Preparation Of Crude Antimicrobiaextract From Bovine Leukocytes And Its Effects On Immunity Of Mice

Bovine blood was collected to extract and isolate antimicrobial components from leukocyte and study the effects of crude extract on immunity of mice.Here are two series in this study.Seriesâ… Preparation of antimicrobial extracts from bovine leukocytes. Extraction of Crude Extract The fresh anticoagulation blood of bovine was collected and lysed by addition of 0.83%ammonium chloride solution to have no red cell under microscopic examination,centrifugation and collect leukocytes sedimentation.Antimicrobial peptides were extracted by 10cetic acid solution at 4℃, high speed centrifugation and collected supernatants and%A freezed-dried.The extract were dissolved by 0.01%acetic acid solution and the crude extract was obtained.Use the radial diffusion plate assay method,the crude extracts's antimicrobial activity against Escherichia.coli O₇₈,Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10231 were revealed.The results showed that there were antimicrobial matters in bovine leukocyte,and there was strong antimicrobial activity against all the bacteria strains above.Isolation of The Crude Extract The crude extract were isolated into two parts, the negative-charged and the positive-charged,by means of Ion-exchange chromatography.To determine whether the ion-exchange fractions display antimicrobial activity,the radial diffusion plate assay method was taken,and the microbes of Escherichia.coli O78,Staphylococcus.aureus 1056MRSA and Candida.albicans ATCC10321 were used as test organisms.The results showed that bovine blood leukocyte antimicrobial peptides were cationic molecules.The antimicrobial activity of the cationic ion-exchange fraction against Escherichia.coli O78 was stronger than that against Staphylococcus aureus 1056MRSA and Candida albicans ATCC10321.Seriesâ…¡Effects of crude extract from bovine leukocyte on immunity of mice The crude extract of bovine leukocyte that was obtained by the above method was used to determined the effects on immunity of mice.The experiment animal was designed into five groups:acetic acid control group,protein control group,low dose group,middle dose group and high dose group.We used 10 mice in each treatment and used 100 mice in the whole process of experiments.The mice were fed timely in each day among the days,some 5 mice among 10 ones were administrated 15 days later and tested immune apparatus index and carbon clearance index(α),and Other 5 mice were administrated 20 days later and tested T cell subsets of peripheral blood.The results showed that compared with control group,in middle dose group the immune apparatus index reached a significant level(p＜0.01) and the carbon immune apparatus index reached a significant level(p＜0.05).And T cell subsets CD3⁺,CD4⁺,CD4⁺/CD8⁺ reached a significant level(p＜0.01).So we can conclude that the immunity of mice couldbe enhanced significantly by feeding leukocyte crude extract of bovine.With feeding leukocyte crude extract of bovine 15 and 20 days,the immune apparatus index,carbon clearance index and T cell subsets CD3⁺,CD4⁺,CD4⁺/CD8⁺ reached a significant level.But compared with 15-day feeding duration,no obvious change happened in the 20-day feeding group(p＞0.05).It can be concluded that the bovine leukocyte crude extract can enhance the immunity levels of mice at an appropriate dose of 7.5mg/mL.