Characterizing fecal bioaerosols in dairy barns using microbial source tracking markers and bovine enteric viruses

Dairy manure contains bacterial and viral pathogens that can infect livestock and humans. These pathogens present a risk of airborne transport when bioaerosols are generated from land-applied manure. The transport range and distribution of fecal bioaerosols around dairy farms could be studied using microbial source tracking (MST) markers and bovine enteric viruses, but their applicability and limitations have not been studied for this purpose. This study investigated the distribution of ruminant-specific MST markers and bovine enteric viruses in air samples from a working dairy farm (Jun 2024-Jan 2025) and assessed their recovery using the InnovaPrep Cub ACD-210 air sampler. Markers included ruminant Bacteroides, Bacteroidales-like CowM3, the bacterial 16S rRNA gene, five bovine-specific enteric viruses, and two bacteriophages (MS2 and phiX174). Sampling locations included indoor, naturally and mechanically ventilated livestock barns and outdoor control locations. Bacterial MST markers were detected more frequently than bovine enteric viruses, on 19/19 sample dates versus 7/19 sample dates, and ruminant Bacteroides occurred at a higher concentration than CowM3, for example, at mean concentrations of 262,803 genomic copies/m³ versus 7,853 genomic copies/m³ in the naturally ventilated barn. Bacteriophages were never detected. The Cub recovered higher concentrations of bacterial markers than comparison gelatin filter button samplers, by average factors of 6.3-21.0. Recovery of viruses by the Cub appeared comparable to button samplers. Results inform the development of bioaerosol sampling strategies on dairy farms, which could be used for biomonitoring during outbreaks and understanding endemic spread of infectious disease on and between livestock farms.IMPORTANCEHighly pathogenic avian influenza (HPAI) is a pressing health concern for the dairy industry, and recent advances have highlighted a need to understand its distribution in fecal bioaerosols on and around dairy farms. Microbial source tracking (MST) markers and bovine enteric viruses could be key tools in advancing this line of research, but their application and limitations with respect to air sampling have not been well-studied. This study assessed the distribution of bacterial MST markers and bovine enteric viruses on a working HPAI-free dairy farm. The results provide a basis for selecting among several competing markers and sampling technologies and highlight areas where additional development is needed to formulate effective bioaerosol sampling strategies for dairy farms.