Immunogenicity evaluation of rBoNT/E nanovaccine after mucosal administration

Objective(s): The Botulism syndrome is caused by types A to G of botulinum neurotoxins. The binding domains of these neurotoxins are immunogenic and considered as appropriate candidate vaccines. Due to the low immunogenicity of recombinant vaccines, there have been many studies on the use of biocompatible carriers such as chitosan nanoparticles for the delivery of these vaccines. The aim of this study was evaluating the efficiency of chitosan nanoparticles as carriers for a candidate vaccine, binding domain of BoNT/E, through oral and intranasal routes.Materials and Methods: Chitosan nanoparticles containing rBoNT/E binding domain, were synthesized via ionic gelation. After administration of the nanoparticles to mice through oral and intranasal routes, antibody titers were assessed by ELISA and, finally, all groups were challenged by active botulinum neurotoxin type E.Results: The groups that received nanoparticles containing the antigen, through oral and intranasal routes, and the group that received the bare antigen orally, were able to tolerate 5×102 folds of MLD. The intranasally immunized mice by the bare antigen were able to tolerate 2×103 folds of the toxin’s MLD. Conclusion: It seems that the use of chitosan nanoparticles has no significant effect on the protective immunization of the mice against botulinum BoNT/E in either route (P>0.05), even intranasal administration of the bare antigen gives better mice immunization against the toxin

Biodegradation of Chlorpyrifos and Diazinon Organophosphates by Two Bacteria Isolated from Contaminated Agricultural Soils

Introduction: The organophosphate insecticides such as chlorpyrifos and diazinon have been widely used to control insects in home, agriculture, and veterinary. These compounds are a threat to public health and the environment; on the other hand, they have low degradation rate and therefore can be stable for a long time in soil. A major factor determining the fate of organophosphate insecticides in soil and water is biodegradation. Materials and methods: Two diazinon and chlorpyrifos degrading bacterial strains were isolated from pesticides contaminated soils. The isolated bacterial strains were identified by 16S ribosomal RNA gene and fatty acid methyl ester analysis. Results: Strong correlation was seen between microbial growth and the two organophosphates degradation. On average, bacterial strain 1 and 2 degraded 88.27% and 82.45% of initial applied diazinon in medium and degraded 81.07% and 88.35 % of initial applied chlorpyrifos during 20 days, respectively. The isolated bacterial strains were identified as Acinetobacter and Pseudomonas sp. The highest diazinon degradation were found by Acinetobacter and the highest chlorpyrifos degradation were found by Pseudomonas when cultivated in the mineral salt medium. Discussion and conclusion: The identified pure bacterial strains utilized chlorpyrifos and diazinon as a source of carbon. They were able to degrade most of the parental molecule in 20 days. Therefore, the isolated bacterial strains may have the potential for use in the bioremediation of diazinon and chlorpyrifos-contaminated soils

Computational analysis and gene cloning: design and preparation of a multi subunit vaccine consisting of EspA, Stx2B and Intimin antigens against enterohaemrrhagic <em>Escherichia coli</em>

16-26Escherichia coli O157:H7 is an intestinal pathogen that made diarrhoea, haemolytic uremia syndrome (HUS) and hemorrhagic colitis (HC) in patients. Roles of EspA and Intimin at the beginning of bacteria colonization in intestine are critical. Destruction of protein synthesis route with shiga toxins of E. coli O157:H7 is mediated through B-subunit of toxins. In this study, in silico approaches were performed to design a suitable construct from EspA, Intimin and Stx2B and a recombinant chimeric antigen was produced. Bioinformatics analyses such as physicochemical data, mRNA folding, 3D structures of chimera and various immunoinformatic data, such as linear and conformational B-cell epitopes, T-cell epitopes were reported according to authentic data base. The chimeric gene was prepared as synthetic construct after designing and cloning. The validation result showed that 83.9% residues lie in favoured or additional allowed region of the Ramachandran plot. Epitope prediction results proved very good distribution of conformational B-cell epitopes in the 3D structure of chimera. The identified T-cell epitopes are apt to bind MHC molecules. A good quantity of recombinant chimeric antigen was achieved in host cells. From in silico approach, an appropriate multi subunit vaccine candidate was designed and prepared for immunological examinations

Subcloning and Assessment of the Expression of Synthetic Botulinum Neurotoxin Type B Binding Domain (BD/B)

Background and Objectives: Botulism syndrome is caused by Clostridium botulinum neurotoxin. This neurotoxin has 7 serotypes, from A to G. The best way to prevent botulism syndrome caused by BoNT/B is immunization with recombinant binding domains of BoNT/B (due to containing sufficient epitopes to stimulate the immune system). In this study, the expression of the BoNT/B binding domain as a candidate vaccine, was investigated. Methods: At first, the sequence of the BoNT/B binding domain gene was obtained from NCBI website with accession number of EF028399.1. After codon optimization, the gene was synthesized in pUC18 vector. Then, the gene was subcloned in pET32a(+) expression vector that carries an ampicillin selection marker. PCR, enzymatic digestion, and sequencing were used to confirm subcloning accuracy, and E. coli BL21(DE3) was used for the expression analysis. The accuracy of protein expression was evaluated by electrophoresis and western blotting. Results: PCR reaction, enzymatic digestion, and sequencing confirmed that the gene of interest has been subcloned appropriately in the vector. The expression analysis by SDS-PAGE and subsequently western blotting, showed that the protein of interest is not expressed in the mentioned expression strain. Conclusion: Although the gene was successfully subcloned in pET32a(+) vector, no significant expression of the gene was observed

Identification of E. coli O157:H7 by Using Specific Primers for rfbE and stx2b Genes

Background: E. coli O157:H7 is one of the intestinal pathogens which causes serious lesion in gastrointestinal system. Detection of this bacteria that able to produce toxin and is the major responsible for hospital infection, usually done by culture on sorbitol-MacConkey agar which is time-consuming test. The aim of this study was the preparation a fast and precise method in order to identification of this bacterium by molecular method based on PCR technique. Material and Methods: rfbE and stx2b genes were selected for proprietary identification of E. coli O157:H7 and the amplification of them were performed by PCR following designing specific primers. Sorbitol-MacConkey agar was used to verification of growth ability of selected colonies during PCR. Results: By appearance of the bonds belong to rfbE and stx2B genes on agarose gel, the ability of designed primers in gene detection in samples of E .coli O157:H7 was verified. Colonies which selected during PCR have growth potency on sorbitol-MacConkey agar medium. Conclusion: It was revealed that we can prepare a fast, precise and relative comfortable method for detection of E. coli O157:H7 strain by using PCR technique and specific primers than other available methods

Subcloning and Assessment of the Expression of Synthetic Gene of Botulinum Neurotoxin Type A Binding Domain (BD/A)

Background and Objectives: Botulism syndrome is caused by Clostridium botulinum neurotoxin. This neurotoxin has seven serotypes ranging from A to G. The best way to prevent botulism syndrome caused by Botulinum neurotoxin (BoNT), is using recombinant vaccine made from its binding domain (due to having sufficient epitopes to stimulate immune system). In this study, the binding domain of BoNT serotype A (BoNT/A), was investigated. Methods: Initially, BoNT/A gene with accession number CP000727.1, was obtained from GenBank and was codon optimized according to the codon usage of E. coli. Then, the sequence was synthesized in pET28a plasmid and then subcloned in pGEX-4T-1 expression plasmid. The subcloning was done using PCR with Pfu DNA polymerase and then double digestion with XmaI and XhoI restriction enzymes. E. coli BL21 strain was used as the expression host. The selected marker for pGEX-4T-1 was ampicillin. Results: PCR and restriction digestion with the mentioned enzymes confirmed the subcloning process. The assessment of gene expression was performed by SDS-PAGE and western blotting (using horse anti-BoNT/A (and then glutathione affinity chromatography was performed. Although, the subcloning was performed successfully, no protein expression was observed.&nbsp; Conclusion: According to the findings of this study, it seems that other hosts, such as eukaryotic hosts should be used for recombinant expression of BoNT/A binding domain

Assessment of the safety of chicken egg yolk antibody (IgY) consumption by lipid peroxidation marker in mice

Production of antibodies in chickens (IgY) has significantly attracted attention of scientists. Numerous publications have reported use of IgY in diagnosis, therapy and prophylaxis. Production of antigen-specific antibodies in chicken can help treat and prevent infectious diseases. The aim of this study was to assess the safety  of IgY( anti  E. coli O157:H7)  on the antioxidant system in mice .Therefore in  this study, three different  doses of IgY against E. coli O157:H7 (0.9375, 1.875 and  3.75 g / kg)  were administrated through oral route to 18 mice (treated groups) and PBS to the control group  and 14 days after administration, blood samples were collected from the mice. Serum malondialdehyde (MDA) level and catalase, glutathione peroxidase and superoxide dismutase activity were measured using commercial kits. Oral administration of IgY against E. coli O157:H7 in  doses  of 0.9375, 1.875 and 3.75 g / kg  caused no  deaths and showed no toxic effects on mice. In this study, after 14 days of  IgY administration there were no significant changes in the activity of antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase) and  MDA  serum level   compared to the control group. Our findings revealed that oral administration of IgY against E. coli O157:H7 does not show any toxic effects and does not   disturb the antioxidant system in mice . These findings could be indicative of safety of oral administration  of  IgY in mice