Air-liquid interface (Ali) systems allow the establishment of a culture environment more representative of that in vivo than other culture systems. They are useful for performing mechanistic studies of respiratory epithelial cells as drug permeation barriers and can be used to study the interactions between hosts and respiratory pathogens. However, there have been few studies concerning Ali cultures of primary swine tracheal epithelial cells (STECs) and an immortalized STEC line, and the differences between these two systems remain poorly defined. In this study, we established Ali culture systems for primary STECs and for immortalized STEC line, and we systematically compared the differentiation capacities and immunological functions of these systems for the first time. Under Ali culture conditions, immortalized STEC line and primary STECs could survive for at least forty days, formed tight junctions and differentiated into stratified cells. They both possessed complete abilities to produce mucin and inflammatory cytokines and develop cilia. However, in contrast to primary STECs, which had a heterogeneous morphology, Ali-cultured immortalized STEC line appeared to be a homogenous population. The formation of tight junctions in Ali-cultured primary STECs was superior to that in immortalized STEC line. In addition, cilia in Ali-cultured immortalized STEC line were more pronounced, but their duration of expression was shorter than in primary STECs. Ali-cultured primary STECs and immortalized STEC line systems possessing complete abilities to undergo ciliary differentiation and inflammatory cytokine production were established for the first time in this study, and several differences in morphology and the formation of tight junctions and cilia were observed between these two systems. These two systems will be important tools for drug screening studies, as well as for detailed analyses of the interactions between hosts and respiratory pathogens.

Mycoplasma hyopneumoniae is an important respiratory pathogen that causes great economic losses to the pig industry worldwide. Although some putative virulence factors have been reported, pathogenesis remains poorly understood. Herein, we evaluated the relative abundance of proteins in virulent 168 (F107) and attenuated 168L (F380) M. hyopneumoniae strains to identify virulence-associated factors by two-dimensional electrophoresis (2-DE). Seven proteins were found to be ≥ 1.5-fold more abundant in 168, and protein–protein interaction network analysis revealed that all seven interact with putative virulence factors. Unexpectedly, six of these virulence-associated proteins are encoded by core rather than accessory genomic elements. The most differentially abundant of the seven, fructose-1,6-bisphosphate aldolase (FBA), was successfully cloned, expressed and purified. Flow cytometry demonstrated the surface localisation of FBA, recombinant FBA (rFBA) mediated adhesion to swine tracheal epithelial cells (STEC), and anti-rFBA sera decreased adherence to STEC. Surface plasmon resonance showed that rFBA bound to fibronectin with a moderately strong KD of 469 nM. The results demonstrate that core gene expression contributes to adhesion and virulence in M. hyopneumoniae, and FBA moonlights as an important adhesin, mediating binding to host cells via fibronectin.

Background/Objectives: Mycoplasma hyopneumoniae (Mhp), porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) are major contributors to porcine respiratory disease complex (PRDC), causing substantial economic losses. Methods: This study evaluated a commercial PCV2-Mhp bivalent vaccine, Cirbloc^® M Hyo (Vaccine A), containing recombinant PCV2 Cap protein and inactivated Mhp antigen, vs. two reference bivalent vaccines (B and C) in an Mhp challenge model. Eighty-two healthy 3-week-old specific-pathogen-free piglets were randomly allocated to five groups (three vaccinated, unvaccinated challenged control, unvaccinated unchallenged control). Pigs received a single intramuscular vaccination at 3 weeks of age and were challenged intratracheally with virulent Mhp lung homogenate five weeks later. Clinical signs, serology, lung lesions, histopathology, and Mhp DNA detection in lungs were assessed. Results: No vaccine-related adverse reactions occurred. Vaccine A induced earlier and higher Mhp-IgG seroconversion before challenge than reference vaccines. Following challenge, Vaccine A significantly reduced mean lung lesion scores compared with the unvaccinated challenged control (2.28 ± 2.37 vs. 6.56 ± 4.03; p < 0.05), with markedly reduced peribronchiolar inflammation and lymphoid hyperplasia and the lowest Mhp DNA detection rate among challenged groups. Conclusions: Cirbloc^® M Hyo provided enhanced protection against experimental Mhp challenge, as shown by reduced lung lesions and milder histopathological changes, supporting its use for comprehensive PRDC management.