminococcus bovis strains with unique characteristics.
This study presents JE7A12T, an isolate from the ruminal content of a dairy cow. The genus Ruminococcus was first described by A. Kaars Sijpesteijn with Ruminococcus flavefaciens as the type strain (Sijpesteijn A K, Kaars Sijpesteijn A. Vol. 15, Antonie van Leeuwenhoek. 1949. p. 49-52; Sijpesteijn A K. J Gen Microbiol. 1951 November; 5(5 Suppl.):869-79). Previously, Ruminococcus have been isolated from the rumen and gastrointestinal tract of a wide variety of animals including humans (Ezaki T. Ruminococcus [Internet]. Bergey's Manual of Systematics of Archaea and Bacteria. 2015. p. 1-5). The genus is polyphyletic and divided into two groups. Ruminococcus group 1 includes the type strain Ruminococcus flavefaciens, Ruminococcus albus, Ruminococcus bromii, and Ruminococcus callidus. Ruminococcus group 2 species have recently undergone taxonomic re-classification with many species being reassigned to different genera. It is now believed that true members of the genus Ruminococcus are the species found in group 1 (Liu C et al. Vol. 58, International Journal of Systematic and Evolutionary Microbiology. 2008. p. 1896-902). The following description pertains to the isolation and the classification of a novel group 1 amylolytic species, strain JE7A12T, of the genus Ruminococcus.
JE7A12T was recovered from the rumen content of a healthy, Holstein dairy cow obtained from Dairy Experts (Tulare, Calif., USA). After 48 hours of anaerobic incubation at 37-39° C., JE7A12T displays off-white-colored colonies on supplemented Bacto Tryptic Soy Broth (TSB-FAC) (BD, San Jose, Calif., USA). Gram-staining was performed as described by Jones et al. (Jones D. Manual of Methods for General Bacteriology [Internet]. Vol. 34, Journal of Clinical Pathology. 1981. p. 1069-1069). Cell morphology was observed under Accu-Scope EXC-350 light microscope at 1000× magnification using cells grown for 48 h at 37° C. on TSB+FAC. Consistent with previous descriptions of the genus, JE7A12T is a strictly anaerobic coccoid, commonly found in pairs and chains (Ezaki T. Ruminococcus [Internet]. Bergey's Manual of Systematics of Archaea and Bacteria. 2015. p. 1-5). Although isolated from rumen content, JE7A12T does not require rumen fluid for growth.
Carbohydrate fermentation of JE7A12T was qualitatively measured using the API 50CH carbon panel (BioMérieux, Marcy-l'Étoile, France). JE7A12T cells were grown to late exponential phase and recovered by centrifugation at 3,000×g for 10 minutes. Cells were resuspended and 0.017% (wt/vol) bromocresol purple added as a pH indicator for acidification of carbohydrates (Avgustin G et al. Int J Syst Bacteriol. 1997 April; 47(2):284-8.). Closely related Ruminococcus strains derived from the bovine rumen are unable to ferment glucose, fructose, galactose while their human derived counterparts are able to utilize these carbon sources (Mukhopadhya I et al. Environ Microbiol. 2018 January; 20(1):324-36). Therefore, fermentation of glucose, fructose, and galactose in combination with genomic data could act to differentiate JE7A12T from closely related, rumenally derived, Ruminococcus.
Metabolite production was measured using a Waters Acquity UPLC Q System with RI detector. The column used was a Phenomenex 00H-0138-KO Rezex ROA Organic Acid H+(8%) operated at 60° C. The mobile phase was 0.00325 N H2S04 at 0.5 mL/min. Pure standards were used for calibration at varying concentrations. JE7A12T produces acetate as a major fermentation product as well as ethanol and glycerol as minor products. Major fermentation product comparison between JE7A12T and other species in the genus Ruminococcus are shown in Table 14 below.
R. albus
R. bromii
R. callidus
R. champanellensis
R. flavefaciens
R. gauvreauii
R. gnavus
R. lactaris
R. torques
The data shown in Table A for R. albus, R. bromii, R. flavefaciens, R. callidus, R. gnavus, R. lactaris, R. torques as represented in paper from Ezaki (Ezaki T. Ruminococcus [Internet]. Bergey's Manual of Systematics of Archaea and Bacteria. 2015. p. 1-5). R. champanellensis data as represented by Chassard et al. (Chassard C et al. Ruminococcus champanellensis sp. nov., a cellulose-degrading bacterium from human gut microbiota [Internet], Vol. 62, International Journal of Systematic and Evolutionary Microbiology. 2012. p. 138-43). R. gauvreauii data as represented by Domingo et al. (Domingo M-C et al. Ruminococcus gauvreauii sp. nov., a glycopeptide-resistant species isolated from a human faecal specimen [Internet]. Vol. 58, International Journal of Systematic and Evolutionary Microbiology. 2008. p. 1393-7).
The 16S rRNA gene was amplified from JE7A12T using 27F and 534R primers (Lane et al., 1991; Muyzer et al., 1992) and paired-end sequenced (2×300 bp) on an Illumina Miseq. The resulting sequence was quality trimmed and compared to the NCBI database. The closest neighbors to JE7A12T based on sequence similarity were Ruminococcus bromii (90°), Butyricicoccus pullicaecorum (89%), and Colidextribacter massiliensis (89%). Whole genome sequence of JE7A12T was generated using hybrid methods as described by Jain et al. Nat Biotechnol. 2018 April; 36(4):338-45.). Whole genome size and GC content were compared between JE7A12T and phylogenetically close neighbors as well as other members of the genus Ruminococcus. The results are shown in Table 15 below. GC content of JE7A12T should act as a differentiating characteristic for the species as it is lower than the any other member in the genus.
Ruminococcus bovis JE7A12T
Anaeromassilibacillus sp. An172 (GCA_002160515)
Clostridium sp. (GCA_000431855)
Clostridium sp. (GCA_000435335)
Eubacterium sp. (GCA_000436775 )
Eubacterium sp. (GCA_000437975 )
Ruminococcus albus DSM 20455 (GCA_000179635)
Ruminococcus bromii YE282 (GCA_900101355)
Ruminococcus callidus ATCC 27760
Ruminococcus champanellensis DSM 18848
Ruminococcus flavefaciens ATCC 19208
Ruminococcus gauvreauii DSM 19829
Ruminococcus gnavus AGR2154 (GCA_000526735)
Ruminococcus lactaris ATCC 29176
Ruminococcus sp. (GCA_000433495)
Ruminococcus torques ATCC 27756
To further investigate taxonomic identity whole genome average nucleotide identity (AM) was compared between JE7A12T and closely related whole genomes (Richter M K Rosselló-Móra R Proc Natl Acad Sci USA. 2009 Nov. 10; 106(45):19126-31.). Genomes used for comparison were selected based on phylogenetic proximity. Additionally, all current species of Ruminococcus were included in the ANI analysis. The results are shown in
The best match from a cultured genome with standing nomenclature was Ruminococcus bromii. Though the two strains are still genetically distant, with 88% sequence similarity between the two, but with only 2.1% coverage of the genome. The closest overall match is an uncultured Eubacterium with no standing nomenclature. Whole genome nucleotide dissimilarity should be used as a strong differentiator of JE7A12T from the other taxa in the genus.
16S based phylogeny was computed by the neighbor-joining method using MEGA X (Kumar S et al. Mol Biol Evol. 2018 Jun. 1; 35(6):1547-9.). JE7A12T was placed in a dendrogram of all Ruminococcus isolates available in the RDP database (Cole J R et al. Nucleic Acids Res. 2014 January; 4:D633-42.). The resulting dendrogram is presented in
Description of Ruminococcus bovis sp. nov.
Ruminococcus bovis (bo.vis. bos, bovis L. m. gen. n. of the cow)
Ruminococcus bovis is an obligate anaerobe, catalase negative, and oxidase negative bacterium. It gram stains gram-variable (
The type strain (PTA-125917, NRRL B-67764) originally collected as JE7A12, was isolated from rumen content of a healthy, Holstein cow from Dairy Experts (Tulare, Calif., USA).
This study presents JE7A12T, an isolate from the ruminal content of a dairy cow. Phenotypic and genotypic traits of the isolate were explored. JE7A12T was found to be a strictly anaerobic, catalase negative, oxidase negative, coccoid bacterium that grows in chains. API 50 CH carbon source assay showed growth on D-glucose, D-fructose, D-galactose, glycogen, and starch. HPLC showed acetate as the major fermentation product as result of carbohydrate fermentation. Phylogenetic analysis of JE7A12T based on 16S rRNA nucleotide sequence and whole genome amino acid sequence show a divergent lineage from the closest neighbors in the genus Ruminococcus. 16S sequence comparison, whole genome average nucleotide identity (ANI), and GC content data suggest that JE7A12T represents a novel species for which we propose the name Ruminococcus bovis sp nov. with JE7A12T as the type strain.
R. bovis (Ascusb_5) was subjected to a series of preservation challenges and recoveries in order to improve yield through a serial preservation process.
R. bovis was subjected to three rounds of Preservation by Vaporization (PBV) challenges. Briefly, an aliquot from a glycerol stock was streaked onto a growth plate. After an appropriate incubation time, a single colony was selected and used to inoculate a seed tube of Tryptic Soy Broth. The seed tube inoculate was cultured to allow bacterial expansion and the expanded bacterial culture was then used to inoculate the main fermentation culture. The bacterial cells were cultured in the main fermentation culture until mid-stationary phase. Loading sugars are included, if necessary. After 40 hours, cells were harvested and combined with preservation solutions to produce a preservation mixture.
For preservation, 100 μL of each preservation mixture was dispensed into a 2 mL serum vial, which was then sealed with a lyophilization cap and placed the vials in an aluminum lyophilizer block. The vials were frozen at −80° C. for at least one hour and then the vials were transferred to the lyophilizer in the aluminum block. Lypholization caps were changed to the open position and the following lyophilization program was executed:
(a) Freeze at −17° C. at atmospheric pressure for 30 minutes
(b) Freeze at −17° C. at 1000 mTorr for 15 minutes
(c) Freeze at −17° C. at 300 mTorr for 15 minutes
(d) Incubate at 30° C. at 300 mTorr for 24 hours
(e) Incubate at 40° C. at 300 mTorr for 24 hours
(f) Hold at 25° C.
All vials are then removed from the lyophilizer and rehydrated in the following manner:
(a) 1 mL of sterile PBS is added to each vial (effectively a 10× dilution to the initial preservation mixture) and reconstituted by slowly pipetting up and down. This mixture was then diluted 6 additional logs (for a total dilution of E-07) and a 5 μL aliquot from each vial was spot plated for CFU determination.
(b) A separate aliquot of the reconstituted PBV product was streaked onto a plate as the starting plate (a “rescue” plate) for re-inoculation in subsequent.
A second and third round of PBV is then performed according to the protocol described above, using the “rescue” plates as the initial source of bacteria for inoculation of the seed tube.
The results from Round 1-3 for Ascusb_5 are presented in Table 17 below. As shown, there was a dramatic increase in the Survival % of Colony Forming Units (CFU)/mL for Ascusb_5 of SEQ ID NO: 2108 from Round 1 (RCB) to Round 2 (Rescue 1).
The genomes of the RCB isolate and the Round 3 isolate of Ascusb_5 were sequenced to determine any genomic changes as a result of the serial passage. Briefly, DNA was isolated from R. bovis using a Qiagen Powersoil Pro kit. Short read sequencing libraries were prepared from the isolated DNA using the Nextera XT kit (Illumina, San Diego, Calif.) by the manufacturer's recommended protocol. Libraries were sequenced on an Illumina MiSeq (1×300 bp). Reads were mapped to the reference genome using bowtie2 (Langmead B, Salzberg S. (2012) Fast gapped-read alignment with Bowtie 2. Nature Methods. 9: 357-359) and analyzed for mutations using breseq (Deatherage D E, Barrick J E. (2014) Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol. Biol. 1151: 165-188).
A summary of the mutations is presented in Table 18 below. Mutations 7 and 8 are silent mutations and unlikely to result in significant effects. Mutations 2, 3, 5, and 6 affect either integrases or transposases and are unlikely to affect preservation tolerance. Mutation 1 is likely the key mutation resulting in the improvement of preservation tolerance in Ascusb_5. It occurs 4 bp upstream of the Galactose operon repressor, GalR-LacI. This key protein represses transcription of a host of genes related to carbohydrate uptake and metabolism. As cryoprotectant uptake, often in the form of non-reducing sugars, is a key step in preservation tolerance, a change in the regulation of sugar uptake could result in a dramatic improvement in preservation tolerance. The phosphomannomutase could provide another key mutation, perhaps disrupting the metabolism of preservation sugars and enabling intracellular accumulation. The Ascusb_5 microbe from round 3 did not exhibit any mutations in its 16S nucleic acid sequence (SEQ ID NO: 2108).
R. bovis mutation summary
This study examines the effect of microbial supplementation on feed efficiency, milk yield, and milk compositional characteristics in dairy cows.
A total of 90 multi-parous cows were brought to the Research Barn at least 10 days prior the experimental phase of the study to adapt to facilities and feeding system. Thereafter, cows were blocked based on milk yield and assigned to 1 of 3 groups and followed for 150 days.
The treatment groups are shown in Table 19 below. The control group received a total mixed ration without microbial supplementation.
Clostridium sp. (SEQ ID NO: 28)
Pichia kudriavzevii (SEQ ID NO: 32)
Clostridium sp. (SEQ ID NO: 28)
Pichia kudriavzevii (SEQ ID NO: 32)
Ruminococcus bovis (SEQ ID NO:
Butyrivibrio fibrisolvens (SEQ ID
Cows in all treatment groups shared the same housing space, which is a roof covered pen bedded with sand. Adjacent to this area was a cow traffic alley with feed mangers and waterers providing ad libitum feed and water. Cows were milked twice a day in a double 10 parallel parlor.
All cows were fed the same ration other than the microorganisms included in the feed. A total mixed ration (TMR) was delivered twice a day into 48 feed mangers of which 16 were assigned to each group (Treatment 1, Treatment 2, and control) following a sequential order.
A TMR load was prepared once a day using a feed mixing wagon. The amount of TMR prepared every day for cows in the study was 105% of the previous day average intake. First, feed to be fed to cows assigned to Treatment 1 (105% of the previous day average intake for the study group) was unloaded into a long strip over the floor and 150 g of product was spread along the top of the strip. Second, feed from the same load to be fed to cows assigned to Treatment 2 was unloaded into another feed strip over the floor and 150 g of product was spread along the top of the strip. Third, the last portion of the feed from the same load to be fed to cows assigned to Control was delivered into feed mangers assigned to that study group. An empty wagon returned to the feed preparation area and the strip of feed assigned to Treatment 1 cows was loaded into the wagon, mixed for a minimum of 10 minutes, and delivered into mangers assigned to Treatment 1. Finally, an empty wagon returned to the feed preparation area and the strip of feed assigned to Treatment 2 cows was loaded into the wagon, mixed for a minimum of 10 minutes, and delivered into mangers assigned to Treatment 2.
Feed intake was recorded individually for each cow and continuously with automatic feed mangers placed on weighing cells using the BioControl Controlling and Recording Feed Intake (CRFT) system (Biocontrol, CRFI, Rakkestad, Norway). The CRFI system limits the access of cows to the different mangers depending on the treatment to which were assigned. It allows all cows assigned to the same treatment group to access all feed mangers with that treatment. Records were available from three days before until the end of the experimental phase of the study.
Milk yield: Individual cow milk yield was recorded at each milking using an electronic milk meter (AfiMilk MPC, Afikim, Israel). Records were available from three days before until the end of the experimental phase of the study.
Milk composition: Individual cow milk fat, protein and lactose was measured at each milking by an optical in-line milk component analyzer (AfiLab, Afikim, Israel). Records were available from three days before until the end of the experimental phase of the study. In addition, a composite milk sample was collected from milk meters to be analyzed for milk fat, protein and lactose by the local DHIA association (Tulare DHIA, Tulare, Calif.). Records were available for milk collected at the morning milking the three days before commencement of the experimental phase of the study, and twice a week thereafter.
Body Condition Score and Weight: Cows were weighed individually after the morning milking using a PS-2000 scale (Salter Brecknell, Fairmont, Minn.) on the last day of adaptation phase, and then on experimental days 30, 60, 90, 120 and 150. A 1-5 scoring was assigned for body condition.
Feed analysis: Individual ingredients from the ration and TMR representative samples were analyzed for nutritional and mineral content at DairyExperts Feeds Lab (DairyExperts, Inc, Tulare, Calif.). Ingredients were sampled once during adaptation and once during the experimental phase. TMR was sampled once during adaptation and once a week during the Intervention period.
A number of outcomes were evaluated, including:
(a) Dry Matter Intake (DMI): the feed consumed (Kg) per cow in an as fed basis times the dry matter percentage of the feed obtained from the laboratory analysis;
(b) Daily Milk Yield: calculated as the sum of both morning and afternoon milk weights (Kg);
(c) 3.5% Fat Corrected Milk (FCM): milk yield value corrected for 3.5% fat using formula from NRC (2001): [(0.4324×kg of milk)+(16.216×kg of fat)];
(d) Energy Corrected Milk (ECM): milk yield value corrected for 3.5% fat and 3.2% true protein using formula from NRC (2001): [(0.3246×kg of milk)+(12.86×kg of fat)+(7.04×kg of true protein)];
(e) Milk Components Percentage: daily milk crude protein (%), fat (%), and lactose (%) was calculated as the average of both morning and afternoon readings from the in-line sensor. Also, DHIA results were available from the samples collected at the morning milking on the days previously listed;
(f) Milk Components Yield: obtained by multiplying daily milk crude protein (%), fat (%), lactose (%) by the daily milk yield (Kg);
(g) Feed Efficiency: defined as Kg of 3.5% FCM produced per Kg of DM consumed; and
(h) Body Condition Score and Weight: these variables were analyzed for the measurements taken at different time points.
Table 20 below shows a summary of parameters from cows in control, Treatment 1, and Treatment 2 at the beginning of the trial. ECM, energy corrected milk; DIM, days in milk; and FE, feed efficiency.
Table 21 below shows a summary of the trial results from cows in control, Treatment 1, and Treatment 2. Overall, cows in Treatment 2 performed better than cows in Treatment 1. Trt, treatment; ECM, energy corrected milk; DMI, dry matter intake; and FE, feed efficiency.
Table 22 below shows a summary of the trial results pre-peak (less than 90 days in milk) from cows in control, Treatment 1, and Treatment 2. ECM, energy corrected milk; DMI, dry matter intake, and FE, feed efficiency.
Table 23 below shows a summary of the trial results post-peak (greater than 90 days in milk) from cows in control, Treatment 1, and Treatment 2. ECM, energy corrected milk; DMI, dry matter intake, and FE, feed efficiency.
Overall, cows administered treatment 1 showed an increase in milk yield and energy corrected milk, and an increase in milk fat and protein from weeks 1-9 of the trial. Cows administered treatment 1 also showed increased feed efficiency consistently across the entire trial. Cows administered treatment 2 showed an increase in milk yield and energy corrected milk from weeks 6-24 of the trial, and an increase in milk fat and protein from weeks 5-24 of the trial. Cows administered treatment 2 also showed no significant decreases in body weight and condition scores.
This study examines the effect of microbial supplementation on feed efficiency, milk yield, and milk compositional characteristics.
Seventy-two Holstein dairy cows, between 28-100 days in milk were assigned to one of three treatments (n=24 cows/trt; Control, Treatment 1, and Treatment 2). Animals were evaluated for soundness and removed before beginning trial if they exhibited feet and leg issues, were a three quartered animal or had more than one case of mastitis during calving to beginning of covariate period.
Treatments were blocked and balanced for parity, days in milk, and current milk yield. Parity was defined as primiparous (no more 30% of animals), or multiparous (2nd or greater). Animals were within a range of 15-20 days in milk within the block. The level of milk production was as tight as possible with the goal being within a range of 10 lbs of milk within a block.
Cows were adapted to tie-stalls and then baseline data (covariate) was collected for 2 weeks prior to start of treatments for all cows. Cows remained on their respective treatment diets for 140 days.
The treatment groups are shown in Table 24 below. The control group received a total mixed ration without microbial supplementation.
Clostridium sp. (SEQ ID NO: 28)
Pichia kudriavzevii (SEQ ID NO: 32)
Clostridium sp. (SEQ ID NO: 28)
Pichia kudriavzevii (SEQ ID NO: 32)
Ruminococcus bovis (SEQ ID NO:
Butyrivibrio fibrisolvens (SEQ ID
A top-dress for each cow was produced daily by adding the 5 g treatment to approximately 150 g of the carrier ground corn. Treatments were top-dressed on the feed and mixed into the top 3-6 inches of the total mixed ration (TMR). Treatments were color-coded and marked on the stalls and containers delivering the top-dress to the cows. Personnel changed gloves between treatments.
Treatments were packed into a daily packet of approximately 132 g, with each packet containing enough product for each cow in the treatment group plus 10% overage. Packets were stored at 4° C.
Each day, a fresh packet was opened. 5 g was weighed out for each cow individually and mixed into approximately 150 g of ground corn. Once completed, the packet was resealed and labeled with the date opened before storing at 4° C. Approximately every 50 days, one unused packet was assayed for quality control.
The basal TMR was delivered to the barn and CALAN Data Rangers were used to weigh out individual animal feedings. Animals were given approximately 60% of food at first feeding. Cows were fed at a reasonably consistent time each day (approximately 9 am), and then the rest of feed was put in a barrel in front of cows and fed later in day to ensure feed is always available. Individual cow dry matter intake (DMI) was adjusted daily to allow for a 10% feed refusal rate. Cow feeding areas were separated using plastic panels approximately 34 in. high near cows to 20 in. at back of manger area to prevent cross contamination of feed. The test products were hand-fed once daily by top-dressing on each cow's individual TMR diet. Treatments were mixed into the top 3-6 inches of TMR
Dry Matter Intake: Dry matter intakes from −14 to 140 treatment (daily, summarized by week). Diets were offered at ad libitum intake with 10% refusals. Orts were weighed daily and daily intakes were calculated for the duration of the study.
Body Weights: Double body weights were collected at beginning and end of covariate period, every 28 days and at removal from trial. More frequent body weights were allowed and were defined by each trial site but the double body weights were a requirement. Average body weight change was calculated by 28 day periods and overall body weight change was based on body weight at end of covariate.
Body Condition Score (BCS): BCS scores were determined using the 1-5 Elanco scoring system. Two scorers at beginning and end of covariate, every 28 days and at removal from experiment. Average BCS were determined and used for analysis. If BCS was ≥0.5 between scorers, then scorers independently rescored animal.
Milk composition: Once a week on the trial, a milk sample was collected at each milking during a 24 hour period. Milk samples were collected on the same day(s) of the week. Milk samples were not be composited but were sent to DHIA laboratories in Dubuque, Iowa for analysis of milk fat, protein, lactose, total solids, MUN and somatic cell counts.
Milk production: Cows were milked daily at approximately 4:00 am and 3:30 μm in a D-12 parallel milking parlor. Daily milk weights were captured by DairyPlan software. A milking system maintenance including calibration of the meters were performed prior to start of trial. Average daily milk and ECM by week and total treatment period was calculated based on milk and milk composition data collected on the trial.
Feed efficiency: Average daily milk and ECM by week and total treatment period was calculated based on milk and milk composition data collected on the trial. Weekly feed efficiency was calculated as milk/DMI and ECM/DMI.
Feed sampling: Weekly silage samples were collected and composited monthly. Dry matter determinations was conducted on corn silage and wet forages weekly. TMR was adjusted based on these dry matter determinations. Concentrate mixes, forages, and TMR was sampled weekly, composited by months and analyzed for model profile nutrients by chemical methods at Dairy One Forage Laboratory, Ithaca, N.Y.
Health and Reproduction: All health (including mastitis) and reproductive events and treatments was recorded throughout the trial and summarized by treatment. Cows were let out once a day for about 2 hours for exercise and estrus observations were captured during this and other times animals moving to parlor and back.
Rumen Fluid Collection: Rumen fluid was collected from 10 blocks of animals on the experiment. The samples were collected by esophageal tube or rumen canula. For samples collected using an esophageal tube, the first 200-300 mL was discarded to minimize saliva contamination. The pH of the sample was then determined and if the pH was greater than 6.9 than the animal was resampled due to possible saliva contamination.
Table 25 below shows a summary of the trial results from cows in control, Treatment 1, and Treatment 2. Overall, cows in Treatment 2 performed better than cows in Treatment 1. Trt, treatment; trt*time, treatment over time; and ECM, energy corrected milk.
This study examines the effect of microbial supplementation on feed efficiency, milk yield, and milk compositional characteristics.
Ninety Holstein dairy cows, between 70-114 days in milk were assigned to one of three treatments (n=30 cows/trt; Control, Treatment 1 and Treatment 2). Animals were evaluated for soundness and removed before beginning the trial if they had feet or leg issues, were a three quartered animal or had more than one case of mastitis during calving to beginning of covariate period.
Treatments were blocked and balanced for parity, days in milk, and current milk yield. Parity was defined as primiparous (no more 30% of animals) or multiparous (2nd or greater). Animals were within a range of 15-20 days in milk within the block. The level of milk production was as tight as possible with the goal being within a range of 10 lbs of milk within a block.
Cows were adapted to tie-stalls and then a baseline data (covariate) was collected for 2 weeks prior to the start of treatment for all cows. Cows remained on their respective treatment diets for 112 days.
The treatment groups are shown in Table 26 below. The control group received a total mixed ration without microbial supplementation.
Clostridium sp. (SEQ ID NO: 28)
Pichia kudriavzevii (SEQ ID NO: 32)
Ruminococcus bovis (SEQ ID NO:
Butyrivibrio fibrisolvens (SEQ ID
Clostridium sp. (SEQ ID NO: 28)
Pichia kudriavzevii (SEQ ID NO: 32)
Ruminococcus bovis (SEQ ID NO:
Butyrivibrio fibrisolvens (SEQ ID
Treatments were packed into a daily packet of approximately 165 g with each packet containing enough product for each cow in the treatment group plus 10% overage. Packets were be stored at 4° C.
Each day, a fresh packet was opened. 5 g was weighed out for each cow individually and mixed into approximately 150 g of ground corn. Once completed, the packet was resealed and labeled with the date opened before storing at 4° C. Approximately every 50 days, one unused packet was assayed for quality control.
The basal diet was formulated to meet or exceed dairy NRC nutrient requirements for protein, minerals and vitamins. The basal TMR was formulated based on a 2:1 ratio of corn silage to alfalfa haylage and include 10-20% byproducts, as well as ground corn and protein sources to provide 20% forage NDF, 28-32% total NDF, and 30% starch. The basal TMR was delivered to the barn and CALAN Data Rangers were used to weigh out individual animal feedings. Animals were given all of their daily allotment of food at one feeding. Cows were fed at a reasonably consistent time each day (approximately 9 am). Individual cow dry matter intake (DMI) was adjusted daily to allow for a 10% feed refusal rate. Cow feeding areas were separated by hard plastic dividers in the feed manger between cows to prevent cross contamination of feed and the feed mangers were blocked when cows were being released or being brought back to tie stalls. The test products were hand-fed once daily by top-dressing on each cow's individual TMR diet. Treatments were mixed into the top 3-6 inches of TMR.
Dry Matter Intakes: Dry matter intakes from −14 to 112 days of treatment (daily/weekly). Diets were offered at ad libitum intake with 10% refusals. Orts were weighed daily and daily intakes calculated for the duration of the study.
Body Weights: Body weights were collected 3 times per week. The average of body weights collected were used as the body weights for the time periods. Average body weight change was calculated by 28-day periods and overall body weight change based on body weight at end of covariate to end of trial
Body Condition Score (BCS): BCS scores were determined using the 1-5 Elanco scoring system. Two scorers at beginning and end of covariate, every 28 days and at removal from experiment. Average BCS were determined and used for analysis. If BCS was ≥0.5 between scorers, then scorers independently rescored animal.
Milk Composition: Once a week on the trial, a milk sample was collected at each milking of a 48-hour period. Milk samples were collected on the same day(s) of the week. Milk samples were not composited but were sent to DHIA laboratories in East Lansing for analysis of milk fat, protein, lactose, solids not fat, total solids, MUN and somatic cell counts.
Milk Production: Cows were milked daily at approximately 3:00 am and 2:00 μm in a D-7 herringbone milking parlor. Daily milk weights were captured by the Boumatic software. A milking system maintenance including calibration of the meters was performed prior to start of trial. Average daily milk and ECM by week and total treatment period was calculated based on milk and milk composition data collected on the trial.
Feed Efficiency: Average daily milk and ECM by week and total treatment period was calculated based on milk and milk composition data collected during the trial. Weekly feed efficiency was calculated as milk/DMI, ECM/DMI, and total energy captured as milk and body tissue/DMI.
Feed Sampling: Weekly silage samples were collected and composited monthly. A NIR nutrient analysis was determined. Dry matter determinations were conducted on corn silage and wet forages twice weekly by Koster tester. TMR may be adjusted based on these dry matter determinations. Samples of all feeds were collected weekly, composited monthly and analyzed by wet chemistry for DM, NDF, starch, CP, lipid, and ash.
Health and Reproduction; All health (including mastitis) and reproductive events and treatments were recorded throughout the trial and summarized by treatment. Cows were let out once a day for about 2 hours for exercise and estrus observations are captured during this and other times animals moving to parlor and back.
Other Measurements: Total tract digestibility of dry matter, neutral detergent fiber, starch, protein, and lipid was determined on 20 cows from each treatment group at approximately 30 to 50 days on treatment. A total of 8 samples for each cow was collected over 5 consecutive days. During the sampling period, blood glucose, insulin, and non-esterified fatty acids were collected and also on one other day at −1, +2 and +6 h of feeding time as metabolic indicators.
Rumen samples were collected via oro-ruminal sampling on 12 cows per treatment group during pretreatment, approximately 50-60 days on treatment and 90-100 days on treatment.
Table 27 below shows a summary of the trial results from cows in control, Treatment 1, and Treatment 2. Overall, cows in Treatment 2 performed better than cows in Treatment 1. Trt, treatment; and ECM, energy corrected milk.
All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes, including PCT Application Nos. PCT/US2017/012573 and PCT/US2020/020311.
However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
Other subject matter contemplated by the present disclosure is set out in the following numbered embodiments:
Embodiment 1. An orally deliverable composition for increasing milk production or improving milk compositional characteristics in a ruminant, comprising:
wherein a first population of microbes present in the rumen before administration of the ruminant supplement increase in abundance after administration of the ruminant supplement, and
wherein a second population of microbes present in the rumen before administration of the ruminant supplement decrease in abundance after administration of the ruminant supplement.
Embodiment 62. A composition comprising:
This application claims the benefit of priority to U.S. Provisional Application No. 63/002,588, filed on Mar. 31, 2020, hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/025264 | 3/31/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63002588 | Mar 2020 | US |
