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.

The effect of MS14 on innate and cellular immune responses in BALB/c mice.

MS14 is an Iranian natural preparation of herbal-marine source with no obvious toxicity in oral administration, which possesses anti-inflammatory and immunomodulatory effects. In this study, the effect of oral administration of MS14 on nitric oxide (NO) production of peritoneal macrophages and lymphocyte Th1 cytokines and delayed-type hypersensitivity (DTH) test in BALB/c mice were investigated. Peritoneal macrophages were cultured and NO production was measured by Griess method. Viability of macrophages was assayed by MTT (3-(4,5-dimethy-2-lthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) test. Interleukin-2 (IL-2) and INFγ levels in supernatant of spleen lymphocytes culture were assayed by enzyme-linked immunosorbent assay kit. For DTH test the mice were immunized with sheep red blood cell and DTH was measured 24 h after the last immunization of mice. NO production of macrophages has been diminished significantly in MS14 treated group (about 40%) at the presence or absence of stimulators. Macrophage viability had no significant alteration after MS14 administration. However, interferon-γ production of lymphocytes was significantly decreased in MS14 group both at the absence or presence of concanavalin A (ConA; about 50%); IL-2 production declined about 20% at the presence of ConA. In comparison with the control group, MS14 had no statistically significant effect on DTH test. The results have pointed that MS14 may have immunomodulatory potentials at least through its decreasing effect on NO production of macrophages and level of Th1 cytokine pattern.

Distribution and Genetic Diversity of Salmonella enterica in the Upper Suwannee River.

The Suwannee River spans the Florida/Georgia border to the Gulf of Mexico, and contributes to regional irrigation and recreational activities. Association of Salmonella enterica with these resources may result in the contamination of produce and disease outbreaks. Therefore, surface water was examined for the distribution of S. enterica at multiple time points from 4 sites on the upper Suwannee River. Isolates were confirmed by detection of the invA gene, and 96% of all samples were positive for the bacterium. Most probable number enumeration ranged from <18 to 5400 MPN/100 mL. Genetic diversity of these isolates (n=110) was compared to other environmental (n=47) or clinical (n=28) strains and to an online library (n=314) using DiversiLab rep-PCR. All strains showed >60% similarity and distributed into 16 rep-PCR genogroups. Most (74%) of the Suwannee River isolates were clustered into two genogroups that were comprised almost exclusively (97%) of just these isolates. Conversely, 85% of the clinical reference strains clustered into other genogroups. However, some Suwannee River isolates (12%) were clustered with these primarily clinically-associated genogroups, supporting the hypothesis that river water can serve as a disease reservoir and that pathogenic strains may persist or possibly originate from environmental sources.