Source: UNIVERSITY OF MAINE submitted to
ENVIRONMENTAL IMPACTS OF EQUINE OPERATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1009215
Grant No.
(N/A)
Project No.
ME031604
Proposal No.
(N/A)
Multistate No.
NE-1441
Program Code
(N/A)
Project Start Date
Jan 29, 2016
Project End Date
Sep 30, 2019
Grant Year
(N/A)
Project Director
Causey, R.
Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469
Performing Department
Animal and Veterinary Sciences
Non Technical Summary
In the US, over 12 million tons of antibiotics are sold for use in animals annually. Manureand waste bedding from these animals can contain antibiotic residues. In thecultivation of fruits and vegetables grown for human consumption, small farms mayuse composted equine waste as a soil amendment. The potential therefore exists forcontamination of the food supply both by antibiotics in composted animal waste, andby antibiotic resistant bacteria (ABRB) whose survival is promoted by antibiotics. Wewish to show proof-of-concept that antibiotics alter the microflora of compost, potentiallyfavoring ABRB, and possibly preventing inactivation of pathogenic organismsby the composting process. The objectives of this study are to compare microbialcommunities in composted equine waste with and without the antibiotic TrimethoprimSulfadiazine. Specifically we will study composting of: 1. waste from horses receivingantibiotics, 2. antibiotics directly applied to equine waste, and 3. equine wastewith no antibiotics. For each of these three methods the trial will be repeated inthree replicate piles, with samples taken for analysis after 0, 3, 7, 14, 21, 28, and 35days of composting. Microbial activity will be compared using single carbon-sourcesubstrate autilization, temperature profiling and 16S rRNA gene sequencing, Resultswill improve our knowledge of the effects of antibiotics on microbial communities incompost. In particular this seed grant would lead to development of non-pathogenic,traceable antibiotic resistant surrogate organisms which could be used to track ABRBin compost, cultivated vegetables, run-off and the food supply.
Animal Health Component
10%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71238101100100%
Goals / Objectives
Develop suitable manure storage and disposal strategies best adapted to equine farms, particularly focusing on smaller farms (< 20 head per farm)
Project Methods
We will address the impact of a common equine oral antibiotic (Trimethoprim - Sulfadiazine)on the microbial flora in composted equine manure. Specifically, in piles ofcomposted equine manure with and without Trimethoprim - Sulfadiazine we will:1. Determine the effect of Trimethoprim - Sulfadiazine on microbial activity throughcomparing temperature profiles and metabolism of substrates2. Determine the effect of Trimethoprim - Sulfadiazine on microbial community structureby comparing colony counts and 16S ribosomal RNA gene sequence data.The information generated would then serve as a starting point for further studiesto develop best practices to eliminate antimicrobialresistant bacteria in compost intendedfor use on small farms.

Progress 01/29/16 to 09/30/19

Outputs
Target Audience:This work is intended to be of value to all equine industry professionals, veterinarians, organic farmers, pharmaceutical manufacturers and state and federal regulatory agencies. Changes/Problems:Based on reviews from granting agencies wealtered the focus of the study to determine if there is indeed a problem of antibiotic resistance in equine stall waste. We chose to determine the presence of antibiotics resistant to three antibiotics (ampicillin, gentamycin, and ciprofloxacin) that represent three different classes of antibiotics (beta lactamases, aminoglycosides, and fluorinated quinolones) all of are commonly used in human medicine. This was selected as the basis of the dissertation research of Alexandria Poulin. However, she subsequently obtained a teaching position and moved to a different PhD advisor. This inpart was due to reduction in my research activitywhen I assumedincreased administrative duties. What opportunities for training and professional development has the project provided?Alexandria Poulin received her MS in May 2016. Scott Mitchell successfully wrote a summer research grant to fund this work and presented the data at the 10th International Conference on Equine Infectious Diseases in Buenoa Aires, Argentina in April 2016.This project supportedtwo undegraduate senior projects (Jessica Wilson and Andrew Hatch) How have the results been disseminated to communities of interest?Work has been presented at the Waste to Worth conference in Cary NC on April 2017, at the annual multistate meetings of NE1441, at scientific meetings and in the scientific literature. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have developed methods (PCR and ELISA) of detecting the DNA and protein respectively of equine pathogens in compost. This research will assist usindevelopment of best practices of storage of waste bedding. We have used equine manure and compost as a readily available media for testing the longevity of S. equi in the environment. We have shown that the natural microbial community in soiled equine bedding and compost is able to kill S. equi. The moisture content of bedding appears to be a key component of the killing. This work served as a Master's Thesis for Alexandria Poulin (nee Garcia), and components were presented at USDA multistate meeting. We conducteda trial to evaluate the effectiveness of Jones Hamilton Poultry Litter Treatment (Sodium Bisulfate) in ammonia abatement in horse stalls. Findings indicated thatthe hygroscopic nature of the product created moisture in the bedding, which was considered a disadvantage. We alsoevaluated in-paddock composting of horse manure and concluded that it was necessary to use additional stall waste. Possily the added carbon from shavings and nitrogen from urine aided the composting processin the paddock. Horses are administered high levels of antibiotics for therapeutic purposes. We have begun work on addressing the prevalence of antibiotic resistant bacteria in normal equine manure, by examining the presence of gram negative bacteria and coliforms displaying resistance to ampiciliin, gentamicin, and ciprofloxacin in equine stall waste. Specifically, to develop techiques, we have surveyed microbial populations in Tryptic Soy Agar (TSA; reveals Gram + and - bacteria) and MacKonkey's Agar (MAC; reveals only Gram - bacteria), each media being used with and without antibiotics. To date we have investigated the presence of ampicillin resistance, and shown a significant ampicillin resistant population in gram negative microflora in stall waste.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: (** = Graduate Student; * = Undergraduate Student) Poulin A**, Hutchinson M, Dube M, Stokes M, Mitchell S*, Edwards A*, Harvey K*, Myer A*, Causey R, Abatement of Streptococcus equi in Soiled Equine Bedding and Compost, Journal of Equine Veterinary Science. 2018;70:117-122.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poulin A, Causey R. 2017 Abatement of Streptococcus equi in Equine Compost Proceedings of the Waste to Worth Conference, Cary NC April 18-21
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mitchell S, Bernard E, Longo A, Garcia M, Hutchinson M, Causey R. The use of PLFA analysis to detect differences in microbial activity in compost from horses treated with and without antibiotics. 2016. Journal of Equine Veterinary Science, Volume 39 , S5 (Abstract).
  • Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Poulin A. Abatement of Streptococcus equi in equine compost. MS Thesis, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: State Report to Annual Meeting of NE1441, Environmental Impacts of Equine Operations, University of Maryland, College Park, Maryland, August 8, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: State Report to Annual Meeting of NE1441, Environmental Impacts of Equine Operations, University of Maine, Orono, Maine, August 20-22, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: State Report to Annual Meeting of NE1441, Environmental Impacts of Equine Operations, Rutgers University, Columbus, New Jersey, August 19-21, 2019.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:This work is intended to be of value to all equine industry professionals, veterinarians, organic farmers, pharmaceutical manufacturers and state and federal regulatory agencies. Changes/Problems:Based on reviews from granting agencies we have altered the focus of the study to determine if there is indeedproblem of antibiotic resistance in equine stall waste. We have therefore chosen to determine the presence of antibiotics resistant to threeantibiotics (ampicillin, gentamycin, and ciprofloxacin)that represent three different classes of antibiotics (beta lactamases, aminoglycosides, and fluorinated quinolones) all ofare commonly used in human medicine. What opportunities for training and professional development has the project provided?This work constitutes part of theDissertation Research for Alexandria Poulin who ispursuing a PhD. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We will extend testing to gentamicin and ciprofloxacin, and to compareresults between fresh stall waste, stall waste from an active pile at140 F or greater, and from a pile that has been allowed to cure for several months and has cooledto ambient temperature.

Impacts
What was accomplished under these goals? We have begun work on addressing the prevalence of antibiotic resistant bacteria innormal equine manure, byexaminingthe presence ofgram negative bacteria and coliforms displayingresistance to ampiciliin, gentamicin, and ciprofloxacin in equine stall waste. Specifically, todeveloptechiques, we have begun to survey microbial populations inTryptic Soy Agar (TSA; revealsGram + and - bacteria) and MacKonkey's Agar (MAC; reveals onlyGram - bacteria), each media being used with and without antibiotics.To date we have investigated the presence of ampicillin resistance, and shown a significant ampicillinresistant population ingram negativemicroflora in stall waste.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: (** = Graduate Student; * = Undergraduate Student) Poulin A**, Hutchinson M, Dube M, Stokes M, Mitchell S*, Edwards A*, Harvey K*, Myer A*, Causey R, Abatement of Streptococcus equi in Soiled Equine Bedding and Compost, Journal of Equine Veterinary Science. 2018;70:117-122.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: State Report to Annual Meeting of NE1441, Environmental Impacts of Equine Operations, University of Maine, Orono, August 20 ⿿ 22, ME 2018. Poulin A, Knight C.


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

Outputs
Target Audience:This work is intended to be of value to all equine industry professionals, veterinarians, organic farmers, pharmaceutical manufacturers and state and federal regulatory agencies. At the Waste to Worth Conference in Cary NC in April 18-21, we presented data from Alexandria Poulin's Master's Thesis. This allowed us to establishe relationships with Jones Hamilton corporation and the University of Florida. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project currently supports two undegraduate senior projects (Jessica Wilson and Andrew Hatch), and is the basis of Alexandria Poulin's PhD research. How have the results been disseminated to communities of interest?Work to date has been presented at the Waste to Worth conference in Cary NC on April 2017 and at the annual multistate meeting of NE1441. What do you plan to do during the next reporting period to accomplish the goals?The next meeting of the multistate group NE1441 is scheduled for August 21-22, 2018 at the University of Maine, hosted by the PD (Causey).

Impacts
What was accomplished under these goals? We are currently conducting a trial to evaluate the effectiveness of Jones Hamilton Poultry Litter Treatment (Sodium Bisulfate) in ammonia abatement in horse stalls. We are also conducting a project to evaluate the in paddock composting of horse manure using additional stall waste added to the paddock.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Poulin A, Causey R. 2017 Abatement of Streptococcus equi in Equine Compost Proceedings of the Waste to Worth Conference, Cary NC April 18-21


Progress 01/29/16 to 09/30/16

Outputs
Target Audience:This work is intended to be of value to all equine industry professionals, veterinarians, organic farmers, pharmaceutical manufacturers and state and federal regulatory agencies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Alexandria Poulin received her MS in May 2016 working on this project. Scott Mitchell successfully wrote a summer research grant to fund this work and presented the data at the 10th International Conference on Equine Infectious Diseases in Buenoa Aires, Argentina in April 2016. How have the results been disseminated to communities of interest?They have been presented at national meetings and in conference proceedings. What do you plan to do during the next reporting period to accomplish the goals?Alexandria Poulin has commenced a PhD prgram looking at the transport of pathogens from waste bedding into vegetables, and how this transport can be mitigated by composting.

Impacts
What was accomplished under these goals? The Impacts of this project may be summarized as follows: Horses are administered high levels of antibiotics for therapeutic purposes. These antibiotics are frequently excreted in urine and feces, and contaminate waste bedding. Because composted horse bedding is used as fertilizer there is a chance these antibiotics may find their way into the food supply. In addition, the antibiotics may contribute to the development of antibiotic resistant bacteria. We have shown changes in the microbes in composted bedding from horses treated with antibiotics. In addition, we are also interested in detection of potential pathogens in compost. For this reason we have developed methods (PCR and ELISA) of detecting the DNA and protein respectively of equine pathogens in compost. This research will allow us to develop best practices of storage of waste bedding. The following is a more detailed account of these accomplishments. A study was carried out to determine the impact of a common oral antibiotic administered to horses (sulphadiazine trimethoprim), on the microbial community of feces from horses receiving antibiotics. A herd of 15 horses aged received the recommended dose of a commercial oral antibiotic (12.5 g of sulfadiazine and 2.5 g trimethoprim per 1100 lb horse SID) for 4 days. Manure was collected from all horses from days 2 to 4 of antibiotic administration and composted in a static windrow, sampled at 0, 2, and 7 days. Samples were submitted at each time point for phospholipid fatty acid (PLFA) analysis. In samples from horses not treated with these antibiotics, gram-positive fatty acids accounted for a larger percentage of the microbial community than gram-negative bacterial fatty acids. In samples from treated horses, both gram-positive and gram-negative bacterial fatty acid content stayed constant over time. This suggests the presence of Sulfadiazine-trimethoprim in compost inhibits the growth of gram-positive bacteria in comparison to gram-negative bacteria. While preliminary, these data suggest that administration of oral antibiotics to horses alters the microflora in composted equine stall waste. We also wished to determine the relative merits of PCR, ELISA and culture in surveillance for S. equi. We have been able to establish capability for all three assays in our laboratory. We have used equine manure and compost as a readily available media for testing the longevity of S. equi in the environment. We have been able to show that the natural microbial community in soiled equine bedding and compost is able to kill S. equi. The moisture content of bedding appears to be a key component of the killing. This work served as a Master's Thesis for Alexandria Poulin (nee Garcia), and components were presented at USDA multistate meeting (NE 1441 Environmental Impacts of Equine operations) in 2015 and 2016. Work on compost led to a USDA grant submission in 2015.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Mitchell S, Bernard E, Longo A, Garcia M, Hutchinson M, Causey R. The use of PLFA analysis to detect differences in microbial activity in compost from horses treated with and without antibiotics. 2016. Journal of Equine Veterinary Science , Volume 39 , S5 (Abstract).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Poulin A. Abatement of Streptococcus equi in equine compost. MS Thesis, 2016.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: State Report to Annual Meeting of NE!441, Environmental Impacts of Equine Operations, University of Maryland, College Park, Maryland, August 8, 2016.