Methods of administering compositions of bacillys subtilis PB6, butyric acid with zinc, and optionally red clover, to reduce liver abscesses in cattle

Abstract

The present invention relates to administering an animal feed additive or supplement that contains a combination of Bacillus subtilis PB6, butyric acid and zinc, and optionally red clover, in an amount effective to decrease the incidence and severity of liver abscesses in weaned beef and dairy steers.

Description

[0001] METHODS^OF^ADMINISTERING^COMPOSITIONS^OF^BACILLYS^SUBTILIS^PB6,^BUTYRIC^ ACID^WITH^ZINC,^AND^OPTIONALLY^RED^CLOVER,^TO^REDUCE^LIVER^ABSCESSES^IN^ CATTLE^^ CROSS‐REFERENCE^TO^RELATED^APPLICATIONS^The present application claims the benefit of priority to United States ProvisionalPatent Application No. 63 / 541,468, entitled “METHODS OF ADMINISTERINGCOMPOSITIONS OF BACILLYS SUBTILIS PB6, BUTYRIC ACID WITH ZINC, ANDOPTIONALLY RED CLOVER, TO REDUCE LIVER ABSCESSES IN CATTLE,” filed September29, 2023, the entire disclosure of which is incorporated by reference herein.BACKGROUND^OF^THE^INVENTION^Liver abscesses are a common and costly condition in the fed cattle industry,affecting approximately 12 to 32% of finished cattle (Amachawadi and Nagaraja, 2016).Severe abscesses have been linked to decreased growth performance, leading toappreciable economic losses for cattle feeders and beef processors. Beef livers are anexport variety meat, and the U.S. exported 76% of beef livers to Egypt (62,119 metric tons)in 2016 (Global Trade Atlas, 2017). The presence of a liver abscesses (LA) can decrease thevalue of beef carcasses by $38 per animal (Brown and Lawrence, 2010) and cost the cattlefeeder and beef processor > $470 million dollars annually (Herrick et al., 2022). Therefore,effective prevention and control strategies are needed to decrease the economic burdenassociated with LA.Two bacterial species are likely associated with the development of LA in cattle,Fusobacterium^necrophorum,^and^Salmonella^enterica^serovar Lubbock^(Amachawadi and

[0002] 1    Nagaraja, 2015). Although the role of^S.^enterica.^in LA formation is not fully understood, ithas been used in combination with F.^necrophorum to successfully induce LA (McDaniel etal., 2023). Indeed, if S.^enterica contributes to the development of LA, supplementationwith Bacillus^subtilis PB6 could decrease LA prevalence in cattle because it has beenreported to decrease Salmonella in the feces of cattle (Broadway et al., 2020; Smock et al.,2020).^Leaky gut could also be a contributing factor to LA formation, but it is unclearwhether pathogens invade the liver through ruminal lesions or leaky tight junctions in thelower gastrointestinal tract (Sanz-Fernandez et al., 2020). Assuming the latter, butyric acidand zinc supplementation might improve tight junction integrity and barrier function in thelower gastrointestinal tract by increasing villus height, crypt depth, and maintainingosmotic pressure (Ohata et al., 2005).Tylosin phosphate, a macrolide antibiotic, is currently administered in commercialfeedlots to reduce incidences of liver abscesses, though it has been reported that there isvariable efficacy among different groups of cattle. (Weinroth et al., 2019). Additionally,growing concerns of antibiotic resistance has prompted urgency in finding alternativesolutions for decreasing the incidence and severity of liver abscesses in cattle. Forinstance, the United States Food and Drug Administration (FDA) banned the use ofantibiotics as growth promoters in cattle feed and drinking water in 2015. These productscan only be used for therapeutic purposes under veterinary oversight (Veterinary FeedDirective, 2015). Due to consumer pressure and this regulation, finding a suitablealternative to tylosin phosphate has been a topic of critical research (Rode et al., 2002;Beauchemin and McGinn, 2006; Dehghani et al., 2019).

[0003] 2     ^ BRIEF^DESCRIPTION^OF^THE^INVENTION^The present invention relates to administering a feed additive or supplement tocattle that is a combination of Bacillus^subtilis PB6, butyric acid and zinc, and optionally redclover, to decrease the incidence and severity of liver abscesses in weaned beef and dairysteers. Another aspect of the present invention relates to administering the feed additive orsupplement containing a combination of Bacillus^subtilis PB6, butyric acid and zinc to Bos^Taurus influenced natural fed cattle in an amount effective to decrease the incidence ofliver abscesses.^ BRIEF^DESCRIPTION^OF^THE^FIGURES^Figure 1 depicts the daily feed disappearance for beef × dairy steers fed ground cornwith or without (CON) Bacillus^subtilis and encapsulated butyric acid and zinc (CV), and CV+ red clover (Trifolium^pratense; CVC) to evaluate the formation of liver abscesses. Errorbars denote the standard errors of least squares means (n = 10 to 15 steers / mean).Treatment, time, and treatment × time (P < 0.01). *Denotes treatment and time difference P< 0.01.^ ^ DETAILED^DESCRIPTION^OF^THE^INVENTION^The present invention relates to administering a combination of Bacillus^subtilis PB6and butyric acid with zinc, with or without red clover or extracts, to decrease the incidenceand severity of liver abscesses in weaned beef and dairy steers.

[0004] 3     According to at least one embodiment, a combination of Bacillus^subtilis PB6 andbutyric acid with zinc is administered to cattle in an amount to reduce the incidence orseverity of LA in cattle. In at least one embodiment, the following ingredients wereadministered: B.^subtilis^PB6 (CLOSTAT Dry® dry, 2.2×108 CFU / g) in an amount rangingfrom between about 0.5g to about 15g / steer daily, for instance 13g / steer daily, butyric acidin an amount ranging from about 0.5 g to about 5g / steer daily, and zinc in an amountranging from about 0.1 to about 0.5g daily, and in certain embodiments, optionally up to30g red clover daily.According to at least one embodiment, a combination of Bacillus^subtilis PB6 andbutyric acid with zinc is administered to cattle in order to improve beef carcass scores. Inat least one embodiment, the following ingredients were administered: B.^subtilis^PB6(CLOSTAT Dry® dry, 2.2×108 CFU / g) in an amount ranging from between about 0.5g toabout 15g / steer daily, for instance 13g / steer daily, butyric acid in an amount ranging fromabout 0.5 g to about 5g / steer daily, and zinc in an amount ranging from about 0.1 to about0.5g daily, and in certain embodiments, optionally up to 30g red clover daily.In at least one embodiment, the butyric acid and zinc is encapsulated, where thecattle were fed encapsulated butyric acid with zinc (3g / daily) to deliver a dosage of about0.75g butyric acid and 0.3g zinc. In alternative embodiments, the butyric acid and zinc maybe delivered in alternative forms, with or without encapsulation. Persons of ordinary skillin the art would appreciate that the dosage may vary based on the specific circumstanceson the feedlot, for instance in certain embodiments, the dosage of butyric acid and zinc maybe increased or decreased. In alternative embodiments, the dosage of PB6 may be

[0005] 4    increased or decreased. In certain embodiments, the additive or supplement combinationfurther comprises red clover or an extract of red clover.According to at least one embodiment, the combination is administered to cattle atleast once a day for at least 25 days before harvest. In alternative embodiments, thecomposition is administered to cattle between about 25 days to about 100 days, forinstance for at least 35 days, at least 40 days, or at least 45 days, at least 50 days, at least 55days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 80 days, atleast 85 days, at least 90 days, at least 95 days, and at least 100 days before harvest.According to at least one embodiment, the combination is administered to the cattlebetween days 150 to 250 days on feed.In at least one embodiment, the combination is administered to cattle for at least 35days before harvest. In at least one embodiment, the combination is administered to cattlefor at least 50 days before harvest. In another embodiment, the combination isadministered to cattle for at least 65 days before harvest. In another embodiment, thecombination is administered to cattle for at least 75 days before harvest.In alternative embodiments, the combination is administered to the cattle at leastonce daily for at least 25 days before the cattle is harvested, wherein the administration ofthe feed additive or supplement occurs daily between days 100 to 300 days on feed, forinstance between days 150 to 250 or days 170 to 200.In at least one embodiment, the combination is administered orally at least oncedaily. In alternative embodiments, the combination is administered twice a day. Inalternative embodiments, the combination is administered up to three times a day.

[0006] 5     ^ EXAMPLE^1^^Materials^and^Methods: Forty beef ^ dairy steers (initial BW 97 ± 11.5 kg) wereindividually housed in a climate-controlled barn. Steers were assigned randomly to 1 of 3treatments: 1) cycled between a low-starch control and high-starch acidotic diet (CON; n =10); 2) cycled between the control and high-starch acidotic diet and supplemented with 13g / steer daily of B.^subtilis^PB6 (CLOSTAT Dry® dry, 2.2×108 CFU / g, Kemin Industries, Inc.,Des Moines, IA) + 3 g / steer daily of butyric acid (0.75g daily) and zinc (0.3g daily; VilliTech,Kemin Industries, Inc; CV; n = 15); or 3) CV + 30 g / steer daily of chopped red clover (CVC;n = 15). All steers were intraruminally inoculated with Fusobacterium^necrophorum subsp.necrophorum and Salmonella^enterica serovar Lubbock on d 20. After euthanasia on d 39,necropsies were performed to assess gross pathology.Results^and^Discussion: Steers in CVC had a greater percentage of normal lung scoresthan steers in the CON in CV treatments (P = 0.04). Liver abscess prevalence did not differamong treatments (P = 0.68); however, liver abscess prevalence decreased 25% in CV vs.CON and 22% in CVC vs. CON. Feed disappearance decreased in all treatments from d 0 to 3and from d 20 to 22 but otherwise increased over time. Steers in CV consumed more feedthan CON and CVC from d 24 to 38 (P < 0.01).Implications^and^Applications: Our data demonstrate the viability of the experimentalmodel to evaluate LA mitigation strategies. Although no statistical differences in LA wereattributable to the supplements evaluated, the decrease in liver abscess prevalence wenoted with CV could be biologically and economically impactful. Further research is neededto elucidate specific mechanisms of abscess prevention via nutritional supplementation.

[0007] 6     All experimental procedures involving live animal subjects were approved by theLivestock Issues Research Unit Institutional Animal Care and Use Committee (approvalnumber: 2023-02).Animal^Management^and^Experimental^Treatment^ApplicationWeaned beef ^ dairy steers (n = 40; initial BW = 97 ^ 11.5 kg) were obtained from acommercial calf ranch in eastern New Mexico. Steers were transported to the USDA-ARSLivestock Issues Research Unit facilities near Lubbock, TX on d -22 relative to the first dietcycle (Table 1). On arrival, steers were individually weighed to nearest 0.2 kg (MP600 loadcells; Tru-Test Group, Auckland, New Zealand), given an ear tag with unique identification,dewormed with fenbendazole (Safeguard, Merck Animal Health, Whitehouse Station, NJ),and administered tildipirosin as a prophylactic antibiotic for the prevention of respiratorydisease (Zuprevo, Merck Animal Health).

[0008] 7     Table^1. Timeline of key events in which beef × dairy steers were fed a low-starch controldiet (CON) and then cycled to a high-starch acidotic diet. Each diet included ground cornwith or without Bacillus^subtilis and encapsulated butyric acid and zinc (CV) or CV + redclover (Trifolium^pratense; CVC) to evaluate the formation of liver abscesses1.^Item DayAdaptation to facility -22 to -18Steers fed basal diet -22 to -1Acidotic cyclesCycle 1 0 to 2Cycle 2 5 to 7Cycle 3 10 to 12Cycle 4 15 to 17Continuous feeding of acidotic diet 18Body weight measurement -16, -9, -2, 5, 12, 19, 26, 33, 38Bacterial inoculation 20Blood collection -2, 12, 26, 38Harvest and gross pathology 391Between cycles 1, 2, and 3, steers remained on the control diet for 2 d. For cycle 4, aheadof inoculation, steers remained on the control diet for 3 d to ensure ruminal pH would notlyse the inoculated F.^necrophorum. The bacterial inoculation included a mixture ofFusobacterium^necrophorum (8.00 × 108 CFU), and Salmonella^enterica (2.67 × 109CFU / mL).Steers were assigned randomly to 1 of 3 experimental treatments balanced by BW:1) steers were^cycled between a low-starch control and high-starch acidotic diet (CON; n =10); 2) steers were^cycled between a low-starch control and high-starch acidotic diet andsupplemented with 13 g / steer daily of B.^subtilis^PB6 (CLOSTAT® Dry , Kemin Industries,Inc., Des Moines, IA) + 3 g / steer daily of butyric acid and zinc (VilliTech, Kemin Industries,Inc; CV; n = 15); and 3)^steers were cycled between a low-starch control and high-starchacidotic diet and as supplemented as with the CV treatment with the addition of 30 g / steerdaily of chopped red clover (CVC; n = 15; Monterey Bay Herb Company, Watsonville, CA).

[0009] 8    All supplements were mixed into a ground corn carrier twice during the study (d -22 and10) and 115 g of the supplement was top dressed and mixed with the diet at the time offeeding at approximately 0800 h. Steers in the CON treatment group received anequivalent amount of ground corn with no added supplement.Diets were formulated to meet nutrient requirements for growing cattle (NASEM,2016). Ingredient and analyzed nutrient composition of the diets are summarized in Table2. Before d 0, all steers were fed the CON diet for 22 d. The diet was offered at 2.5% of BWon a DM basis on the day of arrival, after which steers were fed to appetite targeting adlibitum intake with^ad^libitum access to fresh water. The diets were formulated to providedifferent concentrations of starch and NDF. The control diet was a low-starch growing diet,whereas the acidotic diet was formulated to have more fermentable starch and less in NDFto promote acidotic conditions in the rumen. In all treatments, on d 0, the diet wasswitched from the control to acidotic diet for 3 d, followed by feeding control diet for 2 d.This 5-d cycle was repeated 4 times during the study. Weekly diet samples were obtainedto form a weekly composite for analyses of CP, NDF, total starch, crude fat, Ca, and Pcontents (Servi-Tech Laboratories, Amarillo, TX). A second diet sample was dried at 100 ^Cfor 24 h in a forced-air oven (Thermo Scientific Heratherm OGS100, Thermo FisherScientific, Inc., Waltham, MA) for analysis of DM to calculate DMI. Steers were housed on aslatted floor, so data presented represents feed disappearance and not feed intake becausea small amount might have fallen through the slats. Feed disappearance was recorded asDM fed minus DM of the orts. Daily feed deliveries and orts were weighed to the nearest 5g at 0800 h (UWE AMP-150 NTEP bench scale; Intelligent Weighing Technology, Inc.Democratic Taiwan). In the event of bloat, a flexible tube (0.95 cm internal diameter × 1.5

[0010] 9    m long) was passed orally into the rumen to relieve pressure. Bloat was relieved for 5steers throughout the study (2 from CON, 1 from CV, and 2 from CVC; data not presented).On day 20, steers were gently restrained in a squeeze chute to allow for thecollection of ruminal fluid and fecal samples. Additionally, steers were administered thebacterial inoculation described below while they were restrained. For collection of ruminalfluid and the bacterial inoculation, a flexible tube 1.91-cm diameter was passed through africk-type speculum from the oral cavity to the level of the oropharynx and directed to theesophagus and passed into the rumen. The bacterial inoculation contained ~100 mL eachof S.^enterica^(2.67 × 109 CFU / g) and F.^necrophorum (8.00 × 108 CFU / g). Followinginoculation, the flexible tube was flushed with 100 mL of phosphate buffered saline to clearany residual inoculant. After the bacterial inoculation on day 20, steers were fed theacidotic diet for the remainder of the study. Body weights and rectal temperatures weremeasured on d -16, -9, -2, 5, 12, 19, 26, 34 and at harvest on day 39.

[0011] 10    Table^2. Ingredient and nutrient composition of the low-starch control and acidotic diets fed tobeef × dairy steers to evaluate the formation of liver abscesses1^DietItem Control AcidoticIngredient, % of dry matter (DM)Steam-flaked corn - 54.40Dry-rolled corn 20.20 -Wet corn gluten feed2 46.10 21.90Ground corn - 14.90Ground alfalfa hay 30.20 4.60Limestone 1.50 1.50Urea - 0.70Vitamin and mineral supplement3 2.00 2.00Analyzed nutrient composition4Diet DM, % 72.30 77.90Crude protein, % 18.60 16.80Neutral detergent fiber, % 35.00 15.10Total starch, % 20.70 57.40Crude fat, % 2.90 3.00Ca, % 0.93 0.66P, % 0.61 0.36NEm5, Mcal / kg 1.67 2.29NEg5, Mcal / kg 1.08 1.581Dry matter basis, except diet DM%.2SweetBran; Cargill, Inc. Blair, NE.3Supplement supplied 44.40% crude protein, 5.99% potassium chloride, 3.82% salt, 8.34mg / kg cobalt carbonate, 395.0 mg / kg copper sulfate, 408.0 mg / kg iron sulfate, 764 mg / kgmanganous oxide, 2.92 mg / kg sodium selenate, and 2,490.00 mg / kg zinc sulfate on a DM basis.4Analysis performed by Servi-Tech Laboratories, Amarillo, TX.5NEm and NEg are tabular values (NASEM, 2016).Each morning throughout the experiment, trained personnel visually monitoredsteers for clinical signs of illness using a system described by Step et al. (2008) and Wilsonet al. (2015). Throughout the study, 25 steers were given additional antibiotic therapy,which included 9 steers from CON, 5 steers from CV, and 11 steers from CVC. One steer

[0012] 11    from CON and one steer from CVC were euthanized for chronic respiratory illness on days20 and 24, respectively.Gross^Pathology^Scores^All steers were humanely harvested on day 39. At necropsy, gross pathology wasrecorded on the lungs, rumens, livers, and colons. The lung scoring system developed byTennant et al. (2014) was used, in which lungs were scored as normal, mild, or severedepending on whether there were visible or palpable lesions associated with bovinerespiratory disease (BRD) or mild hyperemia of the cranioventral lobes withoutconsolidation. A mild score indicated lesions associated with BRD in up to 50% of thecranioventral lobe(s). Steers with severe scores had lesions associated with BRD andpleural adhesions within the thoracic cavity or consolidation of 51 to 100% of theircranioventral lobes.Rumens were scored as normal, mild, and severe. A normal score indicated healthyepithelium without ulceration or inflammation, a mild score indicated consolidation ofruminal mucosal surface or denuded papillae, and a severe score indicated active rumenitislesions or ulcers. Livers were scored as either normal or abnormal, with a score of 0indicating no detectable abscess and a score of A- indicating at least 1 detectable abscess, Aindicating at least 2 abscesses and depending on the size and severity of the abscess, andA+ indicating at least 3 liver abscesses and showing the most severe cases of liverabscesses (Brown et al., 1975).Colons were scored on a scale from 1 to 4, in which a score of 1 indicated a colonfree of parasites and bacteria according to Mansfield and Urban (1996). Scores of 2indicated patchy hemorrhage (50% is hemorrhagic) and slightly roughened tissue. A score

[0013] 12    of 3 indicated severe hemorrhage (more than 50% of surface area) and noticeableroughness. A score of 4 indicated the colons were covered in fibrinonecroticpseudomembrane, with sloughed mucosa and lymphoglandular complexes.^^Liver^Abscess^Culturing^Techniques^At necropsy, if a LA was identified, it was aseptically removed from the liver, placedon ice, and shipped overnight to Kansas State University College of Veterinary Medicine todetermine the prevalence of F.^necrophorum and S.^enterica. Culture procedures for F.^necrophorum and S.^enterica were conducted as described by Amachawadi et al. (2017).Briefly, a sterile scalpel was used to cut the capsule of abscesses after they were searedwith a hot spatula. To isolate S.^enterica, the inner wall of the abscess capsule was swabbedand streaked onto three plates blood agar (Remel) and 2 plates of Hektoen Enteric (HE)agar (Becton, Dickinson and Company). As a control, one HE and one blood agar plate wereincubated aerobically within an aerobic glove box (Thermo Fisher Scientific Inc.), and aseparate HE plate and blood agar plate were incubated anaerobically within an anaerobicglove box (Thermo Fisher Scientific Inc.). The Salmonella^was enriched using tetrathionatebroth (Becton Dickinson and Company; for 24 h at 37 ℃) and Rappaport Vassiliadis broth(Becton, Dickinson and Company) for 24 h at 37℃ before isolation on HE agar (incubated24 h at 37 ℃). Presumptive, phenotypic colonies^were agglutinated with S.^entericapolyvalent O antiserum (Becton, Dickinson and Company) allowing for genus confirmation.On confirmation of positive results, B, C1, C2, D1, D2, and E antisera were tested forserogroup classification. Brain heart infusion broth that was pre-reduced with 0.05%cysteine HCl and anaerobically sterilized (BHI-S) was inoculated with presumed F.^necrophorum from an abscess. In addition to microscopic morphology, Rapid-ANA II test

[0014] 13    kits (Thermo Fisher Scientific Inc.) were used for confirmation of the subspecies(necrophorum or funduliforme) determined by sedimentation characteristics in phosphatetest and BHI-S broth (Tan et al., 1994b).^Sample^Processing^for^Salmonella^Prevalence^Fecal samples were collected on days -18, 24, 38, and subiliac and mesenteric lymphnodes, and ileum were collected at necropsy. To determine the prevalence of Salmonella, a25-g sample was mixed with a 1:10 dilution of PBS in a special bag (Seward Ltd.; WestSussex, UK), after which it was blended using a Stomacher® 400 Circulator (Seward Ltd.)for 2 min at 230 rotations per min. Before blending, subiliac lymph nodes were crushedwith a rubber mallet (Arthur et al., 2008). The bags were sealed, gently massaged by hand,and 1 mL of the mixture was enriched by diluting it 1:10 with Tetrathionate Hajna broth(Remel Inc., San Diego, CA) containing iodine and Rappaport-Vassiliadis broth (Oxoid Ltd.,Basingstoke, UK). These enriched mixtures were then placed in an incubator, with some at37°C and others at 42°C and left overnight. After incubation, the enriched cultures weremixed, and a small 10-µL loop was used to streak the enriched material onto xylose lysinedeoxycholate (XLD; Becton, Dickinson and Co., Franklin Lakes, NJ) agar that had novobiocin(25 µg / mL) added. These streaked agar plates were then incubated at 37°C overnight andan additional 24 h at 25°C. Finally, colonies that looked like Salmonella were confirmedusing latex agglutination (Salmonella Test Kit; Oxoid Ltd).Quantitative^Polymerase^Chain^Reaction^and^Tissue^Culture^Methods^Ruminal fluid and tissue, colon content and tissue, ileum tissue, healthy liver tissue,and liver abscesses (if present) were aseptically collected from a consistent anatomicallocation during necropsy. Samples were stored at 4°C before being shipped overnight to

[0015] 14    Kansas State University for quantitative polymerase chain reaction (q-PCR) and culturetissues for determining the prevalence of F.^necrophorum subsp. necrophorum and^funduliforme^within each individual sample.Whole^Genome^Sequencing^of^the^F.^necrophorum^subsp.^necrophorum^Strains^Strains isolated from the liver abscesses and the ruminally inoculated strain F.^necrophorum, 8L1 were cultured, genomic DNA was extracted, and samples submitted forwhole genome sequencing to CosmosID (Germantown, MD). Following the manufacturer'sprotocol, genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen,Germantown, MD). Extracted DNA samples were quantified using the QuantiFluor®dsDNA System (Promega, Madison, WI) chemistry using GloMax Plate Reader Systems(Promega, Madison, WI). ^One nanogram of DNA was used to prepare DNA libraries usingthe Nextera XT DNA Library Preparation Kit (Illumina) and IDT Unique Dual Indexes. Tofragment the genomic DNA, a proportional amount of the Illumina Nextera XTfragmentation enzyme was used. The samples were indexed with dual indices, and thenPCR was performed for 12 cycles to create libraries. AMpure magnetic beads (BeckmanCoulter) were used to purify DNA libraries, and QIAGEN EB buffer was used to elutesamples. QubitTM dsDNA HS Assay Kit and QubitTM 4 fluorometer were used to quantifythe DNA libraries. The libraries were sequenced at Cosmos ID Inc. (Rockville, MD) on anIllumina HiSeq X and NextSeq 2000 platform.Bioinformatics^Analysis^The paired-end reads were processed and trimmed using the Genome Assembly toolprovided by PATRIC (https: / / pubmed.ncbi.nlm.nih.gov / 31667520 / ). Subsequently, single-nucleotide polymorphisms (SNP) from liver abscesses were identified and used to

[0016] 15    construct phylogenetic trees. The DNA sequences obtained from the NCBI GenBank, alongwith SNP found in the core genomes of the inoculated strains and strains isolated from liverabscesses were incorporated into the construction of phylogenetic trees. The maximumlikelihood criterion was employed for the construction of these trees, ensuring accuraterepresentation and classification of the strains (Allard et al., 2012).^Quantitative^PCR^Assay^to^Detect^and^Quantify^F.^necrophorum^in^Ruminal^and^Colonic^Contents^and^Epithelial^Tissues^The detection and quantification of F.^necrophorum^were determined using a real-time quantitative PCR assay targeting the subspecies-specific promoter sequences of theleukotoxin gene, lktA (Zhang et al., 2006).Statistical^AnalysesFeed disappearance and BW was analyzed using PROC GLIMMIX of SAS (SAS Inst.Inc., Cary NC) as a repeated measures with individual steer as the experimental unit. Themodel included the fixed effects of treatment, time, and their interaction, with steer nestedwithin treatment being the subject of repeated measures. Various covariate structureswere tested, and the one resulting in the smallest Akaike and Schwarz Bayesian criteriawas chosen for the analyses. The PROC GLIMMIX procedure was used to assess thedifferences in percent prevalence of Salmonella and liver abscesses and binomialproportions of necropsy scores among treatments. The model included the fixed effect oftreatment. Both models included the Kenward-Rogers adjustment for an unequal numberof experimental units among treatments. The significance level for all analyses was set at α= 0.05, with statistical tendencies indicated at 0.05 < P ≤ 0.10. In cases where the main

[0017] 16    effects were significant, pairwise comparisons were performed (α = 0.05) to examinedifferences among treatments.^ RESULTS^AND^DISCUSSIONBody^Weight^and^Feed^Disappearance^The were no differences in BW among treatments throughout the study (P = 0.75;finalBW = 167, 175, and 170 kg ± 8.0 kg for CON, CV, and CVC, respectively; data not shown).According to Owens et al. (1998), when switching feedlot cattle from a high-forage diet to ahigh-concentrate diet, there can be a negative effect on the ruminal microbial populationsthat can decrease BW gain and increase metabolic disorders such as acidosis. Albornoz et al.(2013) and Zhang et al. (2013) also reported that decreased DMI followed by a return to adlibitum intake predisposes cattle to ruminal acidosis. For feed disappearance (Figure 1), aninteraction between treatment and time was observed (P < 0.01), whereby feeddisappearance was greater (P < 0.01) in CV than CON and CVC on days 22, 28, and 33 to 38.In addition, feed disappearance was greater in CV and CVC than CON on d -6 to 0 (P < 0.01).Steers in CON, CV, and CVC all had decreased feed intake from day 0 to 3 (P < 0.01) and day20 to 22 (P < 0.01). From days 24 to 38, CV consumed more feed than CON or CVC (P < 0.01).Our findings agree with Smith et al. (2021), who reported an increase in DMI when yearlingcrossbred beef steers were fed Bacillus^subtilis. Broadway et al. (2020) reported increasedDMI in weaned Holstein steers when supplemented with Bacillus^subtilis PB6, which agreeswith our results.Gross^Pathology^The CVC steers had a greater proportion of normal lung scores than CON or CV (P =0.04; Table 3). Conversely, no differences were observed in the proportions of mild or severe

[0018] 17    lung scores among treatments (P > 0.15). Although CVC steers had the greatest proportionof normal lungs, 62.5% were therapeutically treated with tulathromycin for BRD relapseafter the initial metaphylaxis was administered. The red clover included in the CVCsupplement contains an isoflavone, biochanin A (Flythe et al., 2010; Harlow et al., 2017;Harlow et al., 2018). Biochanin A might have contributed to the increase in normal lungscores, as it has historically been valued as a respiratory remedy for whooping cough,measles, bronchitis, laryngitis, and tuberculosis in humans (Ko et al., 2011). Nonetheless,the concentration of biochanin A in the red clover was not measured in the current study.While it is important to note that all steers consumed all of their top-dressed supplementdaily, future studies are necessary to better understand the effects of red clover andbiochanin A on lung health in cattle.

[0019] 18    Table^3. Gross pathological evaluation of tissues of beef × dairy steers fed ground corn with orwithout (CON) Bacillus^subtilis and encapsulated butyric acid and zinc (CV), Bacillus^subtilis,encapsulated butyric acid and zinc, and CV plus red clover (Trifolium^pratense; CVC) cycled on anacidotic diet and inoculated with Fusobacterium and Salmonella to evaluate liver abscessformation and severityTreatment1 ^Item CON CV CVC SEM2 P-valueLung3, %Normal 22.2a 6.7a 57.1b 14.00 0.04Mild 66.7 80.0 42.9 15.70 0.15Severe 11.1 13.3 0.0 10.51 0.99Rumen4,%Normal 77.8 80.0 64.3 13.86 0.61Mild 22.2 20.0 35.7 14.86 0.61Severe 0.0 0.0 0.0 - 1.00Liver abscess5,% 77.8 60.0 64.3 13.86 0.680 22.2 40.0 35.7 7.25 0.32A- 0.0 0.0 0.0 -A 22.2 0.0 14.3 13.86 0.32A+ 55.6 60.0 50.0 16.56 0.32No. of abscessesper liver 2.8 2.1 1.4 0.56 0.31Colon6, % 177.8 66.7 85.7 13.86 0.502 22.2 33.3 14.3 13.86 0.503 0.0 0.0 0.0 - -4 0.0 0.0 0.0 - -a,bLeast squares mean in a row that do not have common superscripts differ; P < 0.05.1CON = control diet and acidotic diet with intraruminal bacterial inoculation; CV = control dietand acidotic diet supplemented with ground corn, CLOSTAT® dry, and VilliTech® withintraruminal bacterial inoculation; CVC = CV + red clover with intraruminal bacterial inoculation.2Standard error of least squares means, n = 9 steers in CON; 15 steers in CV and 14 steers in CVC.3Lung scored as normal, mild, and severe. Normal scores were lungs with no palpable lesionsassociated with BRD and no consolidation of cranioventral lobes. Severe scores were lungs withconsolidation of 51 to 100% of cranioventral lobes, with lesions, and any adhesion to thoraciccavity (Thompson et al., 2006; Rezac et al., 2014).

[0020] 19    4Ruminal epithelium scored as normal, mild, or severe. Normal scores indicate healthyepithelium, and lack of inflammation or ulceration. Severe scores indicate active rumenitis withinflammation and lesions.5Liver abscess scored as 0 or abscessed. A score of 0 was a normal liver with no abscesses.6Colons were scored as 1, 2, 3, or 4. A score of 1 indicated a healthy colon unaffected by parasitesor bacteria; the gut was smooth and tan. A score of 4 indicated colons covered withfibrinonecrotic pseudomembrane, possibly denuded, and if not obliterated bypseudomembranes, filled with mucopurulent debris (Mansfield and Urban, 1996).No differences among treatments were noted for ruminal scores (P^= 0.61). Amongthe 38 steers, 10 (CON = 2, CV = 3, and CVC = 5) had signs of rumenitis. Rumenitis ischaracterized by inflammation of the ruminal epithelium and can be caused by variousdietary factors (Nagaraja and Chengappa, 1998). In the current experiment, the acidoticdiet fed and cycled to all treatments aimed to decrease ruminal pH rapidly, to attempt toinduce rumenitis and subsequently facilitate liver abscess development (Nagaraja andChengappa, 1998; Amachawadi and Nagaraja, 2016; McDaniel et al., 2023). The overallrumenitis prevalence among CON, CV, and CVC was 26%, whereas the overall LAprevalence was 67%. Therefore, in the current experiment there was a weak relationshipbetween rumenitis and LA development. These results suggest that alternative routes ofbacterial translocation from the gastrointestinal tract to the liver occurred to develop LA. Apotential route for bacterial translocation is that Salmonella^in the hindgut can traverse theepithelial barrier, most likely in the small or large intestine, enter the lymphatic system,and reach the portal capillary system of the liver to initiate infection (Amachawadi et al.,2017). Additional research is necessary to understand how bacteria are translocated to theliver if not through ruminal lesions.

[0021] 20     F.^necrophorum is known to be one of the most active lysine-degrading bacteriafound on the ruminal epithelium (Russell, 2006; Elwakeel et al., 2013) and can be isolatedfrom ruminal contents of cattle fed various diets (Narayanan et al., 1997). The presence ofruminal endotoxins or F.^necrophorum adhering to the ruminal epithelium with the help ofouter membrane proteins could cause ruminal epithelial damage (Kumar et al., 2013). Theextent of ruminal epithelial damage caused by F.^necrophorum alone is unknown.Interestingly, most of the rumens scored in the present study were given a score of normal,meaning they lacked signs of rumenitis. Therefore, the lack of ruminal lesions does notalign with the only route of translocation of F.^necrophorum^occurring via a ruminal lesion,as there were far more liver abscesses than ruminal lesions. Nonetheless, the techniquesemployed in the present study lack the ability to delineate the precise trajectory of F.^necrophorum and S.^Lubbock invasion within the liver – whether it occurred throughruminal lesions or via compromised tight junctions along the lower gastrointestinal tract.These potential pathways of bacterial translocation warrant further investigation.Liver abscesses were detected in all treatments (Table 3). No differences in LAprevalence (P = 0.32) nor severity (P = 0.68) were noted among treatments, even thoughCV and CVC decreased LA 25% and 22%, respectively, relative to CON. Likewise, number ofabscesses present on each liver did not differ (P = 0.31) among treatments, even thoughthere was a 50% decrease in the number of abscesses per liver for CVC vs. CON. Of the 25abscesses collected and sampled at the time of necropsy, CON 77.8% (n = 7), CV 60.0% (n =9), and CVC 57.1% (n = 8; P^= 0.59), 24 of abscesses yielded F.^necrophorum^subsp.necrophorum.

[0022] 21     No differences among treatments were detected in colon scores (P = 0.50; Table 3).A previous LA challenge study with Holstein steers also reported no difference in colonscores, although abscess prevalence was 15% of the steers in the study (McDaniel et al.,2023). Fusobacterium^^necrophorum subsp. necrophorum was detected within the colontissue among all treatments, but it did not differ (P = 0.49) among treatments. F.^^necrophorum subsp. necrophorum also was isolated from colon contents in CV, but notdetected in other treatments. The colonic epithelium is a crucial physical andimmunological barrier in the lower gastrointestinal tract. Increased hindgut fermentationcan lead to a decrease in the pH of the large intestine, an increase in endotoxinconcentration within the large intestine digesta, and subsequent damage to the hindgutepithelium (Liu et al., 2014; Ye et al., 2016). Previous studies reported that extensivehindgut fermentation can trigger inflammation, potentially caused by amines, endotoxins,or bacteria that breach an impaired intestinal barrier (Gozho et al., 2006; Crawford et al.,2007).Detection^and^Quantification^of^F.^necrophorum^in^Ruminal^and^Colonic^Contents^and^Epithelial^Tissues^In the present study, quantitative PCR analysis allowed for the determination ofabundance and differentiation of F.^necrophorum^subsp.^necrophorum and F.^necrophorum^subsp.^funduliforme in ruminal tissue, ruminal contents, colon tissue, and colon contents.The abundance of F.^necrophorum subsp. necrophorum and funduliforme determined byqPCR are presented in Table 4. For F.^necrophorum subsp. necrophorum, no differenceswere detected in abundance within ruminal tissue or ruminal contents (P = 0.68).

[0023] 22    Similarly, neither colon tissue nor its contents differed in the abundance of F.^necrophorumsubsp. necrophorum (P > 0.47), and no F.^necrophorum subsp. necrophorum was detected inileal tissue or contents. F.^necrophorum subsp. funduliforme concentration did not differamong treatments in ruminal tissue or contents (P > 0.37) or in colon tissue (P > 0.47), andF.^necrophorum subsp. funduliforme was not detected in colon contents, ileal tissue, or ilealcontent.

[0024] 23   Table^4. Abundance of^Fusobacterium^necrophorum subsp. necrophorum^(4a)^orFusobacterium^necrophorum subsp. funduliforme (4b) identified using quantitativepolymerase chain reaction in ruminal, colonic, and ileal tissue and contents of beef ×dairy steers fed ground corn with or without (CON) Bacillus^subtilis and encapsulatedbutyric acid and zinc (CV), and CV plus red clover^(Trifolium^pratense; CVC) to evaluatethe formation of liver abscessesSubsp. necrophorumTreatment1Tissue CON CV CVC SEM2 P-valueRumen tissue3 2.67 3.35 3.59 0.635 0.67Rumencontents4 1.43 1.29 1.88 0.483 0.68Colon tissue3 1.00 0.32 0.97 0.436 0.49Colon contents4 ND 0.24 ND 0.147 0.47Ileum Tissue3 ND ND ND - -Ileum contents4 ND ND ND - -^Subsp. funduliformeTreatment1Tissue CON CV CVC SEM2 P-valueRumen tissue3 1.48 1.92 0.76 0.572 0.37Rumencontents4 2.04 1.65 1.25 0.475 0.60Colon tissue3 0.51 ND 0.31 0.260 0.47Colon contents4 ND ND ND - -Ileum Tissue3 ND ND ND - -Ileum contents4 ND ND ND - -ND = non-detectable.1CON = control diet and acidotic diet with intraruminal bacterial inoculation; CV =control diet and acidotic diet supplemented with ground corn, CLOSTAT® Dry, andVilliTech® with intraruminal bacterial inoculation; CVC = CV + red clover withintraruminal bacterial inoculation.2Standard error of least squares means, n = 9 steers in CON; 15 steers in CV and 14steers in CVC.3Log10 CFU / g of as-is tissue.4Log10 CFU / mL of contents.   Notably, both subspecies were detected among all treatments within the ruminaltissue and ruminal contents and also detected within the colon tissue and colon contents.The steers in our study were subjected to inoculation with F.^necrophorum subsp.necrophorum, which combined with lactic acid production from a high-starch diet couldincrease the abundance of^F.^necrophorum^in the ruminal contents. Fusobacterium^necrophorum, a Gram-negative, anaerobic bacterium commensal in the rumen of cattle,serves as the predominant causative agent of liver abscesses in cattle (Scanlan andHatchcock, 1983; Tan et al., 1996).Nagaraja and Chengappa (1998) noted that once ruminal lesions occur, F.^necrophorum can either create a ruminal lesion or enter the portal vein and be transportedto hepatic tissue, a manifestation underscored by a numerical increase in the prevalence ofsubsp. necrophorum in the ruminal epithelial tissue of CV and CVC vs. CON (Table 5). Theemergence of S.^Lubbock within liver abscesses, as evidenced by its isolation, and thepostulation by Amachawadi and Nagaraja (2016) regarding the conceivable role of a leakygut phenomenon, accentuates a secondary plausible route for the invasion of pathogenicbacteria into the hepatic portal system.Table^5. Bacterial prevalence within liver abscesses collected at the time of harvest of beef ×dairy steers fed ground corn with or without (CON) Bacillus^subtilis and encapsulated butyricacid and zinc (CV), and CV + red clover (Trifolium^pratense; CVC) to evaluate the formation ofliver abscesses^Liver abscess containing bacteria, %Bacteria Treatment1^ CON CV CVC SEM2 P-valueF.^necrophorum^subsp.^77.8 60 57.1 0.75 0.59 Necrophorum^

[0025] 25    F.^necrophorum^subsp.^- - - - - Funduliforme^ T.^pyogenes^ - - - - - Salmonella^ 44.4 40 35.7 0.79 0.81 T.^pyogenes^and^Salmonella^- - - - -F.^necrophorum^subsp.^Necrophorum^and^ 44.4 33.3 28.6 0.81 0.74 Salmonella^F.^necrophorum^subsp.^Necrophorum,^T.^ - - - - - pyogenes^and^ Salmonella^1CON = control diet and acidotic diet with intraruminal bacterial inoculation; CV = controldiet and acidotic diet supplemented with ground corn, ^CLOSTAT® Dry, and VilliTech® withintraruminal bacterial inoculation; CVC = CV + red clover with intraruminal bacterialinoculation.2Standard error of least squares means, n = 9 steers in CON; 15 steers in CV and 14 steers inCVC.Fusobacterium^necrophorum^actively degrades proteins and amino acids, withparticular emphasis on lysine obtained from feed and the ruminal epithelium (Russell,2006; Elwakeel et al., 2013). In forage-fed steers, the population of F.^necrophorum remainsrelatively low, generally less than 1 × 105 per gram of ruminal contents (Tan et al., 1994c).Nonetheless, as intake of starch increases, as in the current study, the population of F.^necrophorum increases, exceeding 1 × 106 per gram of ruminal contents. This increase isassociated with availability of lactate because of the introduction of the high-concentrate,acidotic diet (Coe et al., 1999; Tan et al., 1994a). The increase in starch intake and

[0026] 26     subsequent lactic acid production likely served as a substrate for the proliferation of F.^necrophorum and provided a favorable environment within the rumen of the beef × dairy^steers in the current study.Bacterial^Isolation^from^Liver^Abscesses^There was no difference detected in the percentage of liver abscesses containing F.^necrophorum^subsp. necrophorum^among treatments^(P^= 0.59: Table 5). Moreover, F.^necrophorum subsp.^funduliforme^and Trueperella^pyogenes were not isolated incombination from any liver abscesses. No differences were detected for the percentage ofliver abscesses containing S.^enterica (P^= 0.81). One scar that was collected at the time ofnecropsy (1 steer in CVC) did not yield Fusobacterium^subsp. necrophorum nor subsp.funduliforme but was positive for S.^enterica. Moreover, no differences were observed inthe proportion of liver abscesses that contained a combination of F.^necrophorum subsp.necrophorum and S.^enterica (P = 0.74). No liver abscesses collected during the currentstudy yielded T.^pyogenes^or F.^necrophorum subsp. funduliforme.The ability of F.^necrophorum to migrate to and establish itself in the liver isfacilitated by competitive advantages. Ruminal tissue damage can occur when theseendotoxins are present or when F.^necrophorum adheres to the rumen wall via the outermembrane protein (Kumar et al., 2013), and F.^necrophorum is aerotolerant and exhibitsremarkable oxygen tolerance (Hofstad, 1984), allowing colonization of the ruminalepithelium. The bacterium also can proliferate efficiently at the slightly basic pHcommonly observed at the ruminal epithelial surface (Tan et al., 1994a) in contrast tooverall reticulorumen pH in homeostatic conditions primarily ranging from 5.8 to 6.5Nagaraja and Titgemeyer, 2007). In the current study,^F.^necrophorum subsp. necrophorum

[0027] 27    was present in most liver abscesses. The absence of F.^necrophorum^subsp. funduliformeand T.^pyogenes emphasize the opportunistic ability of F.^necrophorum subsp. necrophorumto outcompete other bacteria.Salmonella^Prevalence^Initially, on d -18 only a small proportion of steers were shedding Salmonella intheir feces, which did not differ among treatments (P = 0.98; Table 6). Subsequently, nodifferences (P > 0.12) in fecal Salmonella shedding on days 2 or 38 were detected. Anotable increase in Salmonella prevalence in feces was evident on d 24 (P^= 0.12). Theincreased fecal Salmonella prevalence in all treatments occurred after ruminal inoculationon day 20, which included S.^enterica^subsp. Lubbock. Furthermore, there were nodifferences in Salmonella prevalence within subiliac or mesenteric lymph nodes amongtreatments after harvest (P > 0.87), and Salmonella^prevalence in the ileum did not differ (P= 0.44) among treatments.Table^6. Salmonella prevalence in fecal samples, subiliac lymph nodes, mesenteric lymphnodes and ileum tissue from beef × dairy steers fed ground corn with or without (CON)Bacillus^subtilis and encapsulated butyric acid and zinc (CV), and CV + red clover(Trifolium^pratense; CVC) to evaluate the formation of liver abscesses.Treatment1Item CON CV CVC SEM2 P-valueFecal d -18,% 0.0 0.0 6.7 6.44 0.98Fecal d 24, 77.8 93.3 85.7 13.87 0.12 %Subiliaclymph 0.0 6.7 0.0 19.96 0.97nodes, %

[0028] 28     Mesentericlymph 11.1 13.3 7.1 10.48 0.87nodes, %Ileum, % 0.0 26.7 7.1 11.42 0.441CON = control diet and acidotic diet with intraruminal bacterial inoculation; CV = controldiet and acidotic diet supplemented with ground corn, ^CLOSTAT® dry, and VilliTech®with intraruminal bacterial inoculation; CVC = CV + red clover with intraruminal bacterialinoculation.2Standard error of least squares means, n = 9 steers in CON; 15 steers in CV and 14 steers inCVC.The exact contribution of Salmonella in LA formation remains unclear, and it hasonly recently been isolated from liver abscesses (Amachawadi and Nagaraja, 2015).Nevertheless, Salmonella can colonize the intestines, particularly the small intestine, andstress can cause disruptions of tight junction barrier function (Boyle et al., 2006), whichcould allow Salmonella to invade the lymphatic system and liver (Rings, 1985). Currentresults indicated that Salmonella was isolated from 40% of liver abscesses, whereasprevious research indicated that Salmonella was only isolated from 25% of naturallyoccurring abscesses (Amachawadi and Nagaraja, 2016). Similar to our results, McDaniel etal. (2023) reported 57% Salmonella prevalence in Holstein steers used in an experimentalmodel to induce liver abscesses. Herrick et al. (2022) reported a breed effect, withHolsteins exhibiting an increased prevalence of Salmonella (40%) compared with beefbreeds (29.5%). Given that McDaniel et al. (2023) reported 57% Salmonella^prevalence inliver abscesses and 40% Salmonella was isolated from liver abscesses in the current studyusing beef × dairy steers, these results support the findings of Herrick et al. (2022) that

[0029] 29    there could be a breed effect associated with the prevalence of Salmonella^within liverabscesses.Although no statistical differences in LA were detected following supplementation,the decrease in liver abscess prevalence observed with CV could be biologically andeconomically impactful to the beef industry. Further research is necessary to not onlydetermine repeatability, but to elucidate the mechanism(s) by which the combination ofthese supplements impact liver abscess formation. Furthermore, these data illustrate thatthe experimental model of acidosis and bacterial inoculation we used can be a viable modelto evaluate novel strategies to mitigate LA thereby allowing producers to make informeddecisions.EXAMPLE^2^Materials^and^Methods: Four feedyards in the high plains region, of the United States ofAmerica, were used for this study. Yards were randomly assigned to treatments; Control,normal diet for natural cattle in the high plains region of the United States (CON, n = 3,572head); Treatment, normal diet for natural cattle in the high plains region of the U.S. andsupplemented 0.5 g / head daily^B.^subtilis^PB6 (CLOSTAT® 500, 6.60×109 CFU / g, KeminIndustries, Inc., Des Moines, IA) + 3 g / head daily of butyric acid (0.75g daily) and zinc (0.3gdaily; VilliTech, Kemin Industries, Inc; CV; n = 4,406 head). Anywhere between 200 – 500head were harvested per treatment from 157 – 233 days on feed.^ ^ ^

[0030] 30     Results^and^Discussion:Liver^abscess^prevalence^The CV treatment increased the prevalence of normal livers from 157 - 233 DOF(“DOF” as used herein refers to Days of Feed, 12 harvest days) by 6.36% and decreased theamount of abnormal livers by 9.18% compared to control (Table 7). When singling outharvest days CV increased the percentage of normal livers by 34.05% on Day 170, 28.35%on Day 205 and 15.38% on Day 226, respectively. It was observed that the combination ofBacillus^subtilis PB6, butyric acid, and zinc are proving to be a useful combination indecreasing the occurrence of abnormal livers in natural fed cattle.Table^7.^Liver abscess prevalence from Bos Taurus influences natural cattle supplementedwith or without (CON) Bacillus^subtilis PB6 and encapsulated butyric acid and zinc (CV).Treatment1Item Days On Feed CON CV %, DifferenceHead 157-233 3572 4406Normal Liver, % 57.28 61.03 6.34Abnormal Liver, % 42.72 38.97 -9.18Head 157 304 304Normal Liver, % 56.91 58.22 2.28Head 164 304 379Normal Liver, % 63.16 67.55 6.72Head 170 342 266Normal Liver, % 48.25 68.05 34.05Head 178 304 380Normal Liver, % 58.88 59.21 0.56Head 184 266 380Normal Liver, % 60.53 66.32 9.13Head 197 304 379Normal Liver, % 63.49 65.96 3.82Head 199 304 380Normal Liver, % 46.38 41.84 -10.29Head 205 304 380Normal Liver, % 55.59 73.95 28.35Head 212 304 380Normal Liver, % 60.86 56.32 -7.75Head 219 304 456Normal Liver, % 64.47 54.39 -16.96

[0031] 31     Head 226 228 456Normal Liver, % 53.95 62.94 15.38Head 233 304 266Normal Liver, % 55.59 59.77 7.251CON = control diet; CV = control diet, CLOSTAT® 500, and VilliTECH®Having described the invention with reference to particular compositions, theories ofeffectiveness, and the like, it will be apparent to those of skill in the art that it is not intendedthat the invention be limited by such illustrative embodiments or mechanisms, and thatmodifications can be made without departing from the scope or spirit of the invention, asdefined by the appended claims. It is intended that all such obvious modifications andvariations be included within the scope of the present invention as defined in the appendedclaims. The claims are meant to cover the claimed components and steps in any sequencewhich is effective to meet the objectives there intended, unless the context specificallyindicates to the contrary.It should be further appreciated that minor dosage and formulation modifications ofthe composition and the ranges expressed herein may be made and still come within thescope and spirit of the present invention.The foregoing description has been presented for the purposes of illustration anddescription. It is not intended to be an exhaustive list or limit the invention to the preciseforms disclosed. It is contemplated that other alternative processes and methods obvious tothose skilled in the art are considered included in the invention. The description is merelyexamples of embodiments. It is understood that any other modifications, substitutions,and / or additions may be made, which are within the intended spirit and scope of thedisclosure. From the foregoing, it can be seen that the exemplary aspects of the disclosureaccomplish at least all of the intended objectives.^

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[0038] 38

Claims

CLAIMS^1. A method of decreasing incidence of liver abscess in cattle on feedlots comprisingthe steps of orally administering to the cattle a supplement that contains an effectiveamount of Bacillus^subtilis PB6, butyric acid and zinc for at least 25 days prior toharvest.

2. The method of claim 1, wherein the supplement further comprises red clover or anextract of red clover.

3. The method of claim 1, wherein the supplement is administered daily for at least 35days prior to harvest.

4. The method of claim 1, wherein the supplement is administered daily for at least 50days prior to harvest.

5. The method of claim 1, wherein the supplement is administered daily for at least 75days prior to harvest.

6. The method of claim 1, wherein the supplement is mixed into animal diet.

7. The method of claim 6, wherein the supplement is top dressed and mixed with thediet at the time of feeding.

8. A method of decreasing severity of liver abscess in cattle on feedlots comprising thesteps of administering a feed additive to the cattle comprising an effective amount ofBacillus^subtilis PB6, butyric acid and zinc for at least 25 days prior to harvest.

9. The method of claim 8, wherein the additive further comprises red clover or anextract of red clover.

10. The method of claim 8, wherein the additive is administered daily for at least 35days prior to harvest.

11. The method of claim 8, wherein the additive is administered daily for at least 50days prior to harvest.

12. The method of claim 8, wherein the additive is administered daily for at least 75days prior to harvest.

13. The method of claim 8, wherein the additive is mixed into animal diet.

14. The method of claim 13, wherein the additive is top dressed and mixed with the dietat the time of feeding.

15. A method of improving scoring of a beef carcass at harvest comprising the steps oforally administering an effective amount of an animal feed supplement containingBacillus^subtilis PB6, butyric acid and zinc to the cattle for at least 25 days prior toharvest.

16. The method of claim 15, wherein the supplement further comprises red clover or anextract of red clover.

17. The method of claim 15, wherein the supplement is administered daily for at least50 days prior to harvest.

18. The method of claim 15, wherein the supplement is administered daily for at least75 days prior to harvest.

19. The method of claim 15, wherein the supplement is mixed into animal diet.

20. The method of claim 19, wherein the supplement is top dressed and mixed with thediet at the time of feeding.^ ^40

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