Study On Isolation, Identification And High-density Fermentation Of Haemophilus Paragallinarum HB Strain

Hpg disease was caused by infection of Haemophilus paragallinarum（Hpg）, it was alsocalled infectious coryza disease. The disease is one of the important bacterial diseases which areharmful to the poultry industry, it can brought huge economic losses to the poultry industry. Inthis research, one haemophilus paragallinarum strain was isolated and identified, haemophilusparagallinarum medium formulation is optimized, large-scale high-density fermentationprocesshad been studied and put into production by using the formula, The study provided a scientificbasis for the vaccine production. The research was summarized as follows.1） Screening of Hpg Hubei isloates, and phylogenetic analysis of its hemagglutiningene.One bacterium was isolated from sinus mucus of disease chicken in Jinmen city, Hubeiprovince. It were identified by bacterial culture characteristics, Gram staining, biochemical tests,susceptibility testing, serotype analysis, regression testing, attack drug protection testing.According to genomic sequences of Hpg, specific primers were synthesized for identification ofHpg. The PCR products was ligated to pMD18-T, then the ligetion product was sequeced, thesequencing results were matched with the reported sequences. The results of biochemical testexplained that nitrate reduction, glucose oxidase, sucrose, growth requires NAD were positive,trehalose, hydrogen sulfide, peroxidase were negative. The sequences homology of Hubei isolatewith the reported genome sequence of Hpg were from98%to100%.Hemagglutinin gene was amplified by PCR from the isolate, the prducts of PCR weresequenced, the sequencing results were compared with the published hemagglutinin gene byanalysis of the genetic evolution. The results showed that the amplified fragment size was1038bp, the homology of Hubei isolate with the published Tianjin, Sd-1strain were100%, thehomology of Hubei isolate with the published hagA, SA-3, E-3C,0083, HP8,221strains were96.0%and96.1%.2）Optimization experiments of culture medium as the isolate by shake flaskCurrently, the effect of infectious coryza vaccines medium is not very satisfactory,enrichment effects can not better meet production requirements. The study, based on semi-synthetic medium of "order", focused on animal serum, serum concentrations of glucose,coenzyme concentration on the impact of the situation throughout the training process. Theresults show that adding chicken serum5%(V/V), the amount of glucose added1g/L,0.1%addition of coenzyme solution1%(V/V), culture was the highest OD value of absorbance,stability, longer duration.3）High-densityfermentation process studies of Hpg HB isloatesHigh-quality vaccines for infectious coryza disease plays a particularly crucial role asprevention of the disease In this experiment, strains of Haemophilus paragallinarum HB wascultivated using10L and200L fermenter by batch fermentation.The inoculation, incubation temperature and pH parameters are grouped fumble, finally thefollowing conclusions are as follows, inoculum5%(V/V), incubation temperature37.5℃, pHvalue of7.2, culture was the highest OD value of absorbance and maximum viable cell count.Cell density OD value obtained and the number of viable cells were15.4,3.18x10~9cfu/ml as anideal result using200L fermentor. The results for large-scale high-density fermentation forhaemophilus paragallinarum provided intentional reference.