QUANTIFYING CATTLE MANURE-AMR PERCEPTIONS AND TREATMENT SYSTEM VARIABILITIES TO DEVELOP A NOVEL COMMUNICATION FRAMEWORK FOR CONVEYING AMR SCIENCE AND MITIGATION OPPORTUNITIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1014699
• Grant No.: 2018-68003-27467
• Project No.: MD-ENST-05570
• Proposal No.: 2017-05570
• Program Code: A4171
• Project Start Date: May 1, 2018
• Project End Date: Apr 30, 2023
• Grant Year: 2018

Project Director
Lansing, S.

Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742

Performing Department
AGNR

Non Technical Summary
Antimicrobial Resistance (AMR) research, outreach, and communication involve multiple stakeholders, yet, to date, there has not been a systematic characterization of AMR perceptions and knowledge, or effective development of AMR communication strategies. In addition, there is evidence suggesting that thermal-based processes are more effective in reducing AMR, however, the relationship between farm-appropriate, higher-temperature manure treatment methods and AMR reductions have not been specifically quantified. This research will address these needs by evaluating AMR perceptions and creating targeted outreach campaigns for farmers, policymakers and other key stakeholders. We will also determine manure matrix variability and create a robust. Science-based, manure sampling protocol for AMR to address the inherent variability of the manure matrixes and test higher-temperature manure treatment methods using the project-developed protocol. The long-term goals of this project are to: 1) provide research-based knowledge of stakeholder perceptions (and misconceptions) of AMR prevalence, conveyance, and reductions in the environment, 2) create and implement communication strategies and tools that best convey AMR knowledge, and 3) research the most promising thermal-based AMR manure treatment technologies in dairy operations based on our created dairy manure sampling protocol for AMR.The results from this project can be used to inform farmers on manure management technologies and how to effectively convey AMR knowledge. The team has unique experience with media outreach and will disseminate scientific research effectively through iterative design involving stakeholder review and feedback, discussions using online social media, reinforcing messages with in-depth web-based information, and in-person presentations and discussions with key stakeholders.

Animal Health Component

0%

Research Effort Categories

Basic

60%

Applied

40%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
403	0210	1040	50%
711	0210	1040	50%

Knowledge Area
403 - Waste Disposal, Recycling, and Reuse; 711 - Ensure Food Products Free of Harmful Chemicals, Including Residues from Agricultural and Other Sources;

Subject Of Investigation
0210 - Water resources;

Field Of Science
1040 - Molecular biology;

Keywords

amr

cattle manure

high temperature treatment

media communication

protocol

stakeholder perception

Goals / Objectives
Antimicrobial Resistance (AMR) research, outreach and communication involve many stakeholders, including farmers, milk and meat processors, researchers/extension educators, veterinarians, food safety experts, consumers, doctors, and policymakers. While there has been much progress in AMR research since launching the AFRI Food Safety AMR program, there has not been a systematic characterization of perceptions and knowledge of AMR presence in the cattle agroecosystems or the development of appropriate outreach strategies needed to effectively communicate with stakeholders. This research will address this need by systematically quantifying AMR knowledge and perceptions and creating targeted outreach campaigns for farmers, who increasingly interact with the public, as well as policymakers and other stakeholders in order to communicate the nuances of AMR prevalence in dairy and beef cattle production. In addition, through our prior research, thermal-based processes have the potential to be more effective in reducing AMR, but systemic relationships between novel higher-temperature manure treatment methods and AMR reductions have not been conducted. We will conduct this research as well as a statistically rigorous characterization of AMR in manure in order to create a manure sampling protocol suitable for accurate and precise quantification of AMR in the inherently heterogeneous and variable manure matrixes and management systems.The long-term goals of this project are to: 1) provide quantified and research-based communication tools to policy makers, farmers, and other agricultural stakeholders who interact with the public, based upon knowledge of stakeholder perceptions (and misconceptions) of AMR prevalence, conveyance, and reductions in the environment, 2) utilize our sampling protocol for AMR to test three of the most promising thermal-based AMR manure treatment technologies in dairy operations, some of which are already used by farms to meet other farm goals, i.e. rotary drum composters for solids treatment and anaerobic digestion, and 3) create communication strategies that best convey science-based knowledge on cattle-AMR dynamics to stakeholders, enabling them to communicate this dynamic issue and make sustainable decision on its mitigation. The results can be used to inform farmers, policy makers, and other stakeholders, on manure management technologies and how to effectively convey AMR knowledge to consumers.The supporting objectives are: Analyze stakeholder AMR perception, knowledge and decision-making, including reasons for variability in farmer antibiotic use and application.Define matrix variability and create sampling requirements/protocols for manure assessment using practical, emerging high-temperature dairy manure treatment technologies for reducing AMR (rotatory drum composting, thermophilic digestion, and thermal hydrolysis pre-treatment), and model the relationship between temperature and treatment, including in AMR runoff from beef open feedlots.Create an effective communication framework based on research into stakeholder's understanding and needs that includes social media, web and media resources, and eXtension material tailored to important stakeholders in the AMR arena, i.e. farmers, veterinarians, food safety experts, and policy makers, resulting in effective and novel strategies for conveying the complicated issues surrounding AMR to stakeholders.Rising concern with antibiotic resistant bacteria has led to a recent proliferation of regulations from federal agencies as well as state level laws. Meanwhile, industry is taking its own approach toward governing antibiotic use through contract mandates, and various consumer groups have increased pressure for farmers to go "antibiotic free". Farmers are situated within this rapidly changing and complex regulatory landscape, and, collectively, employ a wide variety of antibiotic practices. Due to this rapidly changing situation, there is currently little understanding by researchers concerning how current regulations and best practice guidance affect the kinds of antibiotic practices farmers use. Similarly, non-farming stakeholders, i.e. veterinarians, policy makers, industry representatives, often have little understanding concerning how and why farmers engage in the antibiotic practices that they do. There is therefore a need to: a) understand the variety of antibiotic practices of farmers and the drivers behind their decision making, and b) understand key stakeholder attitudes towards AMR. Understanding the diverse practices, perspectives, and decision points among antibiotic stakeholders can become a foundation for better communication about what constitutes best antibiotic practice under specific circumstances.Previous studies (and our on-going work) have found significant variability and lack of correlation between antibiotics and ARG, and there currently is no standardized approach to sample heterogeneous manure matrices for AMR. These efforts also suggest that thermal treatment systems offer greater potential to mitigate antibiotic residues, ARB, and ARG than traditional manure handling systems, and while some testing has been conducted at bench-scale, farm-relevant, pilot-scaled system of thermophilic composting and anaerobic digestion have not been investigated, with no work done exploring thermal hydrolysis. Thermal hydrolysis prior to digestion has been practiced in Europe and at wastewater treatment plants in the U.S., but this technology and further analyses of thermophilic AD could have large impacts on antibiotic destruction. The relationship between temperature, pressure and AMR needs to be systemically investigated. These promising treatments also offer system to develop improved sampling protocols that account for sampling and technical variability.There is a need for farmers, the public, and policymakers to have accurate information on the impact of farm practices on AMR and how AMR is reduced. The effective delivery of this information requires an understanding of stakeholder perceptions and the assessment of delivery approaches, as well as novel media approaches to delivering this content.

Project Methods
Objective 1a: Understand the reasons for variability in farmer antibiotic use and application.Workshop/conference surveys, interviews, and focus groups of antibiotic practices, farmer understanding of regulations and recommended best practices will be conducted. Approximately 30 in-depth interviews will take place. Results from interviews and focus groups will be used to conduct a follow up survey of approximately 200 farmers concerning antibiotic usage and manure management, key factors that go into decisions to change or adopt new practices and what information is used to initiate a change in practice, and what is needed to enable the farmers to communicate with others about AMR.Objective 1b: Characterize differences in perception of AMR among farmers, veterinarians, and advocacy groups. We will conduct six focus groups (of 5-8 people) of diverse non-farming stakeholders. Focus groups are designed to gauge existing knowledge and perceptions concerning attitudes and perceptions of antimicrobial use and antimicrobial resistance. We will conduct thirty semi-structured interviews with a broader set of land use stakeholders in farming communities in the Mid-Atlantic (dairies) and mid-West (beef). Thirty-five will conduct a Q-sort and sort statements on how each statement reflects their thinking concerning best management practices (from most strongly disagree to most strongly agree). Significant factors will be extracted using principal components analysis, and interpreted qualitatively to determine the dominant discourses concerning best practices for antimicrobial use.Objective 2a: Define AMR measurement variability and create sampling protocols for manure assessment. Create extension materials based on these findings. Intensive sampling (n=20) along with technical replicates (n=3) is planned on-farm analyses of a farm with a full-scale, high-temperature bedding recovery unit (BRU) for antibiotics in raw manure (influent pit), SLS liquid effluent, SLS separated solids, and in high temperature composted solids (post-BRU). All samples will be collected, stored on ice, and preserved (as needed) within 48 hours for characterization and antibiotic testing. Manure samples will be characterized for total solids, volatile solids and pH, C:N, NH4, TN, and TP. Select antibiotics (and their breakdown products) will be prioritized for residue testing in Dr. Clifford Rice's laboratory using an LC-MS/MS with an API inlet (electrospray and APCI) coupled to a liquid chromatraph (LC) instrument. Analytes will be identified and quantitated using multiple reaction monitoring methods available on a triple quadrupole mass spectrometer, including parent and daughter ions. A select group of samples will be used for initial ARB and ARG to select for coliforms, Staphylococcus and Streptococcus spp. Colony forming units will be counted, and pathogen communities will be analyzed for ARGs, with total microbial community DNA isolated following methods of collaborator Durso, and quantified for antibiotic resistance genes representing β-lactamase, macrolide, and sulfonamide genes, as well as an integrase gene commonly associated with horizontal gene transfer. Statistical analysis will be conducted to determine significant differences in manure characteristics and antibiotics between manure matrices using analysis of variance (ANOVA) and Tukey-Kramer multiple comparisons. These efforts will be assembled into a draft protocol for AMR sampling that will be circulated as a FactSheet and targeted for publication as an ASABE or APHA standard.Objective 2b: Assess effect of temperature on manure treatment technologies in reducing AMR from rotatory drum composting/bedding recovery unit, thermophilic digestion and thermal hydrolysis pre-treatment.To test the effect of BRU conditions on antibiotic and ARG degradation, 3 drum rotation speeds (controlling retention time) and 3 ventilation rates (controlling temperature) will be tested in a factorial design (9 treatments). The results will be modeled to determine the effect of temperature using a simple regression model. For high-temperature digestion, manure will be tested initially using lab-scale testing, with antibiotics injections at different temperatures and retention times to develop time and temperature antibiotic degradation curves, with post-testing of antibiotic concentration, presence of antibiotic-resistant bacteria, manure volume and moisture content. The experimental design will test four temperatures, starting at 30°C and increasing by 10°C to 60°C. Biogas volume will be measured using a glass, gas-tight syringe equilibrated to atmospheric pressure. Biogas samples will be analyzed for CH4 composition using a gas chromatograph. The results will be modeled to determine the effect of temperature on both methane production and antibiotic mitigation using a multiple regression model. To verify at pilot-scale, six plug-flow digesters (3 m3 each) will be utilized using optimal conditions from lab testing. Influent and effluent samples will be collected twice per week from each digester and tested for biogas, antibiotics, ARB, and ARG. A thermal hydrolysis pre-treatment testing will be conducted using triplicate runs at 4 different temperatures and 4 retention times to determine effect on antibiotics, AMB and AMG with manure and DC WATER sludge.Objective 2c: Modeling the relationship between AMR runoff from beef lots and temperaturePast runoff VFA data will be analyzed and modeled based on antibiotics, ARG, ARB and weather station data to develop a temperature dependent curve based on runoff collected and weather conditions using MACRO 5.1 model, a one-dimensional, process oriented dual-permeability model of water flow and solute transport, in which temperature data can be applied.Objective 3a: Summarize current science-based knowledge of AMR and AMR reduction and treatment, as it relates to dairy and beef agroecosystems and food safety We will conduct an up-to-date review of scientific literature to supplement our data collection in order to publish a review article and additional eXtension deliverables that give specific AMR mitigation details concerning cattle manure.Objective 3b: Identify and assess media approaches of communicating science-based knowledge of AMR to stakeholders, with a survey of farmers, veterinarians and policy makers through eXtension listserve to understand knowledge and perceptions and inform communication outreach objectives. A survey will be developed and conducted with select audiences to assess the anticipated effectiveness of various approaches and distributed.Objective 3c: Develop social media, web resources, and a communication program to extend scientific knowledge on dairy and beef agroecosystem AMR dynamics to a wider audience.Park Productions will: 1) select and develop the appropriate tools for conveying the content on various media platforms (for example, web streaming and social media), 2) design and create a prototype for media resources, 3) produce the initial media prototype, 4) present the prototype to stakeholders for feedback, 5) refine the prototype based on stakeholder feedback, and 6) launch the refined version of media resources on public platforms to extend audience reach.Objective 3d: Create eXtension, workshop and material resources based on social science research to help key stakeholders understand complicated AMR issues, and how to discuss and communicate with the public, i.e. product consumers.We will conduct two workshops and field days and strategically partner with other extension programs to engage specific target audiences (dairy farmers and their advisors, policy makers, and NGOs). We will develop and deliver oral presentation materials at dairy industry meetings, conferences, and other related events, as opportunities become available, and post materials on online platforms.

Progress 05/01/18 to 04/30/23

Outputs
Target Audience:The target audience for the iAMResponsible Project's outreach efforts include livestock producers, food safety experts, extension educators, consumers, veterinarians, medical professionals, and policymakers. Approximately 4000 persons are following one or more of the iAMResponsible Project social media accounts with strong representation by medical professionals, educators, and AMR researchers worldwide. The eight professional vidoes prepared for this project highlighting the project outcomes and educating our target audience on antimicrobial resistance (AMR) have been shown in University classes, disseminated through social media, located on faculty websites, and are part of the iAMR website and iAMR YouTube Page. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The University of Maryland had seven undergraduate students and a PhD student conducting research under this project, receiving his PhD in Spring 2023 from work on this project, and mentoring the undergraduate students on this project. The students that were directly supported from this project over the past 5 years include three African American males (including the PhD student) and one Hispanic female. Students have been trained in quantifying antibiotic resistance and provided lab and field experiences in protocol implementation, data collection, and project management. Lab experiences included chemical, molecular, and microbiological techniques and assessing samples in complex matrices. Students also developed skills in research communication through presentation at various conferences and institution events via poster, oral, and virtual presentations. During the reporting period three master students and three undergraduate students developed or refined STEM communication skills by contributing to the development and distribution of educational materials for the iAMResponsible networks at the University of Nebraska. The continued expansion of media development by the iAMResponsible team has also required several team members to develop new expertise in video production, audio production, and graphic design utilizing programs such as Canva and Adobe Creative Suite, among others. At Cornell University two recent female veterinary graduates were hired as post-docs under a professional services agreement to learn about antimicrobial stewardship associated with mastitis treatment and control. They implemented practices, such as selective dry cow therapy in commercial dairy herds and further trained the veterinary of record for each herd. At UMBC, a female PhD student was funded. She undertook the vast majority of interviews (~90%) and assisted with setting up the online Q Method online survey. She also led the team that conducted the scoping review, which was published in PLOS ONE. Jaime, has completed her fieldwork and is writing her dissertation based on this work. Her anticipated graduation date is Spring 2024. The project also hired a female undergraduate student to assist with the scoping review, where she was trained in bibliographic methods, database management, and the topic of antibiotic perceptions. How have the results been disseminated to communities of interest?Primary dissemination to interested communities has been via social media, websites, online publications, in-course materials, journal articles, and at professional, educational, and community events. Across the four on-line social media outlets (Facebook, Twitter, Instagram, YouTube), dissemination of AMR related materials, has reach > 5,000 people to date. Social media material includes graphics, videos, and highlights. In addition to social media content, the iAMResponsible team launched a podcast series (Tales of the Resistance), which discusses and highlights AMR related research and topics. The iAMResponsible team is also leading a multi-university online course "AMR from a One Health Perspective" which explores the many faceted challenges of AMR with emphasis on developing scientific communication skills in young STEM professionals. During the Spring of 2022, twenty students at six universities (Nebraska, Maryland, North Carolina State, Washington State, Oklahoma State, and Minnesota) participated in the course, with an additional fourteen students participating in the Spring 2023 session. During the final reporting period, iAMResponsible and the Livestock and Poultry Environmental LearningCommunity (LPELC) networks were leveraged to deliver the eight-video series summarizing important takeaways from this project. The videos were shared widely among these networks along with a short questionnaire to determine their impacts on audience perceptions of dairy production and antibiotic use. We have been able to disseminate demonstrated success of selective dry cow therapy to major opinion leading groups, such as the American Association of Bovine Practitioners (AABP), National Mastitis Council (NMC), and Pro-Dairy. This has multiplied the findings throughout the dairy industry and lead to further extension presentations to various state veterinary medical associations and dairy producer groups. The University of Maryland has presented research at various professional conferences and networks, such as American Ecological Engineering Society (AEES), American Society of Microbiology (ASM), and the American Society of Agricultural Biological Engineers (ASABE). Dissemination also includes publications and engagement through department and intuitional events. In 2019, the Human Dimension and Public Perceptions of AMR workshop (funded by NIFA under a separate grant with PI Lansing leading the effort) facilitated new collaborations to bring social scientists and other researchers (across academia, NGOs, and federal personnel) together to discuss the public understanding of communication elements of the AMR field. This convening has resulted in various published papers and networks that nationwide engagement that continued past the conference ending and greatly influenced the outreach effort conducted for this project. The eight professional videos produced by Well Said Media have been published that showcase the work conducted from this grant and highlight the results and takeaway messages. The videos include three AMR education videos, four videos that go into each of the project objectives, and final video that provides an overview of the grant. These videos are currently on YouTube for public viewing as well as distribution through each collaborating institution. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Analyze stakeholder AMR perception, knowledge, and decision-making Over the 5-year granting period, stakeholder engagement was analyzed via various studies. These include Q-Method surveys, literature reviews, and interviews (N = 56) with dairy and beef farm stakeholders, including farmers, consumers, veterinarians, extension agents, and other industry stakeholders such as equipment suppliers and industry advocates. These studies highlighted various stakeholder discourse and research around antibiotic utilization in dairy and beef industries. The Q-method survey was used to gauge different stakeholder (farmers, farm managers, dairy purchasers, veterinarians, extension agents, and consumers) perspectives around the responsible use of antibiotics, with the results identifying three distinct and significant discourses: 1) a farmer-centric discourse; 2) a public- health centric discourse; 3) a pro-naturalism and anti-regulation discourse. The results from this study are published in the journal Agriculture and Human Values. A scoping review of all previous research on consumer perceptions of antibiotic use in animal husbandry as conducted and published in PLOS ONE. Our of 3,560 articles, we identified 124 articles that met our standards for analysis. The current state of the field recognizes that consumers are generally concerned about antibiotic use in animal agriculture, with females, upper class, and highly educated consumers being the most consistently concerned. Drawing on our scoping review, we completed a 1500-person online Qualtrics survey of beef and dairy consumers. This survey tested how different messages about antibiotic use on dairy and beef farms affect a person's willingness to pay for antibiotic free products. Data from this survey is in preparation for publication. Interviews with dairy farmers in Maryland, Pennsylvania, and New York and beef farmers in Nebraska and Maryland were conducted to understand how the Veterinary Farm Directive has impacted farm practices. Data producing technologies were found to play a key role in helping dairy farmers manage mastitis with less antibiotic use. We found that farms adopting data technologies often encounter several key points of friction in data adoption, including intermittent access to the internet and what farmers often perceive as a conflict between what the data says about animal health and their own intuition. The data driven results of farm technologies were perceived as sometimes telling farmers what was obvious to them, or others times the data driven results would contradict famer's intuitive and daily understandings of animal health. Interviews with farmers revealed that, rather than blindly following what the data says, farmers are instead engaged in an ongoing process of constructing knowledge about animal health using a mix of objective data-driven metrics combined with longstanding farm knowledge. Our results indicate that farmers understand advantages to data producing technologies and are adopting data producing technologies, but with an approach that suggests deep skepticism about the overall value of a data driven approach to farm management. The results of this work were presented at the 2023 American Association of Geographers annual conference and is currently under review in the journal Geoforum. Beyond farmer perceptions of data in their work, we demonstrated the potential role of data collection on farm management by analyzing the implementation of selective dry cow therapy (SDCT) on commercial dairy farms. Previous research indicates that under the proper conditions and management a dairy farm could move from blanket therapy at the end of lactation to more prudent use of antibiotics based on algorithms. These algorithms indicated the cow's risk for having or acquiring an intramammary infection. The results indicated that most farms with the right risk profile can move from blanket dry cow therapy to treating only about half of the cows at the end of their lactation (>50% decrease). With this data we were able to further translate these on-farm results to other farmers, veterinarians, and herd advisors through videos, peer-reviewed publication, professional conference presentations, and extension presentations to leading opinion groups. Objective 2: Assessment of thermal manure/ wastewater technologies An antibiotic extraction method was developed for multi-residual, multi-class antibiotic analysis using liquid chromatography in tandem with mass spectrometry method. This method was published in the journal Antibiotics and utilized in subsequent experiments. A mass flow analysis of the bedding recovery unit (BRU) on a dairy farm allowed for quantification of the mass of antibiotics, antibiotic resistance genes (ARG), and antibiotic resistant bacteria (ARB) from solid liquid separation and high temperature rotary drum processing (i.e., BRU). The study indicated that most manure containing AMR factors went untreated following solid-liquid separation, with 95% pumped to a storage lagoon and 5% proceeding to BRU processing. The removal of antibiotic residuals during BRU processes was insignificant, yet the BRU processing was 100% effective in removing the ARB examined. Five (Intl1, sul1, tetQ, tetX and tetM) of the eight ARGs tested were found to have significant reduction (>95%) following the thermophilic composting BRU system. An anaerobic digestion experiment was implemented to understand antibiotic removal at two temperatures (mesophilic at 35°C and thermophilic at 55°C) over 43 days of digestion. The antibiotic erythromycin was more efficiently degraded under mesophilic conditions, with 100% removal by Day 36 compared to 97% reduction for thermophilic conditions after 43 days. Thermophilic conditions had higher oxytetracycline degradation, with 66% removal compared to only 22% removal at mesophilic conditions. Both temperatures were effective in reducing most ARGs (5 out of 8 genes tested), but enrichment of other resistance genes was also documented. Thermal hydrolysis pretreatment of manure and wastewater biosolids prior to digestion was tested to determine if differences in manure and biosolids response to high pressure and high temperature pretreatment. The publications for both tests are under preparation. Objective 3: Create an effective communication framework for AMR outreach: The iAMResponsible team (www.iamrproject.com) works to translate and transfer new research findings for delivery to the target audiences. Over 70 new pieces of outreach content were created for distribution on social media and added to the existing database of media and research related to antimicrobial resistance (AMR). Across four online social media outlets for dissemination of AMR related materials, 5,200 people connected and engaged with one or more of the social media accounts. The iAMResponsible and LPELC networks delivered a series of eight videos that summarized important takeaways from this project. The videos were shared among these networks along with a short questionnaire to determine their impacts on audience perceptions of dairy production and antibiotic use. A total of 142 persons had completed the questionnaire as of July 2023. The iAMResponsible team also lead a multi-university online course 'AMR from a One Health Perspective,' which explores the many faceted challenges of AMR with emphasis on developing scientific communication skills in young STEM professionals. A total of 40 students across six universities (Nebraska, Maryland, North Carolina State, Washington State, Oklahoma State, and Minnesota) participated in the course, over four years. The iAMResponsible team launched a podcast series (Tales of the Resistance), to discuss research findings on AMR in food production systems with general (non-expert audiences), this series has produces bi-weekly episodes covering a wide range of important AMR topics.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Capel MB, Nydam DV, Forrestal A. A prospective descriptive study of field experiences with selective dry cow therapy in New York dairy farms. Veterinary Sciences.
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Lansing, D. M.,; & Barrett, J. (2023). Antibiotic responsibility and agricultural publics: diverse stakeholder perceptions of antibiotic use in animal agriculture. Agriculture and Human Values, 1-14.
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Poindexter, C ., Yarberry, A., Rice, C., Lansing, S. 2022. Quantifying Antibiotic Distribution in Solid and Liquid Fractions of Manure Using a Two Step, Multi-Residue Antibiotic Extraction. Antibiotics.
• Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Poindexter, C.,�Yarberry, A., Rice, C., Georgakakos, C.,�Lansing, S. 2023. Antibiotic Resistance and Antibiotic Partitioning with Manure Separation and a High Temperature Bedding Recovery Unit. Journal of Environmental Management.
• Type: Journal Articles Status: Other Year Published: 2023 Citation: Poindexter, C.,�Yarberry, A., Rice, C., Lansing, S. 2023. Time Series Analysis of Antibiotic Resistance within Diary Manure under Mesophilic and Thermophilic Anaerobic Digestion. Science of the Total Environment.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Barrett, J. (2023). What happens in Europe: Dairy producers anxieties about US adoption of EU veterinary drug policy. Presentation for the American Association of Geographers Annual Meeting. Denver, CO. April, 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Forrestal A, Potter T, Nydam DV, Capel MB. Supporting the Implementation and Monitoring of Selective Dry Cow Therapy (SDCT) on NY State Dairy Farms. 55th Annual Conference of American Association of Bovine Practitioners. Long Beach, CA. September 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lansing, D. and Barrett, J. (2023). Recognizing value: data-based and observational health assessments on US dairies. Presentation for American Association of Geographers Annual Meeting. Denver, CO. April 2023.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Potter T, Nydam DV, Forrestal A, Capel MB. Putting research to the test: Implementing selective dry cow therapy across New York State. 55th Annual Conference of American Association of Bovine Practitioners. Long Beach, CA. September 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Poindexter C., 2023. Fate of antibiotics residues, antibiotic resistant bacteria and antibiotic resistance genes in US diary manure management systems. Oral. ABIOTEC II Webinar, Germany.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poindexter, C., Lansing, S. Yarberry, A., Rice, C., Georgakakos, C., Gooch, C. Lansing, S. 2022. A Mass Balance Approach to Antibiotic Resistance Partitioning in Dairy Manure Through A Continuous High Temperature Rotary Drum Composting Bedding Recovery Unit. Oral. American Society for Agricultural and Biological Engineering. Houston, TX. July 17-20, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Zelt, M and A. Schmidt. Impacts of social media on public awareness and behavior related to antimicrobial resistance. [Poster presentation]. Waste to Worth, April 18-22, 2022. Oregon, Ohio. https://lpelc.org/impacts-of-social-media-on-public-awareness-and-behavior-related-to-antimicrobial-resistance/
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poindexter, C ., Yarberry, A., Rice, C., Lansing, S. 2022. Correlation of Antibiotic Resistance and Temperature During Anaerobic Digestion of Dairy Manure. American Ecological Engineering Society. Oral. Baltimore, MD. June 20- 23, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poindexter, C ., Yarberry, A., Rice, C., Lansing, S. 2022. Comparative Analysis of Mesophilic and Thermophilic Anaerobic Digestion on the Reduction of Antibiotic Resistance within Diary Manure. Poster. American Society of Microbiology. Washington D.C. June 9-13, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poindexter, C ., Yarberry, A., Rice, C., Lansing, S. 2022. Two-step multi-residue antibiotic extraction method for comparison of antibiotics concentrations in manure as it moves through a manure treatment system. Poster. American Society of Mass Spectrometry, Minneapolis, MN. June 6-9, 2022.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Schmidt, A.M, M. Zelt, S. Lansing, R. Tikekar, M. Sharara, J. Harrison, and N. Noyes. Antimicrobial Resistance from a One-Health Perspective: A Multi-Disciplinary University Instruction from Extension Professionals. [Poster presentation]. Waste to Worth, April 18-22, 2022. Oregon, Ohio. https://lpelc.org/antimicrobial-resistance-from-a-one-health-perspective-multi-disciplinary-university-instruction-from-extension-professionals/
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Schmidt, A.M. Im an Expert& Why Arent You Listening to Me?!. [Sponsored Special Session]. Waste to Worth, April 18-22, 2022. Oregon, Ohio. https://lpelc.org/im-an-expert-why-arent-you-listening-to-me/
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Zelt, M, A. Vogel, A.M. Schmidt, and B. Zelt. The iAMResponsible Project Resource Library - evidence-based, antimicrobial resistance-related outreach content for food producers and consumers [Poster presentation]. 2023 NIAMRRE Conference. May 16, 2023. Gainesville, Florida.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Zelt, B., M. Zelt, and A.M. Schmidt. Impacts of iAMResponsible Project social media on public awareness and behavior related to antimicrobial resistance [Poster presentation]. 2023 NIAMRRE Conference. May 16, 2023. Gainesville, Florida.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Schmidt, A.M., M. Zelt, S. Lansing, R. Tikekar, M. Sharara, J. Harrison, and N. Noyes. Antimicrobial Resistance from a One-Health Perspective: A Multi-Disciplinary University Instruction from Extension Professionals. [Conference presentation]. 2023 NIAMRRE Conference. May 16, 2023. Gainesville, Florida.
• Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Barrett, J.; Lansing, D. M. (2023). From cow sense to data sense: hybrid data assemblages on US dairy farms.

Progress 05/01/21 to 04/30/22

Outputs
Target Audience:The target audience for the iAMResponsible Project's outreach efforts include livestock producers, food safety experts, extension educators, consumers, veterinarians, medical professionals, and policymakers. Approximately 4000 persons are following one or more of the iAMResponsible Project social media accounts with strong representation by medical professionals, educators, and AMR researchers worldwide Changes/Problems:Describe major changes/problems in approach and reason(s) for these major changes. If applicable, provide special and/or additional reporting requirements specified in the award Terms and Conditions. DC Water has shut down the use of the pilot scale thermal hydrolysis system. UMD found another collaborator at Virginia Tech University with a similar pilot-scale thermal hydrolysis system that has agreed to participate in this project and use their system. COVID-19 Impacts: Well Said Media has taken over for Park Production team to facilitate multimedia (videography etc.) dissemination of the work conducted within the project. Park Production had to be replaced due to Covid-19 delays and obligations to other projects restricting availability. What opportunities for training and professional development has the project provided?The University of Maryland has involved two college undergraduate students and one PhD student in conducting the research under this project, with the PhD student involved in mentoring the undergraduate students as well. The students include two African American males and one Hispanic female directly supported in this project: one PhD student and two undergraduate students. In addition to extension and research professionals, the iAMResponsible team relies on the assistance of student workers at the University of Nebraska. During the reporting period 1 PhD student, 2 MS students, and 4 undergraduate students contributed to the development and distribution of educational materials for the national networks. All team members are encouraged to participate in course lectures and discussion for the AMR from a one-health perspective online course, providing team members with access to leaders in the fields not only of AMR research but also of science communication and education. The continued expansion of media development by the team has also required several team members to develop expertise in video production, audio production, and graphic design, utilizing programs such as Canva, and the Adobe Creative Suite, among others. At Cornell University, Drs. Tracy Potter and Amber Forrestal, who are also recent veterinary graduates, have been hired as post docs under a professional services agreement to learn about antimicrobial stewardship associated with mastitis treatment and control and then implement practices, such as selective dry cow therapy in commercial dairy herds, and further train the veterinary of record for each herd. UMBC has employed one PhD student and one undergraduate to facilitate data collection and obtain research experiences. How have the results been disseminated to communities of interest?Primary dissemination has been via social media, in course materials, and at professional, educational, and community events. An invited address in the Dairy Sessions at 55th Annual Conference of American Association of Bovine Practitioners was conducted. What do you plan to do during the next reporting period to accomplish the goals?The publication of the antibiotic detection method manuscript in Chemosphere is expected. The developed LC-MS/MS antibiotic method previously described in Objective 2a will be utilized for testing antibiotic reduction in manure and wastewater sludge following thermal hydrolysis treatment and subsequent anaerobic digestion (Objective 2b) in Spring 2022. The method will also be incorporated into future mobile genetic element experiments for holistic assessment of antibiotics and other ecological parameters on gene movement (an extra initiative, not specified in the proposal). Completion and submission of two manuscripts for the BRU study and comparative AD study are expect to Chemosphere and Science of the Total Environment, respectively, in Summer 2022. The metagenomic analyses of ARGs for both studies (Objectives 2b and 2c) is on-going. This will include completion of qualitative polymerase chain reaction (qPCR) experiments, and the M13 sequencing and gene verification of the nine selected primers/genes, 1 mobile genetic element (intl1), 1 macrolide (ermB), 1 beta-lactam (bla-2), 1 sulfonamide (sul-1), 4 tetracycline (tetX, tetM, tetW, tetQ), and 16S for total microbial mass. Pilot-scale thermal hydrolysis digestion trials (Objective 2d) will begin in Spring 2022 using the 50 L thermal hydrolysis unit at Virginia Tech University with manure and wastewater sludge samples tested and compared using varying conditions and antibiotic spikes. Mobile genetic element experiments will be conducted to investigate the impact of waste treatment on the movement of mobile genetic elements in the dissemination of ARGs. In-depth laboratory simulations of waste treatment conditions using a mixture of methods including bacterial culturing, sequencing, and microfluidics will be conducted to monitor mechanisms of horizontal gene transfer and potential influencers. University of Nebraska- Lincoln During the next reporting period, we will continue to expand our content library to incorporate and make accessible research findings from a wider selection of contributors and expand our content creation to new media types. We will establish a regular mailing list/newsletter to promote newly added library materials to a national network of extension professionals, agricultural advisors, and consumer advisors such as doctors, nutritionists, and teachers. The team will seek to develop new partnerships with 4-H and consumer-facing food safety extension professionals to expand its reach with non-scientific audiences. Cornell University In the upcoming year we aim to enroll 20 more herds in selective dry cow therapy in an effort to train more veterinarians of record in this practice as well as normalize the behavior across the dairy farming community. To aid in this effort we are planning shoot educational videos highlighting interactions between veterinarians and farmers with Well Said Media. University of Maryland Baltimore County (UMBC) We plan to present the results at the Waste 2 Worth conference and give personal presentations of the results to selected participants in the survey. We plan to publish the results of the Q survey in a peer reviewed journal in Summer 2022. We plan to deploy the new Qualtrics survey of consumer perspectives of antibiotics, analyze the results, and then submit the results to a peer reviewed journal Well Said Media Well Said has been added as a new partner with the AMR team to produce a series of videos, including the survey studies results and public education & awareness campaigns. Work will include pre-production such as consulting, creating interview questions, storyboarding, travel, and filming (a total of approx. 44 shoot hours over approx. 7 filming days capturing a variety of interviews, B-Roll footage, and aerial (drone) footage at locations, such as Cornell, Maryland, and Nebraska), producing professional voiceovers, creating animated motion graphic elements to be determined as the project develops, and editing approximately 8-10 videos varying in length between 30 seconds - 3 minutes. Closed captions for all videos will also be included.

Impacts
What was accomplished under these goals? Analysis form Q-Method Survey conducted over the past two years with various stakeholders (n=30) discerned three overarching perspectives: farmer-centric, public health centric, and pro-naturalism. Meta-analysis of research articles that focused on consumer perceptions of antibiotic utilization in animal agricultural found upper class, highly educated women to be the most concerned with antibiotics (Objective 1). Analysis of thermal based manure treatment technologies found the BRU system to be effective at eliminating resistant bacteria but not as effective in reducing antibiotics concentrations. The comparative analysis of anaerobic digestion temperature and retention times found antibiotic degradation to be temperature and antibiotic dependent (Objective 2). Our iAMResponsible extension platform expanded and strengthened its reach, with 50 new pieces of content, an expanding webinar series and the development of a podcast show (Objective 3). Additionally, we enrolled 20 dairy herds in selective dry cow therapy and are monitoring antibiotic use, udder health, and milk production outcomes as part of a new initiative/objective added to this project with a change in PI at Cornell University (Objective 3). Objective 1: Analyze stakeholder AMR perception, knowledge, and decision-making Based on information from stakeholder interviews, we designed, implemented, and completed an online Q-method survey (n = 30) to gauge different perspectives around the responsible use of antibiotics. Survey respondents included farmers, farm managers, dairy purchasers, veterinarians, extension agents, and consumers. We analyzed the results using a principal components analysis and identified three distinct and significant discourses: 1) a farmer-centric discourse; 2) a public-health centric discourse; 3) a pro-naturalism and anti-regulation discourse. We conducted an extensive scoping review of all previous research on consumer perceptions of antibiotic use in animal husbandry. After sorting through 3,560 articles that were returned from our database queries, we ultimately reviewed 124 articles that met our standards for inclusion. The current state of the field recognizes that consumers are generally concerned about antibiotic use in animal agriculture, with females, upper class, and highly educated consumers being the most consistently concerned. Less attention has been paid to why consumers are concerned. We will conduct more qualitative work to understanding the differences between consumer's stated concerns and their ultimate purchasing decisions. The results of this scoping review are published in PLOS ONE (Barrett et al. 2021). Drawing on our scoping review, we have begun a study design to test whether various messages around antibiotic use will affect consumer behavior. This will be a large sample survey of consumers using Qualtrics. Objective 2a: Creation of an improved antibiotic extraction method: The finalized liquid chromatography in tandem with triple quadrupole mass spectrometry (LC-MS/MS) method manuscript has been submitted to Chemosphere. This method development has been successfully completed. Objective 2b: Effect of rotary drum composting bedding recovery unit (BRU) on AMR: Mass flow analysis of bedding recovery unit (BRU) management systems was completed using four different substrates: 1) unprocessed manure, 2) liquid fraction generated from a screw press separator, 3) solid separated fraction, and 4) final BRU product. Three antibiotics were detected in the manure: tetracycline, tulathromycin, penicillin-G. Benzyl-penicilloic acid was added as penicillin G metabolite for degradation analysis. Antibiotic concentrations ranged from 0.436 - 4.10 ug/kg. Calculated mass flow rates found that 95% of the mass fractioned with the separated liquid fraction. The remaining 5% of the mass in the separated solid faction contained 11 - 20% of tetracycline and tulathromycin antibiotics. No significant reductions of either antibiotic was found following BRU processing of the separate solids. Solid-liquid separation significantly reduced viable antibiotic resistance bacteria (ARB) in the solid separated fraction, with complete elimination of pathogens following BRU processing. Solid liquid separation prior to BRU processing resulted in the majority of the mass within the separated liquid pumped without treatment to the storage lagoon. The BRU technology has minimal impact on the residual antibiotic concentration within the solid separated fraction but successfully eliminated pathogenic and ARB in the bedding. Objective 2c: Effect of mesophilic and thermophilic anaerobic digestion on AMR: Lab-scale batch anaerobic digestion testing was conducted at mesophilic (35°C) and thermophilic (55°C) conditions, with spiked oxytetracycline, ampicillin, and erythromycin antibiotics.Mesophilic conditions were more efficient at removing erythromycin, with the complete elimination by Day 21 compared to Day 43 under thermophilic conditions. Thermophilic conditions had significantly greater reductions of oxytetracycline (67% loss) compared to 21% in mesophilic conditions after 43 days of incubation.The Beta lactam, ampicillin, had the lowest concentration among all three antibiotics, likely due to the easily hydrolyzed beta lactam ring. By Day 43, mesophilic conditions resulted in significant more degradation than thermophilic conditions. Both conditions were effective at eliminating both fecal indicator species (Enterococci and E. coli) by Day 9. 16S sequencing is under way for comparative analysis of bacterial community diversity following the 43-day digestion. Antibiotic Resistance Gene Analysis: Gene analysis for both studies (Objectives 2b and 2c) is on-going. Ten genes/primers, 1 mobile genetic element (intl1), 1 macrolide (ermB), 1 beta-lactam (bla-2), 2 sulfonamide (sul-1,), 4 tetracycline (tetX, tetM, tetW, tetQ), and 16S for total microbial mass were selected for further progression of qualitative analysis. Lack of primer efficiency for qPCR resulted in the selection of new primers and qPCR troubleshooting. Objective 3: Create an effective communication framework for AMR outreach: The project team continues to grow, with new collaborators from four states previously not directly integrated into the iAMResponsible project network (Texas, Iowa, North Carolina, and Minnesota). The iAMResponsible team further expanded their online course on AMR from a One Health perspective with emphasis on developing scientific communication skills in young STEM professionals. During the Spring of 2021, twenty-four students at five universities (Nebraska, Maryland, North Carolina State, Washington State, and Minnesota) participated in the course. Over 50 new pieces of outreach content were created during the reporting period for distribution on social media and added to the existing library of some 400 pieces of media and research related to AMR curated by the iAMResponsible team and available to the public on the Livestock and Poultry Environmental Learning Community (LPELC) website. Four on-line social media outlets for dissemination of AMR related materials are managed with regular (monthly, weekly, or daily) outputs. In partnership with LPELC, the project team delivered the third webinar in an ongoing series of webinars on AMR impacts on agriculture aimed at agricultural producers and their advisors. In late 2021, the iAMResponsible team launched a podcast series (Tales of the Resistance) to discuss research findings on AMR in food production systems with general (non-expert audiences). Twenty dairy herds in selective dry cow therapy and are monitoring antibiotic use, udder health, and milk production outcomes were enrolled as part of a new initiative/objective added to this project with a change in PI at Cornell University (Objective 3).

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Barrett, J. R., Innes, G. K., Johnson, K. A., Lhermie, G., Ivanek, R., Greiner Safi, A., & Lansing, D. (2021). Consumer perceptions of antimicrobial use in animal husbandry: A scoping review. PLOS ONE, 16(12), e0261010.
• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Poindexter, C.,Yarberry, A., Rice, C., Lansing, S. 2022. Two-Step Multi-Residue Antibiotic Extraction Method for Complex Manure Matrices. Submitted to Chemosphere.
• Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Potter T, Nydam DV, Forrestal A, Capel MB, Thomas MJ, Rapnicki P. Implementing Selective Dry Cow Therapy on Commercial Dairy Farms: heightened antimicrobial stewardship. Invited article to Microorganisms under submission.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Wind, L., Briganti, J.S., Brown, A.M., Neher, T.P., Davis, M.F., Durso, L.M., Spicer, T., Lansing, S., 2021. Finding what is inaccessible: Antimicrobial resistance language use among the One Health domains. Journal of Antibiotics 10 (4): 385. doi: 10.3390/antibiotics10040385
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Supporting the Implementation and Monitoring of Selective Dry Cow Therapy (SDCT) on NY State Dairy Farms. 55th Annual Conference of American Association of Bovine Practitioners. Long Beach, CA.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Schueler, J., Naas, K., Hurst, J., Aga, D.D., Lansing, S., 2021. Effects of on-farm dairy manure composting on tetracycline content and nutrient composition. Journal of Antibiotics 10 (4): 443. doi: 10.3390/antibiotics10040443
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Schueler, J., Lansing, S., Crossette, E., Naas, K., Hurst, J., Raskin, L., Wigginton, K., Aga, D.S., 2021. Tetracycline, sulfadimethoxine, and antibiotic resistance gene dynamics during anaerobic digestion of dairy manure. Journal of Environmental Quality 50(3): 694-705. doi: 10.1002/jeq2.20211
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Lansing, S., 2021. Quantifying cattle manure-AMR perceptions and treatment system variabilities to develop a novel communication framework for conveying AMR science and mitigation opportunities: A4171 Food Safety Challenge Area Mitigating Antimicrobial Resistance. USDA-AFRI Project Directors Meeting. Virtual Meeting. May 3-5, 2021.
• Type: Websites Status: Published Year Published: 2021 Citation: The team created a project brand titled iAMResponsible to represent the project, increase visibility of the project and our efforts, and build trust in project outputs. The iAMR Project has the following on-line outlets in use or development: Twitter: https://twitter.com/i_AMResponsible Facebook: https://www.facebook.com/iAMResponsibleEDU Instagram: https://www.instagram.com/iamresponsibleedu/ YouTube: https://www.youtube.com/channel/UC4cO1Gr80Y8skUWYk_try3g Website: https://iamrproject.unl.edu/
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Poindexter, C., Lansing, S. Yarberry, A., Rice, C., Georgakakos, C., Gooch, C. (2021) High Temperature Rotary Drum Composting and Solid-Liquid Separation Impact on Antibiotic Resistance. American Society for Agricultural and Biological Engineering. (Virtual) (Abstract Accepted) July 12-16, 2021.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Poindexter, C, Yarberry, A., Rice. C., Lansing, S. (2021) Comparative Analysis of High-Temperature Rotary Drum Composting and Thermophilic Anaerobic Digestion Effects on Antibiotic Resistance in Dairy Manure. American Society for Microbiology (Abstract Accepted) June 24, 2021.

Progress 05/01/20 to 04/30/21

Outputs
Target Audience:The target audience for the iAMResponsible Project's outreach efforts include livestock producers, food safety experts, extension educators, consumers, veterinarians, medical professionals, and policymakers. Approximately 3,500 persons are following one or more of the iAMResponsible Project social media accounts with strong representation by medical professionals, educators, and AMR researchers worldwide. Engagement events for social media content totaled just over 3000 during the period of April 2020 to March 2021. Changes/Problems:COVID-19 Impacts: We had plans to present initial findings at the American Association of Geographers Annual Meeting in April 2020 and the American Ecological Engineering Society Meeting in June 2020. Both conferences were canceled due to COVID-19. Due to the global covid-19 pandemic, experiments and analyses in Objective 2 were postponed in 2020 due to campus closures and covid regulations that significantly delayed sample analysis and experiment execution. The thermohydrolysis anaerobic digestion study initially set to take place in summer of 2020 has been postponed for a year, but is planned to be conducted, as proposed, in 2021. Conducting in-person interviews or Q-sorts is no longer feasible in the near future. Fortunately, our Q-sort activity can be done entirely online. Future follow-up interviews can be done using WebEx, phone calls, etc., if needed due to COVID-19. We have already done some interviews this way, so this is not anticipated to be a problem. Change in PI at Cornell: The outreach effort in New York States will be now be led by Daryl V. Nydam. He is a Professor at Cornell in the Dept. of Population Medicine and Diagnostic Science in the College of Veterinary Medicine. He also serves as the Faculty Director of the Cornell Atkinson Center for Sustainability. Curt Gooch, who was a co-PI on the project is on leave from Cornell, and Dr. Nydam has stepped in as the co-PI at this time. A postdoctoral associate will work with Dr. Nydam in each region to: 1) identify farms to enroll in the program; 2) facilitate management meetings between the herd's local veterinarian of record, farm management, and QMPS personal to 2a) conduct baseline mastitis risk assessment, 2b) implement pathogen based treatment of clinical mastitis (PBTCM) and/or selective dry cow therapy (SDCT), 2c) collect data on key performance indicators (e.g. SCC, clinical mastitis, drug savings); 3) write up farm specific reports; and 4) lead peer-reviewed publications and lay publications about antibiotic stewardship. What opportunities for training and professional development has the project provided?iAMResponsible Team members participated in training to improve scientific communication, by attending an August 2020 webinar on methods for improving science and health communication. Team members were also given the opportunity to utilize programs such as Canva, Hootsuite, Camtasia, and Airtable to improve effective communication using images, video, and content management. The University of Maryland has involved on high school student, two college undergraduate students, and one PhD student, who are involved in conduct research under this project, with the PhD student involved in mentoring as well. The students include two African American males directly supported in this project: one PhD student and one undergraduate student, as well as one female Latinx high school intern. The University of Maryland- Baltimore County study has involved one PhD student and two undergraduates mentored. How have the results been disseminated to communities of interest?Primary dissemination of extension materials has been via social media, web-publications, as part of course content for seminar students, and virtual professional conferences. Initial results from our interviews conducted by UMBC were presented at a workshop at Cornell University's College of Veterinary Medicine titled "Understanding Antimicrobial Resistance in Animal Agriculture". Our talk summarized our interview findings by discussing the diverse perceptions of antibiotic use and regulations across the many actors in the dairy and beef industries. The workshop was attended by approximately 100 people from nearby academic and farming communities. We attended the Nebraska Cattlemen's 2019 mid-year conference to engage with stakeholders and to introduce our project and initial findings to the Animal Health and Nutrition committee meeting. What do you plan to do during the next reporting period to accomplish the goals?University of Maryland: The antibiotic detection method manuscript will be completed and submitted to Chemosphere. Moreover, the developed LC-MS/MS antibiotic method previously described in Objective 2a will be utilized for antibiotic in manure and wastewater biosolids following thermal hydrolysis treatment and subsequent anaerobic digestion (Objective 2d). The method will also be incorporated into future mobile genetic element experiments for holistic assessment of antibiotics and other ecological parameters on gene movement. The metagenomic analyses of ARGs for both studies (Objectives 2b and 2c) is currently on-going. This will include completion of qualitative Polymerase Chain Reaction experiments, and the M13 sequencing and gene verification of the 10 selected primers/genes, 1 mobile genetic element (intl1), 1 macrolide (ermB), 1 beta-lactam (bla-2), 2 sulfonamides (sul-1, sul-2), 4 tetracycline (tetX, tetM, tetW, tetQ), and 16S for total microbial mass. Additionally, relationship between ARGs, heavy metal resistance, and related resistance genes primers of interest for gene amplification will be explored. All data from the rotary drum composting study and batch mesophilic and thermophilic digestion studies will be statistically analyzed and prepared for publication, including antibiotics, ARGs, ARBs, heavy metals, and nutrients. Pilot-scale thermal hydrolysis digestion trials (Objective 2d) will begin in 2021 using the 50 L thermal hydrolysis unit at DC Water with manure and wastewater samples tested and compared using varying conditions and antibiotic spikes. Mobile genetic element experiments will be conducted to investigate the impact of waste treatment on the movement of mobile genetic elements in the dissemination of ARGs. In-depth laboratory simulations of waste treatment conditions using a mixture of methods including bacterial culturing, sequencing, and microfluidics will be conducted to monitor mechanisms of horizontal gene transfer and potential influencers. University of Nebraska- Lincoln During the next reporting period, the iAMResponsible team will continue to expand our partnership with the LPELC to create regular content for the lpelc.org website on AMR and improve our AMR library to incorporate and make accessible research findings from a wider selection of contributors. We will seek to develop new partnerships with 4-H and consumer-facing food safety extension professionals to expand our reach with non-scientific audiences. We intend to re-run the surveys we delivered via social media during the spring of 2021 to assess any changes in audience knowledge and perception of AMR over the preceding year. Cornell University To provide outreach to veterinarians and dairy livestock producers, we have developed a plan to make pathogen-based treatment of clinical mastitis (PBTCM) and/or selective dry cow therapy (SDCT) available to any farm in New York State where they are likely to be successful, educating the farmers as well as reducing the incidence of mastitis. Our specific goal is to reach approximately 50,000 - 100,000 more cows on about 50 farms (some farms will milk 150 cows, while others could milk 2000). This would put dairy farming in a leadership position nationwide and ahead of mandates by consumers, advocacy groups, or in fact the government in terms of AMU and AMR. We aim to empower individual dairy farmers, their veterinarian of record, and other advisors to make prudent antibiotic decisions locally in a manner that best suits the well-being of their cows and farms addressing the nuances of individual farms and cows. This project will benefit dairy producers and our industry demonstrating to consumers that dairy farmers are working to control disease on their farms to ensure good animal welfare, are stewards of antibiotic use, and maintain antibiotic decision making at the farm level. In achieving these goals, we will preserve and perhaps increase demand for dairy products, decrease money spent on antibiotics without decreasing health and performance of cows, assist in keeping residues low, and thus increase profits for farmers who continue to make healthful, safe, and affordable dairy products for consumers. University of Maryland Baltimore County (UMBC) For the social science work, we will conduct our Q statement sorting activity with a broad range of stakeholders. Many of our previously interviewed subjects will conduct the Q-sort. Once enough participants have conducted the sort, we will analyze the data using a statistical technique called a factor analysis. The factor analysis will identify common patterns in the sorts, allowing us to "map" the diverse perspectives around the relationship between animal health and antibiotic use and to identify the type of actors that hold these various perspectives. We intend to complete publications based on our interviews and the Q statement activity. We intend to present our findings at conferences.

Impacts
What was accomplished under these goals? Objective 1: Analyze stakeholder AMR perception, knowledge, and decision-making Last year, we conducted 28 interviews with a diverse range of stakeholders including dairy farmers, beef cattle farmers, veterinarians, extension agents etc. that were analyzed and reported the previous year. This year, we expanded our pool of interviewees and conducted 2 focus groups with meat product consumers about their perceptions of antibiotic use in animal agriculture. We found consumers tended to either not know much about antibiotic use or had very negative views of antibiotic use in animal agriculture. We have analyzed the transcripts from all interviews and focus groups and have created a Q-sort concourse. We completed a pilot Q-sort study of this concourse with 13 participants to test the online platform as well as the relevance of the statements that the Q-sort participants were asked to sort. After making a few adjustments to the Q-sort concourse based on the pilot study feedback, we are currently contacting stakeholders for the actual Q-sort survey. We hope to conduct approximately 30 Q-sorts in the next few months. Objective 2a: Creation of an improved antibiotic extraction method: Validation experiments and analysis were conducted for the finalized liquid chromatography in tandem with triple quadrupole mass spectrometry (LC-MS/MS) method that was developed. The method included a two-step extraction process that utilizes ultrasonic and mechanical mixing using two separate solvents (EDTA-McIlvaine followed by methanol addition), followed by a solid phase extraction through a C-18 cartridge, which is eluted with methanol and concentrated using N2 gas. The final concentrate is reconstituted using 1:1 acetonitrile and DI water mixture and stored in the freezer until analysis on the LC-MSMS. The methods total recoveries across manure substrates were 66 - 131% for tetracycline, 56% for sulfonamide, 47 - 55% for macrolides and 0 - 57% for β-lactams using a single extraction process for a complex mixture. All data and statistical analyses on the method development experiments have been completed. The manuscript is currently in final preparation and will be submitted Chemosphere in 2021. Objective 2b: Effect of rotary drum composting bedding recovery unit (BRU) on AMR: Complete analysis of the bedding recovery unit (BRU) management systems required sampling of four different substrates: 1) the unprocessed manure, 2) the liquid fraction generated from a screw press separator, 3) the solid separated fraction, and 4) the final BRU product. The solid fraction was collected from the top of the screw and is used as the influent to the BRU unit. All the samples were collected over a 24-hour periodto show partitioning of antibiotics, bacteria, and their corresponding resistance genes. Thesampling data was collected during the previous year and was analyzed in the laboratory during this period. Supplementary analysis of nutrient (nitrogen and phosphorus) and heavy metals were completed on the manure, the liquids, solids, and BRU samples, with the data analyzed using a correlation analysis of partitioning of antibiotics, bacteria, and their corresponding resistance genes within the sample and their relationships to metal, nutrient, and carbon partitions. The total mass of manure processed during the 24-hour sampling period was 7730 kg/hr. The results showed that 95% (7320 kg/hr) of the mass fractionated with the liquid separated portion, which was piped directly to a nearby storage lagoon, while 5% (410 kg/hr) of the manure continued to the high temperature BRU processing. Our analyses showed that nitrogen and phosphorus concentrations follow a similar partitioning trend as the mass flow, with higher nutrient concentrations associated with the liquid fraction. Zinc, copper, iron, aluminum, manganese, and boron were all detected in the manure. The antibiotics followed a similar pattern as the manure, with 68-78% of the tetracyclines, 68% of tulathromycin, and 40-50% of the beta-lactams partitioning with the liquid separated fraction of the raw manure. The antibiotics fractions that remained in the solid separated manure into the BRU inlet included 12% of the tetracycline, 14% of the 4-epitetracycline, and 20% of the tulathromycin. The manuscript of the study results is currently being written for submission during the next study period. Objective 2c: Effect of mesophilic and thermophilic anaerobic digestion on AMR: Dairy manure from the cooperating dairy farm was assessed using lab-scale batch anaerobic digestion testing at mesophilic (35°C) and thermophilic (55°C) conditions, with spiked antibiotics injection of oxytetracycline, ampicillin, and erythromycin, over a 43-day retention period. The effect of time and temperature were evaluated using triplicate bottles that were destructively sampled at 6 time points (Days 0, 3, 9, 21, 32, 43). The samples were analyzed to determine the effect of time on degradation to develop time and temperature antibiotic degradation curves, with post-testing of antibiotic concentration, presence of ARB, ARGs, and manure characteristics, including nutrients and metals. Total biogas and methane production from the manure digestion were also measured. The results showed that 35°Chad greater methane (CH4) production with 259 ml CH4/g VS in the control (no antibiotics added) and 234 ml CH4/g VS in the triplicate samples spiked with the antibiotic mixture. At 55°C, the CH4 production in the control (234 mL CH4/g VS) was much higher than the samples spiked with antibiotics (160 mL CH4/g VS. The antibiotic reduction in 35°C reactors were 21, 84, and 100% for oxytetracycline, ampicillin, and erythromycin, respectively, while the 55°C reactors had reductions of 67, 49, and 97%,respectively, resulting in the 55°C digesters having greater tetracycline degradation, while 35°C conditions had 100% removal of erythromycin and much higher degradation of ampicillin. Objective 3: Create an effective communication framework for AMR outreach: The iAMResponsible team continues its collaboration with the Livestock and Poultry Environmental Learning Community (LPELC). Cooperative efforts with the LPELC included an expansion of research-based resources on AMR for community members and the development of a new focus area for iAMR outreach efforts: science communication; specifically, the provision of both training and resources to extension and other STEM professionals to empower their communication efforts on AMR. The iAMR team further expanded their online course on AMR from a One Health perspective with emphasis on developing scientific communication skills. During the Spring of 2021, twenty-four students at five universities (Nebraska, Maryland, North Carolina State, Washington State, and Minnesota) participated in the course. Four on-line social media outlets for the dissemination of AMR related materials are managed with monthly, weekly, or multi-weekly outputs. Over 50 new pieces of outreach content were created during the reporting period for distribution on social media and added to the existing library of some 400 pieces of media and research related to AMR curated by the iAMR team and available to the public on the LPELC website. During the spring of 2020, the team conducted a series of social media surveys to assess audience knowledge and attitudes toward AMR, food safety, and toward the iAMR Project. Survey results indicate that the team has utilized social media effectively to build a recognized and trusted brand among medical professionals, educators, and AMR researchers worldwide. However, outreach on social media has proved less effective for reaching audiences previously unaware of AMR and its potential impact on food safety.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Oliver, J. P., Hurst, J. J., Gooch, C. A., Stappenbeck, A., Sassoubre, L., & Aga, D. S. (2021). On-farm screw press and rotary drum treatment of dairy manure-associated antibiotic residues and resistance. Journal of Environmental Quality, 50(1), 134143. https://doi.org/10.1002/jeq2.20161
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Schueler, J.; Naas, K.; Hurst, J.; Aga, D.; Lansing, S. Effects of On-Farm Dairy Manure Composting on Tetracycline Content and Nutrient Composition. Antibiotics 2021, 10, 443. https://doi.org/10.3390/antibiotics10040443
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Schueler, J.; Lansing, S.; Crossette, E.; Naas, K.; Hurst, J.; Raskin, L.; Wigginton, K.; Aga, D.; Tetracycline, sulfadimethoxine, and antibiotic resistance gene dynamics during anaerobic digestion of dairy manure Journal of Environmental Quality 2021, 1, 1-12. https://doi.org/10.1002/jeq2.20211.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Poindexter, C., Lansing, S. Yarberry, A., Rice, C., Georgakakos, C., Gooch, C. 2020. Effects of High Temperature Manure Treatment Technologies on Antibiotics, Antibiotic Resistance Genes, and Antibiotic Resistance Bacteria. Oral. American Society for Agricultural and Biological Engineering. Omaha, NE. July 12-15, 2020. (Abstract Accepted)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Poindexter, C., Lansing, S. Yarberry, A., Rice, C., Georgakakos, C., Gooch, C. 2020. Effects of Pasteurization and Anaerobic Digestion on Antibiotics and Antibiotic Resistance. Poster. American Society of Microbiology. Chicago, IL. June 18-24. (Abstract Accepted)
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Poindexter, C., Lansing, S. Yarberry, A., Rice, C., Georgakakos, C., Gooch, C. 2020. The Impact of High Temperature Treatment Technologies on Mitigating Antibiotic Resistance in Dairy Manure. Oral. American Ecological Engineering Society. Columbus, OH. June 9-11, 2020. (Abstract Accepted)
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zelt, M., A.M. Schmidt, Z. Staley, X. Li, B. Wang, and D. Miller. Antibiotic resistance profiles in fallow soil receiving raw, composted or stockpiled beef manure, or inorganic fertilizer. 2020 World Congress on One Health. Nov 3, 2020. Edinburgh, Scotland.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zelt, M., and Schmidt, A.M. (2020) The iAMResponsible Project: Building a communication network to motivate broad action on antimicrobial resistance. 2020 ASABE International Meeting, Omaha, NE. July, 14 2020.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zelt, M. and A.M. Schmidt. The iAMResponsible project: Building a communication network to motivate broad action on antimicrobial resistance. Midwest AMR Consortium. Des Moines, IA. May 26-29, 2020.
• Type: Websites Status: Published Year Published: 2020 Citation: Schmidt, A.M.. Antimicrobial Resistance is Native to the Environment. lpelc.org. June 3, 2020. https://lpelc.org/tag/iamresponsible/page/2/
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Zelt, M., and A.M. Schmidt. Understand. Adapt. Preserve. Increasing knowledge and motivating behavioral changes among food producers and consumers to preserve the efficacy of antibiotics through the iAMResponsible" Project. 2020 World Congress on One Health. Nov 3, 2020. Edinburgh, Scotland.
• Type: Websites Status: Published Year Published: 2020 Citation: Mware, N.A. Antimicrobial Resistance in Developing Countries: Current State and Controlling Strategies. lpelc.org. June 19, 2020. https://lpelc.org/antimicrobial-resistance-in-developing-countries-current-state-and-controlling-strategies/ Nixon, K. Are there alternatives to antibiotics? lpelc.org. July 3, 2020. https://lpelc.org/are-there-alternatives-to-antibiotics/ Zelt, M. How do you like your steak? lpelc.org. August 6, 2020. https://lpelc.org/how-do-you-like-your-steak/
• Type: Websites Status: Published Year Published: 2020 Citation: Schmidt, A.M, K. Nixon, L. Hernandez, M. McKasy, and L. Johnson. Communicating Science Using the Science of Communication. lpelc.org. August 18, 2020. https://lpelc.org/communicating-science-using-the-science-of-communication/
• Type: Websites Status: Published Year Published: 2020 Citation: Harris, A. An array of veterinary antibiotics has been found in water and soil samples. lpelc.org. September 17, 2020. https://lpelc.org/an-array-of-veterinary-antibiotics-has-been-found-in-water-and-soil-samples/
• Type: Websites Status: Published Year Published: 2020 Citation: Zelt, M. Antibiotic resistance higher in environments impacted by human or animal waste. lpelc.org. November 2, 2020. https://lpelc.org/antibiotic-resistance-higher-in-environments-impacted-by-human-or-animal-waste/
• Type: Websites Status: Published Year Published: 2020 Citation: Henning, E. Application of organic fertilizers increases antibiotics in soil. lpelc.org. November 23, 2020. https://lpelc.org/application-of-organic-fertilizers-increases-antibiotics-in-soil/
• Type: Websites Status: Published Year Published: 2020 Citation: Henning, E. Composting can reduce antimicrobial resistance in manure. lpelc.org. December 14, 2020. https://lpelc.org/composting-can-reduce-antimicrobial-resistance-in-manure/
• Type: Websites Status: Published Year Published: 2021 Citation: Trout, Z. The growth of antibiotic resistance has become a serious threat to human health. lpelc.org. February 25, 2021. https://lpelc.org/the-growth-of-antibiotic-resistance-has-become-a-serious-threat-to-human-health/

Progress 05/01/19 to 04/30/20

Outputs
Target Audience:Our target audience for the outreach efforts include livestock producers, food safety experts, extension educators, consumers, veterinarians, medical professionals ,and policymakers. In-person events produced roughly 1000 individual contacts to diverse audiences working in agriculture, scientific research, college students, and community members at locations across the United States. Approximately 3,000 persons are following one or more of the iAMR Project social media accounts with strong representation by medical professionals, educators, and AMR researchers worldwide. Engagement events for social media content totaled just over 9,500 during the period of April 2019 to January 2020. Changes/Problems: We had plans to present initial findings at the American Association of Geographers Annual Meeting in April 2020 and the American Ecological Engineering Society Meeting in June 2020. Both conferences were canceled due to COVID-19. Conducting in-person interviews or Q sorts in no longer feasible in the near future. Fortunately, our Q sort activity can be done entirely online. Future follow up interviews can be done using Webex, phone calls etc. if needed due to COVID-19. We have already done some interviews this way, so this is not anticipated to be a problem. The thermohydrolysis anaerobic digestion study will be conducted once the research labs at University of Maryland re-open, presumably sometime this summer. While data analysis is on-going, laboratory work has been suspended due to COVID-19. What opportunities for training and professional development has the project provided?Team members participated in training to improve scientific communication, specifically by attending two professional conferences emphasizing communication on AMR: Human Dimensions of AMR (Nebraska City, NE, May 30-31st 2019), and 9th Annual National Institute for Animal Agriculture antibiotic symposium (Ames, IA, October 15-17 2019). Members were also given opportunity to utilize programs such as Canva, Adobe Spark, Animaker and Airtable to improve effective communication using images, video and content management. The University of Maryland has involved four college undergraduate students, and two PhD. students who assist, train and conduct research under this project, and one MS student, including three African American males directly supported in this project: one PhD student and two undergraduate students The University of Maryland- Baltimore County has involved one PhD student and two undergraduates mentored. How have the results been disseminated to communities of interest?Primary dissemination has been via social media and at professional, educational, and community events. Initial results from our interviews conducted by UMBC were presented at a workshop at Cornell University's College of Veterinary Medicine titled "Understanding Antimicrobial Resistance in Animal Agriculture". Or talk summarized our interview findings by discussing the diverse perceptions of antibiotic use and regulations across the many actors along dairy and beef industry. The workshop was attended by approximately 100 people from nearby academic and farming communities. We attended the Nebraska Cattlemen's 2019 mid-year conference to engage with stakeholders and to introduce our project and initial findings to the Animal Health and Nutrition committee meeting. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will have the team website fully up and running, creating a more permanent platform to host the resources we have developed. We will partner with the LPELC to create regular content for the lpelc.org website on AMR. We will continue to expand our content library to incorporate and make accessible research findings from a wider selection of contributors and expand our content creation to include new media types such as video or podcasts. We will establish a regular mailing list/newsletter to promote newly added library materials to a national network of extension professionals, agricultural advisors, and consumer advisors such as doctors, nutritionists and teachers. We will continue to search for additional opportunities and collaborations to promote these resource platforms targeted at our intended audience. Evaluation efforts over the next year will be conducted working with the Methodology and Evaluation Research Core (MERC) at the University of Nebraska-Lincoln to facilitate evaluation of existing and new AMR-related resources, content delivery methods, and changes in AMR-related knowledge and behavior among target audiences. Two surveys are currently being developed aimed at (1) evaluating the impact of social media outreach efforts in increasing awareness of AMR and gaging resultant behavioral changes; and (2) assessing needs of extension professionals across the country for development of new resources to support their understanding of AMR and inform the interactions they will have within their own educational and outreach networks. LC-MS/MS antibiotic extraction and detection method developed will be published through the manuscript currently be prepared. The metagenomic analyses of ARGs, including the completion of qualitative Polymerase Chain Reaction experiments, and the M13 sequencing and gene verification of the 10 selected primers/ genes and generation of calibration curves for gene abundance analysis within BRU and batch experiments will be completed. Additionally, relationship between ARGs, heavy metal resistance, and related resistance genes primers of interest for gene amplification will be explored. All data from the rotary drum composting study and batch digestion studies will be statistically analyzed and prepared for publication, including antibiotics, ARGs, ARBs, heavy metals, and nutrients. Pilot scale thermal hydrolysis digestion trials will begin using the 50 L thermal hydrolysis unit at DC Water with manure and wastewater samples tested and compared using varying conditions and antibiotic spikes. Mobile genetic element experiments will be conducted to investigate the impact of waste treatment on the movement of mobile genetic elements in the dissemination of ARGs. Through the conduction of in-depth laboratory simulations of waste treatment conditions using a mixture of methods including bacterial culturing, sequencing, and microfluidics to monitor mechanisms of horizontal gene transfer and potential influencers. For the social science work, we will conduct our Q statement sorting activity with a broad range of stakeholders. Many of our previously interviewed subjects will conduct the sort. Once enough participants have conducted the sort, we will analyze the data using a statistical technique called a factor analysis. The factor analysis will identify common patterns in the sorts, allowing us to "map" the diverse perspectives around the relationship between animal health and antibiotic use and to identify the type of actors that hold these various perspectives. We intend to complete publications based on our interviews and the Q statement activity. We intend to present our findings at conferences.

Impacts
What was accomplished under these goals? Numerous interviews with stakeholders were conducted and showed two distinct perspectives on best practices for animal health (individual vs herd) and the relationship between animal health and antibiotic use. A new, robust method development for antibiotic detection in the complex manure matrix was developed and verified. This method was used to assess the effects on rotary drum composting and anaerobic digestion in decreasing AMR. Analyses showed that both technologies can reduce various factors that contribute to antibiotic resistance, with large reductions in antibiotic resistant bacteria (ARB) during treatment. Our iAMR extension platform is rapidly growing, with a global audience. We have hosted workshops, lectures, and produced hundreds of social media graphics and content items to facilitate connection between researchers, agricultural personnel, and policy makers. As we generate a greater understanding of stakeholder's perspectives on AMR and further quantify the effect of advanced manure technologies on reducing AMR, more informed decisions and policies can be implemented by informed farmers, consumers, and stakeholders. Objective 1: Analyze stakeholder AMR perception, knowledge, and decision-making We conducted 28 interviews with a diverse range of stakeholders including dairy farmers, beef cattle farmers, veterinarians, extension agents, etc. Preliminary results found divergent views on best practices concerning animal health among farmers. Some farmers preferred a proactive herd-based approach toward health and antibiotic use, while others viewed a more individual cow approach. We also found divergent views on the relationship between animal health and antibiotic use. Some of the non-farming stakeholders consider less antibiotic use on the farm was linked to better animal welfare. Whereas, a significant number of farmers viewed less antibiotic more challenging for animal welfare. These contrasting perspectives around the relationship between antibiotics and animal health point to a specific area for education and information sharing. Objective 2a: Creation of an improved antibiotic extraction method A finalized liquid chromatography in tandem with triple quadrupole mass spectrometry (LC-MS/MS) method was developed uses a two-step process that utilizes ultrasonic and mechanical mixing using two separate solvent extractions, EDTA-McIlvaine and methanol and a solid phase extraction through a C-18 cartridge, which is eluted with methanol and concentrated under nitrogen gas. The final concentrate is reconstituted using 1:1 acetonitrile and DI water mix for analysis on the LC-MS/MS. This method was validated for 20 antibiotics covering 4 antibiotic classes. The resulting method achieved beta-lactam recoveries of 5 to 74%, tetracycline recoveries of 54 to 108%, sulfadimethoxine recovery of 49%, and macrolide recoveries of 50 to 97%. This method was used in the rotary drum composting study and batch digestion studies. Objective 2b: Effect of rotary drum composting bedding recovery unit (BRU) on AMR Complete analysis of the rotary drum manure management systems required sampling of four substrates: 1) unprocessed manure, 2) liquid fraction and 3) solid separated fraction of the manure generated by a screw press compression, and 4) the output of the BRU unit where all of the solid manure fraction proceeded. Samples were collected over a 24-hour period, starting with the initial addition of hospital barn manure into the inlet pit of the manure pit to the final 24-hour processing of the bedding product to show partitioning of antibiotics, bacteria and corresponding resistance genes. The total mass, mass flow, and total solids was determined using a corn kernel tracer test. The estimated residence time of BRU was determined to be 13-26 hours. Penicillin G, benzylpenicillioc acid, tulathromycin, tetracycline, and 4-epitetracycline were recovered from the manure. The beta lactams partitioned into the more aqueous substrates, while the tetracyclines and tulathromycin were more distributed in the solid substrate. The ARB analysis was based E.coli and Entercocci counts, with highest bacterial counts in the unprocessed and liquid separated samples. With significantly lower bacterial counts from the separated solids and minimal to no bacterial colonies recovered from the final BRU substrate. Objective 2c: Effect of mesophilic and thermophilic anaerobic digestion on AMR Dairy manure from same farm as the BRU unit was tested using lab-scale batch anaerobic digestion testing at mesophilic (35°C) and thermophilic (55°C) conditions, with spiked antibiotics injection of oxytetracycline, ampicillin, and erythromycin, over a 43-day digestion period. The effect of time and temperature were tested using triplicate bottles that were destructively sampled at 6 time points (Days 0, 3, 9, 21, 32, 43) to develop time and temperature antibiotic degradation curves, with testing of antibiotic concentrations, presence of ARB, antibiotic resistance genes (ARG), and solids and organic content. Total biogas and methane production were measured throughout the batch process. Percent reduction in antibiotics (oxytetracycline, ampicillin, and erythromycin) during mesophilic digestion were 21, 84, and 100%, respectively and 67, 49, and 97%, respectively, during thermophilic digestion. Thermophilic conditions had greater tetracycline degradation, while mesophilic conditions had greater degradation efficiency for the macrolide erythromycin. The ARB analyses showed that both digestion temperature had complete reduction of ARB by Day 9 of the 43-day digestion period. DNA extractions protocols and primer sets were troubleshooted to determine the protocol bested suited for optimal yields across all substrates and suite of genes being analyzed. Ten primers were selected for further progression of qualitative polymerase chain analysis for the BRU unit and the digestion reactor samples. Objective 3: Create an effective communication framework for AMR outreach The iAMR outreach project team was expanded to include institutions in four new states (NY, CA, WA, CO). Collaboration the Livestock and Poultry Environmental Learning Community (LPELC) helped to expand the nationwide capacity to deliver AMR related materials to agricultural producers. Two on-line social media outlets for dissemination of AMR related materials are managed with weekly or multi-weekly outputs. Content includes videos, summaries of published research, and press articles curated by the team to build on and expand audience understanding of AMR. Over 150 pieces of graphical content were created for distribution on social media and print in the past year. These infographics are intended to help illustrate AMR related scientific research, spread awareness of the basic concepts of AMR, describe current public health concerns, and provide information around best practices the intended audience can take to minimize potential AMR-related risks. In partnership with LPELC, the project team delivered the first of a series of webinars on AMR impacts on agriculture aimed at agricultural producers and their advisors. A preliminary evaluation on the impact of outreach efforts on Twitter was conducted. Moving forward, we will work to broaden the project's reach among scientific and medical experts currently following, with focused impact on reaching audiences previously unaware of AMR. Team members from two participating Universities, UMD and UNL, developed an online college AMR course that was jointly delivered in Spring 2020. Nine graduate students enrolled in the course learned about AMR from a One Health perspective and developed scientific communication skills by contributing new materials to the team's outreach database.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Oliver, J., C. Gooch, S. Lansing, J. Schueler, J Hurst, L. Sassoubre, E. Crossette and D. Aga, 2020. Invited Review: Fate of antibiotic residues, antibiotic-resistant bacteria, and antibiotic resistance genes in US dairy manure management systems. Journal of Dairy Science 103:1051-1071. doi: 10.3168/jds.2019-16778.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Glaize, Ayanna, E. Gutierrez-Rodribuez, I. Hanning, S. Diaz-Sanchez, C. Gunter, A H.M. van Viet, W. Watson and S. Thakur, 2019. Transmission of antimicrobial resistant non-O157 Escherichia coli at the interface of animal-fresh produce in sustainable farming environments. International Journal of Food Microbiology, 319. doi: 10.1016/j.ijfoodmicro.2019.108472
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hurst, J.J., J. Oliver, J. Schueler, C.A. Gooch, S. Lansing, E. Crossette, K.R. Wiggington, L. Raskin, D. Aga and L.M. Sassoubre, 2019. Trends in antimicrobial resistance genes in manure blend pits and long-term storage across dairy farms with comparisons to antimicrobial usage and residual concentrations. Environmental Science & Technology 53(5): 2405-2415. doi: 10.1021/acs.est.8b05702.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Lansing D.M and J. Barrett, 2019. Contrasting Perspectives on Prudent Use of Antimicrobials in Agriculture. Seminar on Understanding Antibiotic Use and Resistance in Animal Agriculture. Cornell University, College of Veterinary Medicine. Ithaca, NY. December 5, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Lansing, S., 2019. Anaerobic digestion of manure, algae, food waste, and wastewater for energy production, nutrient transformations, and reduction of antimicrobial resistance. John Hopkins School of Public Health Wolman Seminar. Baltimore, MD. April 9, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Lansing, S., 2019. Anaerobic digestion of waste for meeting sustainability goals. University of Oklahoma Microbiology Earth Month Seminar. Norman, OK. April 12, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Lansing, S., 2019. On-farm anaerobic digestion. Maryland Energy In-Service Training. Ellicott City, MD. October 21, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Lansing, S., C. Cottrell and R. Sharif, 2019. Antimicrobial resistance. University of Maryland Health Center: Student Health Advisory Committee. College Park, MD. November 20, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Lansing, S., 2020. Bioenergy technologies in the food, water, energy nexus. University of Maryland Global STEWARDS Annual Meeting. College Park, MD. January 24, 2020.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Lansing, S., J. Schueler, E. Crossette, K. Naas, J. Hurst, J. Oliver, L. Raskin, K. Wiggington, C. Gooch and D. Aga, 2019. Effect of microbial treatment processes on antimicrobial resistance (AMR): Digestion and composting. American Ecological Engineering Society. Asheville, NC. June 3-6, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Poindexter, C. 2019. The Scoop on Poop Tech: Antibiotic and Manure Management [video webinar]. Managing Manure to Mitigate Antibiotic Resistance. University of Lincoln-Nebraska. Available at: http://learn.extension.org/events/3704. November 15, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Yarberry, A., C. Rice, C. Poindexter, and S. Lansing, 2019. Use of Triple Quadrupole Mass Spectrometry to Characterize Antibiotics in Cow Manure. Poster. American Society of Mass Spectrometry. Atlanta, GA. June 2-6, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Zelt, M., A.M. Schmidt, N. Mware, X. Li, Z. Staley, H. Wilson and G. Erickson. 2019. Antimicrobial resistance in beef feedlot manure as impacted by forage concentration and essential oil in finishing cattle diets. 2019 ASABE International Meeting. Boston, MA. July 9, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2019 Citation: Zelt, M., A.M. Schmidt, Z. Staley, X. Li, B. Wang and D. Miller 2019. Antibiotic resistance profiles in fallow soil receiving raw, composted or stockpiled beef manure, or inorganic fertilizer. 2019 North American Manure Expo. Fair Oaks, IN. August 30, 2019.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Barrett, J., 2020. Animal Health decision-making for U.S. Beef and Dairy: A Political Ecology. American Association of Geographers Annual Meeting. Denver, CO. April 2020. Abstract Accepted. Conference Cancelled due to COVID-19.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: Lansing, D.M., 2020. Knowing Bodies: Antimicrobial governance across agrifood commodity chains. American Association of Geographers Annual Meeting. Denver, CO. April 2020. Abstract Accepted. Conference Cancelled due to COVID-19.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Schmidt, A.M., 2020. iAMResponsible": Educating food producers & consumers about antimicrobial resistance. Scientia. January 8, 2020. doi:10.33548/SCIENTIA460
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Johnson, L., A.M. Schmidt, C. Poindexter, X. Li and M. Zelt, 2019. Managing manure to mitigate antibiotic resistance. Livestock and Poultry Environmental Learning Community Webinar. November 15, 2019.
• Type: Websites Status: Published Year Published: 2020 Citation: Schmidt, A., Zelt, M., 2020. iAMR Project. Available at: http://www.iamrproject.com
• Type: Other Status: Published Year Published: 2019 Citation: Schmidt, A.M., 2019. Research and Outreach Highlight: i(AM)Responsible, a nationwide outreach network on antimicrobial resistance. Nebraska One-Health Newsletter. 1 November 2019.

Progress 05/01/18 to 04/30/19

Outputs
Target Audience: Our target audience for the outreach efforts include livestock producers, veterinarians, extension educators, consumers, and policymakers. Approximately 600 extension educators, university faculty, non-governmental organizations, government agencies, veterinarians, farmers, feed suppliers, and other agro-industry businesses in animal agriculture and allied fields were reached through professional conference outputs and workshops. An October workshop was planned by PI Lansing with two presentations on AMR. The workshop consisted of 85 participants that included a mix of agro-industry businesses (37%), government agencies (23%), Extension/university personnel (20%), NGOs, and farmers. Additionally, a presentation was given at the MidAtlantic Crop Management School to over 100 farmers, extension agents, NGOs, and agro-industry professionals. Approximately 1,400 persons are following one or more of the iAMR Project social media accounts with strong representation by medical professionals, educators and AMR researchers worldwide. Engagement events for social media content totaled just over 3,200 during the first six months of operation (Oct 2018 - March 2019). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The University of Maryland has involved four college undergraduate students, and two PhD. students who assist, train and conduct research under this project, and one MS student, including two African American males directly supported in this project: one PhD student and one undergraduate student. Team members participated in training to improve scientific communication, improve their use of social media, and develop personal and professional "brands". Members were also given opportunity to utilize programs such as Canva, Adobe Spark and Animaker to improve effective communication using images and video. How have the results been disseminated to communities of interest? Primary dissemination has been via social media, at a special conference session targeting a national Extension audience, at the MidAtlantic Crop Management School, a workshop for manure management, academic conferences, and dairy industry conferences. Additionally, we held an Advisory Committee meeting (20 participants) on January 7, 2019 where we received valuable feedback on the project goals, progress to date, and other avenues of inquiry to consider. Due to the government shut down, our USDA colleagues could not join the meeting, but the meeting was recorded and transcribed to allow all project participants to receive the important feedback from the Advisory Committee. What do you plan to do during the next reporting period to accomplish the goals? Stakeholders Perceptions Interviews and surveys of dairy and beef cattle farmers and associated stakeholders will continue. After interviews are analyzed, a set of statements will be presented to stakeholders for a Q-sort analysis (Goal 1#). This Q-sort will be analyzed and results written. Findings will be presented at two conferences and workshops. iAMR During the next reporting period, we will have the team website fully up and running, creating a more permanent platform to host the resources we have developed. Continue to expand our content library to incorporate and make accessible research findings from a wider selection of contributors. Search for additional opportunities and collaborations to promote these resource platforms targeted at our intended audience University of Maryland: LC-MS-Antibiotic Detection/ Extraction Method Development Revisit Karaca method using non-freeze-dried samples, a n-hexane lipid extraction step, and matrix adjusted calibration curves. Determine recoveries using Berendsen method varying final reconstitution pH for better analyte ionization prior to entering MS source. Continue developing and fine-tuning methodology for new UPLC-MS-MS. Continue work with deuterated antibiotics for antibiotic quantification. Metagenomic Analysis Optimize intracellular DNA (iDNA) and extracellular DNA (eDNA) extraction protocols for bulk manure samples. Trouble shoot and select appropriate ARG and related resistance genes primers for gene amplification. Begin DNA extractions from manure collected by field teams Start data analysis to determine relative and absolute abundances of resistance from both metagenomic data and qPCR assays High Temperature Manure Treatment Trials Manure sample collection from the rotary drum composting unit (Cornell, NY) will commence with samples analyzed pre and post-composting. Pilot scale thermal hydrolysis digestion trials will begin (DC water), with manure samples and wastewater samples tested and compared using varying conditions and antibiotics spikes Lab-scale thermophilic digestion (UMD) trials will also be conducted an compared to mesophilic digestion and the effect on AMR.

Impacts
What was accomplished under these goals? Objective 1: A survey was created to assess stakeholder AMR perception, knowledge, and decision-making, changes in antibiotic use since the introduction of the veterinary feed directive, and associated concerns/opportunities with the changing policy. To date, we have 64 survey participants, including dairy farmers, veterinarians, and feed suppliers and have conducted ten interviews with dairy farmers. Data collection is ongoing, with more survey participants expected and additional interviews scheduled. A nationwide team of university extension faculty representing all regions of the country - North Central, Western, Southern, and Northeast - was established and meets monthly via Zoom. Members represent disciplines of food science, veterinary medicine, engineering, animal science, and communications, with producer- and consumer-facing extension appointments in their respective institutions. The team has organized a special conference session at the 2019 Waste-to-Worth Conference to highlight the shared responsibility among all sectors of society to address AMR. Invited speakers from academic and ARS appointments are experts in environmental, agricultural and food safety-related AMR, national and international AMR policy, etc. Participants at the conference session represent university extension, technical service provider agencies, conservationists and university faculty and students working in areas associated with livestock production nationwide. The purpose of this session is to improve understanding of the complex issue of AMR among participants. Objective 2: Antibiotic quantitation method development and multi-residue extraction protocols for antibiotics from manure were explored to support the overarching goal of determining how various thermal treatments affect AMR activity in manure. Method development for the quantitation of 14 chosen antibiotics using a liquid chromatography tandem triple quadrapole mass spectrometer was conducted. A derivitization method using piperidine was investigated to prevent rapid degradation of beta-lactam drugs in manure prior to extraction and testing. To determine the requirements for a sampling protocol, methodologies for sample preparation and extraction were investigated with the goal to develop an expedient and accurate antibiotic measurement protocol. The four extraction methods detailed below were the primary foundation for method development. Each extraction method was experimentally replicated to determine feasibility, data reproduction, and applicability in terms of recoveries for the target antibiotics. Jacobsen and Halling-Sorensen, 2006 The liquid manure was freeze-dried and pulverized, followed by a three-part extraction. This extraction method used accelerated solvent extraction (ASE) for sample organics removal, followed by liquid-liquid extractions using heptane to remove lipids, and a final solid phase extractions (SPE) conducted via stacked SAX-HLB cartridges. Of the antibiotics spiked, six antibiotics were detected with recoveries from 0.36 to 53%. Van Holthoon et al., 2010 The manure was centrifuged to separate the liquid and solid fractions. Liquid Fraction "milk-method": A phosphate buffer solution was added to separated liquid samples, centrifuged, and treated with piperidine for beta-lactam derivatization. Samples were loaded onto Oasis HLB cartridges for filtration. Eluted and evaporated to dryness at 60°C under nitrogen. The samples were then reconstituted in a 2% acetonitrile solution and analyzed. Recoveries ranged from 0% to 678% for the pre-separation spiked liquid samples and 1.3% to 36% for the liquid samples spiked after separation. Solid Fraction "feed-method": After separation from the liquid fraction, the solid fraction was freeze-dried, then ground using a coffee grinder. Phosphate buffer solution was added to samples, centrifuged, and treated with piperidine for beta-lactam derivatization. Samples were subsequently purified using Oasis HLB cartridges. Eluates were dried at 60°C under nitrogen, then reconstituted in a water, acetonitrile, formic acid mix before LC-MS analysis. Antibiotic recoveries of 0% to 152% were observed for the solid samples spiked before separation and 0% to 26% for solid samples spiked after separation. Berendsen et al., 2013 Similar to the van Holthoon et al. (2010) approach, piperidine derivatization was used to increase recovery of beta-lactam pharmaceuticals., with incubation of the piperidine at 60°C for 1 hour. This method was tested three separate times (BER1, BER2, BER3). The BER1 and BER2 samples were non-separated manure, while the BER3 samples were separated. A borate solution and piperidine were added, followed by a n-hexane step for lipid removal. BER2 and BER3 were adjusted to pH 7 and BER3 was adjusted to pH 4 to examine the effect of pH on antibiotic recovery. Oasis HLB cartridges were used for BER1 and BER2 and Strata-X RP cartridges BER3. Eluants were dried at 45°C with nitrogen and reconstituted with 1% piperidine. Sample analytes were determined using matrix adjusted calibration curves. In all three cases, samples were analyzed for all antibiotics as well as for the piperidine derivatized beta-lactams to determine if the method was suitable for the non-derivatized antibiotics and to determine if the beta-lactams were 100% derivatized. Recoveries ranged from 0% to 412% for BER1, 0.23% to 553% for BER2, 0% to 62% for BER3 at pH4, and 0% to 55% for BER3 at pH 7. Karaca et al., 2018 The antibiotics are salted out of the freeze-dried manure using sodium chloride (NaCl). Mcllvaine buffer and Ethylenediaminetetraacetic acid (EDTA) solutions were used to saturate the freeze dried sample after salt (NaCl) was added. Centrifugation was used for separation of solvents. The collected solvent layers were combined and dried under N2 at 45 °C, then reconstituted, and filtered through 0.22 um PTFE filters. Recoveries ranged from 0% to 561%, 0% to 3%, and 0% to 984% for the various antibiotics tested. Objective 3: Three on-line outlets for dissemination of AMR related materials were established, including a project website and two social media accounts. Content for these outlets included existing videos, short summaries of published research, and press articles curated by the team to build on and expand audience understanding of AMR and its risks. Nearly 100 pieces of graphical content were created for distribution on social media and via printed material. These infographics are intended to help illustrate and highlight scientific research findings related to AMR, spread awareness of the basic scientific principles of AMR, describe the current public health concerns related to AMR, and disseminate information that improves confidence among the intended audience of action they can take to minimize potential AMR-related risks. While no evaluation has been conducted to date, a contract has been established with the Methodology and Evaluation Research Core (MERC) at the University of Nebraska-Lincoln to facilitate evaluation of existing and new AMR-related resources, content delivery methods, and changes in AMR-related knowledge and behavior among target audiences.

Publications