Immunogenic Potency of Formalin and Heat Inactivated E. coli O157:H7 in Mouse Model Administered by Different Routes.

BACKGROUND: Enterohemorrhagic Escherichia coli (E. coli) (EHEC) O157:H7 is a major foodborne pathogen causing severe disease in humans worldwide. Cattle are important reservoirs of E. coli O157:H7 and developing a specific immunity in animals would be invaluable. The administration of Whole Cell Vaccines (WCV) is a well-established method of vaccination against bacterial infections. Route of administration, inactivation and using suitable adjuvant have significant effects on the characteristics and efficacy of WCV. METHODS: In the present study, an attempt was made to evaluate the immunogenic potency of heat and formalin inactivated cells administered orally and subcutaneously in mouse model by ELISA. Mice pretreated with streptomycin were used as a model to evaluate the efficacy of subcutaneous versus oral administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. RESULTS: Both forms of inactivated cells induced immune response and hence protection against infectious diseases caused by E. coli O157:H7. However, formalin inactivated cells of E. coli O157:H7 showed superior antigenicity compared to heat inactivated cells. Subcutaneous immunization of mice with both heat and formalin inactivated E. coli O157:H7 induced significant specific levels of IgG antibodies but did not lead to significant antigen-specific IgA rise in feces, whereas oral immunization elicited significant levels of IgG antibodies with some animals developing antigen-specific IgA in feces. CONCLUSION: Inactivated E. coli O157:H7 is highly immunogenic and can induce protective immune responses via oral immunization.

Presenting a rapid method for detection of Bacillus cereus, Listeria monocytogenes and Campylobacter jejuni in food samples.

OBJECTIVES: Listeria monocytogens, Bacillus cereus and Campylobacter jejuni are three toxin producing bacteria over the world, especially in Iran, and it is essential to find a certain, rapid procedure to identify these microorganisms. In this research, these bacteria were simultaneously detected by multiplex PCR technique in foods. MATERIALS AND METHODS: The primary approval of bacterial strains was performed by biochemical tests. PCR primers were designed based on the nucleotide sequences of the NHEB/NHEC gene of B. cereus, the hly gene of L. monocytogenes and the C gene of C. jejuni. The specificity of Multiplex PCR method was determined using seven food poisoning bacteria including Salmonella typhi, Shigella dysentery, Yersinia pestis, Staphylococcus aureus, Clostridium perfringens, Clostridium botulinum and Vibrio cholerae. To confirm the reaction, DNA extraction was performed from 30 food samples (milk), and gene amplification was performed by PCR. The length of amplified fragments was 300 bp, 210 bp and 160 bp for NHEB/NHEC, hly and C genes, respectively. RESULTS: The detection limits of the PCR method were 5, 4 and 3 pg for L. monocytogenes, B. cereus and C. jejuni, respectively. Specifisity test showed that this reaction is spesific to these 3 bacteria. CONCLUSION: In this study, we introduced a new multiplex PCR method for simultsnus detection of L. monocytogens, B. cereus and C. jejuni. These results can be used for detection of other toxin producing bacteria in food.

Immunogenic evaluation of chimeric recombinant protein against ETEC, EHEC and Shigella.

Diarrheal diseases still remain health problem worldwide and out of many bacteria responsible for, Shigella and pathogenic Escherichia cause the most diarrheas in the world. Shigellacause bacterial dysenteries and shigellosis through invasion where the most effective proteins for pathogenesis is Ipac. Critical virulence protein for ETEC infection is CFA/I with two subunits called cfab and cfae. . Attachment of EHEC is the main step of infection and the protein Intimin plays the key role in this function. Protection against the vast majority of responsible pathogens of diarrheas requires development of the combination vaccine against Shigella, ETEC and EHEC. In the present study, a multisubunitprotein (CII) containing immunologically significant parts of CfaB, IpaC and Intimin was designed. The chimeric gene (CII) was codon optimized and analyzed with different bioinformatic servers, then synthesized and expressed in E. coli. Mice, Guinea pig and, Caco-2 Cell line were used as challenge models for EHEC, shigella and ETEC respectively. The chimeric protein induced significant immune response and therefore could be a suitable vaccine candidate against these three pathogens.

A review on strategies for decreasing E. coli O157:H7 risk in animals.

Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that younger children are most prone to this microorganism. Hemolytic Uremic Syndrome (HUS) caused by EHEC, leads to the destruction of red blood cells and kidney failure. The virulence of E.coli O157:H7 is attributed to fimbriae, that facilitate colonization of bacteria within the colon and verotoxins (VT) or Shiga toxins (Stx) that are released into the blood. Although, in most cases, the infection is self-limitedin young children and aged population, it may cause HUS. Therefore, several investigations are performed in order to offer effective therapies and vaccines, which can prevent and treat the infection in appropriate time. As the pathogenesis of this infection is complicated, a multi-targeted strategy is required. Since cattle are the most important reservoir of EHEC and the root of contamination, reducing E. coli O157:H7 at the farm level should decrease the risk of human illness. Several vaccine approaches have been employed with different proper outcomes in animal models, including recombinant proteins (virulence factors such as; Stx1/2, intimin, EspA, fusion proteins of A and B Stx subunits), avirulent ghost cells of EHEC O157:H7, live attenuated bacteria expressing recombinant proteins, recombinant fimbrial proteins. In addition to protein-based vaccines, DNA vaccines have provided proper prevention in the laboratory animal model. This review paper summarizes the previous studies, current status and future perspective of different immunization strategies for eradicating Enterohemorrhagic Escherichia coli O157:H7.

Chitosan-Based Intranasal Vaccine against Escherichia coli O157:H7.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an infectious zoonotic pathogen causing human infections. These infections, in some cases, can lead to hemolytic uremic syndrome and its life-threatening complications and even death worldwide. The first intimate bacterial adhesion, intimin (I), with its own receptor translocated intimin receptor (Tir) and E. coli secreted protein A, acting as Tir conduit, are highly immunogenic proteins for vaccine development against E. coli O157:H7. METHODS: A chimeric trivalent recombinant protein was previously found to be a suitable strategy for developing vaccines against E. coli O157:H7. In this study, the recombinant EIT (rEIT) was used to design a protective EHEC nasal nanovaccine. Chitosan and its water-soluble derivative, trimethylated chitosan (TMC), as muco-adhesive biopolymers, are good candidates for preparation of nanovaccines. Using the electrospraying technique, as a novel method, we could obtain particles of rEIT loaded with chitosan and TMC on a nanometer scale. Mice were immunized with intranasal administration or intrapretoneal injection of rEIT. RESULTS: The rEIT-specific immune responses (IgG and IgA) were measured by indirect ELISA. Only nasal administration of chitosan electrospray and TMC formulation produced significant secretion IgA. Intranasal administration of nanovaccine reduced the duration of bacterial fecal shedding on mice challenged with E. coli O157:H7. CONCLUSION: Since development of mucosal vaccines for the prevention of infectious diseases requires efficient antigen delivery; therefore, this research could be a new strategy for developing vaccine against E. coli O157:H7.

EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

BACKGROUND AND OBJECTIVE: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS). Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium. MATERIALS AND METHODS: A divalent recombinant EspA-Intimin (EI) protein containing EspA120 and Intimin280 attached with a linker was amplified from a trivalent construct and cloned in pET-28a (+) vector. The immunization was conducted in mice after expression and purification of the recombinant EI (rEI). RESULTS: Mice subcutaneously immunized with rEI, elicited significant rEI specific serum IgG antibodies and showed significantly decreased E.coli O157:H7 shedding compared to the control group. CONCLUSION: The chimeric recombinant protein induced strong humoral response as well as protection against oral challenges with live E.coli O157:H7.

A DNA vaccine against Escherichia coli O157:H7.

BACKGROUND: Infection with Escherichia coli O157:H7 rarely leads to bloody diarrhea and causes hemolytic uremic syndrome with renal failure that can be deadly dangerous. Intimin, translocated Intimin receptor (Tir), and enterohemorrhagic E. coli (EHEC) secreted protein A (EspA) proteins are the virulence factors expressed by locus of enterocyte effacement locus of EHEC. This bacterium needs EspA as a conduit for Tir delivery into the host cell and the surface arrayed Intimin, which docks the bacterium to the translocated Tir. METHODS: Here we used triplet synthetic gene (eit) which was designed from three genes: espA coding EspA 120 lacking 36 amino acids from the N-terminal of the protein, eae coding Intimin constructed of 282 amino acids from the C-terminal and tir coding Tir 103, residues 258-361 which interacts with Intimin. The multimeric gene was cloned in two eukaryotic vectors pAAV-multiple cloning site-green fluorescent protein and pCI-neo. The pAAV was used for gene expression assay in cell line 293T and pCI-neo-EIT (EspA, Intimin, Tir) was used as DNA vaccine in mice. Test groups were injected intramuscularly with pCI-neo-EIT four times and mice control group was injected under the same conditions with PBS or pCI-neo vector. RESULTS: The titration of serums showed that BALB/c mice were successfully immunized with DNA vaccine compared to control groups and also they were protected against challenges of live oral using E. coli O157:H7. CONCLUSION: The results suggest that the DNA vaccine could induce protective immunity either alone or in combination with purified antigens to reduce EHEC infection.

Immunogenicity of a plant-derived edible chimeric EspA, Intimin and Tir of Escherichia coli O157:H7 in mice.

Transgenic plants offer the possibility to produce and deliver an oral immunogen on a large-scale with low production costs and minimal purification or enrichment. Cattles are important reservoirs of Escherichia coli O157:H7 and developing a specific immunity in animals would be invaluable. Intimin, Tir, and EspA proteins are the virulence factors expressed by LEE locus of enterohemorrhagic E. coli. We hypothesized that the chimeric recombinant forms of these effectors delivered as an edible-base vaccine would reduce colonization of bacteria in mice. A synthetic gene (eit) composed of espA (e), eae (i) and tir (t) attached by linkers was constructed. The gene was codon optimized and cloned into plant expression vectors adjacent to CaMV35S and FAE promoters for expression in tobacco and canola plants. Of total soluble protein 0.2% and 0.3% (in average) were detected in transgenic tobacco leaves and canola seeds respectively. Mice immunized either subcutaneously or orally with recombinant EIT and challenged with E. coli O157:H7 significantly exhibited reduced bacterial shedding. Application of transgenic plants containing trivalent immunogen is an effective tool for protection against E. coli O157:H7.

Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) comprise an important group of enteric pathogens causing hemorrhagic colitis and hemolytic uremic syndrome. These bacteria need EspA (E) as a conduct for Tir (T) delivery to the host cell and surface arrayed intimin (I) which docks the bacterium to the translocated Tir. This phenomenon leads to attaching and effacing (A/E) lesions. A trivalent recombinant protein called rEIT composed of immunologically important portions of EspA, Intimin and Tir was constructed as a candidate vaccine. For high-level expression, the EIT gene was synthesized with codon bias of E. coli. The immunization was conducted in mice with purified rEIT. The results showed that this chimeric protein induced strong humoral response as well as protection against live challenges using EHEC.

Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains.

Botulinum neurotoxins (BoNTs) are potent toxicant proteins composed of a heavy chain (100 kDa) and a light chain (50 kDa) of seven (A-G) serotypes that is responsible for botulism syndrome. In this study, polypeptides from C-terminal heavy chain of BoNTs serotypes A, B and E to the length of 54, 45 and 48 amino acid respectively were selected, linked together using a hydrophobic linker and expressed in E. coli. The expression efficiency of the chimeric protein was found to be 51%. The chimeric protein was produced in the form of inclusion body (IB) both at two studied temperatures, 30 degrees C and 37 degrees C. This IB was extracted by ultracentrifugation and followed for chimeric protein solubilization and purification using of ultrafiltration and preparative electrophoresis. The purified chimeric protein was characterized using blotting and ELISA. To evaluate the protection ability of this chimeric antigen against their active toxins, it was injected to mice and the antibody titer as well as the extent of protectivity were determined. Mice given three injections (10 microg/mice) of the antigen were protected against an intra-peritoneal administration of 10 LD(50 )of serotypes A and E, but 100 LD(50) of serotype B. We conclude that a significant correlation exists between the antigenic characteristics and protection capability of the chimeric protein prepared in this study.

Cloning, high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E.

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain and a heavy chain (HC) linked through a disulfide bond. In the present study we explored the immunogenicity and protective capability of the most effective part corresponding to 1163-1256 residues of botulinum type E neurotoxin HC gene. DNA encoding the 93 C-terminal amino acid of HC residues was synthesized with optimal codon usage for expression. These DNA fragments were ligated into a pLivSelect vector and subcloned into expression vector pET32a. Recombinant plasmids were then transformed into Escherichia coli strain BL21 DE3. The recombinant protein was purified by nickel affinity gel column chromatography. The HC1163-1256 was identified by antibodies raised against BoNT/E. HC1163-1256 was shown to bind with synaptotagmin and gangliosides, indicating that the expressed and purified HC1163-1256 protein retains a functionally active conformation. The immunization with recombinant protein induced a protection level in mice. The immunization with 2mug of the recombinant protein induced a significant protection level in mice. In conclusion, availability of the recombinant protein provides an effective system to study the biochemical and physical interactions involved during BoNT binding to nerve cells and protection against its toxicity.

A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex.

Developing improved tuberculosis (TB) diagnostics is one of the international research priorities, as TB remains globally a major health threat. Loop-mediated isothermal amplification (LAMP) is a new nucleic acid detection method that can be used in low-resource settings, because it does not require expensive or complex instruments. Using the repetitive insertion sequence IS6110 as a target gene, we developed an efficient LAMP assay, which specifically detects members of the Mycobacterium tuberculosis complex (MTBC). This assay proved 20 times more sensitive than IS6110-based conventional PCR. Moreover, its sensitivity was, respectively, 50 and 20 times higher than the one obtained with the two previously described LAMP assays for M. tuberculosis, based on gyrB and rrs, respectively. Identical sensitivities were obtained for LAMP and nested PCR, but the LAMP assay was more rapid and cost-effective than the latter. Although, our LAMP assay can successfully be performed using a non-denatured template, this results in a 200-fold reduction in the sensitivity of the assay. Moreover, by performing our LAMP assay on 15 clinical sputum samples from TB patients we were able to detect MTB. Taken together, our preliminary results indicate that IS6110-based MTBC-LAMP assay is a promising new TB-diagnostic test, with high sensitivity and that could easily be applied for the diagnosis of TB in a low-resource setting.

Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA.

BACKGROUND: The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. METHODS: The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. RESULTS AND CONCLUSION: The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.

Synthesis, lipophilicity study and in vitro evaluation of some rodenticides as acetylcholinesterase reversible inhibitors.

The anti-AChE activity of phosphoramidates has been noticed for many years. Because of the wide application of phosphoramidates in recent years, there has been a continuing research for synthesis, purification and identification of effective and safe derivatives. In this study some rodenticides with the general formula Me(2)NP(O)(p-OC(6)H(4)-X)(2), where X = H, CH(3), Cl, have been synthesized in water (without organic solvent) and characterized by (31)P, (31)P {(1)H}, (13)C and (1)H NMR spectroscopy. Since lipophilicity has been recognized for its importance in QSAR studies, efforts have been made to determine the logP values. The ability of these rodenticides to inhibit human acetylcholinesterase (hAChE) has been predicted with PASS (Prediction of Activity Spectra for Substances) software (version 1.917) and then has been evaluated by a modified Ellman's assay and spectrophotometric measurements.