Isolation And Identification Of Haemophilus Parasuis And Screening Of The Attenuated Strains

The disease was serious harm to the healthy development of the pig industry. Thepathogens is Haemophilus parasuis which is parasitic on a conventional herd conditional upperrespiratory pathogens, characterized by joint swelling pigs, lameness, fever, dyspnea. Hps cancause disease which is mainly characterzed by serosal inflammation, myocarditis,arthritis andmeningitis and other symptoms. In this study,212suspected from twenty-seven pig farmswere collected for molecular epidemiology, isolation and identification of Hps in the of ShaanxiProvince. Four strains of Haemophilus parasuis were successfully isolated and identified from thesuspected samples. Furthermore, a suitable method for Haemophilus parasuis growth and storagewas established. By the pathogenicity test, measurement and virulence plate agglutination test, apreliminary screening better reactogenicity strains to expect as seed bacterial was developed. Themain research results are as follows:1. From March2012to April2013,212samples from twenty-seven pig farms were collectedin Shaanxi Province. Among which four Hps were ultimately isolated and identified, by bacterialcultivation, morphologic observation, biochemical test, animal test and16S rRNA sequencing.The total isolation percent was1.88%.2. H. parasuis is difficult to be cultivated, which affects the detection of bacteria from clinicalsamples to some extent. The results showed that Hps can not grow in the ordinary agar platemedium, blood agar plate medium and MacConkey agar plate medium and slowly grow onchocolate agar medium. However, H. parasuis can grow very well in tryptone soy agar (TSA)medium supplement with4%calf serum and0.01%NAD (modified TSA, mTSA). The reliablemethod for Hps long-term preservation is mTSA with20%glycerol at final concentration andstored at-80℃,which keep Hps alive at least for three months.3. The pathogenicity test was carried out among six isolates BJ1204, HX1212, BJ1212,YL1301, BJ1001and XA1001,among which BJ1001and XA1001were two isolates kept in ourlaboratory. The pathogenicity test results showed that the virulence of H. parasuis isolates werevary greatly. BJ1204was the most virulent, BJ1212followed, the virulence of HX1212andYL1301were the weakest while other strains were either weak pathogenic or non-pathogenic.Furthmore, the antigen and positive serum of above six Hps were produced by standard method.By the Plate agglutination test, we found the BJ1212isolate had better cross immunogenicity than other isolates, and has great potential as bacterial seed for vaccine R&D. The following work willfocus on BJ1212isolate cross-protection in mouse model, attenuation on NTG mutagenesis plate,and the vaccine protection efficiency based on BJ1212isolate in swine after bacterial challenge.