Showing 3 results for Characterization
B Jafari , Ra Khavari Nejad , F Vaziri , Sd Siadat,
Volume 4, Issue 3 (11-2017)
Abstract
Introduction: Extracellular vesicles (EVs) contain active biological compounds which play important roles in biological processes. The secretion of EVs is a common phenomenon which occurs in archaea, bacteria and mammalian cells. The secretion of bacterial EVs has been discovered in various species of both Gram-negative and Gram-positive bacteria. Faecalibacterium prausnitzii is one of the commensal bacteria in human intestinal tract which has potentially therapeutic effects by secretion of bioactive compounds. Within the last few years, many investigations have been performed with respect to extracting and obtaining EVs and as a result, there are many methods to isolate and characterize EVs. The aim of this study was to isolate EVs from F. prausnitzii strain A2-165 and to characterize their physico-chemical properties. Methods: EVs were isolated from F. prausnitzii strain A2-165 using ultracentrifugation and filtration. The EVs of bacterium were then characterized by Scanning Electron Microscopy, SDS-PAGE, Bradford assay, NanoDrop and Limulus Amebocyte Lysate (LAL) test. Results: The extracted EVs were confirmed with the shape of vesicles and the sizes ranging from ~ 30 to 250 nm. Total protein concentration of EVs were ~ 3 mg/ml from two methods; Bradford and NanoDrop, respectively. Protein profile pattern of F. prausnitzii-derived EVs ranged from 11 up to 245 kDa. Endotoxin measurement was 2.04 EU/ml. Conclusion: The results of the current study demonstrated that EVs purity and conformation were acceptable. However, further investigations are necessary to elucidate the safety, efficacy, practicality and mechanism of action of this bacterium EVs in clinical practices, especially as vaccine delivery vehicles in the field of vaccine research.
M Sekhavati , Sd Siadat , M Noofeli , A Mohebati Mobarez ,
Volume 5, Issue 2 (12-2018)
Abstract
Introduction: In spite of high vaccination coverage, whooping cough (pertussis) is still a worldwide health problem. The main reason for pertussis outbreak is waning immunity of safer acellular vaccines which have replaced the more reactogenic cellular vaccines. A new generation of pertussis vaccines that is potent and safe is desperately needed to control the disease. Previous studies have indicated that outer membrane vesicles (OMVs) obtained from Bordetella pertussis have desirable characteristics which make them a good candidate for application as pertussis vaccine. They contain surface immunogens in a native structure, are self-adjuvant and are easily uptaken by the antigen presenting cells. Methods: B. pertussis Tohama strain was cultured at 35°C in Stainer-Scholte broth. The OMVs were isolated by a new sequential ultracentrifugation method. The extracted OMVs were characterized by electron microscopy, SDS-PAGE and ELISA assays. Results: The existence of pertussis toxin, filamentous haemagglutinin and a 69-kDa antigen in B. pertussis OMVs was verified using an ELISA assay. Electron microscopy showed the size of these OMV’s at 40-200 nm. The ELISA results indicated that the OMVs extracted using this protocol contain major immunogens. Conclusion: We report for the first time a simple protocol for the efficient extraction of B. pertussis OMVs. This protocol can be used in the process of making new generations of B. pertussis vaccines.
Mohammad Majid Ebrahimi, Shahla Shahsavandi, Naser Ghodsian,
Volume 7, Issue 2 (12-2020)
Abstract
Introduction: Poultry vaccines are used to immunize chickens against different diseases. Inactivated vaccines have been widely used to protect poultry against diseases such as infectious bursal disease (IBD). IBD is one of the most important viral immunosuppressive diseases in the poultry industry. This viral disease targets the immune organs. This study was aimed to evaluate the effect of an inactivated IBDV antigen on inducing the humoral immune response in Specific-Pathogen-Free (SPF) chickens. Methods: An infectious strain of bursal disease virus (IBDV) was isolated from an affected chicken bursa of Fabricius. Serological diagnostic tests and molecular experiments were carried out to identify the isolate. Different concentrations of formalin, beta propiolacton (BPL) and binary ethylenimine (BEI) were used for inactivation of IBDV. The samples of IBDV antigen were adjuvanted separately with ISA-70. Three-week-old SPF chickens were divided into four groups. Groups 1, 2, and 3 received 0.5 ml of the adjuvanted antigens subcutaneously and group 4 received PBS as negative control. Blood samples from each group were collected 4 weeks post-inoculation and the targets were measured by ELISA and serum neutralization test (SNT). Results: The lowest concentrations that could fully inactivate the infectivity of IBD virus were 2.5 mM for BEI, 0.15% for BPL and 0.1% for formalin. Examination of the inactivated samples with 0.1% formalin showed a decrease in antigenicity after 12 months. Treatment with 2.5 mM BEI and 0.15% BPL showed no apparent adverse effect on IBDV infectivity and showed a reliable inactivation. In the SPF chickens of all experimental groups, the antibody titers raised against IBDV were detected by ELISA. Conclusion: In the group which the virus was inactivated using BEI, the antigenicity stability was much better than others. Hence, BEI-inactivated IBDV is suggested for preparing more immunogenic, efficient and stable vaccines against IBD.
