Z-005. Identification and Characterization of New Virulence Associated Factors of Haemophilus parasuis

M. Sack, N. Baltes;
Univ. of Vet. Med., Hannover, GERMANY.

Background: Haemophilus (H) parasuis is a common opportunistic pathogen of pigs. It causes Glässer’s disease, a severe inflammation of the serous membranes, but recently has been a cause of meningitis, arthritis, and pneumonia. 15 serotypes are known to date. However, individual strains of each serotype may show differences in pathogenicity. Virulence factors of H. parasuis are largely unknown to date. Methods: In this study, Representational Difference Analysis (RDA) was used to identify new virulence-associated factors. DNA of several serotype reference strains known to possess different characteristics of pathogenicity were subjected to a subtractive hybridization procedure. Remaining serotypes and 17 pathogenic field strains were tested by PCR for the existence of resulting sequences considered characteristic for pathogenicity. Furthermore, the protein patterns 15 of serotype reference strains and of 17 field strains were compared by gel electrophoresis. MALDI and Q-TOF mass spectrometry followed by CODEHOP PCR were used to identify selected proteins. Results: A total of 58 fragments from RDA was sequenced. Among these are a putative hemolysin and transporter protein (HhdA, HhdB) which have not been characterized in H. parasuis. They were shown to be present in all invasive and absent from non-pathogenic reference strains. Another fragment showed similarity to an outer membrane receptor protein involved in iron-uptake and a third one to an autotransporter protein. Q-TOF mass spectrometry de novo sequencing yielded peptide sequences for two proteins: a 42 kDa major outer membrane protein (putative porin) and a large putative autotransporter protein (>150 kDa) which is absent from invasive serotype reference strains 5 and 12. Conclusion: Representational Difference Analysis was used successfully to identify gene fragments which are present exclusively in the tester DNA. Several fragments were found to be exclusively present in strains associated with pathogenicity. The putative hemolysin HhdA and its transporter protein HhdB appear to be associated with invasiveness among serotype reference strains.