Virulence-associated trimeric autotransporters ofHaemophilus parasuisare antigenic proteins expressed in vivo

Abstract

Glässer’s disease is a re-emerging swine disease characterized by a severe septicaemia. Vaccination has been widely used to control the disease, although there is a lack of extended cross-protection. Trimeric autotransporters, a family of surface exposed proteins implicated in host-pathogen interactions, are good vaccine candidates. Members of this family have been described in Haemophilus parasuis and designated as virulence-associated trimeric autotransporters (VtaA). In this work, we produced 15 recombinant VtaA passenger domains and looked for the presence of antibodies directed against them in immune sera by immunoblotting. After infection with a subclinical dose of H. parasuis Nagasaki, an IgG mediated antibody response against 6 (VtaA1, 5, 6, 8, 9 and 10) of the 13 VtaA of the Nagasaki strain was detected, indicating that they are expressed in vivo. IgA production against VtaA was detected in only one animal. VtaA were more likely to be late antigens when compared to early (Omp P5 and Omp P6) and late (YaeT) defined antigens. Antibody cross-reaction with two orthologs of Nagasaki’s VtaA5 and 6, VtaA15 and 16 of strain HP1319, was also detected. No antibodies against VtaA were detected in the sera of animals immunized with a bacterin of the Nagasaki strain, suggesting poor expression in the in vitro conditions used. Taken together, these results indicate that VtaA are good candidate immunogens that could be used to improve H. parasuis vaccines. However, their capacity to confer protective immunity needs to be further studied.

Genomic and antigenic characterization of monomeric autotransporters of Haemophilus parasuis: an ongoing process of reductive evolution

“…All procedures and strains were as described previously (Olvera et al, 2010;Pina et al, 2009). The highly pathogenic H. parasuis Nagasaki strain, four nasal strains (SW114, F9, D74 and HP-3123) and six invasive strains (HP-1205, HP-1302, HP-1319, HP-2163, HP-2269 were tested (Pina et al, 2009).…”

“…For OMP preparation, bacterial cultures were grown to an OD 600 of 0.7 in PPLO agar supplemented with 1 % IsoVitaleX (Becton Dickinson). The whole-cell H. parasuis Nagasaki bacterin was prepared as previously described (Olvera et al, 2010). Briefly, bacteria were used to infect pigs to test the virulence of the strain.…”

“…The gene fragments were cloned in the pASK-IBA33plus plasmid (IBA-BioTAGnologies) and transformed in Escherichia coli Rosetta BL21(DE3)pLysS (Novagen -Merck Chemicals). Induction and production of the recombinant proteins with a C-terminal 66 histidine tag were performed as previously described (Olvera et al, 2010). The peptides were purified using the HisSpinTrap kit (GE Healthcare) and quantified with the BCA Protein Assay kit (Thermo Scientific Pierce).…”

“…Procedures for immunoblotting were as previously described (Olvera et al, 2010). Briefly, after SDS-PAGE, purified proteins were transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare), and revealed using the ECL Advance Western blotting detection kit (GE Healthcare).…”

“…Although there is not a strict correlation between serovars and virulence, serovar 5 is considered to be highly pathogenic (Rapp-Gabrielson et al, 2006). To date, no universal vaccine exists against this pig pathogen but, recently, putative virulence factors and vaccine candidates have been proposed (Olvera et al, 2010) and some of them tested in a mouse model (Zhou et al, 2009a). In part, this was possible due to the analysis of complete (Yue et al, 2009;Xu et al, 2011) and partial (Pina et al, 2009) genome sequences and the derived immunoproteomic work (Zhou et al, 2009b).…”