Isolation And Identification Of Haemophilus Parasuis And The Typing By PCR-RFLP Analysis Of The TbpA Gene

Gl?sser's Disease is a typical bacterial disease, which is caused by Haemophilus parasuis, always causes serofibrinous pleurisy, pericarditis, peritonitis, arthritis and meningitis in piglets. In recent years, the incidence and mortality of the disease increased significantly in the world, creating huge economic losses to the pig industry. Nowadays, many institutions have isolateded lots of Hps isolates and obtained some achievements in its epidemiology. However, these studies only focused in some certain area. The study related to Shaanxi province is still rare. In this research, 5 strains of Haemophilus parasuis were isolated and identified from GuangZhong District of Shaanxi province, and the appropriate preservation conditions for Haemophilus parasuis strains were explored and obtained. Further we performed drug sensitivity test against common antibiotic drugs to the isolates. The PCR-RFLP analysis based on the tbpA gene was used for genetyping of the 5 Haemophilus parasuis isolates. Not only can the isolation, identification and molecular epidemiology of Haemophilus parasuis provide guidance for controlling the disease, but also help revealing its prevalent pattern in the district. The main results are as follows:1. To certify whether the Gl?sser's disease existed in Guanzhong District, from Shaanxi province, we isolated 5 bacteria from the swine which were characterized by polyserositis and fibrinopurrlent polyarthritis. Then we identified them based on the cultural characteristics, morphologic characteristics, biochemical characteristics; primers were designed according to the sequences of Haemophilus parasuis and PCR was conducted to amplify the isolated strains. Amplification product was connected to the T vector and sequenced. Homology of 16S rRNA sequences between the sequences of the isolates and those from GenBank was between 99%～100%. All the above results conformed the isolates to be Haemophilus parasuis. Our findings also suggested that Haemophilus parasuis widely existed in this area, providing certain experimental basis for studies of Haemophilus parasuis disease in this area.2. The conservation conditions of the 5 isolates were also explored. This was performed through inoculating well-growing strains in different mediums and solutions at different temperatures. The preserved strains were recovered and detected whether they still survived every certain time. The results showed that inoculating Haemophilus parasuis strains in TSB medium twice and preserving them in sterilization glycerin with final concentration of 20% at -70℃was the best way of preservation, laying a certain foundation for the further study of Haemophilus parasuis.3. Drug susceptibility pieces method was used to detect sensitivity of the 5 isolates of Haemophilus parasuis against nine common drugs. All of the strains were 100% sensitive to Ofloxacin, Tobramycin and Kanamycin, as well as Rifabutin (40%), while Cefuroxime and Carbenicillin was not sensitive at all. Through which, we found that all the 5 strains were not highly sensitive against the common drugs and different isolates also showed different sensitivity to the same drug, which provided basis for the treatment of Haemophilus parasuis in this region.4. PCR-RFLP genotyping method, which was created by Redondo et al., was used in this research. Three restriction enzymes, TaqⅠ, AvaⅠand AfaⅠ, were used for PCR-RFLP genotyping the turn iron binding protein protein A (tbpA) genes of the 5 strains of Haemophilus parasuis. Four different RFLP gene patterns were obtained, which were named EBC, BBJ, EBJ, EBJ and EAD, respectively. We further compared and analyzed the four genotypes and other genotypes obtained by relevant research institutes, the results showed that the prevalent genotypes of Haemophilus parasuis isolates in Shaanxi province presented high diversity. Our results provided theory basis for molecular epidemiology investigation of Haemophilus parasuis disease in Shaanxi province.We isolated and identified 5 Haemophilus parasuis isolates and took the drug sensitive tests of them. Then the isolates were subjected to be ananlyzed using PCR-RFLP based on the tbpA gene. Through this work, we provided an effective method to study the isolation and identification, molecular character and the control of Haemophilus parasuis disease.