NOVEL GRAS PROBIOTIC BACTERIAL STRAIN TO INHIBIT ACIDOSIS AND LIVER ABSCESSES IN CATTLE

Abstract

The present disclosure provides compositions and methods of using such compositions that reduce the incidence of, duration of, frequency of, or severity of clinical signs associated with or caused by pathogen infection. Representative pathogens include Streptococcus, Fusobacterium, Escherichia, and Arcanobacterium. In general, the composition includes a quantity of at least one Bacillus species.

Claims

1. A composition comprising: at least one bacterial species; and a component selected from the group consisting of one or more additional active ingredients, excipients, dissolution agents, surfactants, antioxidants, antiseptics, preservatives, penetrants, osmoprotectants, cryoprotectants, prebiotics, and any combination thereof.

2. The composition of claim 1, wherein said bacterial species synthesize and/or secrete/excrerte bacteriocins or short peptide sequences that inhibit the growth of one or more pathogens.

3. The composition of claim 1, wherein said bacterial species is a probiotic species.

4. The composition of claim 3, wherein said probiotic species is present in the composition in a form selected from the group consisting of a live bacterial culture, an inactivated or killed bacterial culture, a lyophilized bacterial preparation, a cell culture extract, or any combination thereof.

5. The composition of claim 1, wherein said bacterial species is a Bacillus species.

6. The composition of claim 1, wherein said bacterial species is Bacillus licheniformis, alone or in combination with Bacillus pumilus.

7. The composition of claim 1, wherein said bacterial species has a 16S sequence having at least 80% sequence homology or sequence identity to any one of SEQ ID NOs. 1-3.

8. The composition of claim 1, wherein said bacterial species inhibits the growth of a pathogen.

9. The composition of claim 8, wherein said pathogen is selected from the group consisting of Streptococcus, Fusobacterium, Escherichia, and Arcanobacterium.

10. The composition of claim 9, wherein said pathogen is selected from the group consisting of Streptococcus bovis, Fusobacterium necrophorum, Escherichia coli, and Arcanobacterium pyogenes.

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. The composition of claim 1, wherein said composition is in a form capable of being administered in an administration route selected from the group consisting of oral (po), inhalation, buccal, sublingual, suppository, microbiome transplantation, topical, dermal, and any combination thereof.

18. The composition of claim 1, wherein said composition is in a form selected from the group consisting of liquids, solids, tablets, pills, capsules, solids in a liquid medium, powders, lozenges, straws, sachets, cachets, solutions, elixirs, suspensions, emulsions, solutions, syrups, sprays, aerosols, soft and hard gelatin capsules, sterile packaged powders, combined with or introduced into a food product as a direct fed microbial, ointments, dips, pastes, and any combination thereof.

19. (canceled)

20. A method of decreasing the incidence of, severity of, duration of, or frequency of clinical signs of infection from a pathogen selected from the group consisting of Streptococcus, Fusobacterium, Escherichia, and Arcanobacterium, comprising the steps of: administering a therapeutically-effective amount of the composition of claim 1 to an animal in need thereof.

21. The method of claim 20, wherein said animal in need thereof is selected from the group consisting of a human, cow, pig, sheep, deer, goat, horse, or chicken.

22. The method of claim 20, wherein said composition is administered via an administration route selected from the group consisting of oral (po), inhalation, buccal, sublingual, suppository, microbiome transplantation, topical, dermal, and any combination thereof.

23. The method of claim 20, wherein said composition comprises a Bacillus species.

24. (canceled)

25. (canceled)

26. The method of claim 23, wherein administration of said composition comprising said Bacillus species inhibits the growth of said pathogen.

27. The method of claim 20, wherein said pathogen is selected from the group consisting of Streptococcus bovis, Fusobacterium necrophorum, Escherichia coli, and Arcanobacterium pyogenes.

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. The method of claim 20, wherein said composition is in the form of a paste, spray, ointment, or direct fed microbial.

35. The method of claim 34, wherein said composition is applied as a top dressing to animal feed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1A is a photograph illustrating the results from initial screening with Fusobacterium necrophorum ATCC 27852;

[0048] FIG. 1B is a photograph illustrating the results from initial screening with Streptococcus bovis ATCC 700410;

[0049] FIG. 1C is a photograph illustrating the results from a secondary screening of top candidates using Streptococcus bovis ATCC 700410 to further confirm the ability to inhibit Fusobacterium necrophorum ATCC 27852, and identifying that the top isolates, based on 16S rDNA sequencing of the full length gene, were Bacillus licheniformis;

[0050] FIG. 1D is a photograph illustrating the results from a secondary screening of top candidates using Fusobacterium necrophorum ATCC 27852 to further confirm the ability to inhibit Streptococcus bovis ATCC 700410, and identifying that the top isolates, based on 16S rDNA sequencing of the full length gene, were Bacillus licheniformis;

[0051] FIG. 2A is a photograph illustrating an antimicrobial assay against Streptococcus bovis ATCC 700410 using a cell extract from an overnight culture of Bacillus licheniformis to evaluate inhibition of Streptococcus bovis;

[0052] FIG. 2B is a photograph illustrating an antimicrobial assay against Streptococcus bovis ATCC 700410 using lyophilized cells from an overnight culture of Bacillus licheniformis to evaluate inhibition of Streptococcus bovis;

[0053] FIG. 2C is a photograph illustrating an antimicrobial assay against Streptococcus bovis ATCC 700410 using heat killed cells from an overnight culture of Bacillus licheniformis to evaluate inhibition of Streptococcus bovis;

[0054] FIG. 3A is a photograph illustrating an antimicrobial assay demonstrating inhibition against Escherichia coli O157;

[0055] FIG. 3B is a photograph illustrating an antimicrobial assay demonstrating inhibition against Arcanobacterium pyogenes;

[0056] FIG. 4 is a graph illustrating the decrease in liver abscesses in animals receiving an administration of a composition of the disclosure in comparison to animals that did not receive such an administration;

[0057] FIG. 5 is a chart comparing the frequency of animals having liver abscess in animals that received an administration of a composition of the disclosure and in animals that did not receive such an administration; and

[0058] FIG. 6 is a graph illustrating the fold-difference in liver abscess presence in animals that did or did not receive an administration of a composition of the disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0059] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.

Example 1

Methods

Isolation of the Bacterial Strains

[0060] Rumen samples were collected from beef cattle through the rumen fistula using a manual pump from 4 animals. Each animal was on a different diet. Sampling was performed in a manner ensuring the presence of rumen digest particles. The rumen fluid from each animal was mixed together to increase bacterial diversity. Equal amounts (˜10 g) of each rumen sample were mixed together in a 50 ml conical tube and sterile PBS was added to bring the volume to 50 ml. The tube was shaken vigorously for 5 minutes using a vortexer. Aliquots of this sample were incubated at 70° C. for 1 hour to reduce the growth of vegetative cells and to stress the spores in the sample to germinate. The sample was serially diluted (10.sup.1-10.sup.4) and plated on ISP 2, ISP 4, brain heart infusion (BHI) and casein starch agar (Himedia Laboratories, India) with 50 μg/ml cycloheximide (Sigma Aldrich, St Louis, Mo.) and 20 μg/ml Nalidixic acid (Acros Organics, NJ) using a spiral plater that further dilutes the sample as it is plated. The plates were incubated anaerobically at 37° C. for 14 days.

Screening for Isolates Against Streptococcus bovis and Fusobacterium necrophorum

[0061] After incubation of 24 hrs, 48 hrs, 72 hrs and 96 hrs, all separated single colonies were picked into 96 well plates containing brain heart Infusion (BHI) broth (Himedia Laboratories, India) at each time point and were incubated anaerobically and aerobically at 37° C. After the presence of visible growth in the wells, sterile glycerol was added to a final concentration of 25% and the resulting isolates were stored in −80° C. until used for screening experiments.

[0062] Screening for isolates that are inhibitory towards Streptococcus bovis and Fusobacterium necrophorum was performed as follows in low profile bioassay plates (Thermo Scientific). Briefly, 1000 μl of overnight cultures of Streptococcus bovis (ATCC 700410 (JB1)) and 2-day cultures of Fusobacterium necrophorum (ATCC27852), were independently plated on BHI (BD BBL™, Sparks, Md.) plates and were allowed to dry for 15 min. The dried plates were then spotted with the isolates from the 96 well plates using a plate replicator. The Streptococcus bovis test organism was grown on BHI and Fusobacterium necrophorum was grown on cooked meat medium (Hardy Diagnostics). The plates were incubated anaerobically at 37° C. Plates were checked periodically for clear zones around the isolates, which was indicative of the isolate producing an inhibitory product against the test strains (Streptococcus bovis or Fusobacterium necrophorum). Positive isolates were re-grown from the 96-well plates and glycerol stocks were prepared for archiving, which contained 25% glycerol. Additionally, a secondary screening was performed on the positive strains to confirm inhibition of the test strain. The isolates positive for one test organism were tested against the other test organism to identify isolates with the capacity to inhibit both Streptococcus bovis and Fusobacterium necrophorum. The resulting isolates with the capacity to inhibit the growth of both Streptococcus bovis and Fusobacterium necrophorum were identified for further investigation (see FIGS. 1A-1D).

Characterization of the Microbial Strains

[0063] To characterize the microbial isolates and to confirm the identity of the test strains, 16S rRNA gene was sequenced. Briefly, overnight cultures of each strain was use for DNA extraction using the QuickExtract™—Bacteria DNA extraction kit (Epicenter, Madison, Wis.) according to the manufacturer's protocol. The resulting DNA was used for PCR-based amplification using universal 16S rRNA primers (27F and 1492R). The PCR was done using the Terra™ PCR Direct polymerase mix according to the manufacturers protocol (Clonetech Laboratories, Mountain view, CA). The PCR conditions were, 98° C. for 2 min, followed by 30 cycles of 98° C. for 10 sec, 58° C. for 30 sec, and 68° C. for 90 sec. A final extension of 68° C. for 4 min was used at the end of the 30 cycles. The PCR products were visualized using agarose gel electrophoresis to ensure a single product. The resulting 1500 bp PCR product was purified using shimp alkaline phosphatase and exonuclease and was sequenced from both directions using the 27F and 1492 primers. Sequencing was performed at Eurifins Genomics according to their protocol. The sequencing results generated were matched against the NCBI non-redundant database and the ribosomal database project to identify the taxonomic classification of the unknown isolates and also to verify the taxonomy of the test strains.

[0064] Based on the 16S sequencing results, two of the isolates were identified as Bacillus licheniformis UNL07 which is on the US Federal Drug administration's (FDA's) generally accepted as safe (GRAS) list and was identified for further testing.

Whole Genome Sequencing of Bacillus licheniformis UNL07

[0065] Overnight cultures of B. licheniformis were used for DNA extraction using the QuickExtract™—Bacteria DNA extraction kit (Epicenter, Madison, Wis.) according to the manufacturer's protocol. The resulting DNA was used for library preparation for whole genome sequencing using the Illumina MiSeq system. Briefly, 600 ng of total DNA was used for Illumina library preparation as described by the manufacturer using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Single barcoded adapters were used for the library preparation. The resulting libraries were subjected to size selection (fragment range of 500-1200 bp) using sonication and the Pippin Prep System with 1.5% gel cassettes (Sage Science). The resulting libraries were pooled and sequenced on the Illumina MiSeq sequencing platform using the 250 bp paired-end sequencing strategy to obtain a depth of at least 50× coverage of each genome. Bridge amplification and sequencing will be performed as described by the manufacturer.

Bioinformatic Analysis of the Bacillus licheniformis UNL07 Genome

[0066] Sequences generated from MiSeq runs was demultiplexed and processed independently. Briefly, Trimmomatic, version 0.33, was used with a custom adaptor fasta file to trim off adaptor sequences and retain sequences with a minimum length of 130 bp. Artificial duplicates were removed using cd-hit-454. Additionally, a 30 bp sliding window with a quality threshold of 30 was used to trim low quality reads. Ambiguous bases were removed using the ‘fastq_filter’ command in USEARCH.

[0067] All resulting reads were assembled with SPAdes. Unpaired reads were also used as input with kmer lengths of 21, 33, 55, 77, 99, and 127, under the ‘single cell setting (--sc)’ within SPAdes. The resulting contigs were further analyzed using KmerFinderJS, which can be found on the internet at the omictools/kmerfinderjs-tool website, to identify bacterial species information based on the whole genome and to identify the similarity to closest genomes in the databases. The results of this analysis is shown in Table 1.

TABLE-US-00001 TABLE 1 Bacillus Lichenoformis Total Total query template coverage coverage Total Template p_Value [%] [%] depth Description TAXID Species GCF_0018960 1.01E−22 87.05 96.71 0.97 NZ_CP018249.1 1856406 Bacillus sp. 25.1_ASM189 H15-1 602vl GCF_0032538 1.01E−22 86.88 95.89 0.96 NZ_CP021970.11402 1402 Bacillus licheniformis 15.1_ASM325 CBA7132 381vl GCF_0020740 1.01E−04 86.66 94.11 0.94 NZ_CP014795.1 1402 Bacillus licheniformis 75.1_ASM207 407vl GCF_0022368 1.01E−22 86.34 93.65 0.94 NZ_CP022477.1 1402 Bacillus licheniformis 95.1_ASM223 689vl GCF_0017261 1.01E−22 85.74 91.79 0.91 NZ_CP017247.1 1402 Bacillus licheniformis 25.1_ASM172 612vl GCF_0023932 1.01E−22 44.02 45.68 0.46 NZ_CP023168.1 1648923 Bacillus paralicheniformis 25.1_ASM239 322vl GCF_0018958 1.01E−22 10.45 11.21 0.12 NZ_CP018197.1 561879 Bacillus safensis 85.1_ASM189 588vl GCF_0019386 1.01E−22 9.82 10.55 0.11 NZ_CP015607.1 561879 Bacillus safensis 65.1_ASM193 866vl GCF_0008008 1.01E−22 9.01 10.05 0.11 NZ_CP010075.1 756828 Bacillus sp. 25.1_ASM800 WP8 82vl GCF_9001869 1.01E−06 6.3 6.73 0.07 NZ_LT906438.1 1408 Bacillus pumilus 55.149386E 02

Preparing Cell Extracts for Antimicrobial Assays

[0068] Based on published studies and previous methods, the cell extracts of the two isolates were purified as described in Berditsch et al. Briefly, 5 ml of the cultures were centrifuged at 3000×g for 15 min at room temperature. The cell pellet was resuspended in the lysis buffer (150 mM NaCl and 20 mM HCL) and was incubated at 80° C. for 15 min, followed by addition of absolute ethanol (1:1 mix—final concentration of 50%). The slurry was then incubated at room temperature with gentle horizontal agitation for 1 hour. Following, extraction with ethanol, the slurry was centrifuged and the supernatant which contained the ethanoic cell extract was collected.

Antimicrobial Assays Against Streptococcus bovis, Fusobacterium Necrophorum, Escherichia coli O157 and Arcanobacterium pyogenes

[0069] Both the ethanolic cell extract and live cells were used for agar disk diffusion antimicrobial assays. The Kirby-Bauer Disk Diffusion Susceptibility test was performed as described previously with the exception of using Mueller Hinton agar plates with 5% sheep blood (purchased from BD BBL™, Sparks, Md.) instead of regular Mueller Hinton agar plates without blood. This was done because of the fastidious nature of Fusobacterium necrophorum. All incubations for the antimicrobial assays were performed in an anaerobic jar at 37° C. within the anaerobic chamber (See, FIGS. 2A-2C). Additionally, live culture assays using Escherichia coli O157 and Arcanobacterium pyogenes was performed to evaluate the ability of B. licheniformis to inhibit growth of these pathogens (FIGS. 3A and 3B).

Example 2

[0070] Feeding Trial Using the Composition as a Direct Fed Microbial

[0071] In order to assess the effectiveness of a composition in accordance with the present disclosure, a feeding trial using the composition as a direct fed microbial was performed. The diet feed consisted of 30.93% dry-rolled corn, 14.30% corn silage, 53.18% WDGS, and 1.59% of a mineral/vitamin supplement. Five groups of cattle (35-36 head of cattle in each group) were allowed to eat the diet feed and acted as controls for the study. The diet was fed for 140 day feeding period after the cattle were adapted to the finishing diet. The treatment of DFM was imposed on the base diet above where, cattle (n=150) were fed DFM at a concentration of 1 billion cells per head per day. Five other groups of cattle (35-36 head of cattle in each group) were fed the same feed diet but to which a quantity of the composition was applied to render a direct fed microbial. The study design and results are provided below in Table 2. Test subjects were examined for the presence of liver abscesses at the end of the study. The results (as shown in FIG. 4) demonstrated that 16.67% of the control animals developed liver abscesses while just 2.27% of the animals that were fed the direct fed microbial composition developed liver abscesses. FIGS. 5 and 6 further illustrate that test subjects fed the direct fed microbial experienced a significant reduction in the incidence of liver abscesses.

TABLE-US-00002 TABLE 2 Total Total Pen Treatment Head/pen Count A+ A− A % A+ A− A 101 DFM 35 4 3 1 0 11.43 8.57 2.86 0 102 Control 35 4 2 2 0 11.43 5.71 5.71 0 103 DFM 36 6 5 1 0 16.67 13.89 2.78 0 104 DFM 36 6 5 1 0 16.67 13.89 2.78 0 105 Control 36 4 3 1 0 11.11 8.33 2.78 0 106 Control 36 4 3 1 0 11.11 8.33 2.78 0 107 DFM 35 7 5 2 0 20.00 14.29 5.71 0 108 DFM 35 5 4 1 0 14.29 11.43 2.86 0 109 Control 35 11 7 2 2 31.42 20.00 5.71 5.71 110 Control 35 6 5 1 0 17.14 14.29 2.86 0

Example 3

[0072] Feeding Trial Incorporating Bacillus licheniformis and Bacillus pumilus

[0073] A feeding trial was conducted at United States Meat Animal Research Center. A cohort of n=300 cattle were fed a diet containing 71.75% dry rolled corn, 9% corn stalks, 15% wet distillers grains, 0.75% urea and 3.5% vitamin supplement with monensisn. The diet was fed for 240 day feeding period. The treatment of DFM was imposed on the base diet above where, cattle (n=150) were fed DFM at a concentration of 1 billion cells per head per day. The DFMs were fed in two stages, Bacillus pumilus was only fed for the first 140 days. After further in vitro analysis, a cocktail of Bacillus pumilus and Bacillus licheniformis was fed together, each at a concentration of 1 billion cells per head per day. Before feeding the mixture of the two DFMs, rumen samples were collected through esophageal tubing to evaluate Fusobacterium populations in both the treatment and control animals. The DFM was top dressed on the feed bunks by spraying the feed bunks after feed delivery.

Harvesting the DFM

[0074] Harvesting of DFM occurred by growing DFM in BHI at for 48 hrs, shaking, at 37° C. for 48 hours in one-liter volumes with 48 hours. Cell counting was performed using Flow Cytometry. Invitrogen's Live/Dead Assay (Life Technologies Corporation Eugene, Oreg.) stained the cells for viewing and the protocol was followed according to manufactures' directions. Spherotech 5.14 μm beads were used as a reference while conducting the flow cytometry. (Sphero ACFP-50-5 Spherotech Inc. Lake Forest, Ill.). Twenty percent glycerol was added to the culture prior to −80° C. storage. Flow cytometry was used to calculate the cfu's present in each mL. If need, culture was diluted in order to reach a concentration of one billion cells per mL.

[0075] The results of this trial showed that the DFM provided significant benefits and protection against infection and accompanying clinical signs or symptoms of the pathogens discussed herein.