IMPROVED CATTLE FEED EFFICIENCY VIA BACTERIOPHAGE-MEDIATED MICROBIOME MANIPULATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: SMALL BUSINESS GRANT
• Reporting Frequency: Annual
• Accession No.: 1028840
• Grant No.: 2022-33610-37855
• Cumulative Award Amt.: $600,000.00
• Proposal No.: 2022-04378
• Project Start Date: Sep 1, 2022
• Project End Date: Aug 31, 2025
• Grant Year: 2022
• Program Code: [8.3]- Animal Production & Protection

Recipient Organization
SENTINEL ENVIRONMENTAL GROUP, LLC
5250 JACKWOOD STREET
HOUSTON,TX 770961313

Performing Department
(N/A)

Non Technical Summary
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 goal of this project is to develop a phage-based feed additive that increases cattle feed efficiency and is compatible with current feed manufacturing and cattle production processes.To achieve broad coverage of bacterial strains found in contemporary production settings, Streptococcus phages will be subjected to in vitro host range expansion protocols. Fusobacterium phages will be subjected to in vitro protocols designed to enhance their killing efficiencies against resistant bacteria. Phage thermal stability and their potential for thermal adaptation will then be assessed. Subsequently, promising candidates will undergo yield optimization and production scale up. Finally, efficacy and optimal treatment strategies will be determined using in vitro studies and animal trials. Successful completion of this project would be economically beneficial for producers, and would help mitigate the environmental impacts of animal agriculture while establishing a generalizable, cost-effective framework for livestock microbiome engineering.

Animal Health Component

70%

Research Effort Categories

Basic

20%

Applied

70%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
302	3310	1100	100%

Knowledge Area
302 - Nutrient Utilization in Animals;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1100 - Bacteriology;

Keywords

feed efficiency

feedlot

cattle

bacteriophage

microbiome engineering

Goals / Objectives
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.

Project Methods
Objective 1: Isolate S. bovis phages and characterize their life cycles.Task 1A: Isolate S. bovis phages: Rumen fluid and cattle fecal samples will be used to inoculate enrichment cultures amended with 1% starch or 50 mM maltose, which are known to select for the growth of S. bovis. Phage pools will be prepared from each enrichment culture using a fraction-based approach to help maximize phage recovery. Briefly, this involves centrifugation to clarify the supernatant, followed by filtration through a 0.45 μm PVDF filter. One third of the filtrate is then passed through a 0.22 μm filter (fraction contains enveloped and nonenveloped phages < 220 nm), and another third is chloroform treated (1:10) to lyse residual bacteria (contains nonenveloped phages < 450 nm).Task 1B: Characterize phage life cycles: Phage life cycle will be assessed in several ways, including examination of colony morphology, response to high phage concentrations, genome sequencing, and the capacity to form lysogens. In general, lytic phages form clear plaques while temperate phages tend to form turbid plaques. Thus, phages forming clear plaques are preferentially collected during isolation. We have also found it useful to assess differences in phage production using low and high multiplicities of infection (MOI) as the probability of entering lysogeny often increases at higher MOIs.Objective 2: Expand phage host ranges and efficacy through in vitro evolution.Task 2A: Host range expansion. The Appelmans protocol is a commonly used method for generating phage variants with novel lytic host ranges, and has been repeatedly found to improve phage therapy outcomes. While our F. varium phages have relatively broad strain coverage, our S. bovis phages appear to have a narrow host range, which is consistent with prior reports. Thus, we will apply Appelmans protocol to our S. bovis phages to expand their host ranges. Briefly, this consists of exposing individual bacterial strains to serial dilutions of phage cocktails and subsequently collecting the lysates from all completely lysed wells plus the first dilution displaying regrowth after an overnight incubation. The lysates and then combined, centrifuged, and filter sterilized. This process is then repeated until the combined phage lysate is able to form plaques on each bacterial strain.Task 2B: Improving phage killing efficiency. Phage killing efficiency can also be enhanced using Appelmans protocol, or other similar methods that rely on phage recombination to generate large numbers of novel variants that are then selected for on the basis of lytic ability. In addition to these methods, we will use coevolutionary phage training on our lytic F. varium phages to generate phage populations more efficient at infecting contemporary bacterial strains and avoiding the development of resistance.Objective 3: Assess and improve phage thermal stability.Task 3A: Assess phage thermal stability. Compound feed manufacturing requires short term exposure to temperatures between 70 - 80°C, whereas cattle rations prepared in feed mills may be 65 - 70°C when finished. In general, most phages are sensitive to temperatures above 60°C. We will determine the thermal stability of select phages by monitoring changes in viable phage counts after incubation at relevant temperatures over time (e.g., 65°C for 1 hour with 5 min sampling intervals).Task 3B: In vitro thermal adaptation. Serially passing phages while applying thermal selection reproducibly increases thermal stability. To accelerate mutagenesis, phage suspensions will be treated with a chemical mutagen prior to amplification in a susceptible host. Phage lysates will then be filter sterilized and subject to heat selection by incubation at 50°C for 1 hour in a thermocycler. Surviving phages will then be amplified and titered to complete the cycle.Task 3C: Evaluate impact of microencapsulation on thermal stability. While phages typically display some propensity for thermal adaptation, the practical limits of such methods may not be sufficient to maintain viability even for short periods at temperatures above 70°C. However, encapsulated phages were recently demonstrated to withstand 95°C for up to 2 mins. As such, we will explore the potential for microencapsulation to improve thermal stability. Several methods will be assessed, including pullulan-trehalose drop cast films, chitosan encapsulation, and alginate encapsulation.Objective 4: Optimize and scale up phage production.Task 4A: Optimize phage yields. Maximizing production yields is critical to ensuring the financial viability of phage-based biocontrol. Factors that will be investigated during optimization experiments include the manufacturing host, host density at inoculation, phage multiplicity of infection (MOI), temperature, pH, agitation, harvest time, and media composition.Task 4B: Scale up phage production. Batch systems are widely used for phage production due to their simplicity and relatively high yield, and we will use these for initial production efforts while gradually shifting to a two-stage cycling system. Our approach is to develop a semi-continuous process that capitalizes on the advantages of both batch (high phage titers, robustness) and continuous (high volumetric throughput, reduced downtime per production run) production systems. Briefly, production hosts are grown in the first stage to the appropriate optical density (OD) under optimal culture conditions. The majority of this culture is then transferred to the second stage and equilibrated to the infection temperature (e.g., 30°C), which slows bacterial growth, and phages are added at a predetermined MOI. Simultaneously, fresh media is added to the culture remaining in the first stage to initiate production of new hosts for a second cycle. The infective process is monitored by OD600 and the culture is harvested once population-wide lysis has taken place. Immediately after harvest, the new first stage culture is transferred to the second stage and the process is repeated.Objective 5: Assess efficacy and safety of phage cocktails.Task 5A: In vitro batch studies. Phage cocktail efficacy against F. varium and S. bovis as well as potential off-target effects will be assessed using anaerobic batch cultures inoculated with rumen fluid and amended with phage cocktails. Media will be prepared using fresh or cryopreserved rumen fluid and buffer at a 1:4 ratio. Batch cultures will be sparged with anaerobic gas (5% H2: 10% CO2: 85% N2) and maintained at 39 °C in the dark with shaking. Cocktails will be diluted such that individual phages will be added to batch cultures at concentrations of 0, 105 or 108 phages/mL. Cultures will be sampled every 2 hours for 8 hours after each phage application and again after 24 hours. Samples from each batch will be analyzed by both real-time qPCR (for total 16S rRNA, S. bovis and F. varium abundance) and 16S rRNA gene sequencing (for community analysis).Task 5B: In vivo studies. We will utilize 10 ruminally-cannulated steers to facilitate temporal sampling and monitoring of rumen microbiome composition after phage treatment. This will allow us to test the efficacy of different treatment strategies at preventing the development of phage resistance over longer time periods. Tests will be conducted using various doses and time intervals between each dose.

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:The primary target audiences reached for the current project period were cattle feedlot management, industry professionals (consultants and producers), and university and government researchers (including students). Interactions with these audiences were through research collaborations, consulting, and conference presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided numerous training and professional development opportunities, for both the students and staff involved. It has supported three full-time employees and enabled their training in numerous techniques ranging from phage isolation and characterization to metagenomic sequencing and analysis. Additionally, it has supported a part-time employee/student and served as the foundation for their thesis work. Five other students were also involved in the animal trial, which required conducting some novel procedures and presented unique educational opportunities. All key personnel were also able to attend the ASM Microbe 2023 conference and the 2023 Evergreen Phage conference, which provided substantial professional development opportunties. Additionally, key personnel and collaborators participated in preparing two new peer-reviewed publications this project period. How have the results been disseminated to communities of interest?Project results have been disseminated in the following manner: Two publications in leading industry journals (PHAGE and ACS Agricultural Science & Technology) Oral presentation at Microbiota Days 2023 Depositing sequencing data in a public database (GenBank) Discussions with academic and industrial experts What do you plan to do during the next reporting period to accomplish the goals?While we have met the success metric for Objectives 2, 3, 4, and 5, several challenges remain to be overcome this next project period. For Objective 1, our success metric was to isolate and characterize at least 6 genetically distinct lytics phages against S. bovis. Currently, most of our S. bovis phages fall into 3 genetically distinct groups, while we have three individual phages that each appear unique. However, two of these phages have proven difficult to sequence despite multiple attempts. Thus, for the next project period, we will focus on characterizing these three phages, and potentially isolating additional phages to expand our current library. We will also continue to improve phage production methods, particularly for several phages that do not reproduce well in liquid broth. These phages do produce high titers when grown in double layer agar, but this technique is not very scalable. We intend to test several different methods to enhance production, including the use of semi-solid or viscous media and microcarrier beads that can serve as substrate for bacterial attachment.

Impacts
What was accomplished under these goals? This project period marked significant progress toward characterizing and improving bacteriophages targeting Streptococcus bovis/Streptococcus equinus complex (SBSEC) bacteria. Below is a detailed summary of achievements across the project objectives for the current project period: Objective 1: Isolate additional S. bovis complex phages and characterize life cycles. Substantial work was completed to expand and characterize our SBSEC phage library. We isolated over 25 phages, though some were unstable or duplicates of previously identified strains, resulting in a focused characterization of 17 unique phages. These phages were categorized into types A, B, C, and others based on phenotypic traits, including host range, optimal amplification conditions, and bacterial growth inhibition in vitro. Host range analysis showed that despite high phage specificity, our SBSEC phages could lyse bacterial isolates from geographically diverse sources. These results suggest broader potential applications, with host range further expandable using established methods like Appleman's protocol. Genomic sequencing of 13 phages revealed a high degree of genetic similarity within certain groups (>99% identity for types B and C), with genome sizes ranging from 33.8 to 40.5 Kbp and GC content from 37.1% to 39.4%. These values closely align with the GC content of SBSEC genomes (~37%). Ongoing work is focused on elucidating the links between small genomic differences and observed phenotypic traits, providing insights into phage-host interactions and informing future applications. Objective 2: Expand phage host ranges and efficacy through in vitro evolution. We built on previous successes in expanding host range through Appleman's protocol by conducting in vitro evolution experiments to increase phage virulence. Six SBSEC phages underwent 30 rounds of serial passaging with bacterial cultures, with growth clearance and plaque-forming ability assessed every 10 rounds. Among these, one phage (PY1) showed dramatic improvement in bacterial suppression. While two phages (Mushu, PY7) showed minimal or no improvement, and two (PY20, PY3) lost viability, these results provide valuable data on the variability of phage adaptability. The study highlights the potential for enhancing phage efficacy through targeted training, with implications for developing more robust phage formulations. Objective 3: Assess and improve phage thermal stability. Thermal adaptation experiments were conducted to address the limited heat tolerance of two lytic phages, CSJC and Mushu, which previously lost viability after 10 minutes at 65°C. By applying heat-exposure protocols combined with sodium pyrophosphate (SPP) and ethyl-methanesulfonate (EMS), we successfully increased their thermal resistance. The improved phages demonstrated significantly greater stability under heat stress compared to their parental counterparts, representing a key step toward developing phages suitable for varied environmental and processing conditions. Objective 4: Scale up and optimize production of Streptococcus and Fusobacterium phages. We optimized protocols to achieve higher phage titers for SBSEC bacteriophages, focusing on adjusting multiplicity of infection (MOI) and initial cell concentrations in liquid cultures. These efforts resulted in 2-3 fold higher titers for most phages, enabling more efficient production. Three phages, however, remained challenging to amplify outside of plaque assays. For Fusobacterium varium phages, previously reported high titers were maintained, though further increases were not observed. These results demonstrate progress in scaling up phage production and improving yields for future applications.

Publications

• Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2024 Citation: Schwarz, Cory, Jacques Mathieu, Jenny Laverde Gomez, Marina Tikhonova, T. G. Nagaraja, and Pedro JJ Alvarez. "Detection of Tylosin Resistance in Fusobacterium necrophorum subspecies necrophorum." ACS Agricultural Science & Technology (2024).
• Type: Peer Reviewed Journal Articles Status: Accepted Year Published: 2024 Citation: Schwarz, Cory, Jacques Mathieu, Jenny Laverde Gomez, Megan R. Miller, Marina Tikhonova, Clark Hamor, and Pedro JJ Alvarez. "Isolation and Characterization of Six Novel Fusobacterium necrophorum Phages." PHAGE (2024).

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:The primary target audiences reached for the current project periodwerecattle feedlot management, industry professionals (consultants andproducers), and university and government researchers (including students). Interactions with these audiences were through research collaborations, consulting, and conference presentations. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided numerous training and professional development opportunities, for both the students and staff involved. It has supported three full-time employees and enabled their training in numerous techniques ranging from phage isolation and characterization to metagenomic sequencing and analysis. Additionally, it has supported a part-time employee/student and served as the foundation for their thesis work. Five other students were also involved in the animal trial, which required conducting some novel procedures and presented unique educational opportunities. All key personnel were also able to attend the ASM Microbe 2023 conference and the 2023 Evergreen Phage conference, which provided substantial professional development opportunties. Additionally, key personnel and collaborators participated in preparing a publication onsome of our initial findings. How have the results been disseminated to communities of interest?Project results have been disseminated in the following manner: Publication in a leading industry journal (Journal of Animal Science) Invited symposium at ASAS 2023 Invited oral presentation at Microbiota Days 2023 Poster presentation at ASM Microbe 2023 Poster presentation at Evergreen 2023 Depositing sequencing data in a public database (GenBank) Discussions with academic and industrial experts What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, our primary focus will be on optimizing and modifying various adaptive laboratory evolution (ALE) methods to help advance our efforts at improving phage killing efficiencies, expanding phage host ranges, and increasing thermal stability. There is relatively little research regarding the use of ALE for phages and it is clear that much more work is remaining in this area. A particular area we intend to focus on is a quantitative analysis of the rationale behind certain methods, such as the Appleman's protocol. Though there are many potential parameters to optimize for this and similar assays, little work has been done in that regard. Improving our understanding of individual phage mutation rates andgrowth kinetics under various conditions will help ensure we are able to capture desirable mutations as they might arise. Additionally, we will assess how different combinations of phages and bacterial strains impact the results of select ALE methods. For example, in Appleman's protocol, is it better to use phages with high similarity to promote more frequent recombination, or more genetically diverse phages that have the potential for producing a broader range of potential outcomes or novel functionalities?How does the number of phages used in each cocktail impact the results? Is it beneficial to use mixed species cultures containing both susceptible and non-susceptible hosts? Our experience to date suggests that improving ALE method efficiency will be critical for commercializing off-the-shelf phage products and we believe focusing our efforts on this is key to advancing this project as well as the broader field of phage-based microbiome engineering.

Impacts
What was accomplished under these goals? Substantial progress towards accomplishing the overall project goals was made this project period, with several objectives having been completed. Major accomplishments included the isolation and characterization of nine new phages that are able to infect S. bovis complex species (objective 1: 80% complete), the successful scaling up of four F. varium phages and six S. bovis phages to greater than 1010phages/mL (objective 4: 50% complete), and completion of a small-scale animal trial to evaluate phage efficacy and safety in vivo(objective 5: 100% complete). Other accomplishments include successfully expanding the host range of our S. bovis phage collection (objective 2: 25% complete), and assessing the thermal stability of numerous F. varium and S. bovisphages (objective 3: 33% complete). Specific details are discussed below. Objective 1: Isolate additionalS. bovisphages and characterize life cycles. Our goal for this objective was to isolate 6 new S. bovis complex phages, and we were able to isolate 9 from independent samples, bringing our total to 15 S. bovis complex phages. We have completed genome sequencing for 6 of these and are currently in the process of sequencing the remaining genomes. Of the 6 genome-sequenced phages, one is temperate while the remaining 5 are lytic. Objective 2:Expand phage host ranges and efficacy through in vitro evolution. In general, our F. varium phages have relatively broad host ranges (infect multiple strains) but a limited ability to inhibit host growth (regrowth after ~7 h). In contrast, our S. bovis complex phages have strong inhibitory activity (up to 30 h without bacterial growth), but a fairly narrow host range. Thus, we conducted adaptive laboratory evolution with the intent of increasing F. varium phage killing efficiencies and broadening S. bovis phage host ranges. Specifically, 6 F. varium phages were subjected to a phage training protocol (TRAIN30) in conjunction with various hosts. After 30 cycles,we foundthat 4 phages had increased inhibitory activity (6 -32% reduction in area under the curve (AUC) relative tothe parent phage (p<0.05)). One phage performed worse than its parentphage by 13%(p<0.05). One phage improved after the training, but not in a statistically significant manner (4% less bacterial growth). We also attempted to accelerate mutagenesis using the mutagen ethyl methanesulfonate (EMS). After 5 rounds (CAVE5 protocol), we observed one phage with a 6% increase in inhibitory activity (p<0.05), while the others showed non statistically signficant changes in activity. We did not observe any changes in phage yields in liquid culture amplifications of any of the F. variumphages after either the TRAIN30 or CAVE5 method. However, average yields range from 1 - 6 x1010 PFU/mL of lysate, which exceeds our production objectives. A modified Appleman's protocol was used to facilitate S. bovis complex host range expansion. A total of 30 cycles was performed using a combination ofthree phage-sensitive S. bovis strains,four phage-resistant strains, and five phages. After 10 rounds, plate reader assays and spot tests indicated one resistant strain was being inhibited, and by 30 rounds all four resistant strains were clearly being lysed by the pooled phage cocktail. However, further investigation of several phage isolates was impaired by their apparent instability as their activity was lost over very short periods of time (days), despite numerous attempts. We are currently developing new protocols to address such instability issues during adaptation. Objective 3: Assess and improve phage thermal stability. The thermal stability of allS. boviscomplex and F. variumphages was evaluated at 65C at various time intervals up to 60 minutes. Four out of six F. variumphages presented a titer loss of only 2-log after60 mins at 65C. One phage dropped by 5-log after 60 minutes and only one phagedroppedbelow the limit of detection after 30 mins at 65C. Considering theirhigh innate thermal stability, thermal adaptation ofF. variumphages was not pursued. In contrast, thermal adaptation of two S. boviscomplex phages was attempted since they were found to be very temperature sensitive. A 5-log decrease in titer was observed after only 5 mins at 65C with titers droppingto undetectable levels after 10 mins. We used two methods to try to increase thermal stability: In theSPP-55method, phages were exposed to 50 mM sodium pyrophosphate (SPP) at 55C for 30 mins, and subsequentlyamplified in their corresponding host. This method had previously been successful in our laboratory to increase phage thermal stability. However, after 6 rounds, there was no detectable improvement in stability at 65C for either phage. In the EMS-55method, phages were exposed to the mutagen EMS prior to amplification. Subsequent exposure to 55C for 30 minutes and amplification of survivors was performed to selectfor mutants with increased thermal stability. However, similarly, after 9 rounds, neither phageshowed increased thermal stability at 65C. Currently we are exploring new methods for thermal adaptation, including encapsulation, which has been found to be very effective for certain phages. Objective 4: Scale up and optimize production ofStreptococcusandFusobacteriumphages. To date, we have achieved production yields of 1010 PFU/mL for all phages tested up to the 1 L scale. Additional efforts to scale to 10 L will be performed with fully adapted phages. No challenges are foreseen. Objective 5: Assess safety and efficacy of phage cocktails. A small-scale animal study was performed using ten cannulated calves at a USDA facility. One calf was removed from the study prior to phage treatment due to illness. Three calves were provided PBS only as a control. The other six were split into low dose (108phages) and high dose (1010 phages) treatment groups. TwoF. varium phage cocktails containing two lytic phages eachwere provided twice daily (rotated).The ruminal contents were sampled prior to study initiation and then at various intervals over two weeks using an adaptive sampling regimen. Data from this study is still being analyzed, though no safety issues were observed or identified during necroscopy.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Schwarz, C., Mathieu, J., Gomez, J. L., Miller, M. R., Tikhonova, M., Nagaraja, T. G., & Alvarez, P. J. (2023). Unexpected finding of Fusobacterium varium as the dominant Fusobacterium species in cattle rumen: potential implications for liver abscess etiology and interventions. Journal of Animal Science, 101, skad130.