ANTIBIOTIC LOADING TO THE ENVIRONMENT FROM DAIRY FARMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1002130
• Project Start Date: Jan 7, 2014
• Project End Date: Sep 30, 2018
• Program Code: [(N/A)]- (N/A)

Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061

Performing Department
Dairy Science

Non Technical Summary
Excretion of antibiotics by livestock and their subsequent activity and persistence in soils is important because their accumulation in manure and soils may provide a potentially significant contributor to antibiotic resistance. Also, the excretion of large portions of dosed antibiotic to manure raises obvious questions about use of that manure as fertilizer in organic food systems. Much more research effort is needed on excretion rates, persistence and environmental activity of agricultural antibiotics and their metabolites.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	0110	1100	50%
402	3410	1100	50%

Knowledge Area
133 - Pollution Prevention and Mitigation; 402 - Engineering Systems and Equipment;

Subject Of Investigation
0110 - Soil; 3410 - Dairy cattle, live animal;

Field Of Science
1100 - Bacteriology;

Keywords

antibiotic resistance

antibiotic excretion

dairy cattle

Goals / Objectives
Evaluate the timecourse dynamics of excretion of antibiotics by dairy cows during and after antibiotic therapy;Identify physico-chemical factors (e.g., temperature, light exposure, pH) most effective in degrading antibiotics in manure; andEvaluate manure management strategies as tools to degrade antibiotics in environmental reservoirs.

Project Methods
Objective I: Evaluation of change in, and temporal pattern of, excretion of antibiotics following antibiotic therapyIa. Antibiotics used for mastitis therapyNine healthy, peak lactation dairy cows will be used in this study. Three cows will be used as negative controls with no antibiotic treatment. Three cows will be treated therapeutically with pirlimycin (Pirsue®; intramammary dose typical for clinical mastitis; two doses of 50 mg each per quarter, 24 h apart). Three cows will be treated therapeutically with cephapirin sodium (ToDay®; one dose of 200 mg per quarter).Ib. Antibiotics used prophylactically at the end of lactationTwelve end-of-lactation cows will be used in this study. Three cows will be used as negative controls with no antibiotic treatment. Three will be treated with single dose intramammary dry cow therapy 300 mg cephapirin activity per quarter (Tomorrow®), 3 cows with Ceftiofur 500 mg per quarter (Spectramast®) and 3 cows with 1 million IU penicillin plus 1 g streptomycin per quarter (Quartermaster®).Ic. Antibiotics used for metritis infectionNine healthy, peak lactation dairy cows will be used for this study. Three cows will be used as negative controls with no antibiotic treatment. Three cows will be treated with Excede® (ceftiofur crystalline free acid; two doses of 300 mg/100 lb body weight, 3 d apart), and 3 cows with penicillin at 18 IU/lb IM once daily for three days.Housing, feeding, sample collection and analysis, common to all studiesCows will be housed in a metabolism barn throughout the study. Twenty-four hours prior to treatment, cows will be moved to the metabolism barn for acclimation and fitted with urinary catheters to allow separate collection of feces and urine. On this day fecal samples will be collected from the rectum into sterile containers and spot samples of urine will be collected to serve as baseline (will be used as covariate for statistical analysis).Twenty-four hours after being moved to the metabolism barn cows will be treated with the assigned antibiotic or sterile saline as control. Total collection (feces and urine) will be conducted from d 1 to 7 post-treatment. Additionally, fecal and urine samples will be collected from the rectum into sterile containers on d 1, 3, 5, 7, 14, and 28 post-treatment. Samples will be stored at -20° C immediately after collection.Fecal and urine samples will be extracted and subject to liquid chromatography tandem mass spectrometry (HPLC/MS/MS) to quantify concentration of antibiotics and metabolites using EPA methods 1694. Antibiotic concentration data will be integrated with mass excretion of urine and feces to quantify extent and time course of antibiotic excretion.Statistical analysis In each study, the main effect of treatment, day, and treatment by day interaction on antibiotic excretion will be evaluated using PROC GLIMMIX in SAS. Pretreatment data for each cow will be used as covariate for that cow.Objective II: Evaluation of physical factors (e.g., temperature, light exposure, pH) most effective in reducing persistence of antibioticsUnderlying mechanisms of the effects of manure treatments on antibiotics depend on temperature, pH, and light exposure. One antibiotic from each of experiments Ia, Ib and Ic will be selected for further study based on their rate of use in the dairy industry and extent of excretion and administered to healthy cows (n=3 per treatment). Feces from cows receiving the antibiotics will be collected on d 3 post treatment into sterile containers. Another set of fecal samples will be collected from cows receiving no antibiotics. Manure will be subjected to different temperatures and pH under light and dark conditions (Table 1).Table 1: Experimental plan (manure incubation)Feces*Temperature, ºCpHLight/darkSampling time, dNo antibiotic10, 25, 35, 555, 7, 9, 12Light/dark0, 1, 3, 7, 14, 28, 56, 90Antibiotic 110, 25, 35, 555, 7, 9, 12Light/dark0, 1, 3, 7, 14, 28, 56, 90Antibiotic 210, 25, 35, 555, 7, 9, 12Light/dark0, 1, 3, 7, 14, 28, 56, 90Antibiotic 310, 25, 35, 555, 7, 9, 12Light/dark0, 1, 3, 7, 14, 28, 56, 90A separate set of incubations will be conducted using dry cow feces without added water to evaluate the impact of water content on antibiotic degradation. The combination of physical factors and the experimental setup will be similar to that described above. The first set of analyses will include samples from four timepoints (d 0, 3, 7, and 28 = 128) and the results will determine the need for analysis of samples from remaining timepoints.Statistical analysis. The main effect of the fixed variables (temperature, pH, light, time) and their interaction on antibiotic degradation in manure will be evaluated in SAS using PROC GLIMMIX.Objective III: Identification of most effective practical management practices based on the underlying mechanisms identified in Objective IISelection of manure management practice and relevant parameters will be based on the results of incubation studies described in Objective II; as a starting point we propose the following experiments.IIIa -anaerobic liquid storage. The fate of antibiotics in manure (collected during the cow study described in Objective II) will be evaluated by storing liquid manure anaerobically for a period of 18 weeks. Eight 1100 L tanks will be used to simulate manure storage pits. Nine hundred L of manure from each treatment will be added to replicate storage tanks at the beginning of each storage period. The manure will be stored in a temperature controlled room with temperature managed based on average weekly temperatures (derived from the analysis of the local weather data for the past 10 years). A 10 mL aliquot will be sampled on d 1, 3, 7, 10 and 14, and then every two weeks for antibiotics analysis. There will be 4 manures, 13 timepoints, and 2 replicates (4 × 13 × 2 = 104 samples).IIIb - Stockpile. Manure will be obtained from cows receiving dry cow therapy to construct replicate stockpiles, each 15 m long, 1 m wide and 0.5 m high. Stockpiles will be left untouched for 18 wk except during temperature recording and sample collection. Each stockpile will be divided into 3 sampling regions (~4 m long) each separated by 1 m long non-sampling regions. Samples (50 g/location) will be collected on d 1, 3, 7, 10 and 14, and then every two weeks. Samples will be stored immediately at -20°C until antibiotic analysis.IIIC - composting. The influence of composting on antibiotic residues will be evaluated using laboratory-scale composters as described by others (Arikan et al., 2009b; Wang et al., 2012b). Fresh feces from dairy cows receiving no antibiotic, and manure from the health cows treated with cephapirin, pirlimycin, and ceftiofur will be used in this study. Feces will be mixed with straw (< 1 cm long) and hardwood chips (< 1 cm particle size) in a ratio (w/w) to achieve 40% total solids. An aliquot (? 2 kg) of mixed manure will be loaded in duplicate composters for each type of feces and will be incubated for 18 weeks. Temperature and pH will be monitored daily. Samples will be collected on d 1, 3, 7, 10 and 14, and then every two weeks.Statistical analysis. The main effect of manure management practices, day, and their interaction will be evaluated using PROC GLIMMIX in SAS. The model will include fixed effect of management practices, day, and their interactions.

Progress 01/07/14 to 09/30/18

Outputs
Target Audience:The target audiences are dairy and beef farmers, veterinarians and other technical support personnel, federal agencies (USDA, FDA, EPA others), and policy/decision-makers. Also graduate and undergraduate students studying in an array of relevant fields. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A total of six undergraduate students had the opportunity to work one-on-one with a mentor. A total of 13 undergraduate students had the opportunity to review the relevant literature in a small group weekly class/journal club with a research mentor. One graduate student was trained, earned an MS degree and had the opportunity to present research at a national meeting. One post-doctoral researcher was trained and had the opportunity to present resercch at a national meeting. How have the results been disseminated to communities of interest?Extension publications were developed and distributed. Course modules targeted at undergraduate and graduate students were developed and distributed. Peer-reviewed research publications were published. Invited lectures were presented. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? More than 70% of antibiotics sold in the US are used in animal agriculture and societal concern is increasing about the contribution of this to the medical challenge of antibiotic resistance. Our interdisciplinary research group seeks to act proactively to identify critical control points, from farm to fork, to reduce the spread of antibiotic resistance. One of our research activities is measurement of the timecourse of excretion of antibiotics from treated animals. This will support efforts to isolate antibiotic-containing manure, allowing manure treatment interventions. The target audiences are dairy and beef farmers, veterinarians and other technical support personnel, federal agencies (USDA, FDA, EPA others), and policy/decision-makers. My research group has demonstrated that excretion of therapeutically dosed antibiotics by beef and dairy cattle is rapid (within hours or days of treatment). This suggests that only manure from 3 to 5 days post-treatment is a risk factor for spread of antibiotics to the ecosystem. If sick cows are segregated from the herd for treatment (as is usual practice) segregation of their manure is relatively simple. Our research has demonstrated that composting of manure from antibiotic treated beef and dairy cattle is effective in degrading many, although not all, antibiotics, within 7 weeks. Our work has resulted in increased knowledge about the relative impact of various best management practices on flow of antibiotics through critical control points in agro-ecosystems, increased opportunities for students to engage in experimental research, and increased interdisciplinary collaboration and stakeholder involvement. Expected long term impacts include improved science-based policy regarding the management of antibiotics used in livestock, sustained therapeutic effectiveness of antibiotics, a safer food supply, and reduced health costs due to fewer cases of antibiotic resistance development in population. In this period: 1. Experiments were conducted and samples were analyzed to quantify excretion of pirlimycin, cephapirin and ceftofur from dairy cows treated with recommended dosages. 2. Experiments were conducted and samples were analyzed from experiments conducted to identify physico-chemical factors (e.g., temperature, light exposure, pH) most effective in degrading antibiotics in manure. 3. Experiments were conducted and samples were analyzed from experiments conducted to evaluate manure management strategies as tools to degrade antibiotics in environmental reservoirs. 4. Data was analyzed from the above experiments. Following intramammary infusion, cephapirin and pirlimycin were excreted in feces and urine for at least 3 d following administration. Prolonged excretion (days rather than hours) suggests that manure from cows receiving intramammary antibiotic preparations should be segregated and treated to remove antibiotic residues. Such treatment systems would need only treat relatively small quantities of manure because on dairy farms, animals receiving antibiotics are typically housed separately from the main herd (e.g. the dry cow pen or hospital pen). Generation of quantitative data on mass and timecourse of excreted antibiotics will help the dairy industry be proactive in developing pragmatic science-based solutions to this emerging environmental challenge. Data was generated on the effect of composting on degradation of antibiotics in beef and dairy cattle manure. The study provides a comprehensive, head-to-head, replicated comparison of the effect of static and turned composting on typical antibiotics used in dairy cattle in their actual excreted form and corresponding influence on composting efficacy. Manure from dairy cows (with or without pirlimycin and cephapirin administration) were composted at small-scale (wet mass: 20-22 kg) in triplicate under static and turned conditions following "federal guidelines". Thermophilic temperature (>55°C) was attained and maintained for 3 d in all composts, with no measureable effect of compost method on the pattern, rate, or extent of disappearance of the antibiotics examined. Disappearance of all antibiotics, except pirlimycin, followed bi-phasic first-order kinetics. Near-complete removal of pirlimycin (100%) was achieved. Both static and turned composting were generally effective for reducing three common dairy antibiotic residuals excreted in manure, with no apparent negative impact of antibiotics on the composting process.

Publications

• Type: Other Status: Published Year Published: 2017 Citation: Maurer, Knowlton and Vallaton. 2017. What is the Veterinary Feed Directive? Virginia Cooperative Extension publication number HORT-262NP
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Knowlton K. F. and P. P. Ray. Competing priorities: Antibiotic use in a time of increasing antibiotic resistance. In Proceedings of the Virginia State Feed Association and Feed Management Cow College, Roanoke, VA. February 2018.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Knowlton K. F. and M. A. G. von Keyserlingk. To treat or not to treat: Public attitudes on the therapeutic use of antibiotics in the dairy industry. World Conference on Animal Production, Vancouver, BC, CAN.
• Type: Other Status: Published Year Published: 2018 Citation: H. T. V. Le, P. Ray, K. Knowlton, R. O. Maguire, and K. Xia. 2018. H. T. V. Le, P. Ray, K. Knowlton, R. O. Maguire, and K. Xia. 2018. Environmental fate of antibiotics - Impact of manure land application methods. Abstracts of Papers of the American Chemical Society. Vol. 255. 1155 16th ST, NW, Washington, DC 20036: Amer Chemical Soc.
• Type: Other Status: Published Year Published: 2017 Citation: Gleason, Maurer, Knowlton and Vallaton. 2017. Battling Resistance: Judicious Antibiotic Use in Beef and Dairy Cattle. Virginia Cooperative Extension publication number DASC-106NP

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Target audiences reached in this reporting period include extension specialists, policy and decision makes and academia (faculty and university students). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduates and one graduate student received training. How have the results been disseminated to communities of interest?Published in peer-reviewed journal. Presented to advisory group of policymakers, extension specialists and scientists. What do you plan to do during the next reporting period to accomplish the goals?Finish analysis of effect of anaerobic digestion on degradation of antibiotics in dairy manure. Prepare and submit publications on these results.

Impacts
What was accomplished under these goals? Data was generated on the effect of composting on degradation of antibiotics in beef and dairy cattle manure. The study provides a comprehensive, head-to-head, replicated comparison of the effect of static and turned composting on typical antibiotics used in beef and dairy cattle in their actual excreted form and corresponding influence on composting efficacy. Manure from steers (with or without chlortetracycline, sulfamethazine, and tylosin feeding) and dairy cows (with or without pirlimycin and cephapirin administration) were composted at small-scale (wet mass: 20-22 kg) in triplicate under static and turned conditions following "federal guidelines". Thermophilic temperature (>55°C) was attained and maintained for 3 d in all composts, with no measureable effect of compost method on the pattern, rate, or extent of disappearance of the antibiotics examined, except tylosin. Disappearance of all antibiotics, except pirlimycin, followed bi-phasic first-order kinetics. However, individual antibiotics displayed different fate patterns in response to the treatments. Reduction in concentration of chlortetracycline (71 to 84%) and tetracycline (66 to 72%) was substantial, while near-complete removal of sulfamethazine (97 to 98%) and pirlimycin (100%) was achieved. Tylosin removal during composting was relatively poor. Both static and turned composting were generally effective for reducing most beef and dairy antibiotic residuals excreted in manure, with no apparent negative impact of antibiotics on the composting process, but with some antibiotics apparently more recalcitrant than others.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Wepking, C., B. Avera, B. Badgley, J. E. Barrett, J. Franklin, K. F. Knowlton, P. P Ray, C. Smitherman and M.S. Strickland. 2017. Exposure to dairy manure leads to greater antibiotic resistance and increased mass-specific respiration in soil microbial communities. Proc. Royal Soc.B 284: DOI: 10.1098/rspb.2016.2233
• Type: Book Chapters Status: Published Year Published: 2017 Citation: Littier, H., L. Chambers, and K. F. Knowlton. 2017. Animal agriculture as a contributor to the global challenge of antibiotic resistance. CAB Rev. 12 (003). doi: 10.1079/PAVSNNR201712003
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ray, P.P., C. Chen, K. F. Knowlton, A. Pruden and K. Xia. 2017. Fate and effect of antibiotics in beef and dairy manure during static and turned composting. J. Environ. Qual. 46: 45-54. DOI:10.2134/jeq2016.07.0269

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Target audiences reached in this reporting period include extension specialists, policy and decision makes and academia (faculty and university students). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two undergraduate students and one post-doctoral researcher received training. How have the results been disseminated to communities of interest?Accepted for publication in peer-reviewed journal. Presented to advisory group of policymakers, extension specialists and scientists. What do you plan to do during the next reporting period to accomplish the goals?Finish analysis of effect of anaerobic digestion on degradation of antibiotics in dairy manure. Prepare and submit publication on these results.

Impacts
What was accomplished under these goals? Data was generated on the effect of composting on degradation of antibiotics in beef and dairy cattle manure. The study provides a comprehensive, head-to-head, replicated comparison of the effect of static and turned composting on typical antibiotics used in beef and dairy cattle in their actual excreted form and corresponding influence on composting efficacy. Manure from steers (with or without chlortetracycline, sulfamethazine, and tylosin feeding) and dairy cows (with or without pirlimycin and cephapirin administration) were composted at small-scale (wet mass: 20-22 kg) in triplicate under static and turned conditions following "federal guidelines". Thermophilic temperature (>55°C) was attained and maintained for 3 d in all composts, with no measureable effect of compost method on the pattern, rate, or extent of disappearance of the antibiotics examined, except tylosin. Disappearance of all antibiotics, except pirlimycin, followed bi-phasic first-order kinetics. However, individual antibiotics displayed different fate patterns in response to the treatments. Reduction in concentration of chlortetracycline (71 to 84%) and tetracycline (66 to 72%) was substantial, while near-complete removal of sulfamethazine (97 to 98%) and pirlimycin (100%) was achieved. Tylosin removal during composting was relatively poor. Both static and turned composting were generally effective for reducing most beef and dairy antibiotic residuals excreted in manure, with no apparent negative impact of antibiotics on the composting process, but with some antibiotics apparently more recalcitrant than others.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kulesza, S. B., R. O. Maguire, K. Xia, J. Cushman, K. Knowlton, and P. Ray. 2016. Manure injection affects the fate of pirlimycin in surface runoff and soil. Journal of environmental quality 45:511-518.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Partha Ray, Chaoqi Chen, Katharine F. Knowlton, Amy Pruden, and Kang Xia. Fate and effect of antibiotics in beef and dairy manure during static and turned composting. J. Environ. Qual (in press)

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audiences reached in this reported period include extension specialists and agents, dairy farmers, policy- and decision-makers, and academia including faculty and university students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One post-doctoral researcher and 2 undergraduate students have been trained. How have the results been disseminated to communities of interest?Two research papers have been published, and a lecture delivered in 3 settings. What do you plan to do during the next reporting period to accomplish the goals?Research on the effects of manure treatment will be completed, samples analyzed and results published.

Impacts
What was accomplished under these goals? Research is underway to evaluate the effect of physico-chemical factors (e.g., temperature, light exposure, pH) most effective in degrading antibiotics in manure. A manuscript summarizing the results is in development. Research is underway to evaluate the effect of manure treatment strategies on antibiotic persistance in manure.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Kulesza, S. B, J. Cushman, K. Knowlton, R. O. Maguire , P. Ray, and K. Xia. 2015. Manure injection impacts fate of pirlimycin in surface runoff and soil. J. Environ. Qual.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Chambers, L., Ying Yang, Heather Littier, Partha Ray, Tong Zhang, Amy Pruden, Michael Strickland, and Katharine Knowlton. 2015. Metagenomic analysis of antibiotic resistance genes in dairy cow feces following therapeutic administration of third generation cephalosporin. PLOS-ONE 10.8 (2015) E0133764

Progress 01/07/14 to 09/30/14

Outputs
Target Audience: Animal and dairy scientists through presentation of results at a national meeting. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two undergraduate students had the opportunity to work one-on-one with a mentor. One graduate student had the opportunity to present research results at a national meeting. One post-doctoral researcher had the opportunity to present research results at a national meeting. How have the results been disseminated to communities of interest? Three abstracts were presented (1 by a graduate student and 2 by a post-doc) at the national meeting of the American Dairy Science Association. The audience included other animal and dairy scientists. One paper has been published in a peer-reviewed journal on the results of this research. What do you plan to do during the next reporting period to accomplish the goals? Additional experiments will be conducted and sample analysis completed. Additional research presentations and publications will be presented.

Impacts
What was accomplished under these goals? 1. Experiments were conducted to evaluate the timecourse dynamics of excretion of antibiotics by dairy cows during and after antibiotic therapy. 2. Experiments were conducted to identify physico-chemical factors (e.g., temperature, light exposure, pH) most effective in degrading antibiotics in manure. 3. Experiments were begun to evaluate manure management strategies as tools to degrade antibiotics in environmental reservoirs.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Caudle, L., H. Littier, A. Pruden-Bagchi, X. Feng, and K. F. Knowlton. 2014. Effect of prophylactic and therapeutic antibiotic administration on fecal excretion of antibiotic resistance genes by dairy cows. J. Dairy Sci. 97 (Suppl. 2): 169
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Ray, P., C. Shang, K. Xia, and K. F. Knowlton. 2014. Development and validation of a UPLC-MS/MS method to monitor cephapirin excretion in dairy cows following intramammary infusion. PlOS-One 9 (11): e112343. DOI: 10.1371/journal.pone.0112343
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Ray, P. P., K. F. Knowlton, C. Shang and K. Xia. 2014. Quantification of cephapirin in dairy cow feces and urine using solid phase extraction (SPE) coupled with ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) . J. Dairy Sci. 97 (Suppl. 2): 747.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Ray, P. P., K. F. Knowlton, C. Shang and K. Xia. 2014. Method development and application: Solid phase extraction (SPE) clean-up and ultra performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) quantification of pirlimycin in dairy cow feces and urine. J. Dairy Sci. 97 (Suppl. 2): 748.