Preparation and Infection of Bovine Precision-Cut Lung Slices (b-PCLS) as an Ex Vivo Model for Respiratory Pathogens.

Precision-cut lung slices (PCLS) represent a physiologically relevant ex vivo model that bridges the gap between simplified in vitro systems and in vivo animal studies by preserving native lung architecture, cellular diversity, and functional responses. This model provides a valuable platform for investigating respiratory tract infections and tissue-level host-pathogen 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-cut lung slices (b-PCLS). The workflow includes lung inflation with 1.5% low-melting-point 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-PCLS 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 Pasteurella multocida and bovine coronavirus (BCoV), along with standardized sampling strategies for downstream analyses. Finally, we provide an optimized RNA extraction protocol that yields high-quality RNA from both control and inoculated b-PCLS, supporting downstream host and pathogen transcriptomic analyses. Collectively, these protocols expand the applicability of large-animal 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-level host-pathogen interactions in bovine lungs. Overall, this ex vivo system offers a valuable tool for bovine respiratory research, assay development, and molecular analysis. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation, maintenance, and viability assessment of b-PCLS Basic Protocol 2: Inoculation of b-PCLS with Pasteurella multocida Basic Protocol 3: Inoculation of b-PCLS with bovine coronavirus Basic Protocol 4: Extraction of RNA from culture supernatants and b-PCLS.