PREVALENCE OF ANTIMICROBIAL RESISTANCE IN COW-CALF OPERATIONS ON CALIFORNIA PASTURE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1025939
• Project Start Date: Feb 17, 2021
• Project End Date: Jun 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Population Health & Reproduction

Non Technical Summary
The use of antimicrobial drugs in livestock has come under increased scrutiny as a major contributor to antimicrobial resistance (AMR) in both livestock and humans. As a precaution, FDA and the state of California made legislative changes to increase veterinary oversight on the use of medically important antimicrobial drugs in livestock and mandated the monitoring for AMR in livestock. However, limited information is available on AMR status on cow-calf operations in California. Our goal is to quantify the burden of antimicrobial resistance determinants in fecal bacteria shed by cattle of different age groups and management systems and understand the risk factors for fecal shedding of pathogens carrying AMR genes. We will conduct a cross-sectional study on a convenience sample of 6 herds enrolled from Northern California based on willingness to participate and good record keeping. Fecal samples from 10 animals per ranch will collected for DNA extraction and analysis for relative abundances of different bacterial types and resistance determinants between different animal groups, and determine the effect of treatment as well as herd level management practices on bacterial resistance. The outcome of this study will establish AMR benchmarks in beef animals and generate knowledge on how the bacterial resistance may change during calf hood and into adulthood for cattle on pasture or range in California.

Animal Health Component

25%

Research Effort Categories

Basic

50%

Applied

25%

Developmental

25%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
307	0790	1080	50%
311	4099	1060	50%

Knowledge Area
311 - Animal Diseases; 307 - Animal Management Systems;

Subject Of Investigation
4099 - Microorganisms, general/other; 0790 - Rangelands, other;

Field Of Science
1060 - Biology (whole systems); 1080 - Genetics;

Keywords

antimicrobial resistance

cow-calf operations

beef cattle

metagenomics

Goals / Objectives
The goal of this project is to quantify the burden of antimicrobial resistance determinants in fecal bacteria shed by beef cattle of different age groups and management systems and understand the risk factors for fecal shedding of pathogens carrying AMR genes. The specific objectives this project are as follows:1) Sequence metagenomes of individual and pooled fecal samples from beef calves and cows on pasture or range in California2) Assess the prevalence of antimicrobial resistant genes in bacteria shed in the feces of cattle of different age, groups and management systems.

Project Methods
Herd enrolment and samplingAnimal Care and Use Committee approval will be obtained. A convenience sample of 6 herds in northern California will be enrolled in the study based on willingness to participate, presence of animal facilities, good records, and organic status. Ten animals per ranch will be sampled either as animals are processed through a chute (e.g. during pregnancy examination) or if they are observed defecating on pasture. Herds with a minimum of 100 animal will be enrolled. Samples will consist of approximately 50 g of feces collected from the rectum or picked up off the ground from an area of the fecal pad not in contact with the ground. Sampling will be blocked by age groups; five samples from dams (COW) and five samples from calves (CALF). If animals are going through a chute, they will be sampled based on a random numbers list, otherwise, the first animals that are observed defecating will be sampled. Samples collected in sterile whirl packs will be transported on ice to the Davis lab (Maier Lab). During the farm visits, an in-person interview will be conducted to collect information on the age of the animals and antibiotic treatments in the herd, if applicable.DNA extraction and next-generation sequencingDNA will be extracted from all samples with the Qiagen DNeasy PowerSoil Kit. In addition, a pooled samples (POL) from five calves or dams will be prepared for each herd by mixing equal amounts of fecal matter from five individual animals (IND). Purity and concentration of extracted DNA will be measured with a spectrophotometer (NanoDrop™ OneC). Library preparation, sample purification and normalization will be conducted as previously described using and following Illumina preparations kits and purification kits [8].Next-Generation Sequencing: DNA fragments will be paired-end sequenced (150 base pair reads) in multiplexed pools on the Illumina HiSeq 4000 platform at the UC Davis DNA Technologies & Expression Analysis Core Laboratory. Data will also be quality-filtered and aligned by the Core facility. Data will be uploaded to the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server, were it will be processed for quality control and annotated, as previously described [8].Data analysis: Data will be processed using SAS and JMP (SAS Institute Inc., Cary, NC). Relative abundances of different bacterial taxa and microbial functions in each sample will be used as covariates in discriminant analysis models to determine which bacterial species are most important to differentiate between COW and CALF groups, and POL and IND group. Taxa and resistance gene functions retained after discriminant analysis will be evaluated by a screening analysis using the false discovery rate to correct for multiple comparisons. Taxa and gene functions with a significantly different relative frequency between treatment groups will be selected for further analysis using a multivariate mixed effects logistic regression model (Pereira et al., 2018). In addition to treatment group effect, multiple models will be used to evaluate the effect of herd level management practices collected through the in-person interview

Progress 02/17/21 to 06/30/21

Outputs
Target Audience:The target audience will include the beef cow-calf producers, veterinarians and livestock extension advisors. California is the 4th largest beef producer in the country with over 5 million beef cattle (including cattle in feedlots, bulls, cows, calves and growing young stock). Information collected will contribute to the understanding of the prevalence of antimicrobial resistance in the California cow-calf operationsthat has not been assessed. Study outcome will be disseminated to the target audience through the University of California Extension network via organized workshops. Additional outreach avenues will inlcude presenatation at the California Cattlemen's Association yearly meeting, newsletters publication as well as online sources. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One lab staff was trained in analysis of next generation sequencing data for microbial communities profiling 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? Nothing Reported

Impacts
What was accomplished under these goals? The following have been accomplished: 1. Fecal sample collection for 30 cows and 30 calves from 6 different cow-calf operations in northern California 2. Questionnaier survey of the sample cow-calf operations to capture information antimicrobial drug use and general management practices 3. DNA extraction from all the fecal samples for metagenomic analysis. The DNA have been submitted to the UC Davis Genomic center for next generation sequencing

Publications

Progress 02/17/21 to 06/30/21

Outputs
Target Audience: Nothing Reported Changes/Problems:The short project implementation timeline and the slow down of research activities due to COVID-19 pandemic hindered timely completion of the project objectives. Most of the critical activities have been completed and plan to continue with the data analysis, publication and extension activities beyond the project date. What opportunities for training and professional development has the project provided?One laboratory staff working on the project was trained in high-through put sequencing (HTS) data analysis for microbial community profiling. The one week training was offered by the UC Davis Bioinformatics Core facility. 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?1. Complete analysis of the sequecning data 2. Prepare a manuscript to publish the study outcomes 3. Conduct extension meetings to disseminate the study outcomes

Impacts
What was accomplished under these goals? 1. Field work completed. A total of 60 fecal samples; 6 cow-calf operations x 10 samples each operation. the 10 samples from each operation was taken for 5 calves and 5 adult cows. In total, we collected fecal samples from 30 adult cows and 30 calves. 2. Farm survey completed. A questionnaier survey was completed for each of the sample farms to capture information on antimicrobial drug use and general management practices. 3. DNA extraction for metagenomic analysis completed. DNA samples were extracted from all the 60 samples for metagenomic analysis. The DNA samples have been submitted to the UC Davis Genomic center for next generation sequencing.

Publications