The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions

Area of Science:

• Veterinary Science
• Gastroenterology
• Infectious Disease Research

Background:

• Gastrointestinal (GI) infections in sheep impact animal health and productivity.
• Studying host-pathogen interactions in the sheep GI tract is challenging due to its inaccessibility.
• Zoonotic pathogens in sheep GI infections pose risks to human health.

Purpose of the Study:

• To develop a reliable in vitro culture system for ovine gastric and intestinal epithelial organoids.
• To establish a model for studying host-pathogen interactions in sheep GI infections.
• To reduce and replace animal use in GI pathogen research, aligning with the 3Rs principles.

Main Methods:

• Generation of ovine abomasal and ileal epithelial organoids.
• Characterization of organoid structure and stability through serial passage.
• RNA sequencing (RNA-seq) to compare organoid and tissue transcriptomes.
• Cryopreservation and resuscitation of organoid lines.
• Pathogen challenge experiments using helminths (Teladorsagia circumcincta) and bacteria (Salmonella enterica).
• Inversion of organoid polarity for apical surface pathogen exposure.

Main Results:

• Ovine abomasal and ileal organoids form stable, self-organizing 3D structures.
• Organoid transcriptomes reflect their tissue of origin and remain stable over serial passages.
• Organoids can be cryopreserved, enabling long-term storage and reducing animal numbers.
• Successful infection of organoids by the helminth Teladorsagia circumcincta.
• Demonstrated pathogen interaction with inverted polarity organoids using Salmonella Typhimurium.

Conclusions:

• A robust in vitro system for ovine gastric and intestinal organoids has been established.
• These organoids serve as a valuable model for studying sheep GI infections and host-pathogen dynamics.
• The developed organoid system supports the 3Rs principles by reducing reliance on live animals for research.