EFFECT OF FEEDING DAIRY CALVES MILK CONTAINING ANTIMICROBIAL RESIDUES ON THE EMERGENCE OF ANTIMICROBIAL RESISTANT ENTERIC BACTERIA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0230577
• Project Start Date: Oct 1, 2012
• Project End Date: Sep 30, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Vet Population Medicine & Diagnostic Science

Non Technical Summary
The proposed study supports the USDA National Institute of Food and Agriculture (NIFA) strategic plan (2007-2012) which includes reducing the incidence of foodborne illnesses and contaminants through research, education and extension (Objective 4.1); it also supports Cornell University's applied Research and Extension priorities (FY12-16) which include food safety, emphasizing causes of microbiological contamination and microbiological resistance, and global food security and hunger, highlighting increased use of sustainable practices resulting in improved or protected soil, air and water quality and production of safe food. In the last report by the Centers for Disease Control and Prevention (CDC) for foodborne disease outbreaks in the United States, non-typhoical Salmonella was the most common cause of outbreak-related hospitalization and was responsible for more than half of multistate outbreaks. Shiga toxin-producing Escherichia coli (STEC) was the second most common cause of outbreak-related hospitalizations. Compared with susceptible strains, multidrug resistant Salmonella pose an increased threat to public health causing severe illness and an increased risk of hospitalization. According to the FDA summary report on antimicrobials sold or distributed for use in food-producing animals and the IMS Nationals Sales Perspectives for antibacterial drugs, more than 79% of antimicrobials in the United States are used in food-producing animals. On dairy farms, pre-weaned calves are an important source for antibiotic resistance and have been identified as having 23 times higher odds of presenting multidrug resistant E. coli isolates compared to cows. In the Unites States approximately 33% of dairy farms feed calves "waste milk", the non-salable milk originated from cows being treated with antibiotics. Advantages of feeding calves waste milk include greater weight gain and an estimated savings of $0.69 per calf per day when compared to feeding milk without antibiotic residues. To evaluate the effect of feeding calves waste milk on the amplification of multidrug resistance, our study proposes to quantify the concentrations of residual antibiotics in waste milk from Upstate NY dairy farms and use this information to formulate laboratory-made waste milk that mimics waste milk from commercial dairy farms. This formulated waste milk will be fed to pre-weaned calves which will be monitored for antimicrobial resistant Salmonella and E. coli and for shifts in the fecal micro biota and antibiotic resistance genes using epidemiological, microbiological and modern metagenomic technics (culture-independent studies of the collective set of genomes of mixed microbial communities). The results from our study will generate information to allow dairy producers, veterinarians and other public health professionals to make informed decisions and develop cohesive strategies to reduce the spread and persistence of multidrug resistant bacteria, benefiting both animal and human health.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
307	3410	1170	10%
307	3450	1040	20%
311	3410	1040	20%
311	3410	1100	20%
722	3410	1040	10%
722	3410	1100	10%
722	3410	1170	10%

Knowledge Area
311 - Animal Diseases; 722 - Zoonotic Diseases and Parasites Affecting Humans; 307 - Animal Management Systems;

Subject Of Investigation
3410 - Dairy cattle, live animal; 3450 - Milk;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1170 - Epidemiology;

Keywords

salmonella

escherichia coli

antibiotic resistance

zoonoses

food safety

calves

milk

Goals / Objectives
The goal of this project is to determine the effect of feeding dairy calves non-salable milk containing antimicrobial residues ("waste milk") on the emergence and endurance of antimicrobial resistant enteric bacteria. The main objectives of our proposed project will be: define the concentration of antimicrobial drugs present in the "waste milk" fed to pre-weaned calves in representative dairy farms in Upstate New York and use the data as a reference in the design of a controlled trial where we will measure the impact from feeding pre-weaned dairy calves waste milk on the dissemination and persistence of antimicrobial resistant Salmonella and E. coli and on the shift of the fecal microbiota and antibiotic resistance genes. We expect to find that feeding dairy calves milk containing low concentrations of antimicrobial drugs increases the frequency of antimicrobial resistance genes and antimicrobial resistant Salmonella and E. coli on dairy farms, thereby creating a health hazard for both animals and humans. By using metagenomic techniques (culture-independent technique for studying the collective set of genomes of mixed microbial communities), we expect to demonstrate that feeding waste milk to pre-weaned calves allows resistant bacteria to thrive, as shown in previous research where the fecal E. coli population increased in swine receiving feed-added antimicrobials. We also anticipate that feeding calves low levels of antimicrobial drugs in the milk will increase the diversity of antibiotic resistance genes compared to calves receiving non-medicated whole milk. Reducing the transmission of agents causing foodborne diseases such as multidrug resistant Salmonella is a challenge for the food industry and public health professionals. This is currently of particular relevance in New York State where resistant Salmonella Dublin, a host-adapted serovar in cattle, is becoming more prevalent. The results of the proposed studies will provide a solid basis for public health professionals to make science based assessments, estimate risks and implement strategic solutions to reduce the dissemination of antimicrobial resistance, benefiting both animal and human health. In addition, farm management practices that result in reduced antimicrobial resistance, decreased incident of food-borne illnesses, and treatment failure will extend the effectiveness of currently available antibiotics and prolong the return on investment for those drugs, benefiting pharmaceutical companies, the food production industry and consumers and also attract future investments for the development of new effective drugs by the private sector.

Project Methods
The first phase of our project will consist of collecting non-salable milk ("waste milk") samples from a representative number of dairy farms in Upstate New York and use commercially available methods to test for the presence of antibiotic residues in the milk. Samples testing positive for antibiotics will be further analyzed using Liquid Chromatography Mass Spectrometry, a technique that quantifies antibiotics present in milk in nanograms per milliliter (ng/ml). The results from the antibiotic quantification will be used to create a database of concentrations for the antibiotics identified in waste milk. This dataset will be used in a controlled trial where one group of pre-weaned calves will receive whole milk with antibiotics added at concentrations identified in the waste milk, and one group will receive non-medicated whole milk. Fecal samples collected from these calves will be used to isolate E. coli and Salmonella which will be tested for antimicrobial susceptibility to 12 antibiotics using Kirby-Bauer disk diffusion agar assay in accordance with the guidelines published by the Clinical and Laboratory Standards Institute (CLSI). Statistical analysis of the data from the antimicrobial susceptibility test for E.coli and Salmonella isolates will be done in SAS (SAS Inst., Inc., Cary, NC). The impact of feeding pre-weaned calves with milk containing residual levels of antibiotic on fecal microbiota and antibiotic resistance genes will be addressed by retrieving frozen fecal samples collected from pre-weaned calves in the controlled trial and analyzing these samples using modern culture-independent techniques. For this procedure we will extract DNA from the fecal samples and amplify the 16S rRNA gene for individual samples using a unique 10-base barcode primer. These individual barcoded bacterial 16S rRNA genes will then be prepared for pyrosequencing at the Cornell University Life Sciences Core Laboratories Center using Roche 454 GS-FLX System Titanium Chemistry. Selected fecal DNA samples will also have the bacterial community explored by using metagenomic whole genome shotgun sequencing. The Ribosomal Database Project (RDP) classifier will be used to assign 16S rRNA gene sequences of each sample to the bacterial taxonomy. Using Basic Local Alignment Search Tool (BLAST), the pyrosequensing data will be used to examine the nucleotide collection databases. The metagenomic DNA contigs will be analyzed using MEGAN, a computer program that allows analysis of large metagenomic data sets. The metagenomic DNA will also be analysed against the Antibiotic Resistant Gene Database (ARGD). Through these timely proposed studies we will directly benefit dairy farmers, veterinarians and other professionals in the dairy industry by providing scientific data to allow evaluation and implementation of sustainable on-farm management practices that will reduce the dissemination of antimicrobial resistance and generate safe food. To increase the impact of the results of the project we plan to present the data at national or international level scientific meetings or conferences and publish the results in a recognized scientific journal.

Progress 10/01/12 to 09/30/15

Outputs
Target Audience:Dairy farmers, veterinarians, and NYS dairy industry professionals will be able to use the data to develop and implement management strategies to reduce and control the spread of antimicrobial resistance on the dairy farm. This information will also be of significance for farmers and veterinarians when considering the potential health and economic advantages and disadvantages of feeding calves waste milk. Public health professionals, legislators, and researchers in the dairy industry interested in increased food safety are also included in the target audience. Changes/Problems:TheWhole Genome Sequencing part of the project is not yet complete. Part of the delay isrelated tothe online MG-RASTserver being off-line for a monthand a half while the site was being updated. Sequencing for this part of the project hasbeen done and data is currently under analysis. Analysis of the WGS data will be completed, and a manuscript will be written and submitted for publication in a peer-reviewed journal. What opportunities for training and professional development has the project provided?Funds for this project directly supported the graduate training of a Latino-American PhD student, Dr. Richard Pereira, who benefitted by developing his abilities to plan and execute a research project, developing knowledge and skills related to conducting metagenomics studies, and analyzing and interpreting the results from these studies. In addition it gave him an opportunity to improve his manuscript writing skills. The calf trials conducted during this study provided an opportunity for 5 veterinary students to learn about calf management, and how to plan and conduct a calf trial. How have the results been disseminated to communities of interest?Our results have been published in peer-reviewed journals and disseminated through presentations at national and international conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Our study found that feeding waste milk to preweaned dairy calves has the potential to at least temporarily increase the selection of resistance to drug of critical importance to both animal and human health. To eliminate this risk, an initial option would be to discontinue the practice at dairy farms. Disadvantages include the possibility of polluting the environment with drug residues by disposing of the waste milk, disposal of a nutritious food source, and an increase in the costs of feeding preweaned dairy calves. A reasonable assessment to determine whether to continue or discontinue feeding waste milk to dairy calves must take into account the health hazards to animals and humans, and economic advantages and disadvantages. An alternative to reduce the negative impacts of drug residues present in waste milk fed to calves is to use methods to degrade these drugs to concentrations below the minimum selective concentration which may decrease the selection for a given resistance determinant. Additionally, the microbiota of preweaned calves fed milk with drug residues easily discriminated at the genus level on a weekly microbial profile. Nevertheless, analysis comparing the abundance of taxon between calves receiving or not milk containing drug residues showed significant differences only at the genus levels, but not at the phylum, class, order and family level.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: In vivo selection of resistant E. coli after ingestion of milk with added drug residues Richard Van Vleck Pereira, Julie D Siler, Rodrigo Carvalho Bicalho, Lorin D Warnick PLoS ONE 12/2014; 9(12):e115223. DOI:10.1371/journal.pone.0115223 � 3.23 Impact Factor
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Multiresidue screening of milk withheld for sale at dairy farms in central New York State R V Pereira, J D Siler, R C Bicalho, L D Warnick Journal of Dairy Science 01/2014; 97(3). DOI:10.3168/jds.2013-7421 � 2.57 Impact Factor
• Type: Journal Articles Status: Awaiting Publication Year Published: 2016 Citation: Ingestion of milk containing very low concentration of antimicrobials: longitudinal effect on fecal microbiota composition in preweaned calves Pereira RV, Lima S, Siler JD, Foditsch C, Warnick LD, Bicalho RC Submitted to PLoS One  accepted

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The target audience for efforts during this reporting period was public health professionals, legislators, dairy farmer workers and owners, veterinarians, researchers and other professionals in the dairy industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The funds for this project are directly supporting the graduate training of a Latino-American PhD student, Richard V. Pereira, which has benefitted from the activities, proposed on this period by developing his abilities to plan and execute research projects. Some specific skills include hands on laboratory experience with next-generation sequencing approaches and analysis of data using advance statistical methods. In addition these studies have given an opportunity to improve his manuscript writing skills. The skills he as obtained during these projects will be vital to increase his statistical, epidemiological and genomic skills, and prepare him to become a competitive and independent researcher. Furthermore the calf trials conducted during this period have provided an opportunity for 5 veterinarian students to learn about calf management, and how to plan and conduct a calf trial. How have the results been disseminated to communities of interest? These results have been disseminated through peer-reviewed articles and presentation of results at conferences of national and international impact. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Controlled trial evaluating the effects of feeding milk with residual concentrations of antibiotics to pre-weaned dairy calves. The objective of this study is to determine effect on selection of antimicrobial resistant E.coli from feeding dairy calves milk containing residual concentration of antimicrobials. As a results from the finding in aim one (screening waste milk for drugs), we decided to add ceftiofur to list of drugs added to the milk of calves in the treatment group (being fed the simulated "waste milk"); prior to this study we did not expect to observed such a high prevalence of ceftiofur in waste milk. For this study calves were in a controlled feeding trial from birth to 6 weeks of age. Calves in the treatment group were fed raw milk added drug residues based on results from aim one. The manuscript for this study has been submitted and accepted for publication in the open-access journal PLoS ONE. Overall our findings were that calves ingesting milk with drug residues had a significant increase in resistance to ceftiofur and to other antimicrobials belonging to three different drug classes when compared to control calves fed milk not containing detectable drug residues. Moreover, there was an increase in the prevalence of fecal E. coli resistant to 3 or more antimicrobial drugs in calves fed milk with drug residues when compared to control calves. The magnitude of these findings was unexpected considering that the concentration of the drugs added to the milk ranged from 53 (tetracycline) to 3200 (ampicillin) times below the minimum inhibitory concentration (the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation) for resistant E. coli. These findings are of marked relevance not only for the dissemination of antimicrobial resistance in dairy calves, but also because they raise questions about the consequences of routine ingestion of drugs residues by humans and other animals. 2. Addressing the impact of feeding preweaned calves with milk containing residual levels of antibiotic on fecal microbiota and antibiotic resistant genes. The objective of this study is to evaluate the impact of feeding preweaned calves waste milk on the shift of the fecal microbiota and antibiotic resistant genes over time. For this study DNA extraction and purification, and library preparation for 16S rRNA metagenomic sequencing have been completed. Sequencing of samples for the 16S rRNA metagenomic analysis is ongoing.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Pereira RV, Siler J, Bicalho RC, Warnick LD. In vivo selection of resistant E. coli after ingestion of milk with added drug residues. Accepted November, 26, 2014.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Pereira RV, Siler JD, Bicalho RC, Warnick LD. Effect of feeding preweaned dairy calves raw milk added very low antimicrobial concentrations on the resistance of commensal fecal Escherichia coli. XXVIII World Buiatrics Congress. Cairns, Australia. July, 2014.

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: The target audience for which efforts for this reporting period were all sectors of the dairy industry, including dairy farm workers (which are largely composed by Hispanics workers), farm owners, veterinarians, researchers and other professionals in the dairy industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The funds for this project directly supporting the graduate training of a Latino-American PhD student, Richard Pereira, which has benefitted from the activities proposed on this period by developing his abilities to plan and execute a research projects. In addition it has given an opportunity to improve manuscript writing skills (he was responsible for writing and submitting the waste milk article accepted for publication) Furthermore the calf trial initiated during this period has provided an opportunity for 8 veterinarian students to learn about calf management, and how to plan and conduct a randomized research trial. How have the results been disseminated to communities of interest? These results have been disseminated through a peer-reviewed article and presentation of results at a conference of national impact and one regional symposium. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Quantification of antimicrobial drugs in the waste milk from dairy farms in upstate New York. The objective of this study was to determine the concentration of antimicrobial drugs present in the “waste milk” fed to preweaned calves in representative dairy farms in Upstate New York. This study was completed and generated vital information of drugs present in the waste milk, providing also their concentrations. Our results revealed that the most prevalent drug identified in waste milk samples was ceftiofur, followed by penicillin and ampicillin. The results from this study are vital for further studies evaluating the impacts of the disposal and use of waste milk on the selection of antimicrobial resistance in dairy calves fed waste milk and on the effect on the environment in cases when this milk is discarded. 2. Controlled trial evaluating the effects of feeding milk with residual concentrations of antibiotics to pre-weaned dairy calves. The objective of this study is to determine effect on selection of antimicrobial resistant E.coli from feeding dairy calves milk containing residual concentration of antimicrobials. As a results from the finding in study 1 ( screening waste milk for drugs), we decided to add ceftiofur to list of drugs added to the milk of calves in the treatment group (being fed the simulated “waste milk”); prior to this study we did not expect to observed such a high incidence of ceftiofur in waste milk. We have initiated the calf feeding trial and estimate that these trials will be completed by April 2014. Although we currently do not have enough calves enrolled in the study to draw conclusions, the preliminary results from our findings indicate that antimicrobial susceptibility profile for E. coli isolates cultured from samples from calves being the milk added subinhibitory concentrations of antimicrobial drugs have higher incidence of resistance to most of the antimicrobials tested when compared to E. coli isolates from the control calves being fed milk not containing residual concentrations of antimicrobial drugs.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Pereira RV, Siler J, Bicalho RC, Warnick LD. Multi-residues screening of milk withheld for sale at dairy farms in Central New York. Journal Dairy of Science. Accepted for publication Nov. 2013.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Pereira RV, Siler J, Bicalho RC, Warnick LD. Multi-residues screening of milk withheld for sale at dairy farms in Central New York. 12th Annual Biological and Biomedical Sciences (BBS) Symposium, Ithaca, NY
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Pereira RV, Siler J, Bicalho RC, Warnick LD. Multi-residues screening of milk withheld for sale at dairy farms in Central New York. 2013 Conference for Research Workers in Animals Diseases (CRWAD), Chicago, Illinois