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This model provides a valuable platform for investigating respiratory tract infections and tissue\u2010level host\u2013pathogen interactions in bovine lung tissue, including those involving pathogens associated with bovine respiratory disease (BRD), a leading cause of morbidity, mortality, and economic loss in the global cattle industry. While PCLS protocols are well established for murine and human lungs, standardized and detailed methodologies optimized for bovine lung tissue remain scarce. Here, we present a comprehensive, reproducible protocol for generating, culturing, assessing viability, inoculating, and extracting RNA from bovine precision\u2010cut lung slices (b\u2010PCLS). The workflow includes lung inflation with 1.5% low\u2010melting\u2010point agarose, vibratome slicing, and culture conditions that preserve bronchiolar architecture and ciliary activity. Viability is assessed longitudinally using ciliary activity scoring and a metabolic assay, demonstrating that b\u2010PCLS remain viable and highly active for at least 7 days in culture. We also describe inoculation procedures applicable to bacterial and viral pathogens relevant to BRD, exemplified by\n                    <jats:italic>Pasteurella multocida<\/jats:italic>\n                    and bovine coronavirus (BCoV), along with standardized sampling strategies for downstream analyses. Finally, we provide an optimized RNA extraction protocol that yields high\u2010quality RNA from both control and inoculated b\u2010PCLS, supporting downstream host and pathogen transcriptomic analyses. Collectively, these protocols expand the applicability of large\u2010animal PCLS for research on respiratory infections, support the principles of replacement and reduction in animal experimentation, and provide a complementary platform for controlled investigation of tissue\u2010level host\u2013pathogen interactions in bovine lungs. Overall, this\n                    <jats:italic>ex vivo<\/jats:italic>\n                    system offers a valuable tool for bovine respiratory research, assay development, and molecular analysis. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Generation, maintenance, and viability assessment of b\u2010PCLS\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Inoculation of b\u2010PCLS with\n                    <jats:italic>Pasteurella multocida<\/jats:italic>\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Inoculation of b\u2010PCLS with bovine coronavirus\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 4<\/jats:bold>\n                    : Extraction of RNA from culture supernatants and b\u2010PCLS\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70459","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T14:22:06Z","timestamp":1790259726000},"source":"Crossref","is-referenced-by-count":0,"title":["Preparation and Infection of Bovine Precision\u2010Cut Lung Slices (b\u2010PCLS) as an\n                    <i>Ex Vivo<\/i>\n                    Model for Respiratory Pathogens"],"prefix":"10.1002","volume":"6","author":[{"given":"Asal","family":"Ahmadi","sequence":"first","affiliation":[{"name":"Department of Paraclinical Sciences, Faculty of Veterinary Medicine Norwegian University of Life Sciences, \u00c5s  Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ghazal","family":"Ahmadi","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Ecology, Agriculture, and Biotechnology University of Inland Norway  Hamar Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vilde","family":"Galleberg\u2010Wahlberg","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Sciences, Faculty of Veterinary Medicine Norwegian University of Life Sciences, \u00c5s  Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abdolrahman","family":"Khezri","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Ecology, Agriculture, and Biotechnology University of Inland Norway  Hamar Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veslem\u00f8y Sunniva","family":"Oma","sequence":"additional","affiliation":[{"name":"Department of Production Animal Clinical Sciences, Faculty of Veterinary Medicine Norwegian University of Life Sciences, \u00c5s  Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jochen","family":"Meens","sequence":"additional","affiliation":[{"name":"Institute of Microbiology University of Veterinary Medicine Hannover  Hannover Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mette","family":"Myrmel","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Sciences, Faculty of Veterinary Medicine Norwegian University of Life Sciences, \u00c5s  Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann\u2010Katrin","family":"Llarena","sequence":"additional","affiliation":[{"name":"Department of Paraclinical Sciences, Faculty of Veterinary Medicine Norwegian University of Life Sciences, \u00c5s  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