Immunoproteomic identification of 11 novel immunoreactive proteins of Riemerella anatipestifer serotype 2

Riemerella anatipestifer (RA) is one of the most important bacterial pathogens of ducks and other avian species worldwide. Twenty-one serotypes of RA have been identified, with RA serotype two (RA2) being reported as one of the most predominant serotypes underlying infections in China. Current approaches to the control of RA are hindered by the absence of effective vaccines, particularly those that exhibit cross-protection between different serotypes. In this study, a combination of two-dimensional electrophoresis, Western blot analysis and mass spectrometry were used to identify the antigenic proteins of RA2. A total of 16 immunoreactive proteins, representing 12 distinct proteins, were identified. These included OmpA, a known immunogenic protein of RA, as well as novel immunogens. PCR analysis also indicated that genes corresponding to each of the 12 distinct proteins were conserved among different RA serotypes. Eleven genes encoding these proteins were cloned and expressed in Escherichia coli BL21 (DE3). Eight of the 11 expressed proteins were able to react with hyperimmune rabbit serum against RAf153. One of these, recombinant elongation factor G, responded to RA2 sera but not RA1, whereas recombinant OmpA responded to both RA1 and RA2 sera. These data form a basis for the development of vaccine for both homologous and heterogeneous RA serotypes in addition to the production of target antigens for the development of diagnostic antibodies with the potential to distinguish between RA serotypes.