Immune responses following neonatal vaccination with conserved F4 fragment of VtaA proteins from virulent Glaesserella parasuis adjuvanted with CAF®01 or CDA.

Glaesserella parasuis is a Gram-negative bacterium that colonizes the upper airways of swine, capable of causing a systemic infection called Glässer's disease. This disease is more frequent in young post-weaning piglets. Current treatments against G. parasuis infection are based on the use of antimicrobials or inactivated vaccines, which promote limited cross-protection against different serovars. For this reason, there is an interest in developing novel subunit vaccines with the capacity to confer effective protection against different virulent strains. Herein, we characterize the immunogenicity and the potential benefits of neonatal immunization with two different vaccine formulations based on the F4 polypeptide, a conserved immunogenic protein fragment from the virulence-associated trimeric autotransporters of virulent G. parasuis strains. With this purpose, we immunized two groups of piglets with F4 combined with cationic adjuvant CAF®01 or cyclic dinucleotide CDA. Piglets immunized with a commercial bacterin and non-immunized animals served as control groups. The vaccinated piglets received two doses of vaccine, at 14 days old and 21 days later. The immune response induced against the F4 polypeptide varied depending on the adjuvant used. Piglets vaccinated with the F4+CDA vaccine developed specific anti-F4 IgGs, biased towards the induction of IgG1 responses, whereas no anti-F4 IgGs were de novo induced after immunization with the CAF®01 vaccine. Piglets immunized with both formulations displayed balanced memory T-cell responses, evidenced upon in vitro re-stimulation of peripheral blood mononuclear cells with F4. Interestingly, pigs immunized with F4+CAF®01 controlled more efficiently a natural nasal colonization by a virulent serovar 4 G. parasuis that spontaneously occurred during the experimental procedure. According to the results, the immunogenicity and the protection afforded by F4 depend on the adjuvant used. F4 may represent a candidate to consider for a Glässer's disease vaccine and could contribute to a better understanding of the mechanisms involved in protection against virulent G. parasuis colonization.

Time course Haemophilus parasuis infection reveals pathological differences between virulent and non-virulent strains in the respiratory tract.

Haemophilus parasuis is a common inhabitant of the upper respiratory tract of pigs and the etiological agent of Glässer's disease. However, the host-pathogen interaction remains to be well understood. In this work, 33 colostrum-deprived pigs were divided in 4 groups and each group was inoculated intranasally with a different H. parasuis strain (non-virulent strains SW114 and F9, and virulent strains Nagasaki and IT29755). Animals were necropsied at different times in order to determine the location of the bacteria in the respiratory tract of the host during infection. An immunohistochemistry method was developed to detect H. parasuis in nasal turbinates, trachea and lung. Also, the co-localization of H. parasuis with macrophages or neutrophils was examined by double immunohistochemistry and double immunofluorescence. Virulent strains showed a biofilm-like growth in nasal turbinates and trachea and were found easily in lung. Some virulent bacteria were detected in association with macrophages and neutrophils, but also inside pneumocyte-like cells. On the other hand, non-virulent strains were seldom detected in nasal turbinates and trachea, where they showed a microcolony pattern. Non-virulent strains were essentially not detected in lung. In conclusion, this work presents data showing differential localization of H. parasuis bacteria depending on their virulence. Interestingly, the intracellular location of virulent H. parasuis bacteria in non-phagocytic cells in lung could allow the persistence of the bacteria and constitute a virulence mechanism.

Differential interactions of virulent and non-virulent H. parasuis strains with naïve or swine influenza virus pre-infected dendritic cells.

Pigs possess a microbiota in the upper respiratory tract that includes Haemophilus parasuis. Pigs are also considered the reservoir of influenza viruses and infection with this virus commonly results in increased impact of bacterial infections, including those by H. parasuis. However, the mechanisms involved in host innate responses towards H. parasuis and their implications in a co-infection with influenza virus are unknown. Therefore, the ability of a non-virulent H. parasuis serovar 3 (SW114) and a virulent serovar 5 (Nagasaki) strains to interact with porcine bone marrow dendritic cells (poBMDC) and their modulation in a co-infection with swine influenza virus (SwIV) H3N2 was examined. At 1 hour post infection (hpi), SW114 interaction with poBMDC was higher than that of Nagasaki, while at 8 hpi both strains showed similar levels of interaction. The co-infection with H3N2 SwIV and either SW114 or Nagasaki induced higher levels of IL-1ß, TNF-α, IL-6, IL-12 and IL-10 compared to mock or H3N2 SwIV infection alone. Moreover, IL-12 and IFN-α secretion differentially increased in cells co-infected with H3N2 SwIV and Nagasaki. These results pave the way for understanding the differences in the interaction of non-virulent and virulent strains of H. parasuis with the swine immune system and their modulation in a viral co-infection.