OBJEVTIVETo study the effect of spvB/spvC gene on Salmonella virulence and the Host immune.

METHODSSTM.211, STM.211-Delta;spvB, STM.211-Delta;spvC, STM.211-Delta;spvB.spvC and PBS were infected with 0.2 mL 10(5) CFU corresponding strain respectively by intraperitoneal. We observed the mental status, movement, diarrhea, weight, pelage changed hair of the infected mouse. Then the level of IL-10, IL-12, IFN-γ were detected by ELISA. Finally, we observe the pathological changes of liver and spleen with the general view and the microscope.

RESULTSInfection symptoms of STM.211, STM.211-Delta;spvB and STM.211-Delta;spvC were significantly worse than PBS group, but there was no significant difference between STM.211-spvB.spvC group and PBS group. The secretion of IFN-γ and IL-12 of STM.211, STM.211-Delta;spvB, STM.211-Delta;spvC group were significantly lower than those in the STM.211-Delta;spvB.spvC group (P<0.05), but IL-10 secretion was significantly higher than STM.211-Delta;spvB.spvC group (P<0.05). There were no statistical significance among the STM.211, STM.211-Delta;SpvB, STM.211-Delta;spvC groups (P>0.05).

CONCLUSIONSSalmonella virulence can be affected obviously by spvB combined with spvC gene, but not by spvB or spvC. spvB/spvC gene can inhibit the TH1 cytokines (IFN-γ and IL-12) secretion but promote the TH2 cytokines (IL-10) expression, leading immune response trend to TH2 shift. It shows that spvB/spvC gene can help the bacteria evade the host immune defenses, leading to aggravation of infection.

Animals ; Cytokines ; immunology ; Interleukin-12 ; Mice ; Salmonella ; genetics ; pathogenicity ; Salmonella Infections ; immunology ; Virulence ; Virulence Factors ; genetics

OBJECTIVETo construct the eukaryotic expression plasmid containing lvgA gene flanked with CpG motifs of Legionella pneumophila for its expression in NIH3T3 cells.

METHODSlvgA gene flanked with CpG motifs of Legionella pneumophila was amplified by PCR. The PCR products was inserted into the eukaryotic expression plasmid pcDNA3.1/myc-his(+) to construct the recombinant plasmid pclvgA/CpG, which was subsequently transfected into NIH3T3 cells via lipofection. Immunofluorescence analysis was carried out to detect the transient expression of the plasmid in the cells.

RESULTSSequence analysis showed that the recombinant plasmid pclvgA/CpG contained the lvgA/CpG fragment with a length of 657 bp, encoding a protein of 27.7 Ku. Immunofluorescence analysis identified the transient expression of the recombinant plasmid pclvgA/CpG in NIH3T3 cells.

CONCLUSIONThe lvgA gene flanked with CpG motifs of Legionella pneumophila has been constructed successfully, and the transient expression of the recombinant plasmid pclvgA/CpG can be detected in NIH3T3 cells.

Animals ; Bacterial Vaccines ; genetics ; CpG Islands ; genetics ; Legionella pneumophila ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection ; Virulence Factors ; biosynthesis ; genetics

OBJECTIVE: To evaluate the efficacy of cis-2-dodecenoic acid (BDSF) in the treatment and prevention of vaginal candidiasis in vivo. METHODS: The activities of different concentrations of BDSF against the virulence factors of Candida albicans (C. albicans) were determined in vitro. An experimental mouse model of Candida vaginitis was treated with 250 μmol/L BDSF. Treatment efficiency was evaluated in accordance with vaginal fungal burden and inflammation symptoms. RESULTS: In vitro experiments indicated that BDSF attenuated the adhesion and damage of C. albicans to epithelial cells by decreasing phospholipase secretion and blocking filament formation. Treatment with 30 μmol/L BDSF reduced the adhesion and damage of C. albicans to epithelial cells by 36.9% and 42.3%, respectively. Treatment with 200 μmol/L BDSF completely inhibited phospholipase activity. In vivo mouse experiments demonstrated that BDSF could effectively eliminate vaginal infection and relieve inflammatory symptoms. Four days of treatment with 250 μmol/L BDSF reduced vaginal fungal loads by 6-fold and depressed inflammation. Moreover, BDSF treatment decreased the expression levels of the inflammatory chemokine-associated genes MCP-1 and IGFBP3 by 2.5- and 2-fold, respectively. CONCLUSION BDSF is a novel alternative drug that can efficiently control vaginal candidiasis by inhibiting the virulence factors of C. albicans.
Animals ; Candida albicans ; drug effects ; metabolism ; pathogenicity ; physiology ; Candidiasis, Vulvovaginal ; drug therapy ; genetics ; immunology ; microbiology ; Chemokine CCL2 ; genetics ; immunology ; Disease Models, Animal ; Fatty Acids, Monounsaturated ; administration & dosage ; Female ; Fungal Proteins ; genetics ; metabolism ; Humans ; Insulin-Like Growth Factor Binding Protein 3 ; genetics ; immunology ; Mice ; Virulence ; drug effects ; Virulence Factors ; genetics ; metabolism

In the present study, Staphylococcus (S.) hyicus strains isolated in Russia (n = 23) and Germany (n = 17) were investigated for the prevalence of the previously described genes sheta and shetb. Sheta was detected in 16 S. hyicus strains. Sheta-positive strains were mainly found among strains isolated from exudative epidermitis, and frequently together with the exfoliative toxin-encoding genes exhD and exhC. Partial sequencing of sheta in a single S. hyicus strain revealed an almost complete match with the sheta sequence obtained from GenBank. None of the S. hyicus strains displayed a positive reaction with the shetb-specific oligonucleotide primer used in the present study. According to the present results, the exotoxin encoding gene sheta seems to be distributed among S. hyicus strains in Russia and Germany. The toxigenic potential of this exotoxin, which does not have the classical structure of a staphylococcal exfoliative toxin, remains to be elucidated.
Animals ; Cattle ; Cattle Diseases/epidemiology/microbiology ; DNA Primers ; Dog Diseases/epidemiology/microbiology ; Dogs ; Epidermitis, Exudative, of Swine/epidemiology ; Exfoliatins/*genetics/immunology ; Germany ; Pneumonia/epidemiology/veterinary ; Russia ; Staphylococcal Infections/immunology/veterinary ; Staphylococcus aureus/genetics/*pathogenicity ; Swine ; Swine Diseases/epidemiology ; Virulence/*genetics ; Virulence Factors/genetics/immunology

Dermonecrotic toxin (DNT) is identified as one of the most important virulence factor of Bordetella bronchiseptica. The complete coding sequence (4 356 bp) of the dnt gene was cloned into the prokaryotic expression vector pET-28a, and expressed in the Eschierichia coli BL21 (DE3) under IPTG (Isopropyl-beta-D-thiogalactopyranoside) induction. The recombinant His6-DNT protein showed immunological reactivity in the Western-blot analysis. The recombinant protein was purified from crude lysates of BL21 harboring pET-DNT with the purity of 93.2%. His6-DNT showed the dermonecrotic effects in the infant mouse assay. However, rabbit anti-serum against recombinant DNT protein could neutralize the dermonecrotic effects of native DNT to the infant mice in vivo. These findings suggest that the recombinant DNT protein retained the characteristics and immunogenicity of native DNT. Furthermore, this approach could be used to induce active immunity and serum immunoglobulin for production of a passive therapeutic reagent. In this study, we have shown that the recombinant His6-DNT protein retained the characteristics of native DNT of B. bronchiseptica, which built a good foundation for the further research on the structure and function of DNT.
Animals ; Animals, Newborn ; Bordetella bronchiseptica ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Mice ; Neutralization Tests ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Transglutaminases ; biosynthesis ; genetics ; Virulence Factors, Bordetella ; biosynthesis ; genetics

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.
ADP Ribose Transferases ; genetics ; Animals ; Bacterial Toxins ; genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Exotoxins ; genetics ; Female ; Gene Expression ; Immunization ; Influenza A virus ; immunology ; physiology ; Lung ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Viral Matrix Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Virulence Factors ; genetics

Verocytotoxic Escherichia (E.) coli strains are responsible for swine oedema disease, which is an enterotoxaemia that causes economic losses in the pig industry. The production of a vaccine for oral administration in transgenic seeds could be an efficient system to stimulate local immunity. This study was conducted to transform tobacco plants for the seed-specific expression of antigenic proteins from a porcine verocytotoxic E. coli strain. Parameters related to an immunological response and possible adverse effects on the oral administration of obtained tobacco seeds were evaluated in a mouse model. Tobacco was transformed via Agrobacteium tumefaciens with chimeric constructs containing structural parts of the major subunit FedA of the F18 adhesive fimbriae and VT2e B-subunit genes under control of a seed specific GLOB promoter. We showed that the foreign Vt2e-B and F18 genes were stably accumulated in storage tissue by the immunostaining method. In addition, Balb-C mice receiving transgenic tobacco seeds via the oral route showed a significant increase in IgA-positive plasma cell presence in tunica propria when compared to the control group with no observed adverse effects. Our findings encourage future studies focusing on swine for evaluation of the protective effects of transformed tobacco seeds against E. coli infection.
Administration, Oral ; Agrobacterium tumefaciens ; Animals ; Antigens, Bacterial/genetics/metabolism ; Bacterial Vaccines/administration & dosage/adverse effects/*pharmacology ; Edema Disease of Swine/*immunology/microbiology ; Escherichia coli Infections/immunology/microbiology/*veterinary ; Escherichia coli Proteins/*genetics/metabolism ; Female ; Fimbriae Proteins/genetics/metabolism ; Genetic Engineering ; Intestines/immunology/microbiology/pathology ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plants, Genetically Modified/*genetics/metabolism ; Seeds/genetics/metabolism ; Shiga Toxin 2/genetics/metabolism ; Shiga-Toxigenic Escherichia coli/genetics/immunology/*pathogenicity ; Swine ; Tobacco/*genetics/metabolism ; Virulence Factors/genetics/metabolism

AIMTo elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.

METHODOLOGYA genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)).

RESULTSP. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate.

CONCLUSIONThe combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Anti-Bacterial Agents ; pharmacology ; Chromosome Mapping ; DNA Transposable Elements ; genetics ; Drug Resistance, Bacterial ; genetics ; E-Selectin ; analysis ; immunology ; Endothelial Cells ; immunology ; microbiology ; Gene Deletion ; Humans ; Lipid A ; analysis ; immunology ; Lipopolysaccharides ; analysis ; immunology ; Mutagenesis, Insertional ; genetics ; Open Reading Frames ; genetics ; Phosphoric Monoester Hydrolases ; genetics ; physiology ; Polymyxin B ; pharmacology ; Porphyromonas gingivalis ; enzymology ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Toll-Like Receptor 4 ; analysis ; immunology ; Virulence Factors ; physiology

The threshold hypothesis of attenuated lentiviral vaccine considers that the type of host response to infections of lentiviruses depends on the viral load. To evaluate the correlation between viral loads of the attenuated vaccine strain of equine infectious anemia virus (EIAV) and their effects to induce protective immunity, longitudinal plasma viral loads in groups of horses inoculated with either an attenuated EIAV vaccine strain (EIAV(DLV125)) or sub-lethal dose of an EIAV virulent strain (EIAV(LN40)) were compared. Similar levels of plasma viral loads ranging from 10(3)-10(5) copies/mL were detected from samples of these two groups of animals (P > 0.05) during 23 weeks post the inoculation. However, different responses to the challenge performed thereafter with lethal dose of the EIAV virulent strain were observed from the groups of horses inoculated with either EIAV(DLV125) or sub-lethal dose of EIAV(LN40). The protective efficiency was 67% (3 of 4 cases) and 0 (none of 2 cases), respectively. Our results implicate that the viral load of EIAV attenuated vaccine is not the primary factor, or at least not the solo primary factor, to determine the establishment of immune protection.
Animals ; Equine Infectious Anemia ; blood ; immunology ; prevention & control ; Horses ; Immunization ; methods ; Infectious Anemia Virus, Equine ; immunology ; pathogenicity ; RNA, Viral ; blood ; genetics ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Vaccines, Attenuated ; administration & dosage ; immunology ; Viral Load ; Viral Vaccines ; administration & dosage ; immunology ; Virulence ; immunology

A recombinant immunotoxin named CEA/PE38/KDEL was constructed, which was composed of anti-CEA single-chain Fv and the truncated and modified form of Pseudomonas exotoxin (PE38/KDEL). The CEA/PE38/KDEL immunotoxin was expressed in the E. coli strain BL21 (DE3)-star as inclusion bodies. The denatured inclusion bodies were purified with Ni-NTA chelate agarose, then the constant gradient dialysis was used to perform the refolding of the CEA/PE38/KDEL immunotoxin. Results of FACS and MTT assay indicate that the refolded immunotoxins keep potent and specific cytotoxicity to tumor cells bearing CEA antigens.
ADP Ribose Transferases ; biosynthesis ; genetics ; pharmacology ; Antibodies ; genetics ; metabolism ; pharmacology ; Antineoplastic Agents ; metabolism ; pharmacology ; Bacterial Toxins ; biosynthesis ; genetics ; pharmacology ; Carcinoembryonic Antigen ; immunology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Exotoxins ; biosynthesis ; genetics ; pharmacology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; Immunotoxins ; genetics ; isolation & purification ; metabolism ; pharmacology ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Virulence Factors ; biosynthesis ; genetics ; pharmacology