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ARS Home » Plains Area » Clay Center, Nebraska » U.S. Meat Animal Research Center » Genetics, Breeding, and Animal Health Research » Research » Research Project #435841

Research Project: Recent Advances in Sequencing Technologies to Characterize the Respiratory Microbiome and Virome Associated with Bovine Respiratory Disease

Location: Genetics, Breeding, and Animal Health Research

Project Number: 3040-31000-100-15-I
Project Type: Interagency Reimbursable Agreement

Start Date: Jul 15, 2019
End Date: Jul 14, 2022

Objective:
The overall goal of this proposal is to profile respiratory pathogens in the upper respiratory tract of cattle throughout the production life-cycle of the animal. To determine this, microbiome and virome profiles at time points prior to weaning in addition to after the animals enter the feedlot and are likely to be diagnosed with BRDC, will be evaluated. Populations of cattle in the US and Ireland will be evaluated across multiple years and cattle source locations, to provide a clearer picture of the relationships of profiles in animals that develop BRDC or not. The objectives of this proposal are: 1) investigate the prevalence and distribution of the respiratory microbiome and virome associated with BRDC in beef and dairy herds in Ireland and in beef herds at the US Meat Animal Research Center (US MARC); 2) develop methods to accurately identify the infectious agents of BRDC using next generation sequencing (NGS), third generation sequencing (TGS), bioinformatics technologies, and high-throughput sensitive and rapid diagnostics; and 3) elucidate the dynamics of secondary viral and bacterial infection by monitoring experimentally virus infected animals in longitudinal studies.

Approach:
We will sample the upper respiratory tract of animals as they progress from birth to feedlot, and produce microbiome profiles with high-throughput 16S rRNA sequence and specific viral pathogens with qRT-PCR. Upper respiratory tract samples will be collected from all animals in the study (approximately 800 each year) at three time points, including prebreeding (approximately 70 days of age), preconditioning (140 days), and weaning (160 days). Additional samples will be collected for animals diagnosed with Bovine Respiratory Disease (BRD; symptoms including lethargy, nasal discharge, elevated body temperature, and distressed breathing) at time of diagnosis along with control cohorts that do not display symptoms and have not be diagnosed with BRD previously. In addition to evaluation of respiratory pathogen profiles at the previously identified time points, respiratory tissues in calves with severe BRD will be evaluated. Three to six weeks after weaning, animals treated for BRD symptoms will be evaluated for severity based upon respiration rate, BRD symptoms, and rectal temperature. Animals with severe symptoms will be euthanized and sent to necropsy to collect samples from several organs. Approximately 15 severe BRD animals will be targeted. In addition, one control animal (untreated and asymptomatic) will also be euthanized and sent to necropsy for every three affected animals. Lung (multiple lobes) and lung lavage samples, trachea, bronchial tubes, lymph nodes, and lung abscesses will be collected and swabbed for microbial samples. Nasal swabs (4 at each sampling point described previously, 2 from each nostril) will be collected from the upper nasal cavity using 6-inch swabs. For animals diagnosed with BRD in the feedlot, 6” and deep nasal pharyngeal swabs will be collected. Total DNA will be extracted from each swab using a commercial kit that our lab has previously used with success. DNA will then be amplified through PCR for the full 16S rRNA variable region using standard PCR and sequencing with the MinION. Total RNA will be extracted and reverse transcribed for evaluation of presence of viral pathogens including BOCV, BRSV, BVDV and BoHV-1 using a real-time RT-PCR multiplex assay. Microbiome profiles will be created using public database of 16S rRNA sequence in Qiime and webMGA, and used to determine if animals treated for BRDC have different pathogen profiles at the species level relative to control cohorts at the time points prior to weaning and at time of diagnosis with BRD.