Approaches to study the pig nasal microbiota in the context of the host

Abstract

The animal microbiota is the ecological community of microorganisms living in multiple body sites. In particular, the nasal microbiota plays an important role in host health as it constitutes a barrier for respiratory pathogens. Interactions among the pig nasal commensal bacteria and with the nasal epithelium are determinant for pathogen exclusion, but no systematic approach was available to elucidate such interactions. This thesis provides new tools for exploring the pig nasal microbiota within its host context. First, we studied whether an intensive antibiotic treatment to sows could prevent the transfer of nasal microbiota to piglets, as previous work highlighted that sows are the main source of piglet microbiota. The treatment of sows with ceftiofur alone or in combination with tulathromycin diminished the bacterial load in the nasal cavity of sows and piglets, causing dysbiosis in the piglets' nasal microbiota, which showed unusual taxa. However, the transmission of the microbiota was not entirely prevented, which limits the use of this piglet model for microbiota studies. Additional strategies are needed to develop germ-free pigs, while ensuring that the conditions remain easy to implement. Next, we aimed to establish in vitro models to study the network of the piglet nasal microbiota. For this, we developed the Porcine Nasal Consortium (PNC8), a rationally designed synthetic community of 8 strains representing the most prevalent and/or abundant genera found in the nasal microbiota of healthy piglets. We found that PNC8 members varied in their ability to grow across 23 different in vitro conditions, which suggests that they harbour distinct metabolic functions. In addition, we confirmed the existence of interactions, such as the cooperation among Rothia nasimurium and Staphylococcus aureus with Glaesserella parasuis (virulent and non-virulent strains). In conclusion, PNC8 represents a valuable tool for examining the interaction network in the piglet nasal microbiota under in vitro defined conditions, as well as to elucidate its role in respiratory pathogen exclusion. To explore the nasal microbiota-host interplay in a controlled in vitro system, we developed for the first time Porcine Nasal Organoids (PNOs), which showed similar structure and cell types than the in vivo pig nasal mucosa. We confirmed that some PNC8 members (Moraxella pluranimalium, R. nasimurium and G. parasuis) as well as a virulent G. parasuis strain adhered and colonized the PNOs, although at different levels. We also examined microbial-microbial and microbial-host interactions in this system by co-culturing R. nasimurium with the remaining 3 strains. Besides, PNOs reacted to the bacteria secreting different cytokines. M. pluranimalium and G. parasuis stimulated the production of proinflammatory cytokines, while R. nasimurium induced the secretion of INFγ and diminished the proinflammatory effect produced by the other bacteria. We conclude that PNOs recapitulate the in vivo nasal mucosa and can be useful to study host-microbe interactions in an in vivo-like system, contributing to the 3Rs principles. In summary, this thesis provides new approaches to explore the nasal microbiota network under controlled conditions that mimic the in vivo environment of the piglet's nose. This work will contribute to understand the role of microbiota in pathogen exclusion.

Date of Award	4 Jul 2025
Original language	English
Supervisor	Ana Maria Florencia Correa Fiz (Director), Virginia Aragón Fernández (Director) & Rosa Marina Sibila Vidal (Director)