Adrian Muwonge ¹ Priscilla F. Gerber ² Bryan A. Wee ¹ Jill Thomson ³ JJ Wang ⁴ Patrick G. Halbur ⁵ Tanja Opriessnig ^5,6*

• ¹ The Digital One Health Laboratory, Roslin Institute, University of Edinburgh, Edinburgh, Scotland, United Kingdom
• ² Department of Infectious Diseases and Public Health, City University of Hong Kong, Kowloon, Hong Kong, SAR China
• ³ Veterinary and Analytical Laboratory, Scotland’s Rural College Veterinary Services, Midlothian, United Kingdom
• ⁴ Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, China Agricultural University, Beijing, Beijing Municipality, China
• ⁵ Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa, United States
• ⁶ Moredun Research Institute, Penicuik, Scotland, United Kingdom

Pathogen introduction and transmission at the farm, regional, or national level are associated with reduced animal welfare and negative impacts on herd economics. Ongoing infectious disease surveillance, active or passive, is therefore of high importance. For optimal resolution, each pig is sampled individually, for example by collecting blood or nasal swabs. In recent years, oral fluids have become very useful for population surveillance at the pen level. Another alternative is sampling the air to capture pathogens circulating across the entire barn via bioaerosols.This study aimed to examine the potential utility of bioaerosol metagenomics for pathogen detection on pig farms. Methods Bioaerosols via automated air sampler, and oral fluid via pen-based ropes, were collected from each of two Scottish indoor pig farms. All samples were subjected to conventional routine bacterial isolation, and total genomic nucleic acids were extracted for PCR screening for three pig DNA viruses, three bacterial Mycoplasma species and an RNA virus and Illumina shotgun metagenomic sequencing.Oral fluids contained more DNA compared to bioaerosol samples. DNA integrity exhibited limited impact on PCR or sequence yield. While Streptococcus suis could be cultured from a single oral fluid sample, reads mapped to S. suis were detectable in all metagenomic samples. Other bacterial pig pathogens, including Mycoplasma hyorhinis, M. hyopneumoniae and M. hyosynoviae, were detected in oral fluid and aerosols by PCR and metagenomics. One of the two farms was PRRSV positive, and the virus was detectable via PCR in oral fluids but not in bioaerosols. Antimicrobial resistance (AMR) gene profiles had less variation between bioaerosols and oral fluids. Some identified AMR genes had strikingly similar abundance overall.Overall these findings indicate that there is potential utility of bioaerosol metagenomics for pathogen surveillance on pig farms; however, more research is needed for technical and cost optimization to allow for routine pathogen detection on livestock farms.