(12) Patent Application Publication (10) Pub. No.: US 2017/0196922 A1 Embree Et Al

US 20170196922A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0196922 A1 Embree et al. (43) Pub. Date: Jul. 13, 2017

(54) METHODS FOR IMPROVING MILK Publication Classification PRODUCTION BY ADMINISTRATION OF (51) Int. Cl. MICROBAL CONSORTA A636/064 (2006.01) A23K 40/35 (2006.01) (71) Applicant: Ascus Biosciences, Inc., San Diego, A23K 50/10 (2006.01) CA (US) A6IR 9/00 (2006.01) A6II 35/742 (2006.01) (72) Inventors: Mallory Embree, Vista, CA (US); (52) U.S. Cl. Luke Picking, San Diego, CA (US); CPC ...... A61K 36/064 (2013.01); A61K 9/0053 Grant Gogul, Cardiff, CA (US); Janna (2013.01); A61K 9/0019 (2013.01); A61 K Tarasova, San Diego, CA (US) 35/742 (2013.01); A23K 50/10 (2016.05); A23K 40/35 (2016.05) (21) Appl. No.: 15/400,484 (57) ABSTRACT The disclosure relates to isolated microorganisms—includ ing novel strains of the microorganisms—microbial consor (22) Filed: Jan. 6, 2017 tia, and compositions comprising the same. Furthermore, the disclosure teaches methods of utilizing the described micro organisms, microbial consortia, and compositions compris Related U.S. Application Data ing the same, in methods for modulating the production and (60) Provisional application No. 62/276,142, filed on Jan. yield of milk and milk components in ruminants. In particu 7, 2016, provisional application No. 62/276,531, filed lar aspects, the disclosure provides methods of increasing on Jan. 8, 2016, provisional application No. 62/334, desirable components of milk in ruminants. Furthermore, the 816, filed on May 11, 2016, provisional application disclosure provides for methods of modulating the rumen No. 62/415,908, filed on Nov. 1, 2016. microbiome. Patent Application Publication Jul. 13, 2017. Sheet 1 of 21 US 2017/O196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 2 of 21 US 2017/O196922 A1

3x&&&&&&&&&&&&&&&&&&&&&&&&& Patent Application Publication Jul. 13, 2017. Sheet 3 of 21 US 2017/O196922 A1

pe sees sees spp. xppe spot see pop box ope, ope

b. e. ex sex 3.8 x: x- E. S. Patent Application Publication Jul. 13, 2017. Sheet 4 of 21 US 2017/O196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 5 of 21 US 2017/O196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 6 of 21 US 2017/O196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 7 of 21 US 2017/O196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 8 of 21 US 2017/O196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 9 of 21 US 2017/O196922 A1

-{}-

3.JOSOA §§§?§§§§¿?§§§§§{{}}:$?? Patent Application Publication Jul. 13, 2017. Sheet 10 of 21 US 2017/0196922 A1

8.IOOS OW Patent Application Publication Jul. 13, 2017. Sheet 11 of 21 US 2017/O196922 A1

3.00S ON Patent Application Publication Jul. 13, 2017. Sheet 12 of 21 US 2017/0196922 A1 3ss &

coS ON Patent Application Publication Jul. 13, 2017. Sheet 13 of 21 US 2017/O196922 A1

#nuouqomeonele:

3.IOSOIN Patent Application Publication Jul. 13, 2017. Sheet 14 of 21 US 2017/0196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 16 of 21 US 2017/O196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 17 of 21 US 2017/O196922 A1

£209

LuongVNHÂ¡ind

99009|1009

Patent Application Publication Jul. 13, 2017. Sheet 18 of 21 US 2017/0196922 A1

s a S

isei 388 is: Patent Application Publication Jul. 13, 2017. Sheet 19 of 21 US 2017/0196922 A1

Patent Application Publication Jul. 13, 2017. Sheet 20 of 21 US 2017/0196922 A1

38. & : & x

scies in

Ex: 3 &: | a: afsix sax seasy goaquinn Patent Application Publication Jul. 13, 2017. Sheet 21 of 21 US 2017/0196922 A1

US 2017/O 196922 A1 Jul. 13, 2017

METHODS FOR IMPROVING MILK selective breeding; however, the need for more efficient PRODUCTION BY ADMINISTRATION OF production of milk and milk components per animal is MICROBAL CONSORTA required. 0006 Identifying compositions and methods for sustain CROSS-REFERENCE TO RELATED ably increasing milk production and modulating milk com APPLICATIONS ponents of interest while balancing animal health and well being have become imperative to satisfy the needs of every 0001. This application claims the benefit of priority to day humans in an expanding population. Increasing the U.S. Provisional Application No. 62/276,142, filed Jan. 7, worldwide production of milk and further modulating desir 2016; U.S. Provisional Application No. 62/276,531, filed able milk components by Scaling up the total number of Jan. 8, 2016; U.S. Provisional Application No. 62/334,816, livestock on dairy farms would not only be economically filed May 11, 2016; and U.S. Provisional Application No. infeasible for many parts of the world, but would further 62/415,908, filed Nov. 1, 2016; each of which is herein result in negative environmental consequences. incorporated by reference in its entirety. 0007 Thus, meeting global milk and milk component yield expectations, by simply scaling up current high-input FIELD agricultural systems utilized in most of the developed world is simply not feasible. 0002 The present disclosure relates to isolated and bio 0008. There is therefore an urgent need in the art for logically pure microorganisms that have applications, inter improved methods of increasing milk production and further alia, in dairy production. The disclosed microorganisms can increasing yield of desirable milk components. be utilized in their isolated and biologically pure States, as well as being formulated into compositions. Furthermore, SUMMARY OF THE DISCLOSURE the disclosure provides microbial consortia, containing at least two members of the disclosed microorganisms, as well 0009. In some aspects, the present disclosure provides as methods of utilizing said consortia. Furthermore, the isolated microbes, including novel strains of microbes, pre disclosure provides for methods of modulating the rumen sented in Table 1 and/or Table 3. microbiome. 0010. In other aspects, the present disclosure provides isolated whole microbial cultures of the microbes identified STATEMENT REGARDING SEQUENCE in Table 1 and Table 3. These cultures may comprise LISTING microbes at various concentrations. 0011. In some aspects, the disclosure provides for utiliz 0003. The sequence listing associated with this applica ing one or more microbes selected from Table 1 and/or Table tion is provided in text format in lieu of a paper copy, and 3 to increase a phenotypic trait of interest in a ruminant. is hereby incorporated by reference into the specification. Furthermore, the disclosure provides for methods of modu The name of the text file containing the sequence listing is lating the rumen microbiome by utilizing one or more ASBI 002 02US SeqList ST25.txt. The text file is 893 kb, microbes selected from Table 1 and/or Table 3. was created on Oct. 31, 2016, and is being submitted 0012. In some embodiments, a microbial consortium electronically via EFS-Web. comprises at least two microbial strains selected from Table 1 and/or Table 3. In some embodiments, a microbial con BACKGROUND sortium comprises at least one microbial strain selected from Table 1 and/or Table 3. In a further embodiment, a microbial 0004. The global population is predicted to increase to consortium comprises at least two microbial strains, wherein over 9 billion people by the year 2050 with a concurrent each microbe comprise a 16S rRNA sequence encoded by a reduction in the quantity of land, water, and other natural sequence selected from SEQID NOs: 1-30 and 2045-2103 or resources available per capita. Projections indicate that the an ITS sequence selected from SEQ ID NOS:31-60 and average domestic income will also increase, with the pro 2104-2107. In an additional embodiment, a microbial con jected rise in the GDP of China and India. The desire for a sortium comprises at least one microbial strain, wherein diet richer in animal-source proteins rises in tandem with each microbe comprise a 16S rRNA sequence encoded by a increasing income, thus the global livestock sector will be sequence selected from SEQ ID NOs: 1-30 and 2045-2103, charged with the challenge of producing more milk using or an ITS sequence selected from SEQ ID NOS:31-60 and fewer resources. The Food and Agriculture Organization of 2104-2107. the United Nations predict that 70% more food will have to 0013. In some embodiments, the microbial consortia of be produced, yet the area of arable land available will the present disclosure comprise at least two microbial decrease. It is clear that the food output per unit of resource strains, wherein each microbe comprises a 16S rRNA input will have to increase considerably in order to Support sequence encoded by a sequence selected from SEQ ID the rise in population. NOs: 1-30, SEQ ID NOs:61-1988, or SEQ ID NOs:2045 0005 Milk and milk components from lactating rumi 2103; or an ITS sequences selected from SEQ ID NOS:31 nants are predominantly utilized in the preparation of food 60, SEQ ID NOs: 1989-2044, or SEQ ID NOS:2104-2107. stuffs in many different forms. Nevertheless, milk and milk 0014. In one embodiment, the microbial consortium com components find numerous alternative applications in non prises at least two microbial strains comprising Ascusb 7. food areas such as the manufacture of glues, textile fibers, Ascusb 32, Ascusf 45, and Ascusf 24. In a further embodi plastic materials, or in the production of ethanol or methane. ment, the microbial consortium comprises at least one There have been many strategies to improve milk production microbial Strain comprising Ascusb 7, Ascusb 32, Ascusf and content in ruminants through nutritional modulations, 45, and Ascusf 24. In one embodiment, the microbial con hormone treatments, changes in animal management, and sortium comprises at least two microbial strains comprising US 2017/O 196922 A1 Jul. 13, 2017

Ascusb 7. Ascusb 32, Ascusf 45, and Ascusf 24. In a istering the composition to the animal. In further embodi further embodiment, the microbial consortium comprises at ments, the animal is a ruminant, which may further be a cow. least one microbial strain comprising Ascusb 7. Ascusb. 32. 0019. In some embodiments, the composition is admin Ascusf 45, and Ascusf 24. In one embodiment, the micro istered at least once per day. In a further embodiment, the bial consortium comprises at least two microbial strains composition is administered at least once per month. In a comprising Ascusb 7. Ascusb. 1801, Ascusf 45, and further embodiment, the composition is administered at least Ascusf 24. In a further embodiment, the microbial consor once per week. In a further embodiment, the composition is tium comprises at least one microbial Strain comprising administered at least once per hour. Ascusb 7. Ascusb. 1801, Ascusf 45, and Ascusf 24. In one 0020. In some embodiments, the administration com embodiment, the microbial consortium comprises at least prises injection of the composition into the rumen. In some two microbial strains comprising Ascusb 7, Ascusb. 268, embodiments, the composition is administered anally. In Ascusf 45, and Ascusf 24. In a further embodiment, the further embodiments, anal administration comprises insert microbial consortium comprises at least one microbial Strain ing a Suppository into the rectum. In some embodiments, the comprising Ascusb 7. Ascusb. 268, Ascusf 45, and Ascusf composition is administered orally. In some aspects, the oral 24. In one embodiment, the microbial consortium comprises administration comprises administering the composition in at least two microbial strains comprising Ascusb 7. Ascusb combination with the animals feed, water, medicine, or 232, Ascusf 45, and Ascusf 24. In a further embodiment, vaccination. In some aspects, the oral administration com the microbial consortium comprises at least one microbial prises applying the composition in a gel or viscous Solution strain comprising Ascusb 7. Ascusb 232, Ascusf 45, and to a body part of the animal, wherein the animal ingests the Ascusf 24. In one embodiment, the microbial consortium composition by licking. In some embodiments, the admin comprises at least two microbial strains comprising Ascusb istration comprises spraying the composition onto the ani 7, Ascusb_32, Ascusf 45, and Ascusf 249. In a further mal, and wherein the animal ingests the composition. In embodiment, the microbial consortium comprises at least Some embodiments, the administration occurs each time the one microbial strain comprising Ascusb 7. Ascusb. 32. animal is fed. In some embodiments, the oral administration Ascusf 45, and Ascusf 249. In one embodiment, the micro comprises administering the composition in combination bial consortium comprises at least two microbial strains with the animal feed. comprising Ascusb 7. Ascusb 32, Ascusf 45, and Ascusf 0021. In some embodiments, the at least one improved 353. In a further embodiment, the microbial consortium trait is selected from the group consisting of an increase of comprises at least one microbial strain comprising Ascusb fat in milk, an increase of carbohydrates in milk, an increase 7, Ascusb_32, Ascusf 45, and Ascusf 353. In one embodi of protein in milk, an increase of vitamins in milk, an ment, the microbial consortium comprises at least two increase of minerals in milk, an increase in milk volume, an microbial strains comprising Ascusb 7. Ascusb 32, Ascusf improved efficiency in feed utilization and digestibility, an 45, and Ascusf 23. In a further embodiment, the microbial increase in polysaccharide and lignin degradation, an consortium comprises at least two microbial Strains com increase in fatty acid concentration in the rumen, pH balance prising Ascusb 7. Ascusb 32, Ascusf 45, and Ascusf 23. In in the rumen, a reduction in methane emissions, a reduction one embodiment, the microbial consortium comprises at in manure production, improved dry matter intake, an least two microbial strains comprising Ascusb. 3138 and increase in energy corrected milk (ECM) by weight and/or Ascusf 15. In a further embodiment, the microbial consor Volume, an improved efficiency of nitrogen utilization, and tium comprises at least one microbial Strain comprising any combination thereof, wherein said increase or reduction Ascusb_3138 and Ascusf 15. In one embodiment, the at is determined by comparing against an animal not having least one microbial strain comprises Ascusb_3138. In been administered said composition. another embodiment, the at least one microbial strain com 0022. In some embodiments, the increase in fat in milk is prises Ascusf 15. an increase in triglycerides, triacylglycerides, diacylglycer 0015. In one embodiment, a composition comprises a ides, monoacylglycerides, phospholipids, cholesterol, gly microbial consortium of the present disclosure and an colipids, and/or fatty acids. In some embodiments, an acceptable carrier. In a further embodiment, a composition increase of carbohydrates is an increase in oligosaccharides, comprises a microbial consortium of the present disclosure lactose, glucose, and/or glucose. In some embodiments, an and acceptable carrier. In a further embodiment, the micro increase in polysaccharide degradation is an increase in the bial consortium is encapsulated. In a further embodiment, degradation of cellulose, lignin, and/or hemicellulose. In the encapsulated microbial consortium comprises a polymer. Some embodiments, an increase in fatty acid concentration In a further embodiment, the polymer may be selected from is an increase in acetic acid, propionic acid, and/or butyric a saccharide polymer, agar polymer, agarose polymer, pro acid. tein polymer, Sugar polymer, and lipid polymer. 0023. In some embodiments, the at least two microbial 0016. In some embodiments, the acceptable carrier is strains or the at least one microbial strain present in a selected from the group consisting of edible feed grade composition, or consortia, of the disclosure exhibit an material, mineral mixture, water, glycol, molasses, and corn increased utility that is not exhibited when said strains occur oil. In some embodiments, the at least two microbial strains alone or when said strains are present at a naturally occur forming the microbial consortium are present in the com ring concentration. In some embodiments, compositions of position at 10° to 10" cells per gram of said composition. the disclosure, comprising at least two microbial strains as taught herein, exhibit a synergistic effect on imparting at 0017. In some embodiments, the composition may be least one improved trait in an animal. In some embodiments, mixed with livestock feed. the compositions of the disclosure—comprising one or more 0018. In some embodiments, a method of imparting at isolated microbes as taught herein—exhibit markedly dif least one improved trait upon an animal comprises admin ferent characteristics/properties compared to their closest US 2017/O 196922 A1 Jul. 13, 2017 naturally occurring counterpart. That is, the compositions of 0027. In one embodiment, a substantially pure culture of the disclosure exhibit markedly different functional and/or an isolated microbial strain may comprise any one of the structural characteristics/properties, as compared to their strains or microbes of the present disclosure. closest naturally occurring counterpart. For instance, the 0028. In one embodiment, a method of modulating the microbes of the disclosure are structurally different from a microbiome of a ruminant comprises administering a com microbe as it naturally exists in a rumen, for at least the position of the present disclosure. In a further embodiment, following reasons: said microbe can be isolated and purified, the administration of the composition imparts at least one such that it is not found in the milieu of the rumen, said improved train upon the ruminant. In one embodiment, the microbe can be present at concentrations that do not occur at least one improved trait is selected from the group in the rumen, said microbe can be associated with acceptable consisting of an increase of fat in milk, an increase of carriers that do not occur in the rumen, said microbe can be carbohydrates in milk, an increase of protein in milk, an formulated to be shelf-stable and exist outside the rumen increase of vitamins in milk, an increase of minerals in milk, environment, and said microbe can be combined with other an increase in milk volume, an improved efficiency in feed microbes at concentrations that do not exist in the rumen. utilization and digestibility, an increase in polysaccharide Further, the microbes of the disclosure are functionally and lignin degradation, an increase in fatty acid concentra different from a microbe as it naturally exists in a rumen, for tion in the rumen, pH balance in the rumen, a reduction in at least the following reasons: said microbe when applied in methane emissions, a reduction in manure production, an isolated and purified form can lead to modulation of the improved dry matter intake, an increase in energy corrected rumen microbiome, increased milk production, and/or milk (ECM) by weight and/or volume, and an improved improved milk compositional characteristics, said microbe efficiency of nitrogen utilization; wherein said increase or can be formulated to be shelf-stable and able to exist outside reduction is determined by comparing against an animal not the rumen environment, Such that the microbe now has a having been administered said composition. In an additional new utility as a Supplement capable of administration to a embodiment, the modulation of the microbiome is a ruminant, wherein the microbe could not have such a utility decrease in the proportion of the microbial strains present in in its natural state in the rumen, as the microbe would be the microbiome prior to the administration of the composi unable to survive outide the rumen without the intervention tion, wherein the decrease is measured relative to the micro of the hand of man to formulate the microbe into a shelf biome of the ruminant prior to the administration of the stable state and impart this new utility that has the afore composition. mentioned functional characteristics not possessed by the 0029. In one embodiment, the method of increasing fat in microbe in its natural state of existence in the rumen. milk is an increase in triglycerides, triacylglycerides, dia 0024. In one embodiment, the disclosure provides for a cylglycerides, monoacylglycerides, phospholipids, choles ruminant feed Supplement capable of increasing a desirable terol, glycolipids, and/or fatty acids. phenotypic trait in a ruminant. In a particular embodiment, 0030. In one embodiment, the method of increasing car the ruminant feed Supplement comprises: a microbial con bohydrates is an increase in oligosaccharides, lactose, glu sortium of the present disclosure at a concentration that does cose, and/or galactose. not occur naturally, and an acceptable carrier. In one aspect, 0031. In one embodiment, the method of increasing poly the microbial consortium is encapsulated. saccharide degradation is an increase in the degradation of 0025. In one embodiment, an isolated microbial strain is lignin, cellulose, pectin and/or hemicellulose. selected from any one of the microbial strains in Table 1 0032. In one embodiment, the method of increasing fatty and/or Table 3. In one embodiment, an isolated microbial acid concentration is an increase in acetic acid, propionic strain is selected from the group consisting of Ascusb 7 acid, and/or butyric acid. deposited as Bigelow Accession Deposit No. Pat 0033. In one embodiment, the method of modulation of ent201612011; Ascusb 32 deposited as Bigelow Accession the microbiome is an increase in the proportion of the at least Deposit No. Patent201612007; Ascusb 82 deposited as Big one microbial strain of the microbiome, wherein the increase elow Accession Deposit No. Patent201612012; Ascusb. 119 is measured relative to a ruminant that did not have the at deposited as Bigelow Accession Deposit No. Pat least one microbial Strain administered. ent201612009; Ascusb. 1801 deposited as Bigelow Acces 0034. In one embodiment, the method of modulation of sion Deposit No. Patent201612009; Ascusf 206 deposited the microbiome is a decrease in the proportion of the as Bigelow Accession Deposit No. Patent201612003: microbial strains present in the microbiome prior to the Ascusf 23 deposited as Bigelow Accession Deposit No. administration of the composition, wherein the decrease is Patent201612014: Ascusf 24 deposited as Bigelow Acces measured relative to the microbiome of the ruminant prior to sion Deposit No. Patent201612004: Ascusf 45 deposited as the administration of the composition. Bigelow Accession Deposit No. Patent201612002; Ascusf 0035. In one embodiment, a method of increasing resis 208 deposited as Bigelow Accession Deposit No. Pat tance of cows to the colonization of pathogenic microbes ent201612003: Ascusb_3138 deposited as NRRL Accession comprises administering a composition of the present dis Deposit No. B-67248; and Ascusf 15 deposited as NRRL closure, resulting in the pathogenic microbes being unable to Accession Deposit No. Y-67249. colonize the gastrointestinal tract of a cow. In another 0026. In one embodiment, an isolated microbial strain of embodiment, a method for treating cows for the presence of the present disclosure comprises a polynucleotide sequence at least one pathogenic microbe comprises the administra sharing at least 90% sequence identity with any one of SEQ tion of a microbial consortium of the present disclosure and ID NOS:1-2107. In another embodiment, an isolated micro an acceptable carrier. In a further embodiment, the admin bial strain of the present disclosure comprises a polynucle istration of the microbial consortium or microbial compo otide sequence sharing at least 90% sequence identity with sition results in the relative abundance of the at least one any one of SEQ ID NOS:1-60 and 2045-2107. pathogenic microbe to decrease to less than 5% relative US 2017/O 196922 A1 Jul. 13, 2017

abundance in the gastrointestinal tract. In another embodi Research Service (ARS) Culture Collection (NRRL(R), ment, the administration of the microbial consortium or located at 1815 N. University St. Peoria, Ill. 61604, USA. microbial composition results in the relative abundance of Some microorganisms described in this application were the at least one pathogenic microbe to decrease to less than deposited with the Bigelow National Center for Marine 1% relative abundance in the gastrointestinal tract. In Algae and Microbiota, located at 60 Bigelow Drive, East another embodiment, the administration of the microbial Boothbay, Me. 04544, USA. consortium or microbial composition results in the patho ASC-01 (NRRL B-67248) and ASC-02 (NRRLY-67249) were deposited on genic microbe being undetectable in the gastrointestinal this date tract. 0038. The deposits were made under the terms of the 0036. In one embodiment, the microbial compositions Budapest Treaty on the International Recognition of the and/or consortium comprise bacteria and/or fungi in spore Deposit of Microorganisms for the Purposes of Patent Pro form. In one embodiment, the microbial compositions and/ cedure. The NRRL(R) and/or Bigelow National Center for or consortium of the disclosure comprise bacteria and/or Marine Algae and Microbiota accession numbers for the fungi in whole cell form. In one embodiment, the microbial aforementioned Budapest Treaty deposits are provided in compositions and/or consortium of the disclosure comprise Table 3. The accession numbers and corresponding dates of bacteria and/or fungi in lysed cell form. In some aspects of deposit for the microorganisms described in this Application formulating microbes according to the disleosure, the are separately provided in Table 25. microbes are: fermented->filtered-scentrifuged-sly 0039. The strains designated in the below tables have ophilized or spray dried->and optionally coated (i.e. a been deposited in the labs of Ascus Biosciences, Inc. since “fluidized bed step'). at least Dec. 15, 2015. 0040. In Table 1, the closest predicted hits for taxonomy Budapest Treaty on the International Recognition of the of the microbes are listed in columns 2, and 5. Column 2 is Deposit of Microorganisms for the Purpose of Patent the top taxonomic hit predicted by BLAST, and column 5 is Procedures the top taxonomic hit for genus+species predicted by 0037. Some microorganisms described in this Applica BLAST. The strains designated in the below table have been tion were deposited on Apr. 25, 2016', with the United deposited in the labs of Ascus BioSciences, Inc. since at least States Department of Agriculture (USDA) Agricultural Dec. 15, 2015. US 2017/O 196922 A1 Jul. 13, 2017

IHT8IVIL US 2017/O 196922 A1 Jul. 13, 2017

US 2017/O 196922 A1 Jul. 13, 2017

US 2017/O 196922 A1 Jul. 13, 2017 10

US 2017/O 196922 A1 Jul. 13, 2017 11

ZHT8IVIL US 2017/O 196922 A1 Jul. 13, 2017 12

US 2017/O 196922 A1 Jul. 13, 2017 13

US 2017/O 196922 A1 Jul. 13, 2017 14

US 2017/O 196922 A1 Jul. 13, 2017 15

US 2017/O 196922 A1 Jul. 13, 2017 16

TABLE 3 TABLE 3-continued Bacteria of the present disclosure. Bacteria of the present disclosure. Predicted Closest Predicted Closest Taxa of Isolated Strain Sequence Taxa of Isolated Strain Sequence Microbes Designation Identifier Microbes Designation Identifier Corynebacterium Ascusb 3 61 Bacteroides Ascusb 1218 32 Prevoteia Ascusb 50 62 Coprococcus Ascusb. 1239 33 Connaimonas Ascusb 90 63 AnaerovOrax Ascusb 1269 34 Cliostridium XIVa. Ascusb 117 64 Pseudoflavonifactor Ascusb 1296 35 Hippea Ascusb. 171 65 Pseudoflavonifactor Ascusb 1296 36 AnaerovOrax Ascusb. 177 66 Prevoteia Ascusb 1298 37 Cliostridium XIVa. Ascusb. 179 67 Lachnospiracea incertae sedis Ascusb. 1304 38 Runneiibacilius Ascusb. 224 68 Roseburia Ascusb. 1320 39 Cliostridium XIVa. Ascusb. 234 69 Prevoteia Ascusb. 1330 40 Lachnospiracea incertae sedis Ascusb. 274 70 Coprococcus Ascusb 955 O8 Prevoteia Ascusb. 276 71 Prevoteia Ascusb 958 09 AnaerovOrax Ascusb. 293 72 Cliostridium XIVa Ascusb 980 10 Pseudoflavonifactor Ascusb 327 73 Prevoteia Ascusb 982 11 Prevoteia Ascusb. 337 74 Caioneiia Ascusb 990 12 Cliostridium XIVa. Ascusb. 357 75 Meihanobrevibacter Ascusb. 993 13 Cliostridium XIVa. Ascusb 357 76 Riminococcits Ascusb. 1013 14 Coprococcus Ascusb. 361 77 Lachnospiracea incertae sedis Ascusb 1021 15 Pyramidobacter Ascusb. 388 78 Coprococcus Ascusb 1033 16 Syntrophococcits Ascusb. 425 79 Cliostridium XIVa. Ascusb. 1090 17 Prevoteia Ascusb. 444 8O Lachnospiracea incertae sedis Ascusb. 1108 18 Cliostridium XIVa. Ascusb. 456 81 Prevoteia Ascusb. 1113 19 Prevoteia Ascusb. 492 82 AnaerovOrax Ascusb. 1114 2O Roseburia Ascusb. 523 83 Asterolepiasma Ascusb. 1116 21 Cliostridium XIVa. Ascusb 526 84 Cliostridium XIVa. Ascusb. 1118 22 Lachnospiracea incertae sedis Ascusb 570 85 Cattiobacter Ascusb. 1123 23 Cliostridium XIVa. Ascusb. 584 86 Lachnospiracea incertae sedis Ascusb. 1128 24 Acidothermis Ascusb 605 87 Roseburia Ascusb. 1152 25 Adlercreutzia Ascusb. 606 88 Cliostridium XIVa. Ascusb. 1166 26 Prevoteia Ascusb. 617 89 Acinetobacter Ascusb 1170 27 Lachnospiracea incertae sedis Ascusb 635 90 Bacteroides Ascusb 1176 28 Proteiniciasticum Ascusb 642 91 Erysipeiothrix Ascusb 1182 29 Lachnospiracea incertae sedis Ascusb 647 92 Coprococcus Ascusb. 1199 30 AnaerovOrax Ascusb 656 93 Cliostridium XIVa. Ascusb. 1201 31 Prevoteia Ascusb 669 94 Bacteroides Ascusb 1218 32 Bacteroides Ascusb 681 95 Coprococcus Ascusb. 1239 33 Cliostridium III Ascusb. 704 96 AnaerovOrax Ascusb 1269 34 Prevoteia Ascusb. 706 97 Pseudoflavonifactor Ascusb 1296 35 Acinetobacter Ascusb. 717 98 Pseudoflavonifactor Ascusb 1296 36 Erysipeiothrix Ascusb 752 99 Prevoteia Ascusb 1298 37 Bacteroides Ascusb 790 OO Lachnospiracea incertae sedis Ascusb. 1304 38 Cliostridium XIVa. Ascusb 797 O1 Roseburia Ascusb. 1320 39 Butyrivibrio Ascusb. 802 O2 Prevoteia Ascusb. 1330 40 Eubacterium Ascusb. 805 O3 Riminococcits Ascusb. 1336 41 Prevoteia Ascusb 828 O4 Atopobium Ascusb. 1341 42 Eubacterium Ascusb 890 05 Eubacterium Ascusb. 1347 43 Prevoteia Ascusb 909 O6 Robinsonieia Ascusb 1355 44 Lachnospiracea incertae sedis Ascusb 924 O7 Neisseria Ascusb 1357 45 Coprococcus Ascusb 955 O8 Riminococcits Ascusb. 1362 46 Prevoteia Ascusb 958 09 Prevoteia Ascusb. 1364 47 Cliostridium XIVa. Ascusb 980 10 Slackia Ascusb. 1389 48 Prevoteia Ascusb 982 11 Prevoteia Ascusb 1400 49 Caioneiia Ascusb 990 12 Cliostridium XIVa. Ascusb. 1410 50 Meihanobrevibacter Ascusb. 993 13 Bacteroides Ascusb. 1417 51 Riminococcits Ascusb. 1013 14 Anaerorhabdus Ascusb. 1426 52 Lachnospiracea incertae sedis Ascusb 1021 15 Bacteroides Ascusb. 1433 53 Coprococcus Ascusb 1033 16 Prevoteia Ascusb. 1439 S4 Cliostridium XIVa. Ascusb. 1090 17 Corynebacterium Ascusb. 1440 55 Lachnospiracea incertae sedis Ascusb. 1108 18 Atopobium Ascusb. 1468 56 Prevoteia Ascusb. 1113 19 Streptophyta Ascusb. 1473 57 AnaerovOrax Ascusb. 1114 2O Prevoteia Ascusb. 1485 58 Asterolepiasma Ascusb. 1116 21 Roseburia Ascusb 1490 59 Cliostridium XIVa. Ascusb. 1118 22 Prevoteia Ascusb 1492 60 Cattiobacter Ascusb. 1123 23 Prevoteia Ascusb. 1528 61 Lachnospiracea incertae sedis Ascusb. 1128 24 Eubacterium Ascusb. 1538 62 Roseburia Ascusb. 1152 25 Rhodocista Ascusb 1543 63 Cliostridium XIVa. Ascusb. 1166 26 Prevoteia Ascusb 1546 64 Acinetobacter Ascusb 1170 27 Cliostridium XIVa. Ascusb. 1553 65 Bacteroides Ascusb 1176 28 Prevoteia Ascusb. 1554 66 Erysipeiothrix Ascusb 1182 29 Prevoteia Ascusb. 1571 67 Coprococcus Ascusb. 1199 30 Streptophyta Ascusb. 1578 68 Cliostridium XIVa. Ascusb 1201 31 Ochrobactrum Ascusb 1580 69 US 2017/O 196922 A1 Jul. 13, 2017 17

TABLE 3-continued TABLE 3-continued Bacteria of the present disclosure. Bacteria of the present disclosure. Predicted Closest Predicted Closest Taxa of Isolated Strain Sequence Taxa of Isolated Strain Sequence Microbes Designation Identifier Microbes Designation Identifier Asaccharobacter Ascusb. 19353 874 Cliostridium XIVa. Ascusb. 20449 945 Kordia Ascusb. 19371 875 Cyanobacteria Ascusb. 20456 946 Riminococcits Ascusb. 19376 876 Lactobacilius Ascusb. 20463 947 Cliostridium III Ascusb. 19379 877 Cliostridium XIVa. Ascusb. 20529 948 Ethanoigenens Ascusb. 19392 878 Prevoteia Ascusb. 20534 949 Cliostridium XIVa. Ascusb. 19412 879 Prevoteia Ascusb. 20540 950 Barnesieiia Ascusb. 19414 88O Marinobacter Ascusb. 20569 951 Eubacterium Ascusb. 19444 881 Butyricimonas Ascusb. 20576 952 Prevoteia Ascusb. 19457 882 Prevoteia Ascusb. 20594 953 Anaerophaga Ascusb. 19496 883 Dongia Ascusb. 20595 954 Acetitomaculum Ascusb. 19498 884 AnaerovOrax Ascusb. 20639 955 Prevoteia Ascusb. 19503 885 Butyricimonas Ascusb. 20757 956 Cliostridium III Ascusb. 19507 886 Cryptanaerobacter Ascusb 20826 957 Marinoscium Ascusb. 19558 887 Papillibacter Ascusb. 20904 958 Pedobacter Ascusb. 19568 888 Clostridium sensu stricto Ascusb. 20938 959 Prevoteia Ascusb. 19579 889 Escherichia Shigella Ascusb. 20943 960 Prevoteia Ascusb. 19613 890 Butyricicoccus Ascusb. 20986 961 AnaerovOrax Ascusb. 19633 891 Prevoteia Ascusb 21013 962 Cliostridium XIVa. Ascusb. 19658 892 Lachnospiracea incertae sedis Ascusb 21027 963 Cliostridium IV Ascusb. 19662 893 Thermotaiea Ascusb 21035 964 Lachnospiracea incertae sedis Ascusb. 19681 894 Cohaesihacier Ascusb 21042 96S Clost ridium sensu stricto Ascusb. 19694 895 Cliostridium XVIII Ascusb 21043 966 Lishizhenia Ascusb. 19698 896 Lachnospiracea incertae sedis Ascusb 21085 967 Pedobacter Ascusb. 19700 897 Spirochaeta Ascusb 21095 968 Howardeia Ascusb. 19731 898 Cliostridium XIVa. Ascusb. 21112 969 Roseburia Ascusb. 19745 899 Hydrogenoanaerobacterium Ascusb. 21147 970 Cliostridium XIVa. Ascusb. 19754 900 Cliostridium IV Ascusb. 21151 971 AnaerovOrax Ascusb. 19765 901 Papillibacter Ascusb. 21160 972 Lentisphaera Ascusb. 19772 902 Sporosarcina Ascusb. 21190 973 Prevoteia Ascusb. 19778 903 Selenomonas Ascusb. 21219 974 Saccharofermenians Ascusb. 19779 904 Papillibacter Ascusb. 21229 975 Cyanobacteria Ascusb. 19818 905 Lachnospiracea incertae sedis Ascusb. 21244 976 Proteiniphilum Ascusb. 19824 906 Cliostridium XIVa. Ascusb. 21271 977 Schwartzia Ascusb. 19855 907 Saccharofermenians Ascusb. 21297 978 Anaerorhabdus Ascusb. 19884 908 Cliostridium IV Ascusb. 21309 979 Robinsonieia Ascusb. 19885 909 Lachnospiracea incertae sedis Ascusb. 21348 98O Cliostridium IV Ascusb. 19904 910 Cliostridium IV Ascusb. 21425 981 Erysipelotrichaceae incertae sedis Ascusb. 19936 911 Lachnospiracea incertae sedis Ascusb. 21436 982 Flavobacterium Ascusb. 1995.0 912 Desulfotomaculum Ascusb. 21466 983 Pedobacter Ascusb. 19955 913 Pedobacter Ascusb. 21484 984 Cliostridium III Ascusb. 19982 914 Anaeroplasma Ascusb 21546 985 Selenomonas Ascusb. 20001 915 Cliostridium IV Ascusb 21585 986 Rhizobium Ascusb 20027 916 Treponema Ascusb 21595 987 Victivais Ascusb 20044 917 Mogibacterium Ascusb. 21601 988 Butyricimonas Ascusb 20062 918 Parabacteroides Ascusb. 20064 919 Adhaeribacter Ascusb 20067 920 Eubacterium Ascusb 20086 921 BRIEF DESCRIPTION OF THE FIGURES Acidobacteria Ascusb 20100 922 Treponema Ascusb 20104 923 Cliostridium XIVa. Ascusb 20108 924 0041 FIG. 1 shows a general workflow of one embodi Cliostridium XIVa. Ascusb 20135 925 ment of the method for determining the absolute abundance Schwartzia Ascusb 20143 926 of one or more active microorganism strains. Prevoteia Ascusb 20162 927 Selenomonas Ascusb 20172 928 0042 FIG. 2 shows a general workflow of one embodi Beijerinckia Ascusb 20219 929 ment of a method for determining the co-occurrence of one Eubacterium Ascusb 20221 930 or more, or two or more, active microorganism strains in a Adhaeribacter Ascusb 20251 931 Verrucomicrobia Ascusb 20264 932 sample with one or more metadata (environmental) param Desulfobulbus Ascusb 20275 933 eters, followed by leveraging cluster analysis and commu Bacteroides Ascusb 20278 934 nity detection methods on the network of determined rela Runneiibacilius Ascusb 20291 935 tionships. Agarivorans Ascusb 20293 936 Cliostridium XIVa. Ascusb. 20306 937 0043 FIG. 3A, FIG. 3B, and FIG. 3C depict the results Selenomonas Ascusb. 20312 938 of a field trial in which dairy cows were administered a Verrucomicrobia Ascusb. 20365 939 composition comprising Ascusb. 3138 and Ascusf 15: FIG. Prevoteia Ascusb. 20368 940 Spirochaeta Ascusb. 20392 941 3A reveals the average number of pounds of milk fat Selenomonas Ascusb. 20405 942 produced over time: FIG. 3B reveals the average number of Spiroplasma Ascusb. 20424 943 pounds of milk protein produced over time; and FIG. 3C Pedobacter Ascusb. 20440 944 reveals the average number of pounds of energy corrected milk (ECM) produced over time. The vertical line intersect US 2017/O 196922 A1 Jul. 13, 2017 30 ing the data points in each of FIG. 3A, FIG. 3B, and FIG. 3C 0059 FIG. 19 depicts the correlation of the absolute cell marks the day at which administration of the microbial count with activity filter of target strain Ascus 713 to bioconsortia ceased. pounds (lbs) of milk fat produced. 0044 FIG. 4 depicts the milk yield (kg) daily means (no 0060 FIG. 20 depicts the absolute cell count with activity fill) and covariate adjusted weekly least square means (Solid filter of target strain Ascus 7 and the pounds (lbs) of milk fat fill)-SEM of cows assigned either to Control (circle) or produced over the course of an experiment. Inoculated (trapezoid) by intervention period study days. 0061 FIG. 21 depicts the correlation of the relative 004.5 FIG.5 depicts the milk crude protein yield (CP, kg) abundance with no activity filter of target strain Ascus 3038 daily means (no fill) and weekly least square means (Solid to pounds (lbs) of milk fat produced. fill)-SEM of cows assigned either to Control (circle) or Inoculated (trapezoid) by Intervention period study days. DETAILED DESCRIPTION 0046 FIG. 6 depicts the milk fat yield (kg) daily means Definitions (no fill) and weekly least square means (solid fill)-SEM of cows assigned either to Control (circle) or Inoculated (trap 0062) While the following terms are believed to be well eZoid) by Intervention period study days. understood by one of ordinary skill in the art, the following 0047 FIG. 7 depicts the energy corrected milk yield definitions are set forth to facilitate explanation of the (ECM, kg) daily means (no fill) and weekly least square presently disclosed subject matter. means (solid fill)-SEM of cows assigned either to Control 0063. The term “a” or “an” may refer to one or more of (circle) or Inoculated (trapezoid) by Intervention period that entity, i.e. can refer to plural referents. As such, the study days. terms 'a' or “an”, “one or more' and “at least one' are used 0048 FIG. 8A and FIG. 8B depict the shared percent interchangeably herein. In addition, reference to “an ele similarity (percent identity) among the bacteria (FIG. 8A) ment' by the indefinite article “a” or “an does not exclude and fungi (FIG. 8B) of Table 1. The data points represent the the possibility that more than one of the elements is present, greatest percent similarity pairing for each strain. unless the context clearly requires that there is one and only one of the elements. 0049 FIG. 9 depicts the MIC score distribution for 0064 Reference throughout this specification to “one rumen bacteria and milk fat efficiency. embodiment”, “an embodiment”, “one aspect', or “an 0050 FIG. 10 depicts the MIC score distribution for aspect” means that a particular feature, structure or charac rumen fungi and milk fat efficiency. teristic described in connection with the embodiment is 0051 FIG. 11 depicts the MIC score distribution for included in at least one embodiment of the present disclo rumen bacteria and dairy efficiency. Sure. Thus, the appearances of the phrases "in one embodi 0052 FIG. 12 depicts the MIC score distribution for ment” or “in an embodiment” in various places throughout rumen fungi and dairy efficiency. this specification are not necessarily all referring to the same 0053 FIG. 13 depicts the MIC score distribution for embodiment. Furthermore, the particular features, struc rumen bacteria and milk fat efficiency with four species of tures, or characteristics can be combined in any Suitable bacteria and their MIC scores, in which the species have manner in one or more embodiments. been evaluated in 3" party studies. The lower the MIC score, 0065. As used herein, in particular embodiments, the the less likely the species/strains are capable of positively terms “about' or “approximately' when preceding a numeri modulating milk fat efficiency in dairy cows. cal value indicates the value plus or minus a range of 10%. 0054 FIG. 14 depicts an undegraded carbon source (Day 0066. As used herein the terms “microorganism’ or O) and a degraded carbon source (Day 7), as utilized in the “microbe' should be taken broadly. These terms are used insoluble carbon Source assays. interchangeably and include, but are not limited to, the two 0055 FIG. 15 depicts a decrease in the number of cows prokaryotic domains, Bacteria and Archaea, eukaryotic exhibiting greater than 200,000 somatic cell counts (SSC)/ fungi and protists, as well as viruses. In some embodiments, mL milk in dairy cows that were administered a microbial the disclosure refers to the “microbes’ of Table 1 or Table 3, compostion of the present disclosure versus dairy cows that or the “microbes’ incorporated by reference. This charac were not administered a microbial composition of the pres terization can refer to not only the predicted taxonomic ent disclosure. microbial identifiers of the table, but also the identified strains of the microbes listed in the table. 0056 FIG. 16 depicts a diagram that exemplifies how the 0067. The term “microbial consortia” or “microbial con diet influences the production of volatile fatty acids which in sortium” refers to a subset of a microbial community of turn modulate milk production, body condition, growth, etc. individual microbial species, or strains of a species, which Reproduced from Moran, 2005. Tropical dairy farming: can be described as carrying out a common function, or can feeding management for Small holder dairy farmers in the be described as participating in, or leading to, or correlating humic tropics (Chapter 5), Landlinks Press, 312 pp. with, a recognizable parameter, Such as a phenotypic trait of 0057 FIG. 17 depicts a schematic diagram that illustrates interest (e.g. increased milk production in a ruminant). The an example process flow for use with an exemplary microbe community may comprise two or more species, or strains of interaction analysis and selection system, including the a species, of microbes. In some instances, the microbes determination of MIC scores discussed throughout the pres coexist within the community symbiotically. ent disclosure. 0068. The term “microbial community” means a group of 0058 FIG. 18 depicts the non-linearity of pounds of milk microbes comprising two or more species or strains. Unlike fate produced over the course of an experiment to determine microbial consortia, a microbial community does not have to rumen microbial community constituents that impact the be carrying out a common function, or does not have to be production of milk fat in dairy cows. participating in, or leading to, or correlating with, a recog US 2017/O 196922 A1 Jul. 13, 2017

nizable parameter, Such as a phenotypic trait of interest (e.g. denotes that said culture is substantially free (within scien increased milk production in a ruminant). tific reason) of other living organisms and contains only the 0069. As used herein, “isolate, “isolated, “isolated individual microbe in question. The culture can contain microbe,” and like terms, are intended to mean that the one varying concentrations of said microbe. The present disclo or more microorganisms has been separated from at least Sure notes that isolated and biologically pure microbes often one of the materials with which it is associated in a particular “necessarily differ from less pure or impure materials. See, environment (for example soil, water, animal tissue). e.g. In re Bergstrom, 427 F.2d 1394, (CCPA 1970)(discuss 0070 Microbes of the present disclosure may include ing purified prostaglandins), see also. In re Bergy, 596 F.2d spores and/or vegetative cells. In some embodiments, 952 (CCPA 1979) (discussing purified microbes), see also, microbes of the present disclosure include microbes in a Parke-Davis & Co. v. H.K. Mulford & Co., 189 F. 95 viable but non-culturable (VBNC) state. See Liao and Zhao (S.D.N.Y. 1911) (Learned Hand discussing purified adrena (US Publication US2015267163A1). In some embodiments, line), affd in part, rev’d in part, 196 F. 496 (2d Cir. 1912), microbes of the present disclosure include microbes in a each of which are incorporated herein by reference. Further biofilm. See Merritt et al. (U.S. Pat. No. 7,427,408). more, in some aspects, the disclosure provides for certain 0071. Thus, an "isolated microbe' does not exist in its quantitative measures of the concentration, or purity limi naturally occurring environment; rather, it is through the tations, that must be found within an isolated and biologi various techniques described herein that the microbe has cally pure microbial culture. The presence of these purity been removed from its natural setting and placed into a values, in certain embodiments, is a further attribute that non-naturally occurring state of existence. Thus, the isolated distinguishes the presently disclosed microbes from those strain or isolated microbe may exist as, for example, a microbes existing in a natural state. See, e.g., Merck & Co. biologically pure culture, or as spores (or other forms of the V. Olin Mathieson Chemical Corp., 253 F.2d 156 (4th Cir. strain) in association with an acceptable carrier. 1958) (discussing purity limitations for vitamin B12 pro 0072. As used herein, “spore' or “spores' refer to struc duced by microbes), incorporated herein by reference. tures produced by bacteria and fungi that are adapted for 0076. As used herein, “individual isolates' should be Survival and dispersal. Spores are generally characterized as taken to mean a composition, or culture, comprising a dormant structures, however spores are capable of differen predominance of a single genera, species, or strain, of tiation through the process of germination. Germination is microorganism, following separation from one or more the differentiation of spores into vegetative cells that are other microorganisms. The phrase should not be taken to capable of metabolic activity, growth, and reproduction. The indicate the extent to which the microorganism has been germination of a single spore results in a single fungal or isolated or purified. However, “individual isolates' can bacterial vegetative cell. Fungal spores are units of asexual comprise Substantially only one genus, species, or strain, of reproduction, and in Some cases are necessary structures in microorganism. fungal life cycles. Bacterial spores are structures for surviv 0077. As used herein, “microbiome' refers to the collec ing conditions that may ordinarily be nonconductive to the tion of microorganisms that inhabit the digestive tract or survival or growth of vegetative cells. gastrointestinal tract of an animal (including the rumen if 0073. As used herein, “microbial composition” refers to said animal is a ruminant) and the microorgansims’ physical a composition comprising one or more microbes of the environment (i.e. the microbiome has a biotic and physical present disclosure, wherein a microbial composition, in component). The microbiome is fluid and may be modulated Some embodiments, is administered to animals of the pres by numerous naturally occurring and artificial conditions ent disclosure. (e.g., change in diet, disease, antimicrobial agents, influx of 0074 As used herein, “carrier”, “acceptable carrier', or additional microorganisms, etc.). The modulation of the “pharmaceutical carrier refers to a diluent, adjuvant, excipi microbiome of a rumen that can be achieved via adminis ent, or vehicle with which the compound is administered. tration of the compositions of the disclosure, can take the Such carriers can be sterile liquids, such as water and oils, form of: (a) increasing or decreasing a particular Family, including those of petroleum, animal, vegetable, or synthetic Genus, Species, or functional grouping of microbe (i.e. origin; Such as peanut oil, Soybean oil, mineral oil, Sesame alteration of the biotic component of the rumen microbiome) oil, and the like. Water or aqueous solution saline solutions and/or (b) increasing or decreasing volatile fatty acids in the and aqueous dextrose and glycerol Solutions are preferably rumen, increasing or decreasing rumen pH, increasing or employed as carriers, in some embodiments as injectable decreasing any other physical parameter important for Solutions. Alternatively, the carrier can be a solid dosage rumen health (i.e. alteration of the abiotic component of the form carrier, including but not limited to one or more of a rumen mircrobiome). binder (for compressed pills), a glidant, an encapsulating 0078. As used herein, “probiotic” refers to a substantially agent, a flavorant, and a colorant. The choice of carrier can pure microbe (i.e., a single isolate) or a mixture of desired be selected with regard to the intended route of administra microbes, and may also include any additional components tion and standard pharmaceutical practice. See Hardee and that can be administered to a mammal for restoring micro Baggo (1998. Development and Formulation of Veterinary biota. Probiotics or microbial inoculant compositions of the Dosage Forms. 2" Ed. CRC Press. 504 pg.); E. W. Martin invention may be administered with an agent to allow the (1970. Remington's Pharmaceutical Sciences. 17' Ed. microbes to survive the environment of the gastrointestinal Mack Pub. Co.); and Blaser et al. (US Publication tract, i.e., to resist low pH and to grow in the gastrointestinal US20110280840A1). environment. In some embodiments, the present composi 0075. In certain aspects of the disclosure, the isolated tions (e.g., microbial compositions) are probiotics in some microbes exist as isolated and biologically pure cultures. It aspects. will be appreciated by one of skill in the art, that an isolated 0079. As used herein, “prebiotic” refers to an agent that and biologically pure culture of a particular microbe, increases the number and/or activity of one or more desired US 2017/O 196922 A1 Jul. 13, 2017 32 microbes. Non-limiting examples of prebiotics that may be I0087. The term “marker” or “unique marker” as used useful in the methods of the present disclosure include herein is an indicator of unique microorganism type, micro fructooligosaccharides (e.g., oligofructose, inulin, inulin organism Strain or activity of a microorganism Strain. A type fructans), galactooligosaccharides, amino acids, alco marker can be measured in biological samples and includes hols, and mixtures thereof. See Ramirez-Farias et al. (2008. without limitation, a nucleic acid-based marker Such as a Br. J. Nutr. 4:1-10) and Pool-Zobel and Sauer (2007.J. Nutr. ribosomal RNA gene, a peptide- or protein-based marker, 137:2580-2584 and supplemental). and/or a metabolite or other small molecule marker. 0080. The term “growth medium' as used herein, is any 0088. The term “metabolite' as used herein is an inter medium which is Suitable to Support growth of a microbe. mediate or product of metabolism. A metabolite in one By way of example, the media may be natural or artificial embodiment is a small molecule. Metabolites have various including gastrin Supplemental agar, LB media, blood functions, including in fuel, structural, signaling, stimula serum, and tissue culture gels. It should be appreciated that tory and inhibitory effects on enzymes, as a cofactor to an the media may be used alone or in combination with one or enzyme, in defense, and in interactions with other organisms more other media. It may also be used with or without the (such as pigments, odorants and pheromones). A primary addition of exogenous nutrients. metabolite is directly involved in normal growth, develop 0081. The medium may be amended or enriched with ment and reproduction. A secondary metabolite is not additional compounds or components, for example, a com directly involved in these processes but usually has an ponent which may assist in the interaction and/or selection important ecological function. Examples of metabolites of specific groups of microorganisms. For example, antibi include but are not limited to antibiotics and pigments such otics (such as penicillin) or sterilants (for example, quater as resins and terpenes, etc. Some antibiotics use primary nary ammonium salts and oxidizing agents) could be present metabolites as precursors, such as actinomycin which is and/or the physical conditions (such as salinity, nutrients (for created from the primary metabolite, tryptophan. Metabo example organic and inorganic minerals (such as phospho lites, as used herein, include Small, hydrophilic carbohy rus, nitrogenous salts, ammonia, potassium and micronutri drates; large, hydrophobic lipids and complex natural com ents such as cobalt and magnesium), pH, and/or tempera pounds. ture) could be amended. I0089. As used herein, the term “genotype” refers to the 0082. As used herein, the term “ruminant includes mam genetic makeup of an individual cell, cell culture, tissue, mals that are capable of acquiring nutrients from plant-based organism, or group of organisms. food by fermenting it in a specialized stomach (rumen) prior I0090. As used herein, the term “allele(s) means any of to digestion, principally through microbial actions. Rumi one or more alternative forms of a gene, all of which alleles nants included cattle, goats, sheep, giraffes, yaks, deer, relate to at least one trait or characteristic. In a diploid cell, antelope, and others. the two alleles of a given gene occupy corresponding loci on 0083. As used herein, the term “bovid' includes any a pair of homologous chromosomes. Since the present member of family Bovidae, which include hoofed mammals disclosure, in embodiments, relates to QTLS, i.e. genomic Such as antelope, sheep, goats, and cattle, among others. regions that may comprise one or more genes or regulatory I0084. As used herein, “energy-corrected milk” or “ECM’ sequences, it is in some instances more accurate to refer to represents the amount of energy in milk based upon milk "haplotype' (i.e. an allele of a chromosomal segment) volume, milk fat, and milk protein. ECM adjusts the milk instead of “allele’, however, in those instances, the term components to 3.5% fat and 3.2% protein, thus equalizing “allele' should be understood to comprise the term “haplo animal performance and allowing for comparison of pro type'. Alleles are considered identical when they express a duction at the individual animal and herd levels over time. similar phenotype. Differences in sequence are possible but An equation used to calculate ECM, as related to the present not important as long as they do not influence phenotype. 0091. As used herein, the term “locus' (loci plural) disclosure, is: means a specific place or places or a site on a chromosome ECM=(0.327xmilk pounds)+(12.95xfat pounds)+(7. where for example a gene or genetic marker is found. 2xprotein pounds) 0092. As used herein, the term “genetically linked refers 0085. As used herein, “improved' should be taken to two or more traits that are co-inherited at a high rate broadly to encompass improvement of a characteristic of during breeding such that they are difficult to separate interest, as compared to a control group, or as compared to through crossing. a known average quantity associated with the characteristic 0093. A “recombination' or “recombination event as in question. For example, “improved milk production asso used herein refers to a chromosomal crossing over or ciated with application of a beneficial microbe, or consortia, independent assortment. The term “recombinant” refers to of the disclosure can be demonstrated by comparing the milk an organism having a new genetic makeup arising as a result produced by an ungulate treated by the microbes taught of a recombination event. herein to the milk of an ungulate not treated. In the present 0094. As used herein, the term “molecular marker” or disclosure, “improved does not necessarily demand that the “genetic marker” refers to an indicator that is used in data be statistically significant (i.e. p-0.05); rather, any methods for visualizing differences in characteristics of quantifiable difference demonstrating that one value (e.g. the nucleic acid sequences. Examples of Such indicators are average treatment value) is different from another (e.g. the restriction fragment length polymorphism (RFLP) markers, average control value) can rise to the level of “improved.” amplified fragment length polymorphism (AFLP) markers, I0086. As used herein, “inhibiting and suppressing and single nucleotide polymorphisms (SNPs), insertion muta like terms should not be construed to require complete tions, microsatellite markers (SSRs), sequence-character inhibition or Suppression, although this may be desired in ized amplified regions (SCARs), cleaved amplified poly Some embodiments. morphic sequence (CAPS) markers or isozyme markers or US 2017/O 196922 A1 Jul. 13, 2017

combinations of the markers described herein which defines 0100. As used herein, the term “homozygous' means a a specific genetic and chromosomal location. Markers fur genetic condition existing when two identical alleles reside ther include polynucleotide sequences encoding 16S or 18S at a specific locus, but are positioned individually on cor rRNA, and internal transcribed spacer (ITS) sequences, responding pairs of homologous chromosomes in the cell of which are sequences found between Small-subunit and large a diploid organism. Conversely, as used herein, the term subunit rRNA genes that have proven to be especially useful "heterozygous' means a genetic condition existing when in elucidating relationships or distinctions among when two different alleles reside at a specific locus, but are compared against one another. Mapping of molecular mark positioned individually on corresponding pairs of homolo ers in the vicinity of an allele is a procedure which can be gous chromosomes in the cell of a diploid organism. performed by the average person skilled in molecular 0101. As used herein, the term "phenotype” refers to the biological techniques. observable characteristics of an individual cell, cell culture, 0095. The primary structure of major rRNA subunit 16S organism (e.g., a ruminant), or group of organisms which comprise a particular combination of conserved, variable, results from the interaction between that individuals genetic and hypervariable regions that evolve at different rates and makeup (i.e., genotype) and the environment. enable the resolution of both very ancient lineages such as 0102. As used herein, the term "chimeric' or “recombi domains, and more modern lineages such as genera. The nant when describing a nucleic acid sequence or a protein secondary structure of the 16S subunit include approxi sequence refers to a nucleic acid, or a protein sequence, that mately 50 helices which result in base pairing of about 67% links at least two heterologous polynucleotides, or two of the residues. These highly conserved secondary structural heterologous polypeptides, into a single macromolecule, or features are of great functional importance and can be used that re-arranges one or more elements of at least one natural to ensure positional homology in multiple sequence align nucleic acid or protein sequence. For example, the term ments and phylogenetic analysis. Over the previous few “recombinant can refer to an artificial combination of two decades, the 16S rRNA gene has become the most otherwise separated segments of sequence, e.g., by chemical sequenced taxonomic marker and is the cornerstone for the synthesis or by the manipulation of isolated segments of current systematic classification of bacteria and archaea nucleic acids by genetic engineering techniques. (Yarza et al. 2014. Nature Rev. Micro. 12:635-45). 0103) As used herein, a “synthetic nucleotide sequence' 0096. A sequence identity of 94.5% or lower for two 16S or 'synthetic polynucleotide sequence' is a nucleotide rRNA genes is strong evidence for distinct genera, 86.5% or sequence that is not known to occur in nature or that is not lower is strong evidence for distinct families, 82% or lower naturally occurring. Generally, such a synthetic nucleotide is strong evidence for distinct orders, 78.5% is strong sequence will comprise at least one nucleotide difference evidence for distinct classes, and 75% or lower is strong when compared to any other naturally occurring nucleotide evidence for distinct phyla. The comparative analysis of 16S Sequence. rRNA gene sequences enables the establishment of taxo 0104. As used herein, the term “nucleic acid” refers to a nomic thresholds that are useful not only for the classifica polymeric form of nucleotides of any length, either ribo tion of cultured microorganisms but also for the classifica nucleotides or deoxyribonucleotides, or analogs thereof. tion of the many environmental sequences. Yarza et al. 2014. This term refers to the primary structure of the molecule, and Nature Rev. Micro. 12:635-45). thus includes double- and single-stranded DNA, as well as 0097. As used herein, the term “trait” refers to a charac double- and single-stranded RNA. It also includes modified teristic or phenotype. For example, in the context of some nucleic acids such as methylated and/or capped nucleic embodiments of the present disclosure, quantity of milk fat acids, nucleic acids containing modified bases, backbone produced relates to the amount of triglycerides, triacylglyc modifications, and the like. The terms “nucleic acid' and erides, diacylglycerides, monoacylglycerides, phospholip “nucleotide sequence' are used interchangeably. ids, cholesterol, glycolipids, and fatty acids present in milk. 0105. As used herein, the term “gene' refers to any Desirable traits may also include other milk characteristics, segment of DNA associated with a biological function. including but not limited to: predominance of short chain Thus, genes include, but are not limited to, coding sequences fatty acids, medium chain fatty acids, and long chain fatty and/or the regulatory sequences required for their expres acids; quantity of carbohydrates such as lactose, glucose, Sion. Genes can also include non-expressed DNA segments galactose, and other oligosaccharides; quantity of proteins that, for example, form recognition sequences for other Such as caseins and whey, quantity of vitamins, minerals, proteins. Genes can be obtained from a variety of Sources, milk yield/volume; reductions in methane emissions or including cloning from a source of interest or synthesizing manure; improved efficiency of nitrogen utilization; from known or predicted sequence information, and may improved dry matter intake; improved feed efficiency and include sequences designed to have desired parameters. digestibility; increased degradation of cellulose, lignin, and 0106. As used herein, the term “homologous' or “homo hemicellulose; increased rumen concentrations of fatty acids logue' or “ortholog” is known in the art and refers to related Such as acetic acid, propionic acid, and butyric acid; etc. sequences that share a common ancestor or family member 0098. A trait may be inherited in a dominant or recessive and are determined based on the degree of sequence identity. manner, or in a partial or incomplete-dominant manner. A The terms “homology,” “homologous.” “substantially simi trait may be monogenic (i.e. determined by a single locus) lar and "corresponding Substantially are used interchange or polygenic (i.e. determined by more than one locus) or ably herein. They refer to nucleic acid fragments wherein may also result from the interaction of one or more genes changes in one or more nucleotide bases do not affect the with the environment. ability of the nucleic acid fragment to mediate gene expres 0099. In the context of this disclosure, traits may also sion or produce a certain phenotype. These terms also refer result from the interaction of one or more mammalian genes to modifications of the nucleic acid fragments of the instant and one or more microorganism genes. disclosure Such as deletion or insertion of one or more US 2017/O 196922 A1 Jul. 13, 2017 34 nucleotides that do not substantially alter the functional (1997) PNAS 94:4504-4509: Crameri et al.(1998) Nature properties of the resulting nucleic acid fragment relative to 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458. the initial, unmodified fragment. It is therefore understood, For PCR amplifications of the polynucleotides disclosed as those skilled in the art will appreciate, that the disclosure herein, oligonucleotide primers can be designed for use in encompasses more than the specific exemplary sequences. PCR reactions to amplify corresponding DNA sequences These terms describe the relationship between a gene found from cDNA or genomic DNA extracted from any organism in one species, subspecies, variety, cultivar or strain and the of interest. Methods for designing PCR primers and PCR corresponding or equivalent gene in another species, Sub cloning are generally known in the art and are disclosed in species, variety, cultivar or strain. For purposes of this Sambrook et al. (1989) Molecular Cloning: A Laboratory disclosure homologous sequences are compared. “Homolo Manual (2nd ed., Cold Spring Harbor Laboratory Press, gous sequences” or "homologues' or “orthologs are Plainview, N.Y.). See also Innis et al., eds. (1990) PCR thought, believed, or known to be functionally related. A Protocols: A Guide to Methods and Applications (Academic functional relationship may be indicated in any one of a Press, New York); Innis and Gelfand, eds. (1995) PCR number of ways, including, but not limited to: (a) degree of Strategies (Academic Press, New York); and Innis and sequence identity and/or (b) the same or similar biological Gelfand, eds. (1999) PCR Methods Manual (Academic function. Preferably, both (a) and (b) are indicated. Homol Press, New York). Known methods of PCR include, but are ogy can be determined using Software programs readily not limited to, methods using paired primers, nested primers, available in the art, such as those discussed in Current single specific primers, degenerate primers, gene-specific Protocols in Molecular Biology (F. M. Ausubel et al., eds., primers, vector-specific primers, partially-mismatched prim 1987) Supplement 30, section 7.718, Table 7.71. Some ers, and the like. alignment programs are MacVector (Oxford Molecular Ltd, 0111. The term “primer' as used herein refers to an Oxford, U.K.), ALIGN Plus (Scientific and Educational oligonucleotide which is capable of annealing to the ampli Software, Pennsylvania) and AlignX (Vector NTI, Invitro fication target allowing a DNA polymerase to attach, thereby gen, Carlsbad, Calif.). Another alignment program is serving as a point of initiation of DNA synthesis when Sequencher (Gene Codes, Ann Arbor, Mich.), using default placed under conditions in which synthesis of primer exten parameters. sion product is induced, i.e., in the presence of nucleotides 0107 As used herein, the term “nucleotide change” refers and an agent for polymerization Such as DNA polymerase to, e.g., nucleotide Substitution, deletion, and/or insertion, as and at a suitable temperature and pH. The (amplification) is well understood in the art. For example, mutations contain primer is preferably single stranded for maximum efficiency alterations that produce silent Substitutions, additions, or in amplification. Preferably, the primer is an oligodeoxyri deletions, but do not alter the properties or activities of the bonucleotide. The primer must be sufficiently long to prime encoded protein or how the proteins are made. the synthesis of extension products in the presence of the 0108. As used herein, the term “protein modification' agent for polymerization. The exact lengths of the primers refers to, e.g., amino acid Substitution, amino acid modifi will depend on many factors, including temperature and cation, deletion, and/or insertion, as is well understood in the composition (A/T vs. G/C content) of primer. A pair of art bi-directional primers consists of one forward and one 0109 As used herein, the term “at least a portion' or reverse primer as commonly used in the art of DNA ampli "fragment of a nucleic acid or polypeptide means a portion fication such as in PCR amplification. having the minimal size characteristics of such sequences, or 0112 The terms “stringency” or “stringent hybridization any larger fragment of the full length molecule, up to and conditions’ refer to hybridization conditions that affect the including the full length molecule. A fragment of a poly stability of hybrids, e.g., temperature, Salt concentration, pH, nucleotide of the disclosure may encode a biologically formamide concentration and the like. These conditions are active portion of a genetic regulatory element. A biologically empirically optimized to maximize specific binding and active portion of a genetic regulatory element can be pre minimize non-specific binding of primer or probe to its pared by isolating a portion of one of the polynucleotides of target nucleic acid sequence. The terms as used include the disclosure that comprises the genetic regulatory element reference to conditions under which a probe or primer will and assessing activity as described herein. Similarly, a hybridize to its target sequence, to a detectably greater portion of a polypeptide may be 4 amino acids, 5 amino degree than other sequences (e.g. at least 2-fold over back acids, 6 amino acids, 7 amino acids, and so on, going up to ground). Stringent conditions are sequence dependent and the full length polypeptide. The length of the portion to be will be different in different circumstances. Longer used will depend on the particular application. A portion of sequences hybridize specifically at higher temperatures. a nucleic acid useful as a hybridization probe may be as Generally, stringent conditions are selected to be about 5°C. short as 12 nucleotides; in some embodiments, it is 20 lower than the thermal melting point (Tm) for the specific nucleotides. A portion of a polypeptide useful as an epitope sequence at a defined ionic strength and pH. The Tm is the may be as short as 4 amino acids. A portion of a polypeptide temperature (under defined ionic strength and pH) at which that performs the function of the full-length polypeptide 50% of a complementary target sequence hybridizes to a would generally be longer than 4 amino acids. perfectly matched probe or primer. Typically, stringent con 0110 Variant polynucleotides also encompass sequences ditions will be those in which the salt concentration is less derived from a mutagenic and recombinogenic procedure than about 1.0 M Na+ ion, typically about 0.01 to 1.0 MNa+ such as DNA shuffling. Strategies for such DNA shuffling ion concentration (or other salts) at pH 7.0 to 8.3 and the are known in the art. See, for example, Stemmer (1994) temperature is at least about 30° C. for short probes or PNAS 91:10747-10751; Stemmer (1994) Nature 370:389 primers (e.g. 10 to 50 nucleotides) and at least about 60° C. 391; Crameri et al. (1997) Nature Biotech. 15:436-438: for long probes or primers (e.g. greater than 50 nucleotides). Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. Stringent conditions may also be achieved with the addition US 2017/O 196922 A1 Jul. 13, 2017

of destabilizing agents such as formamide. Exemplary low include anaerobic conditions, certain chemicals, the pres stringent conditions or “conditions of reduced stringency’ ence of light, acidic or basic conditions, etc. include hybridization with a buffer solution of 30% forma 0117. As used herein, a “tissue specific' promoter is a mide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2XSSC promoter that initiates transcription only in certain tissues. at 40° C. Exemplary high Stringency conditions include Unlike constitutive expression of genes, tissue-specific hybridization in 50% formamide, 1M NaCl, 1% SDS at 37° expression is the result of several interacting levels of gene C., and a wash in 0.1 xSSC at 60° C. Hybridization proce regulation. As such, in the art sometimes it is preferable to dures are well known in the art and are described by e.g. use promoters from homologous or closely related species to Ausubel et al., 1998 and Sambrook et al., 2001. In some achieve efficient and reliable expression of transgenes in embodiments, stringent conditions are hybridization in 0.25 particular tissues. This is one of the main reasons for the M. Na2HPO4 buffer (pH 7.2) containing 1 mM Na2EDTA, large amount of tissue-specific promoters isolated from 0.5-20% sodium dodecyl sulfate at 45° C., such as 0.5%, particular tissues found in both scientific and patent litera 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, ture. 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%, followed by 0118. As used herein, the term “operably linked’ refers to a wash in 5xSSC, containing 0.1% (w/v) sodium dodecyl the association of nucleic acid sequences on a single nucleic sulfate, at 55° C. to 65° C. acid fragment so that the function of one is regulated by the 0113. As used herein, “promoter” refers to a DNA other. For example, a promoter is operably linked with a sequence capable of controlling the expression of a coding coding sequence when it is capable of regulating the expres sequence or functional RNA. The promoter sequence con sion of that coding sequence (i.e., that the coding sequence sists of proximal and more distal upstream elements, the is under the transcriptional control of the promoter). Coding latter elements often referred to as enhancers. Accordingly, sequences can be operably linked to regulatory sequences in an "enhancer is a DNA sequence that can stimulate pro a sense or antisense orientation. In another example, the moteractivity, and may be an innate element of the promoter complementary RNA regions of the disclosure can be oper or a heterologous element inserted to enhance the level or ably linked, either directly or indirectly, 5' to the target tissue specificity of a promoter. Promoters may be derived in mRNA, or 3' to the target mRNA, or within the target their entirety from a native gene, or be composed of different mRNA, or a first complementary region is 5' and its comple elements derived from different promoters found in nature, ment is 3' to the target mRNA. or even comprise synthetic DNA segments. It is understood 0119) As used herein, the phrases “recombinant con by those skilled in the art that different promoters may direct struct”, “expression construct”, “chimeric construct”, “con the expression of a gene in different tissues or cell types, or struct’, and “recombinant DNA construct” are used inter at different stages of development, or in response to different changeably herein. A recombinant construct comprises an environmental conditions. It is further recognized that since artificial combination of nucleic acid fragments, e.g., regu in most cases the exact boundaries of regulatory sequences latory and coding sequences that are not found together in have not been completely defined, DNA fragments of some nature. For example, a chimeric construct may comprise variation may have identical promoter activity. regulatory sequences and coding sequences that are derived 0114. As used herein, a “constitutive promoter is a from different sources, or regulatory sequences and coding promoter which is active under most conditions and/or sequences derived from the same source, but arranged in a during most development stages. There are several advan manner different than that found in nature. Such construct tages to using constitutive promoters in expression vectors may be used by itself or may be used in conjunction with a used in biotechnology, such as: high level of production of vector. If a vector is used then the choice of vector is proteins used to select transgenic cells or organisms; high dependent upon the method that will be used to transform level of expression of reporter proteins or scorable markers, host cells as is well known to those skilled in the art. For allowing easy detection and quantification; high level of example, a plasmid vector can be used. The skilled artisan production of a transcription factor that is part of a regula is well aware of the genetic elements that must be present on tory transcription system; production of compounds that the vector in order to successfully transform, select and requires ubiquitous activity in the organism; and production propagate host cells comprising any of the isolated nucleic of compounds that are required during all stages of devel acid fragments of the disclosure. The skilled artisan will also opment. Non-limiting exemplary constitutive promoters recognize that different independent transformation events include, CaMV 35S promoter, opine promoters, ubiquitin will result in different levels and patterns of expression promoter, alcohol dehydrogenase promoter, etc. (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), and thus that 0115. As used herein, a “non-constitutive promoter' is a multiple events must be screened in order to obtain lines promoter which is active under certain conditions, in certain displaying the desired expression level and pattern. Such types of cells, and/or during certain development stages. For screening may be accomplished by Southern analysis of example, tissue specific, tissue preferred, cell type specific, DNA, Northern analysis of mRNA expression, immunob cell type preferred, inducible promoters, and promoters lotting analysis of protein expression, or phenotypic analy under development control are non-constitutive promoters. sis, among others. Vectors can be plasmids, viruses, bacte Examples of promoters under developmental control include riophages, pro-viruses, phagemids, transposons, artificial promoters that preferentially initiate transcription in certain chromosomes, and the like, that replicate autonomously or tissues. can integrate into a chromosome of a host cell. A vector can 0116. As used herein, “inducible' or “repressible' pro also be a naked RNA polynucleotide, a naked DNA poly moter is a promoter which is under chemical or environ nucleotide, a polynucleotide composed of both DNA and mental factors control. Examples of environmental condi RNA within the same strand, a poly-lysine-conjugated DNA tions that may affect transcription by inducible promoters or RNA, a peptide-conjugated DNA or RNA, a liposome US 2017/O 196922 A1 Jul. 13, 2017 36 conjugated DNA, or the like, that is not autonomously (i.e., activity) reads is greater than normalized first marker replicating. As used herein, the term “expression” refers to (cell count) reads; (5) log2 fold change between activity and the production of a functional end-product e.g., an mRNA or quantity or cell count; (6) normalized second marker (activ a protein (precursor or mature). ity) reads is greater than mean second marker (activity) 0120 In some embodiments, the cell or organism has at reads for entire sample (and/or sample set); and/or any least one heterologous trait. As used herein, the term “het magnitude threshold described above in addition to a sta erologous trait” refers to a phenotype imparted to a trans tistical threshold (i.e., significance testing). The following formed host cell or transgenic organism by an exogenous example provides thresholding detail for distributions of DNA segment, heterologous polynucleotide or heterologous RNA-based second marker measurements with respect to nucleic acid. Various changes in phenotype are of interest to DNA-based first marker measurements, according to one the present disclosure, including but not limited to modify embodiment. ing the fatty acid composition in milk, altering the carbo 0.125. As used herein “shelf-stable” refers to a functional hydrate content of milk, increasing an ungulate's yield of an attribute and new utility acquired by the microbes formu economically important trait (e.g., milk, milk fat, milk lated according to the disclosure, which enable said proteins, etc.) and the like. These results can be achieved by microbes to exist in a useful/active state outside of their providing expression of heterologous products or increased natural environment in the rumen (i.e. a markedly different expression of endogenous products in organisms using the characteristic). Thus, shelf-stable is a functional attribute methods and compositions of the present disclosure. created by the formulations/compositions of the disclosure 0121. As used herein, the term “MIC means maximal and denoting that the microbe formulated into a shelf-stable information coefficient. MIC is a type of nonparamentric composition can exist outside the rumen and under ambient network analysis that identifies a score (MIC score) between conditions for a period of time that can be determined active microbial strains of the present disclosure and at least depending upon the particular formulation utilized, but in one measured metadata (e.g., milk fat). Further, U.S. appli general means that the microbes can be formulated to exist cation Ser. No. 157217,575, filed on Jul. 22, 2016 (issued as in a composition that is stable under ambient conditions for U.S. Pat. No. 9,540,676 on Jan. 10, 2017) is hereby incor at least a few days and generally at least one week. Accord porated by reference in its entirety. ingly, a “shelf-stable ruminant Supplement' is a composition 0122) The maximal information coefficient (MIC) is then comprising one or more microbes of the disclosure, said calculated between strains and metadata 3021a, and between microbes formulated in a composition, Such that the com strains 3021b, as seen in FIG. 17. Results are pooled to position is stable under ambient conditions for at least one create a list of all relationships and their corresponding MIC week, meaning that the microbes comprised in the compo scores 3022. If the relationship scores below a given thresh sition (e.g. whole cell, spore, or lysed cell) are able to impart old 3023, the relationship is deemed/identified as irrelevant one or more beneficial phenotypic properties to a ruminant 3023b. If the relationship is above a given threshold 3023, when administered (e.g. increased milk yield, improved the relationship deemed/identified as relevant 2023a, and is milk compositional characteristics, improved rumen health, further subject to network analysis 3024. The following code and/or modulation of the rumen microbiome). fragment shows an exemplary methodology for Such analy sis, according to one embodiment: Isolated Microbes I0126. In some aspects, the present disclosure provides isolated microbes, including novel strains of microbes, pre Read total list of relationships file as links sented in Table 1 and Table 3. threshold = 0.8 I0127. In other aspects, the present disclosure provides for i in range(len(links)): isolated whole microbial cultures of the microbes identified if links >= threshold in Table 1 and Table 3. These cultures may comprise multiplieri = 1 microbes at various concentrations. else multiplieri = 0 I0128. In some aspects, the disclosure provides for utiliz end if ing one or more microbes selected from Table 1 and Table links temp = multiplier links 3 to increase a phenotypic trait of interest in a ruminant. final links = links templinks temp = 0 savetXt(output file, final links) I0129. In some embodiments, the disclosure provides iso output file.close() lated microbial species belonging to taxonomic families of Clostridiaceae, Ruminococcaceae, Lachnospiraceae, Acidaminococcaceae, Peptococcaceae, Porphy 0123 Based on the output of the network analysis, active romonadaceae, Prevotellaceae, Neocallimastigaceae, Sac strains are selected 3025 for preparing products (e.g., charomycetaceae, Phaeosphaeriaceae, Erysipelotrichia, ensembles, aggregates, and/or other synthetic groupings) Anaerolinaeceae, Atopobiaceae, Botryosphaeriaceae, containing the selected Strains. The output of the network Eubacteriaceae, Acholeplasmataceae, Succinivibrionaceae, analysis can also be used to inform the selection of strains Lactobacillaceae, Selenomonadaceae, Burkholderiaceae, for further product composition testing. and Streptococcaceae. 0.124. The use of thresholds is discussed above for analy 0.130. In further embodiments, isolated microbial species ses and determinations. Thresholds can be, depending on the may be selected from genera of family Clostridiaceae, implementation and application: (1) empirically determined including Acetanaerobacterium, Acetivibrio, Acidamino (e.g., based on distribution levels, setting a cutoff at a bacter; Alkahphilus, Anaerobacter; Anaerostipes, number that removes a specified or significant portion of low Anaerotruncus, Anoxynatronium, Bryantella, Butyricicoccus, level reads); (2) any non-zero value; (3) percentage/percen Caldanaerocella, Caloramator; Caloranaerobacter, Camini tile based; (4) only strains whose normalized second marker cella, Candidatus Arthromitus, Clostridium, Coprobacillus, US 2017/O 196922 A1 Jul. 13, 2017 37

Dorea, Ethanologenbacterium, Faecalibacterium, Gar 0142. In further embodiments, isolated microbial species ciella, Guggenheimella, Hespellia, Linningia, Natronin may be selected from genera of family Selenomonadaceae cola, Oxobacter; Parasporobacterium, Sarcina, Soehngenia, including Anaerovibrio, Centipeda, Megamonas, Mit Sporobacter, Subdoligranulum, Tepidibacter, Tepidimicro suokella, Pectinatus, Propionispira, Schwartzia, Selenomo bium, Thermobrachium, Thermohalobacter, and Tindallia. nas, and Zymophilus. 0131. In further embodiments, isolated microbial species 0143. In further embodiments, isolated microbial species may be selected from genera of family Ruminococcaceae, may be selected from genera of family Burkholderiaceae including Ruminococcus, Acetivibrio, Sporobacter; Anaero including Burkholderia, Chitinimonas, Cupriavidus, Lau filium, Papillibacter, Oscillospira, Gemmiger, Faecalibac tropia, Limnobacter; Pandoraea, Paraburkholderia, Pauci terium, Fastidiosipila, Anaerotruncus, Ethanolingenens, monas, Polynucleobacter, Ralstonia, Thermothrix, and Acetanaerobacterium, Subdoligranulum, Hydrogenoan Wautersia. aerobacterium, and Candidadus Soleaferrea. 0144. In further embodiments, isolated microbial species 0.132. In further embodiments, isolated microbial species may be selected from genera of family Streptococcaceae may be selected from genera of family Lachnospiraceae, including Lactococcus, Lactovum, and Streptococcus. including Butyrivibrio, Roseburia, Lachnospira, Aceti 0145. In further embodiments, isolated microbial species tomaculum, Coprococcus, Johnsonella, Catonella, may be selected from genera of family Anaerolinaeceae Pseudobutyrivibrio, Syntrophococcus, Sporobacterium, including Aestuariimicrobium, Arachnia, Auraticoccus, Parasporobacterium, Lachnobacterium, Shuttleworthia, Brooklawnia, Friedmanniella, Granulicoccus, Luteococcus, Dorea, Anaerostipes, Hespellia, Marvinbryantia, Oribacte Mariniluteicoccus, Microlunatus, Microprulina, Nauman rium, Morvella, Blautia, Robinsoniella, Cellulosilyticum, nella, Propionibacterium, Propionicicella, Propioniciclava, Lachnoanaerobaculum, Stomatobaculum, Fusicatenibacter, Propioniferax, Propionimicrobium, and Tessaracoccus. Acetatifactor; and Eisenbergiella. 0146 In further embodiments, isolated microbial species 0133. In further embodiments, isolated microbial species may be selected from genera of family Prevotellaceae, may be selected from genera of family Acidaminococca including ParaprevOtella, Prevotella, hallella, Xvlanibacter, ceae, including Acidaminococcus, Phascolarctobacterium, and AlloprevOtella. Succiniclasticum, and Succinispira. 0.147. In further embodiments, isolated microbial species 0134. In further embodiments, isolated microbial species may be selected from genera of family Neocallimastigaceae, may be selected from genera of family Peptococcaceae, including Anaeromyces, Caecomyces, Cyllamyces, Neocal including Desulfotomaculum, Peptococcus, Desulfitobacte limastix, Orpinomyces, and Piromyces. rium, Syntrophobotulus, Dehalobacter; Sporotomaculum, 0.148. In further embodiments, isolated microbial species Desulfosporosinus, Desulfonispora, Pelotomaculum, Ther may be selected from genera of family Saccharomycetaceae, mincola, Cryptanaerobacter, Desulfitibacter, Candidatus including Brettanomyces, Candida, Citeromyces, Cyniclo Desulforudis, Desulfurispora, and Desulfitospora. myces, Debaryomyces, Issatchenkia, Kazachstania (syn. 0135) In further embodiments, isolated microbial species Arxiozyma), Kluyveromyces, Komagataella, Kuraishia, may be selected from genera of family Porphy Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, romonadaceae, including Porphyromonas, Dysgonomonas, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Tannerella, Odoribacter, Proteinphilum, Petrimonas, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharo Paludibacter; Parabacteroides, Barnesiella, Candidatus myces, and Zygotorulaspora. Vestibaculum, Butyricinomas, Macelibacteroides, and 0149. In further embodiments, isolated microbial species Coprobacter: may be selected from genera of family Erysipelotrichaceae, including Erysipelothrix, Solobacterium, Turicibacter, Fae 0136. In further embodiments, isolated microbial species calibaculum, Faecalicoccus, Faecalitalea, Holdemanella, may be selected from genera of family Anaerolinaeceae Holdemania, Dielma, Eggerthia, Erysipelatoclostridium, including Anaerolinea, Bellilinea, Leptolinea, Levilinea, Allobacterium, Breznakia, Bulleidia, Catenibacterium, Longilinea, Ornatilinea, and Pelolinea. Catenisphaera, and Coprobacillus. 0.137 In further embodiments, isolated microbial species 0150. In further embodiments, isolated microbial species may be selected from genera of family Atopobiaceae includ may be selected from genera of family Phaeosphaeriaceae, ing Atopbium and Olsenella. including Barria, Bricookea, Carinispora, Chaetoplea, 0.138. In further embodiments, isolated microbial species Eudarluca, Hadrospora, Isthmosporella, Katumotoa, Lauti may be selected from genera of family Eubacteriaceae tia, Metameris, Mixtura, Neophaeosphaeria, Nodulospha including Acetobacterium, Alkalibacter, Alkalibaculum, eria, Ophiosphaerella, Phaeosphaeris, Phaeosphaeriopsis, Aminicella, Anaerofitstis, Eubacterium, Gardella, and Pseu Setomelanomma, Stagonospora, Teratosphaeria, and doramibacter: Wilmia. 0.139. In further embodiments, isolated microbial species 0151. In further embodiments, isolated microbial species may be selected from genera of family Acholeplasmataceae may be selected from genera of family Botryosphaeriaceae, including Acholeplasma. including Amare nomyces, Aplosporella, Auerswaldiella, 0140. In further embodiments, isolated microbial species Botryosphaeria, Dichomera, Diplodia, Discochora, Dothi may be selected from genera of family Succinivibrionaceae dothia, Dothiorella, Fusicoccum, Granulodiplodia, Guig including Anaerobiospirillum, Ruminobacter. Succinatino nardia, Lasiodiplodia, Leptodothiorella, Leptodothiorella, nas, Succinimonas, and Succinivibrio. Leptoguignardia, Macrophoma, Macrophomina, Nattrassia, 0141. In further embodiments, isolated microbial species Neodeightonia, Neofisicocum, Neoscytalidium, Otthia, may be selected from genera of family Lactobacillaceae Phaeobotryosphaeria, Phomatosphaeropsis, Phyllosticta, including Lactobacillus, Paralactobacillus, Pediococcus, PseudofusicOccum, Saccharata, Sivanesania, and Thyro and Sharpea. Strona. US 2017/O 196922 A1 Jul. 13, 2017

0152. In some embodiments, the disclosure provides iso ferase. In some embodiments, the heterologous polynucle lated microbial species belonging to genera of Clostridium, otide may be operably linked to one or more promoter. Ruminococcus, Roseburia, Hydrogenoanaerobacterium, Saccharofermentans, Papillibacter, Pelotomaculum, Butyri TABLE 4 cicoccus, Tannerella, PrevOtella, Butyricinonas, Piromyces, Candida, Vrystaatia, Orpinomyces, Neocallinastix, and Taxa (largely Genera) of the present disclosure not Phyllosticta. In further embodiments, the disclosure pro known to have been utilized in animal agriculture. vides isolated microbial species belonging to the family of Intestinimonas Anaerolinea Lachnospiraceae, and the order of Saccharomycetales. In Pseudobutyrivibrio Olseneia Etibacterium Catenisphaera further embodiments, the disclosure provides isolated Faecalibacterium Solobacterium microbial species of Candida xylopsoci, Vrystaatia aloei Biatitia Raisonia cola, and Phyllosticta capitalensis. Coprococcus Casaiteia Anaeroplasma Acholepiasma 0153. In some embodiments, a microbe from the taxa Aminiphilus Mitsuokeia disclosed herein are utilized to impart one or more beneficial Alistipes Sharpea properties or improved traits to milk in ruminants. Oscillibacter Neocalinastix Odoribacter Pichia 0154) In some embodiments, the disclosure provides iso Tannereia Candida lated microbial species, selected from the group consisting Hydrogenoanaerobacterium Orpinomyces of Clostridium, Ruminococcus, Roseburia, Hydrogenoan Succini vibrio Sugiyamaeila Ruminobacter Cyllamyces aerobacterium, Saccharofermentans, Papillibacter, Peloto Lachnospira Caecomyces maculum, Butyricicoccus, Tannerella, PrevOtella, Butyrici Sinimarinibacterium Temeiia monas, Piromyces, Pichia, Candida, Vrystaatia, Hydrogenoanaerobacterium Titricibacter Orpinomyces, Neocallinastix, and Phyllosticta. Cliostridium XIVa. Anaerolinea Saccharofermenians Piromyces 0155. In some embodiments, the disclosure provides Butyricicoccus Olseneia novel isolated microbial strains of species, selected from the Papillibacter Cliostridium XICa group consisting of Clostridium, Ruminococcus, Roseburia, Pelotomaculum Erysipelotrichaceae Hydrogenoanaerobacterium, Saccharofermentans, Papilli Lachnospiracea Solobacterium Anaeroplasma Raistonia bacter, Pelotomaculum, Butyricicoccus, Tannerella, Pre Cliostridium Eubacterium votella, Butyricimonas, Piromyces, Pichia, Candida, Vrys Rikenelia Lachnobacterium taatia, Orpinomyces, Neocallinastix, and Phyllosticta. Tannereiia Acholeplasma Particular novel strains of these aforementioned taxonomic Howardelia Selenomonas Butyricimonas Sharpea groups can be found in Table 1 and/or Table 3. Succini vibrio Phyllosticta 0156 Furthermore, the disclosure relates to microbes Ruminobacter Candida xylopsoc Syntrophococcits Candida apicol having characteristics Substantially similar to that of a Pseudobutyrivibrio Saccharomycetales microbe identified in Table 1 or Table 3. Ascomycota Candida rugos 0157. 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 pro duction. Microbial Consortia 0158 For instance, the isolated microbes described in 0.161. In some aspects, the disclosure provides microbial Table 1 and Table 3, or consortia of said microbes, are able consortia comprising a combination of at least any two to increase total milk fat in ruminant milk. The increase can microbes selected from amongst the microbes identified in be quantitatively measured, for example, by measuring the Table 1 and/or Table 3. effect that said microbial application has upon the modula 0162. In certain embodiments, the consortia of the pres tion of total milk fat. ent disclosure comprise two microbes, or three microbes, or 0159. In some embodiments, the isolated microbial four microbes, or five microbes, or six microbes, or seven strains are microbes of the present disclosure that have been microbes, or eight microbes, or nine microbes, or ten or genetically modified. In some embodiments, the genetically more microbes. Said microbes of the consortia are different modified or recombinant microbes comprise polynucleotide microbial species, or different strains of a microbial species. sequences which do not naturally occur in said microbes. In 0163. In some embodiments, the disclosure provides con Some embodiments, the microbes may comprise heterolo sortia, comprising: at least two isolated microbial species gous polynucleotides. In further embodiments, the heterolo belonging to genera of Clostridium, Ruminococcus, Rose gous polynucleotides may be operably linked to one or more buria, Hydrogenoanaerobacterium, Saccharofermentans, polynucleotides native to the microbes. Papillibacter, Pelotomaculum, Butyricicoccus, Tannerella, 0160. In some embodiments, the heterologous polynucle Prevotella, Butyricimonas, Piromyces, Pichia, Candida, otides may be reporter genes or selectable markers. In some Vrystaatia, Orpinomyces, Neocallinastix, and Phyllosticta. embodiments, reporter genes may be selected from any of Particular novel strains of species of these aforementioned the family of fluorescence proteins (e.g., GFP, RFP YFP, and genera can be found in Table 1 and/or Table 3. the like), B-galactosidase, luciferase. In some embodiments, 0164. In some embodiments, the disclosure provides con selectable markers may be selected from neomycin phos sortia, comprising: at least two isolated microbial species, photransferase, hygromycin phosphotransferase, aminogly selected from the group consisting of species of the family coside adenyltransferase, dihydrofolate reductase, aceto of Lachnospiraceae, and the order of Saccharomycetales. lactase synthase, bromoxynil nitrilase, B-glucuronidase, 0.165. In particular aspects, the disclosure provides dihydrogolate reductase, and chloramphenicol acetyltrans microbial consortia, comprising species as grouped in Tables US 2017/O 196922 A1 Jul. 13, 2017 39

5-11. With respect to Tables 5-11, the letters A through I 0170 E Strain designation Ascusb 1801 identified in represent a non-limiting selection of microbes of the present Table 1: disclosure, defined as: 0171 F Strain designation Ascusf 23 identified in Table (0166 A Strain designation Ascusb 7 identified in Table 1: 1: s s G f 24 identified in Table B 3138 identified in 0172 Strain designation Ascus (0167 Strain designation Ascusb 1: Table 1: s (0168 C Strain designation Ascusb 82 identified in 0173 H Strain designation Ascus f 45 identified in Table Table 1: 1; and (0169 D Strain designation Ascusb 119 identified in (0174 I Strain designation Ascus f 15 identified in Table Table 1: 1. TABLE 5

Eight and Nine Strain Consortia

A, B, C, D, E, F, G, H A, B, C, D, E, F, G, I A, B, C, D, E, F, H, I A, B, C, D, E, G, H, I A, B, C, D, F, G, H, I A, B, C, E, F, G, H, I A, B, D, E, F, G, H, I A, C, D, E, F, G, H, I B, C, D, E, F, G, H, I A, B, C, D, E, F, G, H, I

TABLE 6

Seven Strain Consortia

CDHI,— F–FHHE. –T

TABLE 7

Six Strain Consortia

|-F–H O— F–Eo—I —oI ODFE–—IT

TABLE 8

Five Strain Consortia US 2017/O 196922 A1 Jul. 13, 2017

TABLE 9

Four Strain Consortia A, B, C, D A, B, C, E A, B, C, F A, B, C, G A, B, C, H, A, B, C, I A, B, D, E A, B, D, F D, G, H, I A, B, D, G A, B, D, H A, B, D, I A, B, E, F A, B, E, G A, B, E, H A, B, E, I A, B, F, G E, F, G, H A, B, F. H. A. D. F., H A, D, F, I A, D, G, H A, D, G, I A, D, H, I A, E, F, G A, E, F, H E, F, G, I A, B, F, I A, B, G, H A, B, G, I A, B, H, I A, C, D, E A, C, D, F A, C, D, G A, C, D, H E, F, H, I A, C, D, I A, C, E, F A, C, E, G A, C, E, H A, C, E, I A, C, F, G A, C, F, H A, C, F, I E, G, H, I A, C, G, H A, C, G, I A, C, H, I A, D, E, F A, D, E, G A, D, E, H A, D, E, I A, D, F, G F, G, H, I A, E, F, I A, E, G, H A, E, G, I A, E, H, I A, F, G, H A, F, G, I A, F, H, I A, G, H, I D, E, F, H B, C, D, E B, C, D, F B, C, D, G B, C, D, H B, C, D, I B, C, E, F B, C, E, G, B, C, E, H D, E, F, I B, C, E, I B, C, F, G B, C, F, H B, C, F, I B, C, G, H B, C, G, I B, C, H, I B, D, E, F D, E, G, H B, D, E, G, B, D, E, H B, D, E, I B, D, F, G B, D, F, H B, D, F, I B, D, G, H B, D, G, I D, E, G, I B, D, H, I B, E, F, G B, E, F, H B, E, F, I B, E, G, H B, E, G, I B, E, H, I B, F, G, H D, E, H, I B, F, G, I B, F, H, I B, G, H, I C, D, E, F C, D, E, G C, D, E, H C, D, E, I C, D, F, G D, F, G, H C, D, F, H C, D, F, I C, D, G, H C, D, G, I C, D, H, I C, E, F, G C, E, F, H C, E, F, I D, F, G, I C, E, G, H C, E, G, I C, E, H, I C, F, G, H C, F, G, I C, F, H, I C, G, H, I D, E, F, G D, F, H, I

TABLE 10

Three Strain Consortia A, B, C A, B, D A, B, E A, B, F A, B, G A, B, H A, B, I A, C, D A, C, E, G, H, I E, F, H A, C, F A, C, G A, C, H, A, C, I A, D, E A, D, F A, D, G A, D, H A, D, I F, H, I E, F, G A, E, F A, E, G A. E. H. A. E., I A, F, G A, F, H A, F, I A, G, H A, G, I F, G, I D, H, I A, H, I B, C, D B, C, E B, C, F B, C, G B, C, H B, C, I B, D, E B, D, F F, G, H D, G, I B, D, G B, D, H B, D, I B, E, F B, E, G, B, E, H B, E, I B, F, G B. F., H E, H, I E, F, I B, F, I B, G, H B, G, I B, H, I C, D, E C, D, F C, D, G C, D, H C, D, I E, G, I D, G, H C, E, F C, E, G C, E, H C, E, I C, F, G C, F, H C, F, I C, G, H C, G, I E, G, H D, F, I C, H, I D, E, F D, E, G D, E, H D, E, I D, F, G D, F, H

TABLE 11

Two Strain Consortia A, B A, C A, D A E A, F A, G A H A, I B, C B, D B, E B, F B, G. B., H B, I C, D C, E C, F C, G C, H. C., I D, E D, F D, G D, H D, I E, F E, G E H E, I F, G F. H F, I G, H G, I H, I

0.175. In some embodiments, the microbial consortia may American Society for Microbiology, Washington, D.C. be selected from any member group from Tables 5-11. (1980), each of which is incorporated by reference. 0180 Isolation can be effected by streaking the specimen Isolated Microbes—Source Material on a Solid medium (e.g., nutrient agar plates) to obtain a single colony, which is characterized by the phenotypic traits 0176 The microbes of the present disclosure were described hereinabove (e.g., Gram positive/negative, obtained, among other places, at various locales in the capable of forming spores aerobically/anaerobically, cellular United States from the gastrointestinal tract of cows. morphology, carbon Source metabolism, acid/base produc (0177. Isolated Microbes Microbial Culture Techniques tion, enzyme secretion, metabolic secretions, etc.) and to 0.178 The microbes of Table 1 and Table 3 were matched reduce the likelihood of working with a culture which has to their nearest taxonomic groups by utilizing classification become contaminated. tools of the Ribosomal Database Project (RDP) for 16s 0181 For example, for microbes of the disclosure, bio rRNA sequences and the User-friendly Nordic ITS Ectomy logically pure isolates can be obtained through repeated corrhiza (UNITE) database for ITS rRNA sequences. subculture of biological samples, each subculture followed Examples of matching microbes to their nearest taxa may be by streaking onto Solid media to obtain individual colonies found in Lan et al. (2012. PLOS one. 7(3):e32491), Schloss or colony forming units. Methods of preparing, thawing, and and Westcott (2011. Appl. Environ. Microbiol. 77(10):3219 growing lyophilized bacteria are commonly known, for 3226), and Koljalg et al. (2005. New Phytologist. 166(3): example, Gherna, R. L. and C. A. Reddy. 2007. Culture 1063-1068). Preservation, p 1019-1033. In C. A. Reddy, T. J. Beveridge, 0179 The isolation, identification, and culturing of the J. A. Breznak, G. A. Marzluf, T. M. Schmidt, and L. R. microbes of the present disclosure can be effected using Snyder, eds. American Society for Microbiology, Washing standard microbiological techniques. Examples of Such ton, D.C., 1033 pages; herein incorporated by reference. techniques may be found in Gerhardt, P. (ed.) Methods for Thus freeze dried liquid formulations and cultures stored General and Molecular Microbiology. American Society for long term at -70° C. in Solutions containing glycerol are Microbiology, Washington, D.C. (1994) and Lennette, E. H. contemplated for use in providing formulations of the pres (ed.) Manual of Clinical Microbiology. Third Edition. ent disclosure. US 2017/O 196922 A1 Jul. 13, 2017

0182. The microbes of the disclosure can be propagated ciated that the microbial strains may be cultured together in a liquid medium under aerobic conditions, or alternatively when compatible culture conditions can be employed. anaerobic conditions. Medium for growing the bacterial strains of the present disclosure includes a carbon Source, a Isolated Microbes—Microbial Strains nitrogen Source, and inorganic salts, as well as specially 0.184 Microbes can be distinguished into a genus based required Substances such as vitamins, amino acids, nucleic on polyphasic taxonomy, which incorporates all available acids and the like. Examples of Suitable carbon Sources phenotypic and genotypic data into a consensus classifica which can be used for growing the microbes include, but are tion (Vandamme et al. 1996. Polyphasic taxonomy, a con not limited to, starch, peptone, yeast extract, amino acids, sensus approach to bacterial systematics. Microbial Rev Sugars such as glucose, arabinose, mannose, glucosamine, 1996, 60:407-438). One accepted genotypic method for maltose, and the like; salts of organic acids such as acetic defining species is based on overall genomic relatedness, acid, fumaric acid, adipic acid, propionic acid, citric acid, such that strains which share approximately 70% or more gluconic acid, malic acid, pyruvic acid, malonic acid and the relatedness using DNA-DNA hybridization, with 5° C. or like; alcohols such as ethanol and glycerol and the like; oil less AT, (the difference in the melting temperature between or fat such as soybean oil, rice bran oil, olive oil, corn oil, homologous and heterologous hybrids), understandard con sesame oil. The amount of the carbon source added varies ditions, are considered to be members of the same species. according to the kind of carbon Source and is typically Thus, populations that share greater than the aforementioned between 1 to 100 gram(s) per liter of medium. Preferably, 70% threshold can be considered to be variants of the same glucose, starch, and/or peptone is contained in the medium species. Another accepted genotypic method for defining as a major carbon source, at a concentration of 0.1-5% species is to isolate marker genes of the present disclosure, (WV). Examples of suitable nitrogen sources which can be sequence these genes, and align these sequenced genes from used for growing the bacterial strains of the present disclo multiple isolates or variants. The microbes are interpreted as Sure include, but are not limited to, amino acids, yeast belonging to the same species if one or more of the extract, tryptone, beef extract, peptone, potassium nitrate, sequenced genes share at least 97% sequence identity. ammonium nitrate, ammonium chloride, ammonium sulfate, 0185. The 16S or 18S rRNA sequences or ITS sequences ammonium phosphate, ammonia or combinations thereof. are often used for making distinctions between species and The amount of nitrogen source varies according to the type strains, in that if one of the aforementioned sequences share of nitrogen source, typically between 0.1 to 30 gram per liter less than a specified percent sequence identity from a of medium. The inorganic salts, potassium dihydrogen phos reference sequence, then the two organisms from which the phate, dipotassium hydrogen phosphate, disodium hydrogen sequences were obtained are said to be of different species phosphate, magnesium Sulfate, magnesium chloride, ferric or strains. sulfate, ferrous sulfate, ferric chloride, ferrous chloride, 0186 Thus, one could consider microbes to be of the manganous Sulfate, manganous chloride, Zinc sulfate, Zinc same species, if they share at least 80%, 85%, 90%, 95%, chloride, cupric Sulfate, calcium chloride, Sodium chloride, 97%, 98%, or 99% sequence identity across the 16S or 18S calcium carbonate, sodium carbonate can be used alone or in rRNA sequence, or the ITS1 or ITS2 sequence. combination. The amount of inorganic acid varies according 0187 Further, one could define microbial strains of a to the kind of the inorganic salt, typically between 0.001 to species, as those that share at least 80%, 85%, 90%, 95%, 10 gram per liter of medium. Examples of specially required 97%, 98%, or 99% sequence identity across the 16S or 18S Substances include, but are not limited to, Vitamins, nucleic rRNA sequence, or the ITS1 or ITS2 sequence. acids, yeast extract, peptone, meat extract, malt extract, 0188 In one embodiment, microbial strains of the present dried yeast and combinations thereof. Cultivation can be disclosure include those that comprise polynucleotide effected at a temperature, which allows the growth of the sequences that share at least 70%, 75%, 80%, 81%, 82%, microbial strains, essentially, between 20° C. and 46°C. In 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, some aspects, a temperature range is 30° C.-39° C. For 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence optimal growth, in some embodiments, the medium can be identity with any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, adjusted to pH 6.0–7.4. It will be appreciated that commer 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, cially available media may also be used to culture the 25, 26, 27, 28, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, microbial strains, such as Nutrient Broth or Nutrient Agar 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, available from Difco, Detroit, Mich. It will be appreciated 57, 58, 59, 60, 2045, 2046, 2047, 2048, 2049, 2050, 2051, that cultivation time may differ depending on the type of 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, culture medium used and the concentration of Sugar as a 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, major carbon source. 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 0183 In some aspects, cultivation lasts between 24-96 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, hours. Microbial cells thus obtained are isolated using 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, methods, which are well known in the art. Examples include, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, but are not limited to, membrane filtration and centrifugal 2106, and 2107. In a further embodiment, microbial strains separation. The pH may be adjusted using sodium hydroxide of the present disclosure include those that comprise poly and the like and the culture may be dried using a freeze nucleotide sequences that share at least 70%, 75%, 80%, dryer, until the water content becomes equal to 4% or less. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, Microbial co-cultures may be obtained by propagating each 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% strain as described hereinabove. In some aspects, microbial sequence identity with any one of SEQ ID NOS:1-2107. multi-strain cultures may be obtained by propagating two or 0189 Comparisons may also be made with 23S rRNA more of the strains described hereinabove. It will be appre sequences against reference sequences. US 2017/O 196922 A1 Jul. 13, 2017 42

0190. Unculturable microbes often cannot be assigned to present disclosure comprise bacteria in the absence of fungi. a definite species in the absence of a phenotype determina In some embodiments, compositions of the present disclo tion, the microbes can be given a candidatus designation Sure comprise fungi in the absence of bacteria. within a genus provided their 16S or 18S rRNA sequences 0196. Bacterial spores may include endospores and aki or ITS sequences subscribes to the principles of identity with netes. Fungal spores may include statismospores, ballis known species. tospores, autospores, aplanospores, Zoospores, mitospores, 0191) One approach is to observe the distribution of a megaspores, microspores, meiospores, chlamydospores, large number of strains of closely related species in sequence urediniospores, teliospores, oospores, carpospores, tet space and to identify clusters of strains that are well resolved raspores, sporangiospores, Zygospores, ascospores, basidi from other clusters. This approach has been developed by ospores, ascospores, and asciospores. using the concatenated sequences of multiple core (house 0.197 In some embodiments, spores of the composition keeping) genes to assess clustering patterns, and has been germinate upon administration to animals of the present called multilocus sequence analysis (MLSA) or multilocus disclosure. In some embodiments, spores of the composition sequence phylogenetic analysis. MLSA has been used Suc germinate only upon administration to animals of the present cessfully to explore clustering patterns among large numbers disclosure. of strains assigned to very closely related species by current taxonomic methods, to look at the relationships between Microbial Compositions Small numbers of strains within a genus, or within a broader 0.198. In some embodiments, the microbes of the disclo taxonomic grouping, and to address specific taxonomic Sure are combined into microbial compositions. questions. More generally, the method can be used to ask 0199. In some embodiments, the microbial compositions whether bacterial species exist—that is, to observe whether include ruminant feed, such as cereals (barley, maize, oats, large populations of similar strains invariably fall into well and the like); starches (tapioca and the like); oilseed cakes; resolved clusters, or whether in Some cases there is a genetic and vegetable wastes. In some embodiments, the microbial continuum in which clear separation into clusters is not compositions include vitamins, minerals, trace elements, observed. emulsifiers, aromatizing products, binders, colorants, odor 0.192 In order to more accurately make a determination ants, thickening agents, and the like. of genera, a determination of phenotypic traits, such as 0200. In some embodiments, the microbial compositions morphological, biochemical, and physiological characteris of the present disclosure are solid. Where solid compositions tics are made for comparison with a reference genus arche are used, it may be desired to include one or more carrier type. The colony morphology can include color, shape, materials including, but not limited to: mineral earths such pigmentation, production of slime, etc. Features of the cell as silicas, talc, kaolin, limestone, chalk, clay, dolomite, are described as to shape, size, Gram reaction, extracellular diatomaceous earth; calcium sulfate; magnesium sulfate; material, presence of endospores, flagella presence and magnesium oxide; products of vegetable origin such as location, motility, and inclusion bodies. Biochemical and cereal meals, tree bark meal, wood meal, and nutshell meal. physiological features describe growth of the organism at 0201 In some embodiments, the microbial compositions different ranges oftemperature, pH, salinity and atmospheric of the present disclosure are liquid. In further embodiments, conditions, growth in presence of different sole carbon and the liquid comprises a solvent that may include water or an nitrogen sources. One of ordinary skill in the art would be alcohol, and other animal-safe solvents. In some embodi reasonably apprised as to the phenotypic traits that define the ments, the microbial compositions of the present disclosure genera of the present disclosure. include binders such as animal-safe polymers, carboxym 0193 In one embodiment, the microbes taught herein ethylcellulose, starch, polyvinyl alcohol, and the like. were identified utilizing 16S rRNA gene sequences and ITS 0202 In some embodiments, the microbial compositions sequences. It is known in the art that 16S rRNA contains of the present disclosure comprise thickening agents such as hyperVariable regions that can provide species/strain-spe silica, clay, natural extracts of seeds or seaweed, synthetic cific signature sequences useful for bacterial identification, derivatives of cellulose, guar gum, locust bean gum, alg and that ITS sequences can also provide species/strain inates, and methylcelluloses. In some embodiments, the specific signature sequences useful for fungal identification. microbial compositions comprise anti-settling agents such 0194 Phylogenetic analysis using the rRNA genes and/or as modified Starches, polyvinyl alcohol, Xanthan gum, and ITS sequences are used to define “substantially similar the like. species belonging to common genera and also to define 0203. In some embodiments, the microbial compositions “Substantially similar strains of a given taxonomic species. of the present disclosure comprise colorants including Furthermore, physiological and/or biochemical properties of organic chromophores classified as nitroso; nitro; azo, the isolates can be utilized to highlight both minor and including monoazo, bisazo and polyazo; acridine, anthra significant differences between strains that could lead to quinone, azine, diphenylmethane, indamine, indophenol, advantageous behavior in ruminants. methine, oxazine, phthalocyanine, thiazine, thiazole, triaryl 0.195 Compositions of the present disclosure may methane, Xanthene. In some embodiments, the microbial include combinations of fungal spores and bacterial spores, compositions of the present disclosure comprise trace nutri fungal spores and bacterial vegetative cells, fungal vegeta ents such as salts of iron, manganese, boron, copper, cobalt, tive cells and bacterial spores, fungal vegetative cells and molybdenum and zinc. bacterial vegetative cells. In some embodiments, composi 0204. In some embodiments, the microbial compositions tions of the present disclosure comprise bacteria only in the of the present disclosure comprise an animal-safe virucide or form of spores. In some embodiments, compositions of the nematicide. present disclosure comprise bacteria only in the form of 0205. In some embodiments, microbial compositions of vegetative cells. In some embodiments, compositions of the the present disclosure comprise Saccharides (e.g., monosac US 2017/O 196922 A1 Jul. 13, 2017

charides, disaccharides, trisaccharides, polysaccharides, oli 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, gosaccharides, and the like), polymeric saccharides, lipids, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, polymeric lipids, lipopolysaccharides, proteins, polymeric 55, 56, 57, 58, 59, or 60 weeks. proteins, lipoproteins, nucleic acids, nucleic acid polymers, 0210. In some embodiments, the microbial compositions silica, inorganic salts and combinations thereof. In a further are shelf stable at room temperature (68-72 F.) or between embodiment, microbial compositions comprise polymers of 50-77 F. for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, agar, agarose, gelrite, gellan gumand the like. In some 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, embodiments, microbial compositions comprise plastic cap 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, Sules, emulsions (e.g., water and oil), membranes, and 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, artificial membranes. In some embodiments, emulsions or 59, or 60 days. In some embodiments, the microbial com linked polymer Solutions may comprise microbial compo positions are shelf stable at room temperature (68-72 F.) or sitions of the present disclosure. See Harel and Bennett (U.S. between 50-77 F. fora period of at least 1, 2, 3, 4, 5, 6, 7, Pat. No. 8,460,726B2). 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 0206. In some embodiments, microbial compositions of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, the present disclosure occur in a Solid form (e.g., dispersed 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, lyophilized spores) or a liquid form (microbes interspersed 57, 58, 59, or 60 weeks. in a storage medium). 0211. In some embodiments, the microbial compositions 0207. In some embodiments, microbial compositions of are shelf stable at -23-35° F. for a period of at least 1, 2, 3, the present disclosure comprise one or more preservatives. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, The preservatives may be in liquid or gas formulations. The 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, preservatives may be selected from one or more of mono 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, saccharide, disaccharide, trisaccharide, polysaccharide, ace 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the tic acid, ascorbic acid, calcium ascorbate, erythorbic acid, microbial compositions are shelf stable at -23-35° F. for a iso-ascorbic acid, erythrobic acid, potassium nitrate, sodium period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. ascorbate, Sodium erythorbate, sodium iso-ascorbate, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, Sodium nitrate, Sodium nitrite, nitrogen, benzoic acid, cal 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, cium Sorbate, ethyl lauroyl arginate, methyl-p-hydroxyben 47, 48, 49, 50, 51, 52,53,54, 55,56, 57,58, 59, or 60 weeks. Zoate, methyl paraben, potassium acetate, potassium benzo 0212. In some embodiments, the microbial compositions iate, potassium bisulphite, potassium diacetate, potassium are shelf stable at 77-100°F. for a period of at least 1, 2, 3, lactate, potassium metabisulphite, potassium Sorbate, pro 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, pyl-p-hydroxy benzoate, propyl paraben, Sodium acetate, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, Sodium benzoate, sodium bisulphite, sodium nitrite, sodium 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, diacetate, sodium lactate, Sodium metabisulphite, sodium 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the salt of methyl-p-hydroxybenzoic acid, sodium salt of pro microbial compositions are shelf stable at 77-100°F. for a pyl-p-hydroxy benzoic acid, sodium Sulphate, sodium period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. Sulfite, Sodium dithionite, Sulphurous acid, calcium propi 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, onate, dimethyl dicarbonate, natamycin, potassium Sorbate, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, potassium bisulfite, potassium metabisulfite, propionic acid, 47, 48, 49, 50, 51, 52,53,54, 55,56, 57,58, 59, or 60 weeks. Sodium diacetate, sodium propionate, Sodium Sorbate, Sorbic 0213. In some embodiments, the microbial compositions acid, ascorbic acid, ascorbyl palmitate, ascorbyl Stearate, are shelf stable at 101-213 F. for a period of at least 1, 2, butylated hydro-xyanisole, butylated hydroxytoluene 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, (BHT), butylated hydroxyl anisole (BHA), citric acid, citric 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, acid esters of mono- and/or diglycerides, L-cysteine, L-cys 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, teine hydrochloride, gum guaiacum, gum guaiac, lecithin, 53, 54, 55, 56, 57,58, 59, or 60 days. In some embodiments, lecithin citrate, monoglyceride citrate, monoisopropyl cit the microbial compositions are shelf stable at 101-213° F. rate, propyl gallate, Sodium metabisulphite, tartaric acid, fora period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, tertiary butyl hydroquinone, Stannous chloride, thiodipropi 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, onic acid, dilauryl thiodipropionate, distearyl thiodipropi 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, onate, ethoxyquin, Sulfur dioxide, formic acid, or tocopherol 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 (s). weeks. 0208. In some embodiments, microbial compositions of 0214. In some embodiments, the microbial compositions the present disclosure include bacterial and/or fungal cells in of the present disclosure are shelf stable at refrigeration spore form, vegetative cell form, and/or lysed cell form. In temperatures (35-40° F.), at room temperature (68-72 F.), one embodiment, the lysed cell form acts as a mycotoxin between 50-77 F., between -23-35° F., between 70-100°F., binder, e.g. mycotoxins binding to dead cells. or between 101-213° F for a period of about 1 to 100, about 0209. In some embodiments, the microbial compositions 1 to 95, about 1 to 90, about 1 to 85, about 1 to 80, about 1 are shelf stable in a refrigerator (35-40 F) for a period of to 75, about 1 to 70, about 1 to 65, about 1 to 60, about 1 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 55, about 1 to 50, about 1 to 45, about 1 to 40, about 1 to 35, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, about 1 to 30, about 1 to 25, about 1 to 20, about 1 to 15, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, about 1 to 10, about 1 to 5, about 5 to 100, about 5 to 95, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some about 5 to 90, about 5 to 85, about 5 to 80, about 5 to 75, embodiments, the microbial compositions are shelf stable in about 5 to 70, about 5 to 65, about 5 to 60, about 5 to 55, a refrigerator (35-40°F) for a period of at least 1, 2, 3, 4, 5, about 5 to 50, about 5 to 45, about 5 to 40, about 5 to 35, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, about 5 to 30, about 5 to 25, about 5 to 20, about 5 to 15, US 2017/O 196922 A1 Jul. 13, 2017 44 about 5 to 10, about 10 to 100, about 10 to 95, about 10 to 35, 20 to 30, 20 to 25, 25 to 100, 25 to 95, 25 to 90, 25 to 90, about 10 to 85, about 10 to 80, about 10 to 75, about 10 85, 25 to 80, 25 to 75, 25 to 70, 25 to 65, 25 to 60, 25 to 55, to 70, about 10 to 65, about 10 to 60, about 10 to 55, about 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 100, 10 to 50, about 10 to 45, about 10 to 40, about 10 to 35, about 30 to 95, 30 to 90, 30 to 85, 30 to 80, 30 to 75, 30 to 70, 30 10 to 30, about 10 to 25, about 10 to 20, about 10 to 15, about to 65, 30 to 60, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 15 to 100, about 15 to 95, about 15 to 90, about 15 to 85, 35, 35 to 100, 35 to 95, 35 to 90, 35 to 85, 35 to 80, 35 to about 15 to 80, about 15 to 75, about 15 to 70, about 15 to 75, 35 to 70, 35 to 65, 35 to 60, 35 to 55, 35 to 50, 35 to 45, 65, about 15 to 60, about 15 to 55, about 15 to 50, about 15 35 to 40, 40 to 100, 40 to 95, 40 to 90, 40 to 85, 40 to 80, to 45, about 15 to 40, about 15 to 35, about 15 to 30, about 40 to 75, 40 to 70, 40 to 65, 40 to 60, 40 to 55, 40 to 50, 40 15 to 25, about 15 to 20, about 20 to 100, about 20 to 95, to 45, 45 to 100, 45 to 95, 45 to 90, 45 to 85, 45 to 80, 45 about 20 to 90, about 20 to 85, about 20 to 80, about 20 to to 75, 45 to 70, 45 to 65, 45 to 60, 45 to 55, 45 to 50, 50 to 75, about 20 to 70, about 20 to 65, about 20 to 60, about 20 100, 50 to 95, 50 to 90, 50 to 85, 50 to 80, 50 to 75, 50 to to 55, about 20 to 50, about 20 to 45, about 20 to 40, about 70, 50 to 65, 50 to 60, 50 to 55, 55 to 100, 55 to 95, 55 to 20 to 35, about 20 to 30, about 20 to 25, about 25 to 100, 90, 55 to 85, 55 to 80,55 to 75, 55 to 70, 55 to 65, 55 to 60, about 25 to 95, about 25 to 90, about 25 to 85, about 25 to 60 to 100, 60 to 95, 60 to 90, 60 to 85, 60 to 80, 60 to 75, 80, about 25 to 75, about 25 to 70, about 25 to 65, about 25 60 to 70, 60 to 65, 65 to 100, 65 to 95, 65 to 90, 65 to 85, to 60, about 25 to 55, about 25 to 50, about 25 to 45, about 65 to 80, 65 to 75, 65 to 70, 70 to 100, 70 to 95, 70 to 90, 25 to 40, about 25 to 35, about 25 to 30, about 30 to 100, 70 to 85, 70 to 80, 70 to 75, 75 to 100, 75 to 95, 75 to 90, about 30 to 95, about 30 to 90, about 30 to 85, about 30 to 75 to 85, 75 to 80, 80 to 100, 80 to 95, 80 to 90, 80 to 85, 80, about 30 to 75, about 30 to 70, about 30 to 65, about 30 85 to 100, 85 to 95, 85 to 90, 90 to 100, 90 to 95, or 95 to to 60, about 30 to 55, about 30 to 50, about 30 to 45, about 100 weeks. 30 to 40, about 30 to 35, about 35 to 100, about 35 to 95, 0216. In some embodiments, the microbial compositions about 35 to 90, about 35 to 85, about 35 to 80, about 35 to of the present disclosure are shelf stable at refrigeration 75, about 35 to 70, about 35 to 65, about 35 to 60, about 35 temperatures (35-40° F.), at room temperature (68-72 F.), to 55, about 35 to 50, about 35 to 45, about 35 to 40, about between 50-77 F., between -23-35° F., between 70-100°F., 40 to 100, about 40 to 95, about 40 to 90, about 40 to 85, or between 101-213° F. for a period of about 1 to 36, about about 40 to 80, about 40 to 75, about 40 to 70, about 40 to 1 to 34, about 1 to 32, about 1 to 30, about 1 to 28, about 1 65, about 40 to 60, about 40 to 55, about 40 to 50, about 40 to 26, about 1 to 24, about 1 to 22, about 1 to 20, about 1 to to 45, about 45 to 100, about 45 to 95, about 45 to 90, about 18, about 1 to 16, about 1 to 14, about 1 to 12, about 1 to 10, 45 to 85, about 45 to 80, about 45 to 75, about 45 to 70, about about 1 to 8, about 1 to 6, about 1 one 4, about 1 to 2, about 45 to 65, about 45 to 60, about 45 to 55, about 45 to 50, about 4 to 36, about 4 to 34, about 4 to 32, about 4 to 30, about 4 50 to 100, about 50 to 95, about 50 to 90, about 50 to 85, to 28, about 4 to 26, about 4 to 24, about 4 to 22, about 4 to about 50 to 80, about 50 to 75, about 50 to 70, about 50 to 20, about 4 to 18, about 4 to 16, about 4 to 14, about 4 to 12, 65, about 50 to 60, about 50 to 55, about 55 to 100, about about 4 to 10, about 4 to 8, about 4 to 6, about 6 to 36, about 55 to 95, about 55 to 90, about 55 to 85, about 55 to 80, about 6 to 34, about 6 to 32, about 6 to 30, about 6 to 28, about 6 55 to 75, about 55 to 70, about 55 to 65, about 55 to 60, about to 26, about 6 to 24, about 6 to 22, about 6 to 20, about 6 to 60 to 100, about 60 to 95, about 60 to 90, about 60 to 85, 18, about 6 to 16, about 6 to 14, about 6 to 12, about 6 to 10, about 60 to 80, about 60 to 75, about 60 to 70, about 60 to about 6 to 8, about 8 to 36, about 8 to 34, about 8 to 32, about 65, about 65 to 100, about 65 to 95, about 65 to 90, about 8 to 30, about 8 to 28, about 8 to 26, about 8 to 24, about 8 65 to 85, about 65 to 80, about 65 to 75, about 65 to 70, about to 22, about 8 to 20, about 8 to 18, about 8 to 16, about 8 to 70 to 100, about 70 to 95, about 70 to 90, about 70 to 85, 14, about 8 to 12, about 8 to 10, about 10 to 36, about 10 to about 70 to 80, about 70 to 75, about 75 to 100, about 75 to 34, about 10 to 32, about 10 to 30, about 10 to 28, about 10 95, about 75 to 90, about 75 to 85, about 75 to 80, about 80 to 26, about 10 to 24, about 10 to 22, about 10 to 20, about to 100, about 80 to 95, about 80 to 90, about 80 to 85, about 10 to 18, about 10 to 16, about 10 to 14, about 10 to 12, about 85 to 100, about 85 to 95, about 85 to 90, about 90 to 100, 12 to 36, about 12 to 34, about 12 to 32, about 12 to 30, about about 90 to 95, or 95 to 100 weeks 12 to 28, about 12 to 26, about 12 to 24, about 12 to 22, about 0215. In some embodiments, the microbial compositions 12 to 20, about 12 to 18, about 12 to 16, about 12 to 14, about of the present disclosure are shelf stable at refrigeration 14 to 36, about 14 to 34, about 14 to 32, about 14 to 30, about temperatures (35-40 F), at room temperature (68-72 F.), 14 to 28, about 14 to 26, about 14 to 24, about 14 to 22, about between 50-77 F., between -23-35° F., between 70-100°F., 14 to 20, about 14 to 18, about 14 to 16, about 16 to 36, about or between 101-213° F. for a period of 1 to 100, 1 to 95, 1 16 to 34, about 16 to 32, about 16 to 30, about 16 to 28, about to 90, 1 to 85, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 16 to 26, about 16 to 24, about 16 to 22, about 16 to 20, about to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 16 to 18, about 18 to 36, about 18 to 34, about 18 to 32, about to 20, 1 to 15, 1 to 10, 1 to 5, 5 to 100, 5 to 95, 5 to 90, 5 18 to 30, about 18 to 28, about 18 to 26, about 18 to 24, about to 85, 5 to 80, 5 to 75, 5 to 70, 5 to 65, 5 to 60, 5 to 55, 5 18 to 22, about 18 to 20, about 20 to 36, about 20 to 34, about to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 20 to 32, about 20 to 30, about 20 to 28, about 20 to 26, about to 15, 5 to 10, 10 to 100, 10 to 95, 10 to 90, 10 to 85, 10 to 20 to 24, about 20 to 22, about 22 to 36, about 22 to 34, about 80, 10 to 75, 10 to 70, 10 to 65, 10 to 60, 10 to 55, 10 to 50, 22 to 32, about 22 to 30, about 22 to 28, about 22 to 26, about 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 22 to 24, about 24 to 36, about 24 to 34, about 24 to 32, about to 15, 15 to 100, 15 to 95, 15 to 90, 15 to 85, 15 to 80, 15 24 to 30, about 24 to 28, about 24 to 26, about 26 to 36, about to 75, 15 to 70, 15 to 65, 15 to 60, 15 to 55, 15 to 50, 15 to 26 to 34, about 26 to 32, about 26 to 30, about 26 to 28, about 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 100, 28 to 36, about 28 to 34, about 28 to 32, about 28 to 30, about 20 to 95, 20 to 90, 20 to 85, 20 to 80, 20 to 75, 20 to 70, 20 30 to 36, about 30 to 34, about 30 to 32, about 32 to 36, about to 65, 20 to 60, 20 to 55, 20 to 50, 20 to 45, 20 to 40, 20 to 32 to 34, or about 34 to 36 months. US 2017/O 196922 A1 Jul. 13, 2017

0217. In some embodiments, the microbial compositions terms “normally solid’ and “normally liquid” as used herein of the present disclosure are shelf stable at refrigeration refer to the state of a material at desired temperatures for temperatures (35-40 F), at room temperature (68-72 F.), storing the resulting microcapsules. Since fats and hydro between 50-77 F., between -23-35° F., between 70-100°F., genated oils do not, strictly speaking, have melting points, or between 101-213° F for a period of 1 to 361 to 341 to the term “melting point' is used herein to describe the 32 1 to 301 to 281 to 261 to 241 to 22 1 to 20 1 to 181 minimum temperature at which the fusible material becomes to 16 1 to 14 1 to 12 1 to 10 1 to 8 1 to 61 one 4 1 to 24 sufficiently softened or liquid to be successfully emulsified to 364 to 344 to 32 4 to 30 4 to 28 4 to 264 to 24 4 to 22 and spray cooled, thus roughly corresponding to the maxi 4 to 204 to 18 4 to 16 4 to 14 4 to 12 4 to 10 4 to 8 4 to mum temperature at which the shell material has sufficient 66 to 36 6 to 34 6 to 32 6 to 30 6 to 28 6 to 266 to 24 6 integrity to prevent release of the choline cores. “Melting to 22 6 to 20 6 to 18 6 to 16 6 to 14 6 to 12 6 to 10 6 to 8 point” is similarly defined herein for other materials which 8 to 36 8 to 348 to 328 to 308 to 288 to 268 to 24 8 to do not have a sharp melting point. 228 to 208 to 18 8 to 168 to 14 8 to 128 to 10 10 to 36 0222 Specific examples of fats and oils useful herein 10 to 34 10 to 32 10 to 30 10 to 28 10 to 26 10 to 24 10 to (some of which require hardening) are as follows: animal 22 10 to 2010 to 1810 to 16 10 to 14 10 to 12 12 to 36 12 oils and fats, such as beef tallow, mutton tallow, lamb tallow, to 34 12 to 32 12 to 30 12 to 28 12 to 26 12 to 24 12 to 22 lard or pork fat, fish oil, and sperm oil; vegetable oils, such 12 to 2012 to 1812 to 16 12 to 14 14 to 36 14 to 34 14 to as canola oil, cottonseed oil, peanut oil, corn oil, olive oil, 32 14 to 30 14 to 28 14 to 26 14 to 24 14 to 22 14 to 2014 Soybean oil, Sunflower oil, safflower oil, coconut oil, palm to 1814 to 1616 to 36 16 to 34 16 to 32 16 to 30 16 to 28 oil, linseed oil, tung oil, and castor oil; fatty acid monoglyc 16 to 26 16 to 24 16 to 22 16 to 20 16 to 1818 to 36 18 to erides and diglycerides; free fatty acids, such as Stearic acid, 34 18 to 32 18 to 30 18 to 28 18 to 26 18 to 24, 18 to 22 18 palmitic acid, and oleic acid; and mixtures thereof. The to 2020 to 36 20 to 34 20 to 32 20 to 30 20 to 28 20 to 26 above listing of oils and fats is not meant to be exhaustive, 20 to 24 20 to 22 22 to 3622 to 34 22 to 32 22 to 30 22 to but only exemplary. 28 22 to 26 22 to 24 24 to 36 24 to 34 24 to 32 24 to 30 24 0223 Specific examples of fatty acids include linoleic to 28 24 to 26 26 to 36 26 to 34 26 to 32 26 to 30 26 to 28 acid, Y-linoleic acid, dihomo-y-linolenic acid, arachidonic 28 to 36 28 to 34 28 to 32 28 to 30 30 to 3630 to 34 30 to acid, docosatetraenoic acid, vaccenic acid, nervonic acid, 32 32 to 36 32 to 34, or about 34 to 36. mead acid, erucic acid, gondoic acid, elaidic acid, oleic acid, 0218. In some embodiments, the microbial compositions palitoleic acid, Stearidonic acid, eicosapentaenoic acid, Val of the present disclosure are shelf stable at any of the eric acid, caproic acid, enanthic acid, caprylic acid, pelar disclosed temperatures and/or temperature ranges and spans gonic acid, capric acid, undecylic acid, lauric acid, tridecylic of time at a relative humidity of at least 1, 2, 3, 4, 5, 6, 7, acid, myristic acid, pentadecylic acid, palmitic acid, marga 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, ric acid, Stearic acid, nonadecyclic acid, arachidic acid, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, heneicosylic acid, behenic acid, tricosylic acid, lignoceric 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, acid, pentacosylic acid, cerotic acid, heptacosylic acid, mon 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, tanic acid, nonacosylic acid, melissic acid, henatriacontylic 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, acid, lacceroic acid, psyllic acid, geddic acid, ceroplastic 89, 90,91, 92,93, 94, 95, 96, 97, or 98% acid, hexatriacontylic acid, heptatriacontanoic acid, and octatriacontanoic acid. Encapsulation Compositions 0224. Another category of fusible materials useful as 0219. In some embodiments, the microbes or microbial encapsulating shell materials is that of waxes. Representa compositions of the disclosure are encapsulated in an encap tive waxes contemplated for use herein are as follows: Sulating composition. An encapsulating composition pro animal waxes, such as beeswax, lanolin, shell wax, and tects the microbes from external stressors prior to entering Chinese insect wax, vegetable waxes, such as carnauba, the gastrointestinal tract of ungulates. Encapsulating com candelilla, bayberry, and Sugar cane; mineral waxes, such as positions further create an environment that may be benefi paraffin, microcrystalline petroleum, oZocerite, ceresin, and cial to the microbes, such as minimizing the oxidative montan; synthetic waxes, such as low molecular weight stresses of an aerobic environment on anaerobic microbes. polyolefin (e.g., CARBOWAX), and polyol ether-esters See Kalsta et al. (U.S. Pat. No. 5,104,662A), Ford (U.S. Pat. (e.g., Sorbitol); Fischer-Tropsch process synthetic waxes; No. 5,733,568A), and Mosbach and Nilsson (U.S. Pat. No. and mixtures thereof. Water-soluble waxes, such as CAR 4,647,536A) for encapsulation compositions of microbes, BOWAX and sorbitol, are not contemplated herein if the and methods of encapsulating microbes. core is acqueous. 0220 in one embodiment, the encapsulating composition 0225. Still other fusible compounds useful herein are comprises microcapsules having a multiplicity of liquid fusible natural resins, such as rosin, balsam, shellac, and cores encapsulated in a solid shell material. For purposes of mixtures thereof. the disclosure, a “multiplicity' of cores is defined as two or 0226 Various adjunct materials are contemplated for O. incorporation in fusible materials according to the present 0221) A first category of useful fusible shell materials is disclosure. For example, antioxidants, light stabilizers, dyes that of normally solid fats, including fats which are already and lakes, flavors, essential oils, anti-caking agents, fillers, of Suitable hardness and animal or vegetable fats and oils pH stabilizers, Sugars (monosaccharides, disaccharides, tri which are hydrogenated until their melting points are Suf saccharides, and polysaccharides) and the like can be incor ficiently high to serve the purposes of the present disclosure. porated in the fusible material in amounts which do not Depending on the desired process and storage temperatures diminish its utility for the present disclosure. and the specific material selected, a particular fat can be 0227. The core material contemplated herein constitutes either a normally solid or normally liquid material. The from about 0.1% to about 50%, about 1% to about 35%. or US 2017/O 196922 A1 Jul. 13, 2017 46 about 5% to about 30% by weight of the microcapsules. In 0232. In some embodiments, the encapsulating shell of Some embodiments, the core material contemplated herein the present disclosure can be up to 10 um, 20 lum, 30 um, 40 constitutes no more than about 30% by weight of the um, 50 um, 60 um, 70 um, 80 um, 90 um, 100 um, 110 um, microcapsules. In some embodiments, the core material 120 um, 130 um, 140 um, 150 um, 160 um, 170 um, 180 um, contemplated herein constitutes about 5% by weight of the 190 um, 200 um, 210 um, 220 um, 230 um, 240 um, 250 lum, microcapsules. The core material is contemplated as either a 260 um, 270 um, 280 um, 290 um, 300 um, 310 um, 320 um, liquid or Solid at contemplated storage temperatures of the 330 um, 340 m, 350 um, 360 um, 370 um, 380 um, 390 um, microcapsules. 400 um, 410 m, 420 um, 430 um, 440 um, 450 um, 460 um, 0228. The cores may include other additives well-known 470 um, 480 m, 490 um, 500 um, 510 um, 520 um, 530 um, in the pharmaceutical art, including edible Sugars, such as 540 um, 550 um, 560 um, 570 um, 580 um, 590 um, 600 um, Sucrose, glucose, maltose, fructose, lactose, cellobiose, 610 um, 620 um, 630 um, 640 um, 650 um, 660 um, 670 um, monosaccharides, disaccharides, trisaccharides, polysaccha 680 um, 690 um, 700 um, 710 um, 720 um, 730 um, 740 um, rides, and mixtures thereof artificial Sweeteners, such as 750 um, 760 um, 770 um, 780 um, 790 um, 800 um, 810 um, aspartame, saccharin, cyclamate salts, and mixtures thereof 820 um, 830 um, 840 um, 850 um, 860 um, 870 um, 880 um, edible acids, such as acetic acid (vinegar), citric acid, 890 um, 900 um, 910 um, 920 um, 930 um, 940 um, 950 um, ascorbic acid, tartaric acid, and mixtures thereof, edible 960 um, 970 um, 980 um, 990 um, 1000 um, 1010 um, 1020 starches, such as corn starch; hydrolyzed vegetable protein; um, 1030 um, 1040 um, 1050 um, 1060 um, 1070 um, 1080 water-soluble vitamins, such as Vitamin C; water-soluble um, 1090 um, 1100 um, 1110 um, 1120 m, 1130 um, 1140 medicaments; water-soluble nutritional materials, such as um, 1150 lum, 1160 lum, 1170 um, 1180 um, 1190 um, 1200 ferrous Sulfate; flavors; salts; monosodium glutamate; anti um, 1210 um, 1220 Lum, 1230 um, 1240 um, 1250 lum, 1260 microbial agents, such as Sorbic acid; antimycotic agents, um, 1270 um, 1280 um, 1290 um, 1300 um, 1310 um, 1320 Such as potassium Sorbate, Sorbic acid, Sodium benzoate, um, 1330 um, 1340 um, 1350 um, 1360 um, 1370 um, 1380 and benzoic acid; food grade pigments and dyes; and um, 1390 um, 1400 um, 1410 um, 1420 um, 1430 um, 1440 mixtures thereof. Other potentially useful supplemental core um, 1450 um, 1460 Lum, 1470 um, 1480 um, 1490 um, 1500 materials will be apparent to those of ordinary skill in the art. um, 1510 um, 1520 um, 1530 um, 1540 um, 1550 um, 1560 0229. Emulsifying agents may be employed to assist in um, 1570 um, 1580 um, 1590 um, 1600 um, 1610 um, 1620 the formation of stable emulsions. Representative emulsify um, 1630 um, 1640 um, 1650 um, 1660 um, 1670 um, 1680 ing agents include glyceryl monostearate, polysorbate um, 1690 um, 1700 um, 1710 um, 1720 um, 1730 um, 1740 esters, ethoxylated mono- and diglycerides, and mixtures um, 1750 um, 1760 um, 1770 um, 1780 um, 1790 um, 1800 thereof. um, 1810 um, 1820 um, 1830 um, 1840 um, 1850 um, 1860 0230. For ease of processing, and particularly to enable um, 1870 um, 1880 um, 1890 um, 1900 um, 1910 um, 1920 the Successful formation of a reasonably stable emulsion, the um, 1930 um, 1940 um, 1950 um, 1960 um, 1970 um, 1980 viscosities of the core material and the shell material should um, 1990 um, 2000 um, 2010 um, 2020 um, 2030 lum, 2040 be similar at the temperature at which the emulsion is um, 2050 um, 2060 um, 2070 um, 2080 um, 2090 um, 2100 formed. In particular, the ratio of the viscosity of the shell to um, 2110 um, 2120 Lum, 2130 um, 2140 um, 2150 lum, 2160 the Viscosity of the core, expressed in centipoise or compa um, 2170 um, 2180 um, 2190 um, 2200 um, 2210 um, 2220 rable units, and both measured at the temperature of the um, 2230 um, 2240 um, 2250 um, 2260 um, 2270 um, 2280 emulsion, should be from about 22:1 to about 1:1, desirably um, 2290 um, 2300 um, 2310 um, 2320 um, 2330 um, 2340 from about 8:1 to about 1:1, and preferably from about 3:1 um, 2350 um, 2360 um, 2370 um, 2380 um, 2390 um, 2400 to about 1:1. A ratio of 1:1 would be ideal, but a viscosity um, 2410 um, 2420 um, 2430 um, 2440 um, 2450 um, 2460 ratio within the recited ranges is useful. um, 2470 um, 2480 um, 2490 um, 2500 um, 2510 um, 2520 0231 Encapsulating compositions are not limited to um, 2530 um, 2540 um, 2550 um, 2560 um, 2570 um, 2580 microcapsule compositions as disclosed above. In some um, 2590 um, 2600 um, 2610 um, 2620 um, 2630 um, 2640 embodiments encapsulating compositions encapsulate the um, 2650 um, 2660 um, 2670 um, 2680 um, 2690 um, 2700 microbial compositions in an adhesive polymer that can be um, 2710 um, 2720 um, 2730 um, 2740 um, 2750 um, 2760 natural or synthetic without toxic effect. In some embodi um, 2770 um, 2780 um, 2790 um, 2800 um, 2810 um, 2820 ments, the encapsulating composition may be a matrix um, 2830 um, 2840 um, 2850 um, 2860 um, 2870 um, 2880 selected from Sugar matrix, gelatin matrix, polymer matrix, um, 2890 um, 2900 um, 2910 um, 2920 um, 2930 um, 2940 silica matrix, starch matrix, foam matrix, etc. In some um, 2950 um, 2960 um, 2970 um, 2980 um, 2990 um, or embodiments, the encapsulating composition may be 3000 um thick. selected from polyvinyl acetates; polyvinyl acetate copoly mers: ethylene vinyl acetate (EVA) copolymers; polyvinyl Animal Feed alcohols; polyvinyl alcohol copolymers; celluloses, includ ing ethylcelluloses, methylcelluloses, hydroxymethylcellu 0233. In some embodiments, compositions of the present loses, hydroxypropylcelluloses and carboxymethylcellu disclosure are mixed with animal feed. In some embodi lose; polyvinylpyrolidones; polysaccharides, including ments, animal feed may be present in various forms such as starch, modified Starch, dextrins, maltodextrins, alginate and pellets, capsules, granulated, powdered, liquid, or semi chitosans; monosaccharides; fats; fatty acids, including oils; liquid. proteins, including gelatin and Zeins; gum arabics; shellacs; 0234. In some embodiments, compositions of the present vinylidene chloride and vinylidene chloride copolymers: disclosure are mixed into the premix at at the feed mill (e.g., calcium lignoSulfonates; acrylic copolymers; polyvinylacry Carghill or Western Millin), alone as a standalone premix, lates; polyethylene oxide; acrylamide polymers and copo and/or alongside other feed additives such as MONENSIN, lymers; polyhydroxyethyl acrylate, methylacrylamide Vitamins, etc. In one embodiment, the compositions of the monomers; and polychloroprene. present disclosure are mixed into the feed at the feed mill. US 2017/O 196922 A1 Jul. 13, 2017 47

In another embodiment, compositions of the present disclo 46 ml, 47 ml, 48 ml, 49 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 sure are mixed into the feed itself. ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, 600 ml, 700 ml, 0235. In some embodiments, feed of the present disclo 800 ml, 900 ml, or 1,000 ml. Sure may be Supplemented with water, premix or premixes, 0243 In some embodiments, the microbial composition forage, fodder, beans (e.g., whole, cracked, or ground), is administered in a dose comprising a total of or at least, grains (e.g., whole, cracked, or ground), bean- or grain 1018, 1017, 1016, 1015, 10', 10, 1012, 10, 1010, 10°, 108, based oils, bean- or grain-based meals, bean- or grain-based 107, 10, 10, 10, 10, or 10 microbial cells. haylage or silage, bean- or grain-based syrups, fatty acids, 0244. In some embodiments, the microbial compositions Sugar alcohols (e.g., polyhydric alcohols), commercially are mixed with feed, and the administration occurs through available formula feeds, and mixtures thereof. the ingestion of the microbial compositions along with the 0236. In some embodiments, forage encompasses hay, feed. In some embodiments, the dose of the microbial haylage, and silage. In some embodiments, hays include composition is administered such that there exists 10° to grass hays (e.g., Sudangrass, orchardgrass, or the like). 10°, 10 to 10°, 10 to 10°, 10 to 10°, 10° to 10°, 107 alfalfa hay, and clover hay. In some embodiments, haylages to 102, 108 to 102, 10° to 10'2, 100 to 10'2, 10 to 102, include grass haylages, Sorghum haylage, and alfalfa hay 10° to 10'', 10 to 10'', 10 to 10'', 10 to 10'', 10° to 10'', lage. In some embodiments, silages include maize, oat, 107 to 10'', 10 to 10'', 10 to 10'', 100 to 10'', 10° to 109, wheat, alfalfa, clover, and the like. 10 to 100, 10 to 100, 10° to 100, 10° to 100, 107 to 100, 0237. In some embodiments, premix or premixes may be 10 to 100, 10 to 10, 10° to 10, 10 to 10, 10 to 10, utilized in the feed. Premixes may comprise micro-ingredi 10 to 10, 10° to 10, 107 to 10, 108 to 10, 10° to 10, 10 ents such as vitamins, minerals, amino acids; chemical to 10, 10 to 10, 10 to 10, 10° to 10, 107 to 10, 10° to preservatives; pharmaceutical compositions such as antibi 107, 10 to 107, 10 to 107, 10 to 107, 10° to 107, 10° to 10°, otics and other medicaments; fermentation products, and 10 to 10°, 10 to 10°, 10 to 10°, 10° to 10, 10 to 10, 10 other ingredients. In some embodiments, premixes are to 10, 10° to 10, 10 to 10, 10° to 10, 10°, 10'', 109, blended into the feed. 10, 10, 107, 10, 10, 10, 10, or 10° total microbial cells 0238. In some embodiments, the feed may include feed per gram or milliliter of the composition. concentrates Such as soybean hulls, Sugar beet pulp, molas 0245. In some embodiments, the administered dose of the ses, high protein soybean meal, ground corn, shelled corn, microbial composition comprises 10° to 10", 10 to 10", wheat midds, distiller grain, cottonseed hulls, rumen-bypass 10 to 10", 10 to 10, 10° to 10", 107 to 10", 10 to 10, protein, rumen-bypass fat, and grease. See Luhman (U.S. 10° to 10, 100 to 10, 10' to 10, 10'2 to 10", 10' to Publication US20150216817A1), Anderson et al. (U.S. Pat. 10", 10' to 10, 10's to 1018, 106 to 10, 107 to 10', No. 3,484.243) and Porter and Luhman (U.S. Pat. No. 10° to 1012, 10 to 1012, 10 to 102, 10 to 10'2, 10° to 102, 9,179,694B2) for animal feed and animal feed supplements 107 to 10°, 10 to 10°, 10 to 10°, 100 to 10°, 10' to capable of use in the present compositions and methods. 1012, 10° to 10, 10 to 10, 10 to 10, 10 to 10'', 10° 0239. In some embodiments, feed occurs as a compound, to 10'', 107 to 10'', 10 to 10'', 10 to 10'', 100 to 10'', 10? which includes, in a mixed composition capable of meeting to 10, 10 to 10, 10 to 10, 10° to 109,107 to 10", 10 the basic dietary needs, the feed itself, vitamins, minerals, to 100, 10 to 100, 10° to 10, 10 to 10, 10 to 10, 10 amino acids, and other necessary components. Compound to 10, 10° to 10, 107 to 10, 10 to 10, 10° to 10, 10 to feed may further comprise premixes. 10, 10 to 10, 10 to 10, 10° to 10, 107 to 10, 10° to 107, 0240. In some embodiments, microbial compositions of 10 to 107, 10 to 107, 10 to 107, 10° to 107, 10° to 10, 10 the present disclosure may be mixed with animal feed, to 10°, 10 to 10°, 10 to 10°, 10° to 10, 10 to 10, 10 to premix, and/or compound feed. Individual components of 10, 10° to 10, 10 to 10, 10° to 10, 10, 107,10,105, the animal feed may be mixed with the microbial compo 10' 10", 1012, 10'', 100, 10, 10, 107, 10°, 10, 10, 10, sitions prior to feeding to ruminants. The microbial compo or 10 total microbial cells. sitions of the present disclosure may be applied into or on a 0246. In some embodiments, the composition is admin premix, into or on a feed, and/or into or on a compound feed. istered 1 or more times per day. In some aspects, the composition is administered with food each time the animal Administration of Microbial Compositions is fed. In some embodiments, the composition is adminis 0241. In some embodiments, the microbial compositions tered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 of the present disclosure are administered to ruminants via to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 the oral route. In some embodiments the microbial compo to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 sitions are administered via a direct injection route into the to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, gastrointestinal tract. In further embodiments, the direct 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, injection administration delivers the microbial compositions 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, directly to the rumen. In some embodiments, the microbial 6, 7, 8, 9, or 10 times per day. compositions of the present disclosure are administered to 0247. In some embodiments, the microbial composition animals anally. In further embodiments, anal administration is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, is in the form of an inserted Suppository. 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 0242. In some embodiments, the microbial composition 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, is administered in a dose comprise a total of, or at least, 1 ml, 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, ml, 21 ml, 22 ml, 23 ml, 24 ml, 25 ml, 26 ml, 27 ml, 28 ml, 3, 4, 5, 6, 7, 8, 9, or 10 times per week. 29 ml, 30 ml, 31 ml, 32 ml, 33 ml, 34 ml, 35 ml, 36 ml, 37 0248. In some embodiments, the microbial composition ml, 38 ml, 39 ml, 40 ml, 41 m, 42 ml, 43 ml, 44 ml, 45 ml, is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, US 2017/O 196922 A1 Jul. 13, 2017 48

1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, um, 2120 um, 2130 um, 2140 um, 2150 um, 2160 lum, 2170 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, um, 2180 um, 2190 um, 2200 um, 2210 um, 2220 lum, 2230 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, um, 2240 um, 2250 um, 2260 um, 2270 um, 2280 um, 2290 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, um, 2300 um, 2310 um, 2320 um, 2330 um, 2340 um, 2350 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, um, 2360 um, 2370 Lum, 2380 um, 2390 um, 2400 um, 2410 3, 4, 5, 6, 7, 8, 9, or 10 times per month. um, 2420 um, 2430 um, 2440 um, 2450 um, 2460 um, 2470 0249. In some embodiments, the microbial composition um, 2480 um, 2490 um, 2500 um, 2510 um, 2520 um, 2530 is administered 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, um, 2540 um, 2550 um, 2560 um, 2570 um, 2580 um, 2590 1 to 4, 1 to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, um, 2600 um, 2610 Lum, 2620 um, 2630 um, 2640 um, 2650 2 to 5, 2 to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, um, 2660 um, 2670 Lum, 2680 um, 2690 um, 2700 um, 2710 3 to 5, 3 to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, um, 2720 um, 2730 um, 2740 um, 2750 um, 2760 um, 2770 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, um, 2780 um, 2790 um, 2800 um, 2810 um, 2820 um, 2830 6 to 7, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, um, 2840 um, 2850 um, 2860 um, 2870 um, 2880 um, 2890 3, 4, 5, 6, 7, 8, 9, or 10 times per year. um, 2900 um, 2910 um, 2920 um, 2930 um, 2940 um, 2950 0250 In some embodiments, the feed can be uniformly um, 2960 um, 2970 um, 2980 um, 2990 um, or 3000 um coated with one or more layers of the microbes and/or thick. microbial compositions disclosed herein, using conventional 0252. In some embodiments, the microbial cells can be methods of mixing, spraying, or a combination thereof coated freely onto any number of compositions or they can through the use of treatment application equipment that is be formulated in a liquid or solid composition before being specifically designed and manufactured to accurately, safely, coated onto a composition. For example, a Solid composition and efficiently apply coatings. Such equipment uses various comprising the microorganisms can be prepared by mixing types of coating technology Such as rotary coaters, drum a solid carrier with a suspension of the spores until the Solid coaters, fluidized bed techniques, spouted beds, rotary mists, carriers are impregnated with the spore or cell Suspension. or a combination thereof. Liquid treatments such as those of This mixture can then be dried to obtain the desired par the present disclosure can be applied via either a spinning ticles. "atomizer disk or a spray nozzle, which evenly distributes 0253) In some other embodiments, it is contemplated that the microbial composition onto the feed as it moves though the Solid or liquid microbial compositions of the present the spray pattern. In some aspects, the feed is then mixed or disclosure further contain functional agents e.g., activated tumbled for an additional period of time to achieve addi carbon, minerals, vitamins, and other agents capable of tional treatment distribution and drying. improving the quality of the products or a combination 0251. In some embodiments, the feed coats of the present thereof. disclosure can be up to 10um, 20 Jum, 30 Jum, 40 um, 50 um, 0254 Methods of coating and compositions in use of said 60 um, 70 um, 80 um, 90 Lum, 100 um, 110 um, 120 um, 130 methods that are known in the art can be particularly useful um, 140 um, 150 um, 160 um, 170 um, 180 um, 190 um, 200 when they are modified by the addition of one of the um, 210 um, 220 um, 230 um, 240 um, 250 um, 260 um, 270 embodiments of the present disclosure. Such coating meth um, 280 um, 290 um, 300 um, 310 um, 320 um, 330 m, 340 ods and apparatus for their application are disclosed in, for um, 350 Lum, 360 um, 370 um, 380 um, 390 um, 400 m, 410 example: U.S. Pat. Nos. 8,097.245, and 7,998.502; and PCT um, 420 Lum, 430 um, 440 um, 450 um, 460 um, 470 um, 480 Pat. App. Publication Nos. WO 2008/076975, WO 2010/ um, 490 Lum, 500 um, 510 um, 520 um, 530 um, 540 m, 550 138522, WO 2011/094469, WO 2010/111347, and WO um, 560 um, 570 um, 580 um, 590 um, 600 um, 610 Lum, 620 2010/111565 each of which is incorporated by reference um, 630 um, 640 um, 650 um, 660 um, 670 um, 680 um, 690 herein. um, 700 um, 710 um, 720 um, 730 um, 740 um, 750 um, 760 0255. In some embodiments, the microbes or microbial um, 770 um, 780 um, 790 um, 800 um, 810 um, 820 um, 830 consortia of the present disclosure exhibit a synergistic um, 840 um, 850 um, 860 um, 870 um, 880 um, 890 um, 900 effect, on one or more of the traits described herein, in the um, 910 um, 920 um,930 um, 940 um, 950 um, 960 um, 970 presence of one or more of the microbes or consortia coming um, 980 um, 990 um, 1000 um, 1010 um, 1020 um, 1030 into contact with one another. The synergistic effect obtained um, 1040 um, 1050 um, 1060 um, 1070 um, 1080 um, 1090 by the taught methods can be quantified, for example, um, 1100 m, 1110 um, 1120 um, 1130 um, 1140 um, 1150 according to Colby's formula (i.e., (E)=X-Y-CX*Y/100)). um, 1160 um, 1170 um, 1180 um, 1190 Lum, 1200 um, 1210 See Colby, R. S., "Calculating Synergistic and Antagonistic um, 1220 um, 1230 um, 1240 um, 1250 um, 1260 um, 1270 Responses of Herbicide Combinations.” 1967. Weeds. Vol. um, 1280 um, 1290 um, 1300 um, 1310 um, 1320 Lum, 1330 15, pp. 20-22, incorporated herein by reference in its um, 1340 um, 1350 um, 1360 um, 1370 um, 1380 um, 1390 entirety. Thus, “synergistic' is intended to reflect an out um, 1400 um, 1410 um, 1420 um, 1430 um, 1440 Lum, 1450 come/parameter/effect that has been increased by more than um, 1460 um, 1470 um, 1480 um, 1490 um, 1500 um, 1510 an additive amount. um, 1520 um, 1530 um, 1540 um, 1550 um, 1560 um, 1570 0256 In some embodiments, the microbes or microbial um, 1580 um, 1590 um, 1600 um, 1610 um, 1620 Lum, 1630 consortia of the present disclosure may be administered via um, 1640 um, 1650 um, 1660 um, 1670 um, 1680 um, 1690 bolus. In one embodiment, a bolus (e.g., capsule containing um, 1700 um, 1710 um, 1720 um, 1730 um, 1740 um, 1750 the composition) is inserted into a bolus gun, and the bolus um, 1760 um, 1770 um, 1780 um, 1790 um, 1800 um, 1810 gun is inserted into the buccal cavity and/or esophagas of the um, 1820 um, 1830 um, 1840 um, 1850 um, 1860 um, 1870 animal, followed by the release/injection of the bolus into um, 1880 um, 1890 um, 1900 um, 1910 um, 1920 um, 1930 the animals digestive tract. In one embodiment, the bolus um, 1940 um, 1950 um, 1960 um, 1970 um, 1980 um, 1990 gun/applicator is a BOVIKALC bolus gun/applicator. In um, 2000 um, 2010 um, 2020 um, 2030 um, 2040 Lum, 2050 another embodiment, the bolus gun/applicator is a QUAD um, 2060 um, 2070 um, 2080 um, 2090 um, 2100 um, 2110 RICAL gun/applicator. US 2017/O 196922 A1 Jul. 13, 2017 49

0257. In some embodiments, the microbes or microbial 0265. The microorganisms of the disclosure may be consortia of the present disclosure may be administered via isolated in substantially pure or mixed cultures. They may be drench. In one embodiment, the drench is an oral drench. A concentrated, diluted, or provided in the natural concentra drench administration comprises utilizing a drench kit/ap tions in which they are found in the source material. For plicator/syringe that injects/releases a liquid comprising the example, microorganisms from saline sediments may be microbes or microbial consortia into the buccal cavity and/or isolated for use in this disclosure by Suspending the sedi esophagas of the animal. ment in fresh water and allowing the sediment to fall to the 0258. In some embodiments, the microbes or microbial bottom. The water containing the bulk of the microorgan consortia of the present disclosure may be administered in a isms may be removed by decantation after a suitable period time-released fashion. The composition may be coated in a of settling and either administered to the GI tract of an chemical composition, or may be contained in a mechanical ungulate, or concentrated by filtering or centrifugation, device or capsule that releases the microbes or microbial diluted to an appropriate concentration and administered to consortia over a period of time instead all at once. In one the GI tract of an ungulate with the bulk of the salt removed. embodiment, the microbes or microbial consortia are admin By way of further example, microorganisms from mineral istered to an animal in a time-release capsule. In one ized or toxic sources may be similarly treated to recover the embodiment, the composition may be coated in a chemical microbes for application to the ungulate to minimize the composition, or may be contained in a mechanical device or potential for damage to the animal. capsul that releases the mcirobes or microbial consortia all 0266. In another embodiment, the microorganisms are at once a period of time hours post ingestion. used in a crude form, in which they are not isolated from the 0259. In some embodiments, the microbes or microbial Source material in which they naturally reside. For example, consortia are administered in a time-released fashion the microorganisms are provided in combination with the between 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 24, 1 to 25, 1 source material in which they reside; for example, fecal to 30, 1 to 35, 1 to 40, 1 to 45, 1 to 50, 1 to 55, 1 to 60, 1 matter, cud, or other composition found in the gastrointes to 65, 1 to 70, 1 to 75, 1 to 80, 1 to 85, 1 to 90, 1 to 95, or tinal tract. In this embodiment, the Source material may 1 to 100 hours. include one or more species of microorganisms. 0260. In some embodiments, the microbes or microbial 0267 In some embodiments, a mixed population of consortia are administered in a time-released fashion microorganisms is used in the methods of the disclosure. between 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 0268. In embodiments of the disclosure where the micro 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 organisms are isolated from a source material (for example, to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 the material in which they naturally reside), any one or a to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 29, or combination of a number of standard techniques which will 1 to 30 days. be readily known to skilled persons may be used. However, by way of example, these in general employ processes by Microorganisms which a solid or liquid culture of a single microorganism can 0261. As used herein the term “microorganism’ should be obtained in a substantially pure form, usually by physical be taken broadly. It includes, but is not limited to, the two separation on the Surface of a solid microbial growth prokaryotic domains, Bacteria and Archaea, as well as medium or by volumetric dilutive isolation into a liquid eukaryotic fungi, protists, and viruses. microbial growth medium. These processes may include 0262 By way of example, the microorganisms may isolation from dry material, liquid Suspension, slurries or include species of the genera of Clostridium, Ruminococ homogenates in which the material is spread in a thin layer cus, Roseburia, Hydrogenoanaerobacterium, Saccharofer over an appropriate Solid gel growth medium, or serial mentans, Papillibacter, Pelotomaculum, Butyricicoccus, dilutions of the material made into a sterile medium and Tannerella, PrevOtella, Butyricimonas, Piromyces, Pichia, inoculated into liquid or solid culture media. Candida, Vrystaatia, Orpinomyces, Neocallinastix, and 0269. Whilst not essential, in one embodiment, the mate Phyllosticta. The microorganisms may further include spe rial containing the microorganisms may be pre-treated prior cies belonging to the family of Lachnospiraceae, and the to the isolation process in order to either multiply all order of Saccharomycetales. In some embodiments, the microorganisms in the material. Microorganisms can then be microorganisms may include species of any genera dis isolated from the enriched materials as disclosed above. closed herein. 0270. In certain embodiments, as mentioned herein 0263. In certain embodiments, the microorganism is before, the microorganism(s) may be used in crude form and unculturable. This should be taken to mean that the micro need not be isolated from an animal or a media. For organism is not known to be culturable or is difficult to example, cud, feces, or growth media which includes the culture using methods known to one skilled in the art. microorganisms identified to be of benefit to increased milk 0264. In one embodiment, the microbes are obtained production in ungulates may be obtained and used as a crude from animals (e.g., mammals, reptiles, birds, and the like), Source of microorganisms for the next round of the method soil (e.g., rhizosphere), air, water (e.g., marine, freshwater, or as a crude source of microorganisms at the conclusion of wastewater sludge), sediment, oil, plants (e.g., roots, leaves, the method. For example, fresh feces could be obtained and stems), agricultural products, and extreme environments optionally processed. (e.g., acid mine drainage or hydrothermal systems). In a Microbiome Shift and Abundance of Microbes further embodiment, microbes obtained from marine or freshwater environments such as an ocean, river, or lake. In 0271. In some embodiments, the microbiome of a rumi a further embodiment, the microbes can be from the surface nant, including the rumen microbiome, comprises a diverse of the body of water, or any depth of the body of water (e.g., arrive of microbes with a wide variety of metabolic capa a deep sea sample). bilities. The microbiome is influenced by a range of factors US 2017/O 196922 A1 Jul. 13, 2017 50 including diet, variations in animal metabolism, and breed, to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 among others. Most bovine diets are plant-based and rich in to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, complex polysaccharides that enrich the gastrointestinal 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, microbial community for microbes capable of breaking 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, down specific polymeric components in the diet. The end 6, 7, 8, 9, 10, 11, or 12 months. products of primary degradation Sustains a chain of 0277. In some embodiments, the presence of the admin microbes that ultimately produce a range of organic acids istered microbes are detected by sampling the gastrointes together with hydrogen and carbon dioxide. Because of the tinal tract and using primers to amplify the 16S or 18S rDNA complex and interlinked nature of the microbiome, changing sequences, or the ITS rDNA sequences of the administered the diet and thus Substrates for primary degradation may microbes. In some embodiments, the administered microbes have a cascading effect on rumen microbial metabolism, are one or more of those selected from Table 1 or Table 3, with changes in both the organic acid profiles and the and the corresponding rNA sequences are those selected methane levels produced, thus impacting the quality and from SEQ ID NOs: 1-60, SEQ ID NOs:2045-2107 and the quantity of animal production and or the products produced SEQ ID NOs identified in Table 3. by the animal. See Menezes et al. (2011. FEMS Microbiol. 0278. In some embodiments, the microbiome of a rumi Ecol. 78(2):256-265.) nant is measured by amplifying polynucleotides collected 0272. In some aspects, the present disclosure is drawn to from gastrointestinal samples, wherein the polynucleotides administering microbial compositions described herein to may be 16S or 18S rDNA fragments, or ITS rDNA frag modulate or shift the microbiome of a ruminant. ments of microbial rNA. In one embodiment, the micro 0273. In some embodiments, the microbiome is shifted biome is fingerprinted by a method of denaturing gradient through the administration of one or more microbes to the gel electrophoresis (DGGE) wherein the amplified rDNA gastrointestinal tract. In further embodiments, the one or fragments are sorted by where they denature, and form a more microbes are those selected from Table 1 or Table 3. In unique banding pattern in a gel that may be used for some embodiments, the microbiome shift or modulation comparing the microbiome of the same ruminant over time includes a decrease or loss of specific microbes that were or the microbiomes of multiple ruminants. In another present prior to the administration of one or more microbes embodiment, the microbiome is fingerprinted by a method of the present disclosure. In some embodiments, the micro of terminal restriction fragment length polymorphism biome shift or modulation includes an increase in microbes (T-RFLP), wherein labelled PCR fragments are digested that were present prior to the administration of one or more using a restriction enzyme and then sorted by size. In a microbes of the present disclosure. In some embodiments, further embodiment, the data collected from the T-RFLP the microbiome shift or modulation includes again of one or method is evaluated by nonmetric multidimensional scaling more microbes that were not present prior to the adminis (nMDS) ordination and PERMANOVA statistics identify tration of one or more microbes of the present disclosure. In differences in microbiomes, thus allowing for the identifi a further embodiment, the gain of one or more microbes is cation and measurement of shifts in the microbiome. See a microbe that was not specifically included in the admin also Shanks et al. (2011. Appl. Environ. Microbiol. 77(9): istered microbial consortium. 2992-3001), Petri et al. (2013. PLOS one. 8(12):e83424), 0274. In some embodiments, the administration of and Menezes et al. (2011. FEMS Microbiol. Ecol. 78(2): microbes of the present disclosure results in a Sustained 256-265.) modulation of the microbiome such that the administered 0279. In some embodiments, the administration of microbes are present in the microbiome fora period of at microbes of the present disclosure results in a modulation or least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 shift of the microbiome which further results in a desired to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 phenotype or improved trait. to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 0280 According to the methods provided herein, a to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, sample is processed to detect the presence of one or more 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, microorganism types in the sample (FIG. 1, 1001; FIG. 2, 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, 2001). The absolute number of one or more microorganism 6, 7, 8, 9, or 10 days. organism type in the sample is determined (FIG. 1, 1002: 0275. In some embodiments, the administration of FIG. 2, 2002). The determination of the presence of the one microbes of the present disclosure results in a Sustained or more organism types and the absolute number of at least modulation of the microbiome such that the administered one organism type can be conducted in parallel or serially. microbes are present in the microbiome fora period of at For example, in the case of a sample comprising a microbial least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 community comprising bacteria (i.e., one microorganism to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 type) and fungi (i.e., a second microorganism type), the user to 4, 2 to 3, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 in one embodiment detects the presence of one or both of the to 4, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, 5 to 10, organism types in the sample (FIG. 1, 1001; FIG. 2, 2001). 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 10, 6 to 9, 6 to 8, 6 to 7, The user, in a further embodiment, determines the absolute 7 to 10, 7 to 9, 7 to 8, 8 to 10, 8 to 9, 9 to 10, 1, 2, 3, 4, 5, number of at least one organism type in the sample—in the 6, 7, 8, 9, or 10 weeks. case of this example, the number of bacteria, fungi or 0276. In some embodiments, the administration of combination thereof, in the sample (FIG. 1, 1002; FIG. 2, microbes of the present disclosure results in a Sustained 2002). modulation of the microbiome such that the administered 0281. In one embodiment, the sample, or a portion microbes are present in the microbiome fora period of at thereof is subjected to flow cytometry (FC) analysis to detect least 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 the presence and/or number of one or more microorganism to 3, 1 to 2, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 types (FIG. 1, 1001, 1002: FIG. 2, 2001, 2002). In one flow US 2017/O 196922 A1 Jul. 13, 2017

cytometer embodiment, individual microbial cells pass will stain red, whereas cells with undamaged membranes through an illumination Zone, at a rate of at least about will stain green. Fluorescent in situ hybridization (FISH) 300*s', or at least about 500*s', or at least about 1000*s extends epifluorescence microscopy, allowing for the fast 1. However, one of ordinary skill in the art will recognize detection and enumeration of specific organisms. FISH uses that this rate can vary depending on the type of instrument fluorescent labelled oligonucleotides probes (usually 15-25 is employed. Detectors which are gated electronically mea basepairs) which bind specifically to organism DNA in the Sure the magnitude of a pulse representing the extent of light sample, allowing the visualization of the cells using an scattered. The magnitudes of these pulses are sorted elec epifluorescence or confocal laser Scanning microscope tronically into “bins' or “channels.” permitting the display (CLSM). Catalyzed reporter deposition fluorescence in situ of histograms of the number of cells possessing a certain hybridization (CARD-FISH) improves upon the FISH quantitative property (e.g., cell staining property, diameter, method by using oligonucleotide probes labelled with a cell membrane) versus the channel number. Such analysis horse radish peroxidase (HRP) to amplify the intensity of the allows for the determination of the number of cells in each signal obtained from the microorganisms being studied. “bin' which in embodiments described herein is an “micro FISH can be combined with other techniques to characterize organism type' bin, e.g., a bacteria, fungi, nematode, pro microorganism communities. One combined technique is tozoan, archaea, algae, dinoflagellate, virus, Viroid, etc. high affinity peptide nucleic acid (PNA)-FISH, where the 0282. In one embodiment, a sample is stained with one or probe has an enhanced capability to penetrate through the more fluorescent dyes wherein a fluorescent dye is specific Extracellular Polymeric Substance (EPS) matrix. Another to a particular microorganism type, to enable detection via a example is LIVE/DEAD-FISH which combines the cell flow cytometer or some other detection and quantification viability kit with FISH and has been used to assess the method that harnesses fluorescence, such as fluorescence efficiency of disinfection in drinking water distribution microscopy. The method can provide quantification of the systems. number of cells and/or cell Volume of a given organism type 0285. In another embodiment, the sample, or a portion in a sample. In a further embodiment, as described herein, thereof is Subjected to Raman micro-spectroscopy in order flow cytometry is harnessed to determine the presence and to determine the presence of a microorganism type and the quantity of a unique first marker and/or unique second absolute number of at least one microorganism type (FIG. 1, marker of the organism type. Such as enzyme expression, 1001-1002: FIG. 2, 2001-2002). Raman micro-spectroscopy cell surface protein expression, etc. Two- or three-variable is a non-destructive and label-free technology capable of histograms or contour plots of for example, light scattering detecting and measuring a single cell Raman spectrum versus fluorescence from a cell membrane stain (versus (SCRS). A typical SCRS provides an intrinsic biochemical fluorescence from a protein stain or DNA stain) may also be “fingerprint of a single cell. A SCRS contains rich infor generated, and thus an impression may be gained of the mation of the biomolecules within it, including nucleic distribution of a variety of properties of interest among the acids, proteins, carbohydrates and lipids, which enables cells in the population as a whole. A number of displays of characterization of different cell species, physiological Such multiparameter flow cytometric data are in common changes and cell phenotypes. Raman microscopy examines use and are amenable for use with the methods described the scattering of laser light by the chemical bonds of herein. different cell biomarkers. ASCRS is a sum of the spectra of 0283. In one embodiment of processing the sample to all the biomolecules in one single cell, indicating a cells detect the presence and number of one or more microorgan phenotypic profile. Cellular phenotypes, as a consequence of ism types, a microscopy assay is employed (FIG. 1, 1001, gene expression, usually reflect genotypes. Thus, under 1002). In one embodiment, the microscopy is optical identical growth conditions, different microorganism types microscopy, where visible light and a system of lenses are give distinct SCRS corresponding to differences in their used to magnify images of Small samples. Digital images genotypes and can thus be identified by their Raman spectra. can be captured by a charge-couple device (CCD) camera. 0286. In yet another embodiment, the sample, or a por Other microscopic techniques include, but are not limited to, tion thereof is subjected to centrifugation in order to deter scanning electron microscopy and transmission electron mine the presence of a microorganism type and the number microscopy. Microorganism types are visualized and quan of at least one microorganism type (FIG. 1, 1001-1002; FIG. tified according to the aspects provided herein. 2, 2001-2002). This process sediments a heterogeneous 0284. In another embodiment of in order to detect the mixture by using the centrifugal force created by a centri presence and number of one or more microorganism types, fuge. More dense components of the mixture migrate away the sample, or a portion thereof is subjected to fluorescence from the axis of the centrifuge, while less dense components microscopy. Different fluorescent dyes can be used to of the mixture migrate towards the axis. Centrifugation can directly stain cells in Samples and to quantify total cell allow fractionation of samples into cytoplasmic, membrane counts using an epifluorescence microscope as well as flow and extracellular portions. It can also be used to determine cytometry, described above. Useful dyes to quantify micro localization information for biological molecules of interest. organisms include but are not limited to acridine orange Additionally, centrifugation can be used to fractionate total (AO), 4,6-di-amino-2 phenylindole (DAPI) and 5-cyano-2,3 microbial community DNA. Different prokaryotic groups Dytolyl Tetrazolium Chloride (CTC). Viable cells can be differ in their guanine-plus-cytosine (G+C) content of DNA, estimated by a viability staining method such as the LIVE/ so density-gradient centrifugation based on G+C content is DEAD Bacterial Viability Kit (Bac-LightTM) which con a method to differentiate organism types and the number of tains two nucleic acid stains: the green-fluorescent SYTO cells associated with each type. The technique generates a 9TM dye penetrates all membranes and the red-fluorescent fractionated profile of the entire community DNA and propidium iodide (PI) dye penetrates cells with damaged indicates abundance of DNA as a function of G+C content. membranes. Therefore, cells with compromised membranes The total community DNA is physically separated into US 2017/O 196922 A1 Jul. 13, 2017 52 highly purified fractions, each representing a different G+C be used to detect the presence and expression of one or more content that can be analyzed by additional molecular tech unique markers in a sample (FIG. 1, 1003-1004: FIG. 2, niques such as denaturing gradient gel electrophoresis 2003-2004). MS is used for example, to detect the presence (DGGE)/amplified ribosomal DNA restriction analysis (AR and quantity of protein and/or peptide markers unique to DRA) (see discussion herein) to assess total microbial microorganism types and therefore to provide an assessment community diversity and the presence/quantity of one or of the number of the respective microorganism type in the more microorganism types. sample. Quantification can be either with stable isotope 0287. In another embodiment, the sample, or a portion labelling or label-free. De novo sequencing of peptides can thereof is subjected to staining in order to determine the also occur directly from MS/MS spectra or sequence tagging presence of a microorganism type and the number of at least (produce a short tag that can be matched against a database). one microorganism type (FIG. 1, 1001-1002: FIG. 2, 2001 MS can also reveal post-translational modifications of pro 2002). Stains and dyes can be used to visualize biological teins and identify metabolites. MS can be used in conjunc tissues, cells or organelles within cells. Staining can be used tion with chromatographic and other separation techniques in conjunction with microscopy, flow cytometry or gel (such as gas chromatography, liquid chromatography, cap electrophoresis to visualize or mark cells or biological illary electrophoresis, ion mobility) to enhance mass reso molecules that are unique to different microorganism types. lution and determination. In vivo staining is the process of dyeing living tissues, 0289. In another embodiment, the sample, or a portion whereas in vitro staining involves dyeing cells or structures thereof is subjected to lipid analysis in order to determine the that have been removed from their biological context. presence of a microorganism type and the number of at least Examples of specific staining techniques for use with the one microorganism type (FIG. 1, 1001-1002: FIG. 2, 2001 methods described herein include, but are not limited to: 2002). Fatty acids are present in a relatively constant pro gram staining to determine gram status of bacteria, portion of the cell biomass, and signature fatty acids exist in endospore staining to identify the presence of endospores, microbial cells that can differentiate microorganism types Ziehl-Neelsen staining, haematoxylin and eosin staining to within a community. In one embodiment, fatty acids are examine thin sections of tissue, papanicolaou staining to extracted by saponification followed by derivatization to examine cell samples from various bodily secretions, peri give the respective fatty acid methyl esters (FAMEs), which odic acid-Schiff staining of carbohydrates, Masson’s are then analyzed by gas chromatography. The FAME pro trichome employing a three-color staining protocol to dis file in one embodiment is then compared to a reference tinguish cells from the surrounding connective tissue, FAME database to identify the fatty acids and their corre Romanowsky stains (or common variants that include sponding microbial signatures by multivariate statistical Wright's stain, Jenner's stain, May-Grunwald stain, Leish analyses. man stain and Giemsa stain) to examine blood or bone 0290. In the aspects of the methods provided herein, the marrow samples, silver staining to reveal proteins and DNA, number of unique first makers in the sample, or portion Sudan staining for lipids and Conklin's staining to detect thereof (e.g., Sample aliquot) is measured, as well as the true endospores. Common biological stains include acridine abundance of each of the unique first markers (FIG. 1, 1003: orange for cell cycle determination; bismarck brown for acid FIG. 2, 2003). A unique marker is a marker of a microor mucins; carmine for glycogen; carmine alum for nuclei; ganism strain. It should be understood by one of ordinary Coomassie blue for proteins; Cresyl violet for the acidic skill in the art that depending on the unique marker being components of the neuronal cytoplasm; Crystal violet for probed for and measured, the entire sample need not be cell walls; DAPI for nuclei; eosin for cytoplasmic material, analyzed. For example, if the unique marker is unique to cell membranes, some extracellular structures and red blood bacterial strains, then the fungal portion of the sample need cells; ethidium bromide for DNA; acid fuchsine for colla not be analyzed. As described above, in some embodiments, gen, Smooth muscle or mitochondria; haematoxylin for measuring the absolute abundance of one or more organism nuclei; Hoechst stains for DNA; iodine for starch; malachite types in a sample comprises separating the sample by green for bacteria in the Gimenez staining technique and for organism type, e.g., via flow cytometry. spores; methyl green for chromatin: methylene blue for 0291 Any marker that is unique to an organism strain can animal cells; neutral red for Nissl substance; Nile blue for be employed herein. For example, markers can include, but nuclei; Nile red for lipohilic entities; osmium tetroxide for are not limited to, small subunit ribosomal RNA genes lipids; rhodamine is used in fluorescence microscopy; Safra (16S/18S rDNA), large subunit ribosomal RNA genes (23S/ nin for nuclei. Stains are also used in transmission electron 25S/28S rDNA), intercalary 5.8S gene, cytochrome c oxi microscopy to enhance contrast and include phosphotung dase, beta-tubulin, elongation factor, RNA polymerase and stic acid, osmium tetroxide, ruthenium tetroxide, ammonium internal transcribed spacer (ITS). molybdate, cadmium iodide, carbohydrazide, ferric chlo 0292 Ribosomal RNA genes (rDNA), especially the ride, hexamine, indium trichloride, lanthanum nitrate, lead small subunit ribosomal RNA genes, i.e., 18S rRNA genes acetate, lead citrate, lead(II) nitrate, periodic acid, phospho (18S rDNA) in the case of eukaryotes and 16S rRNA (16S molybdic acid, potassium ferricyanide, potassium ferrocya rDNA) in the case of prokaryotes, have been the predomi nide, ruthenium red, silver nitrate, silver proteinate, sodium nant target for the assessment of organism types and strains chloroaurate, thallium nitrate, thiosemicarbazide, uranyl in a microbial community. However, the large Subunit acetate, uranyl nitrate, and Vanadyl Sulfate. ribosomal RNA genes, 28S rDNAs, have been also targeted. 0288. In another embodiment, the sample, or a portion rDNAs are suitable for taxonomic identification because: (i) thereof is subjected to mass spectrometry (MS) in order to they are ubiquitous in all known organisms; (ii) they possess determine the presence of a microorganism type and the both conserved and variable regions; (iii) there is an expo number of at least one microorganism type (FIG. 1, 1001 nentially expanding database of their sequences available for 1002: FIG. 2, 2001-2002). MS, as discussed below, can also comparison. In community analysis of samples, the con US 2017/O 196922 A1 Jul. 13, 2017 served regions serve as annealing sites for the corresponding (rplT), ruvA Holliday junction DNA helicase, ruvB Holliday universal PCR and/or sequencing primers, whereas the junction DNA helicase B, serS seryl-tRNA synthetase, rplu variable regions can be used for phylogenetic differentiation. 50S ribosomal protein L21, rpsR30S ribosomal protein S18. In addition, the high copy number of rDNA in the cells DNA mismatch repair protein MutS. rpsT 30S ribosomal facilitates detection from environmental samples. protein S20, DNA repair protein RecN, frr ribosome recy 0293. The internal transcribed spacer (ITS), located cling factor (frr), recombination protein RecR, protein of between the 18S rDNA and 28S rDNA, has also been unknown function UPF0054, mia A tRNA isopentenyltrans targeted. The ITS is transcribed but spliced away before ferase, GTP-binding protein YchF, chromosomal replication assembly of the ribosomes The ITS region is composed of initiator protein DnaA, dephospho-CoA kinase, 16S rRNA two highly variable spacers, ITS1 and ITS2, and the inter processing protein RimM, ATP-cone domain protein, 1-de calary 5.8S gene. This rDNA operon occurs in multiple oxy-D-xylulose 5-phosphate reductoisomerase, 2C-methyl copies in genomes. Because the ITS region does not code for D-erythritol 2.4-cyclodiphosphate synthase, fatty acid/phos ribosome components, it is highly variable. pholipid synthesis protein PlsX, tRNA(Ile)-lysidine synthetase, dnaG DNA primase (dnaG), ruvC Holliday 0294. In one embodiment, the unique RNA marker can be junction resolvase, rpsP30S ribosomal protein S16, Recom an mRNA marker, an siRNA marker or a ribosomal RNA binase A recA, riboflavin biosynthesis protein RibF, glycyl marker. tRNA synthetase beta subunit, trim U tRNA (5-methylamin 0295 Protein-coding functional genes can also be used omethyl-2-thiouridylate)-methyltransferase, rpmI 50S herein as a unique first marker. Such markers include but are ribosomal protein L35, hemEuroporphyrinogen decarboxy not limited to: the recombinase A gene family (bacterial lase, Rod shape-determining protein, rpmA 50S ribosomal RecA, archaea RadA and RadB, eukaryotic RadS1 and protein L27 (rpmA), peptidyl-tRNA hydrolase, translation Rad57, phage UvsX); RNA polymerase B subunit (RpoB) initiation factor IF-3 (inf(c), UDP-N-acetylmuramyl-tripep gene, which is responsible for transcription initiation and tide synthetase, rpmF 505 ribosomal protein L32, rpIL 50S elongation; chaperonins. Candidate marker genes have also ribosomal protein L7/L12 (rpL), leuS leucyl-tRNA syn been identified for bacteria plus archaea: ribosomal protein thetase, ligA NAD-dependent DNA ligase, cell division S2 (rpsB), ribosomal protein S10 (rps.J), ribosomal protein protein FtsA, GTP-binding protein Typ A, ATP-dependent L1 rplA), translation elongation factor EF-2, translation Clp protease, ATP-binding subunit ClpX, DNA replication initiation factor IF-2, metalloendopeptidase, ribosomal pro and repair protein RecP and UDP-N-acetylenolpyruvoylg tein L22, flh signal recognition particle protein, ribosomal protein L4/Lle (rplD), ribosomal protein L2 (rplB), ribo lucosamine reductase. somal protein S9 (rpsl), ribosomal protein L3 (rplC), phe 0296 Phospholipid fatty acids (PLFAs) may also be used nylalanyl-tRNA synthetase beta subunit, ribosomal protein as unique first markers according to the methods described L14b/L23e (rplN), ribosomal protein S5, ribosomal protein herein. Because PLFAs are rapidly synthesized during S19 (rpsS), ribosomal protein S7, ribosomal protein L16/ microbial growth, are not found in storage molecules and L10E (rplP), ribosomal protein S13 (rpsM), phenylalanyl degrade rapidly during cell death, it provides an accurate tRNA synthetase C. subunit, ribosomal protein L15, ribo census of the current living community. All cells contain somal protein L25/L23, ribosomal protein L6 (rplF), fatty acids (FAs) that can be extracted and esterified to form ribosomal protein L11 (rplK), ribosomal protein L5 (rplE), fatty acid methyl esters (FAMEs). When the FAMEs are ribosomal protein S12/S23, ribosomal protein L29, ribo analyzed using gas chromatography—mass spectrometry, somal protein S3 (rpsC), ribosomal protein S11 (rpsK), the resulting profile constitutes a fingerprint of the micro ribosomal protein L10, ribosomal protein S8, tRNA organisms in the sample. The chemical compositions of pseudouridine synthase B, ribosomal protein L18P/L5E, membranes for organisms in the domains Bacteria and ribosomal protein S15P/S13e, Porphobilinogen deaminase, Eukarya are comprised of fatty acids linked to the glycerol ribosomal protein S17, ribosomal protein L13 (rplM), phos by an ester-type bond (phospholipid fatty acids (PLFAs)). In phoribosylformylglycinamidine cyclo-ligase (rpsE), ribonu contrast, the membrane lipids of Archaea are composed of clease HII and ribosomal protein L24. Other candidate long and branched hydrocarbons that are joined to glycerol marker genes for bacteria include: transcription elongation by an ether-type bond (phospholipid ether lipids (PLELs)). protein NusA (nusA), rpoB DNA-directed RNA polymerase This is one of the most widely used non-genetic criteria to subunit beta (rpoB), GTP-binding protein EngA, rpoC distinguish the three domains. In this context, the phospho DNA-directed RNA polymerase subunit beta", priA primo lipids derived from microbial cell membranes, characterized Some assembly protein, transcription-repair coupling factor, by different acyl chains, are excellent signature molecules, CTP synthase (pyrG), secY preprotein translocase subunit because such lipid structural diversity can be linked to SecY, GTP-binding protein Obg/CgtA, DNA polymerase I, specific microbial taxa. rpsF 30S ribosomal protein S6, poA DNA-directed RNA 0297 As provided herein, in order to determine whether polymerase subunit alpha, peptide chain release factor 1, rpl an organism strain is active, the level of expression of one 50S ribosomal protein L9, polyribonucleotide nucleotidyl or more unique second markers, which can be the same or transferase, tsfelongation factor Ts (tsf), rplQ 50S ribosomal different as the first marker, is measured (FIG. 1, 1004: FIG. protein L17, tRNA (guanine-N(1)-)-methyltransferase 2, 2004). Unique first unique markers are described above. (rplS), rply probable 50S ribosomal protein L25, DNA The unique second marker is a marker of microorganism repair protein RadA, glucose-inhibited division protein A, activity. For example, in one embodiment, the mRNA or ribosome-binding factor A, DNA mismatch repair protein protein expression of any of the first markers described MutL, simpl3 SsrA-binding protein (simp3), N-acetylglu above is considered a unique second marker for the purposes cosaminyl transferase, S-adenosyl-methyltransferase MraW. of this invention. UDP-N-acetylmuramoylalanine-D-glutamate ligase, rplS 0298. In one embodiment, if the level of expression of the 50S ribosomal protein L19, rp1T 50S ribosomal protein L20 second marker is above a threshold level (e.g., a control US 2017/O 196922 A1 Jul. 13, 2017 54 level) or at a threshold level, the microorganism is consid a given DNA fragment. A molecular barcode can be created ered to be active (FIG. 1, 1005; FIG. 2, 2005). Activity is and placed between the adapter sequence and the sequence determined in one embodiment, if the level of expression of of interest in multiplex reactions, allowing each sequence to the second marker is altered by at least about 5%, at least be assigned to a sample bioinformatically. about 10%, at least about 15%, at least about 20%, at least 0305 Illumina/Solexa sequencing produces average read about 25%, or at least about 30%, as compared to a threshold lengths of about 25 basepairs (bp) to about 300 bp (Bennett level, which in some embodiments, is a control level. et al. (2005) Pharmacogenomics, 6:373-382: Lange et al. 0299 Second unique markers are measured, in one (2014). BMC Genomics 15, p. 63; Fadrosh et al. (2014) embodiment, at the protein, RNA or metabolite level. A Microbiome 2, p. 6: Caporaso et al. (2012) ISME J, 6, p. unique second marker is the same or different as the first 1621-1624; Bentley et al. (2008) Accurate whole human unique marker. genome sequencing using reversible terminator chemistry. 0300. As provided above, a number of unique first mark Nature, 456:53-59). This sequencing technology is also ers and unique second markers can be detected according to sequencing-by-synthesis but employs reversible dye termi the methods described herein. Moreover, the detection and nators and a flow cell with a field of oligos attached. DNA quantification of a unique first marker is carried out accord fragments to be sequenced have specific adapters on either ing to methods known to those of ordinary skill in the art end and are washed over a flow cell filled with specific (FIG. 1, 1003-1004, FIG. 2, 2003-2004). oligonucleotides that hybridize to the ends of the fragments. 0301 Nucleic acid sequencing (e.g., gDNA, cDNA, Each fragment is then replicated to make a cluster of rRNA, mRNA) in one embodiment is used to determine identical fragments. Reversible dye-terminator nucleotides absolute abundance of a unique first marker and/or unique are then washed over the flow cell and given time to attach. second marker. Sequencing platforms include, but are not The excess nucleotides are washed away, the flow cell is limited to, Sanger sequencing and high-throughput sequenc imaged, and the reversible terminators can be removed so ing methods available from Roche/454 Life Sciences, Illu that the process can repeat and nucleotides can continue to mina/Solexa, Pacific Biosciences, Ion Torrent and Nano be added in subsequent cycles. Paired-end reads that are 300 pore. The sequencing can be amplicon sequencing of bases in length each can be achieved. An Illumina platform particular DNA or RNA sequences or whole metagenome/ can produce 4 billion fragments in a paired-end fashion with transcriptome shotgun sequencing. 125 bases for each read in a single run. Barcodes can also be 0302 Traditional Sanger sequencing (Sanger et al. used for Sample multiplexing, but indexing primers are used. (1977) DNA sequencing with chain-terminating inhibitors. (0306 The SOLiD (Sequencing by Oligonucleotide Liga Proc Natl. Acad. Sci. USA, 74, pp. 5463-5467, incorporated tion and Detection, Life Technologies) process is a by reference herein in its entirety) relies on the selective "sequencing-by-ligation' approach, and can be used with incorporation of chain-terminating dideoxynucleotides by the methods described herein for detecting the presence and DNA polymerase during in vitro DNA replication and is abundance of a first marker and/or a second marker (FIG. 1, amenable for use with the methods described herein. 1003-1004: FIG. 2, 2003-2004) (Peckham et al. SOLiDTM 0303. In another embodiment, the sample, or a portion Sequencing and 2-Base Encoding. San Diego, Calif.: Ameri thereof is Subjected to extraction of nucleic acids, amplifi can Society of Human Genetics, 2007; Mitra et al. (2013) cation of DNA of interest (such as the rRNA gene) with Analysis of the intestinal microbiota using SOLiD 16S Suitable primers and the construction of clone libraries using rRNA gene sequencing and SOLiD shotgun sequencing. sequencing vectors. Selected clones are then sequenced by BMC Genomics, 14(Suppl 5): S16; Mardis (2008) Next Sanger sequencing and the nucleotide sequence of the DNA generation DNA sequencing methods. Annu Rev Genomics of interest is retrieved, allowing calculation of the number of Hum Genet, 9:387-402; each incorporated by reference unique microorganism strains in a sample. herein in its entirety). A library of DNA fragments is 0304 454 pyrosequencing from Roche/454 Life Sciences prepared from the sample to be sequenced, and are used to yields long reads and can be harnessed in the methods prepare clonal bead populations, where only one species of described herein (Margulies et al. (2005) Nature, 437, pp. fragment will be present on the Surface of each magnetic 376-380; U.S. Pat. Nos. 6,274,320; 6,258,568; 6,210,891, bead. The fragments attached to the magnetic beads will each of which is herein incorporated in its entirety for all have a universal P1 adapter sequence so that the starting purposes). Nucleic acid to be sequenced (e.g., amplicons or sequence of every fragment is both known and identical. nebulized genomic/metagenomic DNA) have specific adapt Primers hybridize to the P1 adapter sequence within the ers affixed on either end by PCR or by ligation. The DNA library template. A set of four fluorescently labelled di-base with adapters is fixed to tiny beads (ideally, one bead will probes compete for ligation to the sequencing primer. Speci have one DNA fragment) that are suspended in a water-in-oil ficity of the di-base probe is achieved by interrogating every emulsion. An emulsion PCR step is then performed to make 1st and 2nd base in each ligation reaction. Multiple cycles of multiple copies of each DNA fragment, resulting in a set of ligation, detection and cleavage are performed with the beads in which each bead contains many cloned copies of number of cycles determining the eventual read length. The the same DNA fragment. Each bead is then placed into a SOLiD platform can produce up to 3 billion reads per run well of a fiber-optic chip that also contains enzymes neces with reads that are 75 bases long. Paired-end sequencing is sary for the sequencing-by-synthesis reactions. The addition available and can be used herein, but with the second read of bases (such as A, C, G, or T) trigger pyrophosphate in the pair being only 35 bases long. Multiplexing of release, which produces flashes of light that are recorded to samples is possible through a system akin to the one used by infer the sequence of the DNA fragments in each well. About Illumina, with a separate indexing run. 1 million reads per run with reads up to 1,000 bases in length 0307 The Ion Torrent system, like 454 sequencing, is can be achieved. Paired-end sequencing can be done, which amenable for use with the methods described herein for produces pairs of reads, each of which begins at one end of detecting the presence and abundance of a first marker US 2017/O 196922 A1 Jul. 13, 2017

and/or a second marker (FIG. 1, 1003-1004: FIG. 2, 2003 (1996). Appl Environ Microbiol 62, pp. 3112-3120; Schei 2004). It uses a plate of microwells containing beads to nert et al. (1996). J. Microbiol. Methods 26, pp. 103-117: which DNA fragments are attached. It differs from all of the Schwieger and Tebbe (1998). Appl. Environ. Microbiol. 64. other systems, however, in the manner in which base incor pp. 4870-4876, each of which is incorporated by reference poration is detected. When a base is added to a growing herein in its entirety). In this technique, DNA fragments DNA strand, a proton is released, which slightly alters the such as PCR products obtained with primers specific for the Surrounding pH. Microdetectors sensitive to pH are associ 16S rRNA gene, are denatured and directly electrophoresed ated with the wells on the plate, and they record when these on a non-denaturing gel. Separation is based on differences changes occur. The different bases (A, C, G, T) are washed in size and in the folded conformation of single-stranded sequentially through the wells, allowing the sequence from DNA, which influences the electrophoretic mobility. Rean each well to be inferred. The Ion Proton platform can nealing of DNA strands during electrophoresis can be pre produce up to 50 million reads per run that have read lengths vented by a number of strategies, including the use of one of 200 bases. The Personal Genome Machine platform has phosphorylated primer in the PCR followed by specific longer reads at 400 bases. Bidirectional sequencing is avail digestion of the phosphorylated Strands with lambda exo able. Multiplexing is possible through the standard in-line nuclease and the use of one biotinylated primer to perform molecular barcode sequencing. magnetic separation of one single Strand after denaturation. 0308 Pacific Biosciences (PacBio) SMRT sequencing To assess the identity of the predominant populations in a uses a single-molecule, real-time sequencing approach and given consortium, in one embodiment, bands are excised in one embodiment, is used with the methods described and sequenced, or SSCP-patterns can be hybridized with herein for detecting the presence and abundance of a first specific probes. Electrophoretic conditions, such as gel marker and/or a second marker (FIG. 1, 1003-1004: FIG. 2, matrix, temperature, and addition of glycerol to the gel, can 2003-2004). The PacBio sequencing system involves no influence the separation. amplification step, setting it apart from the other major next-generation sequencing systems. In one embodiment, 0312. In addition to sequencing based methods, other the sequencing is performed on a chip containing many methods for quantifying expression (e.g., gene, protein Zero-mode waveguide (ZMW) detectors. DNA polymerases expression) of a second marker are amenable for use with are attached to the ZMW detectors and phospholinked the methods provided herein for determining the level of dye-labeled nucleotide incorporation is imaged in real time expression of one or more second markers (FIG. 1, 1004; as DNA strands are synthesized. The PacBio system yields FIG. 2, 2004). For example, quantitative RT-PCR, microar very long read lengths (averaging around 4,600 bases) and ray analysis, linear amplification techniques such as nucleic a very high number of reads per run (about 47,000). The acid sequence based amplification (NASBA) are all ame typical “paired-end approach is not used with PacBio, since nable for use with the methods described herein, and can be reads are typically long enough that fragments, through carried out according to methods known to those of ordinary CCS, can be covered multiple times without having to skill in the art. sequence from each end independently. Multiplexing with 0313. In another embodiment, the sample, or a portion PacBio does not involve an independent read, but rather thereof is Subjected to a quantitative polymerase chain follows the standard “in-line' barcoding model. reaction (PCR) for detecting the presence and abundance of 0309. In one embodiment, where the first unique marker a first marker and/or a second marker (FIG. 1, 1003-1004; is the ITS genomic region, automated ribosomal intergenic FIG. 2, 2003-2004). Specific microorganism strains activity spacer analysis (ARISA) is used in one embodiment to is measured by reverse transcription of transcribed ribo determine the number and identity of microorganism strains somal and/or messenger RNA (rRNA and mRNA) into in a sample (FIG. 1, 1003, FIG. 2, 2003) (Ranjard et al. complementary DNA (cDNA), followed by PCR (RT-PCR). (2003). Environmental Microbiology 5, pp. 1111-1120, 0314. In another embodiment, the sample, or a portion incorporated by reference in its entirety for all puposes). The thereof is subjected to PCR-based fingerprinting techniques ITS region has significant heterogeneity in both length and to detect the presence and abundance of a first marker and/or nucleotide sequence. The use of a fluorescence-labeled a second marker (FIG. 1, 1003-1004: FIG. 2, 2003-2004). forward primer and an automatic DNA sequencer permits PCR products can be separated by electrophoresis based on high resolution of separation and high throughput. The the nucleotide composition. Sequence variation among the inclusion of an internal standard in each sample provides different DNA molecules influences the melting behaviour, accuracy in sizing general fragments. and therefore molecules with different sequences will stop 0310. In another embodiment, fragment length polymor migrating at different positions in the gel. Thus electropho phism (RFLP) of PCR-amplified rDNA fragments, other retic profiles can be defined by the position and the relative wise known as amplified ribosomal DNA restriction analysis intensity of different bands or peaks and can be translated to (ARDRA), is used to characterize unique first markers and numerical data for calculation of diversity indices. Bands the abundance of the same in samples (FIG. 1, 1003, FIG. 2, can also be excised from the gel and Subsequently sequenced 2003) (Massol-Deya et al. (1995). Mol. Microb. Ecol. to reveal the phylogenetic affiliation of the community Manual. 3.3.2, pp. 1-18, incorporated by reference in its members. Electrophoresis methods include, but are not entirety for all puposes). rDNA fragments are generated by limited to: denaturing gradient gel electrophoresis (DGGE), PCR using general primers, digested with restriction temperature gradient gel electrophoresis (TGGE), single enzymes, electrophoresed in agarose or acrylamide gels, and stranded-conformation polymorphism (SSCP), restriction stained with ethidium bromide or silver nitrate. fragment length polymorphism analysis (RFLP) or ampli 0311. One fingerprinting technique used in detecting the fied ribosomal DNA restriction analysis (ARDRA), terminal presence and abundance of a unique first marker is single restriction fragment length polymorphism analysis stranded-conformation polymorphism (SSCP) (Lee et al. (T-RFLP), automated ribosomal intergenic spacer analysis US 2017/O 196922 A1 Jul. 13, 2017 56

(ARISA), randomly amplified polymorphic DNA (RAPD), cation of active cells at single-cell resolution is performed DNA amplification fingerprinting (DAF) and Bb-PEG elec with a microscope. MAR-FISH provides information on trophoresis. total cells, probe targeted cells and the percentage of cells 0315. In another embodiment, the sample, or a portion that incorporate a given radiolabelled substance. The thereof is subjected to a chip-based platform such as method provides an assessment of the in situ function of microarray or microfluidics to determine the abundance of a targeted microorganisms and is an effective approach to unique first marker and/or presence/abundance of a unique study the in vivo physiology of microorganisms. A technique second marker (FIG. 1, 1003-1004, FIG. 2, 2003-2004). The developed for quantification of cell-specific Substrate uptake PCR products are amplified from total DNA in the sample in combination with MAR-FISH is known as quantitative and directly hybridized to known molecular probes affixed MAR (QMAR). to microarrays. After the fluorescently labeled PCR ampli 0320 In one embodiment, the sample, or a portion cons are hybridized to the probes, positive signals are scored thereof is subjected to stable isotope Raman spectroscopy by the use of confocal laser Scanning microscopy. The combined with FISH (Raman-FISH) to determine the level microarray technique allows samples to be rapidly evaluated of a second unique marker (FIG. 1, 1004: FIG. 2, 2004). This with replication, which is a significant advantage in micro technique combines stable isotope probing, Raman spec bial community analyses. In general, the hybridization sig troscopy and FISH to link metabolic processes with particu nal intensity on microarrays is directly proportional to the lar organisms. The proportion of stable isotope incorporation abundance of the target organism. The universal high by cells affects the light scatter, resulting in measurable peak density 16S microarray (PhyloChip) contains about 30,000 shifts for labelled cellular components, including protein probes of 16SrRNA gene targeted to several cultured micro and mRNA components. Raman spectroscopy can be used to bial species and “candidate divisions”. These probes target identify whether a cell Synthesizes compounds including, all 121 demarcated prokaryotic orders and allow simultane but not limited to: oil (such as alkanes), lipids (such as ous detection of 8,741 bacterial and archaeal taxa. Another triacylglycerols (TAG)), specific proteins (such as heme microarray in use for profiling microbial communities is the proteins, metalloproteins), cytochrome (such as P450, 0316 Functional Gene Array (FGA). Unlike PhyloChips, cytochrome c), chlorophyll, chromophores (such as pig FGAs are designed primarily to detect specific metabolic ments for light harvesting carotenoids and rhodopsins), groups of bacteria. Thus, FGA not only reveal the commu organic polymers (such as polyhydroxyalkanoates (PHA), nity structure, but they also shed light on the in situ polyhydroxybutyrate (PHB)), hopanoids, steroids, starch, community metabolic potential. FGA contain probes from sulfide, sulfate and secondary metabolites (such as vitamin genes with known biological functions, so they are useful in B12). linking microbial community composition to ecosystem 0321. In one embodiment, the sample, or a portion functions. An FGA termed GeoChip contains >24,000 thereof is subjected to DNA/RNA stable isotope probing probes from all known metabolic genes involved in various (SIP) to determine the level of a second unique marker (FIG. biogeochemical, ecological, and environmental processes 1, 1004: FIG. 2, 2004). SIP enables determination of the Such as ammonia oxidation, methane oxidation, and nitrogen microbial diversity associated with specific metabolic path fixation. ways and has been generally applied to study microorgan 0317. A protein expression assay, in one embodiment, is isms involved in the utilization of carbon and nitrogen used with the methods described herein for determining the compounds. The substrate of interest is labelled with stable level of expression of one or more second markers (FIG. 1, isotopes (such as 'C or 'N) and added to the sample. Only 1004: FIG. 2, 2004). For example, in one embodiment, mass microorganisms able to metabolize the Substrate will incor spectrometry or an immunoassay Such as an enzyme-linked porate it into their cells. Subsequently, 'C-DNA and N immunosorbant assay (ELISA) is utilized to quantify the DNA can be isolated by density gradient centrifugation and level of expression of one or more unique second markers, used for metagenomic analysis. RNA-based SIP can be a wherein the one or more unique second markers is a protein. responsive biomarker for use in SIP studies, since RNA itself 0318. In one embodiment, the sample, or a portion is a reflection of cellular activity. thereof is subjected to Bromodeoxyuridine (BrdU) incorpo 0322. In one embodiment, the sample, or a portion ration to determine the level of a second unique marker thereof is subjected to isotope array to determine the level of (FIG. 1, 1004: FIG. 2, 2004). BrdU, a synthetic nucleoside a second unique marker (FIG. 1, 1004: FIG. 2, 2004). analog of thymidine, can be incorporated into newly syn Isotope arrays allow for functional and phylogenetic screen thesized DNA of replicating cells. Antibodies specific for ing of active microbial communities in a high-throughput BRdU can then be used for detection of the base analog. fashion. The technique uses a combination of SIP for moni Thus BrdU incorporation identifies cells that are actively toring the Substrate uptake profiles and microarray technol replicating their DNA, a measure of activity of a microor ogy for determining the taxonomic identities of active ganism according to one embodiment of the methods microbial communities. Samples are incubated with a C described herein. BrdU incorporation can be used in com labeled substrate, which during the course of growth bination with FISH to provide the identity and activity of becomes incorporated into microbial biomass. The ''C- targeted cells. labeled rRNA is separated from unlabeled rRNA and then 0319. In one embodiment, the sample, or a portion labeled with fluorochromes. Fluorescent labeled rRNA is thereof is subjected to microautoradiography (MAR) com hybridized to a phylogenetic microarray followed by scan bined with FISH to determine the level of a second unique ning for radioactive and fluorescent signals. The technique marker (FIG. 1, 1004: FIG. 2, 2004). MAR-FISH is based on thus allows simultaneous study of microbial community the incorporation of radioactive Substrate into cells, detec composition and specific Substrate consumption by meta tion of the active cells using autoradiography and identifi bolically active microorganisms of complex microbial com cation of the cells using FISH. The detection and identifi munities. US 2017/O 196922 A1 Jul. 13, 2017 57

0323. In one embodiment, the sample, or a portion strain with an environmental parameter within a network, thereof is Subjected to a metabolomics assay to determine wherein the network is a collection of two or more samples the level of a second unique marker (FIG. 1, 1004: FIG. 2, that share a common or similar environmental parameter. In 2004). Metabolomics studies the metabolome which repre another embodiment, the network and/or cluster analysis sents the collection of all metabolites, the end products of method may be applied to determining the co-occurrence of cellular processes, in a biological cell, tissue, organ or two or more active microorganism strains in a sample (FIG. organism. This methodology can be used to monitor the 2, 2008). In another embodiment, the network analysis presence of microorganisms and/or microbial mediated pro comprises nonparametric approaches including mutual cesses since it allows associating specific metabolite profiles information to establish connectivity between variables. In with different microorganisms. Profiles of intracellular and another embodiment, the network analysis comprises link extracellular metabolites associated with microbial activity age analysis, modularity analysis, robustness measures, can be obtained using techniques such as gas chromatogra betweenness measures, connectivity measures, transitivity phy-mass spectrometry (GC-MS). The complex mixture of measures, centrality measures or a combination thereof a metabolomic sample can be separated by Such techniques (FIG. 2, 2009). In another embodiment, the cluster analysis as gas chromatography, high performance liquid chroma method comprises building a connectivity model, Subspace tography and capillary electrophoresis. Detection of metabo model, distribution model, density model, or a centroid lites can be by mass spectrometry, nuclear magnetic reso model and/or using community detection algorithms such as nance (NMR) spectroscopy, ion-mobility spectrometry, the Louvain, Bron-Kerbosch, Girvan-Newman, Clauset electrochemical detection (coupled to HPLC) and radiolabel Newman-Moore, Pons-Latapy, and Wakita-Tsurumi algo (when combined with thin-layer chromatography). rithms (FIG. 2, 2010). 0324. According to the embodiments described herein, 0329. In one embodiment, the cluster analysis method is the presence and respective number of one or more active a heuristic method based on modularity optimization. In a microorganism strains in a sample are determined (FIG. 1, further embodiment, the cluster analysis method is the 1006; FIG. 2, 2006). For example, strain identity informa Louvain method. See, e.g., the method described by Blondel tion obtained from assaying the number and presence of first et al. (2008). Fast unfolding of communities in large net markers is analyzed to determine how many occurrences of works. Journal of Statistical Mechanics: Theory and Experi a unique first marker are present, thereby representing a ment, Volume 2008, October 2008, incorporated by refer unique microorganism strain (e.g., by counting the number ence herein in its entirety for all purposes. of sequence reads in a sequencing assay). This value can be 0330. In another embodiment, the network analysis com represented in one embodiment as a percentage of total prises predictive modeling of network through link mining sequence reads of the first maker to give a percentage of and prediction, collective classification, link-based cluster unique microorganism strains of a particular microorganism ing, relational similarity, or a combination thereof. In type. In a further embodiment, this percentage is multiplied another embodiment, the network analysis comprises dif by the number of microorganism types (obtained at Step ferential equation based modeling of populations. In another 1002 or 2002, see FIG. 1 and FIG. 2) to give the absolute embodiment, the network analysis comprises Lotka-Volterra abundance of the one or more microorganism strains in a modeling. sample and a given Volume. 0331. In one embodiment, relating the one or more active 0325 The one or more microorganism strains are con microorganism strains to an environmental parameter (e.g., sidered active, as described above, if the level of second determining the co-occurrence) in the sample comprises unique marker expression at a threshold level, higher than a creating matrices populated with linkages denoting environ threshold value, e.g., higher than at least about 5%, at least mental parameter and microorganism strain associations. about 10%, at least about 20% or at least about 30% over a 0332. In one embodiment, the multiple sample data control level. obtained at step 2007 (e.g., over two or more samples which 0326 In another aspect of the invention, a method for can be collected at two or more time points where each time determining the absolute abundance of one or more micro point corresponds to an individual sample), is compiled. In organism Strains is determined in a plurality of Samples a further embodiment, the number of cells of each of the one (FIG. 2, see in particular, 2007). For a microorganism strain or more microorganism Strains in each sample is stored in an to be classified as active, it need only be active in one of the association matrix (which can be in Some embodiments, an samples. The samples can be taken over multiple time points abundance matrix). In one embodiment, the association from the same source, or can be from different environmen matrix is used to identify associations between active micro tal sources (e.g., different animals). organism strains in a specific time point sample using rule 0327. The absolute abundance values over samples are mining approaches weighted with association (e.g., abun used in one embodiment to relate the one or more active dance) data. Filters are applied in one embodiment to microorganism strains, with an environmental parameter remove insignificant rules. (FIG. 2, 2008). In one embodiment, the environmental 0333. In one embodiment, the absolute abundance of one parameter is the presence of a second active microorganism or more, or two or more active microorganism strains is strain. Relating the one or more active microorganism related to one or more environmental parameters (FIG. 2, strains to the environmental parameter, in one embodiment, 2008), e.g., via co-occurrence determination. Environmental is carried out by determining the co-occurrence of the strain parameters are chosen by the user depending on the sample and parameter by correlation or by network analysis. (s) to be analyzed and are not restricted by the methods 0328. In one embodiment, determining the co-occurrence described herein. The environmental parameter can be a of one or more active microorganism strains with an envi parameter of the sample itself, e.g., pH, temperature, amount ronmental parameter comprises a network and/or cluster of protein in the sample. Alternatively, the environmental analysis method to measure connectivity of strains or a parameter is a parameter that affects a change in the identity US 2017/O 196922 A1 Jul. 13, 2017 of a microbial community (i.e., where the “identity” of a 0340 Network and cluster based analysis, for example, to microbial community is characterized by the type of micro carry out method step 2008 of FIG. 2, can be carried out via organism strains and/or number of particular microorganism a module. As used herein, a module can be, for example, any strains in a community), or is affected by a change in the assembly, instructions and/or set of operatively-coupled identity of a microbial community. For example, an envi electrical components, and can include, for example, a ronmental parameter in one embodiment, is the food intake memory, a processor, electrical traces, optical connectors, of an animal or the amount of milk (or the protein or fat Software (executing in hardware) and/or the like. content of the milk) produced by a lactating ruminant In one embodiment, the environmental parameter is the presence, Bovine Pathogen Resistance and Clearance activity and/or abundance of a second microorganism strain 0341. In some aspects, the present disclosure is drawn to in the microbial community, present in the same sample. administering one or more microbial compositions 0334. In some embodiments described herein, an envi described herein to cows to clear the gastrointestinal tract of ronmental parameter is referred to as a metadata parameter. pathogenic microbes. In some embodiments, the present 0335) Other examples of metadata parameters include but disclosure is further drawn to administering microbial com are not limited to genetic information from the host from positions described herein to prevent colonization of patho which the sample was obtained (e.g., DNA mutation infor genic microbes in the gastrointestinal tract. In some embodi mation), Sample pH, sample temperature, expression of a ments, the administration of microbial compositions particular protein or mRNA, nutrient conditions (e.g., level described herein further clears pathogens from the integu and/or identity of one or more nutrients) of the Surrounding ment and the respiratory tract of cows, and/or prevent environment/ecosystem), Susceptibility or resistance to dis colonization of pathogens on the integument and in the ease, onset or progression of disease, Susceptibility or resis respiratory tract. In some embodiments, the administration tance of the sample to toxins, efficacy of Xenobiotic com of microbial compositions described herein reduce leaky pounds (pharmaceutical drugs), biosynthesis of natural gut/intestinal permeability, inflammation, and/or incidence products, or a combination thereof. of liver disease. 0336 For example, according to one embodiment, micro 0342. In some embodiments, the microbial compositions organism strain number changes are calculated over multiple of the present disclosure comprise one or more microbes that samples according to the method of FIG. 2 (i.e., at 2001 are present in the gastrointestinal tract of cows at a relative 2007). Strain number changes of one or more active strains abundance of less than 15%, 14%, 13%, 12%, 11%, 10%, over time is compiled (e.g., one or more strains that have 9%, 8%, 7%, 6%. 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or initially been identified as active according to step 2006), O.O1%. and the directionality of change is noted (i.e., negative 0343. In some embodiments, after administration of values denoting decreases, positive values denoting microbial compositions of the present disclosure the one or increases). The number of cells over time is represented as more microbes are present in the gastrointestinal tract of the a network, with microorganism strains representing nodes cow at a relative abundance of at least 0.5%, 1%. 5%, 10%, and the abundance weighted rules representing edges. 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, Markov chains and random walks are leveraged to deter 65%, 70%, 75%, 80%, 85%, 90%, or 95%. mine connectivity between nodes and to define clusters. 0344 Pathogenic microbes of cows may include the Clusters in one embodiment are filtered using metadata in following: Clostridium perfiringens, Clostridium botulinum, order to identify clusters associated with desirable metadata Salmonella typi, Salmonella typhimurium, Salmonella enterica, Salmonella pullorum, Erysipelothrix insidiosa, (FIG. 2, 2008). Campylobacter jejuni, Campylobacter coli, Campylobacter 0337. In a further embodiment, microorganism strains are lari, Listeria monocytogenes, Streptococcus agalactiae, ranked according to importance by integrating cell number Streptococcus dysgalactiae, Corynebacterium bovis, Myco changes over time and strains present in target clusters, with plasma sp., Citrobacter sp., Enterobacter sp., Pseudomonas the highest changes in cell number ranking the highest. aeruginosa, Pasteurella sp., Bacillus cereus, Bacillus 0338 Network and/or cluster analysis method in one licheniformis, Streptococcus uberis, Staphylococcus aureus, embodiment, is used to measure connectivity of the one or and pathogenic strains of Escherichia coli and Staphylococ more strains within a network, wherein the network is a cus aureus. In some embodiments, the pathogenic microbes collection of two or more samples that share a common or include viral pathogens. In some embodiments, the patho similar environmental parameter. In one embodiment, net genic microbes are pathogenic to both cows and humans. In work analysis comprises linkage analysis, modularity analy Some embodiments, the pathogenic microbes are pathogenic sis, robustness measures, betweenness measures, connectiv to either cows or humans. ity measures, transitivity measures, centrality measures or a (0345. In some embodiments, the administration of com combination thereof. In another embodiment, network positions of the present disclosure to cows modulate the analysis comprises predictive modeling of network through makeup of the gastrointestinal microbiome Such that the link mining and prediction, social network theory, collective administered microbes outcompete microbial pathogens classification, link-based clustering, relational similarity, or present in the gastrointestinal tract. In some embodiments, a combination thereof. In another embodiment, network the administration of compositions of the present disclosure analysis comprises differential equation based modeling of to cows harboring microbial pathogens outcompetes the populations. In yet another embodiment, network analysis pathogens and clears cows of the pathogens. In some comprises Lotka-Volterra modeling. embodiments, the administration of compositions of the 0339 Cluster analysis method comprises building a con present disclosure results in the stimulation of host immu nectivity model, Subspace model, distribution model, den nity, and aid in clearance of the microbial pathogens. In sity model, or a centroid model. Some embodiments, the administration of compositions of US 2017/O 196922 A1 Jul. 13, 2017 59 the present disclosure introduce microbes that produce bac 0352. In some embodiments, improving the quantity of teriostatic and/or bactericidal components that decrease or proteins in milk produced by a ruminant, wherein proteins clear the cows of the microbial pathogens. (U.S. Pat. No. include caseins and whey. In some embodiments, proteins of 8,345,010). interest are only those proteins produced in milk. In other 0346. In some embodiments, challenging cows with a embodiments, proteins of interest are not required to be microbial colonizer or microbial pathogen after administer produced only in milk. Whey proteins include immuno ing one or more compositions of the present disclosure globulins, serum albumin, beta-lactoglobulin, and alpha prevents the microbial colonizer or microbial pathogen from lactoglobulin. growing to a relative abundance of greater than 15%, 14%, 0353. In some embodiments, improving the quantity of 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, Vitamins in milk produced by a ruminant is desirable. 1%, 0.5%, 0.1%, or 0.01%. In further embodiments, chal Vitamins found in milk include the fat-soluble vitamins of A, lenging cows with a microbial colonizer or microbial patho D, E, and K; as well as the B vitamins found in the aqueous gen after administering one or more compositions of the phase of the milk. present disclosure prevents the microbial colonizer or micro 0354. In some embodiments, improving the quantity of bial pathogen from colonizing cows. minerals in milk produced by a ruminant is desirable. 0347 In some embodiments, clearance of the microbial Minerals found in milk include iron, Zinc, copper, cobalt, colonizer or microbial pathogen occurs in less than 25 days, magnesium, manganese, molybdenum, calcium, phospho less than 24 days, less than 23 days, less than 22 days, less rous, potassium, Sodium, chlorine, and citric acid. Trace than 21 days, less than 20 days, less than 19 days, less than amounts of the following may be found in milk: aluminum, 18 days, less than 17 days, less than 16 days, less than 15 arsenic, boron, bromine, cadmium, chromium, fluorine, days, less than 14 days, less than 13 days, less than 12 days, iodine, lead, nickel, selenium, silicon, silver, strontium, and less than 11 days, less than 10 days, less than 9 days, less Vanadium. than 8 days, less than 7 days, less than 6 days, less than 5 0355. In some embodiments, improving the milk yield days, less than 4 days, less than 3 days, or less than 2 days and milk volume produced by a ruminant is desirable. In post administration of the one or more compositions of the some embodiments, it is further desirable if the increase in present disclosure. milk yield and Volume is not accompanied by simply an 0348. In some embodiments, clearance of the microbial increase in Solute volume. 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 0356. In some embodiments improving energy-corrected days, 5-25 days, 5-20 days, 5-15 days, 5-10 days, 10-30 milk (ECM) is desirable. In further embodiments, improving days, 10-25 days, 10-20 days, 10-15 days, 15-30 days, 15-25 ECM amounts to increasing the calculated ECM output. In days, 15-20 days, 20-30 days, 20-25 days, or 25-30 days post some embodiments, the ECM is calculated as follows: administration of the one or more compositions of the ECM=(0.327xmilk pounds)+(12.95xfat pounds)+(7.2xpro present disclosure. tein pounds). 0357. In some embodiments, improving the efficiency Improved Traits and digestibility of animal feed is desirable. In some embodiments, increasing the degradation of lignocellulosic 0349. In some aspects, the present disclosure is drawn to components from animal feed is desirable. Lignocellulosic administering microbial compositions described herein to components include lignin, cellulose, and hemicellulose. ruminants to improve one or more traits through the modu 0358. In some embodiments, increasing the concentra lation of aspects of milk production, milk quantity, milk tion of fatty acids in the rumen of ruminants is desirable. quality, ruminant digestive chemistry, and efficiency of feed Fatty acids include acetic acid, propionic acid, and butyric utilization and digestibility. acid. In some embodiments, maintaining the pH balance in 0350. In some embodiments, improving the quantity of the rumen to prevent lysis of beneficial microbial consortia milk fat produced by a ruminant is desirable, wherein milk is desirable. In some embodiments, maintaining the pH fat includes triglycerides, triacylglycerides, diacylglycer balance in the rumen to prevent a reduction of beneficial ides, monoacylglycerides, phospholipids, cholesterol, gly microbial consortia is desirable. colipids, and free fatty acids. In further embodiments, free fatty acids include short chain fatty acids (i.e., C4:0, C6:0. 0359. In some embodiments, decreasing the amount of and C8:0), medium chain fatty acids (i.e., C10:0, C10:1, methane and manure produced by ruminants is desirable. C12:0, C14:0, C14:1, and C15:0), and long chain fatty acids 0360. In some embodiments, improving the dry matter (i.e., C16:0, C16:1, C17:0, C17:1, C18:0, C18:1, C18:2, intake is desirable. In some embodiments, improving the C18:3, and C20:0). In further embodiments, it is desirable to efficiency of nitrogen utilization of the feed and dry matter achieve an increase in milkfat efficiency, which is measured ingested by ruminants is desirable. by the total weight of milk fat produced, divided by the 0361. In some embodiments, the improved traits of the weight of feed ingested. The weight of milk fat produced is present disclosure are the result of the administration of the calculated from the measured fat percentage multiplied by presently described microbial compositions. It is thought the weight of milk produced. that the microbial compositions modulate the microbiome of 0351. In some embodiments, improving the quantity of the ruminants such that the biochemistry of the rumen is carbohydrates in milk produced by a ruminant is desirable, changed in Such a way that the ruminal liquid and solid wherein carbohydrates include lactose, glucose, galactose, Substratum are more efficiently and more completely and oligosaccharides. Tao et al. (2009. J. Dairy Sci. degraded into Subcomponents and metabolites than the 92:2991-3001) disclose numerous oligosaccharides that may rumens of ruminants not having been administered micro be found in bovine milk. bial compositions of the present disclosure. US 2017/O 196922 A1 Jul. 13, 2017 60

0362. In some embodiments, the increase in efficiency tial equation based modeling of populations. In yet another and the increase of degradation of the ruminal Substratum embodiment, network analysis comprises Lotka-Volterra result in an increase in improved traits of the present modeling. disclosure. 0369. The environmental parameter can be a parameter of the sample itself, e.g., pH, temperature, amount of protein in Mode of Action: Digestibility Improvement in Ruminants the sample. Alternatively, the environmental parameter is a 0363 The rumen is a specialized stomach dedicated to parameter that affects a change in the identity of a microbial the digestion of feed components in ruminants. A diverse community (i.e., where the “identity” of a microbial com microbial population inhabits the rumen, where their pri munity is characterized by the type of microorganism strains mary function revolves around converting the fibrous and and/or number of particular microorganism strains in a non-fibrous carbohydrate components into useable sources community), or is affected by a change in the identity of a of energy and protein (FIG. 16). Cellulose, in particular, microbial community. For example, an environmental forms up to 40% of plant biomass and is considered indi parameter in one embodiment, is the food intake of an gestible by mammals. It also is tightly associated with other animal or the amount of milk (or the protein or fat content structural carbohydrates, including hemicellulose, pectin, of the milk) produced by a lactating ruminant In one and lignin. The cellulolytic microbes in the rumen leverage embodiment, the environmental parameter is the presence, extensive enzymatic activity in order break these molecules activity and/or abundance of a second microorganism strain down into simple Sugars and volatile fatty acids. This in the microbial community, present in the same sample. In enzymatic activity is critical to the extraction of energy from Some embodiments, an environmental parameter is referred feed, and more efficient degradation ultimately provides to as a metadata parameter. more energy to the animal. The soluble Sugars found in the 0370. Other examples of metadata parameters include but non-fibrous portion of the feed are also fermented into gases are not limited to genetic information from the host from and Volatile fatty acids such as butyrate, propionate, and which the sample was obtained (e.g., DNA mutation infor acetate. Volatile fatty acids arising from the digestion of both mation), Sample pH, sample temperature, expression of a the fibrous and non-fibrous components of feed are ulti particular protein or mRNA, nutrient conditions (e.g., level mately the main source of energy of the ruminant. and/or identity of one or more nutrients) of the Surrounding 0364 Individual fatty acids have been tested in ruminants environment/ecosystem), Susceptibility or resistance to dis in order to identify their impacts on varying aspects of ease, onset or progression of disease, Susceptibility or resis production. tance of the sample to toxins, efficacy of Xenobiotic com 0365 Acetate: Structural carbohydrates produce large pounds (pharmaceutical drugs), biosynthesis of natural amounts of acetate when degraded. An infusion of acetate products, or a combination thereof. directly into the rumen was shown to improve the yield of milk, as well as the amount of milk fat produced. Acetate EXAMPLES represents at least 90% of acids in the peripheral blood it is possible that acetate can be directly utilized by mammary Example I tissue as a source of energy. See Rook and Balch. 1961. Brit. J. Nutr. 15:361-369. 0366 Propionate: Propionate has been shown to increase Increase Total Milk Fat, Milk Protein, and milk protein production, but decrease milk yield. See Rook Energy-Corrected Milk (ECM) in Cows and Balch. 1961. Brit. J. Nutr. 15:361-369. 0371. The methods of Example I aim to increase the total 0367 Butyrate: An infusion of butyrate directly into the amount of milk fat and milk protein produced by a lactating rumen of dairy cows increases milk fat production without changing milk yield. See Huhtanen et al. 1993. J. Dairy Sci. ruminant, and the calculated ECM. 76:1114-1124. 0372. The methodologies presented herein based upon utilizing the disclosed isolated microbes, consortia, and Network Analysis compositions comprising the same-demonstrate an increase in the total amount of milk fat and milk protein 0368. A network and/or cluster analysis method, in one produced by a lactating ruminant. These increases were embodiment, is used to measure connectivity of the one or realized without the need for further addition of hormones. more strains within a network, wherein the network is a collection of two or more samples that share a common or 0373) In this example, a microbial consortium compris similar environmental parameter. In one embodiment, net ing two isolated microbes, Ascusb. 3138 (SEQ ID NO:28: work analysis comprises linkage analysis, modularity analy deposited as NRRL Y-67248) and Ascusf 15 (SEQ ID sis, robustness measures, betweenness measures, connectiv NO:32; deposited as NRRL Y67249), was administered to ity measures, transitivity measures, centrality measures or a Holstein cows in mid-stage lactation over a period of five combination thereof. In another embodiment, network weeks. analysis comprises predictive modeling of network through 0374. The cows were randomly assigned into 2 groups of link mining and prediction, social network theory, collective 8, wherein one of the groups was a control group that classification, link-based clustering, relational similarity, or received a buffer lacking a microbial consortium. The sec a combination thereof. In another embodiment, network ond group, the experimental group, was administered a analysis comprises mutual information, maximal informa microbial consortium comprising Ascusb_3138 (SEQ ID tion coefficient (MIC) calculations, or other nonparametric NO:28) and Ascusf 15 (SEQ ID NO:32) once per day for methods between variables to establish connectivity. In five weeks. Each of the cows were housed in individual pens another embodiment, network analysis comprises differen and were given free access to feed and water. The diet was US 2017/O 196922 A1 Jul. 13, 2017

a high milk yield diet. Cows were fed ad libitum and the feed TABLE 12 was weighed at the end of the day, and prior day refusals Dry matter intake, milk production and composition, body were weighed and discarded. Weighing was performed with weight (BW) gain, and rumen pH least square means (SEM) a PS-2000 scale from Salter Brecknell (Fairmont, Minn.). of cows assigned to Control and Inoculated groups. 0375 Cows were cannulated such that a cannula Treatment extended into the rumen of the cows. Cows were further provided at least 10 days of recovery post cannulation prior Control Inoculated to administering control dosages or experimental dosages. Outcome

0376 Each administration consisted of 20 ml of a neutral Dry matter intake, kg 26.2 2.8 30.2 - 1.2 buffered saline, and each administration consisted of Milk yield, kg 25.7 1.9 30.6 - 1.9 approximately 10 cells suspended in the saline. The control FCM, kg 27.7 2.5 32.5 2.5 ECM, kg 27.2 24 32.1 2.4 group received 20 ml of the saline once per day, while the Milk components, % experimental group received 20 ml of the saline further comprising 10 microbial cells of the described microbial Crude Protein 3.08: 0.06 3.27 0.11 Fat 3.87 0.08 4.06 O.O8 consortium. LactOSe 4.64 - 0.10 4.73 O.O3 0377 The rumen of every cow was sampled on days 0, 7, Milk components yield, kg 14, 21, and 35, wherein day 0 was the day prior to microbial Crude Protein O.80 O.O7 O.97 O.O7 administration. Note that the experimental and control Fat 1.01 - 0.10 1.20 - 0.10 MUN, mg/dL 6.17 O.60 7.41 - 0.45 administrations were performed after the rumen was FCMADMI 1.22 O.O7 1.10 O.O7 sampled on that day. Daily sampling of the rumen, begin Body weight gain, kg/day O.78 0.44 146 0.43 ning on day 0, with a pH meter from Hanna Instruments Rumen pH 6.24 O.09 6.05 - 0.09 (Woonsocket, R.I.) was inserted into the collected rumen fluid for recordings. Rumen sampling included both particu 0382 Table 12 reveals the effects of daily administration late and fluid sampling from the center, dorsal, Ventral, of an Ascus microbial consortium on the performance of anterior, and posterior regions of the rumen through the multiparous Holstein cows (between 60 and 120 days in cannula, and all five samples were pooled into 15 ml conical milk). Marked differences between the control and inocu vials containing 1.5 ml of stop solution (95% ethanol, 5% lated treatments were observed. The inoculated group expe phenol). A fecal sample was also collected on each sampling rienced increases in all parameters except FCM/DMI and day, wherein feces were collected from the rectum with the rumen pH. The weekly values at the beginning of the use of a palpation sleeve. Cows were weighed at the time of intervention period when cows were still adapting to the each sampling. treatment are included in the calculations. 0378 Fecal samples were placed in a 2 ounce vial, stored (0383 FIGS. 4-7 demonstrate the significant effects of the frozen, and analyzed to determine values for apparent neu microbial consortia on dairy cows for daily milk yield, daily tral detergent fibers (NDF) digestibility, apparent starch milk crude protein yield, daily milk fat yield, and daily digestibility, and apparent protein digestibility. Rumen Sam energy corrected milk yield over time. After an initial pling consisted of sampling both fluid and particulate por adaptation period, during which the microbes were observed tions of the rumen, each of which was stored in a 15 ml to colonize the rumen, the production characteristics of the conical tube. Cells were fixed with a 10% stop solution (5% inoculated treatment group increased and diverged from the phenol/95% ethanol mixture) and kept at 4° C. and shipped control group. to Ascus Biosciences (Vista, Calif.) on ice. 0384 FIG. 3A demonstrates that cows that were admin 0379 The milk yield was measured twice per day, once istered the microbial consortia exhibited a 20.9% increase in in the morning and once at night. Milk composition (% fats the average production of milk fat versus cows that were and % proteins, etc.) was measured twice per day, once in administered the buffered solution alone. FIG. 3B demon the morning and once at night. Milk samples were further strates that cows that were administered the microbial con analyzed with near-infrared spectroscopy for protein fats, sortia exhibited a 20.7% increase in the average production Solids, analysis for milk urea nitrogen (MUN), and Somatic of milk protein versus cows that were administered the cell counts (SCC) at the Tulare Dairy Herd Improvement buffered solution alone. FIG. 3C demonstrates that cows that Association (DHIA) (Tulare, Calif.). Feed intake of indi were administered the microbial consortia exhibited a 19.4% vidual cows and rumen pH were determined once per day. increase in the average production of energy corrected milk. The increases seen in FIG. 3A-C became less pronounced 0380 A sample of the total mixed ration (TMR) was after the administration of the consortia ceased, as depicted collected the final day of the adaptation period, and then by the vertical line intersecting the data points. Successively collected once per week. Sampling was per (0385 FIG. 14 clearly identifies the effect of microbial formed with the quartering method, wherein the samples consortia on the Somatic cell count in the milk. The experi were stored in vacuum sealed bags which were shipped to mental group of cows receiving the microbial consortia Cumberland Valley Analytical Services (Hagerstown, Md.) exhibited a decrease in the number of cows with greater than and analyzed with the NIR1 package. 200,000 somatic cells/ml of milk. In the field of dairy 0381. The final day of administration of buffer and/or farming, the SCC is a strong indicator of milk quality. The microbial bioconsortia was on day 35, however all other majority of Somatic cells found in milk are leukocytes, measurements and samplings continued as described until immune cells that accumulate in a particular tissue/fluid in day 46. increasing numbers usually due to an immune response to a US 2017/O 196922 A1 Jul. 13, 2017 62 pathogen. Generally, the lower the SCC the higher the 04.05 Each administration consists of 5 ml of a neutral quality of milk. Dosogne et al. 2011. J. Dairy Sci. 86(3): buffered saline, and each administration consists of approxi 828-834. mately 10 cells suspended in the saline. The control group receives 5 ml of the saline once per day, while the experi Example II mental groups receive 5 ml of the Saline further comprising 10 microbial cells of the described consortia. Increase Total Milk Fat and Milk Protein in Cows 0406. The rumen of every cow is sampled on days 0, 7, 14, 21, and 35, wherein day 0 is the day prior to microbial 0386. In certain embodiments of the disclosure, the pres administration. Note that the experimental and control ent methods aim to increase the total amount of milkfat and administrations are performed after the rumen has been milk protein produced by a lactating ruminant. sampled on that day. Daily sampling of the rumen, begin 0387. The methodologies presented herein based upon ning on day 0, with a pH meter from Hanna Instruments utilizing the disclosed isolated microbes, consortia, and (Woonsocket, R.I.) is inserted into the collected rumen fluid compositions comprising the same have the potential to for recordings. Rumen sampling included both particulate increase the total amount of milk fat and milk protein and fluid sampling from the center, dorsal, Ventral, anterior, produced by a lactating ruminant. These increases can be and posterior regions of the rumen through the cannula, and realized without the need for further addition of hormones. all five samples were pooled into 15 ml conical vials 0388. In this example, seven microbial consortia com containing 1.5 ml of stop solution (95% ethanol, 5% phe prising isolated microbes from Table 1 are administered to nol). A fecal sample is also collected on each sampling day, Holstein cows in mid-stage lactation over a period of six wherein feces are collected from the rectum with the use of weeks. The consortia are as follows: a palpation sleeve. Cows are weighed at the time of each 0389 Consortium 1–Ascusb 7, Ascusb 32, Ascusf 45, sampling. and Ascusf 24: 04.07 Fecal samples are placed in a 2 ounce vial, stored 0390 Consortium 2 Ascusb 7. Ascusb. 1801, Ascusf frozen, and analyzed to determine values for apparent NDF 45, and Ascusf 24, digestibility, apparent starch digestibility, and apparent pro 0391 Consortium 3–Ascusb 7, Ascusb. 268, Ascusf tein digestibility. Rumen sampling consists of sampling both 45, and Ascusf 24, fluid and particulate portions of the rumen, each of which is 0392 Consortium 4—Ascusb 7, Ascusb. 232, Ascusf stored in a 15 ml conical tube. Cells are fixed with a 10% 45, and Ascusf 24, stop solution (5% phenol/95% ethanol mixture) and kept at 0393 Consortium 5—Ascusb 7. Ascusb 32, Ascusf 45, 4°C. and shipped to Ascus Biosciences (Vista, Calif.) on ice. and Ascusf 249; 0408. The milk yield is measured twice per day, once in 0394 Consortium 6–Ascusb 7, Ascusb 32, Ascusf 45, the morning and once at night. Milk composition (% fats and and Ascusf 353; and % proteins, etc.) is measured twice per day, once in the 0395 Consortium 7 Ascusb 7, Ascusb 32, Ascusf 45, morning and once at night. Milk samples are further ana and Ascusf 23. lyzed with near-infrared spectroscopy for protein fats, Sol 0396 Consortium 8 Ascusb_3138, Ascusb. 1801, ids, analysis for milk urea nitrogen (MUN), and somatic cell Ascusf 45, and Ascusf 15. counts (SCC) at the Tulare Dairy Herd Improvement Asso 0397 Consortium 9 Ascusb. 3138, Ascusb. 268, ciation (DHIA) (Tulare, Calif.). Feed intake of individual Ascusf 45, and Ascusf 15. cows and rumen pH are determined once per day. 0398 Consortium 10 Ascusb. 3138, Ascusb. 232, 04.09. A sample of the total mixed ration (TMR) is Ascusf 23, and Ascusf 15. collected the final day of the adaptation period, and then 0399 Consortium 11—Ascusb 7. Ascusb_3138, Successively collected once per week. Sampling is per Ascusf 15, and Ascusf 249. formed with the quartering method, wherein the samples are 04.00 Consortium 12—Ascusb 7. Ascusb_3138, stored in vacuum sealed bags which are shipped to Cum Ascusf 45, and Ascusf 15. berland Valley Analytical Services (Hagerstown, Md.) and 04.01 Consortium 13—Ascusb 3138, Ascusb 32, analyzed with the NIR1 package. Ascusf 15, and Ascusf 23. 0410. In some embodiments, the percent fats and percent 0402 Consortium 14—Ascusb_3138 and Ascusf 15. protein of milk in each of the experimental cow groups is 0403. The cows are randomly assigned into 15 groups of expected to demonstrate a statistically significant increase 8, wherein one of the groups is a control group that receives over the percent fats and percent protein of milk in the a buffer lacking a microbial consortium. The remaining control cow group. In other embodiments, the increase is not seven groups are experimental groups and will each be expected to be statistically significant, but it is expected to administered one of the thirteen microbial bioconsortia once be still quantifiable. per day for six weeks. Each of the cows are held in individual pens to mitigate cross-contamination and are Example III given free access to feed and water. The diet is a high milk yield diet. Cows are fed twice per day and the feed will be Shifting the Microbiome of Ruminants weighed at each feeding, and prior day refusals will be 0411. In certain embodiments of the disclosure, the pres weighed and discarded. Weighing is performed with a ent methods aim to modulate the microbiome of ruminants PS-2000 scale from Salter Brecknell (Fairmont, Minn.). through the administration of one or more microbes to the 0404 Cows are cannulated such that a cannula extends gastrointestinal tract of ruminants. into the rumen of the cows. Cows are further provided at 0412. The methodologies presented herein based upon least 10 days of recovery post cannulation prior to admin utilizing the disclosed isolated microbes, consortia, and istering control dosages or experimental dosages. compositions comprising the same have the potential to US 2017/O 196922 A1 Jul. 13, 2017

modulate the microbiome of ruminants. The modulation of embodiments, the increase is not expected to be statistically a ruminant's gastrointestinal microbiome may lead to an significant, but it is expected to be still quantifiable. increase of desirable traits of the present disclosure. 0413. In this example, the microbial consortia of Table 5 Example IV are administered to Holstein cows over a period of six weeks. Milk Fat Produced Versus Absolute Abundance of 0414. The cows are randomly assigned into 37 groups of Microbes 8, wherein one of the groups is a control group that receives 0421 Determine rumen microbial community constitu a buffer lacking a microbial consortium. The remaining ents that impact the production of milk fat in dairy cows. thirty-six groups are experimental groups and will each be 0422. Eight lactating, ruminally cannulated, Holstein administered one of the thirty-six microbial consortia once cows were housed in individual tie-stalls for use in the per day for six weeks. Each of the cows are held in experiment. Cows were fed twice daily, milked twice a day, individual pens to mitigate cross-contamination and are and had continuous access to fresh water. One cow (cow given free access to feed and water. The diet is a high milk 4201) was removed from the study after the first dietary yield diet. Cows are fed twice per day and the feed will be Milk Fat Depression due to complications arising from an weighed at each feeding, and prior day refusals will be abortion prior to the experiment. weighed and discarded. Weighing is performed with a 0423 Experimental Design and Treatment: The experi PS-2000 scale from Salter Brecknell (Fairmont, Minn.). ment used a crossover design with 2 groups and 1 experi 0415 Cows are cannulated such that a cannula extends mental period. The experimental period lasted 38 days: 10 into the rumen of the cows. Cows are further provided at days for the covariate/wash-out period and 28 days for data least 10 days of recovery post cannulation prior to admin collection and sampling. The data collection period con istering control dosages or experimental dosages. sisted of 10 days of dietary Milk Fat Depression (MFD) and 0416 Each administration consists of 5 ml of a neutral 18 days of recovery. After the first experimental period, all buffered saline, and each administration consists of approxi cows underwent a 10-day wash out period prior to the mately 10 cells suspended in the saline. The control group beginning of period 2. receives 5 ml of the saline once per day, while the experi 0424 Dietary MFD was induced with a total mixed ration mental groups receive 5 ml of the saline further comprising (TMR) low in fiber (29% NDF) with high starch degrad 10 microbial cells of the described consortia. ability (70% degradable) and high polyunsaturated fatty acid levels (PUFA, 3.7%). The Recovery phase included two 0417. The rumen of every cow is sampled on days 0, 7, diets variable in starch degradability. Four cows were ran 14, 21, and 35, wherein day 0 is the day prior to adminis domly assigned to the recovery diet high in fiber (37% tration. Note that the experimental and control administra NDF), low in PUFA (2.6%), and high in starch degradability tions are performed after the rumen has been sampled on that (70% degradable). The remaining four cows were fed a day. Daily sampling of the rumen, beginning on day 0, with recovery diet high in fiber (37% NDF), low in PUFA (2.6%), a pH meter from Hanna Instruments (Woonsocket, R.I.) is but low in starch degradability (35%). inserted into the collected rumen fluid for recordings. 0425. During the 10-day covariate and 10-day wash out Rumen sampling included both particulate and fluid Sam periods, cows were fed the high fiber, low PUFA, and low pling from the center, dorsal, Ventral, anterior, and posterior starch degradability diet. regions of the rumen through the cannula, and all five 0426 Samples and Measurements: Milk yield, dry matter samples were pooled into 15 ml conical vials containing 1.5 intake, and feed efficiency were measured daily for each ml of stop solution (95% ethanol, 5% phenol). A fecal animal throughout the covariate, wash out, and sample sample is also collected on each sampling day, whereinfeces collection periods. TMR samples were measured for nutrient are collected from the rectum with the use of a palpation composition. During the collection period, milk samples sleeve. Cows are weighed at the time of each sampling. were collected and analyzed every 3 days. Samples were 0418 Fecal samples are placed in a 2 ounce vial, stored analyzed for milk component concentrations (milk fat, milk frozen, and analyzed to determine values for apparent NDF protein, lactose, milk urea nitrogen, Somatic cell counts, and digestibility, apparent starch digestibility, and apparent pro Solids) and fatty acid compositions. tein digestibility. Rumen sampling consists of sampling both 0427 Rumen samples were collected and analyzed for fluid and particulate portions of the rumen, each of which is microbial community composition and activity every 3 days stored in a 15 ml conical tube. Cells are fixed with a 10% during the collection period. The rumen was intensively stop solution (5% phenol/95% ethanol mixture) and kept at sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours 4°C. and shipped to Ascus Biosciences (Vista, Calif.) on ice. after feeding during day 0, day 7, and day 10 of the dietary 0419. The samples of fluid and particulate portions of the MFD. Similarly, the rumen was intensively sampled 0, 2, 4, rumen, as well as the fecal samples are each evaluated for 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours after feeding on day microbiome fingerprinting utilizing the T-RFLP method 16 and day 28 of the sample collection period. Rumen combined with nMDS ordination and PERMANOVA sta contents were analyzed for pH, acetate concentration, tistics. butyrate concentration, propionate concentration, isoacid 0420. In some embodiments, the ruminal and fecal concentration, and long chain and CLA isomer concentra microbiome in each of the experimental cow groups is tions. Rumen sampling included both particulate and fluid expected to demonstrate a statistically significant change in sampling from the center, dorsal, Ventral, anterior, and the microbiomes over the microbiomes in the control cow posterior regions of the rumen through the cannula, and all group as well as the 0 day microbiome samples, wherein the five samples were pooled into 15 ml conical vials. change is a significant increase in the proportion of microbes 0428 Rumen Sample Preparation and Sequencing: After administered in the experimental administrations. In other collection, rumen samples were centrifuged at 4,000 rpm in US 2017/O 196922 A1 Jul. 13, 2017 64 a swing bucket centrifuge for 20 minutes at 4° C. The produced. MIC scores of the microbes of the present dis Supernatant was decanted, and an aliquot of each rumen closure are recited in Table 1. The greater the MIC score, the content sample (1-2 mg) was added to a sterile 1.7 mL tube greater the ability of the microbe to confer an increase in the prefilled with 0.1 mm glass beads. A second aliquot was weight of milk fat produced by a cow collected and stored in an empty, sterile 1.7 mL tube for cell counting. Example V 0429 Rumen samples in empty tubes were stained and put through a flow cytometer to quantify the number of cells Comparative Analysis of MIC Scores From of each microorganism type in each sample. Rumen samples Published Work of Other with glass beads were homogenized with bead beating to lyse microorganisms. DNA and RNA was extracted and Groups purified from each sample and prepared for sequencing on 0434 Utilizing Ascus Biosciences technology, the per an Illumina Miseq. Samples were sequenced using paired formance of currently available microbial feed additive end chemistry, with 300 base pairs sequenced on each end products was predicted. of the library. 0435 Direct-fed microbial products that claim to 0430 Sequencing Read Processing and Data Analysis: enhance dairy performance are openly available on the Sequencing reads were quality trimmed and processed to market. Some of these products contain microorganism identify bacterial species present in the rumen based on a strains that are native rumen microorganisms (Megasphaera marker gene, 16S rDNA, or ITS1 and/or ITS2. Count elsdenii), or are within 97% sequence similarity of native datasets and activity datasets were integrated with the rumen microorganisms. We have identified the species of sequencing reads to determine the absolute cell numbers of microbes utilized in these products, and calculated their active microbial species within the rumen microbial com MIC score with respect to milk fat efficiency (FIG. 13). As munity. Production characteristics of the cow over time, evidenced by the curve in FIG. 13, all of the assayed strains including pounds of milk produced, were linked to the that were available fell below the threshold used to define distribution of active microorganisms within each sample useful and non-useful strains, as describe above. The spe over the course of the experiment using mutual information. cies/strain closest to the cutoff, Prevotella bryantii, has 0431 Tests cases to determine the impact of count data, shown a positive effect in one study. activity data, and count and activity on the final output were Lactobacillus plantarum. MIC 0.28402 run by omitting the appropriate datasets from the sequencing 0436 The calculated MIC predicts that Lactobacillus analysis. To assess the impact of using a linear correlation plantarum is poorly associated with milk fat efficiency, and rather than the MIC on target selection, Pearson’s coeffi the art discloses that an inoculation of L. plantarum yields cients were also calculated for pounds of milk fat produced no increase in milk fat product, and at least one study as compared to the relative abundance of all microorganisms discloses that some strains of L. plantarum create molecules and the absolute abundance of active microorganisms. that cause milk fat depression. See Lee et al. 2007. J. Appl. Microbiol. 103(4):1140-1146 and Mohammed et al. 2012. J. Results Dairy Sci. 95(1):328-339. 0432 One component of the Ascus Biosciences technol Lactobacillus acidophilus. MIC 0.30048 ogy utilized in this application leverages mutual information 0437. The calculated MIC predicts that Lactobacillus to rank the importantice of native microbial Strains residing acidophilus is poorly associated with milkfat efficiency, and in the gastrointestinal tract of the animal to specific animal the art discloses that the administration of L. acidophilus to traits. The maximal information coefficient (MIC) scores are dairy cows/calves had no effect of various aspects of milk calculated for all microorganisms and the desired animal yield/milk component yield. See Higginbotham and Bath. trait. Relationships were scored on a scale of 0 to 1, with 1 1993.J. Dairy Sci. 76(2): 615-620; Abu-Tarboush et al. 1996. represending a strong relationship between the microbial Animal Feed Sci. Tech. 57(1-2):39-49; McGilliard and Stall strain and the animal trait, and 0 representing no relation ings. 1998. J. Dairy Sci. 81(5):1353-1357; and Raeth-Knight ship. A cut-off based on this score is used to define useful et al. 2007. J. Dairy Sci. 90(4): 1802-1809: But see Boyd et and non-useful microorganisms with respect to the improve al. 2011. 94(9):4616-4622 (discloses an increase in milk ment of specific traits. FIG. 9 and FIG. 10 depict the MIC yield and milk protein yield). While Boyd et al. does disclose score distribution for rumen microbial strains that share a an increase in milk and milk protein yield, the controls of relationship with milkfat efficiency in dairy cows. The point this single study do not appear to adequately isolate the the where the curve shifts from exponential to linear (-0.45-0.5 presence of L. acidophilus as the cause of the increase. The for bacteria and -0.3 for fungi) represents the cutoff between body of prior art contradicts the finding of Boyd et al. useful and non-useful microorganism strains pertaining to Megasphaera elsdenii: MIC 0.32548 milkfat efficiency. FIG. 11 and FIG. 12 depict the MIC score 0438. The calculated MIC predicts that Megasphaera distributions for rumen microbial strains that share a rela elsdenii is poorly associated with milkfat efficiency, and the tionship with dairy efficiency. The point where the curve art provides Substantial evidence to suggest that Megaspha shifts from exponential to linear (-0.45-0.5 for bacteria and era elsdenii has no positive effect upon milk fat efficiency, ~0.25 for fungi) represents the cutoff between useful and but multiple references provide evidence to suggest that it non-useful microorganism Strains. has a negative effect on milk fat efficiency. See Kim et al. 0433. The MICs were calculated between pounds of milk 2002. J. Appl. Micro. 92(5):976-982: Hagg. 2008. Disser fat produced and the absolute abundance of each active tation, University of Pretoria. 1-72; Hagg et al. 2010. S. microorganism. Microorganisms were ranked by MIC score, African. J. Animal Sci., 40(2):101-112: Zebeliet al. 2011. J. and microorganisms with the highest MIC scores were Dairy Res. 79(1):16–25; Aikman et al. 2011. J. Dairy Sci. selected as the target species most relevant to pounds of milk 94(6):2840-2849; Mohammed et al. 2012. J. Dairy Sci. US 2017/O 196922 A1 Jul. 13, 2017

95(1):328-339; and Cacite and Weimer. 2016. J. Animal Sci. cannula, and all five samples were pooled into 15 ml conical Poster Abstract. 94(sup. 5):784. vials containing 1.5 ml of stop solution (95% ethanol, 5% Prevotella bryantii: MIC 0.4016.1 phenol) and stored at 4°C. and shipped to Ascus BioSciences 0439. The calculated MIC predicts that Prevotella bry (Vista, Calif.) on ice. antii is not highly associated with milkfat efficiency, and the 0447. A portion of each rumen sample was stained and art provides evidence that P. bryantii administered during put through a flow cytometer to quantify the number of cells Subacute acidosis challenge in midlactation dairy cows has of each microorganism type in each sample. A separate no apparent effect on milk yield, whereas administration of portion of the same rumen sample was homogenized with the microbe to dairy cows in early lactation yields improved bead beating to lyse microorganisms. DNA and RNA was milk fat concentrations. See Chiquette et al. 2012. J. Dairy extracted and purified from each sample and prepared for Sci. 95(10):5985-5995, but see Chiquette et al. 2008.91 (9): sequencing on an Illumina Miseq. Samples were sequenced 3536-3543; respectively. using paired-end chemistry, with 300 base pairs sequenced on each end of the library. The sequencing reads were used Example VI to quantify the number of cells of each active, microbial member present in each animal rumen in the control and Shift in Rumen Microbial Composition after experimental groups over the course of the experiment. Administration of a Microbial Composition 0448 Ascusb_3138 and Ascusf 15 both colonized, and 0440 The methods of the instant example aim to increase were active in the rumen after -3-5 days of daily adminis the total amount of milk fat and milk protein produced by a tration, depending on the animal. This colonization was lactating ruminant, and the calculated energy corrected milk observed in the experimental group, but not in the control (ECM). group. The rumen is a dynamic environment, where the 0441 The methodologies presented herein based upon chemistry of the cumulative rumen microbial population is utilizing the disclosed isolated microbes, consortia, and highly intertwined. The artificial addition of Ascusb. 3138 compositions comprising the same-demonstrate an and Ascuf 15 could have effects on the overall structure of increase in the total amount of milk fat and milk protein the community. To assess this potential impact, the entire produced by a lactating ruminant. These increases were microbial community was analyzed over the course of the realized without the need for further addition of hormones. experiment to identify higher level taxonomic shifts in 0442. In this example, a microbial consortium compris microbial community population. ing two isolated microbes, Ascusb_3138 (SEQ ID NO:28) 0449 Distinct trends were not observed in the fungal and Ascusf 15 (SEQ ID NO:32), was administered to Hol populations over time, aside from the higher cell numbers of Stein cows in mid-stage lactation over a period of five weeks. Ascusf 15 in the experimental animals. The bacterial popu 0443) The cows were randomly assigned into 2 groups of lations, however, did change more predictably. To assess 8, in which one of the groups was a control group that high level trends across individual animals over time, per received a buffer lacking a microbial consortium. The sec cent compositions of the microbial populations were calcu ond group, the experimental group, was administered a lated and compared. See microbial consortium comprising Ascusb. 3138 (SEQ ID 0450 Table 13. Only genera composing greater than 1% NO:28) and Ascusf 15 (SEQ ID NO:32) once per day for of the community were analyzed. The percent composition five weeks. Each cow was housed in an individual pen and of genera containing known fiber-degrading bacteria, was given free access to feed and water. The diet was a high including Ruminococcus, was found to increase in experi milk yield diet. Cows were fed ad libitum and the feed was mental animals as compared to control animals. Volatile weighed at the end of each day, and prior day refusals were fatty acid-producing genera, including Clostridial cluster weighed and discarded. Weighing was performed with a XIVa, Clostridium, Pseudobutyrivibrio, Butyricimonas, and PS-2000 scale from Salter Brecknell (Fairmont, Minn.). Lachnospira were also found at higher abundances in the 0444 Cows were cannulated such that a cannula experimental animals. The greatest shift was observed in the extended into the rumen of the cows. Cows were further genera Prevotella. Members of this genus have been shown provided at least 10 days of recovery post cannulation prior to be involved in the digestion of cellobiose, pectin, and to administering control dosages or experimental dosages. various other structural carbohydrates within the rumen. 0445. Each administration consisted of 20 ml of a neutral Prevotella sp. have further been implicated in the conversion buffered saline, and each administration consisted of of plant lignins into beneficial antioxidants (Schogor et al. approximately 10 cells suspended in the saline. The control PLOS One. 9(4):e87949 (10 p.)). group received 20 ml of the saline once per day, while the 0451. To more directly measure quantitative changes in experimental group received 20 ml of the saline further the rumen over time, cell count data was integrated with comprising 10 microbial cells of the described microbial sequencing data to identify bulk changes in the population at consortium. the cell level. Fold changes in cell numbers were determined 0446. The rumen of every cow was sampled on days 0, 7, by dividing the average number of cells of each genera in the 14, 21, and 35, wherein day 0 was the day prior to microbial experimental group by the average number of cells of each administration. Note that the experimental and control genera in the control group. See Table 13. The cell count administrations were performed after the rumen was analysis captured many genera that fell under the threshold sampled on that day. Daily sampling of the rumen, begin in the previous analysis Promicromonospora, Rhodopirel ning on day 0, with a pH meter from Hanna Instruments lula, Olivibacter, Victivallis, Nocardia, Lentisphaera, (Woonsocket, R.I.) was inserted into the collected rumen Eubacteiru, Pedobacter, Butyricimonas, Mogibacterium, fluid for recordings. Rumen sampling included both particu and Desulfovibrio were all found to be at least 10 fold higher late and fluid sampling from the center, dorsal, Ventral, on average in the experimental animals. PrevOtella, Lach anterior, and posterior regions of the rumen through the nospira, Butyricicoccus, Clostridium XIVa, Roseburia, US 2017/O 196922 A1 Jul. 13, 2017 66

Clostridium sensu stricto, and Pseudobutyrivibrio were Example VII found to be ~1.5 times higher in the experimental animals. Analysis of Rumen Microbes for Volatile Fatty Acid Production and Carbon Source Use TABLE 13 A. Volatile Fatty Acid (VFA) Production Family and Genus Level Analysis of Shifts in Bacterial Populations 0452. To assess the ability of the strains to produce volatile fatty acids, High Performance Liquid Chromatog Experi raphy (HPLC) was utilized to measure the conventration sof Control Varia- mental Varia acetate, butyrate, and propionate in spent media. M2GSC Taxonomy (%) tion (%) tion media was used in an assay mimicking rumen conditions as closely as possibile. Family Level Analysis 0453 For pure cultures, a single colony from each of the desired strains (from anaerobic agar plates) was inoculated into M2GSC media. A medium blank (control) was also Prevotellaceae 15.27 6.43 18.62 S.63 prepared. Cultures and the medium blank were incubated at Ruminococcaceae 16.40 5.14 17.84 6.44 37° C. until significant growth was visible. An optical Lachnospiraceae 23.85 7.63 24.58 6.96 density (OD600) was determined for each culture, and the Genus Level Analysis strain ID was confirmed with Illumina sequencing. An aliquot of culture was subjected to sterile filtration into a Prevoteia 16.14 S.98 1914 5.27 washed glass 15 ml sample vial and analyzed by HPLC: Clostridium XIVa. 12.41 5.35 12.83 4.81 HPLC assays were performed at Michigan State University. Lachnospiracea incertae sedis 3.68 1.68 3.93 1.33 Enrichments that exhibited growth were also stained and cell Riminococcits 3.70 2.21 3.82 1.82 counted to confirm that the individual strains within each Clostridium IV 3.02 1.87 3.51 1.74 enrichment grew. Strains often appeared in multiple enrich ments, so the enrichment with the highest amount of growth Butyricimonas 1.68 1.35 1.83 2.38 for the strain (i.e. the highest increase in cell number of that Clostridium sensu stricto 1.52 O.65 1.81 O.S3 strain) is reported in Table 15. Pseudobutyrivibrio 1.OO O.64 1.42 1.03 0454. Due to the vast complexity of metabolisms and Citrobacter 0.71 1.86 1.95 3.OO microbial lifestyles present in the rumen, many rumen Selenomonas 1.04 O.83 1.34 O.86 microorganisms are incapable of axenic growth. In order to Hydrogeno- 1.03 1.08 1.11 O.78 assay these organisms for desirable characteristics, enrich anaerobacterium ments cultures were established under a variety of condi tions that mimicked particular features of the rumen envi ronment. Diluted rumen fluid (/100 dilution) was inoculated into M2GSC or M2 media supplemented with a variety of TABLE 1.4 carbon Sources including Xylose (4 g/L), mannitol (4 g/L). glycerol (4 g/L), Xylan (2 g/L), cellobiose (2 g/L), arabinose Analysis of Fold Changes in Bacterial cells (4 g/L), mannose (4 g/L), rhaminose (2 g/L), maltose (2 Genus Fold change (experimental control) g/L), maltose (2 g/L), and molasses. Rumen fluid was also Sometimes omitted from the recipe. Additions including Promicromonospora 22619.50 Rhodopinellula 643.31 amino acids, Volatile fatty acids, and antibiotics, were also Olivibacter 394.O1 varied across the enrichments. A medium blank (control) Victivais 83.97 was also prepared. Cultures and the medium blank were Nocardia 73.81 incubated at 37° C. until significant growth was visible. An Lentisphaera 57.70 optical density (OD600) was determined for each culture, Eubacterium SO.19 Pedobacter 26.15 and the strain IDs were confirmed with Illumina sequencing. Butyricimonas 15.47 An aliquot of culture was subjected to sterile filtration into Mogibacterium 15.23 a washed glass 15 ml sample vial and analyzed by HPLC: Desulfovibrio 13.55 HPLC assays were performed at Michigan State University. Anaeroplasma 8.84 Sharpea 8.78 Enrichments that exhibited growth were also stained and cell Erysipelotrichaceae incertae sedis 5.71 counted to confirm that the individual strains within each Saccharofermenians S.O.9 enrichment grew. Strains often appeared in multiple enrich Parabacteroides 4.16 ments, so the enrichment with the highest amount of growth Papillibacter 3.63 Citrobacter 2.95 for the strain (i.e., the highest increase in cell number of that Lachnospiracea incertae sedis 2.27 strain) is reported in Table 15. Prevoteia 1.60 0455 Concentrations of acetate, butyrate, and propionate Butyricicoccus 1.95 were quantified for the medium blanks as well as the sterile Clostridium XIVa. 1.47 Roseburia 1.44 filtered culture samples for both pure strain and enrichment Pseudobutyrivibrio 1.43 experiments. HPLC parameters were as follows: Biorad Clostridium sensu stricto 1.29 Aminex HPX-87H column, 60° C., 0.5 ml/minute mobile Selenomonas 1.25 phase 0.00325 N HSO 500 psi, 35C RI detector, 45 Olseneia 1.04 minute run time, and 5 uL injection volume. Concentrations of acetate, butyrate, and propionate for both pure cultures and enrichments are reported in Table 15. US 2017/O 196922 A1 Jul. 13, 2017 67

TABLE 1.5 TABLE 15-continued Volatile Fatty Acid Production of Microbial Strains Volatile Fatty Acid Production of Microbial Strains as Analyzed with HPLC, Normalized to 1 OD as Analyzed with HPLC, Normalized to 1 OD Sample ID Acetate (gL) Propionate (gL) Butyrate (gL) Sample ID Acetate (gL) Propionate (gL) Butyrate (gL) Ascusb. 5 3.59 O.OO O.OO Ascusb 6589 547 26.95 -0.60 1.54 4.08 O.O3 Ascusb 12103 O.94 O.OO O.OO Ascusb 7 Ascusb. 14245 1.76 O.OO O.OO Ascusb. 11 -6.88 -0.28 -0.04 Ascusb. 20083 27.13 34.SS 3.31 Ascusb. 26 6.10 7.57 1.38 Ascusb. 20187 740 7.36 7.91 Ascusb. 27 O.S9 148 4.98 Ascusb. 32 6.10 7.57 1.38 Ascusb 36 4.30 O.68 O.OO Ascusb. 79 2.00 O.OO O.OO B. Soluble Carbon Source Assay Ascusb 82 6.10 7.57 1.38 0456 To assess the ability of the strains to degrade Ascusb. 89 1.69 4.2O 0.27 various carbon sources, an optical density (OD600) was Ascusb. 101 1.45 -0.21 O.OO used to measure growth of strains on multiple carbon Ascusb 102 2.00 O.OO O.OO Sources over time. Ascusb 104 27.13 34.SS 3.31 Ascusb. 111 1.69 4.2O 0.27 0457 For pure isolates, a single colony from each of the Ascusb. 119 1.54 4.08 O.O3 desired strains (from anaerobic agar plates) was inoculated Ascusb 125 10.97 S.68 4.69 into M2GSC media. A medium blank (control) was also Ascusb. 145 1.69 4.2O 0.27 prepared. Strains were inoculated into a carbon Source assay Ascusb 149 O.OO O.OO O.47 anaerobically, wherein the assay was set up in a 2 mL sterile Ascusb. 159 7.05 4.52 1.42 96-well plate, with each well containing RAMM salts, Ascusb. 183 O.OO O.OO 2.03 Vitamins, minerals, cysteine, and a single carbon Source. Ascusb. 187 10.97 S.68 4.69 Carbon Sources included glucose, Xylan, lactate, Xylose, Ascusb. 190 740 7.36 7.91 Ascusb. 199 11.36 1.17 7.65 mannose, glycerol, pectin, molasses, and cellobiose. Cells Ascusb. 205 6.10 7.57 1.38 were inoculated such that each well started at an OD600 of Ascusb. 232 7.83 1.15 3.19 0.01. Optican densities were read at 600 nm with the Ascusb. 268 2.00 O.OO O.OO Synergy H4 hybrid plate reader. The strain IDs were con Ascusb. 278 7.05 4.52 1.42 firmed with Illumina sequencing after all wells were in Ascusb 329 7.83 1.15 3.19 stationary phase. Ascusb 368 1.69 4.2O 0.27 0458 As in the volatile fatty acid assay above, enrich Ascusb 374 7.83 1.15 3.19 ments were also used to assay carbon Source degradation. Ascusb. 411 1.69 4.2O 0.27 Ascusb. 546 4.30 O.68 O.OO Diluted rumen fluid (/100 dilution) was inoculated into Ascusb 728 2.36 O.OO O.OO M2GSC or M2 media supplemented with a variety of carbon Ascusb. 765 -11.63 O.OO O.OO Sources including Xylose (4 g/L), mannitol (4 g/L), glycerol Ascusb. 810 1.54 4.08 O.O3 (4 g/L), Xylan (2 g/L), cellobiose (2 g/L), arabinose (4 g/L). Ascusb 812 2.00 O.OO O.OO mannose (4 g/L), rhaminose (2 g/L), maltose (2 g/L). Ascusb 817 1.16 O.OO O.09 maltose (2 g/L), and molasses. Rumen fluid was also some Ascusb 826 O42 O.OO O.S1 times omitted from the recipe. Additions including amino Ascusb 880 -0.12 O.OO O.OO acids, Volatile fatty acids, and antibiotics, were also varied Ascusb. 913 10.97 S.68 4.69 Ascusb 974 4.30 O.68 O.OO across the enrichments. A medium blank (control) was also Ascusb. 1069 O.OO O.OO 2.32 prepared. Cultures and the medium blank were incubated at Ascusb 1074 7.05 4.52 1.42 37° C. until significant growth was visible. An optical Ascusb 1295 54 4.08 O.O3 density (OD600) was determined for each culture, and the Ascusb. 1367 740 7.36 7.91 strain IDs were confirmed with Illumina sequencing. Enrich Ascusb. 1632 54 4.08 O.O3 ments that exhibited growth were also stained and cell Ascusb. 1674 O.68 O.30 O.OO counted to confirm that the individual strains within each Ascusb. 1763 69 4.2O 0.27 enrichment grew. Ascusb. 1780 32 O.OO O.21 C. Insoluble Carbon Source Assay Ascusb. 1786 69 4.2O 0.27 Ascusb. 1801 26.95 -0.60 0459. To assess the ability of the strains to degrade Ascusb. 1812 4.08 O.O3 insoluble carbon Sources, visual inspection was leveraged to Ascusb. 1833 1.15 3.19 qualitatively determine a strain's degradation capabilities. Ascusb. 1850 O.OO O.21 0460 For pure cultures, a single colony from each of the Ascusb. 2090 4.08 O.O3 desired strains (from anaerobic agar plates) was inoculated Ascusb. 2124 4.2O 0.27 into anaerobic Hungate tubes containing Lowe's semi Ascusb. 2511 O.OO O.11 defined media with cellulose paper, starch, or grass as the Ascusb. 2530 1.17 7.65 sole carbon source. (Lowe et al. 1985. J. Gen. Microbiol. Ascusb. 2597 O.68 O.OO 131:2225-2229). Enrichment cultures using a /100 dilutiopn Ascusb. 2624 O.OO O.OO Ascusb. 2667 1.46 1.02 of rumen fluid were also set up using the same medium Ascusb. 2836 O.OO O.21 conditions. Cultures were checked visually for degradation Ascusb. 3003 O.OO O.11 of insoluble carbon sources (See FIG. 14). Strain ID was Ascusb. 3138 O.OO 2.50 confirmed using Illumina sequencing. Enrichments that Ascusb 3504 4.2O 0.27 exhibited growth were also stained and cell counted to Ascusb. 3881 4.52 1.42 confirm that the individual strains within each enrichment grew. US 2017/O 196922 A1 Jul. 13, 2017 68

TABLE 16 Analysis of Degradation of Various Soluable and Non-Soluable Carbon Sources by Strains of the Present Disclosure Strain ID D-Glucose Xylan Lactate D-Xylose D-Mannose Glycerol Pectin Molasses Cellobiose Cellulose Starch

Ascusb. 5 ------Unknown Unknown Ascusb 7 ------Unknown Unknown Ascusb. 11 ------Unknown Unknown Ascusb. 26 ------Unknown Unknown Ascusb. 27 -- Unknown Unknown Ascusb. 32 ------Unknown Unknown Ascusb 36 -- -- Unknown Unknown Ascusb. 79 ------Unknown Unknown Ascusb 82 ------Unknown Unknown Ascusb. 89 -- -- Unknown Unknown Ascusb. 101 -- -- Unknown Unknown Ascusb 102 ------Unknown Unknown Ascusb 104 -- Unknown Unknown Ascusb. 111 ------Unknown Unknown Ascusb. 119 -- -- Unknown Unknown Ascusb 125 ------Unknown Unknown Ascusb. 145 -- -- Unknown Unknown Ascusb 149 ------Unknown Unknown Ascusb. 159 ------Unknown Unknown Ascusb. 183 ------Unknown Unknown Ascusb. 187 ------Unknown Unknown Ascusb. 190 ------Unknown Unknown Ascusb. 199 -- -- Unknown Unknown Ascusb. 205 -- -- Unknown Unknown Ascusb. 232 -- -- Unknown Unknown Ascusb. 268 -- Unknown Unknown Ascusb. 278 ------Unknown Unknown Ascusb 329 -- Unknown Unknown Ascusb 368 -- Unknown Unknown Ascusb 374 ------Unknown Unknown Ascusb. 411 -- Unknown Unknown Ascusb. 546 -- -- Unknown Unknown Ascusb 728 ------Unknown Unknown Ascusb. 765 -- -- Unknown Unknown Ascusb. 810 -- -- Unknown Unknown Ascusb 812 -- Unknown Unknown Ascusb 817 ------Unknown Unknown Ascusb 826 ------Unknown Unknown Ascusb 880 ------Unknown Unknown Ascusb. 913 ------Unknown Unknown Ascusb 974 -- -- Unknown Unknown Ascusb. 1069 -- Unknown Unknown Ascusb 1074 ------Unknown Unknown Ascusb 1295 ------Unknown Unknown Ascusb. 1367 ------Unknown Unknown Ascusb. 1632 -- Unknown Unknown Ascusb. 1674 ------Unknown Unknown Ascusb. 1763 -- -- Unknown Unknown Ascusb. 1780 ------Unknown Unknown Ascusb. 1786 -- Unknown Unknown Ascusb. 1801 -- -- Unknown Unknown Ascusb. 1812 -- Unknown Unknown Ascusb. 1833 ------Unknown Unknown Ascusb. 1850 ------Unknown Unknown Ascusb. 2090 -- -- Unknown Unknown Ascusb. 2124 -- Unknown Unknown Ascusb. 2511 ------Unknown Unknown Ascusb. 2530 -- -- Unknown Unknown Ascusb. 2597 -- -- Unknown Unknown Ascusb. 2624 -- Unknown Unknown Ascusb. 2667 ------Unknown Unknown Ascusb 2836 ------Unknown Unknown Ascusb. 3003 ------Unknown Unknown Ascusb. 3138 ------Unknown Unknown Ascusb 3504 -- -- Unknown Unknown Ascusb. 3881 -- -- Unknown Unknown Ascusb 6589 -- Unknown Unknown Ascusb 12103 -- -- Unknown Unknown Ascusb. 14245 ------Unknown Unknown Ascusb 20083 -- Unknown Unknown Ascusb 20187 -- -- Unknown Unknown Ascusf 15 -- -- Unknown -- -- Unknown ------Ascusf 22 Unknown Unknown Unknown -- US 2017/O 196922 A1 Jul. 13, 2017 69

TABLE 16-continued Analysis of Degradation of Various Soluable and Non-Soluable Carbon Sources by Strains of the Present Disclosure Strain ID D-Glucose Xylan Lactate D-Xylose D-Mannose Glycerol Pectin Molasses Cellobiose Cellulose Starch Ascusf 23 -- Unknown Unknown Unknown -- -- Ascusf 24 Unknown Unknown Unknown -- Ascusf 25 -- Unknown Unknown Unknown -- Ascusf 38 Unknown Unknown Unknown -- Ascusf 45 -- Unknown Unknown Unknown ------Ascusf 77 -- Unknown -- Unknown Unknown ------Ascusf 94 -- -- Unknown -- Unknown Unknown ------Ascusf 108 -- Unknown Unknown Unknown -- Ascusf 206 Unknown Unknown Unknown -- Ascusf 208 Unknown Unknown Unknown -- Ascusf 307 -- Unknown Unknown Unknown ------Ascusf 334 -- -- Unknown -- -- Unknown Unknown ------Ascusf 353 -- Unknown -- Unknown Unknown -- Ascusf 1012 Unknown Unknown Unknown --

TABLE 17 TABLE 19 M2GSC and M2 Media Recipes RAMM Salts Media Recipe M2GSC M2 Component g/500 mL Component Amount Component Amount KH2PO O.11 KHPO O.08 Beef Extract 5 g NaHCO 4 g NHCI O.26S Yeast Extract 1.25 g HCl-L-cysteine 0.3 g NaHCO, O6 NaHCO 2 g (NH4)2SO 0.10 g DI HO 500 mL. Cellobiose 1 g MgSO47H2O 0.005 g Starch 1 g KHPO 0.05 g Glucose 1 g KHPO. 0.05 g 0462. After sterilization (autoclave) added: 2 mL of 250x (NH4)2SO (1M) 2.55 mL DI H2O Up to 1000 mL. MgSO,7H2O (0.25M) O.288 mL. modified Wolfe’s vitamin mix, 10 mL of 50x modified KHPO (1M) 1 mL Wolfe's mineral mix, 5 mL of 100 mM cysteine. KHPO (1M) 1.275 mL. Clarified Rumen Fluid 50 mL. Example VIII HC-L-cysteine 0.3 g DI HO Up to 500 mL. Determination of Maximal Information Coefficient (MIC) Scores for Microbe Strains Relevant to Pounds of Milk Produced TABLE 1.8 Experimental Design and Materials and Methods Modified Wolfe's Media Recipes 0463) Objective: Determine rumen microbial community 250X Modified Wolfe's Modified Wolfe's constituents that impact the production of milk fat in dairy Vitamin Mix Mineral Solution COWS. Component g/200 mL Component g/L 0464 Animals: Eight lactating, ruminally cannulated, Holstein cows were housed in individual tie-stalls for use in Pyridoxine-HCl O.S MgSO 7H2O 140 p-Aminobenzoic O.25 Nitrillotriacetic acid 10.96 the experiment. Cows were fed twice daily, milked twice a Lipoic Acid O.216 NaCl SO.O6 day, and had continuous access to fresh water. One cow (cow Nicotinic Acid O.252 MnSOHO 24.99 1) was removed from the study after the first dietary Milk Riboflavin O.O13 CaCl2 5 Fat Depression due to complications arising from an abor Thiamine HCL O.25 CoCl2 6H2O 4.997 tion prior to the experiment. Calcium - DL - O.1 FeSO, 7HO 4.997 Pantothenate 0465 Experimental Design and Treatment: The experi Biotin O.O44 ZnSO 7H2O S.OO3 ment used a crossover design with 2 groups and 1 experi Folic Acid O.OO4 AIK(SO), 12 H2O O.S mental period. The experimental period lasted 38 days: 10 Vitamin B12 O.OO7 CuSO 5HO O499 HBO O.498 days for the covariate/wash-out period and 28 days for data NaMoO 2H2O O.SO3 collection and sampling. The data collection period con DI HO 1 L sisted of 10 days of dietary Milk Fat Depression (MFD) and 18 days of recovery. After the first experimental period, all cows underwent a 10-day wash out period prior to the 0461 All media was prepared with anaerobic water beginning of period 2. (boiled DI HO for 15 minutes then cooled to room tem 0466 Dietary MFD was induced with a total mixed ration perature in a water bath while sparging with N. All media (TMR) low in fiber (29% NDF) with high starch degrad was adjusted to a pH of 6.8 with 2M HC1. 10 mL of media ability (70% degradable) and high polyunsaturated fatty acid was then aliquoted into 15 mL hungate tubs, and the tubes levels (PUFA, 3.7%). The Recovery phase included two were then sparged with 80% N. 20% CO for 3 minutes. diets variable in starch degradability. Four cows were ran US 2017/O 196922 A1 Jul. 13, 2017 70 domly assigned to the recovery diet high in fiber (37% identify bacterial species present in the rumen based on a NDF), low in PUFA (2.6%), and high in starch degradability marker gene. Count datasets and activity datasets were (70% degradable). The remaining four cows were fed a integrated with the sequencing reads to determine the abso recovery diet high in fiber (37% NDF), low in PUFA (2.6%), lute cell numbers of active microbial species within the but low in starch degradability (35%). rumen microbial community. Production characteristics of 0467. During the 10-day covariate and 10-day wash out the cow over time, including pounds of milk produced, were periods, cows were fed the high fiber, low PUFA, and low linked to the distribution of active microorganisms within starch degradability diet. each sample over the course of the experiment using mutual 0468 Samples and Measurements: Milk yield, dry matter information. Maximal information coefficient (MIC) scores intake, and feed efficiency were measured daily for each were calculated between pounds of milk fat produced and animal throughout the covariate, wash out, and sample the absolute cell count of each active microorganism. Micro collection periods. TMR samples were measured for nutrient organisms were ranked by MIC score, and microorganisms composition. During the collection period, milk samples with the highest MIC scores were selected as the target were collected and analyzed every 3 days. Samples were species most relevant to pounds of milk produced. analyzed for milk component concentrations (milk fat, milk 0473. Tests cases to determine the impact of count data, protein, lactose, milk urea nitrogen, Somatic cell counts, and activity data, and count and activity on the final output were Solids) and fatty acid compositions. run by omitting the appropriate datasets from the sequencing 0469 Rumen samples were collected and analyzed for analysis. To assess the impact of using a linear correlation microbial community composition and activity every 3 days rather than the MIC on target selection, Pearson’s coeffi during the collection period. The rumen was intensively cients were also calculated for pounds of milk fat produced sampled 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours after feeding during day 0, day 7, and day 10 of the dietary as compared to the relative abundance of all microorganisms MFD. Similarly, the rumen was intensively sampled 0, 2, 4, and the absolute cell count of active microorganisms. 6, 8, 10, 12, 14, 16, 18, 20, and 22 hours after feeding on day 16 and day 28 during the recovery period. Rumen contents Results and Discussion were analyzed for pH, acetate concentration, butyrate con centration, propionate concentration, isoacid concentration, 0474) Relative Abundances vs. Absolute Cell Counts and long chain and CLA isomer concentrations. 0475. The top 15 target species were identified for the 0470 Rumen Sample Preparation and Sequencing: After dataset that included cell count data (absolute cell count, collection, rumen samples were centrifuged at 4,000 rpm in Table 21) and for the dataset that did not include cell count a swing bucket centrifuge for 20 minutes at 4° C. The data (relative abundance, Table 20) based on MIC scores. Supernatant was decanted, and an aliquot of each rumen Activity data was not used in this analysis in order to isolate content sample (1-2 mg) was added to a sterile 1.7 mL tube the effect of cell count data on final target selection. Ulti prefilled with 0.1 mm glass beads. A second aliquot was mately, the top 8 targets were the same between the two collected and stored in an empty, sterile 1.7 mL tube for cell datasets. Of the remaining 7, 5 strains were present on both counting. lists in varying order. Despite the differences in rank for 0471 Rumen samples with glass beads (1 aliquot) were these 5 strains, the calculated MIC score for each strain was homogenized with bead beating to lyse microorganisms. the identical between the two lists. The two strains present DNA and RNA was extracted and purified from each sample on the absolute cell count list but not the relative abundance and prepared for sequencing on an Illumina Miseq. Samples list, ascus 111 and ascus 288, were rank 91 and rank 16, were sequenced using paired-end chemistry, with 300 base respectively, on the relative abundance list. The two strains pairs sequenced on each end of the library. Rumen samples present on the relative abundance list but not the absolute in empty tubes (2" aliquot) were stained and put through a cell count list, ascus 102 and ascus 252, were rank 50 and flow cytometer to quantify the number of cells of each rank 19, respectively, on the absolute cell count list. These microorganism type in each sample. 4 strains did have different MIC scores on each list, thus 0472. Sequencing Read Processing and Data Analysis: explaining their shift in rank and Subsequent impact on the Sequencing reads were quality trimmed and processed to other strains in the list. TABLE 20 Top 15 Target Strains using Relative Abundance with no Activity Filter Target Strain MIC Nearest Taxonomy

ascus 7 0.97384 d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605) ascus 82 0.97.173 d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermenians (0.0073) ascus 209 0.95251 d: Bacteria(1.0000), p: TM7(0.9991), g: TM7 genera incertae sedis(0.8645) ascus 126 0.91477 d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1242), g: Saccharofermenians (0.0073) ascus 1366 0.897 13 d: Bacteria(1.0000), p: TM7(0.9445), g: TM7 genera incertae sedis(0.0986) ascus 1780 0.89466 d: Bacteria(0.9401), p: Bacteroidetes(0.4304), c: Bacteroidia(0.0551), o: Bacteroidales(0.0198), f: Prevotellaceae(0.0067), g: Prevoteila (0.0052) US 2017/O 196922 A1 Jul. 13, 2017 71

TABLE 20-continued Top 15 Target Strains using Relative Abundance with no Activity Filter Target Strain MIC Nearest Taxonomy ascus 64 0.89453 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8823), o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus 299 0.88979 d: Bacteria(1.0000), p: TM7 (0.9963), g: TM7 genera incertae sedis(0.5795) ascus 102 0.87095 d: Bacteria(1.0000), p: Firmicutes (0.9628), c: Clostridia(0.8317), o: Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g: Saccharofermenians(0.0283) ascus 1801 0.87038 d: Bacteria(0.8663), p: Bacteroidetes (0.2483), c: Bacteroidia(0.0365), o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus 295 0.86724. d: Bacteria(1.0000), p: SR1(0.9990), g: SR1 genera incertae sedis(0.9793) ascus 1139 0.8598 d: Bacteria (1.0000), p: TM7(0.9951), g: TM7 genera incertae sedis(0.4747) ascus 127 0.84082 d: Bacteria(1.0000), p: TM7(0.9992), g: TM7 genera incertae sedis(0.8035) ascus 341 0.8348 d: Bacteria(1.0000), p: TM7(0.9992), g: TM7 genera incertae sedis(0.8035) ascus 252 0.82891 d: Bacteria(1.0000), p: Firmicutes (0.9986), c: Clostridia(0.9022), o: Clostridiales(0.7491), f: Lachnospiraceae(0.3642), g: Lachnospiracea incertae sedis(0.0859)

TABLE 21 Top 15 Target Strains using Absolute cell count with no Activity Filter Target Strain MIC Nearest Taxonomy ascus 7 0.97384 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605) ascus 82 0.97173 d: Bacteria(1.0000), p: Firmicutes (0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermenians(0.0073) ascus 209 0.95251 d: Bacteria(1.0000), p: TM7(0.9991), g: TM7 genera incertae sedis(0.8645) ascus 126 0.91701 d: Bacteria(1.0000), p: Firmicutes (0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1242), g: Saccharofermenians(0.0073) ascus 1366 0.89713 d: Bacteria (1.0000), p: TM7(0.9445), g: TM7 genera incertae sedis(0.0986) ascus 1780 0.89466 d: Bacteria(0.9401), p: Bacteroidetes (0.4304), c: Bacteroidia(0.0551), o: Bacteroidales(0.0198), f: Prevotellaceae(0.0067), g: Prevotella(0.0052) ascus 64 0.89453 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8823), o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus 299 0.88979 d: Bacteria(1.0000), p: TM7 (0.9963), g: TM7 genera incertae sedis(0.5795) ascus 1801 0.87038 d: Bacteria(0.8663), p: Bacteroidetes (0.2483), c: Bacteroidia(0.0365), o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus 295 0.86724. d: Bacteria(1.0000), p: SR1(0.9990), g: SR1 genera incertae sedis(0.9793) ascus 1139 0.8598 d: Bacteria (1.0000), p: TM7(0.9951), g: TM7 genera incertae sedis(0.4747) ascus 127 0.84082 d: Bacteria(1.0000), p: TM7(0.9992), g: TM7 genera incertae sedis(0.8035) ascus 341 0.8348 d: Bacteria(1.0000), p: TM7(0.9992), g: TM7 genera incertae sedis(0.8035) ascus 111 0.83358 d: Bacteria(1.0000), p: Firmicutes (0.7947), c: Clostridia(0.4637), o: Clostridiales(0.2335), f: Ruminococcaceae(0.1062), g: Papillibacter(0.0098) ascus 288 0.82833 d: Bacteria(0.7925), p: Bacteroidetes (0.2030), c: Bacteroidia (0.0327), o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042)

0476 Integration of cell count data did not always affect ascus 341, and ascus 252 were deemed target strains prior the final MIC score assigned to each strain. This may be to applying activity data. These eight strains (53% of the attributed to the fact that although the microbial population initial top 15 targets) fell below rank 15 after integrating did shift within the rumen daily and over the course of the activity data. A similar trend was observed for the absolute 38-day experiment, it was always within 107-10 cells per cell count case. Ascus 126, ascus 1366, ascus 1780, ascus milliliter. Much larger shifts in population numbers would 299, ascus 1139, ascus 127, and ascus 341 (46% of the initial top 15 targets) fell below rank 15 after activity dataset undoubtedly have a broader impact on final MIC scores. integration. Inactive Species vs. Active Species 0479. The activity datasets had a much more severe effect 0477. In order to assess the impact of filtering strains on target rank and selection than the cell count datasets. based on activity data, target species were identified from a When integrating these datasets together, if a sample is dataset that leveraged relative abundance with (Table 22) found to be inactive it is essentially changed to a “0” and not and without (Table 20) activity data as well as a dataset that considered to be part of the analysis. Because of this, the leveraged absolute cell counts with (Table 23) and without distribution of points within a sample can become heavily (Table 21) activity data. altered or skewed after integration, which in turn greatly 0478 For the relative abundance case, ascus 126, ascus impacts the final MIC score and thus the rank order of target 1366, ascus 1780, ascus 299, ascus 1139, ascus 127, microorganisms. US 2017/O 196922 A1 Jul. 13, 2017 72

TABLE 22 Top 15 Target Strains using Relative Abundance with Activity Filter Target Strain MIC Nearest Taxonomy ascus 7 0.97384 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605) ascus 82 0.93391 d: Bacteria(1.0000), p: Firmicutes (0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermenians(0.0073) ascus 102 0.87095 d: Bacteria(1.0000), p: Firmicutes (0.9628), c: Clostridia(0.8317), o: Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g: Saccharofermenians(0.0283) ascus 209 0.84421 d: Bacteria(1.0000), p: TM7(0.9991), g: TM7 genera incertae sedis(0.8645) ascus 1801 0.82398 d: Bacteria(0.8663), p: Bacteroidetes (0.2483), c: Bacteroidia(0.0365), o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus 372 0.81735 d: Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes (0.8623), o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0190) ascus 26 0.81081 d: Bacteria(1.0000), p: Firmicutes (0.9080), c: Clostridia(0.7704), o: Clostridiales(0.4230), f: Ruminococcaceae(0.1942), g: Clostridium IV(0.0144) ascus 18O 0.80702 d: Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes (0.8623), o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0237) ascus 32 0.7846 d: Bacteria(1.0000), p: Firmicutes (0.7036), c: Clostridia(0.4024), o: Clostridiales(0.1956), f: Ruminococcaceae(0.0883), g: Hydrogenoanaerobacterium(0.0144) ascus 288 0.78229 d: Bacteria(0.7925), p: Bacteroidetes (0.2030), c: Bacteroidia(0.0327), o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042) ascus 64 0.77514 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8823), o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus 295 0.76639 d: Bacteria(1.0000), p: SR1(0.9990), g: SR1 genera incertae sedis(0.9793) ascus 546 0.76114 d: Bacteria(1.0000), p: Firmicutes (0.6126), c: Clostridia(0.2851), o: Clostridiales(0.1324), f: Clostridiaceae 1 (0.0208), g: Clostridium sensu stricto(0.0066) ascus 233 0.75779 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8756), o: Clostridiales (0.5860), f: Ruminococcaceae(0.3642), g: Ruminococcus(0.0478) ascus 651 0.74837 d: Bacteria(1.0000), p: Firmicutes (0.7947), c: Clostridia(0.4637), o: Clostridiales(0.2335), f: Ruminococcaceae(0.0883), g: Clostridium IV(0.0069)

TABLE 23 Top 15 Target Strains using Absolute cell count with Activity Filter Target Strain MIC Nearest Taxonomy ascus 7 0.97384 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605) ascus 82 0.93391 d: Bacteria(1.0000), p: Firmicutes (0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermenians(0.0073) ascus 209 0.84421 d: Bacteria(1.0000), p: TM7(0.9991), g: TM7 genera incertae sedis(0.8645) ascus 1801 0.82398 d: Bacteria(0.8663), p: Bacteroidetes (0.2483), c: Bacteroidia(0.0365), o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus 372 0.81735 d: Bacteria(1.0000), p: Spirochaetes(0.9445), c: Spirochaetes (0.8623), o: Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0190) ascus 26 0.81081 d: Bacteria(1.0000), p: Firmicutes (0.9080), c: Clostridia(0.7704), o: Clostridiales(0.4230), f: Ruminococcaceae(0.1942), g: Clostridium IV(0.0144) ascus 102 0.81048 d: Bacteria(1.0000), p: Firmicutes (0.9628), c: Clostridia(0.8317), o: Clostridiales(0.4636), f: Ruminococcaceae(0.2367), g: Saccharofermenians(0.0283) ascus 111 0.79035 d: Bacteria(1.0000), p: Firmicutes (0.7947), c: Clostridia(0.4637), o: Clostridiales(0.2335), f: Ruminococcaceae(0.1062), g: Papillibacter(0.0098) ascus 288 0.78229 d: Bacteria(0.7925), p: Bacteroidetes (0.2030), c: Bacteroidia(0.0327), o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042) ascus 64 0.77514 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8823), o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) ascus 295 0.76639 d: Bacteria(1.0000), p: SR1(0.9990), g: SR1 genera incertae sedis(0.9793) ascus 546 0.76114 d: Bacteria(1.0000), p: Firmicutes (0.6126), c: Clostridia(0.2851), o: Clostridiales(0.1324), f: Clostridiaceae 1 (0.0208), g: Clostridium sensu stricto(0.0066) ascus 32 0.75068 d: Bacteria(1.0000), p: Firmicutes (0.7036), c: Clostridia(0.4024), o: Clostridiales(0.1956), f: Ruminococcaceae(0.0883), g: Hydrogenoanaerobacterium(0.0144) ascus 651 0.74837 d: Bacteria(1.0000), p: Firmicutes (0.7947), c: Clostridia(0.4637), o: Clostridiales(0.2335), f: Ruminococcaceae(0.0883), g: Clostridium IV(0.0069) ascus 233 0.74409 d: Bacteria(1.0000), p: Firmicutes (0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860), f: Ruminococcaceae(0.3642), g: Ruminococcus(0.0478) US 2017/O 196922 A1 Jul. 13, 2017 73

Relative Abundances and Inactive vs. Absolute Cell Counts between two distributions. Over the course of the experi and Active ment, the pounds of milk fat produced changed nonlinearly 0480 Ultimately, the method defined here leverages both (FIG. 18). This particular function may be better represented cell count data and activity data to identify microorganisms and approximated by mutual information than correlations. highly linked to relevant metadata characteristics. Within the To investigate this, the top target strains identified using top 15 targets selected using both methods (Table 23, Table correlation and mutual information, Ascus 713 (FIG. 19) 20), only 7 strains were found on both lists. Eight strains and Ascus 7 (FIG. 20) respectively, were plotted to deter (53%) were unique to the absolute cell count and activity mine how well each method predicted relationships between list. The top 3 targets on both lists matched in both strain as the strains and milk fat. If two variables exhibit strong well as in rank. However, two of the three did not have the correlation, they are represented by a line with little to no same MIC score on both lists, suggesting that they were dispersion of points when plotted against each other. In FIG. influenced by activity dataset integration but not enough to 19, Ascus 713 correlates weakly with milk fat, as indicated upset their rank order. by the broad spread of points. Mutual information, again, Linear Correlations vs. Nonparametric Approaches measures how similar two distributions of points are. When 0481 Pearson’s coefficients and MIC scores were calcu Ascus 7 is plotted with milk fat (FIG. 20), it is apparent that lated between pounds of milk fat produced and the absolute the two point distributions are very similar. cell count of active microorganisms within each sample The Present Method in Entirety vs. Conventional (Table 24). Strains were ranked either by MIC (Table 24a) Approaches or Pearson coefficient (Table 24b) to select target strains 0483 The conventional approach of analyzing microbial most relevant to milk fat production. Both MIC score and communities relies on the use of relative abundance data Pearson coefficient are reported in each case. Six strains with no incorporation of activity information, and ultimately were found on both lists, meaning nine (60%) unique strains ends with a simple correlation of microbial species to were identified using the MIC approach. The rank order of metadata (see, e.g., U.S. Pat. No. 9,206,680, which is herein strains between lists did not match—the top 3 target strains incorporated by reference in its entirety for all purposes). identified by each method were also unique. Here, we have shown how the incorporation of each dataset 0482. Like Pearson coefficients, the MIC score is incrementally influences the final list of targets. When reported over a range of 0 to 1, with 1 Suggesting a very tight applied in its entirety, the method described herein selected relationship between the two variables. Here, the top 15 a completely different set of targets when compared to the targets exhibited MIC scores ranging from 0.97 to 0.74. The conventional method (Table 24a and Table 24c). Ascus Pearson coefficients for the correlation test case, however, 3038, the top target strain selected using the conventional ranged from 0.53 to 0.45—substantially lower than the approach, was plotted against milk fat to visualize the mutual information test case. This discrepancy may be due strength of the correlation (FIG. 21). Like the previous to the differences inherent to each analysis method. While example, Ascus 3038 also exhibited a weak correlation to correlations are a linear estimate that measures the disper milk fat. sion of points around a line, mutual information leverages 0484 Table 24: Top 15 Target Strains using Mutual probability distributions and measures the similarity Information or Correlations TABLE 24a MIC using Absolute cell count with Activity Filter Target Pearson Strain MIC Coefficient Neares Taxonomy ascus 7 O.97384 O2S282SO2 : Bacteria.( .0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), O Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0605) ascus 82 O.93391 O-42776647 : Bacteria.( .0000), p: Firmicutes(0.8349), c: Clostridia(0.5251), O Clostridiales(0.27 4), f: Ruminococcaceae(0.1062), g: Saccharofermenians(0.0073) ascus 209 O84421 : Bacteria.( .0000), p: TM7(0.9991), g: TM7 genera incertae sedis(0.8645) ascus 1801 O.82398 : Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365), O Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), 9. Butyricimonas(0.0047) ascus 372 O.81735 0.341.72258 : Bacteria.( .0000), p: Spirochaetes(0.9445), c: Spirochaetes(0.8623), O Spirochaetales(0.5044), f: Spirochaetaceae(0.3217), 9. Spirochaeta(0.0190) ascus 26 O.8108.1 O.S3OO298 : Bacteria.( .0000), p: Firmicutes(0.9080), c: Clostridia(0.7704), O Clostridiales(0.4230), f: Ruminococcaceae(0.1942), 9. Clostridium IV(0.0144) ascus 102 O.81048 0.35456932 : Bacteria.( .0000), p: Firmicutes(0.9628), c: Clostridia(0.8317), O Clostridiales(0.4636), f: Ruminococcaceae(0.2367), 9. Saccharofermenians(0.0283) ascus 111 O.79035 0.4588.1805 : Bacteria.( .0000), p: Firmicutes(0.7947), c: Clostridia(0.4637), O Clostridiales(0.2335), f: Ruminococcaceae(0.1062), 9. Papillibacter(0.0098) ascus 288 O.78229 O.46522045 : Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327), O Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), 9. Butyricimonas(0.0042) US 2017/O 196922 A1 Jul. 13, 2017 74

TABLE 24a-continued MIC using Absolute cell count with Activity Filter Target Pearson Strain MIC Coefficient Nearest Taxonomy ascus 64 O.77S14 0.454.17OSS Bacteria(1.0000), p: Firmicutes(0.9922), c. Clostridia(0.8823), Clostridiales(0.6267), f: Ruminococcaceae(0.2792), Ruminococcus(0.0605) ascus 295 O.76639 O.24972263 Bacteria(1.0000), p: SR1 (0.9990), g: SR1 genera incertae sedis(0.9793) ascus 546 O.76114 O.23819838 Bacteria(1.0000), p: Firmicutes(0.6126), c. Clostridia(0.2851), Clostridiales(0.1324), f: Clostridiaceae 1 (0.0208), Clostridium sensu stricto(0.0066) ascus 32 0.75068 0.5179697 Bacteria(1.0000), p: Firmicutes(0.7036), c. Clostridia(0.4024), Clostridiales(0.1956), f: Ruminococcaceae(0.0883), Hydrogenoanaerobacterium(0.0144) ascus 651 O.74837 O.27656645 Bacteria(1.0000), p: Firmicutes(0.7947), c. Clostridia(0.4637), Clostridiales(0.2335), f: Ruminococcaceae(0.0883), Clostridium IV(0.0069) ascus 233 O.744O9 O.3609SO98 Bacteria(1.0000), p: Firmicutes(0.9922), c. Clostridia(0.8756), Clostridiales(0.5860), f: Ruminococcaceae(0.3642), Ruminococcus(0.0478)

TABLE 24b Correlation using Absolute cell count with Activity Filter Target Pearson Strain MIC Coefficient Nearest Taxonomy ascus 713 0.71066 0.5305876 d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1062), g: Saccharofermenians(0.0073) ascus 26 0.81081 0.53.00298 d: Bacteria(1.0000), p: Firmicutes(0.9080), c: Clostridia(0.7704), o: Clostridiales(0.4230), f: Ruminococcaceae(0.1942), g: Clostridium IV(0.0144) ascus 1801 O.82398 OS 182261 : Bacteria(0.8663), p: Bacteroidetes(0.2483), c: Bacteroidia(0.0365), o: Bacteroidales(0.0179), f: Porphyromonadaceae(0.0059), g: Butyricimonas(0.0047) ascus 32 0.75068 0.5179697 d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.4024), o: Clostridiales(0.1956), f: Ruminococcaceae(0.0883), g: Hydrogenoanaerobacterium(0.0144) ascus 119 0.6974 0.4968678 d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8756), o: Clostridiales(0.5860), f: Ruminococcaceae(0.3217), g: Ruminococcus(0.0478) ascus 13899 0.64556 0.48739454 d: Bacteria (1.0000), p: Actinobacteria(0.1810), c: Actinobacteria(0.0365), o: Actinomycetales(0.0179), f: Propionibacteriaceae(0.0075), g: Microlunatus(0.0058) ascus 906 0.49256 0.484.18677 d: Bacteria(1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.2714), f: Ruminococcaceae(0.1242), g: Papillibacter(0.0098) ascus 221 0.44006 0.47305903 d: Bacteria(1.0000), p: Bacteroidetes(0.9991), c: Bacteroidia(0.9088), o: Bacteroidales(0.7898), f: Prevotellaceae(0.3217), g: Prevoteila (0.0986) ascus 1039 0.65629 0.46932846 d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.2851), o: Clostridiales(0.1324), f: Ruminococcaceae(0.0329), g: Clostridium IV(0.0069 ascus 288 0.78229 0.46522045 d: Bacteria(0.7925), p: Bacteroidetes(0.2030), c: Bacteroidia(0.0327), o: Bacteroidales(0.0160), f: Porphyromonadaceae(0.0050), g: Butyricimonas(0.0042) ascus 589 O.40868 0.4651165 : Bacteria (1.0000), p: Firmicutes(0.9981), c: Clostridia(0.9088), o: Clostridiales(0.7898), f: Lachnospiraceae(0.5986), g: Clostridium XIVa (0.3698) ascus 41 0.67227 0.46499047 d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.3426), o: Clostridiales(0.1618), f: Ruminococcaceae(0.0703), g: Hydrogenoanaerobacterium(0.0098) ascus 111 0.79035 0.4588.1805 d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.4637), o: Clostridiales(0.2335), f: Ruminococcaceae(0.1062), g: Papillibacter(0.0098) ascus 205 0.72441 0.45684373 d: Bacteria (1.0000), p: Firmicutes(0.6126), c: Clostridia(0.3426), o: Clostridiales(0.1618), f: Peptococcaceae 2(0.0449), g: Pelotomaculum(0.0069) ascus 64 0.77514 0.454.17055 d: Bacteria(1.0000), p: Firmicutes(0.9922), c: Clostridia(0.8823), o: Clostridiales(0.6267), f: Ruminococcaceae(0.2792), g: Ruminococcus(0.0605) US 2017/O 196922 A1 Jul. 13, 2017 75

TABLE 24c Correlation using Relative Abundance with no Activity Filter Target Pearson Strain MIC Coefficient Nearest Taxonomy ascus 3038 0.56239 0.6007549 d: Bacteria(1.0000), p: Firmicutes(0.9945), c: Clostridia(0.8623), o: C ostridiales(0.5044), f: Lachnospiraceae(0.2367), g: Clostridium XIVa (0.0350) ascus 1555 0.66965 0.59716415 d: Bacteria(1.0000), p: Firmicutes(0.7947), c: Clostridia(0.3426), o: C ostridiales(0.1618), f: Ruminococcaceae(0.0449), g: Clostridium IV(0.0073) ascus 1039 0.68563 0.59292555 d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.2851), o: C ostridiales(0.1324), f: Ruminococcaceae(0.0329), g: Clostridium IV(0.0069) ascus 1424 0.55509 0.57589555 d: Bacteria(1.0000), p: Firmicutes(0.8897), c: Clostridia(0.7091), o: Clostridiales(0.3851), f: Ruminococcaceae(0.1422), g: Papilibacter(0.0144) ascus 378 0.77519 O.5671971 : Bacteria (1.0000), p: Firmicutes(0.8349), c: Clostridia(0.5251), o: Clostridiales(0.27 4), f: Ruminococcaceae(0.1062), g: Saccharofermenians (0.0073) ascus 407 0.69783 0.56279755 d: Bacteria(1.0000), p: Firmicutes(0.7036), c: Clostridia(0.3426), o: Clostridiales(0.1618), f: Clostridiaceae 1 (0.0329), g: Clostridium sensu stricto(0.0069) ascus 1584 0.5193 0.5619939 d: Bacteria(1.0000), p: Firmicutes(0.9945), c: Clostridia(0.8756), o: Clostridiales(0.5860), f: Lachnospiraceae(0.3217), g: Coprococcus(0.0605) ascus 760 0.61363 0.55807924 d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851), o: Clostridiales(0.1324), f: Clostridiaceae 1 (0.0208), g: Clostridium sensu stricto(0.0066) ascus 1184 0.70593 0.5578006 d: Bacteria(1.0000), p: “Bacteroidetes (0.9992), c: “Bacteroidia (0.8690), o: “Bacteroidales' (0.5452), f: Bacteroidaceae(0.1062), g: Bacteroides (0.0237) ascus 7394 0.6269 0.5557023 : Bacteria (1.0000), p: Firmicutes(0.9939), c: Clostridia(0.7704), o: Clostridiales(0.4230), f: Lachnospiraceae(0.1422), g: Clostridium XIVa(0.0350) ascus 1360 0.57343 0.5535785 : Bacteria (1.0000), p: Firmicutes(0.9992), c: Clostridia(0.9351), o: Clostridiales(0.8605), f: Lachnospiraceae(0.7052), g: Clostridium XIVa(0.2649) ascus 3175 0.53565 0.548.64305 d: Bacteria(1.0000), p: “Bacteroidetes (0.9991), c: “Bacteroidia (0.8955), o: “Bacteroidales (0.7083), f: “Prevotellaceae (0.1942), g: Prevotella(0.0605) ascus 2581 0.68361 0.5454486 d: Bacteria(1.0000), p: “Spirochaetes'(0.9445), c: Spirochaetes(0.8623), o: Spirochaetales (0.5044), f: Spirochaetaceae(0.3217), g: Spirochaeta(0.0237) ascus 531 0.71315 0.5400517 d: Bacteria(1.0000), p: Firmicutes(0.6126), c: Clostridia(0.2851), o: Clostridiales(0.1324), f: Clostridiaceae 1 (0.0208), g: Clostridium sensu stricto(0.0066) ascus 1858 0.65165 0.5393882 d: Bacteria(1.0000), p: “Spirochaetes (0.9263), c: Spirochaetes(0.8317), o: Spirochaetales (0.4636), f: Spirochaetaceae(0.2792), g: Spirochaeta(0.0237)

Numbered Embodiments of the Disclosure 0492 4. The shelf-stable ruminant supplement accord ing to claim 1, wherein the purified population of 0485 Subject matter contemplated by the present disclo Pichia fungi comprises a fungi as deposited at NRRL sure is set out in the following numbered embodiments: Y-67249. 0486 1. A shelf-stable ruminant supplement capable of 0493 5. The shelf-stable ruminant supplement accord increasing milk production or improving milk compo ing to claim 1, further comprising: sitional characteristics in a ruminant, comprising: 0494 i. a purified population of bacteria that com prises a bacteria with a 16S nucleic acid sequence 0487 a) a purified population of Pichia fungi com that is at least about 97% identical to a nucleic acid prising a fungi with an ITS nucleic acid sequence sequence selected from the group consisting of: SEQ that is at least about 97% identical to SEQ ID NO: ID NOs: 1-30 and 2045-2103, and/or 32; and 0495 ii. a purified population of fungi that com 0488 b) a shelf-stable carrier suitable for ruminant prises a fungi with an ITS nucleic acid sequence that administration, is at least about 97% identical to a nucleic acid 0489 wherein the purified population of Pichia sequence selected from the group consisting of: SEQ fungi of a) is present in the Supplement in an amount ID NOs: 31, 33-60 and 2104-2107. effective to increase milk production or improve 0496 6. The shelf-stable ruminant supplement accord milk compositional characteristics in a ruminant ing to claim 5, wherein the purified population of administered the Supplement, as compared to a rumi bacteria comprises a bacteria with a 16S nucleic acid nant not administered the Supplement. sequence that is at least about 99% identical to a nucleic acid sequence selected from the group consisting of: 0490 2. The shelf-stable ruminant supplement accord SEQ ID NOs: 1-30 and 2045-2103. ing to claim 1, wherein the purified population of 0497 7. The shelf-stable ruminant supplement accord Pichia fungi comprises a fungi with an ITS nucleic acid ing to claim 5, wherein the purified population of fungi sequence that is at least about 99% identical to SEQID comprises a fungi with an ITS nucleic acid sequence NO: 32. that is at least about 99% identical to a nucleic acid 0491 3. The shelf-stable ruminant supplement accord sequence selected from the group consisting of: SEQ ing to claim 1, wherein the purified population of ID NOs: 31, 33-60 and 2104-2107. Pichia fungi comprises a fungi with an ITS nucleic acid 0498 8. The shelf-stable ruminant supplement accord sequence comprising SEQ ID NO: 32. ing to claim 5, wherein the purified population of US 2017/O 196922 A1 Jul. 13, 2017 76

bacteria comprises a bacteria with a 16S nucleic acid increase of vitamins in milk, an increase of minerals in sequence selected from the group consisting of: SEQ milk, or combinations thereof. ID NOS: 1-30 and 2045-2103. 0512 22. The shelf-stable ruminant supplement 0499 9. The shelf-stable ruminant supplement accord according to claim 1, wherein the ruminant adminis ing to claim 5, wherein the purified population of fungi tered the supplement exhibits at least a 1% increase in comprises a fungi with an ITS nucleic acid sequence the average production of milk fat(s), milk protein(s), selected from the group consisting of: SEQID NOs: 31, energy-corrected milk, or combinations thereof. 33-60 and 2104-2107. 0500 10. The shelf-stable ruminant supplement 0513. 23. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of according to claim 1, wherein the ruminant adminis bacteria comprises a bacteria with a 16S nucleic acid tered the supplement exhibits at least a 10% increase in sequence that is at least about 97% identical to SEQID the average production of milk fat(s), milk protein(s), NO: 28. energy-corrected milk, or combinations thereof. 0501 11. The shelf-stable ruminant supplement 0514 24. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of according to claim 1, wherein the ruminant adminis bacteria comprises a bacteria with a 16S nucleic acid tered the supplement exhibits at least a 20% increase in sequence that is at least about 99% identical to SEQID the average production of milk fat(s), milk protein(s), NO: 28. energy-corrected milk, or combinations thereof. 05.02 12. The shelf-stable ruminant supplement 0515. 25. A composition suitable for administration to according to claim 5, wherein the purified population of a ruminant and capable of increasing milk production bacteria comprises a bacteria with a 16S nucleic acid or improving milk compositional characteristics in a sequence comprising SEQ ID NO: 28. ruminant, comprising: 0503 13. The shelf-stable ruminant supplement according to claim 5, wherein the purified population of 0516 a) a purified population of fungi as deposited bacteria comprises a bacteria as deposited at NRRL at NRRL Y-67249; and B-67248. 0517 b) a carrier suitable for ruminant administra 0504 14. The shelf-stable ruminant supplement tion, according to claim 5, wherein both a purified popula 0518 wherein the purified population of fungi of a) tion of bacteria i) and a purified population of fungi ii) is present in the composition in an amount effective are present in the Supplement. to increase milk production or improve milk com 0505 15. The shelf-stable ruminant supplement positional characteristics in a ruminant administered according to claim 1, formulated for administration to the composition, as compared to a ruminant not a COW. administered the composition. 0506 16. The shelf-stable ruminant supplement 0519 26. A composition suitable for administration to according to claim 1, wherein the Supplement is stable a ruminant and capable of increasing milk production under ambient conditions for at least one week. or improving milk compositional characteristics in a (0507 17. The shelf-stable ruminant supplement according to claim 1, formulated as an: encapsulation, ruminant, comprising: tablet, capsule, pill, feed additive, food ingredient, food 0520 a) a purified population of fungi as deposited additive, food preparation, food Supplement, consum at NRRL Y-67249; able solution, consumable spray additive, consumable 0521 b) a purified population of bacteria as depos Solid, consumable gel, injection, Suppository, bolus, ited at NRRL B-67248; and drench, or combinations thereof. 0508. 18. The shelf-stable ruminant supplement 0522 c) a carrier suitable for ruminant administra according to claim 1, wherein the purified population of tion, Pichia fungi is present in the ruminant Supplement at a 0523 wherein the purified population of fungi of a) concentration of at least 10 cells. and purified population of bacteria of b) are present (0509 19. The shelf-stable ruminant supplement in the composition in an amount effective to increase according to claim 1, wherein the ruminant adminis milk production or improve milk compositional tered the Supplement exhibits an increase in milk characteristics in a ruminant administered the com production that leads to a measured increase in milk position, as compared to a ruminant not administered yield. the composition. 0510 20. The shelf-stable ruminant supplement 0524. The aforementioned compositions have markedly according to claim 1, wherein the ruminant adminis different characteristics and/or properties not possessed by tered the Supplement exhibits an increase in milk any individual bacteria or fungi as they naturally exist in the production and improved milk compositional charac rumen. The markedly different characteristics and/or prop teristics that leads to a measured increase in energy erties possessed by the aforementioned compositions can be corrected milk. structural, functional, or both. For example, the composi 0511. 21. The shelf-stable ruminant supplement tions possess the markedly different functional property of according to claim 1, wherein the ruminant adminis being able to increase milk production or improve milk tered the Supplement exhibits an improved milk com compositional characteristics, when administered to a rumi positional characteristic selected from the group con nant, as taught herein. Furthermore, the compositions pos sisting of an increase in milk fat(s), an increase in milk sess the markedly different functional property of being protein(s), an increase of carbohydrates in milk, an shelf-stable. US 2017/O 196922 A1 Jul. 13, 2017 77

Numbered Embodiments of the Disclosure 0539 b) a carrier suitable for ruminant administra tion, 0525. Subject matter contemplated by the present disclo 0540 wherein the ruminant administered the effec sure is set out in the following numbered embodiments: tive amount of the composition exhibits a shift in the 0526 1. A composition capable of modulating the microbiome of the rumen. rumen microbiome of a ruminant, comprising: 0541 8. The method according to claim 7, wherein a 0527 a) a purified population of Pichia fungi com population of microbes present in the ruminant's rumen prising a fungi with an ITS nucleic acid sequence before administration of the composition increase in that is at least about 97% identical to SEQ ID NO: abundance after administration of the composition. 32; and 0542 9. The method according to claim 7, wherein a 0528 b) a carrier Suitable for ruminant administra population of microbes present in the ruminant's rumen tion, before administration of the composition decrease in abundance after administration of the composition. 0529 wherein the purified population of Pichia fungi 0543. 10. The method according to claim 7, wherein a of a) is present in the composition in an amount first population of microbes present in the ruminants effective to cause a shift in the microbiome of the rumen before administration of the composition rumen of a ruminant administered the composition. increase in abundance after administration of the com 0530 2. The composition according to claim 1, position and wherein a second population of microbes wherein a population of microbes present in the rumi present in the ruminants rumen before administration nants rumen before administration of the composition of the composition decrease in abundance after admin increase in abundance after administration of the com istration of the composition. position. 0544 11. The method according to claim 7, wherein 0531. 3. The composition according to claim 1, the rumen microbiome of the ruminant administered wherein a population of microbes present in the rumi the composition is shifted to include an increased nants rumen before administration of the composition presence of fiber-degrading genera, Volatile fatty acid decrease in abundance after administration of the com producing genera, structural carbohydrate-digesting position. genera, or combinations thereof. 0532 4. The composition according to claim 1, 0545 12. The method according to claim 7, wherein wherein a first population of microbes present in the the rumen microbiome of the ruminant administered ruminants rumen before administration of the compo the composition is shifted according to the disclosure sition increase in abundance after administration of the and data presented in Example 6 and Table 13 or Table composition and wherein a second population of 14. microbes present in the ruminants rumen before 0546. The aforementioned compositions have markedly administration of the composition decrease in abun different characteristics and/or properties not possessed by dance after administration of the composition. any individual bacteria or fungi as they naturally exist in the 0533 5. The composition according to claim 1, rumen. The markedly different characteristics and/or prop wherein the rumen microbiome of the ruminant admin erties possessed by the aforementioned compositions can be istered the composition is shifted to include an structural, functional, or both. For example, the composi increased presence of fiber-degrading genera, Volatile tions possess the markedly different functional property of fatty acid-producing genera, structural carbohydrate being able to modulate the rumen microbiome, when admin digesting genera, or combinations thereof. istered to a ruminant, as taught herein. 0534 6. The composition according to claim 1, Numbered Embodiments of the Disclosure wherein the rumen microbiome of the ruminant admin istered the composition is shifted according to the 0547 Subject matter contemplated by the present disclo disclosure and data presented in Example 6 and Table sure is set out in the following numbered embodiments: 13 or Table 14. 0548 1. A method for increasing milk production or 0535 7. A method for modulating the rumen microbi improving milk compositional characteristics in a ome of a ruminant, comprising administering to a ruminant, comprising: 0549 a) administering to a ruminant an effective ruminant an effective amount of a composition com amount of a shelf-stable ruminant Supplement com prising: prising: 0536 a) a purified microbial population, said puri 0550 i. a purified microbial population that com fied microbial population comprising: prises a bacteria with a 16S nucleic acid sequence, 0537 i. a purified population of bacteria that and/or a fungi with an ITS nucleic acid sequence, comprises a bacteria with a 16S nucleic acid which is at least about 97% identical to a nucleic sequence that is at least about 97% identical to a acid sequence selected from the group consisting nucleic acid sequence selected from the group of: SEQ ID NOs: 1-60 and 2045-2107, said bac consisting of: SEQID NOs: 1-30 and 2045-2103, teria having a MIC score of at least about 0.4 and and/or said fungi having a MIC score of at least about 0538 ii. a purified population of fungi that com 0.2; and prises a fungi with an ITS nucleic acid sequence 0551 ii. a shelf-stable carrier suitable for rumi that is at least about 97% identical to a nucleic acid nant administration, sequence selected from the group consisting of 0552 wherein at least one of the bacteria or fungi SEQ ID NOs: 31-60 and 2104-2107; and are capable of converting a carbon Source into a US 2017/O 196922 A1 Jul. 13, 2017

Volatile fatty acid selected from the group consisting 0567 14. The method according to claim 1, wherein of acetate, butyrate, propionate, or combinations the purified microbial population comprises a fungi thereof, and with an ITS nucleic acid sequence selected from the 0553 wherein at least one of the bacteria or fungi group consisting of: SEQ ID NOs: 31-60 and 2104 are capable of degrading a soluble or insoluble 2107. carbon Source; and 0568 15. The method according to claim 1, wherein 0554 wherein the ruminant administered the effec the purified microbial population comprises a bacteria tive amount of the shelf-stable ruminant supplement with a 16S nucleic acid sequence and a fungi with an exhibits an increase in milk production or improved ITS nucleic acid sequence that is at least about 97% milk compositional characteristics, as compared to a identical to a nucleic acid sequence selected from the ruminant not administered the ruminant Supplement. group consisting of: SEQ ID NOs: 1-60 and 2045 0555 2. The method according to claim 1, wherein the 2107. ruminant is a cow. 0569 16. The method according to claim 1, wherein 0556) 3. The method according to claim 1, wherein the the purified microbial population comprises a bacteria ruminant Supplement is stable under ambient condi with a 16S nucleic acid sequence that is at least about tions for at least one week. 97% identical to SEQ ID NO: 28. 0557. 4. The method according to claim 1, wherein the 0570) 17. The method according to claim 1, wherein ruminant Supplement is formulated as an: encapsula the purified microbial population comprises a fungi tion, tablet, capsule, pill, feed additive, food ingredient, with an ITS nucleic acid sequence that is at least about food additive, food preparation, food Supplement, con 97% identical to SEQ ID NO: 32. Sumable solution, consumable spray additive, consum 0571 18. The method according to claim 1, wherein able solid, consumable gel, injection, Suppository, the purified microbial population comprises a Pichia bolus, drench, or combinations thereof. fungi as deposited at NRRL Y-67249. 0558 5. The method according to claim 1, wherein the 0572) 19. The method according to claim 1, wherein ruminant Supplement is encapsulated in a polymer or the purified microbial population only contains organ carbohydrate. isms that are members of a group selected from: 0559) 6. The method according to claim 1, wherein 0573 Intestinimonas, Anaerolinea, Pseudobutyrivi administering comprises: feeding the ruminant Supple brio, Olsenella, Eubacterium, Catenisphaera, Fae ment to a ruminant. calibacterium, Solobacterium, Blautia, Ralsonia, 0560 7. The method according to claim 1, wherein Coprococcus, Casaltella, Anaeroplasma, Achole administering comprises: injecting the ruminant plasma, Aminiphilus, Mitsuokella, Alistipes, Supplement into a ruminant. Sharpea, Oscillibacter, Neocallimastix, Odoribacter, 0561 8. The method according to claim 1, wherein the Pichia, Tannerella, Candida, Hydrogenoanaerobac purified microbial population is present in the ruminant terium, Orpinomyces, Succinivibrio, Sugiyamaella, supplement at a concentration of at least 10° cells. Ruminobacter; Lachnospira, Caecomyces, Sini 0562 9. The method according to claim 1, wherein the marinibacterium, Tremella, Hydrogenoanaerobacte purified microbial population comprises a bacteria with rium, Turicibacter; Clostridium XIVa, Anaerolinea, a 16S nucleic acid sequence that is at least about 97% Saccharofermentans, Butyricicoccus, Olsenella, identical to a nucleic acid sequence selected from the Papillibacter, Clostridium XIa, Pelotomaculum, group consisting of SEQ ID NOs: 1-30 and 2045 Erysipelotrichaceae incertae sedis, Lachnospira 2103. cea incertae sedis, Solobacterium, Anaeroplasma, 0563. 10. The method according to claim 1, wherein Ralstonia, Clostridium sensu stricto, Eubacterium, the purified microbial population comprises a fungi Rikenella, Lachnobacterium, Tannerella, Achole with an ITS nucleic acid sequence that is at least about plasma, Howardella, Selenomonas, Butyricinonas, 97% identical to a nucleic acid sequence selected from Sharpea, Succinivibrio, Ruminobacter; Candida, the group consisting of: SEQ ID NOs: 31-60 and Syntrophococcus, Pseudobtgrivibrio, Orpinomyces, 2104-2107. Cyllamyces, Saccharomycetales, Phyllosticta, Asco 0564) 11. The method according to claim 1, wherein mycota, and Piromyces. the purified microbial population comprises a bacteria 0574 20. The method according to claim 1, wherein with a 16S nucleic acid sequence that is at least about the ruminant administered the effective amount of the 99% identical to a nucleic acid sequence selected from ruminant Supplement exhibits an increase in milk pro the group consisting of: SEQID NOs: 1-30 and 2045 duction that leads to a measured increase in milk yield. 2103. 0575 21. The method according to claim 1, wherein 0565 12. The method according to claim 1, wherein the ruminant administered the effective amount of the the purified microbial population comprises a fungi ruminant Supplement exhibits an increase in milk pro with an ITS nucleic acid sequence that is at least about duction and improved milk compositional characteris 99% identical to a nucleic acid sequence selected from tics that leads to a measured increase in energy-cor the group consisting of: SEQ ID NOs: 31-60 and rected milk. 2104-2107. 0576 22. The method according to claim 1, wherein 0566 13. The method according to claim 1, wherein the ruminant administered the effective amount of the the purified microbial population comprises a bacteria ruminant Supplement exhibits an improved milk com with a 16S nucleic acid sequence selected from the positional characteristic selected from the group con group consisting of SEQ ID NOs: 1-30 and 2045 sisting of an increase in milk fat(s), an increase in milk 2103. protein(s), an increase of carbohydrates in milk, an