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Effect of Feeding Dairy Calves Milk Containing Antimicrobial Residues on the Emergence of Antimicrobial Resistant Enteric Bacteria


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.

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<p>NON-TECHNICAL SUMMARY:<br/> 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.
<p>APPROACH:<br/> 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.
<p>PROGRESS: 2012/10 TO 2013/09<br/>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

Warnick, Lorin; Pereira, Richard; Bicalho, Rodrigo
Cornell University
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