Extraction Of Swainsonine From Metarhizium Anisopliae And Effection On Newcastle Disease Vaccine

Subject: The purpose is seeking a effective technique for the extraction and isolation swainsonine from culture solution of Metarhizium anisopliae,searching for a new source of swainsonine, reducing clinical application cost of swainsonine, and realize swainsonine officinal value. Offering new theory base to effective therapy and research for hunman and animal disease.Methods:①Research on the technical of swainsonine extraction . Metarhizium anisopliae was preserved, passaged,isolated and purified on potato dextrose agar (PDA)medium in a stated time, Inoculating Metarhizium anisopliae on Czapek′s medium, the culture conditions is that: temperture is 25～27℃,pH is 6.5～6.9 ,comparative humidity is 45%～55%,culture time is 14～16 days.Then Metarhizium anisopliae culture solution was obtained and pretreated. Swainsonine was isolated from culture solution by using the method of extraction, column chromatography, decompression sublimation. Production was identified by TLC and GC method.②Experimental study on antimicrobial effects of extractives from Metarhizium anisopliae in vitro. In order to study the bacteriostatic activity of extractives from Metarhizium anisopliae, agar diffusion and tube dilution method were used to observe its bacteriostatic efficacy in vitro.Choosing the five common pathogenic bacterium to obtain the different kinds of extraction antibacterial effect in vitro.③100 14-day-old chickens were randomly assigned into equal five groups. the chickens in three experimental groups were intramuscularly injected respectively with Secondary metabolites of Metsrhizium anisopliae at high, mediumand, low dose and vaccinated with Newcastle disease vaccine. two control groups were administrated with no adjuvant(NA) and normal group. The 1 st to 4rd four groups were immunized with the LaSota live vaccine against Newcastle disease virus(NDV)for two times with 14 days interval. Before and on days continuous 6 weeks after vaccination, 10 chickens were sampled randomly from each group for the dynamic change of serum HI antibody were determined. cellular numbers of leucocyte in peripheral blood were detected, On days 42 after vaccination, 5 chickens per group were collected to the weights of whole body,spleen,bursa and thymus were weighted for calculation of immune organ index.Proliferative response of spleen lymphocyte was examined by MTT method. Results:①Cultured transferring Metarhizium anisopliae on potato dextrose agar(PDA) medium is suitable; Metarhizium anisopliae grew well on modified Czapek′s medium; Using gas chromatography(GC) could detecte swainsonine that in white powder which was isolated from fermentatoin broth.;②The result indicate that the five different pathogenic bacteriums was suppressed by all the 6 kinds of extractives from Metarhizium anisopliae at some level;③The secondary metabolites of Metarhizium anisopliae at dosage of 1-2 mg/kg could increased antibody titer,organ index and cellular number in blood. The proliferative activity of spleen lymphocyte was promoted by secondary metabolites of Metsrhizium anisopliae alone or being combined with LPS and PHA-P.The cellular number in blood,organ index,antibody titer and the proliferative activity of lymphocyte were decreased at the dosage of 4mg/kg.