Study On Selection Of Riemerelia Anatipestifer Strains And Optimization Of High Density Fermentation Of The Isolaets

Riemerella anatipestifer disease, namely the infectious serositis of duck, is one ofthe most important infectious disease which caused duck farming economic loss, it’swidely distributed in the world country and duck feeding area. This research is basedon the11strains of Riemerella anatipestifer separated from a duck farm, through aseries of tests in the dominant serotype strains with strong pathogenicity and goodimmunogenicity as the study object, through the contrast test to determine the mediumwhich have an enrichment effect, using single factor test and research the orthogonaltest of Riemerella anatipestifer liquid culture medium formula, and discussed theoptimum medium composition of Riemerella anatipestifer growth, and theoptimization results are applied to the high density fermentation, optimization offermentation conditions, lay a good foundation for vaccine development ofhigh-quality, efficient next. The main research contents are as follows.1. Isolated and identification of Riemerella anatipestifer and screening ofvaccine strains.The Duck infectious serositis of suspected cases from Beijing duck farm isolatedand identification of Riemerella anatipestifer, and further analyzed its pathogenicityand immunogenicity. The results show, successfully isolated11strains of bacteria,and can be divided into three serotypes, infection of type I and type II; cytotoxicitytest indicated that the11isolates of14day old Cherry Valley duckling pathogenicdifferences, from4/10to10/10range; immunogenicity test results showed thatisolates JX-2, JX-6and the JX-10based vaccine protection against challenge infectionrate can reach more than80%. It should consider the use of containing the strains of3serotypes made polyvalent vaccine vaccination on the farm.2. Screening and optimization of enrichment liquid medium for Riemerellaanatipestifer.This study compares the effect of four medium enrichment including improved Martin broth, modified yeast broth, P-L broth and tryptone soya peptone broth. Theresults show that, the improved yeast broth enrichment effect is the best in the fourkinds of medium, determination of growth curve of bacteria, strain JX-2in modifiedyeast broth culture of24h reached growth peak, bacteria living bacteria number up to166×108CFU/ml. With improved yeast broth for the optimization of culture mediumformula based on the single factor experiment and orthogonal experiment ofRiemerella anatipestifer liquid, the results of single factor experiment showed growthhydrolyzed milk protein, yeast extract, soy peptone, pig stomach, cleavage of blood,horse serum and other components plays a big role in the bacterial concentration;orthogonal test results showed that when the selected20%yeast extract5%; pigstomach digestion solution10%;8%soybean peptone; get horse serum5%thebacterial concentration was highest.3. Optimization and validation of high density fermentation culture processfor Riemerella anatipestifer.The research application of small fermentor, the impact factor of Riemerellaanatipestifer fermentation: ventilation (L/S.L), speed (RPM), medium pH on the basisof3levels orthogonal experiment of the3factors, optimization of high densityfermentation of Riemerella anatipestifer culture process, high density fermentationconditions optimization results display:0.1L/S.L ventilation; speed150R/min;medium pH of7.4the number of live bacteria and bacteria concentration was highest.The optimization of fermentation process of formulation and optimization, massproduction of3batches of Riemerella anatipestifer semi-finished antigen in GMPworkshop, living bacteria number is about30%higher than that before optimization,thus the research provides experimental basis for the study of high densityfermentation production of Riemerella anatipestifer antigen.