Cattle production is the most economically important US agricultural activity, but the industry is under pressure to improve sustainability as beef production uses more land and energy than other major protein sources. Improving feed efficiency is arguably the best approach for enhancing sustainability, as much of the cost and environmental footprint of cattle production is due to feed production. Though numerous factors influence cattle feed efficiency, many recent studies have demonstrated that rumen microbiome composition plays a key role. Phages are promising tools for altering microbiomes, and we recently developed phage libraries active against Streptococcus and Fusobacterium - rumen genera that have substantial influence on feed efficiency. Accordingly, the primary goal of this project is to develop a phage-based feed additive that increases cattle feed efficiency through suppression of select species within these two genera, and that is compatible with current feed manufacturing and cattle production processes. To achieve this goal, the following technical objectives are proposed:1. Isolate additional S. bovis phages and characterize life cycles.Hypothesis: Sequential feeding of two or more broad host coverage, lytic phage formulations will be needed to maintain long-term suppression of target rumen bacteria.Significance: Bacterial resistance to individual phages as well as phage cocktails can occur relatively rapidly, and it may be difficult (or impossible) to eradicate many species from the rumen using conventional treatment strategies. Instead, adaptive approaches that rely on rotating several formulations can be used to maintain chronic target inhibition without the need for eradication. However, this strategy requires a continual supply of phages as well as isolate monitoring programs to address the emergence of resistant strains and assess ongoing efficacy.Success metric: Isolate and characterize at least 6 genetically distinct lytic phages active against S. bovis.2. Expand phage host ranges and efficacy through in vitro evolution.Hypothesis: In vitro methods that promote phage genome recombination during co-infection of a host will produce some novel phages with expanded host ranges and/or killing efficiencies.Significance: Phage-amended feed additives must be able to control the growth of most strains encountered in a production setting without prior knowledge of strain prevalence or distribution. To achieve such broad host coverage, products can either use large numbers of narrow host range phages, or smaller numbers of broad host range phages. However, the use of broad host range phages is highly preferred for efficacy as well as reducing production costs.Success metric: Development of two phage cocktails (containing ≤ 6 phages) that significantly inhibit the growth of ≥ 75% of rumen isolates for the target species.3. Assess and improve phage thermal stability.Hypothesis: Combining mutagenesis with heat selection will enable the development of phages capable of maintaining viability during manufacturing, storage, and implementation.Significance: During manufacturing, phages can be exposed to temperatures up to 70 - 80 °C for short periods (seconds to minutes). Additionally, ration temperatures can reach similar temperatures during feed mill processing. Therefore, phages that remain viable at high temperatures are desirable and their thermal stability should be known to inform manufacturing and application strategies.Success metric: Adapt three or more native rumen phages to survive temperatures of 70 °C for two minutes or 60 °C for thirty minutes with no more than a one-log reduction in titer.4. Scale up and optimize production of Streptococcus and Fusobacterium phages.Hypothesis: We will be able to produce at least 1014 phages per 10 L fermentation.Significance: Reported phage production yields span six orders of magnitude. Production yields at the bottom of this range are unlikely to be financially viable for bovine rumen microbiome engineering. Conversely, high production yields would facilitate pricing flexibility and enable more complex formulations with improved functions.Success metric: Optimize yields to obtain at least 1010 phages/mL and scale up production to 10 L reactors while maintaining this yield.5. Assess efficacy and safety of Streptococcus and Fusobacterium phage cocktails.Hypothesis: Cattle treated daily with two sequentially rotated phage cocktails (~1010 PFU or less) will have sustained and significant reductions in target bacteria populations.Significance: Reducing ruminal concentrations of either Fusobacterium or Streptococcus species would validate whether a causal relationship exists between these bacteria and cattle feed efficiency. Additionally, doses of 1010 phages per animal would demonstrate financial viability, and achieving selective microbial control within cattle rumen would broadly validate phages as effective microbiome engineering agents.Success metric: Use phages to achieve statistically significant reductions in target bacteria concentrations in cattle rumen without negative health or performance consequences.Successful completion of these objectives would provide supporting data for a feedlot trial, which will be used to assess our product in a production setting with sufficient statistical power to demonstrate impact on feed efficiency.