Isolation And Identification Of Haemophilus Parasuis Serovar4,5and Cloning And Sequence Analysis The Neu,Omp2,PaLA,D15Gene

Haemophilus parasuis (Haemophilus parasuis, HPS) is one of the Pasteurella Haemophilus members,it is considered to be the major pathogen of leather Glaser’s disease (Glaesser’ s Disease). Founding in the investigation of large-scale breeding farms, HPS has become a main bacteria which seriously affect the pig industry at home and abroad.Studies have shown that in mixed infection with porcine reproductive and respiratory syndrome virus and other pathogenic can cause immunosuppression,often increase the incidence of infection and result in great economic loss, it also seriously threat to the healthy development of the pig industry worldwide. In order to know the genetic variation of the Sichuan Province of HPS, this study isolated two Haemophilus parasuis from clinical cases,cloning and sequence analysis its virulence genes in order to research the prevention and treatment of HPS and its clinical genetic mutation tracking, which may also lay a foundation of new efficient vaccines.Use the bacteriology test method and PCR separation and identification HPS from suspected infection of the lungs which are from large-scale pig farms SuiNing and MeiShan, and isolated2strains of suspected HPS, with the morphology, dyeing and cultivating characteristics testing, biochemical tests, serum type test and16SrDNA sequence analysis, identify these2strains are HPS,and called them HPSmeish (meishan plant) and HPSsuin (SuiNing), their serum type are respectively type5type and type4. Animal pathogenic test show that type4and type5are strong pathogenic, antimicrobial susceptibility test show that these two strains have strong drug-resistance, HPSsuin only sensitive to amikacin, ampicillin-salbactam, fortum and furazolidone; HPSmeish only sensitive to ampicillin-salbactam,amoxicillin,norfloxacin and enoxacin.In order to compare to the homology and antigenicity of4virulence genes of Haemophilus parasuis (HPS) type4, type5:(neuraminidase) Neu,(outer membrane protein P2) Omp2, PaLA, D15,our experimental design4pairs of specific primers, using PCR technique to clone these genes, clone into PMD19-T carrier, and transform these genes into DH5a, identify them with PCR, then sequencing and analysis. The results showed that the PalA of type4and type5are homology highly amount to99.1%, nucleotide sequence mutation is very small, only3mutantional sites; the Nue of two serotype homology is98.4%,the mutation is very dispersed; the homology of two strains of D15and reference strains Huhe0123are all above99%. Omp2sequencing results of Type4shows that amplified target fragment is1086bp (containing Omp2gene), and compare with HPS SW124strain which was has published4type Omp2gene (1077bp), the results showed that:Omp2of4type of Haemophilus parasuis Sichuan isolates is a relatively stable gene, their nucleotide homology are97%, amino acid homology are92.5%, the mainly variable region is concentrated in the four base sequences:110-156,180-202,448-463,580-605.Compared with the amplified target fragment (1098bp containing Omp2gene) isolated from Omp2sequencing of type5and the Type HPS Naqasaki Omp2gene (1077bp), the result showed that type5of Haemophilus parasuis Sichuan isolates Omp2relatively stable gene, nucleotide homology were94.8%, amino acid homology as of89.9%. The variation of areas are mainly concentrated in the three base sequences:380-400,400-425,803-818.