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Haemophilus parasuis (HPS) is a kind of V factor-dependent, non-hemolytic, gram-negative, rod-shaped bacterium, which belongs to the family of Pastteuerllaeeae. It is a commensal organism of the upper respiratory tract of conventional pigs, but under appropriate conditions can invade and cause severe systemic disease, Glasser’s disease, which is characterized by fibrinous polyserositis, arthritis and meningitis. HPS frequently coinfects with other bacterial agents such as porcine reproductive, respiratory syndrome virus and porcine circovirus, causes the disease which effects the health of naive and young pigs in conventional swine herds, and results in great economic losses in the swine industry worldwide. Thus, it has received much attention in recent years. On account of its complex pathogenesis and unclear virulence factors, exploring the pathogenesis and searching for virulence factors of this bacterium is particularly important.In the study, HPS isolates were isolated and identified from the samples in Shanghai. Studying the difference of expression of the proteins between cultivation conditions of HPS under the normal and mimicking the in vivo pathogenic environment, and using mass spectrometry to identify different proteins, which could provide clue for searching for the new virulence factors, and establish the basis of developing the new subunit vaccines.1. According to the clinical manifestation, pathological change and cultural character, morphous and staining of HPS, initial idetification was carried out.102 suspectable isolates were obtained from the 4124 samples, which were collected from Shanghai area during 2008.2 to 2011.4. Furthermore,61 HPS isolates of 102 were detected by specific PCR and with 16S rRNA gene primers combined with biochemical test results. The isolating rate of HPS is 1.48%, the strains mainly isolated from clinical samples with the 70.5% isolating rate, the rest are from the samples of pig farms and slaughterhouses. Susceptibility test results of the aboved showed that all HPS isolates were susceptible to cephalosporins, quinolone, and were severely resistant to novobiocin, amikacin, lincomycin, erythromycin and gentamicin. The serotyping results demonstrated that the most prevalent serovar was the serovar 5 (34.4%), then the serovar 2,4,13 and 14, and 11 isolates of 61 could not be serotyped.2. According to the previous studies, changing the cultivation condition of the bacteria can be used to simulate the growth condition in the host, which results in the expression of the relevant virulence factors, so that the relevant virulence factors could express. In this experiment, serovar 5 HPS clinical isolate was as the material, mimicking the growth condition in diseased pigs in vitro by changing the cultivation condition and then the total proteins of bacteria were extracted under normal culturing in vitro and that in the mimicking condition, respectively. Then three times of two-dimensional electrophoresis were proceed, and 3 groups two-dimensional electrophoresis figures of proteins were obtained. By analysis of ImageMaster V6.01 software,43 differential protein spots with 1.5 times diversity were screeninged in the gel figures of pH 4～7, in which 15 protein spots were with more than 3 times diversity and 6 protein spots exist only in the latter.3. Analysis of the difference of proteins expression of HPS under the two different cultivation conditions, total 21 protein spots, which were more than 3 times diversity and completely different, were cut respectively in the gels and the relevant different proteins were identified using mass spectrometry. Finally,10 proteins were identified successfully, containing sulphate adenylytransferase, pyruvic acid dehydrogenase, succinyl CoA synthase, outer membrane protein, translation elongation factor TU, urase,3-GAPDH, TRWC,3-O acyl reductase and phosphotransferase system of bacteria. These results provided theoretical basis for searching for the new virulence factors and studying the pathogenesis of HPS.
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