Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.
This study was designed to examine the chemical composition of essential oil and in vitro antioxidant and antimicrobial activity of the essential oil and methanol extracts of Eucalyptus largiflorens F. Muell. The chemical composition of the hydrodistilled essential oil of the leaves of E. largiflorens was analyzed by gas chromatography and gas chromatography/mass spectrometry. The main constituents of the oil were found to be 1,8-cineole (23.1%), cryptone (15.1%), 4-allyloxyimino-2-carene (11.2%), and 4-terpineol (9.6%). The essential oil showed strong antibacterial activity against the studied microorganisms, except for P. aeruginosa, while polar subfraction of methanol extract had moderate antimicrobial activity and the nonpolar subfraction of methanol extract did not show any antimicrobial activity. In contrast, the extract showed better activity than the essential oil in antioxidant activity assays employed, e.g., in a 2,2-diphenyl-1-picryl-hydrazyl system. Also, the polar subfraction of methanol extract (15.1 ± 0.7 µg/ml) showed the highest radical-scavenging activity. In the second case, the inhibition capacity (%) of the nonpolar subfraction (92.7% ± 2.5) was found to be the stronger one. Finally, the total amounts of phenol constituents in the polar subfraction (142.6 ± 0.9 µg/mg), nonpolar subfraction (68.6 ± 0.4 µg/mg), and the oil (36.0 ± 0.6 µg/mg) were determined.
Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10(4) LD(50). The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections.
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