Progress 12/01/18 to 11/30/19
Target Audience: Scientists, Students, Researchers & Extension/Outreach professionals were reached through national and international professional meetings(e.g.ACS, ASABE,AEES,EDAR), university seminars, classroom trainings, and dairy manure extension events (Maryland Dairy Farmer FieldDay). Industryprofessionalswerereachedthroughprofessionalmeetings(BiocycleEastandMarylandDairyFarmerField Day) and directcorrespondences. Farmerswerereachedthroughon-farmdiscussionsandfielddayevents,aidedbythedevelopmentofExtensionFact Sheets and other outreachmaterials. HighSchoolandundergraduatestudentswerereachedthroughinternshipopportunitiesthatincludedlaboratory instruction and experiential fieldwork learningopportunities. GraduateStudentsandfacultyatScientificconferences Changes/Problems:We have experienced some challenges and loss of major personnel that caused a significant delay in our project. First, due to severe weather conditions, our sampling did not begin until late 2016 and continued throughout 2017.Therefore, analysis of samples were also delayed.Second, our liquid chromatography/mass spectrometry broke and took a few months to be repaired, which further delayed our analysis.Third, we lost two major personnel: Co-PI Dr. Lauren Sassoubre had a maternity leave in 2018 and eventually left the University at Buffalo after her baby was born.The postdoctoral fellow working at Cornell University also left Cornell to take a faculty position in August 2018.We need to finish the analysis of the manure samples for antibiotics and antibiotic resistance genes, write papers for scientific journals, and complete the fact sheets that we are preparing for the dairy farm extension work.We are applying the novel quantitative metagenomics method developed in this project to the remainder of the collectedfarm samples and land-applied samples.This requires extensive bioinformatics analyses, which we expect to take several months to complete. To contextualize the ARG results from this study, we are conducting a systematic review of the literature on ARGs in farm systems and land-applied manure and through various treatment processes and will write a review article on the results.Finally, we will prepare manuscripts for publications in scientificjournals and additional fact sheets for outreach. What opportunities for training and professional development has the project provided?1. Jerod Hurst (Ph.D. Student, UB)was trained to learn LC/MS/MS for the analysis of antibiotics, and qPCR for the analysis of antibiotic resistance genes. He attended and presented at scientific conferences including the American Chemical Society National meeting,the International Meeting on Environmental Dimensions of Antimicrobial resistance, Pittsburg Conference, and the International Mass Spectrometry Conference. 2. Kayla Naas (MS student, UB)was trained to learn LC/MS/MS for the analysis of antibiotics, and presented a poster at the American Chemical Society National meeting. 3. Emily Crossette (Ph.D. Student, UM)attended a 9-day workshop at the Woods Hole Marine Microbiology Laboratory to study computational microbial ecology methods. 4. Dr.Jason Oliver (Postdoc, Cornell)attended and presented on project work at the 2017 ASABE annual international meeting. How have the results been disseminated to communities of interest? University at Buffalo: We presented data at the followingconferences: Dr.AgapresentedasinvitedspeakerattheSymposiumforAntibioticsandAntibioticsResistanceGenesinthe Environment, Nanjing University, Nanjing, China, June 14-16,2017 Three posters were presented by graduate students at the 4thInternational Symposium on the Environmental Dimension of Antibiotic Resistance (EDAR) Conference, Lansing, Michigan. August 13-17,2017 Dr. Aga presented at the AmericanChemicalSociety,Washington,D.C.August20-24,2017.OralPresentationtitled:"AssessingDairyManure Management Strategies for the Removal of Antimicrobials and Spread of Antimicrobial ResistantGenes". Dr. Aga was invited as Keynote Speaker at the Conference onHalting Antimicrobial Resistance Dissemination in Aquatic Environments(September 16-19, 2018), at Monte Verita, Ascona, Switzerland, where she gave a presentation on "Global reconnaissance of micropollutants in wastewater and surface waters". Dr, Aga spent 3 weeks at the World Health Organization (WHO), at the United Nations (UN) headquarters in Geneva, Switzerland, to assist in the preparation of the technical report on "Water, sanitation and hygiene (WASH) and Antimicrobial Resistance (AMR): Preventing Infection and Preparing for a Post-Antibiotic World", which is a critical component of the WHO Global Action Plan on Antimicrobial Resistance. Dr. Aga co-organized a workshop with WHO personnel to bring global experts on AMR and address critical issues in combatting AMR spread, held in Hamburg, Germany, in May 8-9, 2019. Dr. Aga co-organized the symposium on "Non-target Dr. Aga was Plenary speaker or Invited Keynote Speaker at the following conferences, where she discussed issue on AMR in Agriculture and Environment: Plenary Speaker,Annual Philippine-American Academy for Science and Engineering Symposium,Manila, Philippines, May 23-24, 2019. Invited Speaker,5thInternational Symposium on the Environmental Dimension of Antibiotic Resistance, The University of Hong Kong, Hong Kong, June 9-14, 2019. Plenary Speaker,3rdInternational Conference on Bioresources, Energy, Environment, and Materials Technology, Hong Kong Polytechnic University, Hong Kong, June 12-15, 2019. Invited Speaker, "Target and Non-target Analysis of Chemicals of Emerging Concerns", Chemistry Department, Hong Kong Baptist University, Hong Kong, June 17-18, 2019. Keynote Speaker,6thInternational Workshop on Frontiers in Environmental Chemical Research, POSTECH, Pohang,Korea, October 25, 2019. "Analysis and Treatment of Antimicrobials in Animal Manure: Challenges and Solutions". Keynote Speaker,11thMicropol and Ecohazard Conference, International Water Association, Seoul National University, Seoul, Korea, October 20-23, 2019. Invited Speaker, "Global Reconnaissance of Antibiotics using Target and Non-target LC-MS/MS Analysis", Chemistry Department, Khon Kaen University, Khon Kaen, Thailand, October 28, 2019. Invited Speaker, "Global Reconnaissance of Antibiotics usingTarget and Non-target LC-MS/MS Analysis", Chemistry Department, Ramkhamhaeng University, Bangkok,Thailand, October 29, 2019. Keynote Speaker,12thInternational Conference on the Challenges in Environmental Science and Engineering, Kaohsiung City, Taiwan, November 3-6, 2019. University ofMichigan: PreliminarymetagenomicworkwassharedattheUNCWaterMicrobiology/IWAHealth-RelatedWaterMicrobiologyjoint conference in Chapel Hill,NC, May 2017 E. Crossette shared the results of the metagenomic quantification study as an oral presentation at the UNC Water Microbiology conference, Chapel Hill, NC May 2018 and May 2019 E. Crossette presented a poster and poster pitch at the International Symposium on Microbial Ecology, Leipzig, Germany, August 2018. K. Langenfeld gave an oral presentation on the quantitative metagenomics method for virus-associated ARGs at the International Water Association Health Related Microbiology conference in Vienna, Austria. Thebioinformaticmethodsforthemetagenomicstudywaspresentedinaposterpresentationentitled"Quantitative metagenomic approach to monitor the dynamics of antimicrobial resistance gene (ARG) profiles through engineered treatmentsystems"atthe"EnvironmentalDimensionofAntimicrobialResistance"SymposiuminLansing,Michigan. Intracellular/Extracellular DNA extraction methods were presented at a poster session at the Borchardt Conference in AnnArbor,Michigan.Theposterwasentitled"Extra-andintracellularantimicrobialresistancegenes(ARGs)andtheirfatein dairy cow manure treatmentstructures." Preliminary findings from the virus DNA extraction methods were shared in poster presentations entitled "Quantitative Analysis of dsDNA Phage Through Wastewater Treatment Plants" at the "Environmental Dimension of Antimicrobial Resistance"SymposiuminLansing,Michiganandatthe"AssociationofEnvironmentalEngineeringandScienceProfessors" Annual Conference in Ann Arbor,Michigan. Cornell University and Maryland Lansing, D., 2018. Public Awareness of Antibiotic Resistance and Antibiotic Regulation.Opportunities and Challenges in Anaerobic Digestion: Maryland and the Northeast US Experience. Annapolis, MD. October 2, 2019. Lansing, D., 2019. Research Survey - Antibiotic Management Decisions and the VFD. Cornell Pro-Dairy Herd Health and Nutrition Conference. Syracuse, NY April 9, 2019. Lansing, D., 2019. Remaking Life Along the Commodity Chain. American Association ofGeographers Annual Meeting. Washington, DC. April 5, 2019. Lansing, S., 2018. Antibiotic resistance, manure technologies, and value-added treatment. Opportunities and Challenges in Anaerobic Digestion: Maryland and the Northeast Experience. Annapolis, MD. October 2, 2018. Lansing, S., 2018. Manure Management and Contaminants: Effects of Antibiotics and Pathogens. 2018 Mid-Atlantic Crop Management School. Ocean City, MD. November 13 - 15, 2018. Lansing, S., Schueler, J., Crossette, E., Naas, K., Hurst, J., Raskin, L., Wiggington, K., Gooch, C., Aga, D.,2019. Fate of antimicrobials during dairy manure management and processing. Waste to Worth. Minneapolis, MN. April 22-26, 2019. Schueler, J., Lansing, S., Felton, G., Hurst, J., Naas, K., Aga, D., Crossette, E., Raskin, L., Wiggington, K., Oliver, J., Gooch, C., 2018. Fate and transport of antibiotics, genes, and nutrients in anaerobic digestion systems (poster). Bioresource Technology for Bioenergy, Bioproducts & Environmental Sustainability. Sitges, Spain. September 16-19, 2018. Professionalmeetings,seminars,andteleconferenceshavebeenusedtodisseminatepreliminaryphaseIresultsto Scientists,Researchers,Extensionpersonnel,Veterinaries,AgriculturalEngineers,andDairyIndustryprofessionals Factsheetshavebeenpreparedtodisseminateinformationonantimicrobialresistanceasitrelatestodairyproductionto farmers and theirstakeholders Currently,reportsthatsummarizePhaseIeffortsarebeingpreparedforthecollaboratingfarmsinadvanceofsit-down meetings to discuss the project's Phase Ifindings What do you plan to do during the next reporting period to accomplish the goals?We have experienced some challenges and loss of major personnel that caused a significant delay in our project. First, due to severe weather conditions, our sampling did not begin until late 2016 and continued throughout 2017.Therefore, analysis of samples were also delayed.Second, our liquid chromatography/mass spectrometry broke and took a few months to be repaired, which further delayed our analysis.Third, we lost two major personnel: Co-PI Dr. Lauren Sassoubre had a maternity leave in 2018 and eventually left the University at Buffalo after her baby was born.The postdoctoral fellow working at Cornell University also left Cornell to take a faculty position in August 2018.We need to finish the analysis of the manure samples for antibiotics and antibiotic resistance genes, write papers for scientific journals, and complete the fact sheets that we are preparing for the dairy farm extension work.We are applying the novel quantitative metagenomics method developed in this project to the remainder of the collectedfarm samples and land-applied samples.This requires extensive bioinformatics analyses, which we expect to take several months to complete. To contextualize the ARG results from this study, we are conducting a systematic review of the literature on ARGs in farm systems and land-applied manure and through various treatment processes and will write a review article on the results.Finally, we will prepare manuscripts for publications in scientificjournals and additional fact sheets for outreach.
What was accomplished under these goals?
Objective 1 Samples were taken every six weeks over a 15-month period from participating farms in Maryland (MD1, MD2, MD3), New York (NY1, NY2, NY3, NY4, NY5, NY6), and Pennsylvania (PA1, PA2) and analyzed for the levels of antimicrobials,ARBandARG. Antimicrobial analysis of tetracyclines, sulfonamides, and macrolides revealed detection of sulfonamides in 6% of the total samples. Tetracyclines were detected with a higher frequency of 86%, suggesting higher environmental stability and use of tetracyclines on dairy farms compared to sulfonamides. Macrolides were not detected in any manure samples. The following analyses were conducted to determine ifcorrelationsexistbetweenmanurecharacteristicsandantimicrobialtransformations:pH, moisture content, total and volatile solids (TV, VS), nutrients (TKN, TKP, NH4), and volatile fatty acids. Extraction efficiency studies for bacterial DNA confirmed efficiencies ranged from 60-120% in manures. We developed a reliable and novel untargeted quantitative methods for ARGs in these systems. PhaseIsampling,manuresystemmonitoring,andantibioticusagemonitoringwascompletedinSeptember2017, samples have been analyzed, and the team is moving forward with statistical analysis of the collecteddata. 2016 antibiotic usage has been summarized for NYS, PA and MD farms. ARGs were identified and quantified from metagenomic data. The metagenomeshavebeenassembledtocomparebothARGannotationwithshortmetagenomicreadsandannotationwith assembled genes. ARG presence in untreated and storage lagoon manure samples for each dairy farm and each collection date was evaluated using qPCRand customized Qiagen Microbial DNA qPCR Arrays. No correlation was observed between int1 normalized copies per gram manure and total antibiotic concentration (Spearman correlation,p=0.082). Customized Qiagen Microbial DNA qPCR Arrays were used to evaluate the presence of nine ARGs simultaneously. Anaerobic digestion among various farms provided no substantial decrease in the concentration of total tetracyclines after treatment. Composting techniques among two farm provided decreases in the concentration of total tetracyclines, suggesting the formation of further degradation products that were not included in the analytical method. Phase II Sampling focused on Cold Springs Farm, denoted as MD3 in this project.The farm utilizes solid-liquid separation, tank and lagoon storage, and composting for their manure management. This focused study sampled a compost windrow made up of the packed bedding from their sick and lame cow barns, managed using the dairy farm's typical practices on a 1000 cow US dairy. Total Kjeldahl nitrogen increased from 15.3 to 18.4 g/kg during the composting period due to decreases in solids, but also potential inhibition of N-mineralization from the present antimicrobials. Physio-chemical results indicate that antimicrobials could impact nitrogen transformations in compost, which should be a consideration of farm nutrient management of sick cow bedding. Objective 2. It was found in this study that antimicrobial trends at the field scale did not follow the trends of degradation found in previous pilot scale and bench scale literature. Tetracycline concentrations in the AD effluent were not significantly reduced compared to the AD influent at any of the farms. The potential of ADto degrade antimicrobials and ARGs in manure before field application was investigated using tetracycline (TC) and sulfadimethoxine (SDM). During the AD process, there was >99% reduction in the SDM concentration in all reactors spiked with SDM. The reactors containing TC additions showed more variability in antimicrobial concentrations, with 0-96% reduction observed during AD. Gene analyses before and after the AD process showed that thetetMgene copies decreased during AD and correlated with declines in TC, however, reductions in SDM concentrations (> 99%) did not correlate with decreases insul1gene copies. In the reactors containing 10 mg/L of either TC or SDM, a 7.8% reduction in methane (CH4) production was observed, while no methane reduction was observed in the reactors 1, 2, or 3. This study showed that flush system manure management led to much lower concentrations of manure nutrients compared to scrape system farms, and farm co-digestion led to slightly higher TP values in manure. Objective 3. This study presented an analysis of the occurrence of antimicrobials and ARGs in soil and manure amended soil from a lab-scale study, while also investigating the uptake or transfer of antimicrobials and ARGs in youngZea mays L.. Antimicrobial uptake was observed inZea mays L.grown under lab-scale conditions with variable behavior observed between the sulfonamides and tetracyclines. In general, sulfonamides had greater uptake than tetracyclines. ARGs were quantified in soil after manure fertilization, however, ARGs were not detected in theZea mays L.samples. ARG prevalence in manure-amended soil significantly decreased forOXA-1andtetOafter 21 days of exposure, howeversul1prevalence increased during the exposure period.?It was found that the manure-amended soil control and spiked manure-amended soil gene copies normalized by 16S rRNA gene copies did not show statistical differences among the ARG prevalence forOXA-1,sul1,andtetOfor day 0 (p-values of 0.438, 0.056, and 0.281, respectively) and day 21 (p-values of 0.235, 0.091, and 0.552, respectively). Objective 4. ?Fact Sheet targeted at the general public: Antibiotic Resistance and Dairy Production in 2016. Five-part Fact Sheet series on "Antibiotic Residues in Dairy Manure"in 2017. The series includes: Part 1: Critically important antimicrobials labeled for dairy use, Part 2: Sampling dairy manure for antibiotic detection, Part 3: Laboratorymethodsforextractingantibioticresiduesfromdairymanure,Part4:Laboratorymethodsforanalyzingantibiotic residues extracted from dairy manure, Part 5: What is known about antibiotic residues in dairymanure? Fact Sheet targeted at dairy farmers:Antibiotic Resistance and Dairy Production - Frequently Asked Questions in 2016. Cornell spoke to a class of 4thand 5thyear veterinary students in May 2017 on the topic of antimicrobial resistance and dairy manure systems in a course titled "Veterinary Perspectives on Pathogen Control in AnimalManure" In October 2017 a Field Day and Tour was hosted by UMD at the Central Maryland Research and Education Center- Clarksville Facility (Ellicott City, MD). The event including four talks from various research experts and extension/outreach professionals, including Curt Gooch from Cornell,onmanuremanagement. The event included 59participants. In October 2018, a workshop was planned by UMD, 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. Four-part Fact Sheet series on "Antimicrobial Resistance and Dairy Manure Systems" in 2018.The series includes Part 1:Antibiotic resistant genes - prevalence in dairy manure, Part 2:Antibiotic resistant genes - Measuring ARG in diary manure, Part 3:Antibiotic resistant bacteria - prevalence in dairy manure, and Part 4:Antibiotic resistant bacterial - measuring ARB in dairy manure. Fact Sheet:Antimicrobial resistance and Dairy Manure Systems:Solid-liquid separation - partitioning of antibiotic resides in 2019. Cornell developed and posted all project extension materials on a new AMR web site:https://prodairy.cals.cornell.edu/environmental-systems-mgt/environmental-considerations/amr/in 2019. Cornell provide handouts and met with NIFA officials at the 2019 Annual Agricultural Research Exhibition and Reception in Washington, DC.
1. Hurst, J.J.; Oliver, J.P.; Schueler, J.; Gooch, C.; Lansing, S.; Crossette, E.; Wigginton, K.; Raskin, L.; Aga, D.S.; Sassoubre, L.M. 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 and Technology 2019, 53, 2405-2415.
2. Mullen, R.A.; Hurst, J.J.; Naas, K. M; Sassoubre, L.M.; Aga, D.S. Assessing uptake of antimicrobials by Zea mays L. and prevalence of antimicrobial resistance genes in manure-fertilized soil. Science of The Total Environment, 2019, 646, 409-415.
3. Oliver, J.P.; Schueler, J.E.; Gooch, C.A.; Lansing, S.; Aga, D.S. Performance quantification of manure management systems at 11 northeastern US dairy farms. Applied Engineering in Agriculture, 2018, 34, 973-1000.
4. Oliver, J.P., Hurst, J.J., Schueler, J., Gooch, C.A., Sassoubre, L. M., Lansing, S., Aga, D.S. 2019. The fate of medically important antibiotic residues, antibiotic resistant bacteria and antibiotic resistance genes in the manure management systems of modern US dairy operation. Invited Review: Journal of Dairy Science, accepted.
Progress 12/01/16 to 11/30/17
Target Audience: Scientists, Researchers & Extension/Outreach professionals were reached through national and international professional meetings (Waste-to-Worth, ASABE, AEES, EDAR), seminars (Cornell Dairy Center of Excellence), classroom trainings (Cornell Vet School), and dairy manure extension events (Maryland Dairy Farmer Field Day). Industry professionals were reached through professional meetings (Biocycle East and Maryland Dairy Farmer Field Day) and direct correspondences. Farmers were reached through on-farm discussions and field day events, aided by the development of Extension Fact Sheets and other outreach materials. High School and undergraduate students were reached through internship opportunities that included laboratory instruction and experiential fieldwork learning opportunities. Graduate Students and faculty at Scientific conferences such as the American Chemical Society (Washington DC) and EDAR (Michigan State University), and at the Antibiotic Resistance Conference at the Nanjing University, China Changes/Problems: The antibiotics data collected from the 11 farms in June and August 2016 were removed from the average results due to a change in the LC/MS/MS method of analyzing the antibiotics after August 2016; The modification in the method was to improve detection limit and reproducibility of analysis which turned out to be very challenging due to the wide ranging nature of manure samples from various farms. The complexity of each manure sample varied from farm to farm, therefore we had to optimize the method after observing the results from June and August 2016 were not reproducible due to matrix effects. What opportunities for training and professional development has the project provided?Graduate Student Emily Crossette attended a 9-day workshop at the Woods Hole Marine Microbiology Laboratory to study computational microbial ecology methods. How have the results been disseminated to communities of interest? 1. University at Buffalo: We presented data at the following conferences: Dr. Aga presented as invited speaker at the Symposium for Antibiotics and Antibiotics Resistance Genes in the Environment, Nanjing University, Nanjing, China, June 14-16, 2017 Three posters were presented at the4th International Symposium on the Environmental Dimension of Antibiotic Resistance (EDAR) Conference, Lansing, Michigan.August 13-17, 2017. American Chemical Society, Washington, D.C. August 20-24, 2017. Oral Presentation titled: "Assessing Dairy Manure Management Strategies for the Removal of Antimicrobials and Spread of Antimicrobial Resistant Genes". 2. University of Michigan: Preliminary metagenomic work was shared at the UNC Water Microbiology/IWA Health-Related Water Microbiology joint conference in Chapel Hill, NC. The bioinformatic methods for the metagenomic study was presented in a poster presentation entitled "Quantitative metagenomic approach to monitor the dynamics of antimicrobial resistance gene (ARG) profiles through engineered treatment systems" at the "Environmental Dimension of Antimicrobial Resistance" Symposium in Lansing, Michigan. Intracellular/Extracellular DNA extraction methods were presented at a poster session at the Borchardt Conference in Ann Arbor, Michigan. The poster was entitled "Extra- and intracellular antimicrobial resistance genes (ARGs) and their fate in dairy cow manure treatment structures." Preliminary findings from the virus DNA extraction methods were shared in poster presentations entitled "Quantitative Analysis of dsDNA Phage Through Wastewater Treatment Plants" at the "Environmental Dimension of Antimicrobial Resistance" Symposium in Lansing, Michigan and at the "Association of Environmental Engineering and Science Professors" Annual Conference in Ann Arbor, Michigan. 3. Cornell University and Maryland (Extension Part) Professional meetings, seminars, and teleconferences have been used to disseminate preliminary phase I results to Scientists, Researchers, Extension personnel, Veterinaries, Agricultural Engineers, and Dairy Industry professionals Fact sheets have been prepared to disseminate information on antimicrobial resistance as it relates to dairy production to farmers and their stakeholders Currently, reports that summarize Phase I efforts are being prepared for the collaborating farms in advance of sit-down meetings to discuss the project's Phase I findings What do you plan to do during the next reporting period to accomplish the goals? Phase II sampling focusing in on one New York farm and one Maryland farm to conduct a more intensive exploration of specific treatment processes including bedding recovery and composting and the effect of time and temperature on antibiotic degradation. Bench and pilot-scale experiments will be conducted to focus in and explore the specific contribution of anaerobic digestion to antimicrobial degradation and resistant gene and bacteria generation. Phase I results will be used to calibrate and create a model to track antimicrobial passage through the different farm manure systems. Further analyze antibiotic data for eight sampling events for correlations to antibiotic usage and possible removal across manure management systems. Extract and analyze for antibiotics in focused sampling on bedding recovery and anaerobic digestion units to assess absolute removal. Continue investigation of degradation of beta-lactam antibiotics in dairy manure by analyzing degradation exposure samples. Investigate possible transformation products of parent beta-lactams by high resolution mass spectrometry. Complete second crop uptake exposure experiments for antibiotic and ARG's. Prepare a manuscript for publication describing uptake of antibiotics and presence of ARG's in maize when grown with manure amended soil. Sample collection is being planned to target specific processes on dairy farms participating in the study. Samples from this round of collection will be analyzed for both antimicrobials and the int1, sul1, tetO and beta-lactam associated resistance genes. Intracellular DNA (iDNA) and extracellular DNA (eDNA) co-extraction processes will be finalized. This will involve the quantification of contaminating DNA fractions using internal standard DNA with qPCR. These internal standards include a virus, bacteria, and an extracellular plasmid. The final bioinformatic processes will be completed for themetagenomic ARG quantification method,which includes quantification of the internal standard genes recovered through sequencing and the identification of ARG on assembled DNA sequences. Additionally, the quantification method will be compared to qPCR, a common approach for gene quantification. PrepareManuscripts for publications in scientific journals
What was accomplished under these goals?
Objective 1.Evaluate the fate of AMR During Phase I sampling, samples were taken every six weeks over a year long period from participating farms in Maryland (MD1, MD2, MD3), New York (NY1, NY2, NY3, NY4, NY5, NY6), and Pennsylvania (PA1, PA2) and analyzed to determine the fate and transport of antimicrobials, ARB and ARG. Each farm utilizes a different manure management system, but the manure was sampled at each point in their management practice (i.e. inception, before and after separation, compost or digestion, before lagoon storage and prior to field application). Manure samples were freeze-dried and homogenized; 100 mg of dried manure solids were analyzed using LC/MS/MS.Antimicrobial analysis of tetracyclines, sulfonamides, and macrolides revealed detection of sulfonamides in 6% of the total samples. Tetracyclines were detected with a higher frequency of 86%, suggesting higher environmental stability and use of tetracyclines on dairy farms compared to sulfonamides. Macrolides were not detected in any manure samples, which reflects the low reported average antibiotic usage of macrolides per month (i.e. 1-5 cows are treated with erythromycin per month). Screening for the presence of beta-lactam antibiotics was performed using a commercial ELISA kit. Screening efforts using ELISA for the analysis of ceftiofur and penicillin G yielded no detection for manure samples which were chosen to screen based on antibiotic usage data from six of the NY farms. These farms reported high usage of beta-lactam antibiotics, however, no parent compounds of the beta-lactam molecules were detected. In addition to antimicrobials, ARB and ARG analyses, the following analyses were conducted on the samples to determine if correlations exist (and can be modeled) between manure characteristics and antimicrobial transformations: pH, moisture content, total and volatile solids (TV, VS), nutrients (TKN, TKP, NH4, PO4), and volatile fatty acids (VFAs). Manure system performance data was collected from each farm (e.g. manure produced, manure digested, AD temperature, biogas production, rotary drum composter temperature) as well as drug treatment records were collected from each farm and along with several meetings with farm herdsman daily drug use is being summarized for each farm. Phase I sampling, manure system monitoring, and antibiotic usage monitoring was completed in September 2017, samples have been analyzed, and the team is moving forward with statistical analysis of the collected data. 2016 antibiotic usage has been summarized for NYS, PA and MD farms, with summary of all data on-going. Herd health and antibiotic administration on farms is variable. Administration data is being compared to antibiotic residual data to see if there are correlations between administration spikes and concentrations in the sampled manure. Under the first goal, ARGs were identified and quantified from metagenomic data. The data will reveal most abundant ARG in the samples and samples containing the most ARGs. This will inform ARGs to track throughout treatment. Do date, the metagenomes have been assembled to compare both ARG annotation with short metagenomic reads and annotation with assembled genes. Read-mapping to the CARD database was completed and an algorithm is being developed to convert relative abundances of reads to copies of an ARG/volume of sample. ARG presence in untreated and storage lagoon manure samples for each dairy farm and each collection date (Sept'16, Oct'16, Dec'16, March'17, June'17, and Aug'17) was evaluated using quantitative polymerase chain reaction (qPCR) and customized Qiagen Microbial DNA qPCR Arrays. The samples analyzed thus far represent ARG concentrations prior to treatment and/or field application.Temporal and location-based variability among each farm and the collection dates reveal no trends. However, the temporal variability observed may be related to climatic conditions and further data analysis is underway. No correlation was observed between int1 normalized copies per gram manure and total antibiotic concentration (Spearman correlation, p=0.082). In addition to qPCR, a Customized Qiagen Microbial DNA qPCR Arrays were used to evaluate the presence of nine ARGs simultaneously. Nine total genes were targeted (ermB, mefA, ereB, tetA, OXA-2, CTX-M-1, VEB-1, aada1, and aadac-6-lb) which represent resistance mechanisms to the following antimicrobial classes: macrolides, tetracyclines, beta-lactams, and aminoglycosides. DNA extracted from untreated manure from each of the 11 farms over six collection dates (Sept'16, Oct'16, Dec'16, March'17, June'17, and Aug'17) and storage drawdown samples were analyzed for the nine ARGs. Objective 2. Bench-scale and pilot-scale Anaerobic Digestion experiments A face-to-face project collaborators meeting was hosted at the EDAR meeting in Lansing, MI to discuss Phase I project status and future directions for Phase II of the project. In addition, another face-to-face meeting between the Cornell and UB teams occurred in Buffalo in September to discuss the best approach for Phase I data analysis. Phase II sampling strategies are being planned, with farmer meetings conducted and scheduled to work out the logistics. Experimental design for bench and pilot scale experiments is being finalized. The bench-scale experiment will be conducted over the winter with the AD pilot scale experiment occurring in Spring 2018. To help meet Goal 2, extraction methods for different forms of ARGs have been investigated. ARGs in the intracellular or extracellular fractions may behave differently in manure which could cause one fraction to persist longer than another. So far, DNA internal standards have been developed to assess recovery of DNA in each fraction and contamination. Additionally, methods to minimize the virus DNA contamination from iDNA/eDNA fractions have also been evaluated. Another graduate student, Kathryn Langenfeld, is developing DNA and RNA extraction methods for viruses from manure. Two foreign phage are used as internal standards to determine the absolute recovery of viral nucleic acid extraction methods. Objective 3. Crop uptake experiments Field soils and long-term storage manures were collected from a NY farm on August 2017 for crop uptake experiment Objective 4. Hydraulic modelling effort Phase I sampling information will be used for model calibration. This will begin in 2018. Objective 5. Extension and education activities A five-part Fact Sheet series on "Antibiotic Residues in Dairy Manure" is near completion. The series includes: Part 1: Critically important antimicrobials labeled for dairy use, Part 2: Sampling dairy manure for antibiotic detection, Part 3: Laboratory methods for extracting antibiotic residues from dairy manure, Part 4: Laboratory methods for analyzing antibiotic residues extracted from dairy manure, Part 5: What is known about antibiotic residues in dairy manure? Cornell spoke to a class of 4th and 5th year veterinary students in May 2017 on the topic of antimicrobial resistance and dairy manure systems in a course titled "Veterinary Perspectives on Pathogen Control in Animal Manure" In October 2017 a Field Day and Tour was hosted by UMD at the Central Maryland Research and Education Center-Clarksville Facility (Ellicott City, MD). The event including four talks from various research experts and extension/outreach professionals on manure management. A farm tour was given where two posters were presented at various locations talking about antibiotic administration on farm and antibiotic detection levels throughout the manure trail (Figure 4). A manure injection demonstration was also provided. The event included 59 participants.
Conference Papers and Presentations
1. Arikan, O., Rice, C., Mulbry, W., Lansing, S., 2017. The fate and effect of lasalocid during anaerobic digestion of dairy manure. Progress in Biogas IV International Conference. Stuttgart, Germany. March 9, 2017.
2. Oliver JP, Gooch CA. Effectiveness of dairy manure management practices at controlling antimicrobial resistance. Biocycle East Coast. Baltimore, MD. Apr. 4-7, 2017.
3. Oliver JP, Gooch C. 2017. Effectiveness of different dairy manure management practices in controlling the spread of antibiotics and antibiotic resistance. Waste-to-Worth Annual Conference, Cary, NC. Apr. 18-21, 2017.
4. Schueler J, Lansing S. Fate and Transport of Antibiotics and Nutrients in Manure Management Systems. American Ecological Engineering Society. Athens, GA. May 22-25, 2017.
5. Oliver JP, Schueler J, Hurst J, Crossette E, Langenfeld K, Gooch C, Lansing S, Felton G, Wigginton K, Raskin L, Sassoubre L, Aga D. 2017. Antibiotics and antibiotic resistant bacteria and genes in northeastern dairy manure management systems Project overview and preliminary findings from an 11 farm case study. ASABE International Meeting, Spokane, Washington. July 16-19, 2017.
6. Lansing, S., Oliver JP, Schueler J, Gooch C, Felton G, Raskin L, Wigginton K, Crossette E, Langenfeld K, Hurst J Sassoubre L, Aga D. 2017. The Effect of Manure Collection and Inherent Variability on Antibiotic Testing of Dairy Manure Systems (poster). 4th International Symposium on the Environmental Dimensions of Antibiotic Resistance. Lansing, MI. August 13-17, 2017.
7. Jerod Hurst, Jason Oliver, Jenna Schueler, Stephanie Lansing, Lauren Sassoubre, Diana Aga. Assessing Dairy Manure Management Strategies for Removal of Antimicrobials, (poster). 4th International Symposium on the Environmental Dimensions of Antibiotic Resistance. Lansing, MI. August 13-17, 2017.
8. Jerod J. Hurst, Lauren Sassoubre, and Diana S. Aga. "Assessing Dairy Manure Management Strategies for Removal of Antimicrobials and Spread of Antimicrobial Resistant Genes", (oral), 254th ACS National Meeting, Washington, DC, August 20-24, 2017.
Progress 12/01/15 to 11/30/16
Target Audience:Chemists, Agricultural Engineers, Farmers Changes/Problems:
What opportunities for training and professional development has the project provided? The University of Maryland participants had involved two African American high school interns on this project, two college undergraduate students, and one Master Students who have assisted with and trained under this project. The University at Buffalo housed Benjamin Davis as part of the Research for Undergraduate Education (REU) program (Summer 2016). The student assisted with research for the USDA project. Through this process, the student learned how we collect samples at the dairy farms, how manure is analyzed for antibiotics, and how manure is analyzed for ARBs and ARGs. His REU experience culminated with a poster presentation. The University of Michigan sent Emily Crossette to the Gordon Research Conference: Environmental Sciences Water, which promoted networking between scientists evaluating antimicrobial resistance gene dissemination in the environment. Additionally, the grant supported a student's attendance of an R-workshop which enhance skills in data processing and coding relevant for microbial community analysis. The workshop also facilitated networking between professionals and researchers in the field of microbiology. The Cornell efforts are being led by a postdoctoral associate. This individual has worked with farm personal and veterinarians to understand the dynamics of farm antibiotic usage and record keeping. Under the supervision of Curt Gooch and with farm personal this individual has sharpened their understanding of the various manure treatment systems and their operational parameters. Additionally, this induvial has attended several professional engineering trainings, outreach/extension events, and is gaining project management skills. Throughout the 2016 sampling season Benjamin Davis, Mitchell Mayville, and Jerod Hurst regularly shadowed Jason Oliver at participating farms during sampling to improve their knowledge of the dynamic manure treatment systems, the sampling process and its subtleties. Approximately 30 private and public NYS professional engineers working in the areas of livestock nutrient management and manure treatment systems were updated on the issue of antibiotic resistance in dairy systems at the May 2016 training meeting. A meeting is being planned for spring 2017 with collaborating NYS dairies their operators and herdsmen to discuss different approached to improve antibiotic treatment recording. How have the results been disseminated to communities of interest? A kickoff meeting was conducted with each participating farmer, followed by several one-on-one meetings with our participant farmers throughout the past seven months to discuss their manure management systems and antibiotic usage and transformations on-farm. In addition, see presentations given in Year 1 and events planned for Year 2 in Goal 5 section above Preliminary work was shared with the scientific community through a poster presentation at the Gordon Research Conference, "The forms and persistence of antimicrobial resistance genes (ARG) in dairy farm manure treatment processes". What do you plan to do during the next reporting period to accomplish the goals?A. University of Maryland Finalize bench-scale and pilot-scale design and implement the experiments. Implement one large field day and several smaller county-extension based field days on the participating farms. Begin calibrating the hydraulic model to each farm using data collected in Years 1 and 2. University of Michigan During the next reporting period, the metagenomic data will be processed and relative and absolute abundance of resistance genes will be compared from both metagenomic data and qPCR assays. Additionally, intracellular DNA (iDNA) and extracellular DNA (eDNA) extraction processes will be optimized. Similar to the bulk DNA extraction kit comparison, the iDNA/eDNA extraction methods will be compared on the basis of yield, absolute abundance of spiked intracellular or extracellular DNA, and purity. Additionally, contamination of intracellular DNA into the extracellular fraction and vice-versa will be quantified and minimized. A viral DNA extraction method will be established and used to quantify the fraction of resistance genes in the viral community of the sample. C. Cornell University Integrate all field data to evaluate the effect of the various manure treatment systems on antibiotic residues. Complete the study investigating the effect of bedding recovery systems on pathogenic ARB. Launch a webpage for the project. In coordination with the UMD team, execute the on-farm meetings and various extension events that are planned and are in planning. Complete and publish the review of the impacts of dairy production on antibiotic resistance D. Univeristy at Buffalo Begin to extract DNA from the manure collected by the field teams. Based on the results from collaborators at University of Michigan, we have decided on an extraction kit for the entire project to be consistent across laboratories. We are in the process of deciding what assays to use for the antimicrobial resistance genes. Complete our laboratory experiments investigating the effect of lime on ARBs, ARGs and antibiotics. We plan to prepare a manuscript with this data. Continue experiments investigating which antimicrobial resistance genes are carried by which bacteria cultured on AccuMast plates. The development of new methods to analyze compounds of interest that have been identified by farm drug usage. The target antibiotics of these new methods include: ampicillin, ceftiofurpenicillin G procaineenrofloxacin, hetacillin potassium, and Amoxicilin Trihydrate . The majority of these target compounds of interest are beta-lactams, which are known to be highly unstable in the environment, degradation and transformation products of these antibiotics will be investigated using high resolution mass spectrometry if parent compounds are not seen in manure samples.
What was accomplished under these goals?
Major Goal 1 Samples were taken every six weeks over the past seven months from participating farms in Maryland (MD1, MD2, MD3), New York (NY1, NY2, NY3, NY4, NY5, NY6), and Pennsylvania (PA1, PA2) and analyzed to determine the fate and transport of antimicrobials, ARB and ARG. Each farm utilizes a different manure management system, but the manure was sampled at each point in their management practice (i.e. inception, before and after separation, compost or digestion, before lagoon storage and prior to field application). Initially, project personnel met with the farmers to better understand their manure management practices (lagoon storage time, solid separation, digester operation, etc.), their antibiotic usage (type, dosage rate, number of cows treated per day/month), and manure field application practices. Manure samples were freeze-dried and homogenized. 100 mg of dried manure solids were added to polypropylene centrifuge tubes. Each sample was spiked with 50mL of surrogate solution containing d4-sulfa-methoxazole, phenyl-13C6-sulfamethazine, demeclocycline, and13C-erythromycin (500 ng mL-1) and allowed to equilibrate for 30 minutes. Solids were suspended with 5 mL of 20:30:50 acetonitrile-methanol-0.1 M EDTA-McIlvaine buffer (pH = 4; v/v/v), vortexed for 30 seconds, ultrasonicated (40 kHz, 120 W) for 10 minutes and centrifuged at approximately 4000gfor 10 min.The supernatant was decanted into a 500-mL HDPE bottle and the solids were extracted twice more. Extracts were diluted with 400 mL of NANOpure water to reduce percentage organic content (≤4.0%) and the pH was adjusted to 4.0±0.2 with H3PO4. Diluted extracts were subjected to SPE with tandem NH2-HLB cartridges. Loaded cartridges were washed with 10 mL of water/methanol (95:5, v/v), the NH2 cartridges were removed, and the HLB cartridges were dried under vacuum.The HLB cartridges were eluted with 10 mL of methanol and evaporated to 200mL under N2at 30°C. Extracts were reconstituted to 1 mL with water/methanol (95:5, v/v) plus 0.1% acetic acid solution, and the mixture was vortexed. A 200-mL aliquot of the extract was placed into a vial insert and spiked with 10mL of 500 ng mL−1spiking solution and ISTD. A second aliquot of 200mL was spiked only with 10mL of 500 ng mL−1ISTD, d10-carbamazepine, and minocycline. Both aliquots were centrifuged at 7000gfor 5 min to remove anyfine particles from thefinal extract before analysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS). In addition to antimicrobials, ARB and ARG analyses, the following analyses were conducted on the samples in order to determine if correlations exist (and can be modeled) between manure characteristics and antimicrobial transformations: pH, moisture content, total and volatile solids (TV, VS), nutrients (TKN, TKP, NH4, PO4), and volatile fatty acids (VFAs). The following data has been produced over the last 7 months and will continue to be produced for the next year: 1) antibiotics usage and total antibiotic load averaged on a monthly basis, 2) nutrients, solids, pH and VFAs, 3) antibiotic. Manure system performance data was collected from each farm (e.g. manure produced, manure digested, AD temperature, biogas production, rotary drum composter temperature) as well as drug treatment records were collected from each farm and along with several meetings with farm herdsman daily drug use is being summarized for each farm. Initial analysis of preliminary antibiotic mitigation data is underway. Progress towards the first project goal includes the submission of manure samples for complete genome sequencing. This first task required optimizing the DNA extraction techniques in order to best obtain a high-quality and representative sample of DNA for sequencing and other downstream analysis. The absolute yield, DNA purity, and absolute abundance of spiked bacteria were compared from samples of dairy manure using two different commercial extraction kits. Major Goal 2 Development of the experimental design for the bench-scale and pilot-scale anaerobic digestion experiments begun based on our antibiotic usage data from the farms participating in this study. Major Goal 3 Sassoubre and Oliver are collaborating with Dr. Rodrigo Bicalho to investigate the antimicrobial resistance genes carried by different species and types of bacteria in manure used for bedding. Bicalho is an associate professor at Cornell University and started FERA Animal Health which makes AccuMast plates, a quick, culture-based method to diagnose mastitis on dairy farms. The research will give a better understanding of which bacteria carry ARGs and which ARGs which will inform treatment strategies for inactivating and removing ARBs and ARGs. Major Goal 4 The model framework and data for model calibration is being collected, and the model platform will be built in year 2. Major Goal 5 We will offer programming to help dairy farmers understand the new FDA regulations instated on January 1, 2017 that eliminate the growth promotion use of human medically important antibiotics and expanding the list of feed-grade antibiotics classified as Veterinary Feed Directive (VFD) drugs. Antibiotic resistance and dairy production Fact Sheets were developed for public and farm audiences and will be published soon to a page on the topic at the Cornell Dairy Env. Systems webpage. The extension programming has begun, with a large field-day planned for Spring 2017 in Maryland, and additional smaller programming in the county offices in MD, PA and NY. We also plan to conduct events in conjunction with PA dairy farm events that will occur in Summer 2017, and we will be part of the Vermont Digester Operator's Group event in December 2017. March 23rd, 2016. Presentation titled "Emerging Manure to Energy Technologies - Are Cost Effective Small Scale Digesters Possible" at the USDA Climate Hub Greenhouse Gas Mitigation Workshop by S. Lansing. This included information on manure management styles and how digesters fit into manure management strategies for dairy farmers. This presentation was followed by a live and archived one-hour webinar available for USDA CEU credit (52 participants for credit, 146 live participants total) give on June 1, 2016. Available at: http://www.climatewebinars.net/ webinars/emerging-manure-to-energy-technologies-are-cost-effective-small-scale-digesters-possible Two more presentations on dairy manure technologies were given entitled, "Emerging manure to energy technologies." The first presentation (May 26, 2016) was sponsored by the World Bank in order to share experiences of good management practices in terms of integrated nutrient pollution control and water quality issues to a Romanian delegation. The second presentation was given to the University of Maryland Agriculture Faculty at the Emeritus Recognition Luncheon. May 2016. Presentation titled "Manure treatment and antibacterial impacts" was developed and presented to Applied Agricultural Engineering Continuing Education Series Session No. 14. Nov. 2016. Presentation titled "Antimicrobial resistance from dairy farms: Evaluating the potential of manure management practices to control its spread" was presented at the Agriculture, Food & Environmental Systems In-Service Meeting, and Extension professionals training held annually at Cornell. Abstracts have been submitted to three professional meetings in 2017 (Waste-to-Worth, Biocycle East, and American Society of Agricultural and Biological Engineering). These meeting are typically attended by industry, Extension and academic audiences that specialize in animal agriculture, waste management, composting and anaerobic digestion systems.