Relatedness of Haemophilus parasuis strains and their proteins\u27 possible roles as virulence factors

摘要

Haemophilus parasuis, the causative agent of Glasser\u27s disease, is considered to be a remerging pathogen of swine in high-health status herds. Several molecular typing methods have been suggested as alternatives to serotyping, which failed to identify up to 30% of field isolates. This study compared random amplification of polymorphic DNA (RAPD) profiles and protein profiles of H. parasuis reference strains and field isolates as methods for identifying and grouping virulent and avirulent isolates. DNA and protein profiles of 15 reference strains and 31 field isolates of H. parasuis, analyzed with two computer-based similarity analyses, revealed unique and reproducible DNA and protein fingerprints among the reference strains and field isolates studied. Similarities and differences existed among avirulent, virulent, and highly virulent strains, which grouped according to their pathogenicity. The combination of RAPD and whole cell lysate SDS-PAGE analyses may be useful for studying the epidemiology of H. parasuis .;This is the first report of the isolation and characterization of a myophage, SuMu, from a field strain of H. parasuis. Electron microscopy showed that the bacteriophage had an iscosahedral head and contractile tail. Mass spectrometry of proteins separated by 1-D and 2-D electrophoresis identified twenty four homologues of bacteriophage proteins. Partial DNA sequencing revealed twenty open reading frames corresponding to fourteen proteins, including eight Mu-like homologues. Epidemiology of H. parasuis may be affected by this phage.;Iron-restricted growth studies of H. parasuis were done to explore potential protein differences between virulent and avirulent isolates. Twenty seven iron-regulated proteins that are homologues of other Haemophilus proteins were identified using 2-D electrophoresis when H. parasuis was grown in stressed (iron-restricted) and nonstressed (iron-replete) conditions. Expressed proteins were further analyzed using mass spectrometry, Western blotting, Far Western blotting, and stable isotope labeling with amino acids in cell culture (SILAC). SILAC experiments confirmed the presence of a homologue of H. influenzae\u27s hemoglobin binding protein (HgbA) which was confirmed by probing blots with biotinylated porcine hemoglobin. Some identified H. parasuis proteins were homologues of other known virulence factors of the Pasteurellacae family and may be used in future virulence studies of H. parasuis .